Modern Surgical Care: Physiologic Foundations and Clinical Applications. Volume 1

medwedi.ru MODERN SURGICAL CARE Third Edition Volume 1 medwedi.ru Associate Editors Barbara L. Bass, M.D. Professo...

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MODERN SURGICAL CARE Third Edition Volume 1

medwedi.ru

Associate Editors

Barbara L. Bass, M.D. Professor of Surgery Department of Surgery Weill Medical College of Cornell University New York, New York, USA Carolyn and John F. Bookout Chair Department of Surgery The Methodist Hospital Houston, Texas, USA

Peter J. Fabri, M.D. Professor of Surgery Associate Dean for Graduate Medical Education University of South Florida College of Medicine Tampa, Florida, USA

Carl E. Haisch, M.D. Professor of Surgery Department of Surgery East Carolina University Brody School of Medicine Director of Surgical Immunology and Transplantation Attending Surgeon Pitt County Memorial Hospital Greenville, North Carolina, USA

David W. Mercer, M.D. Professor and Vice Chairman of Surgery Department of Surgery The University of Texas Health Science Center–Houston Chief of Surgery Lyndon Baines Johnson General Hospital Houston, Texas, USA

Ronald C. Merrell, M.D. Professor of Surgery Department of Surgery Virginia Commonwealth University School of Medicine and Medical Center Richmond, Virginia, USA

Stuart I. Myers, M.D. Professor of Surgery Division of Vascular Surgery Department of Surgery Virginia Commonwealth University School of Medicine and Medical Center Attending Surgeon Hunter Holmes McGuire Veterans Affairs Medical Center Richmond, Virginia, USA

MODERN SURGICAL CARE Physiologic Foundations and Clinical Applications

Third Edition Volume 1

editor-in-chief

Thomas A. Miller, M.D. Ammons Professor of Surgery Division of General Surgery Department of Surgery Virginia Commonwealth University School of Medicine and Medical Center Chief of Surgery Hunter Holmes McGuire Veterans Affairs Medical Center Richmond, Virginia, USA

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Informa Healthcare USA, Inc. 270 Madison Avenue New York, NY 10016 © 2006 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-2869-6 (Hardcover) International Standard Book Number-13: 978-0-8247-2869-4 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

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Dedication

DEDICATION

To all trainees in surgery, medical students rotating on surgical services, and surgeons in practice who recognize the importance of understanding the physiologic underpinnings of treating surgical disease in order to ensure optimal patient care.

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Preface

When the first edition of this book was published in 1988 under the title Physiologic Basis of Modern Surgical Care, its goal was to bridge the gap that commonly exists between basic science information regarding human disease and the ability to apply this knowledge to everyday clinical care. In teaching medical students rotating on surgery, as well as residents embarking on surgical careers, many of my colleagues and I, serving on medical school faculties across the country, were all too acutely aware that management of surgical disorders by these trainees was frequently based on memorized schemes or protocols rather than a thorough understanding of the pathophysiology underlying a particular disease. To address this issue, we published the initial edition with the intent to approach surgical disease as a derangement in normal physiology and that the best way to diagnose and treat it was to understand thoroughly this deviation from normal. We were very gratified with the acceptance of the book by the surgical community and its adoption by many program directors as essential reading to prepare residents for various examinations, such as the annual in-service training examination in surgery and the board certification examination following completion of surgical training. We also received many positive comments from established surgeons regarding its usefulness in preparing for recertification exams. Because of the wide acceptance of the first edition as an important vehicle to train house officers in the physiologic underpinnings of surgical disease, a second edition was published in 1998 under the title Modern Surgical Care: Physiologic Foundations and Clinical Applications. The title change was made to more adequately reflect the linkage between physiology and clinical care. We have been especially pleased with the continuing acceptance of the second edition as an important educational tool and were extremely grateful when Informa Healthcare (formerly Marcel Dekker, Inc.) asked us to produce a third edition. In addition to being thoroughly updated, the third edition reflects the capable assistance and counsel of a group of associate editors who have contributed greatly in streamlining chapters, minimizing repetitive material, adding new chapters to include cutting-edge material, and selecting a more diverse and mainstream authorship who are the current leaders in their fields. Their recommendations were responsible for the inclusion of 91 new authors

not involved in the previous editions. These associate editors include Drs. Barbara Bass, Jeff Fabri, Carl Haisch, Dave Mercer, Ron Merrell, and Stuart Myers. To each of them, I offer my utmost thanks and gratitude. As reflected in the previous editions, the needs of the general surgeon continue to be emphasized in the present edition. Further, the goal of this third edition remains the same, namely to approach surgical disease as a derangement in normal physiology, which needs to be corrected to as near normal as possible to effectively manage and treat the underlying disorder. This book is divided into nine parts, with the first part devoted to information pertinent to the body as a whole, and the eight remaining sections focused on specific organ systems or themes. Diseases affecting the reproductive organs, disorders of the head and neck (other than the thyroid and parathyroid glands), and disorders of the musculoskeletal system are not specifically dealt with because the current practice of surgery only rarely involves these disciplines. Finally, this book is not meant to replace standard textbooks of surgery and, accordingly, is not a comprehensive discussion of all surgical diseases. For less common types of surgical problems or those not regularly encountered in the practice of general surgery, such books should be consulted. I would personally like to offer my sincere appreciation to the folks at Informa Healthcare who have worked with us during the production of the book. I have found everyone with whom I have collaborated to be extremely professional, cordial, and helpful in making sure that this third edition meets the goals that we have set. I would especially like to recognize two people with whom I have had a very close working relationship. The first is Joe Stubenrauch who has been the project editor. He has more than met my expectations and has demonstrated unusual adaptability when I thought something should be done for which initially he and I had differing viewpoints. The second individual is Joanne Jay who was responsible for the final editing of each chapter and preparing the galley proofs. Her attention to detail has been exemplary. I have been very pleased with the outstanding quality of her editorial review. To both of these individuals I say ‘‘Thank you!’’ They made the process of finalizing this book most pleasurable.

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Thomas A. Miller, M.D.

Contents

6. Surgical Infection: Principles of Management and Antibiotic Usage / 127

Preface / v Contributors / xiii

VOLUME 1 PART ONE: GENERAL CONSIDERATIONS IN THE MANAGEMENT OF SURGICAL PATIENTS

1. Metabolic Response to Starvation, Stress, and Sepsis / 1 Joseph F. Amaral, Michael D. Caldwell, and Thomas A. Miller Introduction / 1 Body Composition / 1 Normal Metabolism: Intermediary Metabolism and Substrate Interactions / 8 Neuroendocrine Regulatory Mechanisms / 15 Metabolic Response in Starvation, Injury, and Sepsis / 21 Cytokines / 26 Summary / 28 References / 28

2. Pathophysiology of Fluid and Electrolyte Disorders / 33 Peter J. Fabri and Mark Bloomston Introduction / 33 Maintenance of the Internal Milieu / 33 Homeostatic Control Mechanisms / 35 Pathophysiology and Treatment of Specific Electrolyte and Acid/Base Abnormalities / 38 Principles of Fluid Therapy / 45 Summary / 46 References / 46

3. Surgical Nutrition / 49 Rosemary A. Kozar, Margaret M. McQuiggan, and Frederick A. Moore Introduction / 49 Metabolic Response to Stress vs. Starvation / 49 Rationale for Nutritional Support / 50 Initiation of Nutritional Support / 50 Enteral Nutrition / 53 Total Parenteral Nutrition / 56 Disease-Specific Nutrition / 60 Summary / 61 References / 61

Kathryn M. Verbanac, Lorita Rebellato, and Carl E. Haisch Introduction / 65 An Overview of the Immune System / 65 The Immunocompromised Surgical Patient / 76 Summary / 86 References / 87

Yuan Zhai and Rafik M. Ghobrial Introduction / 91 Allograft Rejection / 91 Immunosuppressive Therapy / 112 Clinical Transplant Outcomes / 120 Summary / 124 References / 124

7. Hemostasis and Thrombosis in the Surgical Patient / 157 Stuart I. Myers, Mark R. Jackson, Michael Sobel, and G. Patrick Clagett Introduction / 157 Mechanisms of Hemostasis / 157 Regulation of Hemostasis / 159 Inherited Disorders of Coagulation / 161 Inherited Qualitative Platelet Disorders / 163 Acquired Disorders of Hemostasis / 164 Acquired Disorders of Platelet Function / 164 Bleeding in the Surgical Patient / 165 Congenital Disorders of Hypercoagulability / 167 Acquired Hypercoagulable Disorders / 168 Antithrombotic Therapy / 170 Therapeutic Agents / 171 Summary / 174 References / 174

8. Pathophysiology of Shock / 181

4. The Immune System and the Immunocompromised Patient / 65

5. Physiologic Basis of Transplantation / 91

Christina Paylan and Rodney Durham Introduction / 127 Pathophysiology of Surgical Infections / 127 Systemic Inflammatory Response / 128 Overall Approach to Sepsis / 129 Definitions of Surgical Infections / 130 Evaluation of Suspected Infection in the Surgical Patient / 131 Role of the Laboratory in Infection Diagnosis / 131 Types of Postoperative Infections / 133 Pathogens Responsible for Surgical Infection / 140 Antibiotics in the Management of Infection / 141 Clinical Use of Antibiotics / 148 Summary / 153 References / 153

Ajai K. Malhotra Introduction / 181 Definition of Shock / 181 Cardiovascular Physiology and Types of Shock / 182 Pathophysiologic Response to Shock / 184 Management Considerations / 190 The Future / 193 Summary / 193 References / 194

9. Neoplastic Disease: Pathophysiology and Rationale for Treatment / 197 Gregory Kennedy and John E. Niederhuber Introduction / 197 Basic Concepts of Cancer Biology / 197 Biologic Rationale for Therapy / 202 Summary / 208 References / 208

10. The Physiology of Anesthesia and Pain / 213 Charles Williams and Denise Lester Introduction / 213 The Physiology of Anesthesia / 213 Preoperative Decisions / 213

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Contents Regional Anesthesia / 214 General Anesthesia / 215 Conscious Sedation / 217 Anesthetic Implications of Selected Disease States / 217 Anesthesia Emergencies / 218 The Physiology of Pain and Analgesia / 219 The Physiology of Nociception / 219 Pain Measurement / 221 The Management of Acute Postoperative Pain / 221 Summary / 224 References / 224

15. Gastric Physiology and Acid-Peptic Disorders / 333 Kenneth S. Helmer and David W. Mercer Introduction / 333 Normal Physiology / 333 Gastric Physiology / 338 Abnormal Physiology / 346 Summary / 362 References / 362

& Small and Large Intestine

16. Physiology of Digestion and Absorption / 369 11. Sepsis and the Syndrome of Multiple Organ Failure / 227 Lena M. Napolitano Introduction / 227 Sepsis: Definitions / 227 Incidence and Outcomes of Sepsis / 229 Pathophysiology of Sepsis / 230 Genetic Variability in Sepsis / 232 Treatment Strategies in Sepsis / 234 Multiple Organ Dysfunction and Failure: Definitions / 244 Incidence and Outcome of MODS / 244 Pathophysiology of MODS / 246 Potential Treatment Strategies for Reduction of MODS / 246 Summary / 247 References / 248

12. Application of Cellular and Molecular Biology in Modern Surgical Practice / 253 Huiping Zhou and Jian-Ying Wang Introduction / 253 Basic Genetic Mechanisms / 253 Cellular and Molecular Biological Technology: From Recombinant DNA to Transgenic Animals / 259 The Molecular Organization of the Cells / 263 Novel Treatment Strategies in Modern Surgical Care / 267 Summary / 269 References / 269

13. Physiologic Principles in Preparing Patients for Surgery / 271 Henry J. Schiller, Kara C. Kort, and Lelan F. Sillin Introduction / 271 General Aspects of Preoperative Preparation / 271 Specific Aspects of Preoperative Preparation / 273 Risks of Hematologic Disease / 286 Prophylaxis Against Thromboembolism / 289 HIV Infection and AIDS / 289 Summary / 290 References / 290

Bobby S. Glickman and Jon S. Thompson Introduction / 369 Fluid and Electrolyte Secretion and Absorption / 369 Overview of Digestion / 371 Protein Absorption / 372 Carbohydrate Absorption / 374 Fat Absorption / 376 Vitamins and Minerals / 377 Regulation / 378 Diarrhea and Malabsorption / 378 Summary / 379 References / 379

17. Circulation and Vascular Disorders of the Splanchnic Vascular Bed / 381 Stuart I. Myers and Patricia A. Lowry Introduction / 381 Normal Anatomy and Collateral Circulation / 381 Physiology of Intestinal Circulation / 384 Reperfusion Injury of the Intestine / 386 Clinical Evaluation of Intestinal Blood Flow / 392 Radiologic Evaluation of Patients with Acute Mesenteric Ischemia / 393 Intraoperative Assessment of Intestinal Viability / 396 Diseases That Affect the Visceral Vessels / 397 Miscellaneous Diseases That Affect the Viscera / 403 Summary / 406 References / 408

18. Inflammatory Disorders of the Small Bowel and Colon / 415 Douglas J. Turner and Barbara L. Bass Introduction / 415 Inflammatory Bowel Disease / 415 Appendicitis / 419 Meckel’s Diverticulum / 421 Jejunoileal Diverticuli / 421 Colonic Diverticular Disease / 422 Clostridium difficile Colitis / 423 Radiation Enteritis / 423 Summary / 424 References / 424

PART TWO: THE ALIMENTARY TRACT & Liver, Biliary Tract, Pancreas, and Spleen & Esophagus and Stomach

14. Physiologic Dysfunction of the Esophagus / 295 Nahid Hamoui and Peter F. Crookes Introduction / 295 Anatomy and Physiology / 295 Symptoms of Esophageal Disease / 296 Physical Examination / 297 Investigations / 297 Esophageal Diseases / 304 Esophageal Motor Disorders / 316 Esophageal Emergencies / 321 Esophageal Tumors / 323 Summary / 328 References / 328

19. Hepatic Physiology / 427 Jose M. Prince and Timothy R. Billiar Introduction / 427 Embryology / 427 Histology / 428 Anatomy / 429 Hepatic Functions / 431 Metabolic Homeostasis / 432 Pathophysiology / 435 Diagnostic Testing / 436 Regeneration / 438 Future / 438 Summary / 438 References / 439

Contents

20. Portal Hypertension / 443

26. The Anatomy, Physiology, and Differential Diagnosis of Acute Abdominal Pain / 539

Alexander S. Rosemurgy and Emmanuel E. Zervos Introduction / 443 Anatomy / 443 Variceal Hemorrhage / 443 Summary / 452 References / 452

21. Calculous Disease of the Gallbladder and Common Bile Duct / 455 Lillian S. Kao and Terrence H. Liu Introduction / 455 Epidemiology / 455 Bile Physiology / 455 Pathogenesis of Gallstones / 459 Clinical Manifestations of Gallstone Disease / 461 Treatment / 463 Summary / 465 References / 465

22. Normal Exocrine Function and Inflammatory Diseases of the Pancreas / 469 David J. Bentrem and Raymond J. Joehl Introduction / 469 Embryology / 469 Gross Anatomy / 469 Acinus / 470 Pancreatic Exocrine Function / 470 Control of Pancreatic Secretion / 471 Acute Pancreatitis / 471 Treatments / 475 Summary / 479 References / 479

23. The Jaundiced Patient / 483 Attila Nakeeb and Henry A. Pitt Introduction / 483 Bilirubin Metabolism / 483 Classification of Jaundice / 483 Pathophysiology of Jaundice / 484 Diagnostic Approach / 487 Patient Management / 492 Benign Disease / 495 Malignant Disease / 497 Summary / 499 References / 499

24. The Spleen / 503 Haytham M. A. Kaafarani and Kamal M. F. Itani Introduction / 503 Gross Anatomy / 503 Embryology / 503 Histology / 504 Physiology and Function of the Spleen / 505 Hematologic Disorders and Splenectomy / 507 Malignancies and Splenectomy / 511 Miscellaneous Conditions / 513 Splenic Trauma / 515 Splenectomy / 520 References / 522

Kathryn A. Richardson, Ryan M. Wolfort, and Richard H. Turnage Introduction / 539 Types of Abdominal Pain / 539 Anatomy and Physiology of Abdominal Pain / 539 Pathophysiologic Stimuli for Somatic and Visceral Nociceptors / 541 Evaluation of Patients with Acute Abdominal Pain / 541 Prototypical Examples of Acute Abdominal Pain / 547 Abdominal Pain in Special Patient Groups / 550 Summary / 553 References / 553

27. Neoplastic Disorders of the Gastrointestinal Tract / 555 Carlos A. Murillo, Kenneth J. Woodside, Lindsey N. Jackson, and B. Mark Evers Introduction / 555 Cellular and Molecular Biology of GI Cancers / 555 Neoplastic Diseases of the Stomach, Small Bowel, and Colorectum / 562 Summary / 579 References / 580

28. Mechanical Disorders of the Stomach, Duodenum, and Intestine / 587 Sean P. Harbison and Daniel T. Dempsey Introduction / 587 Mechanical Disorders of the Stomach and Duodenum / 587 Mechanical Disorders of the Small Bowel / 590 Mechanical Disorders of the Colon / 595 Summary / 597 References / 597

29. Physiologic Derangements of the Anorectum and the Defecatory Pelvic Floor / 599 Janette Gaw and Walter E. Longo Introduction / 599 Anorectal Anatomy / 599 Normal Physiology of the Anus and Rectum / 603 Colonic Function and Anorectal Physiology Testing / 604 Disordered Anorectal Physiology / 605 Neoplasms of the Anal Canal and Anal Margin / 611 Summary / 613 References / 613

30. Derangements in Gastrointestinal Function Secondary to Previous Surgery / 617 Jeannie F. Savas, Thomas A. Miller, and David W. Mercer Introduction / 617 Gastric Dysfunction / 617 Intestinal Dysfunction / 625 Summary / 628 References / 629 Index / I-1

VOLUME 2 PART THREE: THE CARDIOTHORACIC SYSTEM

& Other Conditions

& Lung

25. Gastrointestinal Hemorrhage / 527

31. Pathobiology of Surgically Relevant Pulmonary Disease / 631

Kevin Bruen and Leigh Neumayer Introduction / 527 Initial Evaluation and Management / 527 Upper Intestinal Bleeding / 528 Lower Intestinal Bleeding / 532 Occult Bleeding / 535 Summary / 536 References / 536

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Daniel G. Tang, Jonathan Kiev, and Neri M. Cohen Introduction / 631 Anatomy and Physiology / 631 Perioperative Pulmonary Assessment / 636 Common Pulmonary Disorders / 637 Neoplastic Conditions / 643 Lung Transplantation / 645

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Contents Summary / 646 References / 647

& Heart

32. Normal Cardiac Function / 649 Andrew C. Fiore and Andrew S. Wechsler Introduction / 649 Molecular Mechanisms in Contraction and Relaxation / 649 Mechanics of Isolated Muscle / 651 Function of the Intact Heart / 653 Heart Failure / 660 Summary / 661 References / 661

33. Heart Failure and Resuscitation / 663 Heinrich Taegtmeyer Introduction / 663 Heart Failure / 663 Resuscitation / 671 Summary / 673 References / 674

34. Mechanical Support for the Failing Heart: Current Physiologic Concepts of Management / 677 Sina L. Moainie and Bartley P. Griffith Introduction / 677 Cardiac Support in End-Stage Heart Failure / 677 Summary / 683 References / 683

35. Congenital Heart Lesions / 685 Ralph S. Mosca and Edward L. Bove Introduction / 685 Adjustments in the Circulation After Birth / 685 Congestive Heart Failure / 685 Obstructive Lesions / 686 Left-to-Right Shunts / 689 Right-to-Left Shunts / 691 Inadequate Mixing / 693 Hypoplastic Left Heart Syndrome / 695 Summary / 695 References / 696 Further Reading / 697

38. Urinary Tract Obstruction / 767 J. Robert Ramey and Deborah T. Glassman Introduction / 767 The Upper Urinary Tract / 767 The Lower Urinary Tract / 770 Summary / 772 References / 772

39. Neurogenic Lower Urinary Tract Dysfunction / 775 Hari Siva Gurunadha Rao Tunuguntla and Unyime O. Nseyo Introduction / 775 Anatomy and Physiology of Continence and Micturition / 775 Pathophysiology of LUT Dysfunction / 780 Definition of Common Terms in Neurogenic Voiding Dysfunction / 781 Specific Neurologic Lesions / 781 Classification of NLUTD / 783 Diagnosis of NLUTD / 783 Management of NLUTD / 787 Device Therapy / 791 Urinary Diversion / 792 Quality of Life / 792 Follow-Up / 792 Summary / 793 References / 793

PART FIVE: THE CENTRAL AND PERIPHERAL NERVOUS SYSTEMS

40. Pathophysiology and Management of Head Injury / 795 Egon M. R. Doppenberg, M. Ross Bullock, and William C. Broaddus Introduction / 795 Pathophysiology / 795 General Considerations in the Care of the Head-Injured Patient / 798 Specific Management of the Head-Injured Patient / 799 Monitoring the Injured Brain / 802 Summary / 802 References / 802

41. Spinal Cord Injury / 805 36. Acquired Cardiac Disorders / 699 Dipin Gupta, Andrew C. Fiore, and Glenn J. R. Whitman Introduction / 699 Ischemic Heart Disease / 699 Valvular Heart Disease / 709 Heart Failure / 715 Cardiac Dysrhythmias / 716 Pericardial Disease / 719 Cardiac Tumors / 721 Summary / 721 References / 722

Kangmin Lee and R. Scott Graham Introduction / 805 Epidemiology / 805 Pathophysiology / 805 Evaluation / 807 Classic Injury Patterns / 807 Imaging / 812 Management of Acute SCI / 812 Rehabilitation / 814 Advanced Therapies / 814 Restoration of Function / 814 Summary / 814 References / 814

PART FOUR: THE URINARY SYSTEM

37. Urine Formation: From Normal Physiology to Florid Kidney Failure / 725 Akinsan Dosekun, John R. Foringer, and Bruce C. Kone Introduction / 725 Overview of Renal Physiology / 725 Acute Kidney Failure / 735 Chronic Kidney Disease / 740 Renal Transplantation / 755 Summary and Conclusions / 761 References / 761

42. Injuries to Peripheral Nerves / 817 Irvine G. McQuarrie, Thomas C. Chelimsky, and Karen Bitzer Introduction / 817 Anatomy and Physiology / 817 Pathology / 820 Assessment of the Deficit / 821 Treatment Approach / 824 Rehabilitation After Neurorrhaphy / 826 Summary / 828 References / 828

Contents

PART SIX: THE PERIPHERAL VASCULAR SYSTEM

PART SEVEN: THE ENDOCRINE SYSTEM

43. Physiology of Arterial, Venous, and Lymphatic Flow / 831

49. Calcium and Phosphorus Metabolism and the Parathyroid Gland / 927

Dennis F. Bandyk and Paul A. Armstrong Introduction / 831 Peripheral Arterial System / 831 The Venous System / 841 Lymphatic System / 846 Summary / 847 References / 848 Further Readings / 848

Fiemu E. Nwariaku Introduction / 927 Calcium Homeostasis / 927 Hormonal Regulation of Extracellular Calcium Concentration / 928 Disorders of Calcium Metabolism / 931 Phosphate Metabolism / 937 The Parathyroid Glands / 938 Multiple Endocrine Neoplasia Syndromes / 944 Summary / 945 References / 945

44. Aorta and Arterial Disease of the Lower Extremity / 849 Christopher K. Zarins and Sheila M. Coogan Introduction / 849 Atherosclerosis / 849 Pathophysiologic Processes Affecting the Aorta and Lower Extremity Arteries / 851 Arterial Occlusive Disease of the Aorta and Peripheral Arteries / 852 Evaluation of Peripheral Vascular Occlusive Disease / 854 Treatment of Peripheral Vascular Occlusive Disease / 856 Aneurysmal Disease of the Aorta / 862 Peripheral Artery Aneurysms / 865 Complications of Vascular Procedures / 865 Summary / 866 References / 866

45. Cerebrovascular Disease and Upper-Extremity Vascular Disease / 869 Bruce L. Gewertz and James E. McKinsey Introduction / 869 Cerebral Blood Flow / 869 Clinical Presentation of Cerebrovascular Disease / 871 Types of Cerebrovascular Disease / 872 Upper-Extremity Vascular Disease / 876 Summary / 880 References / 880

46. Venous and Lymphatic Abnormalities of the Limbs / 883 Jose R. Parra and Julie A. Freischlag Introduction / 883 Anatomy / 883 Venous Physiology / 884 Venous Disorders of the Lower Extremity / 884 Venous Disorders of the Upper Extremity / 891 Lymphedema / 892 Summary / 894 References / 894

50. Pituitary Dysfunction / 947 Henry Ty and Kathryn Holloway Introduction / 947 Anatomy of the Pituitary Gland / 947 Physiology of the Pituitary Gland / 948 Hormones of the Adenohypophysis / 948 Hormones of the Neurohypophysis / 950 Regulation of Hormone Secretion / 950 Hypopituitarism / 952 Sellar and Parasellar Lesions / 954 Syndrome of Inappropriate Secretion of ADH / 960 Summary / 961 References / 961

51. Adrenal Glands / 965 Maria A. Kouvaraki, Douglas B. Evans, Ana O. Hoff, and Jeffrey E. Lee Introduction / 965 Embryology, Anatomy, and Histology / 965 Physiology / 966 Neoplasms of the Adrenal Gland / 968 Controversies in the Surgical Management of Adrenal Disease / 982 Adrenal Insufficiency / 983 References / 984

52. The Thyroid Gland / 989 Ronald C. Merrell and Lucian Panait Introduction / 989 Thyroid Anatomy / 989 Physiology / 990 Assessment of Patients with Thyroid Disease / 993 Sick Euthyroid Syndrome / 994 Hyperthyroidism (Thyrotoxicosis) / 995 Hypothyroidism / 997 Thyroiditis / 997 Thyroid Nodule / 998 Thyroid Cancer / 999 Summary / 1003 References / 1003

47. Diseases of the Thoracic Aorta / 897 Michael P. Macris and O. Howard Frazier Introduction / 897 Intrinsic Thoracic Aortic Disease / 897 Traumatic Pseudoaneurysms / 902 Summary / 903 References / 904

53. Endocrine Pancreas / 1005

48. Secondary Hypertension: Pathophysiology and Operative Treatment / 907 James C. Stanley and Gerard M. Doherty Introduction / 907 Adrenal Disease and Hypertension / 907 Renal Artery Occlusive Disease and Hypertension / 912 Summary / 922 References / 922

Ronald C. Merrell, Giacomo P. Basadonna, and Cristiana Rastellini Introduction / 1005 Anatomy and Embryology of the Islets / 1005 Physiology of the Islets / 1007 Islets in Health and Disease / 1010 Endocrine Tumors of the Pancreas / 1016 Summary / 1020 References / 1021

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Contents

54. Multiple Endocrine Neoplasia: Types 1 and 2 / 1025 Frank J. Quayle and Jeffrey F. Moley Introduction / 1025 Multiple Endocrine Neoplasia Type 1 / 1025 Multiple Endocrine Neoplasia Type 2 / 1028 Summary / 1032 References / 1032

Metabolic Alterations / 1091 Organ System Alterations / 1093 Resistance to Infection / 1095 Pulmonary Consequences of Thermal Injury / 1096 Physiologic Considerations in Managing the Burn Patient / 1097 Summary / 1101 References / 1102

PART EIGHT: THE INTEGUMENT AND BODY WALL

55. The Biology of Wound Healing / 1035 Dorne R. Yager and Ashley E. Ducale Introduction / 1035 Phases of Healing / 1035 Other Aspects of Repair / 1038 Wound-Healing Pathologies / 1038 Wound Management / 1040 Summary / 1044 References / 1044

56. Breast: Physiologic Considerations in Normal, Benign, and Neoplastic States / 1047 Rakhshanda Layeeque and V. Suzanne Klimberg Introduction / 1047 Physiology of Development / 1047 Pregnancy / 1050 Physiology of Lactation / 1051 Lactogenesis / 1052 Breast-Feeding / 1053 Physiology of Involution / 1053 Clinical Approach to Breast Pathology / 1054 Work-Up of Common Clinical Symptoms / 1055 Treatment of Benign Breast Pathology / 1057 Treatment of Malignant Breast Pathology / 1058 Summary / 1062 References / 1062

57. Hernias of the Abdominal Wall and Its Contents / 1067 Philip E. Donahue Introduction / 1067 Concepts and Definitions / 1067 Pathophysiology of Hernia Development / 1068 Types of Abdominal Wall Hernias / 1070 Summary / 1082 References / 1083

58. Pathophysiology of Thermal Injury / 1085 Ronald M. Barton, Evan R. Kokoska, David J. Wainwright, and Donald H. Parks Introduction / 1085 The Burn Wound / 1085

PART NINE: SPECIAL PHYSIOLOGIC CONSIDERATIONS

59. Physiologic Problems in the Pediatric Surgical Patient / 1107 Daniel J. Ostlie, Shawn D. St. Peter, Sheilendra S. Mehta, and George K. Gittes Introduction / 1107 Physiologic Considerations / 1107 Glucose, Fluid, and Electrolyte Management / 1107 Thermoregulation / 1109 Pulmonary and Cardiac Transitional Physiology / 1109 Nutritional Support / 1110 Specific Pediatric Surgical Conditions / 1111 Summary / 1123 References / 1124

60. Physiologic Considerations in the Elderly Surgical Patient / 1129 Ronnie Ann Rosenthal and Melissa F. Perkal Introduction / 1129 Physiology and Pathology of Aging / 1129 Preoperative Evaluation, Risk Assessment, and Outcome / 1141 Summary / 1144 References / 1145

61. Surgery for Morbid Obesity / 1149 Eric J. DeMaria, Ramzi Alami, and Robert E. Brolin Introduction / 1149 Etiology and Pathophysiology / 1149 Risks and Complications of Severe Obesity / 1150 Treatment of Severe Obesity / 1151 Patient Management / 1156 Results of Surgical Treatment / 1158 Summary / 1161 References / 1161 Index / I-1

Contributors

Ramzi Alami, MD Fellow in Advanced Laparoscopic Surgery, Department of Surgery, Stanford University Medical Center, Palo Alto, California, U.S.A. Joseph F. Amaral, MD President and CEO, Rhode Island Hospital, Professor of Surgery, Brown University Medical School, Providence, Rhode Island, U.S.A. Paul A. Armstrong, DO Clinical Assistant Professor of Surgery, Department of Surgery, University of South Florida College of Medicine, Tampa, Florida, U.S.A. Dennis F. Bandyk, MD Professor of Surgery, Director, Division of Vascular Surgery, University of South Florida College of Medicine, Tampa, Florida, U.S.A. Ronald M. Barton, MD Associate Professor of Surgery Emeritus, Director of the Burn Center Emeritus, Division of Plastic Surgery, Department of Surgery, Virginia Commonwealth University School of Medicine and Medical Center, Richmond, Virginia, U.S.A. Giacomo P. Basadonna, MD Professor of Surgery, Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts, U.S.A. Barbara L. Bass, MD Professor of Surgery, Department of Surgery, Weill Medical College of Cornell University, New York, New York; Carolyn and John F. Bookout Chair, Department of Surgery, The Methodist Hospital, Houston, Texas, U.S.A. David J. Bentrem, MD Assistant Professor of Surgery, Department of Surgery, Surgical Oncology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, U.S.A. Timothy R. Billiar, MD The George Vance Foster Professor and Chair, Department of Surgery, Presbyterian University Hospital, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, U.S.A. Karen Bitzer, OTR/L, CHT Musculoskeletal Division Coordinator, Department of Rehabilitation Services, University Hospitals of Cleveland, Cleveland, Ohio, U.S.A. Mark Bloomston, MD Fellow in Surgical Oncology, Ohio State University Medical Center, Columbus, Ohio, U.S.A. Edward L. Bove, MD Helen and Marvin Kirsh Professor of Surgery, Professor and Section Head, Cardiac Surgery, Director, Pediatric Cardiac Surgery, University of Michigan, C. S. Mott Children’s Hospital, Ann Arbor, Michigan, U.S.A.

of Bariatric Surgery, University Medical Center at Princeton, Princeton, New Jersey, U.S.A. Kevin Bruen, MD Resident in Surgery, Department of Surgery, University of Utah School of Medicine, Salt Lake City, Utah, U.S.A. M. Ross Bullock, MD, PhD Reynolds Professor, Department of Neurosurgery, Virginia Commonwealth University Health System, Richmond, Virginia, U.S.A. Michael D. Caldwell, MD, PhD Director of Marshfield Medical Research and Education Foundation, Director of the Wound Healing Clinic, Marshfield, Wisconsin, U.S.A. Thomas C. Chelimsky, MD Associate Professor of Neurology, Department of Neurology, Case Western Reserve University School of Medicine, Cleveland, Ohio, U.S.A. G. Patrick Clagett, MD Professor and Chairman, Division of Vascular and Endovascular Surgery, Department of Surgery; Ian and Bob Pickens Distinguished Professorship in Medical Science; Director, Center for Vascular Disease, University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A. Neri M. Cohen, MD, PhD Chief, Division of Thoracic Surgery, Greater Baltimore Medical Center Health Care, Baltimore, Maryland, U.S.A. Sheila M. Coogan, MD Assistant Professor of Surgery, Vascular Surgery Service, Palo Alto Veterans Affairs Hospital, Stanford University School of Medicine, Palo Alto, California, U.S.A. Peter F. Crookes, MD Associate Professor of Surgery, Director of Bariatric Surgery Program, Department of Surgery, University of Southern California Keck School of Medicine, Los Angeles, California, U.S.A. Eric J. DeMaria, MD Professor and Chief, Endoscopy and Bariatric Surgery, Vice Chair, Network General Surgery, Chief, Duke General Surgery at Durham Regional Hospital, Duke University Medical Center, Durham, North Carolina, U.S.A. Daniel T. Dempsey, MD Professor and Chairman, Department of Surgery, Temple University School of Medicine, Philadelphia, Pennsylvania, U.S.A. Gerard M. Doherty, MD Norman W. Thompson Professor of Surgery, Head, Section of General Surgery; Chief, Division of Endocrine Surgery, University of Michigan, Ann Arbor, Michigan, U.S.A.

William C. Broaddus, MD, PhD Hord Professor, Department of Neurosurgery, Virginia Commonwealth University Health System; Chief of Neurosurgery, Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, Virginia, U.S.A.

Philip E. Donahue, MD Professor of Surgery, Department of Surgery, University of Illinois Medical Center at Chicago; Chairman, Division of General Surgery, John H. Stroger, Jr. Hospital of Cook County, Chicago, Illinois, U.S.A.

Robert E. Brolin, MD Adjunct Professor of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, Director

Egon M. R. Doppenberg, MD Former Chief Resident, Department of Neurosurgery, Virginia Commonwealth University

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Contributors

Health System, Richmond, Virginia; presently in neurosurgery private practice in Chicago, Illinois, U.S.A. Akinsan Dosekun, MD Associate Professor of Internal Medicine, Department of Internal Medicine, The University of Texas Medical School at Houston, Houston, Texas, U.S.A. Ashley E. Ducale, MPT, PhD Post-Doctoral Fellow, Department of Surgery, Wound Healing Laboratory, Virginia Commonwealth University School of Medicine, Richmond, Virginia, U.S.A. Rodney Durham, MD Professor of Surgery, Department of Surgery, Division of Trauma and Critical Care, University of South Florida College of Medicine, Tampa General Hospital, Tampa, Florida, U.S.A. Douglas B. Evans, MD Professor of Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, U.S.A. B. Mark Evers, MD Professor and Robertson-Poth Distinguished Chair in General Surgery, Department of Surgery, The University of Texas Medical Branch, Galveston, Texas, U.S.A. Peter J. Fabri, MD Professor of Surgery, Associate Dean for Graduate Medical Education, University of South Florida College of Medicine, Tampa, Florida, U.S.A. Andrew C. Fiore, MD Professor of Surgery, Division of Cardiothoracic Surgery, Department of Surgery, St. Louis University School of Medicine; Chief of Cardiothoracic Surgery, Cardinal Glennon Children’s Hospital, St. Louis, Missouri, U.S.A. John R. Foringer, MD Assistant Professor of Internal Medicine, Department of Internal Medicine, The University of Texas Medical School at Houston, Houston, Texas, U.S.A. O. Howard Frazier, MD Professor of Surgery, University of Texas Health Science Center at Houston; Professor of Surgery, Baylor College of Medicine; Chief of Cardiopulmonary Transplantation; Director of Cardiovascular Surgery Research, Texas Heart Institute, Houston, Texas, U.S.A. Julie A. Freischlag, MD William Stewart Halstead Professor of Surgery, Chair, Department of Surgery; Surgeon-in-Chief, Johns Hopkins Medical Center, Baltimore, Maryland, U.S.A.

Deborah T. Glassman, MD Clinical Assistant Professor, Department of Urology, Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A. Bobby S. Glickman, MD Instructor of Surgery, Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska, U.S.A. R. Scott Graham, MD Associate Professor of Neurosurgery, Department of Neurosurgery, Virginia Commonwealth University School of Medicine and Medical Center, Richmond, Virginia, U.S.A. Bartley P. Griffith, MD Professor of Surgery, Chief, Division of Cardiac Surgery, Director, Heart and Lung Transplantation, Maryland Heart Center, University of Maryland Medical Center, Baltimore, Maryland, U.S.A. Dipin Gupta, MD Fellow, Cardiovascular Surgery, New York University School of Medicine, New York, New York, U.S.A. Carl E. Haisch, MD Professor of Surgery, Department of Surgery, East Carolina University Brody School of Medicine, Director of Surgical Immunology and Transplantation; Attending Surgeon, Pitt County Memorial Hospital, Greenville, North Carolina, U.S.A. Nahid Hamoui, MD Assistant Professor of Surgery, Department of Surgery, University of Southern California Keck School of Medicine, Los Angeles, California, U.S.A. Sean P. Harbison, MD Associate Professor of Surgery, Department of Surgery, Temple University School of Medicine, Philadelphia, Pennsylvania, U.S.A. Kenneth S. Helmer, MD Assistant Professor of Surgery, Department of Surgery, University of Texas Medical School at Houston, Houston, Texas, U.S.A. Ana O. Hoff, MD Assistant Professor of Endocrinology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, U.S.A. Kathryn Holloway, MD Professor of Neurosurgery, Department of Neurosurgery, Virginia Commonwealth University Health System; Neurosurgical Director of the Southeast Parkinson’s Disease Research, Education and Clinical Care Center of Excellence (PADRECC) at Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, Virginia, U.S.A.

Janette Gaw, MD Former Chief Resident, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut; presently in colorectal surgery private practice, Fort Myers, Florida, U.S.A.

Kamal M. F. Itani, MD Professor of Surgery, Boston University Medical Center, Chief of Surgery, VA Boston Health Care System, Boston, Massachusetts, U.S.A.

Bruce L. Gewertz, MD Dallas B. Phemister Professor and Chair, Department of Surgery, The University of Chicago, Chicago, Illinois, U.S.A.

Lindsey N. Jackson, MD Resident in General Surgery, Department of Surgery, The University of Texas Medical Branch, Galveston, Texas, U.S.A.

Rafik M. Ghobrial, MD, PhD Professor of Surgery, Director, Liver, Pancreas and Small Bowel Transplantation, The DumontUCLA Transplant Center, David Geffen School of Medicine of the University of California at Los Angeles, Los Angeles, California, U.S.A.

Mark R. Jackson, MD Former Associate Professor of Surgery, Division of Vascular Surgery, University of Texas Southwestern Medical School, Dallas, Texas; presently in vascular surgery private practice, St. Francis Hospital, Greenville, South Carolina, U.S.A.

George K. Gittes, MD Surgeon-in-Chief, Children’s Hospital of Pittsburgh, Professor of Surgery, Division Chief, Pediatric Surgery, Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, U.S.A.

Raymond J. Joehl, MD Professor of Surgery, Program Director General Surgery Residency, Department of Surgery, Loyola University Medical Center, Maywood; Chief, Surgical Service and Manager, Surgery Service Line, The Charles B. Puestow Surgical Service, Edward Hines, Jr VA Hospital, Hines, Illinois, U.S.A.

Contributors

xv

Haytham M. A. Kaafarani, MD Resident in Surgery, Department of Surgery, University of South Florida Medical School, Tampa, Florida, U.S.A.

Walter E. Longo, MD Professor and Vice Chairman, Chief, Division of General Surgery, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut, U.S.A.

Lillian S. Kao, MD Assistant Professor of Surgery, Department of Surgery, University of Texas Medical School at Houston, Houston, Texas, U.S.A.

Patricia A. Lowry, MD Associate Professor of Radiology, Department of Radiology, Virginia Commonwealth University School of Medicine and Medical Center, Richmond, Virginia, U.S.A.

Gregory Kennedy, MD Chief Resident in Surgery, Department of Surgery, University of Wisconsin Medical School, Madison, Wisconsin, U.S.A.

Michael P. Macris, MD Medical Director, Cardiovascular Surgery, Memorial Hermann Northwest Hospital, Houston, Texas, U.S.A.

Jonathan Kiev, MD Assistant Professor of Surgery, Division of Cardiothoracic Surgery, Department of Surgery, Virginia Commonwealth University School of Medicine and Medical Center, Richmond, Virginia, U.S.A. V. Suzanne Klimberg, MD Professor of Surgery, Department of Surgery, Director, Breast Surgical Oncology, University of Arkansas for Medical Services and the Arkansas Cancer Research Center, Little Rock, Arkansas, U.S.A. Evan R. Kokoska, MD Assistant Professor of Surgery, Department of Surgery–Pediatric Surgery Service, University of Arkansas for Medical Sciences, Little Rock, Arkansas, U.S.A. Bruce C. Kone, MD The James T. and Nancy B. Willerson Chair, Chairman, Department of Medicine, Professor of Internal Medicine and of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, Houston, Texas, U.S.A. Kara C. Kort, MD Assistant Professor of Surgery, Department of Surgery, State University of New York–Upstate Medical University, Syracuse, New York, U.S.A. Maria A. Kouvaraki, MD, PhD Fellow in Endocrine Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, U.S.A. Rosemary A. Kozar, MD, PhD Associate Professor of Surgery, Department of Surgery, University of Texas Medical School at Houston, Houston, Texas, U.S.A. Rakhshanda Layeeque, MD Assistant Professor of Surgery, Department of Surgery, Surgical Oncology Section, University of Massachusetts Memorial Medical Center, Worcester, Massachusetts, U.S.A. Jeffrey E. Lee, MD Professor of Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, U.S.A. Kangmin Lee Resident in Neurosurgery, Department of Neurosurgery, Virginia Commonwealth University Health System, Richmond, Virginia, U.S.A.

Ajai K. Malhotra, MD Assistant Professor of Surgery, Division of Trauma and Critical Care, Department of Surgery, Virginia Commonwealth University School of Medicine and Medical Center, Richmond, Virginia, U.S.A. James F. McKinsey, MD Associate Professor of Clinical Surgery and Site Chief of Vascular Surgery, Columbia University of New York Presbyterian Hospital System, New York, New York, U.S.A. Irvine G. McQuarrie, MD, PhD Associate Professor of Neurosurgery and Neuroscience, Department of Surgery, School of Medicine, Case Western Reserve University; Cleveland VA Medical Center, Cleveland, Ohio, U.S.A. Margaret M. McQuiggan MS, RD, CNSD Clinical Instructor, Department of Surgery, University of Texas Medical School at Houston, Houston, Texas, U.S.A. Sheilendra S. Mehta Former Research Fellow in Pediatric Surgery, Division of Pediatric Surgery, Children’s Mercy Hospital, Department of Surgery, University of Missouri School of Medicine at Kansas City, Kansas City, Missouri, U.S.A. David W. Mercer, MD Professor of Surgery and Vice Chairman, Department of Surgery, The University of Texas Health Science Center–Houston; Chief of Surgery, Lyndon Baines Johnson General Hospital, Houston, Texas, U.S.A. Ronald C. Merrell, MD Professor of Surgery, Department of Surgery, Virginia Commonwealth University School of Medicine and Medical Center, Richmond, Virginia, U.S.A. Thomas A. Miller, MD Ammons Professor of Surgery, Division of General Surgery, Department of Surgery, Virginia Commonwealth University School of Medicine and Medical Center; Chief of Surgery, Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, Virginia, U.S.A. Sina L. Moainie, MD Resident in Cardiothoracic Surgery, Maryland Heart Center, University of Maryland Medical Center, Baltimore, Maryland, U.S.A. Jeffrey F. Moley, MD Professor of Surgery and Chief, Endocrine and Oncologic Surgery, Washington University School of Medicine; Associate Director, Alvin J. Siteman Cancer Center; Attending Surgeon, Barnes-Jewish Hospital, St. Louis, Missouri, U.S.A.

Denise Lester, MD Assistant Professor, Department of Anesthesiology, Virginia Commonwealth University School of Medicine and Medical Center; Director, Chronic Pain Clinic, Anesthesiology Service, McGuire Veterans Affairs Medical Center, Richmond, Virginia, U.S.A.

Frederick A. Moore, MD Professor of Surgery and Vice Chairman, Department of Surgery, Medical Director, Trauma Services, University of Texas Medical School at Houston, Houston, Texas, U.S.A.

Terrence H. Liu, MD Associate Clinical Professor of Surgery, Residency Program Director, UCSF–East Bay Surgery Program, University of California San Francisco School of Medicine, East Bay Campus, Oakland, California, U.S.A.

Ralph S. Mosca, MD Associate Professor of Surgery, Columbia University College of Physicians and Surgeons; Associate Attending Surgeon, New York Presbyterian Hospital/Columbia University Medical Center, New York, New York, U.S.A.

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Contributors

Carlos A. Murillo, MD Resident Instructor, Department of Surgery, Texas Tech University Health Sciences Center, El Paso School of Medicine, El Paso, Texas, U.S.A.

Jose M. Prince, MD Surgical Resident, Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, U.S.A.

Stuart I. Myers, MD Professor of Surgery, Division of Vascular Surgery, Department of Surgery, Virginia Commonwealth University School of Medicine and Medical Center; Attending Surgeon, Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, Virginia, U.S.A.

Frank J. Quayle, MD Resident in Surgery, Department of Surgery, Division of General Surgery, Washington University School of Medicine, St. Louis, Missouri, U.S.A.

Attila Nakeeb, MD Associate Professor of Surgery, Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A. Lena M. Napolitano, MD Professor of Surgery, Director, Surgical Critical Care; Associate Chair, Department of Surgery, University of Michigan Health System, Ann Arbor, Michigan, U.S.A. Leigh Neumayer, MD Professor of Surgery, Program Director, Utah Building Interdisciplinary Research Careers in Women’s Health, University of Utah Medical Center, Salt Lake City, Utah, U.S.A. John E. Niederhuber, MD Acting Director, National Cancer Institute, Bethesda, Maryland; Former Director, University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin, U.S.A. Unyime O. Nseyo, MD Adjunct Professor, Department of Urology, University of Florida School of Medicine; Chief, Urology Section, Malcom Randall VA Medical Center, Gainesville, Florida, U.S.A. Fiemu E. Nwariaku, MD Associate Professor and Vice Chairman, Division of Gastrointestinal and Endocrine Surgery, Department of Surgery, University of Texas Southwestern Medical Center, Dallas, Texas, U.S.A. Daniel J. Ostlie, MD Associate Professor of Surgery, Division of Pediatric Surgery, Children’s Mercy Hospital, Department of Surgery, University of Missouri School of Medicine at Kansas City, Kansas City, Missouri, U.S.A. Lucian Panait, MD Former Postdoctoral Research Fellow, Department of Surgery, Virginia Commonwealth University School of Medicine, Richmond, Virginia, U.S.A. Donald H. Parks, MD Professor and Chief, Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Texas Medical School at Houston, Houston, Texas, U.S.A. Jose R. Parra, MD Former Assistant Professor of Surgery, Division of Vascular Surgery, Department of Surgery, Johns Hopkins Medical Center, Baltimore, Maryland, U.S.A.

J. Robert Ramey, MD Chief Resident in Urology, Department of Urology, Thomas Jefferson University, Philadelphia, Pennsylvania, U.S.A. Cristiana Rastellini, MD Associate Professor, Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts, U.S.A. Lorita Rebellato, PhD Associate Professor of Pathology, Department of Pathology, East Carolina University Brody School of Medicine, Greenville, North Carolina, U.S.A. Kathryn A. Richardson, MD Assistant Professor, Department of Surgery, Louisiana State University Health Sciences Center, Shreveport, Louisiana, U.S.A. Alexander S. Rosemurgy, MD Professor of Surgery and Medicine, Director, Division of General Surgery, Surgical Director, Tampa General Hospital Digestive Disorders Center, University of South Florida College of Medicine, Tampa, Florida, U.S.A. Ronnie Ann Rosenthal, MS, MD Associate Professor of Surgery, Yale University School of Medicine, New Haven; Chief, Surgical Service, Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut, U.S.A. Shawn D. St. Peter, MD Assistant Professor of Surgery, Division of Pediatric Surgery, Children’s Mercy Hospital, Department of Surgery, University of Missouri School of Medicine at Kansas City, Kansas City, Missouri, U.S.A. Jeannie F. Savas, MD Associate Professor of Surgery, Division of General Surgery, Department of Surgery, Virginia Commonwealth University School of Medicine and Medical Center; Attending Surgeon, Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, Virginia, U.S.A. Henry J. Schiller, MD Associate Professor of Surgery, Department of Surgery, Mayo Clinic School of Medicine, Rochester, Minnesota, U.S.A. Lelan F. Sillin, MD, MS(Ed) Professor of Surgery, Vice Chair for Educational Affairs, Department of Surgery, University of Rochester Medical Center, Rochester, New York, U.S.A.

Christina Paylan, MD Former Fellow in Trauma and Critical Care, Department of Surgery, University of South Florida College of Medicine, Tampa General Hospital, Regional Trauma Center, Tampa, Florida, U.S.A; presently in plastic surgery private practice, Tampa, Florida, U.S.A.

Michael Sobel, MD Professor and Vice Chairman, Department of Surgery, University of Washington; Professor and Chief, Puget Sound Veterans Affairs Health Care System, Seattle, Washington, U.S.A.

Melissa F. Perkal, MD Assistant Professor of Surgery, Director, Veterans Affairs Hospital Surgical Intensive Care Unit, Director, Surgical Preceptor Program, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut, U.S.A.

James C. Stanley, MD Handleman Professor of Surgery, Section of Vascular Surgery, Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan, U.S.A.

Henry A. Pitt, MD Professor and Vice Chairman, Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A.

Heinrich Taegtmeyer, MD, DPhil Professor of Medicine, Division of Cardiology, Department of Internal Medicine, University of Texas Medical School at Houston, Houston, Texas, U.S.A.

Contributors

xvii

Daniel G. Tang, MD Chief Resident in General Surgery, Department of Surgery, Virginia Commonwealth University School of Medicine and Medical Center, Richmond, Virginia, U.S.A.

Glenn J. R. Whitman, MD Professor of Surgery, Division of Cardiothoracic Surgery, Temple University School of Medicine, Philadelphia, Pennsylvania, U.S.A.

Jon S. Thompson, MD Professor and Vice Chairman, Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska, U.S.A.

Charles Williams, MD Associate Professor, Vice Chair for Veterans Affairs, Department of Anesthesiology, Virginia Commonwealth University School of Medicine and Medical Center; Chief, Anesthesiology Service, Hunter Holmes McGuire Veterans Affairs Medical Center, Richmond, Virginia, U.S.A.

Hari Siva Gurunadha Rao Tunuguntla, MD, MBBS, MS (Surgery), MCh Resident, Department of Urology, University of Miami, Miller School of Medicine, Miami, Florida, U.S.A. Richard H. Turnage, MD Professor of Surgery and Chairman, Department of Surgery, Louisiana State University Health Sciences Center, Shreveport, Louisiana, U.S.A. Douglas J. Turner, MD Assistant Professor of Surgery, Division of General Surgery, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Henry Ty, MD Former Chief Resident in Neurosurgery, Department of Neurosurgery, Virginia Commonwealth University Health System, Richmond, Virginia; presently in neurosurgery private practice, North Andover, Massachusetts, U.S.A.

Ryan M. Wolfort, MD Resident in Surgery, Department of Surgery, Louisiana State University Health Sciences Center, Shreveport, Louisiana, U.S.A. Kenneth J. Woodside, MD Chief Resident in General Surgery, Department of Surgery, The University of Texas Medical Branch, Galveston, Texas, U.S.A. Dorne R. Yager, PhD Associate Professor of Surgery, Physiology and Biochemistry, Director, Wound Healing Laboratory, Department of Surgery, Virginia Commonwealth University School of Medicine, Richmond, Virginia, U.S.A.

Kathryn M. Verbanac, PhD Professor of Surgery, Division of Transplantation, Department of Surgery, East Carolina University Brody School of Medicine, Greenville, North Carolina, U.S.A.

Christopher K. Zarins, MD Chidester Professor of Surgery, Chief, Division of Vascular Surgery, Stanford University Medical Center, Palo Alto, California, U.S.A.

David J. Wainwright, MD Associate Professor of Surgery, Division of Plastic and Reconstructive Surgery; Department of Surgery, University of Texas Medical School at Houston, Houston, Texas, U.S.A.

Emmanuel E. Zervos, MD Assistant Professor of Surgery, Department of Surgery, Surgical Oncology, University of South Florida College of Medicine, Tampa General Hospital, Tampa, Florida, U.S.A.

Jian-Ying Wang, MD, PhD Professor of Surgery and Pathology, Associate Chair for Basic Research, Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland, U.S.A. Andrew S. Wechsler, MD Stanley K. Brockman Professor and Chairman, Department of Cardiovascular Medicine and Surgery, Drexel University College of Medicine, Philadelphia, Pennsylvania, U.S.A.

Yuan Zhai, MD Assistant Professor of Surgery, Department of Surgery, Section of Liver and Pancreas Transplantation, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, California, U.S.A. Huiping Zhou, PhD Assistant Professor, Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, Virginia, U.S.A.

medwedi.ru

PART ONE: General Considerations in the Management of Surgical Patients

1 Metabolic Response to Starvation, Stress, and Sepsis Joseph F. Amaral, Michael D. Caldwell, and Thomas A. Miller

INTRODUCTION The metabolic response to stress, starvation, and sepsis should be viewed as a complex series of neuroendocrine reflexes resulting in an integrated attempt by the organism to mobilize energy substrates, to preserve oxygen and substrate delivery, and to maintain essential body functions (1). These alterations involve changes in the distribution and use of water, proteins, fats, and carbohydrates. As such, an understanding of normal body composition is essential to make discussion of the metabolic response meaningful.

BODY COMPOSITION Body composition has been defined by Moore (2) as ‘‘the study of the total mass and volume of body components in relation to body size, body configuration, age, sex, disease, and concentration changes.’’ It can be considered from several aspects, including tissue anatomy, chemical composition, and metabolic structure (Fig. 1), with the latter two being the most important with regard to energy stores and ‘‘intermediary metabolism.’’

Chemical Composition

for the relatively high boiling point of water, its relatively high freezing point, and its high specific heat (the energy required to raise the temperature of 1 g of a substance by 1 C). This latter property allows water to exert a major role in temperature regulation, because a large amount of heat is lost as water evaporates (perspiration) and a large amount of heat is required to raise the temperature of water. The extensive hydrogen bonding of water also makes it important in establishing the tertiary structure of proteins and other molecules. Total body water (TBW) is divisible into two components: intracellular water (ICW) and extracellular water (ECW). These two components are separated by cell membranes. Measurement of body fluid compartments uses dilution techniques, based on the concept that a substance that distributes itself equally and exclusively throughout a given compartment (e.g., TBW) can be used to determine the volume of that compartment. This procedure is done by injecting a known amount of the substance and measuring its concentration at steady state, as well as measuring the amount excreted during the period of time required to reach steady state. Thus: Volume ¼

All living organisms may be considered as complex and organized arrangements of fats, proteins, carbohydrates, minerals, and electrolytes in an aqueous environment. Although the percentage of each of these chemical groups varies among and within species, by far the largest component of all living organisms is water.

Amount injected Amount excreted Steady state concentration

Because the amount lost is usually negligible, the convention is to use: Volume ¼

Amount injected Steady state concentration

Water Water is an important substance involved in virtually all physiologic processes. Its physiochemical properties result largely from its electron structure. The hydrogen ions have a slightly positive charge, whereas the oxygen molecule, with its lone pair of electrons, has a negative charge. This difference allows extensive hydrogen bonding and a relatively high dipole moment (5) that causes the water molecules to orient themselves in an external electric field in such a way that they act as an electric buffer. When ionic substances are placed in water, a hydrational shell forms around the individual ions, reducing their electrochemical attraction and thus forming a solution (5). Water thus keeps electrolytes and other polar molecules in solution and allows them independent motion, which is critical to nutrient transport. The extensive hydrogen-bonding capacity of water molecules causes them to orient themselves in a highly organized structure similar to ice. This structure accounts

Numerous substances are available that allow measurement of various fluid compartments (Fig. 2). Generally, the TBW is the most accurately measured compartment. The TBW varies with age, sex, body build, physical activity, disease, and state of hydration. The TBW can be measured accurately using deuterium oxide or tritiated water. For a healthy man, TBW constitutes approximately 60% of total body weight, and for a healthy woman, it constitutes approximately 50% (Table 1). The differences in the proportion of TBW reflect the quantity of skeletal muscle and adipose tissue present in the two sexes. Adipose tissue contains little ICW, whereas skeletal muscle has one of the largest water contents of all tissues (Table 2). Women, with their larger adipose tissue stores and smaller skeletal muscle mass, have less TBW than men. Similarly, the TBW of young, lean athletes is greater than that of elderly, obese nonathletes. The TBW as a percentage of body weight decreases steadily with age (Table 1). Newborns have the highest

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2

Part One: General Considerations

Table 1 Distribution of Total Body Water in Infants, Children, and Adults 100

CHO Mineral

Other

Other nitrogen

Protein

Vital organs

Fat

Blood Nerve

Hydrogen Carbon

Fat

Skin Adipose

% 50

Extracellular water

Extracellular mass

Intracellular water

Body cell mass

Oxygen

Bone

Muscle

0 TISSUE

CHEMICAL

METABOLIC MOLECULAR

Figure 1 Body composition in adult man. Source: From Refs. 3, 4.

percentage, with 75% to 80% of body weight representing water. This percentage decreases during the first few months after birth to approximately 65% of the body weight, where it remains for the remainder of infancy and childhood. This reduction in TBW is primarily the result of reduction in ECW (Fig. 3). Until the age of 12, no difference in TBW is noted between boys and girls. With advancing age, TBW as a percentage of body weight decreases to a low of 52% and 47% in males and females, respectively (2). This results primarily from a decrease in

ECF

ICF

PlasRBC ma ISF

RBC Plasma

Cr54

T1824

131

I ECF

Inulin Sucrose

Mannitol S2O3

TBW (%) Age

Men

Women

0–1 day 1–10 days 1–3 mo 3–6 mo 6–12 mo 1–2 yr 2–3 yr 3–5 yr 5–10 yr 10–16 yr 17–39 yr 40–59 yr 60 þ yr

79 74 72.3 70.1 60.4 58.7 63.5 62.2 61.5 58.9 60.6 54.7 51.5

79 74 72.3 70.1 60.4 58.7 63.5 62.2 61.5 57.3 50.2 46.7 45.5

Abbreviation: TBW, total body water. Source: Adapted from Ref. 7.

ICW, because ECW remains unchanged (5). The size of the ECW space depends on the method used for determining it. Large molecules such as insulin, mannitol, or sucrose appear to underestimate the ECW compartment because of the slower diffusion of these larger molecules into noncellular spaces (5). An ECW space of 15% to 16% of body weight is usually reported with these methods (5). Assessment of the ECW with small molecules such as 35SO4,82Br, and 24Na appears to overestimate the ECW space because of the ability of these small ions to diffuse into cells. An ECW space of 21% to 27% of body weight is usually reported when these methods are used (5). In general, it is estimated that the ECW occupies 20% of the body weight (and therefore 30–40% of TBW). It is divided into plasma (5% of body weight) and interstitial fluid (15% of body weight). The interstitial fluid occupies a rapidly equilibrating functional space between cells and a slowly equilibrating (or nonequilibrating) space composed of epithelial cell secretions, connective tissue, joint space, and cerebrospinal fluids, the so-called transcellular space (9). The functional interstitial fluid accounts for 90%, and the transcellular space accounts for 10% of the total interstitial fluid. The transcellular space should not be equated with the ‘‘third space.’’ The transcellular fluids are a normal component of the ECW and do not affect the functional volume of the extracellular space. The third space results

SO4 Br

Table 2 Distribution of Water in the Various Tissues of a 70 kg Man

Cl Na SCN D 2O

TBW

THO Antipyrine

RBC Plas- ISF ma ECF

Conn. tissue ICF

Figure 2 Substances used to measure the body fluid compartments. Abbreviations: ECF, extracellular fluid; ICF, intracellular fluid; ISF, interstitial fluid; RBC, red blood cell; TBW, total body water; THO, titrated water. Source: From Ref. 6.

Tissue Skin Muscle Skeleton Brain Liver Heart Lungs Kidneys Spleen Blood Intestine Adipose tissue Source: From Ref. 8.

Water (%)

Body weight (%)

Liters of water per 70 kg

72 75.6 22 74.8 68.3 79.2 79 82.7 75.8 83 74.5 10

18 41.7 15.9 2 2.3 0.5 0.7 0.4 0.2 8 1.8 10

9.07 22.1 2.45 1.05 1.03 0.28 0.39 0.25 0.1 4.65 0.94 0.7

Chapter 1: Metabolic Response to Starvation, Stress, and Sepsis

3

Table 3 Electrolyte Composition of the Body Fluid Compartments Serum Serum water (mEq/L) (mEq/L)

100 90

Electrolytes

BODY WEIGHT (%)

80

Total body water (TBW)

70 60

Intracelluar fluid (ICF)

50 40

Extracellular fluid (ECF)

30 20

+

+

+

10 2 4 6 8 Birth 6 12 3yr 6yr 9yr 12yr 15yr Adult mo mo AGE

Figure 3 Distribution of body fluids in children as a function of age and sex. Source: From Ref. 6 and courtesy of Pediatrics 1961; 28:169.

Interstitial fluid (mEq/L)

Intracellular fluid (muscle) (mEq/kg of H2O)

Cations Sodium (Naþ) Potassium (Kþ) Calcium (Ca2þ) Magnesium (Mg2þ)

142 4 5 2

152.7 4.3 5.4 2.2

145 4

10 156 3.3 26

Total cations

153

165

149

195

102 26

109.7 28

114 31

2 8

2

2.2

95

1 6 16

1.1 6.5 17.2

20

Anions Chloride (Cl) Bicarbonate (HCO2 3 ) Phosphate (HPO2 4 ) Sulfate (SO2 4 ) Organic acids Protein Total anions

153

165

55 145

180þ

Source: Adapted from Ref. 7.

from abnormalities in the permeability of cells, such as the abnormalities seen after ischemia and those seen with inflammation that increases the size of the extracellular space but not, a priori, the volume of the ECW. Third-space size is proportional to the severity of the injury. Because fluid and electrolytes in the third space are derived from functional extracellular fluid, the increase in size of the space reduces the functional extracellular volume. Direct measurement of the ICW compartment is extremely difficult, because substances that equilibrate only in the intracellular space have not been defined. Consequently, the ICW space is estimated as the difference between TBW and ECW. The ICW space varies from individual to individual. In healthy normal adults, it constitutes approximately 30% to 40% of body weight (55% of TBW). Because fat has little ICW and skeletal muscle has the highest percentage of ICW, athletic muscular individuals have a higher proportion of ICW; but women, the obese, and the elderly have a smaller muscle mass and therefore have a smaller percentage of their body weight as ICW. The electrolyte composition of the various fluid compartments is noted in Table 3. The major cations are sodium and potassium, and the major anions are chloride and bicarbonate. The absolute amount of a particular ion can only be measured by cadaveric analyses, but the total exchangeable amount of an ion can be estimated by dilution with a labeled

form of the compound. Total exchangeable sodium is not equivalent to total body sodium. A large amount, approximately 1000 mEq, is present in a nonexchangeable form in bone (10). The total exchangeable potassium is more closely equivalent to the total body potassium (TBK) (less than 0.5% is nonexchangeable) (10). Sodium is the major extracellular cation, and potassium is the major intracellular cation (Table 4). The ECW space is approximated by the total body sodium, the ICW space by the TBK, and the TBW by the sum of total body sodium and TBK. Moore (10) has used this relationship to estimate TBW from total exchangeable sodium (Nae) and potassium (Ke) using the following formula: TBM ¼

ðNae þ Ke Þ þ 70 ð2LÞ 163

The differing ionic compositions of the various fluid compartments are the result of variations in the permeabilities and active transport mechanisms present great diversity among the transcellular fluids in this regard (Table 5).

Fat (Lipids) Until the 1950s, lipids, the second largest chemical constituent of the body, were considered to be relatively inert

Table 4 Total and Exchangeable Amounts of Electrolytes in Fluid Compartments of Adult Man Sodium

Potassium

Compartment Total extracellular Total intracellular Total body Total exchangeable Total body intracellular concentration (milliequivalent per liter intracellular water)

Magnesium

Chloride

Bicarbonate

27.8 5.2 33 33 14.4

6.8 5.9 12.7 12.7 16.4

(mEq/kg of body weight) 52.8 5.2 58 41 14.4

2.5 51.3 53.8 52.8 14.3

21.8 8.2 30 3.4, 4.9, 10a 22.8

a

Equilibrated for 24, 48, and 89 hours, respectively. Total exchangeable magnesium is a function of time of equilibration. Source: From Ref. 11.

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Table 5 Mean Electrolyte Composition of Transcellular Fluids Fluid Saliva Gastric juice Bile Pancreatic juice Ileal fluid Cecal fluid Cerebrospinal fluid Sweat

Na (mEq/L)

K (mEq/L)

Cl (mEq/L)

HCO3 (mEq/L)

Hþ (mEq/L)

33 60 149 141

20 10 5 5

34 130 101 77

0 0 45 92

– 90 – –

129 80 141

11 21 3

116 48 127

29 22 23

– – –

45

5

58

0

–

Source: Adapted from Ref. 12.

substances that served as a source of protection and insulation. They now are recognized as essential components of energy metabolism (providing 9.3 kcal/g), hormonal synthesis (steroids), hormonal regulation and action (prostaglandins), and neural transmission (sphingomyelins). In addition, they are required for general cellular integrity and stability (cell membrane phospholipids and cholesterol). Many of the functions of lipids require fatty acids that cannot be synthesized by human beings. The three major essential fatty acids are arachidonic acid, linoleic acid, and linolenic acid. Total body fat (TBF) varies inversely with TBW in normal individuals. During the first four months of life, there is a decrease in the percentage of TBF, expressed as percent of total body weight (Table 6). After puberty, the TBF content increases, with the increase being greater in girls than in boys. In healthy men, TBF accounts for approximately 25% of body weight, and in healthy women, fat accounts for approximately 35% of body weight. With age, there is an increase in the percentage of body weight occupied by fat. However, it is of note that body weight often remains unchanged with age. Because total body weight equals body fat plus fat-free tissues, a decrease in fat-free tissue is thought to occur with aging (14). The percentage of body weight occupied by fat is also inversely related to the level of physical activity. Muscular athletic individuals have a greater muscle mass and smaller percentage of body fat than sedentary individuals of similar body weight (15). However, these changes are somewhat adaptive. For example, a long-distance swimmer benefits from the buoyancy and insulation provided by fat and often has a greater amount of fat than a long-distance runner of equivalent body build (15). This presumably results from a difference in caloric intake between these two groups. Approximately 50% of the fat in human beings is located in the subcutaneous tissue, but the distribution of the subcutaneous tissue varies with age, sex, and physical activity (14). Children have a large amount of subcutaneous

Table 6 Body Composition of Infants and Children as Function of Age

Age (mo) Birth 4 12 24 36

Whole body (g/100 g)

Body weight (kg)

Water

Protein

Lipid

Other

3.5 7 10.5 13 15

75.1 60.2 59 61 62

11.4 11.4 14.6 15.7 16.4

11 26.3 23.9 20.6 18.3

2.5 2.1 2.5 2.7 3.3

Source: From Ref. 13.

tissue over their triceps, but only a small amount of subcutaneous tissue in their abdominal walls. The distribution is reversed in adults. Changes in the body fat distribution occurring as a result of weight gain are not equally distributed (14). For this reason, serial measurement of an isolated anthropometric index may not adequately reflect body composition changes in malnourished individuals who are being repleted. However, changes in the body fat distribution occurring as a result of weight loss are equally distributed. A 10% loss in triceps skinfold is accompanied by a concomitant 10% loss in the size of the subcutaneous tissues of other areas such as the hips, abdomen, thighs, and breasts (14). TBF is defined as the difference between total body weight and fat-free mass (see the equation given below). Fat contains very little water and virtually no potassium. On the other hand, the total water content of fat-free tissue averages 73.2% of TBW (16), and the potassium content of fat-free tissues averages 68.1 mEq/kg (14). Consequently, fatfree tissue may be approximated from either TBW or total exchangeable potassium (Ke), and the TBF is estimated by inference. Equations for these calculations are: For TBW: TBF ¼ BWt ðTBW=0:732Þ

and

%TBF ¼ 1 ð%TBW=0:732Þ For Ke : TBF ¼ BWt ðKe Þ=68:1 and %TBF ¼ ½BWt ðKe =68:1Þ=BWT where BWt is body weight. It should be noted that these methods assume that the hydration and potassium content of that portion of the body that is fat free is constant. However, these assumptions are not always valid because the hydration of the fat-free tissue can vary considerably (e.g., greatest in edematous states and least in dehydrated states). A third method used in calculating TBF involves the measurement of the specific gravity of the individual by underwater weighing procedure (14,15,17,18). This method is based on the findings that normal human fat has a density of 0.9 g/mL and nonfat tissue has an average density of 1.1 g/mL at 37 C. At 37 C, the density of an average reference male containing 15.3% body fat is 1.064 g/mL (19). With the use of these values, the percentage fat in an individual can be determined by measuring the density (D) in water as indicated by the equation: %Fat ¼ ½ð4:570=DÞ 4:142 100 Although the density of human fat changes only with temperature, the density of fat-free tissue changes with age, degree of obesity, and degree of hydration (14). When estimates of TBF made by densitometric methods are compared to calculations of TBF made from TBW, the densitometric methods give a higher estimate of TBF (15,17). Densitometric methods give a lower estimate of TBF when compared to calculations of TBF made from total exchangeable potassium (17,20). A fourth method used to calculate TBF involves the measurement of the uptake of an inert, highly fat-soluble gas such as cyclopropane or krypton (21). The assumption made in this technique is that the gas will only go into fat cells. Thus it should yield a more accurate measurement of TBF than the other methods noted. When compared to measurements made by TBW determination, similar results

5

Chapter 1: Metabolic Response to Starvation, Stress, and Sepsis

protein turnover per kilogram of body weight are the result of a decrease in skeletal muscle mass. TBP may be estimated by one of two methods: (i) measurement of total exchangeable potassium or (ii) measurement of total body nitrogen (TBN) by neutron activation. TBN is linearly related to total exchangeable potassium in both normal and decreased conditions (26,27). The average exchangeable potassium to nitrogen ratio in tissue is 3 mEq/g nitrogen (10). Thus:

are obtained (22). When compared to total exchangeable potassium measurements or densitometric measurements, lower values are obtained (23). However, no data are available comparing the inert gas method with cadaver analyses.

Proteins Proteins are chains of amino acids. Proteins and amino acids form essential components of all living cells and are involved in virtually all body functions. These molecules serve as enzymes, hormones, neurotransmitters, immunoglobulins, and transport molecules. They are also essential components of all cell membranes and various cellular components, including receptors, transport systems, and contractile elements. As such, they are necessary for the metabolism, growth, regulation, replication, protection, repair, communication, and motion of individual cells and the coordinated function of the entire organism. Consequently, it is somewhat remarkable that total body proteins (TBPs) account for only 15% of the body weight in a healthy man and that over 80% of the TBP is present in skeletal muscle and connective tissue. Unlike fat, there are no storage depots for protein. All the body’s protein is functional. As a result of its numerous and varied functions, protein, unlike fat, undergoes considerable daily turnover. Approximately 2.5% of the TBP (250 g in a 70 kg man) is broken down and resynthesized each day (23). More than half this turnover is accounted for by daily secretory processes, white cell turnover, hemoglobin turnover, muscle-protein turnover, and plasma protein turnover. The total turnover rate of body protein diminishes progressively with age (24). The protein synthesis rate per kilogram of body weight decreases from 25 g/kg/day in the neonate to 7 g in a one-year-old infant. In the average man and woman, protein synthesis is 3.2 and 2.6 g/kg of body weight per day, respectively; in an elderly man and woman, it is 2.6 and 1.9 g, respectively. The synthesis rate of transport protein, such as albumin, remains unchanged with increasing age, but the breakdown rate (and presumably synthesis rate) of skeletal muscle decreases (Table 7). If the total turnover rate of protein is expressed per lean body mass rather than body weight, an increase in turnover is noted with aging. Because the lean body mass decreases with aging as a result of a reduction in skeletal muscle mass and the synthesis rate of albumin is unchanged, it is apparent that the changes in

TBN ¼ Ke =3 where Ke equals exchangeable potassium. TBP is directly proportional to TBN by a factor of approximately 6.25. Thus: TBP ¼ ð6:25ÞðTBNÞ ¼ ð6:25ÞðKe =3Þ ¼ 2:08 Ke TBN can also be measured using neutron activation analysis (26,28). When tissues are irradiated with neutrons from either a cyclotron or a plutonium source, gamma rays specific for a substance capturing the neutron are emitted. For nitrogen, gamma rays of 10.83 meV are emitted. Consequently, measurement of the gamma rays produced after neutron activation allows determination of TBN. In general, the results obtained with either method correlate well with each other.

Carbohydrates Carbohydrates serve as the energy source of the body when energy is rapidly required, providing approximately 4 kcal/g. They also play important roles in cell membrane function and stability (glycoproteins and glycolipids), in hormone function (glycoproteins), and as precursors of lipid and nonessential amino acid synthesis. In addition, the brain, RBCs, WBCs, and wounds are to a large extent glucose-dependent tissues. Of the three major sugars found in the human body (glucose, fructose, and galactose), glucose is the primary carbohydrate. In addition to ingested carbohydrates, glucose is readily available from pyruvate and lactate, gluconeogenic amino acids (alanine and glutamine), the glycerol moiety of lipids, and its storage form, glycogen. Most of the body’s glycogen is stored in the liver, skeletal muscle, and cardiac muscle. Muscle glycogen is used primarily by the muscle itself, because muscle lacks

Table 7 Comparison of Whole Body Protein Breakdown with Estimates of Muscle-Protein Breakdown and Albumin Synthesis in Young and Old Adult Human Beings Whole body protein breakdowna (g/day) Group Males Young Old Females Young Old

Muscle-protein breakdownb (g/day)

Albumin synthesisc (g/day)

Mean age (yr)

Per kg body weight

Per kg BCM

Per g creatinine

Per kg body weight

Per kg BCM

Per g creatinine

Per kg body weight

Per kg BCM

Per g creatinine

22 70

2.94 2.64

6.7 7.5

115 163

0.76 0.53

1.74 1.50

30 32

0.19 0.15

0.39 0.40

7 8.4

20 76

2.35 1.94

6.1 6.6

103 166

0.64 0.31

1.69 1.05

28 26

– –

– –

a

Measured by administration of 15N-glycine. Measured as 3-methylhistidine output in urine and computed as muscle protein. c Measured by administration of 15N-glycine. Abbreviation: BCM, body cell mass. Source: From Ref. 25. b

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6

Part One: General Considerations

glucose-6-phosphatase. In contrast, hepatic glycogen is primarily used in providing glucose to glucose-dependent tissues. Because only a little glycogen is stored in the liver, the hepatic stores of glycogen are rapidly depleted by an overnight fast. Cahill (29) has estimated the total hepatic glycogen content of a 70 kg man to be 75 g, and the total muscle glycogen content to be 105 g. The total carbohydrate content of the body is approximately 300 g (30). Except by cadaver analysis, no method is available to measure total body carbohydrate. However, it is of note that the daily intake of carbohydrate approximates the total body stores.

Lean Body Mass and Body Cell Mass Based on densitometric measurements, Behnke (17) and Behnke and Wilmore (15) proposed the division of total body weight into fat and lean body mass. The lean body mass was defined as that portion of the body mass with the least amount of essential body fat compatible with health. The essential body fat was thought to represent 2% to 10% of the total body weight. However, because the essential body fat cannot be differentiated from the nonessential body fat, most investigators have redefined lean body mass as the portion of body mass devoid of all fat, the so-called fat-free body (14). Although ‘‘fat-free body’’ and ‘‘lean body mass’’ are often used interchangeably, there is a small (2–10%) difference between them (Table 8). This chapter subsequently refers only to the fat-free body, but it should be kept in mind that the same statements are generally true for lean body mass. Because the total body weight is equal to TBF plus the fat-free body, the size of the fat-free body can be determined by the same methods used to determine the size of the TBF.

The fat-free body is divisible into the extracellular mass, composed primarily of water, and the body cell mass (BCM), composed of all the metabolically active cells in the body (2,10). The cells in the BCM are actively involved in energy exchange, protein synthesis, enzyme replication, and morphogenesis (2). Therefore the BCM is composed of the skeletal muscle mass (60%), visceral cell mass (20%), and the peripheral cell mass (20%) (Table 8) (10). The peripheral cell mass includes blood cells and connective tissue cells. Although the BCM cannot be measured directly, it can be calculated from the exchangeable potassium, the exchangeable sodium, the TBN, or the ICW. Because more than 98% of the TBK is intracellular, a linear relationship exists between BCM, TBK, total exchangeable potassium, and ICW. Histochemical analysis has demonstrated that approximately one-fourth of the wet weight of cells is protein (31). Consequently: BCM ¼ ðTBKÞð4Þ ¼ ð2:08 Ke Þð4Þ ¼ 8:33 Ke BCM ¼ ðTBNÞð6:25Þð4Þ ¼ 25ðTBNÞ Because the average cell has 150 mEq of intracellular potassium per liter (10) and because each cell is composed of 25.8% solids (74.2% water) (31), BCM is also equivalent to: Ke ð1000=0:732Þ=150 ¼ ðKe Þð9:10Þ or ICW=0:742 Each of these methods yields a different value for the BCM of a given individual. This is most obvious when

Table 8 Comparison of Body Cell Mass, Lean Body Mass, and Fat-Free Body BCM Anatomy

All body cells Protoplasm Nucleus Membrane [ICK]av ¼ 150 mEq/LICW

LBM

g

g

Function

Composition

Cellular metabolism Respiration Oxidation Synthesis Cretion Mitosis [Ke ECK] f ¼ BCM f ¼ 7.510 or Ke 8.33 ¼ BCM 70 kg man 3200 8.33 ¼ 26.6 kg Calories 2.73.6 kcal/hr/kgBCM

FFB

All body cellsg BCM Plus: Plasma ECF ECF TCF Tendon ECT Fascia Collagen Elastin ECS Dermis Skeleton ‘‘2% ¼ 10% essential lipid’’: Fat Cellular metabolism Support Transport Circulation Protection Integument Density ¼ 1100 % LBM ¼ 100 495/d 450 70 kg man Approximately 50 kg Calories 110 kcal/hr/kgLBM

Same as LBM but no lipid at all

g

Same as LBM

FFB ¼ TBW/f f ¼ 0.695 0.735 70 kg man 36.4/0.732 ¼ 49.7 Calories (same as LBM)

Abbreviations: BCM, body cell mass; ECF, extracellular fluid; ECK, extracellular potassium; ECS, extracellular supporting structure; ECT, total extracellular space; f, coefficient; FFB, fat-free body; ICK, intracellular potassium; ICW, intracellular water; Ke, exchangeable potassium; LBM, lean body mass; TBW, total body water; TCF, total cellular fluid. Source: From Ref. 10.

Chapter 1: Metabolic Response to Starvation, Stress, and Sepsis

2400

× 2100

× ×× × ×

1800 IRME (cal/day)

the two methods presented for total exchangeable potassium are used. However, any of these methods provide accurate estimates of sequential changes in an individual or differences among populations, if the same method is used throughout the study. Measurements of total exchangeable potassium by whole body 40K counting or of TBN by neutron activation are difficult to perform and require equipment that is expensive and not readily available. To avoid these practical problems, Shizgal et al. (32) developed a method to estimate total exchangeable potassium from isotope dilution measurements of TBW and total exchangeable sodium. As noted previously, TBW is approximately equal to the sum of the total exchangeable sodium and potassium. Thus:

7

×

1500 1200

×

900

× ×

× ×× × ××

= Healthy young males = Healthy young females = Healthy elderly males × = Surgical patients

× ×

600 300

TME = –8.99 = 10–5Ke + 250

Nae þ Ke =TBW ¼ R

400 800 1200 1600 2000 2400 2800 3200 3600 4000 4400 4800 Ke (mEq)

where R is constant. Total exchangeable sodium and TBW can be easily measured by isotope dilution with deuterium oxide or tritiated water and 22Na. The constant, R, can be approximated by measurement of the sodium, potassium, and water of whole blood. Thus:

Figure 4 Relationship of total body energy expenditure to the total Ke. Abbreviations: Ke, exchangeable potassium; IRME/TME, total metabolized energy. Source: From Ref. 34.

Ke ¼ ½ðRÞðTBWÞ Nae energy expenditure when compared to total exchangeable potassium because skeletal muscle at rest contributes significantly to total exchangeable potassium but not to total metabolic energy expenditure.

and BCM ¼ 8:33 Ke or 9:10 Ke As might be expected, BCM increases with age until the middle years of life. With advancing age, the percentage of body weight composed of the BCM decreases as a result of a decrease in skeletal muscle mass. In addition, men generally have a greater percentage of body weight composed of BCM than women; athletic individuals have a greater percentage of body weight composed of BCM than sedentary individuals; and lean individuals have a greater percentage of body weight composed of BCM than obese individuals. Thus the BCM varies from 20% of body weight in morbidly obese individuals to 54% in lean athletic men (33). A direct correlation exists between total energy expenditure and BCM, whereas other measurements of body composition such as total body mass and fat-free body demonstrate a variable or poor correlation (34). Kinney et al. (34) have demonstrated an oxygen consumption of 8 to 10 mL oxygen per kilogram BCM and an energy expenditure of 2.7 to 3.6 kcal/kg/hr: As noted in Figure 4, total exchangeable potassium (and therefore BCM) is linearly related to the total metabolic energy expenditure in healthy adults and surgical patients until 1800 cal/day are expended. However, when measurements of total metabolic energy expenditure greater than this are included, the relationship is parabolic. This relationship is believed to reflect the lower resting metabolic rate of skeletal muscles per milliequivalent intracellular potassium when compared to visceral tissues. Skeletal muscle, which provides approximately 50% of the TBK, accounts for only 15% of the body’s resting energy expenditure. On the other hand, visceral tissues, such as brain, heart, and kidneys, which provide only 10% of the TBK, account for 70% of the body’s resting energy expenditure (35). Consequently, individuals with a small BCM have less skeletal muscle mass and a good correlation of total exchangeable potassium with total metabolic energy expenditure. In contrast, individuals with a large BCM have a smaller increase in total metabolic

Changes in Body Composition with Stress, Sepsis, and Starvation The body composition of a human being at any given moment is influenced significantly by the individual’s age, sex, physical activity, and previous nutritional status, as well as by concurrent infections, injuries, and disease processes. Changes produced by age, sex, and physical activity have been discussed at length. Tables 9 and 10 and Box 1 provide a summary of the formulas derived from multiple body composition studies of normal individuals (2,10). These formulas can be applied to any healthy adult under normal circumstances to estimate the components of body composition. Ultimately, body composition is the net result of the total chemical constituents taken in minus the total chemical constituents used and excreted. Because carbohydrates and lipids primarily serve as a source of calories, this relationship can be estimated by: DBC ¼ ½ðCin þ Nin þ WSin Þ ½ðCout þ Nout þ WSout Þ where D is change, BC is body composition, C is calories, N is nitrogen, W is water, and S is solutes (electrolytes and Table 9 Estimation of Total Body Water by Age, Sex, and Body Weight Sex

Age (yr)

TBW (L)

95% conf. limits (%)

Males

16–30 31–60 61–90 16–30 31–90

0.4 (BWt) þ 13 0.4 (BWt) þ 11 0.34 (BWt) þ 12 0.31 (BWt) þ 11.6 0.33 (BWt) þ 8.84

16 17 16 13 21

Females

Abbreviations: TBW, total body water; BWt, body weight. Source: From Ref. 10.

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Part One: General Considerations

Table 10 Estimation of Total Exchangeable Potassium by Age, Sex, and Body Weight Sex

Age (yr)

Exchangeable potassium (mEq)

Males

16–30 31–60 61–90 16–30 31–90 61–90

38 (BWt) þ 735 26 (BWt) þ 1383 27 (BWt) þ 723 18 (BWt) þ 1250 17 (BWt) þ 1176 18 (BWt) þ 757

23 20 16 20 23 29

20–60 61–84

97.4 (TBW) 409 2 þ 77 (TBW)

10 17

Females

By TBW Males and Females

Injury and infection

Normal

95% conf. limits (%)

+110 +100 +90 +80

Abbreviations: BWt, body weight; TBW, total body water. Source: From Ref. 10.

minerals). Under normal steady-state conditions, the quantities of these components taken in equal the quantities used or lost, and there is no net change in body composition (BC ¼ 0). If the quantity of a component taken in is greater than the quantity used, this component either is stored (BC > 0), thus changing body composition, or is lost (BC < 0). For example, if caloric intake is greater than caloric loss or expenditure, energy is stored in the body in the form of lipids and carbohydrates. If water intake is greater than output, water is retained (e.g., edema). Fortunately, regulatory mechanisms exist that protect against an increase in body water (Chapter 2) and other nutrients. Consequently, for a net increase to be seen in TBW, there must be a neuroendocrine alteration present as well. Such is the case after trauma and surgery when elevated secretion rates of aldosterone and vasopressin promote the retention of salt and water. On the other hand, excess nitrogen intake is not stored, and, as already noted, maximum rates of protein synthesis exist in each individual. When the intake of nitrogen exceeds the need, the excess nitrogen is converted into urea and is excreted. There are five basic situations in which intake does not equal output—dehydration and the four catabolic stresses defined by Moore (2) as fasting, starvation, injury, and febrile illness. In dehydration, the loss of water exceeds the intake resulting in a reduction in TBW that is distributed throughout the ICW and ECW. In fasting and in starvation (prolonged fasting), caloric and nitrogen expenditures are the same or less than those of a normal individual, but the

Box 1 Estimation of Body Composition by Sex and Body Weight TBW ¼ 0.7945 (BWt) 0.0024 (BWt)2 0.0015 (age) (BWt) (males) TBW ¼ 0.6981 (BWt) 0.0026 (BWt)2 0.0012 (age) (BWt) (females) ICW ¼ 0.623 (TBW) 0.0016 (age) (TBW) (males) ICW ¼ 0.553 (TBW) 0.0007 (age) (TBW) (females) Fat ¼ BWt TBW/0.732 ECW ¼ TBW ICW Ke ¼ 150 (ICW) þ 4 (ECW) Nae ¼ 163.2 (TBW) Ke 69 Abbreviations: BWt, body weight; ECW, extracellular water; ICW, intracellular water; Ke, exchangeable potassium; Nae, exchangeable sodium; TBW, total body water. Source: From Ref. 2.

+70 +60

Third degree burns >20% BSA

+50 +40 +30 +20 Basal

Resting 10%

+10 Normal _10 _20 _30

Severe infection

Multiple fractures Postoperative Partial starvation

_40

Figure 5 Resting energy expenditure during injury and starvation in man. Abbreviation: BSA, body surface area. Source: From Ref. 36.

intake of these substrates is markedly reduced or absent. As a result there is a loss of total body lipids, carbohydrates, and nitrogen. In patients with injury or febrile illness, caloric and nitrogen expenditures are greater than those of the normal individual. The increase in energy expenditure produced by an injury or infection is in proportion to the severity of the insult. Burns are the most severe injury, and generalized sepsis is the most severe febrile illness (Fig. 5). A healthy adult undergoing an elective operation increases the resting energy expenditure by approximately 10%, but the same individual with a severe burn increases the resting energy expenditure by 40% to 120%, depending on the size and the degree of the burn injury (37). A minor febrile illness or a minor febrile complication after an elective operation increases the resting energy expenditure by 13% for each degree Celsius of temperature elevation (38), but generalized sepsis increases the resting energy expenditure by 15% to 50% (37). The changes in body composition resulting from an increase in caloric and nitrogen expenditure during injury and febrile illness are frequently compounded by reductions in intake as a result of anorexia and ileus. However, if the increased measurements are adequately met by exogenous substrate sources (enteral or parenteral), little change in the body composition occurs (10,28,39,40).

NORMAL METABOLISM: INTERMEDIARY METABOLISM AND SUBSTRATE INTERACTIONS Body composition remains in a steady state when four essential conditions are met: (i) energy is supplied in sufficient quantities to meet the metabolic demands of all the body’s tissues, (ii) carbohydrates are supplied in sufficient quantities to meet the requirements of glucosedependent tissues, such as those of the brain, RBCs, and WBCs, (iii) nitrogen is supplied in sufficient quantities to meet the obligatory synthesis of protein, and (iv) water

Chapter 1: Metabolic Response to Starvation, Stress, and Sepsis

and solutes (electrolytes and minerals) are supplied in sufficient quantities to replace daily obligatory losses (water and electrolyte metabolism). In addition, these conditions must be met in the face of varying dietary intakes and varying daily energy requirements. This achievement is possible as a result of numerous substrate-to-substrate interactions (e.g., conversion of protein to carbohydrates) and the neuroendocrine regulation of intermediary metabolism.

Energy Metabolism All metabolic processes in cells either produce energy (exergonic reactions) or use energy (endergonic reactions). The energy required for the operation of all biologic processes in mammalian cells is derived from the inherent energy present in the structure of organic molecules (41). The chemical energy produced by the processing of these organic molecules is transferred to the phosphate bonds of purine nucleotides and other molecules with phosphate bonds such as phosphagens. As noted in Table 11, the hydrolysis of the phosphate bonds of adenosine triphosphate (ATP) or its precursors releases a considerable amount of energy that can be used to drive other biologic processes. The formation of these compounds with high-energy phosphate (HEP) group transfer potential is usually a result of the transfer of reducing equivalents from the substrate by reduction of nicotinamide adenine dinucleotide (oxidized form) (NADþ), flavoproteins, and other coenzymes followed by coupled oxidative phosphorylation of adenosine diphosphate (ADP) in the mitochondria (42). Although ATP serves as a carrier of chemical energy in all living cells, it is not a reservoir of energy (41). The intracellular concentrations of ATP are small, highly regulated, and rapidly depleted. Reservoirs of energy (phosphagens), such as phosphocreatine, do exist in some cells (41). These reservoirs accept HEP bonds when the intracellular concentration of ATP is high and transfer a phosphate group to ADP nucleotides when the availability of ATP is low (41). The intracellular concentrations of adenine nucleotides also provide the cell with a sensitive control mechanism for regulating energy-producing and energy-using processes in cells. Atkinson (43) has introduced the concept of energy charge to explain this regulatory mechanism. Adenylate energy charge (EC) represents the balance between energyusing processes and energy-producing processes. It is defined by the equation: EC ¼ ðATP þ 0:5ADPÞ=ðATP þ ADP þ AMPÞ A normal energy charge signals that energy-producing processes and energy-using processes are in balance. If the 0

Table 11 Energy Released (Go ) During Hydrolysis of High-Energy Phosphate Compounds 0

Reaction

Go (J/mol)

ATP þ H2O ! ADP þ Pi ADP þ H2O ! AMP þ Pi ATP þ H2O ! AMP þ PPi PPi þ H2O ! 2Pi AMP þ H2O ! A þ Pi

36,800 36,000 40,600 31,800 12,600

Abbreviations: A, adenine; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; Pi, inorganic phosphate; PPi, inorganic pyrophosphate. Source: From Ref. 41.

9

energy charge is increased, energy-producing processes are exceeding energy-using processes, and a resultant reduction of energy-producing processes occurs. If the energy charge is decreased, energy-using processes are exceeding energyproducing processes, resulting in a decrease in energy-using processes that may jeopardize cell survival. The major energy-producing processes include the catabolism of carbohydrates, proteins, and lipids. Each of these substrate groups can provide part of the energy present in their structure through cytoplasmic catabolic reactions (e.g., glycolysis). The remainder of the available energy present in these substrates is released during oxidation of the remaining carbon fragments in the intramitochondrial tricarboxylic acid (TCA) cycle. The final common pathway into the TCA cycle for carbohydrates, proteins, and lipids is through the formation of acetyl coenzyme A(CoA) (Figs.6–8). Foreachmolecule ofacetylCoA completely oxidized in the TCA cycle, two molecules of carbon dioxide, three molecules of nicotinamide adenine dinucleotide (reduced form) (NADH), one molecule of flavin adenine dinucleotide (FADH), and one molecule of guanosine triphosphate (GTP) are produced. In total, 12 HEPs are formed primarily by way of transfer of the reducing equivalents from NADH and FADH to oxygen in the electron transport system with the subsequent phosphorylation of ADP linked to this oxidative process (oxidative phosphorylation). Unlike carbohydrates and lipids, amino acids may directly enter the TCA cycle at one of the intermediate reactions (Fig. 6).

Carbohydrate Metabolism In the cytoplasm of all cells, one molecule of glucose is catabolized to pyruvate through the Embden–Meyerhof pathway to yield two molecules of pyruvate, two molecules of ATP, and one molecule of NADH (Fig. 7). The completion of glycolysis (i.e., glucose to lactate) uses one molecule of NADH in the conversion of pyruvate to lactate. Conversely, the conversion of pyruvate to acetyl-CoA produces one molecule of NADH. Because the oxidation of acetyl-CoA in the TCA cycle produces 15 HEPs, the complete oxidation of one molecule of glucose to carbon dioxide and water produces 26 HEPs [(2 12) þ 2] and four molecules of NADH. The latter molecules produce 12 HEPs (three HEPs per molecule of NADH) through coupled oxidative phosphorylation. Consequently, the total energy produced in the complete oxidation of one molecule of glucose to carbon dioxide and water is equivalent to 38 HEPs. This result is in contrast to glycolysis where only two HEPs are produced in the conversion of glucose to lactate. As noted in Fig. 7, there are two nonreversible reactions in glycolysis: (i) the conversion of glucose to glucose 6-phosphate, catalyzed by hexokinase, and (ii) the conversion of phosphoenolpyruvate to pyruvate, catalyzed by pyruvate kinase. These reactions are irreversible because they lose a considerable amount of energy as heat. It is the presence of these two nonreversible reactions that drives glucose to pyruvate. In addition, phosphofructokinase (PFK) and pyruvate kinase act as the major regulators of glycolysis (45). Once the catabolism of glucose has begun, it rapidly proceeds to pyruvate. Under aerobic conditions, most tissues oxidatively decarboxylate pyruvate to acetyl-CoA and then oxidize the acetyl-CoA in the TCA cycle. Under an anaerobic condition, pyruvate cannot be decarboxylated, and it is converted instead to lactate. As a result, elevated tissue and plasma concentrations of lactate (and pyruvate) are characteristic of ischemia and anoxia. Some tissues with a paucity of mitochondria, such as erythrocytes and leukocytes, are capable of glycolysis only. These cells lack

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10

Part One: General Considerations

Protein Glycogen

Proteolysis

Glucose Lactate

Glycolysis Pyruvate

Beta-oxidation

Fatty acyl-CoA

Phenylalanine Tyrosine Leucine Lysine Tryptophan

Cystine Glycine Alanine Serine Threonine

Fatty acid

Acetoacetyl-CoA

Acetyl-CoA

Cholesterol Oxaloacetate Malate Fumarate Succinate

Citrate

TCA cycle (15 ATP/TURN)

Ketone bodies

Isocitrate Alpha-ketoglutarate

Succinyl-CoA Glutamate

Phenylalanine Tyrosine

Isoleucine Methionine Valine

Aspartate Asparagine

the ability to oxidize pyruvate and acetyl-CoA even under aerobic conditions. Therefore they derive all their energy from conversion of glucose to pyruvate and lactate. Carbohydrates other than glucose can also be metabolized through glycolysis. For example, fructose, galactose, mannose, and triose sugars can enter glycolysis after modification by endergonic reactions. Similarly, pentose sugars may also enter the glycolytic pathway. As noted earlier, the total carbohydrate stores of the human body are limited and are rapidly depleted (31). In addition, RBCs, WBCs, and the brain are glucose-dependent tissues that are unable to use nonglucose energy substrates. Thus glucose must be made continuously available. The synthesis of glucose through a process called gluconeogenesis can proceed from lactate, pyruvate, and amino acids. Gluconeogenesis is not simply the reversal of glycolysis, because the unidirectional reactions make glycolysis irreversible. However, gluconeogenic tissues, such as the liver and the kidney, contain four enzymes that essentially allow glycolysis to proceed in reverse fashion from pyruvate (and lactate) to glucose (Fig. 8). The first of these enzymes, pyruvate carboxylase, in the presence of ATP, carbon dioxide, and biotin, converts pyruvate to oxaloacetate in the mitochondria. [Although it does not act as a cofactor, acetyl-CoA must be present in excess for this reaction to proceed (46).] Oxaloacetate is then converted to phosphoenolpyruvate by the cytoplasmic enzyme phosphoenolpyruvate carboxylase and GTP. Because the oxaloacetate is found in the mitochondria and phosphoenolpyruvate carboxylase in the cytoplasm, oxaloacetate must cross the mitochondrial membranes into the cytoplasm. However, because the mitochondrial membranes are relatively impermeable to oxaloacetate, it is thought that oxaloacetate leaves the mitochondria as either malate or aspartate, which

Arginine Histidine Glutamine Proline

Figure 6 Pathways for production and use of acetyl-CoA. Abbreviations: TCA, tricarboxylic acid; CoA, coenzyme A. Source: From Ref. 44.

can be transported through the mitochondrial membranes and then reconverted to oxaloacetate in the cytoplasm (47). Once phosphoenolpyruvate is formed, glycolysis can easily proceed in reverse fashion to fructose-1,6-biphosphatase. The enzyme fructose-1,6-bisphosphatase is required to form fructose 6-phosphate. This enzyme is present in the liver and kidney and to a lesser extent in skeletal muscle. However, it is not present in adipose tissue, smooth muscle, or cardiac muscle (48). Fructose 6-phosphate can then proceed to glucose 6-phosphate by the reversible glycolytic reaction catalyzed by glucose phosphate isomerase, but the conversion of glucose 6-phosphate to glucose requires the last of the gluconeogenic enzymes, glucose 6-phosphatase. This enzyme is present in the liver and kidney but not in skeletal, smooth, or cardiac muscle. Any glucose 6-phosphate that might be formed in skeletal muscle must be converted to glycogen, be used in glycolysis, or be used in the hexose monophosphate shunt, because skeletal muscle cannot release free glucose as a result of the absence of glucose 6-phosphatase. The glucose 6-phosphate formed in the liver and kidney can be converted to glucose and released into the circulation. Because there is a constant production of lactate and pyruvate in aerobic glycolytic tissues, in all tissues during anaerobic conditions, and from the reticuloendothelial system during sepsis and locally inflamed tissues, a constant source of lactate and pyruvate is available to gluconeogenic tissues such as the liver and kidney. In the liver and kidney, these substrates can be converted back to glucose and released into the circulation. The newly formed glucose is then available to glucose-dependent tissues for reconversion to lactate in the so-called Cori cycle (Fig. 9). However, it should be noted that this reconversion does not result in a net increase in glucose carbon, because lactate is itself derived from glucose (49).

Chapter 1:

Metabolic Response to Starvation, Stress, and Sepsis

Glycogen

11

Pyruvate + CO2 ATP Pyruvate carboxylase ADP

Glucose 1-phosphate

Oxaloacetate

ATP ADP

GTP Glucose

Glucose 6-phosphate Hexokinase

Phosphoenolpyruvate carboxylase

GDP

Glucose phosphate isomerase

Phosphoenolpyruvate

Fructose 6-phosphate ATP Phosphofructokinase

ADP

Fructose 1,6-diphosphate

Glyceraldehyde 3-phosphate NAD

Pi

NADH

Triose phosphate

Triose phosphate isomerase Dihydroxyacetone phosphate

Phosphoglycerate kinase Phosphoglyceromutase Enolase

ADP

Lactate dehydrogenase

Phosphoenolpyruvate ATP Pyruvate kinase ADP

Lactate

Pi Fructose 1,6-bisphosphate

Glyceraldehyde phosphate dehydrogenase

3 phospho-glycerol phosphate ATP

H 2O

Fructose-bisphosphatase Fructose 6-phosphate H2O Pi Glucose 6-phosphatate Glucose 6-phosphatase

Pyruvate NAD Pyruvate dehydrogenase

NADH NAD NADH

Glucose

TCA cycle

Figure 8 Gluconeogenic pathway in the liver and the kidney. Source: From Ref. 44.

CO2

Figure 7 Catabolism of glucose and major carbohydrate precursors. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; NAD, nicotinamide adenine dinucleotide; NADH, reduced form of NAD; Pi, inorganic phosphate; TCA, tricarboxylic acid. Source: From Ref. 44.

Protein and Amino Acid Metabolism Energy and glucose can also be derived from the metabolism of amino acids. Although the partial catabolism and transformation of all a-amino acids to their a-keto acid derivatives can occur in most tissues, the complete oxidation of a-amino acids to urea and carbon dioxide occurs primarily in the liver and secondarily in the kidney (50). In general, the catabolism of all amino acids (except lysine) involves the removal of the a-amino acid group from the carbon skeleton to form ammonia and an a-keto acid. This is followed by the conversion of ammonia to urea and by the conversion of the a-keto acids to TCA-cycle intermediates or precursors (51,52). Removal of the a-amino group can occur by one of three processes: (i) transamination, (ii) oxidative deamination, and (iii) nonoxidative deamination (47,48). The most common mechanism is transamination. Transaminases (aminotransferases) interconvert a pair of amino acids and a pair of a-keto acids (Fig. 10). This process requires the presence of pyridoxal phosphate (vitamin B6) for the transfer of the amino group. These reactions are freely reversible and function both in synthesis and in catabolism. At least 12 of the amino acids undergo transaminations, including the branched-chain amino acids, valine, leucine, and

isoleucine. The most notable transaminases are glutamineoxaloacetic transaminase (or aspartate transaminase) and glutamic-pyruvic transaminase (or alanine transaminase). Through the collective action of all the transaminases, the a-amino groups are usually collected in the form of glutamate or alanine. Because a-ketoglutarate can accept the a-amino group of all the amino acids that are transaminated, including alanine, it serves as the final common amino group acceptor to form glutamate. Mallette et al. (53) have proposed and Felig (54) has expanded the concept of an alanine–glucose cycle similar to the Cori cycle (Fig. 9). In peripheral tissues, amino acids are transaminated with pyruvate to form alanine and an a-keto acid. The alanine is then transported to the liver, where it is transaminated with a-ketoglutarate to form pyruvate and glutamate. The pyruvate can then be converted back to glucose and released into the circulation, where it may be taken up by peripheral tissues and converted to pyruvate and lactate. The oxidative deamination of glutamate by glutamate dehydrogenase is an important mechanism in the liver for the removal of the amino group (Fig. 10). Because a-ketoglutarate is the common acceptor for all transaminases, substantial amounts of glutamate are formed. Consequently, the oxidative deamination of glutamine allows for the regeneration of a-ketoglutarate and the removal of free ammonia. Oxidative deamination of other amino acids is also possible through the action of a-amino

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12

Part One: General Considerations

Figure 9 Glucose–lactate (Cori) cycle and glucose–alanine cycle. Source: From Ref. 1.

acid oxidases that are present in the liver and kidney. However, with the exception of glutamate dehydrogenase, these enzymes do not appear to exert a major physiologic role in humans (50,52). Three amino acids, serine, threonine, and histidine, are primarily deaminated nonoxidatively (Fig. 10) (47). The former two amino acids undergo nonoxidative deamination by dehydration, whereas histidine undergoes direct deamination. The non–a-amino groups of glutamine and asparagine are removed by hydrolytic deamination (47). Free ammonia, even in small concentrations, is poorly tolerated by cells. Four mechanisms exist to handle the free

OXIDATIVE DEAMINATION Alpha-amino acid

Amino acid oxidase [Alpha-imino acid]

Flavin Flavin

H2O

H2

Alpha-Keto acid

ammonia produced by oxidative or nonoxidative deamination, thereby keeping the intracellular (and extracellular) concentration of this substance low. Free ammonia can be added to glutamate by glutamine synthetase to form glutamine. This is the primary mechanism for the elimination of ammonia in brain cells and muscle cells. The free ammonia may also be added to a-ketoglutarate, forming glutamate in the freely reversible reaction catalyzed by glutamate dehydrogenase. The resulting glutamate may be used as an amino acid in protein, as a precursor in arginine and citrulline syntheses, or as an a-amino group donor in transaminase reactions. A third mechanism for ammonia elimination is through its excretion by the kidney. Two-thirds of the ammonia excreted by the kidney is derived from the amide nitrogen of glutamine from renal arterial blood and one-third from the a-amino nitrogen of renal arterial amino acids (55). Although these three mechanisms remove a substantial amount of the ammonia formed, most of it is cleared by the liver, after which it enters into the urea cycle (Krebs– Henseleit cycle) (50). As noted in Figure 11, the urea cycle essentially involves the cleavage of a molecule of urea from arginine. The ammonia is first combined with carbon dioxide in the presence of ATP to form carbamylphosphate. Carbamylphosphate then condenses with ornithine to form citrulline, which, through a series of reactions, forms arginine. The arginine is then cleaved into urea and ornithine, thereby reestablishing the cycle. The net energy cost of this cycle is four HEPs, derived from three molecules of ATP. Atkinson and Bourke (56) have suggested an important role for ureagenesis in the maintenance of pH homeostasis. Because the oxidation of amino acids yields both bicarbonate and ammonium ions, the urea cycle promotes the neutralization of the bicarbonate ion by the proton of the ammonium ion during the formation of carbonyl phosphate. The remaining carbon skeletons of amino acid deamination or transamination are converted either to intermediates of the TCA cycle or to precursors of acetyl-CoA, such as pyruvate and acetoacetate. Consequently, all the carbon skeletons of amino acids can be oxidized in the TCA cycle to carbon dioxide and water. The carbon skeletons of all the amino acids may also be converted to glucose or fat. As such, they may be classified as glucogenic, ketogenic, or glucogenic and ketogenic (Table 12). Seven (alanine, serine, glycine, cysteine, cystine, proline, and hydroxyproline) of the 22 most common amino

NH3

NONOXIDATIVE DEAMINATION BY DEHYDRATION H2 O Amino acid oxidase Serine [Intermediates] Pyruvate H 2O

NH3

Pi Citrulline

2 ADP+Pi

DIRECT NONOXIDATIVE DEAMINATION Histidase Serine

CO2+NH4 ATP

[Histidine-enzyme]

Pyruvate

Aspartate ATP

Carbamyl phosphate

AMP+PPi Ornithine

UREA CYCLE

Argininosuccinate

NH3 HYDROLYTIC DEAMINATION OF NONALPHA AMINO GROUP Arginine

Asparaginase Asparagine

Aspartate

Urea

Fumarate

H2O NH 3

Figure 10 Mechanisms for removal of the amino group. Source: From Ref. 44.

Figure 11 Urea cycle. Abbreviations: ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; PPi, inorganic pyrophosphate. Source: From Ref. 44.

Chapter 1:

Table 12 Pathways for the Use of Amino Acid Carbon Fragments Gluconeogenesis Alanine Arginine Aspartic acid Asparagine Cystine Glutamic acid Glycine Histidine Hydroxyproline Methionine Proline Serine Threonine Valine

Ketogenesis Leucine

Gluconeogenesis and ketogenesis Isoleucine Lysine Phenylalanine Tyrosine Tryptophan

acids in proteins are converted to pyruvate. Depending on the redox state of the cell, the pyruvate can either be used for gluconeogenesis or converted to acetyl-CoA. Five amino acid carbon skeletons (phenylalanine, tyrosine, tryptophan, leucine, and lysine) form acetoacetate that may be converted to acetyl-CoA and either oxidized in the TCA cycle or used in fatty acid–synthesis. In addition, in the process of producing acetoacetate, phenylalanine and tyrosine are also cleaved to fumarate, and tryptophan is cleaved to alanine. Thus, phenylalanine, tyrosine and tryptophan may be used both in glucogenesis and in ketogenesis. Lysine may also be used both in glucogenesis and ketogenesis, but its precursor for gluconeogenesis is not known. In contrast, leucine forms one molecule of acetyl-CoA and one molecule of acetoacetate. Because neither acetyl-CoA nor acetoacetate can be converted to pyruvate, the carbon skeleton of leucine can only be used for ketogenesis or oxidation.

Metabolic Response to Starvation, Stress, and Sepsis

13

It should be apparent that any compound that enters the TCA cycle as acetyl-CoA cannot be used as a precursor of glucose. This relates to the fact that by the time it reaches malate, the acetyl-CoA that entered the TCA cycle has been completely oxidized. However, the carbon skeletons of amino acids that are TCA-cycle intermediates can be used for gluconeogenesis. In addition to phenylalanine and tyrosine, which enter the TCA cycle as fumarate, three amino acids enter as succinyl-CoA (isoleucine, methionine, and valine), and two other amino acids enter as oxaloacetate (aspartate and asparagine) and give amino acids that enter as a-ketoglutarate (glutamate, glutamine, proline, histidine, and arginine) (Fig. 12). Consequently, all these amino acids may be used either in gluconeogenesis or in oxidation. Quantitatively, in the isolated perfused liver, only alanine, serine, threonine, and glycine are used in significant amounts for gluconeogenesis (57). As noted previously, excess nitrogen cannot be stored. When the protein intake is excessive, the amino acids resulting from proteolysis are catabolized to nitrogen and a carbon skeleton. The nitrogen is converted to urea, and the carbon skeleton is converted to glucose, lipid, or carbon dioxide, depending on the needs of the cell and the redox state present. Similarly, when glucose is needed but unavailable, excess ingested proteins or existing body proteins are degraded. Although it may be imperceptible, the use of existing body protein for energy or gluconeogenesis always results in the loss of some cellular function.

Lipid Metabolism The final and greatest source of energy in the body is lipid. Stored in adipose tissues as triglycerides, lipids can be released on demand and transported to most tissues for use as an energy source. Tissues capable of using lipids include the liver, kidney, heart, and skeletal muscle; such use,

Figure 12 Pathways through which the carbon skeletons of a-amino acids enter the tricarboxylic acid cycle.

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Part One: General Considerations

however, must occur under aerobic conditions. Non–lipidusing tissues include erythrocytes, leukocytes, and nerve cells. Triglycerides are composed of three fatty acid–chains linked together by a glycerol molecule. During lipolysis, the fatty acids are sequentially cleaved off the glycerol moiety by lipases. The remaining glycerol moiety can then be used for glucose synthesis or converted to pyruvate (Fig. 7). In contrast, fatty acids themselves cannot be used as substrate for gluconeogenesis, because they are ultimately broken down to acetyl-CoA. The catabolism of fatty acids can be divided into two stages. These stages include b-oxidation in the outer mitochondrial membrane to produce molecules of acetyl-CoA, and the processing of acetyl-CoA in the mitochondria to produce carbon dioxide and energy or ketone bodies (Fig. 13) (58,59). Only the first step in fatty acid–catabolism requires energy. In this step, the enzyme thiokinase adds CoA to a fatty acid, producing a fatty acetyl-CoA (Fig. 13). After a sequence of reactions, the final two carbons on the fatty acetyl-CoA are cleaved, resulting in the production of one molecule of acetyl-CoA and a new fatty acetyl-CoA that is two carbon atoms shorter than the parent fatty acetyl-CoA. This process of b-oxidation yields five HEPs per acetyl-CoA formed and, with even-numbered fatty acid chains, continues until the entire fatty acid has been cleaved to acetyl-CoA. In the case of odd-numbered fatty acid chains, b-oxidation continues until a three-carbon fatty acetyl-CoA (propionyl-CoA) remains. The latter substance may then be converted to succinyl-CoA and enter the TCA cycle. The acetyl-CoA that results from the oxidation of fatty acids can be used in one of the three available pathways (Fig. 13) (58,60). The first involves the intramitochondrial oxidation of acetyl-CoA through the TCA cycle to two molecules of carbon dioxide and 12 HEPs. Thus the total

oxidation of a 20-carbon fatty acid, for example, yields: 169 HEPs ¼ ½ð10 5Þ þ ð10 12Þ 1 The second pathway involves the ketogenic pathway in the liver (Fig. 14). Through the action of the enzyme thiolase, two molecules of acetyl-CoA combine to form acetoacetyl-CoA in a freely reversible reaction. AcetoacetylCoA can then be converted to 3-hydroxy-3-methylglutaryl CoA, the precursor in cholesterol synthesis and ketone body formation. The three ketone bodies, acetoacetate, b-hydroxybutyrate, and acetone, are normally produced and released by the liver. Under conditions in which there is an abundance of hepatic glycogen, b-hydroxybutyrate predominates; under conditions in which the liver glycogen is low, acetoacetate predominates. The ketone bodies that are released by the liver can then be used by a variety of peripheral tissues, such as cardiac and skeletal muscle, as a source of energy by conversion back to acetyl-CoA. The final pathway for use of acetyl-CoA is in the synthesis of fatty acids and triglycerides (Fig. 13). This pathway is stimulated by neuroendocrine mechanisms and low cytoplasmic concentrations of fatty acids. Lipogenesis is a cytoplasmic process that requires malonyl-CoA. MalonylCoA is formed from acetyl-CoA by acetyl-CoA carboxylase. When fatty acid–levels are low, the rate-limiting enzyme in malonyl-CoA formation, acetyl-CoA carboxylase, is stimulated, leading to increased intracellular concentrations of malonyl-CoA (61). In turn, the elevated concentration of malonyl-CoA inhibits carnitine acetyl transferase, the enzyme necessary for transport of acetyl-CoA into the mitochondria (62), resulting in an increased concentration of cytoplasmic acetyl-CoA that can then be used for malonyl-CoA synthesis and ultimately for the synthesis of triglycerides and other lipids. In contrast, when the intracellular concentrations of

Figure 13 Metabolic pathways of fatty acid metabolism in the liver. Abbreviations: GAA, gossypol acetic acid; TCA, tricarboxylic acid. Source: From Ref. 1.

Chapter 1:

Acetyl-CoA Thiolase CoA

SH

Acetoacetyl-CoA H 2O HMG-CoA synthetase

Acetyl-CoA CoA

SH

3-Hydroxy-3-methylglutaryl CoA Acetyl CO2 CoA Acetone

HMG-CoA lyase

Acetoacetate

Beta-hydroxybutyrate dehydrogenase

Steroids

Cholesterol NADH NAD+

Beta-hydroxybutyrate

Bile salts

Cholesterol esters

Figure 14 Ketogenic pathway and cholesterol pathway in the liver. Abbreviations: CoA, coenzyme A; HMG, 3-hydroxy-3-methylglutaryl; NADH, reduced nicotinamide adenine dinucleotide. Source: From Ref. 44.

fatty acids are elevated, the rate-limiting enzyme in malonylCoA synthesis (acetyl-CoA carboxylase) is inhibited. Malonyl-CoA concentrations decrease, thereby stimulating carnitine acetyl transferase and increasing the transport of acetyl-CoA into the mitochondria for oxidation and ketogenesis (62). The inhibition of acetyl-CoA carboxylase also results in the accumulation of cytoplasmic citrate that in turn inhibits glycolysis through inhibition of PFK, the so-called Randle effect (45).

NEUROENDOCRINE REGULATORY MECHANISMS Stimuli and Mechanism of Action of the Neuroendocrine System The pathways of intermediary metabolism and substrateto-substrate interactions noted previously are under the local control of substrate availability, cellular redox potential, and cellular energy availability. The integration of this control is governed by the neuroendocrine system. This system may be thought of as a reflex physiologic network in which alterations in homeostasis are perceived by specialized receptors that are located both peripherally and centrally. The receptors

Metabolic Response to Starvation, Stress, and Sepsis

15

transmit their information to the central nervous system (CNS), where the afferent signals are processed and modulated, resulting in release or inhibition of numerous neuroendocrine effectors that produce physiologic changes aimed at correcting the alterations in homeostasis. In the absence of significant injury, sepsis, or starvation, alterations in homeostasis are small, and the responses of the neuroendocrine system to stimuli are directed at fine tuning and integrating the functioning of the organism. In the presence of significant injury, sepsis, or starvation, the stimuli are multiple and intensified, and the reflexes are directed at an integrated attempt by the organism to preserve oxygen delivery, mobilize energy substrates, and minimize pain (Fig. 15) (1). The major stimuli affecting neuroendocrine reflexes include (i) changes in the circulating body fluids; (ii) changes in the oxygen, hydrogen ion, and carbon dioxide concentrations in tissues and blood; (iii) changes in ambient and core temperature; (iv) changes in substrate availability; (v) emotional arousal; (vi) pain; and (vii) infection. Critical to the initiation of the neuroendocrine response is the perception of the stimulus. Paraplegics do not respond to stimuli below the level of cord transection. This lack of response is thought to be the result of the absence of afferent impulses reaching the brain (63). However, conscious perception of the stimulus is not required. An individual responds to a stimulus in the presence of anesthesia, but the response may not be the same as that in the absence of anesthesia. Changes in the circulating body fluids may result from the direct loss of blood (as in hemorrhage), from the loss of plasma volume (as in third-space losses and dehydration), or from the inability of the body fluids to circulate (as in cardiac failure or pulmonary embolism). The changes in circulating body fluids are sensed by high-pressure baroreceptors in the carotid arteries and aorta and by low-pressure stretch receptors in the right atrium. Under normal conditions, the afferent signals from these receptors exert a tonic inhibition of the release of many hormones and of the activities of the CNS and autonomic nervous system (64). When baroreceptor or stretch-receptor activities decrease (e.g., a decrease in blood pressure or blood volume), the tonic inhibition is released, resulting in the increased secretion of adrenocorticotropic hormone (ACTH), vasopressin, b-endorphin, and growth hormone through central pathways and resulting in the increased secretion of epinephrine, norepinephrine, renin, and glucagon through peripheral

Infection Pain

+

Hemorrhage plus ECF loss

Peripheral nerves

Baroreceptors + +

Hypoxemia

+

Fear and anxiety + + + Central _ nervous system +

+ Endocrine glands + + Sympathetic nervous system

Chemoreceptors – + Cardiac stimulation Vascular resistance Fluid shifts + Respiratory stimulation

medwedi.ru

Figure 15 Overview of the neuroendocrine reflexes induced by shock and trauma. Abbreviation: ECF, extracellular fluid. Source: From Ref. 1.

16

Part One: General Considerations

autonomic neural pathways. These responses bring about further neuroendocrine changes such as the inhibition of insulin secretion by epinephrine (65) and the stimulation of aldosterone secretion by renin and ACTH (66). Changes in blood concentrations of oxygen, hydrogen ion, and carbon dioxide initiate neuroendocrine responses through the activation of peripheral chemoreceptors. The chemoreceptors, which are located in the aortic and carotid bodies, have an extremely high–blood flow rate (67). Under normal conditions, these receptors are not activated. However, changes primarily in oxygen and secondarily in carbon dioxide and in hydrogen ions are sensed by these receptors, which result in the activation of neuroendocrine pathways. Because of the high blood flow through the chemoreceptors, the partial pressure of oxygen (PO2) of arterial blood, chemoreceptor tissue, and venous blood is nearly the same. However, a drop in blood flow increases the oxygen extraction by the chemoreceptor tissue, decreases the venous PO2, and through an unknown mechanism, activates the chemoreceptor (67). Consequently, a decrease in circulating volume or pressure not only inhibits baroreceptors and stretch receptors, but also activates chemoreceptors. Pain and emotion also activate the neuroendocrine system. The former acts through the projections of peripheral nociceptive receptors to the CNS, and the latter acts through projections from the limbic areas of the brain to the hypothalamus and lower brain stem nuclei (68). Through these pathways, pain and emotional arousal bring about increased hypothalamic, autonomic, adrenomedullary, and adrenocortical activities, the so-called fight- or-flight reaction of Cannon (69). Abnormalities in core and ambient temperatures, as well as infection, also stimulate neuroendocrine reflexes. Changes in the core temperature of the body are sensed in the preoptic area of the hypothalamus. These changes may result from alterations in ambient temperature, a loss of the normal insulating barrier of the skin (e.g., burns), or a reduction in hepatic thermogenesis produced by inadequate blood flow or substrate supply or in response to inadequate peripheral vasoconstriction or vasodilation. Infection may also decrease the core temperature through the action of endotoxin. Infection may further stimulate neuroendocrine reflexes through a direct action of endotoxin on the hypothalamus (70) or through secondary changes in blood volume, oxygen concentration, substrate concentrations, and pain. The primary substrate alterations that activate the neuroendocrine system are those induced by changes in the plasma glucose concentration. Plasma glucose alterations are sensed by receptors in the hypothalamus and the pancreas. A decrease in plasma glucose concentration stimulates the release of catecholamines, cortisol, growth hormone, and vasopressin through central mechanisms and stimulates the release of glucagon both by central pathways (autonomic nervous system) and peripheral pathways (direct pancreatic activation) (71). In addition, the secretion of insulin is inhibited through central pathways (autonomic nervous system) and directly by the pancreas itself (65). All these stimuli are commonly produced by injury, sepsis, and starvation. Furthermore, these stimuli rarely occur singly. Generally, the individual perceives multiple stimuli that occur both simultaneously and sequentially. Thus the neuroendocrine response is the summation of all the stimuli the individual perceives and processes. According to classic endocrine feedback mechanisms, the

elevation of serum cortisol resulting from one set of stimuli would be expected to inhibit the release of ACTH by a new set of stimuli. Following most injuries, this is not true. The secretion of ACTH is unchanged or increased (potentiated), and the secretion of cortisol may also increase. The mechanism of action of this physiologic facilitation is incompletely understood, but it appears to take 60 to 90 minutes to be of sufficient magnitude to offset the inhibition, and lasts for at least 24 hours (72). Physiologic facilitation and potentiation have been demonstrated with sequential hemorrhages (73) and repeated operations (74), in response to hypoxia and surgery (75), with pain and hemorrhage (20), and with elevated core temperature and hemorrhage (76). Consequently, the response to an injury or an alteration in homeostasis may be modified by previous stimuli, and the response to a second set of stimuli may be different if they had occurred first. The efferent limb of the neuroendocrine system arises from two primary areas, the hypothalamic–pituitary axis and the autonomic regions of the brain stem. The output from the former region involves the release of numerous pituitary hormones, and the output from the latter region involves changes in the neural activities of the sympathetic and parasympathetic nervous systems. Both sets of output either may cause direct changes in physiologic functions or may stimulate or inhibit the secretion of peripheral endocrine organs. The hormones secreted by endocrine organs and the autacoids produced by tissues fall into one of five chemical classes. These include the fatty acid–derivatives of cholesterol (cortisol and aldosterone) or arachidonic acid (prostaglandins), proteins (insulin and glucagon), glycoproteins [thyroid-stimulating hormone (TSH) and corticotropin], small polypeptides (vasopressin and enkephalin), and the amines (catecholamines and serotonin). All these agents act on cellular receptors that are either on the surface of cell membranes or in the cytoplasm of the cell. These cellular receptors are neither fixed nor unchangeable. Instead, they are in a dynamic state in which the number of receptors on cells can be increased (up regulation) or decreased (down regulation) according to need. Furthermore, the affinity of these receptors for their specific hormone can also be changed (77). Steroid hormones (and possibly thyroxine), which are freely permeable to cell membranes, bind to cytosolic receptors in target cells (78,79). The hormone–receptor complex migrates to the cell nuclei, where it interacts with DNA to modulate the transcription of messenger RNA and ultimately the synthesis of enzymatic, structural, and regulatory proteins (Fig. 16A) (78). This may, in part, explain the one- to two-hour delay in the action of steroid hormones. In contrast, the action of most peptide and amine hormones, which generally bind to cell surface receptors, is faster and of shorter duration. In general, these hormones act either through alterations in the intracellular concentrations of cyclic adenosine monophosphate (cAMP) or calcium, the so-called second messengers (81,82), or through other intermediates (growth hormone through somatomedins). The second messenger system of hormonal action operates primarily through the activation and inactivation of regulatory proteins and enzymes rather than through the synthesis of new proteins. This difference explains the faster onset of action and shorter duration of effect of hormones that operate through this system in contrast to those of steroid and other lipid-soluble hormones. The adrenergic receptor system may be considered the prototype for examining the mechanisms of second

Chapter 1:

Metabolic Response to Starvation, Stress, and Sepsis

17

Figure 16 (A) Proposed mechanism of action of steroid hormones. (B) Proposed mechanism of action of peptide hormones through the second messenger system. Abbreviations: ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; m, molecular; MW, molecular weight. Source: From Refs. 1, 80.

messengers, because all the second messenger pathways known are represented in the four adrenergic receptors (a1, a2, b1, and b2) (Fig. 16B). b1- and b2-receptors (differentiated on the basis of radioligand-binding affinity) both function through the activation of membrane-bound adenylate cyclase, which in turn leads to the production of cAMP (81). The increased intracellular concentration of cAMP activates an inactive protein kinase by attaching to a binding protein on the protein kinase molecule. The attachment of cAMP to the regulatory subunit protein results in the release of an active protein kinase that in turn phosphorylates an inactive phosphorylase kinase to an active form. The active phosphorylase kinase then phosphorylates dephosphoregulatory enzymes, possibly resulting in the activation of the regulatory enzyme (e.g., glycogen phosphorylase) or in its inactivation (e.g., glycogen synthetase) (Fig. 17) (83). In addition, active protein kinase may directly act on dephosphoregulatory enzymes without the activation of phosphorylase kinase. In contrast, a2-receptor activation inhibits membrane-bound adenylate cyclase, thereby decreasing the concentration of cAMP and active protein kinase. Activation of a1-adrenergic receptors results in an increase in phosphatidylinositol turnover that then mediates an increase in intracellular

calcium from intracellular and extracellular sources (81,84). The increase in intracellular calcium activates a calciumbinding protein kinase or phosphorylase kinase (Fig. 16B). The actions of intracellular cAMP and calcium in the coupling of receptor activation with hormonal action (stimulus–response coupling) are not independent. Instead, there is a duality to this system in which the actions of calcium and cAMP are highly interrelated, termed synarchic control by Rasmusen (81). As noted in Fig. 18 and Box 2, there are five basic patterns to the synarchic control of hormone-response coupling through cAMP and calcium. In co-ordinate control, a hormone activates both a calciumactivating receptor and a cAMP-activating receptor, either one of which may produce the response alone. In hierarchal control, separate stimuli activate independently the calcium and cAMP pathways that are both necessary for a given response. In sequential control, the activation of one of the two lines of the system leads to the activation of the other limb. Although the first limb can produce the response, activation of the second limb augments the response. In redundant control, two separate stimuli independently activate the two different limbs of the messenger system, either one of which can produce the response. Finally, in

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Part One: General Considerations

Box 2 Calcium–Cyclic AMP Interactions in Stimulus-Response Coupling

Figure 17 Activation of protein kinase leading to enzymatic or physiologic response. Activation of the dephosphoenzyme by phosphorylation may be brought about either directly by the active protein kinase or indirectly through the activation of a phosphorylase kinase by the active protein kinase. Abbreviations: ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate. Source: From Ref. 80.

antagonist control, one stimulus activates one limb of the messenger system that leads to the response, and a second stimulus activates the second limb, which inhibits the ability of the first limb to produce the response. Although each of these control mechanisms can occasionally be found in cells in pure form, most of the presently known hormone– response coupling mechanisms involve mixed patterns (81).

1. Effects of calcium on cAMP messenger systems a. Stimulates cAMP production—brain, adrenal cortex, pancreatic islets, adrenal medulla, slime mold b. Stimulates cAMP hydrolysis—brain, heart, liver, kidney, fly salivary gland, many other tissues c. Activates phosphoprotein product of cAMP-dependent protein kinase, glycogenolysis in many tissues 2. Effects of cAMP on calcium messenger system a. Increases calcium entry across plasma membrane—heart, synapse b. Increases calcium release from mitochondria—kidney, liver, fly salivary gland, others c. Increases calcium uptake by microsomes—heart, uterus, liver, smooth muscles d. Increases calcium efflux across plasma membrane—smooth muscle, heart e. Decreases sensitivity of response elements to calcium—smooth muscle, heart f. Increases sensitivity of response element to calcium—phosphorylase beta kinase, liver, muscle 3. Interrelated activities a. cAMP-dependent and calcium-dependent protein kinases act upon same protein substrate—liver, brain, adrenal cortex b. Regulate sequential steps in metabolic or transport process—secretion in fly salivary gland, glycogenolysis Abbreviation: cAMP, cyclic adenosine monophosphate. Source: From Ref. 81.

Hormonal Regulation of Metabolism The neuroendocrine system is able to regulate metabolic reactions through three basic processes. First, it may increase substrate availability so that by simple stoichiometry (mass action), reactions proceed in a desired direction. This process can be brought about either by an increase in the plasma

COORDINATE CONTROL ++

R1

Ca

R2

cAMP

Response

H

HIERARCHICAL CONTROL H1

R1

++

Ca

+

Response

SEQUENTIAL CONTROL H

R

Ca

H2

R2

++

Response

+

cAMP

cAMP H

cAMP

R +

Ca

REDUNDANT CONTROL H1

R1

++

Ca

Response

++

ANTAGONISTIC CONTROL ++

H1

R1

Ca

H2

R2

cAMP

–

Response

Response H2

R2

cAMP

Figure 18 Patterns of synarchic regulation by calcium and cyclic adenosine monophosphate. Source: From Ref. 81.

concentration of a substrate or by alterations in blood flow and its distribution. Second, the neuroendocrine effectors can alter the membrane transport properties of cells for a given substrate so that more or less of the substrate enters or leaves the cell. Third, the neuroendocrine effectors can alter the activity or synthesis of key regulatory enzymes that are necessary for reactions to proceed. Most hormones operate through more than one of these processes. For example, insulin stimulates both glycogen synthetase and the transport of glucose into cells. In this manner, insulin not only activates the enzymatic mechanisms necessary for glycogenesis, but also increases the availability of the necessary substrates. It would be futile for a hormone to stimulate opposing processes in a given cell. If a hormone stimulated both glycogenesis and glycogenolysis, there would be no net effect. As a result, most hormones not only activate the enzymes necessary for one metabolic pathway, they also inhibit the enzymes necessary for the opposing process. Thus epinephrine, through an a-mechanism, activates glycogen phosphorylase and inactivates glycogen synthetase, whereas insulin inactivates glycogen phosphorylase and activates glycogen synthetase. The coordinated control of metabolism also requires that a hormone not have opposing actions in different tissues. Thus by increasing amino acid uptake in skeletal muscle and decreasing amino acid degradation in the liver, insulin promotes the availability of an abundant substrate supply for the enzymes of protein synthesis it activates. Cortisol produces an inhibition of amino acid uptake in skeletal muscle, increases amino acid uptake by the liver, and stimulates hepatic gluconeogenic enzymes. These processes ensure that an abundant supply of amino acids is available to the liver for cortisol-stimulated gluconeogenesis.

Chapter 1:

The primary hormones involved in the regulation of metabolism include insulin, cortisol, epinephrine, glucagon, growth hormone, vasopressin, and somatostatin. Insulin is the primary anabolic hormone promoting the synthesis of glycogen, proteins, and lipids. Cortisol, epinephrine, glucagon, and vasopressin are the primary catabolic hormones promoting the breakdown of glycogen, proteins, and lipids and the synthesis of glucose from gluconeogenic amino acids, lactate, and pyruvate. In contrast, the actions of growth hormone initially are anabolic, but its late effects are primarily catabolic.

Insulin, Glucagon, and Somatostatin Insulin, composed of two polypeptide chains, one containing 21 amino acids and the other 30 amino acids, and glucagon, a 29–amino acid polypeptide, are produced and secreted by the pancreatic B cells (beta islets of Langerhans) and A cells (alpha islets of Langerhans), respectively. The secretion of both of these hormones is under the control of at least three mechanisms: (i) circulating substrates (glucose, amino acids, and free fatty acids); (ii) the autonomic nervous system; and (iii) other circulating hormones. Under normal physiologic conditions, glucose is the most important regulator of insulin and glucagon secretion. When the plasma concentration of glucose increases, the secretion of insulin increases, and the secretion of glucagon decreases. When the plasma concentration ofglucosedecreases, the secretion of insulin decreases, and the secretion of glucagon increases. These changes are probably the result of a direct action of glucose on pancreatic islet cells and not a result of neuroendocrine modulation of the pancreas by other neuroendocrine effectors (85). The direct action of glucose on islet cell function may be mediated either through a glucoreceptor on the surface of the islet cell or through the intracellular metabolism of glucose in the islet cells (86). Elevations in the plasma concentration of amino acids stimulate the release of both insulin and glucagon. Most, if not all, of the amino acids increase insulin secretion, but the potency of amino acids in stimulating glucagon secretion is variable (87). In general, more gluconeogenic amino acids appear to stimulate glucagon secretion (87). High concentrations of fatty acids stimulate the secretion of insulin and inhibit the secretion of glucagon. Conversely, low concentrations of free fatty acids inhibit the secretion of insulin and stimulate the secretion of glucagon. The potency of fatty acids in regulating insulin and glucagon secretion is substantially less than that of glucose (87). The stimulation of insulin secretion and the inhibition of glucagon secretion after the administration of an oral glucose load are greater than that following the intravenous administration of glucose (88). Similarly, the stimulation of both insulin and glucagon secretion is greater after an oral protein or amino acid load than it is after the intravenous administration of amino acids and protein (89). This effect is thought to be the result of the higher concentrations of substrate in the pancreas, the potentiation by gastrointestinal hormones of the substrate effect on the pancreas, and the effect of neural input to the pancreas that has been stimulated by eating (87). The gastrointestinal hormones, cholecystokinin, gastrin, vasoactive intestinal peptide, substance P, neurotensin, and gastric inhibitory peptide (GIP), increase the secretion of both insulin and glucagon in pharmacologic concentrations (87,90). Although gastrin does appear to potentiate the release of glucagon and insulin induced by amino acids and GIP in physiologic concentrations and

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19

appears to augment the release of insulin by glucose (87), and the physiologic role of gastrointestinal hormones is not certain. The pancreatic A cells and B cells both have a- and b-adrenergic receptors that alter the secretion of insulin and glucagon. a-Adrenergic stimulation of the pancreas inhibits the secretion of both insulin and glucagon, whereas b-adrenergic stimulation of the pancreas stimulates the secretion of both insulin and glucagon (91,92). However, the a- and b-adrenergic receptor density of A cells and B cells is not the same. The b-adrenergic receptor density of A cells is greater than that of B cells (92). As a result, increased sympathetic stimulation of the pancreas or increased circulating concentrations of epinephrine or norepinephrine increase the secretion of glucagon but decrease the secretion of insulin (91,92). In contrast, isoproterenol infusion increases the secretion of both insulin and glucagon (92). In addition to sympathetic stimulation, the parasympathetic limb of the autonomic nervous system alters pancreatic hormone secretion. Both acetylcholine infusion and direct parasympathetic stimulation of the pancreas increase the secretion of both insulin and glucagon (87). In addition to the gastrointestinal hormones and the autonomic nervous system, other hormones alter the secretion of insulin and glucagon. b-Endorphin appears to directly increase the secretion of insulin and glucagon (93), insulin inhibits the release of insulin and stimulates the release of glucagon (87), and glucagon inhibits the release of glucagon and stimulates the release of insulin (87,94). Insulin and glucagon appear to exert their action both directly on islet cells and by the alterations they produce in circulating substrates (87). Cortisol stimulates the release of insulin and glucagon, but it appears to have no direct activity on the secretory ability of A cells and B cells. Instead, cortisol is believed to increase glucagon secretion through an increase in plasma amino acids and to increase insulin secretion by an increase in plasma glucose. In this regard, both cortisol and epinephrine are able to inhibit the peripheral actions of insulin, and both are thought to exert a major role in insulin resistance (95,96). Somatostatin, a tetradecapeptide, is a potent inhibitor of both insulin and glucagon secretion (90,97). In addition to its location in pancreatic D cells, somatostatin is found in the hypothalamus, limbic system, brain stem, spinal cord, other neural tissue, salivary glands, parafollicular thyroid cells, kidneys, and gastrointestinal tissue (90). Although somatostatin was originally named for its ability to inhibit growth hormone secretion, somatostatin is now recognized to inhibit the secretion of TSH, renin, calcitonin, gastrin, secretin, cholecystokinin, insulin, and glucagon (90). In addition, somatostatinergic nerve fibers are involved in the projection of impulses from peripheral sensory organs to the neuroaxis (90). The role somatostatin exerts in the physiologic regulation of insulin and glucagon secretion is not known precisely. The A, B, and D cells have somatostatin receptors that, when activated, inhibit the secretion of glucagon, insulin, and somatostatin, respectively. Although the mechanism of action of somatostatin is thought to be mediated primarily by the local diffusion of somatostatin from D cells to A cells and B cells (90,97), current evidence suggests that somatostatin reaching the pancreas through the blood stream may be more important (98). The effects of somatostatin on A cells are transient, but the effects on B cells are persistent (97). This persistence may account for the relative hyperglycemia that occurs in patients with somatostatinomas or after the longterm administration of somatostatin (97).

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The physiologic actions of glucagon occur primarily in the liver and are mediated through an increase in intracellular cAMP. The activation of glycogen phosphorylase and the inhibition of glycogen synthetase by glucagon promote the breakdown of glycogen to glucose (glycogenolysis) (45). In addition, glucagon stimulates gluconeogenesis through the stimulation of phosphoenolpyruvate carboxykinase, amino acid transport, and amino acid transamination (45,99). The net result is an increase in hepatic production and release of glucose that under basal conditions accounts for 75% of the glucose produced by the liver (100). Glucagon also exerts an important influence over hepatic lipid metabolism. In addition to stimulating lipolysis in adipose tissue and the liver, glucagon inhibits acetyl-CoA carboxylase, the enzyme that converts acetyl-CoA to malonyl-CoA (87). In turn, the reduction in malonyl-CoA produces inhibition of triglyceride synthesis and activation of carnitine acyl transferase. The latter increases fatty acid transfer to the mitochondria and therefore increases the oxidation of acetyl-CoA and ketogenesis (58,87). Peripheral actions of glucagon include the stimulation of lipolysis in adipose tissue, of glycogenolysis in skeletal muscle, and of myocardial contractility (101–103). However, these actions do not appear to be of physiologic significance in human beings (101–103). As a result of glucagon’s ability to increase hepatic glucose production, mobilize fat, and increase ketogenesis, glucagon is important in normal metabolism and more so in the metabolism of altered states. However, the effects of glucagon are evanescent (104). After 30 to 60 minutes, the activity assigned to glucagon decreases even if plasma glucagon concentrations remain elevated. Therefore it appears that an increase in glucagon concentration rather than the absolute amount of glucagon present is a key determinant of glucagon activity (104). This effect also appears to be true of other cAMP-mediated hormones (the burst effect). The physiologic activity of insulin is primarily in the liver, skeletal muscle, and adipose tissue, but insulin does affect many other peripheral tissues. Notable exceptions include erythrocytes and wounded tissue. Insulin promotes the entry of glucose into cells by stimulating the membrane transport of glucose. The increased intracellular concentrations of glucose are used in glycogen synthesis (stimulation of glycogen synthetase and inhibition of glycogen phosphorylase) and in glycolysis (stimulation of glucokinase, PFK, and pyruvate kinase) to produce energy (45). In addition, insulin inhibits gluconeogenesis through the inhibition of phosphoenolpyruvate carboxylase and the stimulation of PFK and pyruvate kinase (45). Insulin also increases the membrane transport of amino acids into the liver and peripheral tissues. The increased intracellular concentrations of amino acids are used in protein synthesis (stimulation of protein synthesis and inhibition of proteolysis). By inhibiting gluconeogenesis and amino acid oxidation, insulin further directs the intracellular amino acids to protein synthesis (99). In adipose tissue, insulin stimulates lipogenesis and inhibits lipolysis, as it does in the liver. By stimulating lipoprotein lipase, insulin also makes triglycerides more available for uptake from the plasma by adipose tissue. Glycerol synthesis and the action of the pentose-phosphate shunt also are increased by insulin in adipose tissue and the liver. Thus insulin is the primary anabolic hormone promoting the storage of lipid, glucose, and protein.

Although insulin and glucagon oppose each other in the metabolic processes each stimulates, a bihormonal response is necessary for maintenance of glucose homeostasis after a protein meal (90). If insulin were secreted alone in response to a protein meal, the increase in protein synthesis and decrease in hepatic glucose production would result in hypoglycemia. Conversely, if glucagon were secreted alone, the decrease in protein synthesis and the increase in hepatic gluconeogenesis would result in hyperglycemia. However, when a rise in glucagon is accompanied by an increase in insulin, hepatic glucose production remains unchanged and euglycemia is maintained. In this regard, Unger (105) has proposed the insulin/glucagon (I/G) ratio as a quantitative measure of hepatic glucose balance. When the I/G ratio is greater than 5, anabolism and protein synthesis are favored. When the I/G ratio is less than 3, glycogenolysis, gluconeogenesis, and lipolysis are favored. However, the validity of this relationship has been questioned (86).

ACTH, Cortisol, and Epinephrine The primary hormones released in response to any physiologic or psychologic stress are the glucocorticoids and catecholamines. These hormones are in large part responsible for the ‘‘fight-or-flight reaction.’’ The release of cortisol is under the control of ACTH, a 39–amino acid polypeptide released from the chromophobe cells in the anterior pituitary gland. In turn, the release of ACTH is itself under the inhibitory influence of cortisol and the stimulatory influence of corticotropin-releasing factor (CRF) produced by the hypothalamus. The release of CRF (and ACTH–cortisol) is stimulated by all the stimuli noted previously and is potentiated by vasopressin, oxytocin, and angiotensin II (106,107). ACTH acts directly on cells of the adrenal zona fasciculata, stimulating the production and release of cortisol through a cAMPmediated conversion of cholesterol to pregnenolone (108). The catecholamines (epinephrine, norepinephrine, and dopamine) are the prototypical neuroendocrine effectors that act as neurotransmitters and hormones (109). Epinephrine, produced almost exclusively by the adrenal medulla, functions primarily as a hormone, whereas norepinephrine and dopamine function primarily as neurotransmitters (109). Although the adrenal medulla may be viewed as a collection of postganglionic sympathetic neurons without axons that release their neurotransmitters into the general circulation, the activation of the sympathetic nervous system does not occur in an all-or-none fashion, and it is not synonymous with adrenomedullary secretion (110). Similarly, adrenomedullary stimulation is not synonymous with the complete activation of the sympathetic nervous system. Numerous stimuli have been identified that lead to increased secretion of catecholamines from the adrenal medulla (e.g., hypotension, hypoxia, hypoglycemia, pain, and fear), but the exact mechanisms involved in adrenomedullary control remain poorly understood (110). Both cortisol and epinephrine function as ‘‘counterregulatory’’ hormones, mediating catabolic processes throughout the body. In the liver, cortisol inhibits several key glycolytic enzymes (glucokinase, PFK, and pyruvate kinase), the pentose-phosphate shunt, and the actions of insulin (45,108). In addition, cortisol stimulates the hepatic uptake of amino acids, transaminases, and several gluconeogenic enzymes (pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase), as well as potentiating the actions of glucagon and epinephrine (45,95,111). As a result, the production of glucose, lactate, and pyruvate by the liver is increased.

Chapter 1:

The metabolic effects of epinephrine are similar to those of glucagon but are more widespread, affecting peripheral tissues and the liver. In the liver, epinephrine stimulates glycogenolysis (a1-mediated stimulation of glycogen phosphorylase and inhibition of glycogen synthetase) (81), lipolysis (b1-mediated activation of triacylglycerol lipase) (112), ketogenesis (b1-mediated inhibition of acetylCoA carboxylase leading to decreased malonyl-CoA and increased carnitine acyl transferase) (68), and gluconeogenesis (b1-mediated inhibition of PFK and hexokinase by the products of glycolysis and glycogenolysis) (113). Thus epinephrine serves to increase hepatic glucose production and lipid breakdown. Although both glucagon and epinephrine increase glucose production by the liver, glucose use by peripheral tissues is not the same in the presence of epinephrine as it is in the presence of glucagon (99). Glucagon promotes the use of glucose by peripheral tissues through the stimulation of insulin secretion. In contract, epinephrine inhibits both the release and the action of insulin, thereby decreasing glucose use in insulin-dependent peripheral tissues. However, epinephrine serves to increase glucose availability to insulin-insensitive tissues such as the brain, whereas glucagon does not shunt glucose to insulininsensitive tissues (99). In adipose tissue, epinephrine increases lipolysis (b-mediated activation of triacylglycerol lipase). In peripheral tissues, epinephrine stimulates glycogenolysis (a1) and inhibits stimulated glucose uptake through a b2- and a1-mechanism (114,115). As a result of increased substrate availability, glycolysis is increased in skeletal muscle, and large amounts of lactate are produced and released into the circulation. The lactate can then be taken up by the liver for subsequent gluconeogenesis (Cori cycle). Therefore during stressful conditions, epinephrine and cortisol both promote a rise in blood glucose and make glucose more available to glucose-dependent tissues. Both of these hormones also promote the breakdown of lipid and thereby its use as a source of fuel. Whereas the actions of epinephrine are direct, many of the actions of cortisol occur as a result of the potentiation or inhibition of other hormones, the so-called permissive action of cortisol.

Growth Hormone and Vasopressin Growth hormone is a 191–amino acid polypeptide that is released from acidophilic cells in the anterior pituitary gland. Its secretion is under the control of a releasing factor (growth hormone–releasing factor) and by an inhibiting factor (somatostatin) (116). Elevation of blood glucose or free fatty acids stimulates the release of growth hormone (116). In addition, the release of growth hormone is stimulated by vasopressin, ACTH, a-melanocyte–stimulating hormone, and estrogen; release of growth hormone is inhibited by cortisol, thyroxine, and growth hormone itself. In addition to its ability to promote protein synthesis and RNA synthesis and to increase linear growth, growth hormone exhibits an important role in the regulation of metabolic processes. Its effects are biphasic, composed of early effects of three to four hours duration and late effects of longer duration (116). In muscle and liver, growth hormone increases amino acid uptake and protein synthesis (117). In addition, growth hormone stimulates glucose uptake in skeletal muscle and antagonizes the lipolytic effects of catecholamines in adipose tissue while increasing protein synthesis (117). Therefore the early effects of growth

Metabolic Response to Starvation, Stress, and Sepsis

21

hormone are similar to insulin. In fact, growth hormone directly stimulates the secretion of insulin by pancreatic B cells during its early phase (66). The late effects of growth hormone include an increased mobilization of fatty acids and ketone bodies by adipose tissue as a result of increased lipolysis. This action of growth hormone occurs only in the presence of cortisol (118). In addition, growth hormone inhibits insulin-stimulated glucose uptake and use, thereby producing a profound stimulation of insulin release by hyperglycemia (66). Arginine vasopressin (antidiuretic hormone) is a nonapeptide that is released by the posterior pituitary gland. Although released primarily in response to an increase in plasma osmolality and to a reduction in effective circulating volume (98,119), vasopressin release is also stimulated by hypoglycemia through nonosmotic pathways (120). Vasopressin is a powerful stimulator of hepatic glycogenolysis (a-receptor) and also stimulates hepatic gluconeogenesis (121,122). As such, it may exert an important role in elevating the blood glucose after injury and during hypoglycemia.

METABOLIC RESPONSE IN STARVATION, INJURY, AND SEPSIS Fasting and Starvation In the absence of food, fasting humans must supply the energy required for daily activities, glucose for glucosedependent tissue, essential amino acids (Table 12), and essential fatty acids from existing body stores. Cahill (29) has estimated that the average resting 70 kg man using 1800 kcal of energy per day requires 180 g of glucose daily—for the metabolism of nervous tissue (144 g) and for other glycolytic tissue (RBCs, WBCs, and the renal medulla) (36 g) (Fig. 19). Because the available glycogen in the liver is only 75 g (Table 13), this amount will not suffice for either the energy requirements or the glucose needs of a fasting man. Although an additional 150 g of glucose is in skeletal muscle as glycogen, as noted previously, it cannot be released from skeletal muscle as free glucose as a result of the absence of glucose 6-phosphatase. Thus, it is apparent that the energy requirements and glucose requirements of fasting human beings must be supplied from noncarbohydrate sources and by gluconeogenesis. The daily energy requirements can be met by the mobilization of approximately 160 g of triglycerides from adipose tissue in the form of free fatty acids (19). The free fatty acids, as well as ketone bodies produced by the liver, are used throughout the body by nonglycolytic tissues such as the heart, kidney, muscle, and liver. In the liver, energy derived from b-oxidation of fat and from oxidation of acetyl-CoA is used to drive the necessary gluconeogenic processes. Gluconeogenic substrates are available from three sources (Table 14). First, the lipolysis of 160 g of triglycerides releases 16 g of glycerol that can be converted by the liver to glucose. Second, some glucose-dependent tissues (i.e., RBCs and WBCs) convert glucose to lactate and pyruvate that may then be reused in the liver by the Cori cycle to produce new glucose. In addition to the 36 g of lactate and pyruvate produced in this manner, skeletal muscle can also release lactate and pyruvate by the breakdown of glycogen and glucose. Third, approximately 75 g of skeletal muscle protein is degraded daily during starvation, and the resulting amino acids are used in the liver for gluconeogenesis. Consequently, the energy required during brief fasting is derived primarily from adipose tissue. In contrast, the

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Part One: General Considerations

FASTING MAN (24 hours, basal: –1800 calories) FUEL CONSUMPTION

ORIGIN OF FUEL

Nerve Muscle protein 75 g

Amino acids Glycerol 16 g

Adipose tissue Triglyceride 160 g

40 g Fatty acid 160 g

144 g Glucose 180 g 36 g 36 g

Liver Glycogen Gluconeo genesis O2 H2O _P

Ketone 60 g

(Fatty acid) 120 g

glucose required is supplied from lactate, pyruvate, glycogen, and amino acids. During the first two to four days of fasting, there is a rapid increase in the urinary nitrogen excretion from 5 to 7 g/day to approximately 8 to 11 g/day (123). This increase is associated with the previously noted breakdown of 50 to 75 g of protein per day. The rapid proteolysis of skeletal muscle protein does not continue during more prolonged fasting. During the next 20 to 40 days of fasting, the urinary nitrogen excretion begins gradually to decline and eventually reaches its nadir of 2 to 4 g of nitrogen per day (123). This decline is the result of ketoadaptation to starvation. In this process, the brain, which does not normally use ketone bodies for fuel, adapts its metabolism and transport systems to use ketone bodies (124). This adaptation results in a significant reduction in the amount of glucose needed by this glucose-dependent tissue and consequently in the amount of amino acid substrate necessary for gluconeogenesis (Fig. 20). Protein conservation follows with only 20 to 30 g of protein catabolized per day (29). The molecular mechanisms responsible for these adaptive responses are incompletely understood. With respect to skeletal muscle proteolysis, an ATP-ubiquitin-proteasome pathway seems to be involved (125,126). Early in the starvation process, this pathway is activated so that muscle-protein degradation occurs during fasting. As the starvation is prolonged, down-regulation of this process occurs so as to conserve muscle protein stores. Concurrent with these adaptations to starvation is a reduction in the resting energy expenditure by as much as 31% (120). In part, the reduction in resting energy expenditure is the result of a reduction in BCM produced by the breakdown of muscle and other proteins. However, the reduction in body size is less than the reduction in resting energy expenditure (120,127). Other factors that may contribute to the reduction in resting energy expenditure include a reduction in voluntary work, a decrease in body temperature, a decrease in cardiac work, a decrease in sympathetic nervous system activity, and a decrease in muscle activity. The changes in metabolism accompanying fasting and starvation are primarily regulated by decreased concentrations of insulin and increased concentrations of glucagon in response to decreasing glucose concentrations (29,58,123). The decreased insulin concentrations promote an increase in lipolysis in adipose tissue and a decrease in glucose uptake

_P

CO2 + H2O

RBC, WBC, etc. _P Lactate and pyruvate O2 Heart, kidney, muscle, etc.

~P

CO2 + H2O

Figure 19 Flow diagram of fuel metabolism in normal fasted man. Abbreviations: RBC, red blood cell; WBC, white blood cell. Source: From Ref. 29.

in insulin-dependent tissues. The increased concentrations of glucagon promote hepatic gluconeogenesis. These changes (including the decreased secretion of insulin and increased secretion of glucagon) may be further augmented by slight increases in the concentrations of epinephrine, ACTH, cortisol, and growth hormone (103,128). However, an actual increase in these hormones is not necessary, because basal concentrations of the counter-regulatory hormones are unopposed by the reduced secretion of insulin that is stimulated by hypoglycemia. Thus through four major adaptive mechanisms, a reduction in resting energy expenditure, the use of protein for gluconeogenesis, the use of fat for energy, and ketoadaptation of the brain, a human is able to survive for prolonged periods of time without food. As a result of the decreased excretion of urea and nitrogen during prolonged starvation, water intake is also reduced. However, it is apparent that this condition cannot be maintained indefinitely. In the average 70 kg man, there are approximately 170,000 calories and 6000 g of protein (Table 13). If it could all be used, starvation for up to 100 days would be tolerated. However, this is not possible because of the loss of essential body functions as body protein is consumed and not replenished. In fact, acute weight losses of 30% to 40% of body weight are usually fatal and associated with a rapid increase in urinary nitrogen excretion and a rapid decline in plasma glucose (123,129,130).

Table 13 Fuel Composition of Normal Humans Fuel Tissues Fat (adipose triglyceride) Protein (mainly muscle) Glycogen (muscle) Glycogen (liver) Total Circulating fuels Glucose (extracellular fluid) Free fatty acids (plasma) Triglycerides (plasma) Total Source: From Ref. 29.

Weight (kg)

Calories

15 6 0.15 0.075

141,000 24,000 600 300 165,900

0.02 0.0003 0.003

80 3 30 113

Chapter 1:

Metabolic Response to Starvation, Stress, and Sepsis

23

Table 14 Amount of Glucose Produced from Lactate, Glycerol, and Amino Acids During Starvation Grams of glucose produced per day Glucose precursor Glycerola Lactate þ pyruvateb Amino acidsc Total glucose produced from above precursors by liver and kidney cortexd Maximum glucose available for oxidation by the brain (i.e., glycerol and amino acid as precursors)e Fuel requirement of brain (glucose equivalents)f Suggested alternative fuel to glucose for braing

3 or 4 days of starvation

Several weeks of starvation

19 39 41 99

19 39 16 74

60

35

120 Ketone bodies

120 Ketone bodies

a

Amount of glucose produced from glycerol is estimated from the amount of triglyceride hydrolyzed per day. In starvation, 190 g of triglyceride is required to satisfy the caloric needs of the subject. Because glycerol represents 10% of triglyceride, it can provide 19 g of glucose per day. This amount is confirmed by measurement of glycerol uptake by liver and kidney using catheterization techniques. b Amount calculated from glucose 1-C turnover studies in man that gives values between 27 and 58 g/day, and this is not affected by the dietary state. Also, the measurement of lactate and pyruvate uptake by the liver and kidney in man by catheterization techniques estimates glucose formation as 39 g/day. c Amount calculated from nitrogen excreted in urine (100 g protein produces 57 g glucose; 1 g nitrogen is equivalent to 6–25 g protein). In early stages of starvation approximately 12 g nitrogen is excreted per day, but this is decreased in prolonged starvation to 4 to 7 g/day. Catheterization studies in subjects undergoing prolonged starvation indicate an uptake of amino acids by liver and kidney that could theoretically produce 26 g glucose per day. d In prolonged starvation, the hepatic–renal glucose production as measured by catheterization techniques provides an estimate of 86 g glucose per day, which is in good agreement with the 74 g obtained in this calculation. e Catheterization techniques have been used to measure the arterio–venous (A–V) differences across the brain. In prolonged starvation, glucose oxidation by the brain (excluding glucose converted to lactate, which is converted back to glucose in the liver and kidney) is estimated as 24 g/day. f Oxygen uptake or total fuel use is measured by catheterization techniques. g The rate of ketone body uptake by the brain has been estimated from A–V differences using catheterization techniques. These studies strongly suggest that ketone bodies are the alternative fuel to glucose during starvation. Source: From Ref. 45.

Injury and Sepsis Cuthbertson (131,132), in his classic studies of the metabolic response to long-bone fractures, defined two phases of the metabolic response to injury—an ebb or shock phase and a flow phase. Moore (133) subsequently divided the flow phase into catabolic and anabolic stages. The ebb phase, constituting the first several hours after injury, is characterized by hyperglycemia and the restoration of circulatory

volume and tissue perfusion. Once perfusion is restored, the flow phase begins. It is characterized by generalized catabolism, negative nitrogen balance, hyperglycemia, and heat production. The flow phase is the best-studied phase and may last from days to weeks, depending on the severity of the injury, the previous health of the individual, and medical intervention. Finally, once volume deficits have been corrected, pain has been eliminated, wounds have been

FASTING MAN, ADAPTED (5_6 weeks) (24 hours, basal: – 1500 calories) FUEL CONSUMPTION

ORIGIN OF FUEL

O2

47g

Muscle Protein 20 g

Glycerol 15 g Adipose tissue Triglyceride 150 g

O2 30 g

Fatty acid 150 g

H 2O –P

–P CO2 + H2O

44 g

Liver, kidney Glycogen Gluconeogenesis

Amino acids

Nerve

Glucose 80 g 50 g

Ketone 57g

(Fatty acid) 112 g

36 g

RBC, WBC, etc. –P 14 g

Lactate and pyruvate O2 Heart, kindly muscle, etc.

–P CO2 + H2O

Urine 10 g ketone = ~100 mEq

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Figure 20 Flow diagram of fuel metabolism in starved man after adaptation. Source: From Ref. 29.

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closed, infection has been controlled, and complete oxygenation has been restored, the anabolic phase begins (1). This phase can be divided into a slow but progressive reaccumulation of protein, followed by the reaccumulation of body fat. Because protein synthesis cannot exceed 3 to 5 g of nitrogen per day, the protein repletion phase may be considerably longer than the catabolic phase in which protein is broken down. The posttraumatic state is characterized by starvation, immobilization, and repair. Although starvation and immobilization are both associated with decreased energy requirements, reparative processes increase energy needs. As a result, the overall energy requirements of traumatized and septic individuals are increased. The increase in energy need varies directly with the severity of injury and the complications that develop. In this regard, the most severe injury is the burn, and the most severe complication is sepsis (Fig. 5). Despite the use of protein and carbohydrate for calories, most of the energy used after trauma and after sepsis is derived from fat. This use is reflected in the low respiratory quotients noted after injury and sepsis. For example, Wilmore et al. (134) found respiratory quotients of 0.70 to 0.76 after severe burns. In addition, septic injury appears to have a greater lipid dependence for energy than nonseptic injury (100,135). Increased lipolysis is seen in both the ebb phase and the flow phase of the metabolic response to injury. During the ebb phase, elevated concentrations of cortisol, catecholamines, glucagon, growth hormone, ACTH, increased sympathetic nervous system activity, and depressed concentrations of insulin favor lipolysis. The presence of cortisol appears to be necessary for the remainder of the hormonal agents to be effective (136,137). Elevated concentrations of glycerol and free fatty acids during the ebb phase are well documented (138–140). However, Kovach et al. (141) have noted that elevation of plasma free–fatty acids may not occur after severe hemorrhage as a result of intense vasoconstriction in adipose tissue, producing minimum blood flow. During the flow phase, net lipolysis persists despite an increase in the concentration of insulin. Increased free fatty acids have been documented after trauma, burns, and sepsis (100,133,142–146). The fatty acids are used throughout the body for energy. In both the ebb and flow phases, the high concentration of intracellular fatty acids and the elevated concentration of glucagon inhibit acetyl-CoA carboxylase, thereby decreasing malonyl-CoA concentrations and fatty acid synthesis. In hepatocytes, the decreased concentration of malonyl-CoA also stimulates carnitine acyl transferase, thereby increasing the transport of acetyl-CoA into the mitochondria for oxidation and ketogenesis. However, the activity of ketogenesis after shock, injury, and sepsis is variable and correlates with the severity of injury (144–146). After major injury and sepsis, ketogenesis is low or absent, whereas after minor injury and sepsis, it is increased but to a lesser extent than is seen during nonstressed starvation (142,147). During starvation, the inhibition of acetyl-CoA carboxylase also results in the accumulation of cytoplasmic citrate that in turn inhibits glycolysis through PFK inhibition (Randle effect) (45). However, after shock and major injury, citrate does not accumulate (148,149). This lack of accumulation may play a role in the persistence of glycolysis after injury. Unlike fasting and starvation, hyperglycemia is a hallmark of the response to injury, sepsis, and stress. An increase in blood glucose occurs during both the ebb and

flow phases and is proportional to the severity of the injury (150,151). There is also an increased concentration of lactate, pyruvate, organic phosphates, total amino acids, glycerol, and free fatty acids. Changes in lactate, pyruvate, and alanine have also been found to correlate with the severity of injury (144). The rise in the concentrations of glucose and other solutes contributes to an elevated plasma osmolality after hemorrhage and injury that is thought to be critical in the complete restitution of blood volume and plasma proteins (119,152–155). The hyperosmolality appears to augment the transcapillary refill phase and the plasma protein– restoration phase of blood volume–restitution by mediating the movement of water from cells to the interstitium and ultimately to the plasma (153–155). The metabolic changes in carbohydrate metabolism arise primarily as the result of the actions and interactions of catecholamines, cortisol, glycogen, insulin, growth hormone, and somatostatin (65,95,96,150,156,157). It is apparent that the elevated blood glucose concentration results from increased hepatic production and from impaired peripheral uptake that are under endocrine control. Both the ebb and the flow phases are associated with hyperglycemia, increased gluconeogenesis, and hepatic and peripheral insulin resistance. However, the mechanisms involved in these carbohydrate ‘‘abnormalities’’ are different. During the ebb phase, plasma insulin is clearly depressed in relationship to the degree of hyperglycemia (111,150,158,159). This results from decreased B-cell sensitivity to glucose that is secondary to catecholamines, somatostatin, reduced pancreatic blood flow, and the increased activity of the sympathetic nervous system (92,115,139,160,162). However, during the flow phase, B-cell sensitivity returns to normal, and insulin concentrations rise to more appropriate values. Nevertheless, hyperglycemia persists (111,163). In both the ebb and the flow phases, there is a delayed rate of assimilation of a glucose load, glucosuria, and a resistance to exogenously administered insulin (156,160,164). Despite this ‘‘diabetes of injury,’’ glucose uptake and use by peripheral tissues in both the ebb and the flow phases have been demonstrated consistently to be greater than that under normal circumstances (111,165–169). The resistance to insulin is manifested in a decreased glucose clearance. Consequently, the high plasma glucose concentration and the attendant increase in plasma–tissue glucose concentration gradient appear to overcome the resistance of peripheral tissues to glucose entry. The insulin resistance that develops appears to result from the action of catecholamines, cortisol, and other factors (65,95,96,115,157). Hepatic carbohydrate metabolism is also affected by insulin resistance. During the ebb phase, elevated concentration of catecholamines, cortisol, and glucagon and a decreased concentration of insulin result in rapid glycogenolysis and an outpouring of glucose from the liver. In addition, these hormonal alterations stimulate gluconeogenesis from alanine, lactate, and pyruvate. Growth hormone also is involved in these processes by inhibiting glucose uptake through inhibition of glucokinase. During the flow phase, gluconeogenesis persists despite nearnormal concentrations of insulin. This persistence appears to result from insulin resistance and produces a continued flow of glucose from the liver. Therefore the hyperglycemia that occurs after injury results from a combination of increased glucose production and glucose release and from a peripheral resistance to the entrance of glucose. After injury and during sepsis, glucose must be provided not only to RBCs, WBCs, renal medulla, and

Chapter 1:

neural tissues, but also to wounded tissue (162,170,171). Glucose uptake in wounded tissue is increased by up to 100%. Wounds demonstrate a lack of insulin sensitivity and do not increase their glucose uptake or glycogenesis in response to insulin (60,172,173). The accelerated glucose uptake in wounded tissue and possibly in septic tissue appears to correlate with the degree of inflammatory cellular infiltrate (171). In addition, it has been demonstrated that the accelerated glycolysis of wounded tissue may be aerobic and not anaerobic, as thought previously (170). The aerobic glycolysis proceeds to lactate in the presence of adequate oxygen. Thus oxygen consumption and carbon dioxide production are normal, but lactate production is accelerated. Increased lactate production may be related to an inability of the NADH shuttle to transfer reducing equivalents from the cytoplasm to the mitochondrion (85,174). Metabolic derangements suggestive of aerobic glycolysis have also been seen in septic tissue (175). In this regard, it is of note that aerobic glycolysis is characteristic of the cellular infiltrate (176). As one might expect, negative nitrogen balance and net proteolysis are characteristic of the posttraumatic and the septic states (177,178). However, only 20% of the protein broken down is used for calories (71). The remainder is used in gluconeogenesis. As noted previously, the production of lactate in the presence of oxygen primarily results from the actions of cortisol, glucagon, catecholamines, and the decreased effectiveness of insulin. The rise in urinary nitrogen is associated with an increased excretion of urea, sulfur, phosphorus, potassium, magnesium, and creatinine, suggesting the breakdown of intracellular material (131,179). Isotope dilution studies suggest that this loss of protein results from the loss of cell mass rather than cell number (131). The nitrogen-to-sulfur and nitrogen-to-potassium ratios suggest that this loss occurs mainly from muscle (131). The marked increase in the urinary excretion of 3-methylhistidine during trauma, sepsis, and burns also suggests the importance of skeletal muscle in response (180,181). Analysis of the protein content and the incorporation of radiolabeled amino acids in visceral tissues and skeletal muscle confirm that it is skeletal muscle that is depleted and the visceral tissue (liver and kidney) is spared (182). This is the opposite of nonstress starvation in which visceral protein is used before muscle protein and has been termed visceral translocation of protein (37,182). In fact, one of the molecular tragedies associated with trauma, burns, and especially sepsis is that the proteolytic suppression accompanying prolonged starvation is not observed. Consequently, continued breakdown of protein results. The alterations in plasma amino acids are not well defined during the ebb and flow phases. During the ebb phase, little change in total amino acid concentrations were noted by Elwyn et al. (183) until the late phases of shock. In addition, it appears that these changes result primarily from a decreased hepatic uptake (183) and not an increased peripheral release as was thought previously (184). During the flow phase, alterations in plasma amino acids appear to be related to the severity of injury and the specific type of injury (121,185–187). Alanine, the major gluconeogenic amino acid, appears to be released from peripheral tissues and taken up by the liver for gluconeogenesis. Early in the flow phase, the concentration of alanine in plasma is increased; but as the injury persists, serum alanine decreases, presumably as a result of its lack of availability in peripheral tissues and its continued hepatic uptake.

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25

Branched-chain amino acids and aspartate and asparagine are transaminated in peripheral tissues, and their remaining carbon fragments are used in the TCA cycle. Nonetheless, muscle concentrations of amino acids generally reveal normal or elevated concentrations of all amino acids except alanine, glutamine, and arginine (188). The net catabolism of protein can result from increased catabolism, decreased synthesis, or a combination of the two. Available data on TBP turnover suggest that after injury the net changes in catabolism and synthesis depend on the severity of the injury (189). Elective operations and minor injury appear to result in a decreased rate of synthesis with a normal rate of protein catabolism (118,140). Severe trauma, burns, and sepsis appear to be associated with increases in both synthesis and catabolism but with a greater increase in the latter, resulting in net catabolism (122,189–191). In this regard, it is important to note that the accelerated proteolysis and a high rate of gluconeogenesis persist after injury and during sepsis (185,192). This persistence appears to result from an inhibition of ketoadaptation after injury and sepsis. Unlike starvation, ketogenesis is not prominent, and it does not fuel the brain in significant amounts. Therefore a high requirement for glucose and therefore gluconeogenesis persist. The mechanism for this inhibition of ketoadaptation is not understood presently. Clowes et al. (193) have presented evidence suggesting the involvement of a circulating peptide containing 33 amino acids in this response. In addition, Baracos et al. (194) have proposed that interleukin-1 (IL-1) (a human leukocyte pyrogen) may be responsible for the accelerated proteolysis that accompanies fever and sepsis (see section on ‘‘Cytokines’’). As noted previously, activation of an ubiquitin-proteasome pathway appears to be involved in skeletal muscle degradation during starvation (125,126). In septic states, rates of ubiquitination of muscle proteins are profoundly increased (125,195,196). Further, inhibition of this process significantly reduces proteolysis in septic conditions (197). The net catabolism of protein that occurs after any injury is dependent on the prior nutritional status and intake, sex and age of the individual, and the severity of the injury. Young healthy males lose more protein in response to an injury than do women or the elderly (40). In addition, the urinary excretion of nitrogen is less after a second operation if it closely follows the first (145,178). This decline is presumably the result of a reduction in available protein stores. Finally, negative nitrogen balance can be reduced or virtually eliminated by high caloric and nitrogen supplementation (34,156,191,198,199). Together, these facts suggest that the loss of protein that occurs after injury is not entirely obligatory to the injury, but is also a manifestation of acute starvation (146).

Local Wound Metabolism Injury is associated with a negative bodily balance of many substrates (200–203). Despite injury-induced abnormalities in cofactors and substrates known to be important in wound healing, most wounds heal after injury (203–205). The ability of the wound to heal in the face of varying hormone and substrate supply suggests that the wound has a biologic priority over the host. This concept of biologic priority of healing wounds was described by Moore and Brennan (206), who stated that 75% of wounds or surgical incisions healed to the point of tensile integrity during a period of negative energy and nitrogen balance. The other 25% healed in patients who returned to eating so rapidly that the

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period of negative balance was of minimal duration (206). It is therefore evident that during a period of postinjury hormonal alterations and general catabolism, the wound has a favored status. This concept suggests that the metabolic and functional determinants of normal wound repair are inherent within the local wound environment. This further suggests that the local wound environment is differently sensitive to substrate and hormonal alterations than are other organ systems.

Cellular Infiltrate as a Determinant of Biologic Priority Following injury, there is a local ordered infiltration of inflammatory cells and a characteristic pattern of cellular predominance within the wound over time. This ordered sequence of cellular predominance leads to characteristic alterations in extracellular substrate concentrations, temporal effects of wound fluid on wound cell function, an altered sequence of expression for peptide growth factors, and degradation of classical peptide hormones. It appears that the metabolism of the wound is determined by the cellular infiltrate and is independent of systemic hormonal and substrate alterations. This is supported by the following findings: 1. 2.

3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13.

A direct relationship exists between DNA content of wounded tissue and the cellular infiltrate (207). Wounded tissue metabolizes glucose rapidly through aerobic glycolysis. This unusual form of glycolysis is characteristic of the leukocytes that comprise the cellular infiltrate (170). A time course for glucose uptake and lactate production by wounded tissue appears to correlate with the development of the cellular infiltrate (207). Autoradiographic studies using [3H]-2-deoxyglucose reveal that the majority of the glucose uptake in wounded tissue occurs in the cellular infiltrate (208). Both wounded tissue and macrophages increase their glucose uptake in response to increasing glucose concentrations in the environment, and this glucose clearance is concentration independent. Thus the lack of saturation of glucose uptake of wounded tissue appears to be determined by the macrophages in the cellular infiltrate (209). A high PFK activity of wounded tissue can be explained by the PFK activity of the cellular infiltrate as measured by 5-[3H]-glucose (208). Macrophage glucose uptake and lactate production are independent of local epinephrine concentrations (208). Macrophages have a stimulatory effect on the HEP content of resident host tissue (210). A decreased HEP content of wounded tissue appears to be secondary to the dilutional effect of the cellular infiltrate (211). The presence of macrophages seems to explain the pattern of purine release from wounded muscle (212). Glutamine utilization by the cellular infiltrate appears to explain the decrease in glutamine content of wounded tissue (213). In the isolated perfused system, hormone and substrate profiles similar to those found following injury do not affect wound metabolism. A prior adrenalectomy does not affect wound metabolism despite substantial effects on noninjured tissue.

In addition, other investigators have shown a lack of effect of exogenous insulin on local wound metabolism in an incubated system (171). Thus there are substantial data to support the position of the wound as independent of circulating hormone and substrate concentrations. Yet it is known that several of the more common clinical examples of impaired wound healing involve systemic hormonal and substrate changes. Specifically, diabetes mellitus (214– 217), steroid therapy (218–220), amino acid deprivation (221,222), and hypophysectomy (223) have been shown to impair wound healing. In summary, generalized catabolism, hyperglycemia, persistent gluconeogenesis, protein wasting, negative nitrogen balance, heat production, and loss of the body mass that parallel the severity of the injury are characteristic after trauma and during sepsis. Most of the energy necessary for biologic processes to proceed is derived from fat. The net catabolism of 300 to 500 g of lean body mass per day is apparently required as a source of amino acids for gluconeogenesis. The persistence of the injury, particularly sepsis, through unknown mechanisms produces inhibition of the usual adaptive mechanisms that occur in starvation, resulting in the persistence of a highly catabolic state. This state in turn leads to protein wasting and malnutrition and ultimately in multiple organ failure (224) and death if the stimuli are not eliminated. (For further information on wound healing, the reader is referred to the chapter 55).

CYTOKINES In addition to the important role that neurohumeral mediators have been shown to play in the metabolic response to stress and injury, emerging evidence is also defining a role for cytokines (225,226). Often these two systems appear to work in tandem to carry out a particular response. Because cytokines are primarily proinflammatory agents, the magnitude of their involvement is related to the severity of the underlying stress or injury. For example, in an elective surgical procedure, the wound is the primary locus of the inflammatory response. Thus, cytokines involved in this response are targeted toward healing this wound; accordingly, their access to the systemic circulation is minimal and the resultant systemic response is modest or nonexistent. In a more traumatic situation in which massive tissue destruction has occurred (e.g., motor vehicle accident and blast injury), the local production of cytokines may be profound with an equally excessive egress of these substances into the systemic circulation. In this situation, the systemic response may result in hemodynamic instability and even distant organ failure. Cytokines comprise a family of small proteins or glycoproteins that alter the function of a target cell through an autocrine or paracrine process; rarely this targeting may be accomplished in an endocrine fashion. Many different cells may secrete cytokines but those commonly involved include lymphocytes, macrophages, Kupffer cells of the liver, and various components of the intestinal epithelium. The biologic response induced by cytokines is considerably diverse and will depend upon the particular cytokine secreted and the target cell affected. Since first being discovered in the 1970s, dozens of cytokines have been identified, and it is likely that many more will become recognized in the years to come. Accordingly, a comprehensive review of cytokines and their physiology is beyond the scope of this chapter [the reader is referred to several excellent references for this information

Chapter 1:

(225–228)]. This discussion will focus on those cytokines that have been shown to influence the metabolic response to stress. These include interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor [(TNF)/cachectin], and interferon-gamma. Cytokines are commonly categorized as being proinflammatory, anti-inflammatory, or both. This is based on their ability to invoke local or systemic inflammation and alter the immune response. The four cytokines involved in the metabolic response to stress are primarily proinflammatory, with the exception of IL-6, which possesses both proinflammatory and anti-inflammatory characteristics. Their effects on the immune response will not be discussed in this context, because this function is extensively covered in the chapter on immunity and the immunocompromised patient (Chapter 4). As mentioned above, cytokines can elicit both local and systemic effects. Because they are produced at the site of injury, their major action will be targeted to that site if the wound is uncomplicated. Thus, in situations such as setting a simple fracture, repairing an inguinal hernia, or removing a diseased gallbladder (particularly if done laparoscopically), a systemic response is highly unlikely. Rather, locally released cytokines will be confined to the site of injury to both induce and modulate the healing process. Important healing factors such as enhancement of blood flow through vasodilation, recruitment of neutrophils, monocytes, and other blood elements to the wound site to prevent or contain infection, maintenance of hemostasis through alterations in coagulation, and induction of angiogenesis and cell proliferation to issue adequate healing may all be cytokine mediated (226). TNF, for example, has been shown to induce microvascular angiogenesis, bone remodeling, and fibroblast proliferation (226). IL-1 induces procoagulant activity, collagenase activity, and collagen synthesis. Further, it has been shown to stimulate osteoclastic activity, which may be important in bone remodeling during injury (226). In situations where the injury is more severe, and particularly if extensive devitalization is present, excessive production of cytokines at the wound site results in their entrance into the systemic circulation that can often be measured. When this situation occurs, a variety of metabolic aberrations have been shown to occur. Release of IL-6 into the circulation appears to play a prominent role in many of these metabolic effects. Paramount among them is its induction of the acute phase response (229). This response is characterized by the reprioritization of hepatic synthesis and release of certain proteins into the blood during inflammation and sepsis and following severe injury (230). Thus, albumin and transferrin, which are normally important proteins synthesized by the liver, are reduced in favor of a number of acute phase proteins whose plasma concentrations increase substantially (sometimes as much as 10–1000-fold) (231). Examples of such proteins include C-reactive protein, a2 macroglobulin, a1 acid glycoprotein, amyloid, and fibrinogen. The physiologic role of many acute phase proteins is unknown, while emerging evidence has shown that others act as opsonins, influence coagulation mechanisms, and contain generalized tissue destruction. Acute phase proteins that possess antiprotease activity can significantly reduce tissue destruction from dead or dying cells. Similarly, fibrinogen can elicit thrombus formation to prevent bleeding in wounded tissue. It would appear, therefore, that an important role for the acute phase response is to set into motion a variety of survival mechanisms to contain the injury and/or associated infection and restore homeostasis (232).

Metabolic Response to Starvation, Stress, and Sepsis

27

Other cytokines that also have been shown to induce the acute phase response in the liver include TNF and IL-1 (226,233,234). Their role appears to be less important, and certainly less sustained, than that of IL-6. An explanation for this is the different half-lives of these cytokines. The half-life of TNF is 20 minutes or less whereas that of IL-1 is approximately six minutes (225). In contrast, IL-6 levels in the circulation are detectable within an hour after injury, reach peak levels within four to six hours, and can be sustained for a week or more (225). It may be that these three cytokines interact to initially induce the acute phase response and that IL-6 keeps it sustained. Because glucocorticoids also regulate hepatocyte biosynthetic activity, it has been proposed that such substances may be important cofactors to IL-6 and other cytokines in inducing and modulating the acute phase response (232). In support of this hypothesis is the finding that IL-6, TNF, and IL-1 stimulate the hypothalamic–pituitary–adrenal axis to secrete ACTH and glucocorticoids (235). Thus, there is likely to be a feedback mechanism involving glucocorticoids and cytokines (236,237). It is well known that injury and inflammation induce a metabolic response in skeletal muscle that results in net catabolism. Thus, amino acids released from skeletal muscle catabolism are preferentially taken up by the liver for gluconeogenesis and protein synthesis (especially acute phase proteins), as needed. Although the preponderance of evidence suggests that glucocorticoids are the major mediator of muscle proteolysis, various cytokines may also be involved (232). In differing models of sepsis, IL-1, TNF, and IL-6 have all been shown to stimulate muscle-protein breakdown (238–240). It would appear, therefore, that regulation of muscle-protein degradation following stress or injury is a multifaceted response. Conceivably, glucocorticoids may orchestrate the primary role, but cytokines may modulate this response in specific settings (232,241). During massive tissue destruction or endotoxemia, for example, cytokines may play a key role, whereas in other conditions such as starvation or differing forms of stress, glucocorticoids play the central role. An interesting effect of cytokines on protein catabolism is that induced by TNF. Chronic excessive production of this cytokine can lead to a state of profound cachexia (hence, its other name, cachectin). Administration of TNF to rodents induces nitrogen loss, decreased food intake, depletion of body lipid stores, and excessive weight loss (242–244). In a mouse model, tumors secreting TNF can cause profound weight loss and cachexia (245). IL-1 has also been shown to induce cachexia (242). Whether TNF or IL-1 are responsible, either alone or in combination, for the cachectic state observed in many cancer patients remains to be elucidated. TNF is also thought to be the primary mediator of many of the systemic manifestations of endotoxemia. In various animal models, administration of this cytokine in high doses can induce fever, tachypnea, tachycardia, hypotension, and death, not dissimilar to that observed in patients in septic shock (226). Pathologic findings are also characteristic of those reported in human septic shock, including pulmonary congestion, intestinal infarction, and adrenal necrosis (246). Other effects of cytokines include reductions in iron and zinc levels, which are commonly observed in septic patients. Both TNF and IL-1 have been shown to elicit these effects, which may actually confer a survival benefit by inhibiting the growth of various microorganisms and limiting the production of oxygen radical formation, both of which

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depend on trace metals (226). Finally, interferon-gamma can upregulate the number of TNF receptors on various cells, particularly monocytes. Although this interferon is primarily an immune modulator with antiviral activity, its TNF-related action may enhance cytotoxicity of devitalized tissue and thereby aid in the hearing of wounded tissue (226). It seems clear that cytokines play a major role in the metabolic response to injury and stress that is just now being unraveled. As our knowledge of this role is clarified, modulation of these substances and their physiologic and pathophysiologic mechanisms should prove beneficial in altering responses that are detrimental to recovery and survival.

SUMMARY Starvation, stress from injury or surgical procedures, and sepsis induce a series of metabolic changes that are regulated by neuroendocrine reflexes and cytokines and result in mobilization of substrates from endogenous tissue stores. These metabolic changes ensure that energy is available for vital functions, oxygen delivery is maintained, and reparative processes take place. An understanding of these complex metabolic interactions depends on an appreciation of normal homeostasis and the distribution of body water, proteins, fat, and carbohydrates. The role of each component of body tissue is important in periods of starvation, stress, and sepsis, especially when the ability to replenish endogenous food stores is impaired as a result of either the inability to consume adequate nutrients or the excessive consumption of tissue stores. The mechanisms described in this chapter illustrate the complexity of the metabolic response to stress, the interrelationships between the neuroendocrine responses, cytokine release and substrate mobilization and use, and the importance of adequate energy and tissue stores for survival and repair of the organisms under conditions of nutrient deprivation.

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178. Siegel JH, et al. Physiological and metabolic correlations in human sepsis. Surgery 1979; 86:163. 179. Frawley JP, Artz CP, Howard JM. Muscle metabolism and catabolism in combat casualties. Arch Surg 1955; 71:612. 180. Bilmazer C, et al. Quantitative contribution by skeletal muscle to elevated rates of whole-body protein breakdown in burned children as measured by 3-MEH output. Metabolism 1978; 27:671. 181. Williamson OH, et al. Muscle-protein catabolism after injury in man, as measured by urinary excretion of 3-methyl histidine. Clin Sci Mol Med 1977; 52:527. 182. Ryan NT. Metabolic adaptations for energy production during trauma and sepsis. Surg Clin North Am 1976; S6:1073. 183. Elwyn DH, et al. Interorgan transport of amino acids in hemorrhagic shock. Am J Physiol 1976; 231:377. 184. Engel FL. The significance of the metabolic changes during shock. Ann NY Acad Sci 1956; 55:383. 185. Clowes GHA, Randall H, Cha C. Amino acid and energy metabolism in septic and traumatized patients. JPEN 1980; 4:195. 186. Dale G, et al. The effect of surgical operation on venous plasma free amino acids. Surgery 1977; 81:295. 187. Woolfe LI, Groves AC, Moore JP. Arterial plasma amino acids in patients with serious postoperative infections and in patients with major fractures. Surgery 1976; 79:283. 188. Askanazi J, et al. Muscle and plasma ammo acids following injury. Ann Surg 1980; 192:78. 189. Birkhain RH, et al. Effects of major skeletal trauma on whole body protein turnover in man measured by L-[l,14C]-leucine. Surgery 1980; 88:294. 190. Long CL, et al. Muscle protein catabolism in the septic patient as measured by 3-methyl histidine excretion. Am J Clin Nutr 1977; 30:1349. 191. Stein TP, et al. Changes in protein synthesis after trauma: importance of nutrition. Am J Physiol 1977; 233:E348. 192. O’Donnell TF, et al. Proteolysis associated with a deficit of peripheral energy fuel substrates in septic man. Surgery 1976; 80:192. 193. Clowes GHA, et al. Muscle proteolysis induced by a circulating peptide in patients with sepsis or trauma. N Engl J Med 1983; 308:545. 194. Baracos V, et al. Stimulation of muscle protein degradation and prostaglandin E2 release by leukocyte pyrogen (interleukin-1). N Engl J Med 1983; 308:553. 195. Tiao G, Fagan J, Roegner V, et al. Energy-ubiquitin-dependent muscle proteolysis during sepsis in rats is regulated by glucocorticoids. J Clin Invest 1996; 97:339. 196. Hobler SC, Williams AB, Fischer D, et al. Activity and expression of the 20S proteasome are increased in skeletal muscle during sepsis. Am J Physiol 1999; 277:R434. 197. Tawa NE Jr, Odessey R, Goldberg AL. Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying not skeletal muscles. J Clin Invest 1997; 100:197. 198. Schrier RW, Berl WT, Anderson RJ. Osmotic and nonosmotic control of vasopressin release. Am J Physiol 1979; 236:F321. 199. Stephens RV, Randall HT. Use of a concentrated, balanced, liquid elemental diet for nutritional management of catabolic states. Ann Surg 1969; 170:642. 200. Levenson SM, Green RW, Taylor FH. Ascorbic acid, riboflavin, thiamine and nicotinic acid in relation to severe injury, hemorrhage and infection in the human. Ann Surg 1946; 124:840. 201. Lund CC, Levenson SM, Green RW. Ascorbic acid, thiamine, riboflavin and nicotinic acid in relation to acute burns in man. Arch Surg 1947; 55:557. 202. Cuthbertson DP. Interrelationships of metabolic changes consequent to injury. Br Med Bull 1954; 10:33–37. 203. Moore FD. Metabolic Care of the Surgical Patient. Philadelphia: WB Saunders, 1959. 204. Hunt TK, Van Winkle W. In: Hunt TK, Dunphy JE, eds. Fundamentals of Wound Management. New York: AppletonCentury-Crofts, 1979:2. 205. Levenson SM, Seifter E, Van Winkle W. In: Hunt TK, Dunphy JE, eds. Fundamentals of Wound Management. New York: Appleton-Century-Crofts, 1979:286.

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206. Moore FD, Brennan ME. Manual of Surgical Nutrition, by the Committee on Pre- and Post-operative Care of the American College of Surgeons. Philadelphia: WB Saunders, 1975:169. 207. Daly JM, et al. Does wounded tissue regulate hepatic glucose production. Surg Forum 1987; 38:23. 208. Forster J, et al. Glucose uptake and flux through phosphofructokinase in wounded rat skeletal muscle. Am J Physiol (Endocrinol Metab 19) 1989; 256:E788. 209. Caldwell MD. Carbohydrate and energy metabolism in healing wounds. In: Barbul A, et al., eds. Growth Factors and Other Aspects of Wound Healing: Biological and Clinical Implications. New York: Alan R Liss, 1988:183. 210. Morris A, et al. A macrophage-mediated factor that increases the high energy phosphate content of skeletal muscle. J Surg Res 1985; 38:373. 211. Morris A, et al. Macrophage interaction with skeletal muscle: potential role of macrophages in determining the energy state of healing wounds. J Trauma 1985; 25(8):751. 212. Morris AS, et al. The role of purine metabolism in the macrophage mediated increase in high energy phosphates in skeletal muscle. J Surg Res 1986; 41:339. 213. Caldwell MD. Local glutamine metabolism in wounds and inflammation. Metabolism 1989; 38(suppl 1):34. 214. Goodson WH, Hunt TK. Studies of wound healing in experimental diabetes mellitus. J Surg Res 1977; 22:221. 215. Spanheimer RG, Umpierrez GE, Stumpf V. Decreased collagen production in diabetic rats. Diabetes 1988; 37:371. 216. Yue EK, et al. Effects of experimental diabetes, uremia, and malnutrition on wound healing. Diabetes 1987; 36:295. 217. Rosenberg CS. Wound healing in the patient with diabetes mellitus. Nurs Clin North Am 1990; 25:247. 218. Ehrlich HP, Hunt TK. Effects of cortisone and vitamin A on wound healing. Ann Surg 1968; 167:324. 219. Hunt TK, et al. Effects of vitamin A on reversing the inhibitory effect of cortisone on healing of open wounds in animal and man. Ann Surg 1969; 170:633. 220. Salmela K, et al. The effect of local methylprednisolone on granulation tissue formation. Acta Chir Scand 1980; 146:541. 221. Seifter E, et al. Arginine: an essential amino acid for injured rats. Surgery 1978; 84:224. 222. Barbul A. Arginine: biochemistry, physiology and therapeutic implications. J Parenter Enter Nutr 1986; 10:227. 223. Skottner A, et al. Anabolic and tissue repair functions of recombinant insulin-like growth factor l. Acta Paediatr Scand (Suppl) 1990; 367:63. 224. Hassett J, Border JR. The metabolic response to trauma and sepsis. World J Surg 1983; 7:125. 225. Lin E, Lowry SF, Calvano SE. Mediators of inflammation and injury. In: Norton JA, Bollinger RR, Chang AE, et al., eds. Surgery: Scientific Basis and Evidence-Based Practice. New York: Springer-Verlag, 2001:69–94. 226. Fong Y, Lowry SF. Cytokines and the cellular response to injury and infection. In: Wilmore DW, et al., eds. ACS Surgery: Principles and Practice. New York: Wed MD, 2002:1603–1622. 227. Fink MP. The role of cytokines as mediators of the inflammatory response. In: Townsend CM Jr, ed. Sabiston Textbook, of Surgery. Philadelphia: Elsevier Saunders, 2004:45–66.

228. Bessey PQ. Metabolic response to critical illness. In: Wilmore DW, et al., eds. ACS Surgery: Principles and Practice. New York: Wed MD, 2002:1495–1520. 229. Heinrich PC, Castell JV, Andus T. Interleukin-6 and the acute phase response. Biochem J 1990; 265:621. 230. Koj A. Definition and classification of acute phase proteins. In: Gordon AH, Koj A, eds. The Acute Phase Response to Injury and Infection. New York: Elsevier, 1985:139–147. 231. Kushner I. The acute phase response: an overview. Methods Enzymol 1988; 163:373. 232. Hasselgren PO. Protein metabolism in surgery. In: Norton JA, Bollinger PR, Chang AE, et al., eds. Surgery: Scientific Basis and Evidence-Based Practice. New York: Springer-Verlag, 2001:105–121. 233. Enayati P, Brennan MF, Fong Y. Systemic and liver cytokine activation. Arch Surg 1994; 124:1159. 234. Lin E, Calvano SE, Lowry SF. Cytokine response in abdominal surgery. In: Schein M, Wise L, eds. Cytokines and the Abdominal Surgeon. Austin: Landes, 1998:17–34. 235. Del Rey A, Besedovsky HO. Metabolic and neuroendocrine effects of pro-inflammatory cytokines. Eur J Clin Invest 1992; 22:10. 236. Besedovsky H, Del Rey A, Sorkin E, et al. Immunoregulatory feedback between interleukin-1 and glucocorticoid hormones. Science 1986; 233:652. 237. Hocke GM, Barry D, Fey GH. Synergistic action of interleukin6 and glucocorticoids is mediated by the interleukin-6 response element of the A2-macroglobulin gene. Mol Cell Biol 1992; 12:2282. 238. Zamir O, Hasselgren PO, Higashiguchi T, et al. Tumor necrosis factor (TNF) and interleukin-l (IL-1) induce muscle proteolysis through different mechanisms. Mediat Inflamm 1992; 1:247. 239. Zamir O, Hasselgren PO, Kunkel S, et al. Evidence that tumor necrosis factor participates in the regulation of muscle proteolysis during sepsis. Arch Surg 1992; 127:170. 240. Goodman MN. Interleukin-6 includes skeletal muscle protein breakdown in rats. Proc Soc Exp Biol Med 1994; 205:182. 241. Mealy K, van-Lanschot JJ, Robinson BG, et al. Are the catabolic effects of tumor necrosis factor mediated by glucocorticoids? Arch Surg 1990; 125:42. 242. Fong YM, Moldawer LL, Marano MA, et al. Cachectin/TNF or IL-1 alpha induces cachexia with redistribution of body proteins. Am J Physiol 1989; 256:R659. 243. Tracey KJ, Wei H, Manogue KR, et al. Cachectin/tumor necrosis factor induces cachexia, anemia, and inflammation. J Exp Med 1988; 167:1211. 244. Moldawer LL, Anderson C, Gelin J, et al. Regulation of food intake and hepatic protein synthesis by recombinant-derived cytokines. Am J Physiol 1988; 254:G450. 245. Oliff A, Defeo-Jones D, Boyer M, et al. Tumors secreting human TNF/cachectin induce cachexia in mice. Cell 1987; 50:555. 246. Tracy KJ, Beutler B, Lowry SF, et al. Shock and tissue injury induced by recombinant human cachectin. Science 1986; 234:470.

2 Pathophysiology of Fluid and Electrolyte Disorders Peter J. Fabri and Mark Bloomston

INTRODUCTION The human body can be likened to a sac of electrolyte-rich fluids in which is suspended or dissolved a complex network of solids known collectively as ‘‘organs.’’ The common medium of these fluids is water; the electrolytes are a mixture of primarily monovalent and divalent ions. The total volume of water, known as total body water, accounts for approximately 60% of total body mass. Substances are continually added to and excreted from this aqueous environment, and only through a system of homeostatic, protective mechanisms is the composition and distribution of this fluid-based system maintained. Disease, pharmaceuticals, and medical interventions all have the potential to disrupt the balance of this fluid medium and result in clinically evident fluid and electrolyte disturbances. To achieve the desired goal of preventing or treating such disturbances, the nature, composition, and interrelationships of these fluids and the homeostatic mechanisms that maintain them must be clearly understood.

MAINTENANCE OF THE INTERNAL MILIEU Total Body Water Water is the universal solvent of the human body. The total volume of this substance is subdivided into discrete parts known as intracellular, extracellular, and transcellular water (Fig. 1). These parts are separated by semipermeable membranes equipped with energy-consuming, work-producing pumps that are usually based on an adenosine triphosphatase (ATP-ase) enzyme system. These pumps are able to maintain electrochemical and concentration gradients across membranes, which result in a marked difference in composition between the intracellular and extracellular spaces. Water, on the other hand, passively follows the laws of osmotic and ionic equilibrium. It traverses these membranes freely to maintain an equal number of solute molecules (osmolality) and ionic particles (tonicity) per unit volume on each side of such semipermeable membranes. This difference between the control of solutes/ions and that of water results in marked differences in volume and electrolyte composition of the two spaces. The normal balance can be disturbed by changing the number of solute molecules on either side of the membrane (e.g., hypoalbuminemia and hyperglycemia) or by disrupting its enzyme-based pumps (e.g., shock and digitalis). The extracellular space can be considered an open system in that the alimentary tract serves as a mode of entrance and exit of water and solute, as shown in Figure 1. Similarly, the lungs, kidneys, and skin also serve as excretory conduits. Entrance and excretion of water and solute are

controlled by active systems that are under homeostatic (nervous or hormonal) control. Thus the composition of this open, extracellular system can be maintained within relatively tight limits (1). Nonstandard entrance routes, such as the administration of fluids intravenously, bypass the normal entry control mechanisms and directly add water and solute to the extracellular space. In this circumstance, the intrinsic ability of the excretory systems to maintain osmotic and ionic stability becomes the limiting factor in maintaining ionic stability and composition of the extracellular space. Failure of these excretory control mechanisms, by inability to either conserve or increase loss, threatens the chemical stability of the extracellular fluids. The intracellular space, on the other hand, is a closed space and is represented by the numerous cells floating within the extracellular water in Figure 1. The only route of entrance and exit is across the semipermeable cellular membrane and its contained enzyme systems. Therefore, extracellular space can be thought of as a conduit and buffer zone to the intracellular space. Only by transfer from or to the extracellular water can intracellular composition be changed. This complex system, richly furnished with active transport mechanisms and buffer zones, rigidly protects the stability of the body fluids and maintains constancy of

Figure 1 Conceptualized model of the human body. Note that everything enters into, is excreted from, and is sampled in extracellular water.

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Table 1 Normal Distribution of Body Water as Percent of Body Weight

Total body water Extracellular water Intracellular water a

a

Men (%)

Women (%)

60 15 45

55 15 40

Values are less than equivalent for men because women have a relatively greater amount of adipose tissue.

the internal milieu. Although not depicted accurately in our model, two-thirds of total body water is intracellular and one-third is extracellular. This translates to about 40% and 20% of total body weight for the intracellular and extracellular compartments, respectively (Table 1).

Extracellular Water The extracellular space is composed of the intravascular space (blood cells plus plasma) (comprising about 8% of total body weight) and the extravascular space (interstitial fluid and lymph). Cerebrospinal fluid is a specialized subspace of extracellular fluid. The vascular and extravascular spaces are in relative continuity, separated only by the rather permeable basement membranes of the blood vessel walls. When these vessels are intact, the formed cellular elements of the blood remain contained in the intravascular space, whereas the aqueous, noncellular plasma undergoes continuous ‘‘filtration’’ through the relatively leaky vessel walls into the extravascular space. The extravascular space, however, is continuously ‘‘drained’’ by a system of lymphatic channels that return protein-rich extracellular fluid to the vascular space through the lymphatic ducts. This continuous-cycle system results in moment-to-moment renewing of the interstitial, pericellular space, bringing fresh nutrients from and carrying waste products to the vascular space, which is in direct continuity with normal routes of intake and excretion. The volume of intravascular fluid (blood) is determined by the oncotic effect of blood cells and large molecules such as albumin, as well as the rate of return of lymph. The volume of the extravascular fluid space depends on the balance between ‘‘filtration’’ of plasma and ‘‘drainage’’ of lymph. Plasma is continuously filtered across the vascular pores, particularly at the capillary level. The rate of filtration is governed by Starling’s law, which takes into account net hydrostatic pressure, net oncotic pressure, and pore size (reflectance and permeability). Fluid shifts across the vascular membrane are controlled by the summed effect of hydrostatic and oncotic pressures. Hydrostatic pressure exists in both the vessel (mean capillary pressure) and the tissue (mean tissue pressure). The difference results in a vector force that typically acts to drive fluid into the extravascular space. Simultaneously, this vector is offset by an oncotic pressure vector, which is the net difference between plasma oncotic pressure and tissue oncotic pressure adjusted for a permeability factor (reflectance) that varies as the ‘‘size’’ of the pores changes. Ordinarily, these vectors, on the average, tend to cancel, and there is only a small flux of fluid across the membrane. However, alteration in any of the main forces can lead to marked derangement in fluid distribution and the development of increased interstitial fluid, a condition recognized as clinical edema. The balanced forces in Starling’s equation result in the passage of fluid across the vascular membrane into the interstitial (extravascular) space. Not addressed in Starling’s equation is the ‘‘drainage’’ effect of the lymphatic system that tends to remove filtered plasma

from the interstitium. Obstruction of lymphatics by conditions such as tumor and infection can further impair fluid clearance and result in localized edema or lymphedema.

Osmolality and Tonicity The composition of extracellular water is reflected by the concentration of solutes. Ordinarily, extracellular composition is maintained within narrow limits by accurate renal control mechanisms [e.g., antidiuretic hormone (ADH) and aldosterone]. These control mechanisms, however, tend to respond to the concentration of a given substance rather than to the total amount of all substances. Concentration represents the combined effect of the amount of a given substance (numerator) dissolved in a given amount of water (denominator). Thus abnormalities in concentration may represent changes in the amount of solute, amount of water, or both. The concentration of total solute is most easily measured by plasma osmolality (mOsm/kg). This value indicates the ratio of solute to water in the plasma and in the extravascular space. Because extracellular water is in equilibrium with intracellular water, shifts of membrane permeable water result in maintenance of osmotic equality throughout total body water. In other words, accumulation of intracellular solute is compensated by a shift in water from the extracellular space to the intracellular space until osmolality is equal and vice versa. The concentration of total ions, indicating ionic strength, is approximated by assessing the concentration of the principal extracellular cation, sodium. Changes in sodium concentration represent changes in tonicity, a term related to but not synonymous with osmolality. Typically, tonicity and osmolality change together, and hyperosmolality usually includes hypertonicity (2). However, any substance that has a low molecular weight and a sizable concentration will contribute to serum osmolality. Thus conditions such as hyperglycemia, azotemia, hyperlactatemia, and accumulation of ethanol, which add solute but not ions, will raise osmolality without a change in tonicity. The contributing factors to osmolality can be understood more clearly by determining the osmolal gradient. This represents the difference between calculated (Osm[c]) and measured (Osm[m]) osmolality. Calculated osmolality is estimated by the formula Osm½c ¼ 1:86½Na þ Glu=18 þ BUN=2:8 where Na is serum sodium concentration in mequiv/L, Glu is serum glucose in mg/100 mL, and BUN is blood urea nitrogen in mg/100 mL. [1.86 is twice the osmotic activity coefficient of a monovalent ion; 18 is the molecular weight of glucose divided by 10 (a correction factor to convert mg/dL to mg/L); and 2.8 is the molecular weight of urea nitrogen divided again by the correction factor 10.] Ordinarily the osmolal gradient is less than 10 mOsm/kg where Osmolal gradient ¼ Osm½m Osm½c A gradient greater than 10 mOsm/kg represents the accumulation of some unmeasured, osmotically active (low molecular weight) substance such as lactate, ethanol, or mannitol. A common error in clinical practice is to assume that a change in measured osmolality represents an increase or decrease in water. As might be anticipated, osmolality is a concentration term and therefore can be disrupted by a change in amount of solute (e.g., azotemia) or amount of water (e.g., dehydration) or both (e.g., hyperosmolar coma).

Chapter 2:

Table 2 Electrolyte Composition

Total body content (mequiv/kg) Exchangeable content (mequiv/kg) Intracellular concentration (mequiv/L) Plasma water concentration (mequiv/L) Serum concentration (mequiv/L)

Na

K

Cl

Ca

Mg

67

58

42

940

32

41

44

30

–

–

10

160

3

2

26

152

5

110

5

3

142

5

103

5

–

Because measured osmolality equals the ratio of solute molecules to water: Osm ¼ solute=water

Pathophysiology of Fluid and Electrolyte Disorders

35

only part of the system that is able to ‘‘control’’ its output. Accepting that fact, it is helpful to think of renal homeostasis as ‘‘throwing the baby out with the bath water, and catching the baby before it goes down the drain.’’ In other words, glomerular filtration, the frontline initiator of renal excretion, is relatively nonspecific. It only limits excretion of substances that are associated with large proteins and therefore too big to pass through the renal vascular pores. Glomerular filtrate, therefore, is an ultrafiltrate of plasma. This ultrafiltrate passes through the proximal tubule where approximately 95% of most solutes and water are reabsorbed. In effect, control of excretion to this point is really a question of ‘‘what isn’t reabsorbed.’’ Final modification of urinary composition takes place in the distal tubule and collecting duct, where sodium is, in a matter of speaking, ‘‘exchanged’’ for hydrogen or potassium and the remaining water is either reabsorbed or not (8–11).

Sodium and Water Homeostasis

By rearrangement, the amount of solute determines the osmolality multiplied by total body water (which can be estimated as 60% of total body weight), as shown by the formula: Solute ¼ Osm 0:6 wt Estimation of this variable allows assessment of changes in total body solute, and, by comparison, permits an appraisal of relative changes in body water versus body solute.

Electrolyte Composition As previously indicated, intracellular and extracellular fluids vary in concentration of electrolytes. The total body content of each electrolyte (3) has been estimated by direct assay (4) and by radionuclide exchange (exchangeable ion) (5–7). The distribution of these electrolytes is unequal and results in marked concentration differences throughout the body (Table 2). Most of the difference between total body content (gravimetric) and exchangeable content is accounted for as substance in bone. As a glance at Table 2 will confirm, and contrary to the impression gained from evaluating serum electrolytes, the content of total exchangeable sodium (Na) is roughly equal to the content of total exchangeable potassium (K). The sum of these ions (exchangeable Na þ K) validly estimates total body cations that are roughly equally divided between intracellular and extracellular water. Accordingly, the serum sodium concentration (or conversely the intracellular potassium concentration) represents the ratio of total body cations to total body water, as shown below: Na ðserumÞ ¼ ðNa þ KÞ=ð0:6 wtÞ By a process analogous to osmolality, cross-multiplying serum sodium concentration by an estimate of total body water yields total body exchangeable cations. An estimate, therefore, of total body water, total body solute, and total body cations allows a more detailed serial assessment of fluid and electrolyte balance for patients in whom derangements are suspected.

HOMEOSTATIC CONTROL MECHANISMS General Concepts The kidney is the cornerstone of the homeostatic mechanisms controlling fluid and electrolyte balance. Although it is true that fluid losses from the skin, lungs, and gastrointestinal (GI) tract may be impressive, the kidney is the

Intracellular water is in equilibrium with extracellular water through osmotic and ionic neutrality. A small gradient is accounted for by anion proteins, which results in a concentration inequality referred to as the Gibbs–Donnan distribution. Extracellular water is controlled largely by plasma volume and serum sodium concentration. Sodium and water conservation must be considered together because their control mechanisms are inseparable. Water deficits or excesses are compensated by changes in ADH release from the pituitary, and its effects within the renal collecting duct (12–14). Changes in sodium composition are accompanied by both ADH and mineralocorticoid responses. A decrease in plasma volume stimulates baroreceptors located in sites such as the right atrium and carotid body, as well as in the juxtaglomerular apparatus and macula densa of the kidney (15). The baroreceptor response results in an increase in ADH release from the posterior pituitary and causes decreased loss of solute-free water in the collecting duct of the kidney. Conversely, as the serum sodium concentration falls, ADH release will be inhibited, and further water conservation will be blunted. The simultaneous effects of decreased glomerular filtration leading to decreased sodium delivery to the juxtaglomerular apparatus results in renin release, which subsequently causes cleavage of angiotensinogen into the decapeptide angiotensin I. This latter substance is converted in the lung to the octapeptide angiotensin II by the angiotensin-converting enzyme (ACE). Angiotensin II is a very potent vasoconstrictor that also directly stimulates aldosterone release from the zona glomerulosa of the adrenal cortex. The resultant increase in circulating aldosterone increases sodium reabsorption in the distal tubule of the kidney, in exchange for potassium and/or hydrogen ions. As serum sodium concentration changes, release of ADH from the posterior pituitary is altered by osmoreceptors in the hypothalamus. Very small changes (e.g., 2 mOsm) can predictably result in a measurable change in plasma ADH levels. Corresponding changes in thirst perception and in the permeability of the collecting duct of the kidney to water also occur. In the case of hypernatremia, thirst increases, and maximum retention of solute-free water occurs in the kidney. This can be corroborated by a high measured urine osmolality (usually >500 mOsm). During hyponatremia, ADH release is inhibited, the stimulus for water conservation in the collecting duct ceases, and solute-free water (‘‘free water’’) is excreted, resulting in hypotonic (< 280 mOsm/kg) urine and a return of serum sodium to normal. In addition, the zona glomerulosa

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Part One: General Considerations

of the adrenal appears to be sensitive to changes in serum sodium concentration, resulting in feedback control of aldosterone release.

Potassium Homeostasis Control of serum potassium levels is quite unlike that of sodium. Sodium is controlled at both the intake and excretion level, whereas potassium intake is unregulated. Potassium, then, is controlled entirely by excretion. Potassium is primarily an intracellular ion, with a concentration many fold greater than plasma. Yet, the extracellular concentration is extremely important in maintaining electrochemical gradients across cell membranes that facilitate depolarization of electrically active cells such as cardiac muscle and specialized conduction cells. Therefore control of extracellular potassium is vitally important. Typically, mammalian diets contain very large amounts of potassium, such that conservation of potassium is not usually a problem unless losses are excessive (diuresis and diarrhea) or renal mechanisms are abnormal (hyperaldosteronism). Potassium conservation and excretion tends to be a direct contrast to sodium conservation. Excess potassium, for example, stimulates the release of aldosterone from the adrenal, whereas too little sodium would result in the same response (16,17). While the majority of potassium reabsorption (like sodium) occurs in the proximal tubule, it is excreted in the distal tubule in response to the aldosterone stimulus. Sodium can be reabsorbed almost entirely within the nephron, whereas potassium conservation is less complete until profound body deficits occur. Distal tubular flow rate is also a major factor in potassium homeostasis. As flow rates increase, potassium excretion becomes inappropriately high. Although total body potassium distribution is greatly affected by renal mechanisms, it is also subject to exchange mechanisms at the cell surface of all cells such that, as sodium (or hydrogen) is transported out, potassium reenters the intracellular fluid (probably insulindependent). Likewise, in severe acidosis or alkalosis, plasma potassium levels may change as a result of the fluxes of potassium that are consequent to the hydrogen ion shifts.

Calcium Homeostasis The majority of calcium in the human body is complexed within the rigid crystal lattice of bone. This phase of calcium hydroxyapatite and phosphate is in equilibrium with three other forms of calcium: protein-bound, ionic complex, and ionic. Only the latter, ionic calcium, is physiologically active. In normal physiology, this very small amount of calcium is responsible for a large number of functions, including extracellular, transcellular, and intracellular communication; coordination of protein structure, including clotting factors; electrical triggering of active cells, such as myocytes; second messenger function together with calmodulin; activation of enzymes; and many others. Because of the many life-critical functions of calcium, its concentration is very tightly controlled and the cell is capable of functioning normally in an environment with little calcium intake. Consequently, normal homeostatic mechanisms do an admirable job at maintaining calcium-dependent physiologic processes in a wide variety of situations. Said another way, clinical abnormalities of calcium metabolism are very rare except in specific circumstances. Normal laboratory measurement of calcium assays all three components of nonbone calcium, yet only the ionized fraction is active. Numerous nomograms and algorithms have attempted to allow the physiologic fraction to be estimated based on serum protein concentrations, but

recent studies suggest that such estimates have little clinical utility and that ionized calcium must be measured to identify clinical hyper- or hypocalcemia. Calcium in the serum is regulated by the interplay of two hormones, parathyroid hormone (parathormone, PTH) and 1,25 dihydroxyvitamin D3. Calcitonin, which is important in other animal species, has not been identified as being of importance in calcium homeostasis in humans. PTH is produced as a very large macropeptide, undergoes posttranslational cleavage to the smaller 84–amino acid peptide, and is released by the parathyroid glands. The parathyroid glands, in turn, have calcium receptors (CaR), which recognize the level of ionized calcium in the extracellular fluid within the parathyroids. Even a small decrease in ionized calcium produces an immediate increase in PTH release and, if of long duration, hyperplasia of the parathyroids themselves. Similarly, a small increase in ambient calcium turns off PTH release and, if of long duration, regression of the parathyroids. This increase in PTH increases the mobilization of calcium from bone, increases the resorption of calcium in the renal tubule, and enhances excretion of phosphate in the renal tubule. Additionally, prolonged increase in PTH elevation induces an increase in hydroxylation of vitamin D in the kidney. 1,25 dihydroxyvitamin D3 increases the absorption of both calcium and phosphate from the intestine. A second hormone, parathyroid hormone–related peptide, homologous with PTH, is often produced in tumors, particularly squamous cell neoplasms, and leads to tumoral hypercalcemia. This must be distinguished from an alternative form of malignancy-related hypercalcemia seen in advanced disease where the tumor is actively invading the bone, leading to osteoclast and osteoblast mobilization and subsequent hypercalcemia. Hypercalcemia can also be produced by diseases that cause an increase in active vitamin D, such as lymphoma, granulomatous diseases including sarcoidosis, and excessive intake of vitamin D in the diet or supplements. When a patient is identified with hypercalcemia, the urgency of correction must be established. Mild hypercalcemia or intermittent hypercalcemia rarely produces physiologically important abnormalities or even symptoms and therefore does not require management. Calcium levels above 12 mg/dL have traditionally been considered urgent, requiring aggressive treatment, and above 14 mg/dL, as a medical emergency (hypercalcemic crisis). Saline diuresis and inhibition of bone resorption with biphosphonates or calcitonin is the mainstay of treatment. Hypercalcemia, if persistent, produces a physiologic equivalent of nephrogenic diabetes insipidus, resulting in volume loss and hypernatremia, with the symptoms of polydypsia and polyuria. Treatment with fluids alone will correct the electrolyte abnormalities but will not correct the underlying cause of the hypercalcemia (18). Hypocalcemia is often seen in critically ill individuals and in individuals who have hypoalbuminemia. Because serum calcium levels are often measured in the basic metabolic panel used in hospitalized patients, the treatment of hypocalcemia as an electrolyte problem is often considered. Current evidence suggests that the hypocalcemia seen in hospitalized patients (except hypoparathyroidism following thyroid or parathyroid surgery) is almost never of clinical significance and does not require treatment. Furthermore, evidence is accumulating that PTH release and responsiveness of the CaR are altered as part of the acute injury response, suggesting that hypocalcemia is actually a programmed component of acute injury (19).

Chapter 2:

In the final analysis, ionized calcium is very tightly controlled and is not often a fluid and electrolyte issue. Treatment of asymptomatic hypocalcemia does not appear to be needed and may even be detrimental. Although the discovery of hypercalcemia clearly requires the establishment of a diagnosis, mild hypercalcemia also does not usually require treatment as a fluid and electrolyte issue unless the calcium level is greater than 12 mg/dL.

Acid/Base Conservation With the exception of several specialized body fluids, the pH of body water is very closely guarded in a narrow range. Normal plasma pH (7.4 0.05) generally is representative of the pH of total body water, although the pH of fluids such as cerebrospinal fluid and intracellular fluid may transiently diverge from that of plasma because of differing controlling factors and influences. For practical purposes, however, the pH of all compartments of body water can be assumed to be equal. To appreciate the intricacies of acid/base balance, a solid understanding of the basic chemical concepts of acids, bases, and dissociation is essential. Acids are substances that have the capability of donating protons (hydrogen ions and hydronium ions). Bases, conversely, are substances that have the ability to accept protons during chemical reactions. Both acids and bases are ionic compounds that, when dissolved in water, have the ability to dissociate into cationic and anionic species. For most organic acids and bases in the body, dissociation is only partial, and the ratio of dissociated to nondissociated forms is determined by the dissociation constant. Acids, when dissociated, contribute hydrogen ions (Hþ) and a corresponding anion (An) to the total ionic composition of the solution. Bases typically contribute a cation and a hydroxyl (OH) ion. A typical acid (or base) dissociation can be described by the chemical reaction: HAn ¼ Hþ þ An: The dissociation constant is determined by the ratio of products to reactants; therefore: K ¼ ½Hþ ½An=½HAn; where the brackets represent concentration in solution. Taking the logarithm of both sides of this equation results in a useful and familiar form of the dissociation equation: logK ¼ log Hþ þ logð½An=HAnÞ pK ¼ pH logð½An=½HAnÞ pH ¼ pK þ logð½An=½HAnÞ: The dissociation constant (and consequently the pK) takes into account the simultaneous equilibrium of water. In the human, many acid/base pairs (buffer pairs) exist in simultaneous equilibrium. This means that the ambient pH of the body determines the ratio of anion to dissociated acid for a number of acids present in the body. Although substances such as phosphoric acid, proteins, and amino acids are all present in abundance and could be used as estimates of acid/base status, the carbonic acid/bicarbonate buffer system is most commonly used for this purpose. Bicarbonate is not the major buffer in the human body (pKa ¼ 6.1). In fact, hemoglobin and proteins make up the bulk of the buffering capacity (maintenance

Pathophysiology of Fluid and Electrolyte Disorders

37

of pH within narrow limits). The reason for measuring the bicarbonate/carbonic acid component of the buffer system is clear. Again, the organic acids and bases in extracellular water are in equilibrium with each other, so any pair could be used to evaluate acid/base status. The major homeostatic mechanisms of acid/base control are pulmonary (the excretion of carbon dioxide) and renal (the conservation of bicarbonate and excretion of hydrogen ions). Consequently, this buffer pair reflects the efficacy of the homeostatic mechanisms that are operative in compensating for changes in the gain or loss of acid or base. In addition, bicarbonate and carbon dioxide (pCO2) are easily measured in plasma. The general dissociation equation, when applied to the bicarbonate/carbonic acid buffer system, is known as the Henderson–Hasselbach equation: pH ¼ 6:1 þ logð½HCO 3 =½H2 CO3 Þ; where [HCO 3 ] is the concentration of bicarbonate and [H2CO3] is the concentration of carbonic acid. Because the concentration of carbonic acid is determined by the partial pressure of carbon dioxide, and the solubility of carbon dioxide in water is 0.03 pH ¼ 6:1 þ logð½HCO 3 =0:03 pCO2 Þ and ½Hþ ¼ 24ðpCO2 =½HCO 3 Þ: Current technology allows the direct measurement of pH and pCO2 in arterial (or venous) blood, enabling the estimation of bicarbonate concentration by the above equation. Alternatively, measurement of bicarbonate concentration and pH would enable calculation of pCO2. In normal circumstances, the ratio of [HCO 3]/0.03 pCO2 is 20/L. Other electrolytes enter the body pool only by ingestion (or injection), but acids (and bases) are rarely present in the diet. Acid is a product of metabolism of other substances and is added to total body water as a function of the rate of metabolism and the fractional use of acidproducing metabolites (Table 3). In general, net acid production is approximately 1 mequiv/kg/day (2 to 3 mequiv/kg/day in infants) and is primarily caused by the production of sulfuric acid from metabolism of thiols; phosphoric acid from metabolism of organic phosphates; and other organic acids from the metabolism of proteins, carbohydrates, and fats. Any addition of base (e.g., antacids) or compounds that generate base (e.g., citrate and lactate) will tend to offset the daily endogenous acid load. Table 3 Physiologic Factors Affecting Plasma Acidity Through plasma bicarbonate changes Rate of hydrogen ion input Rate of hydrogen ion or bicarbonate loss (gastrointestinal) Availability of buffers Bicarbonate space of distribution Rate of net renal acid excretion

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Through plasma pCO2 Rate of carbon dioxide production Rate of alveolar ventilation

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Part One: General Considerations

To maintain acid/base equilibrium, the body must excrete a quantity of acid equal to endogenous production (plus any exogenous acid and minus any exogenous base). This is accomplished primarily by renal excretion of fixed acid in the form of phosphates and ammonia. Ammonia is actively produced by metabolism of glutamine in the kidney and subsequently excreted into the renal tubular lumen. Simultaneously, monohydrogen phosphate is filtered at the glomerulus. Hydrogen ions, filtered or secreted, are trapped by the buffering capability of these two proton acceptors and excreted. In addition, through a carbonic anhydrase– dependent system, the tubular epithelial cell is capable of generating a hydrogen ion and a bicarbonate ion from carbonic acid (dissolved carbon dioxide) and reabsorbing the bicarbonate while the hydrogen ion is excreted. This regenerates the bicarbonate pool and facilitates acid/base stability. If the bicarbonate pool becomes excessive, renal excretion of bicarbonate is increased by a complex mechanism dependent on the decreased hydrogen ion in the tubular fluid. In effect, therefore, the kidney is able to directly influence acid excretion by three mechanisms: excretion of phosphate (affected by glomerular filtration and parathyroid hormone), synthesis of ammonia, and control of the directional flow of bicarbonate. The last of the three mechanisms just enumerated requires further comment. Because of the ready availability of carbon dioxide and water (and consequently carbonic acid), the kidney, through carbonic anhydrase, can control the abundance of hydrogen ions and bicarbonate. By directing the excretion of hydrogen ions into the tubular lumen and the return of bicarbonate to plasma, the kidney has a great capacity to excrete acid and control base levels. Only when the renal mechanisms responsible for reabsorption of base or excretion of acid are compromised does the renal contribution to acid/base balance become limited. In normal circumstances, the rate at which the kidney returns bicarbonate to the body is equivalent to the rate of sodium/hydrogen exchange in the distal tubule. Although the proximal tubule is quantitatively the most important site of bicarbonate reabsorption in the kidney, with a small contribution from the loop of Henle, the distal segment is capable of ‘‘fine-tuning’’ the acid/base excretory balance.

Acute Injury Response Since the discovery of interleukin-1 in 1981, we have continued to learn about the system of cytokines and chemokines, which mediates what is often called the ‘‘acute injury response.’’ Current evidence suggests that the macrophage reacting to surface receptor signals from infectious agents, allergens, trauma, chemokines, and other agonists initiates a vigorous biochemical response leading to a cascade of interrelated events that alter metabolism, body temperature, cellular response, hepatic protein synthesis, and renal handling of fluid and electrolytes (20). More recent evidence suggests that many, if not all, of the observations usually referred to as ‘‘sepsis’’ can actually be explained by the human response to activation of a cell receptor (toll-like receptor). This would imply that what historically has been considered to be an effect of gram-negative endotoxin may actually be the host response to the presence of chemical substances (lipopolysaccharide from the cell wall) rather than a toxic effect of the substances themselves. The impact on fluid and electrolyte balance appears to be primarily mediated by a ‘‘nonphysiologic’’ release of ADH and mineralocorticoids. This increase in the substances that control the reabsorption of salt and water in the distal portion of

the nephron results in decreased renal excretion (often manifested as oliguria) in an attempt to protect the volume and composition of extracellular water. The recognition of the importance and ubiquity of this acute injury response may result in a change in our methods of intravenous fluid therapy as we more fully understand the significance of this hormonal response to injury.

Effect of Medications It is beyond the scope of this chapter to address specific effects of drugs used in clinical practice. It is nevertheless important to recognize that commonly used pharmaceutical agents can produce significant fluid and electrolyte abnormalities. One hundred fifty-two separate drugs are identified as having hyponatremia as a major side effect. These are broadly categorized as agents that cause loss of sodium in the kidney (e.g., diuretics) or that enhance the retention of water in the renal collecting duct [e.g., syndrome of inappropriate antidiuretic hormone (SIADH)]. Hypokalemia is also commonly produced by drugs. Loop-active diuretics, amphotericin B, and drugs producing metabolic alkalosis are commonly responsible. It is prudent to consider the possibility of an adverse drug effect early in the course of evaluating new onset fluid and electrolyte abnormalities (21–24).

PATHOPHYSIOLOGY AND TREATMENT OF SPECIFIC ELECTROLYTE AND ACID/BASE ABNORMALITIES To understand abnormalities of electrolyte homeostasis, some attention must be given to normal daily requirements (Table 4) (25). Although it is true that a state of electrolyte balance requires that intake be equal to losses (and losses can be minimized), realistically there will be daily excretion of electrolytes of a fairly predictable magnitude, allowing a range of estimated daily needs. Under normal conditions, water losses occur primarily through urinary excretion, evaporation from the skin, and water losses through the lungs. The latter two sources are referred to as insensible losses because they are not visible or readily measurable and amount to 500 to 800 mL [300 mL/m2 body surface area (BSA)] of water daily, with almost negligible amounts of sodium and chloride. Urine is the major sensible loss (one that is visible and measurable) and averages between 1200 and 1500 mL of water daily, with 10 to 30 mequiv of sodium and 20 to 60 mequiv of potassium. Another sensible loss is water loss through the feces, which is usually quite minimal. Losses greater than those routinely encountered (e.g., diarrhea) will result in corresponding increases in requirements; increases in normal excretion rates (e.g., renal failure) will necessitate a reduction in intake.

Abnormalities of Water Balance Disturbances in the amount and distribution of total body water are common in clinical practice. Because water Table 4 Adult Daily Requirements Normal Water (total) Water (insensible) Sodium Potassium Chloride Calcium Magnesium Source: From Ref. 25.

2

1500 mL/m 500 mL/m2 0.7–3.6 mequiv/kg 0.7–2.1 mequiv/kg 0.7–3.6 mequiv/kg 0.4–1.1 mequiv/kg 0.3–0.7 mequiv/kg

Minimal 870 mL/m2 – 0.3 mequiv/kg 0.3–0.5 mequiv/kg 0.3 mequiv/kg 0.2 mequiv/kg 0.2–0.4 mequiv/kg

Chapter 2:

distributes throughout the body, restricted in its movement only by osmotic and ionic barriers, abnormalities in amount, with maintenance of normal electrolyte concentrations (i.e., isotonic), are frequent. An isotonic increase in total body water results in edema, whereas an isotonic decrease produces clinical dehydration. Because electrolyte conservatory mechanisms are extremely efficient, deviation from isotonicity is uncommon except at the extremes of age, when compensatory mechanisms are less adequate or access to water or salt is restricted. In infants and the elderly, therefore, hypertonic (sodium > 145 mequiv/L) and hypotonic (sodium < 135 mequiv/L) abnormalities are more common and require that attention be given to the volume as well as the concentration problems. It is important to realize that isotonic abnormalities in total body water are more prevalent than other fluid derangements in adults, only because compensatory mechanisms maintain the concentration of important solutes. In circumstances where a coexistent problem compromises these compensatory mechanisms (e.g., renal disease, inappropriate ADH secretion, diuretic use, and adrenal insufficiency), a superimposed fluid loss or gain may very well not be isotonic. Dehydration is an absolute decrease in total body water and usually represents a balanced loss between intracellular and extracellular volume. Dehydration can be assessed on clinical grounds alone, and in fact, there is no readily available ‘‘test’’ that identifies dehydration unless there is a coexistent abnormality of concentration. Blood urea nitrogen and consequently osmolality are frequently elevated, however, and may be supportive of the diagnosis when the creatinine concentration is normal and the blood urea nitrogen/creatinine ratio is greater than 20. Up to a 5% decrease in total body water can escape clinical detection, without appropriate suspicion by history. Thirst is usually present, however, and is an accurate sign of water deficit. Losses greater than 5% usually lead to conditions such as sunken eyes, loss of skin turgor with tenting of presternal skin, and dry mucous membranes. Greater than 10% dehydration will commonly demonstrate hemodynamic changes with tachycardia and postural hypotension. Treatment of dehydration requires an understanding of the composition of the fluid deficits. Isotonic dehydration reflects a loss of all fluid compartments (and their contained electrolytes) and is corrected by intravenously infusing a balanced salt solution such as Ringer’s lactate (Table 5). Concomitant abnormalities in concentration or tonicity are best assessed by the serum sodium concentration; such electrolyte abnormalities should be treated simultaneously with management of the volume deficit. For example, in hypernatremic dehydration (Table 5), water alone as 5% dextrose in water is used for replacement of the water deficit and a balanced salt solution, for volume restoration. The appropriate intravenous fluid should be administered to correct one-half of the estimated abnormality over 24 hours. This

39

Pathophysiology of Fluid and Electrolyte Disorders

approach is used because most deficits develop over a period of days or weeks and the patient has usually adjusted to them. Rapid replacement of losses may actually impose a greater risk than the deficit itself. Edema can be related to an underlying disease (cardiac, renal, or hepatic) or, as is common in modern practice, to abundant or excessive intravenous fluid administration. Excesses in extracellular fluid typically are susceptible to gravity and hence are most pronounced in dependent areas. Conditions such as pretibial and ankle edema are common in the upright patient. Presacral edema or pitting of the skin overlying the iliac crest is more likely in the recumbent patient. Water intoxication represents a specific abnormality of water balance. Ordinarily, large amounts of ingested or administered water can be excreted quantitatively, without a resultant volume excess or ionic dilution. Very marked amounts, however, particularly in a setting of compromised homeostatic mechanisms, can result in edema, hyponatremia, and dilution of other electrolytes as well (for a further discussion, refer to section on hyponatremia). Treatment of fluid excess requires an understanding of the cause and an assessment of the integrity of homeostatic mechanisms, particularly renal, hepatic, and central nervous system (CNS) function. In the setting of normal compensation, simple fluid restriction or decreased administration is likely to be effective. When simultaneous abnormalities in renal, hepatic, adrenal, or cardiac physiology exist, careful attention to intake and output (an ‘‘accountant’’ approach) is indicated. Only by specific accounting of all volumes, concentrations, and amounts can aggravation or creation of abnormalities be prevented.

Abnormalities of Sodium Because sodium is the major cation within extracellular water and is regularly analyzed in the laboratory, it is surprising that abnormalities in sodium concentration are not discovered more frequently. Although hypernatremia and hyponatremia are seen, they are rigorously prevented by compensatory mechanisms within the kidney. In fact, very small changes in sodium concentration (and subsequently osmolality) result in a measurable change in ADH release in the same direction. When renal concentrating mechanisms are defective (2) (e.g., wash out of renal medullary concentration gradient as with diuretics, partial tubular dysfunction from incipient or resolving acute tubular necrosis, or massive sodium loss in interstitial nephritis), abnormalities in serum sodium are more common.

Hyponatremia Virtually all acute, stressful situations (e.g., infection, anesthesia, and surgery) are accompanied by release of ADH and conservation of free water (26,27). This homeostatic mechanism is very effective in preserving extracellular volume, when combined with sodium conservation, which

Table 5 Composition of Common Solutions Used for Intravenous Therapy Solutions 5% Dextrose and water 0.9% Sodium chloride (normal saline) 0.45% Sodium chloride (half-normal saline) 3% Sodium chloride (hypertonic saline) Lactated Ringer’s solution

Glucose (g/L)

Na (mequiv/L)

Cl (mequiv/L)

HCO3 (mequiv/L)

K (mequiv/L)

Ca (mequiv/L)

50 – – – –

– 154 77 513 130

– 154 77 513 109

– – – – 28a

– – – – 4

– – – – 2.7

Exists in solution as lactate and is ultimately metabolized to bicarbonate (HCO3).

a

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depends on a decrease in perfusion pressure or delivered sodium at the juxtaglomerular apparatus (28,29). Consequently, hyponatremia in adult patients at the time of admission for elective surgery is distinctly uncommon. However, once a patient has received intravenous administration of hypotonic fluids (e.g., 0.45% saline), the inability to excrete the ‘‘free’’ water due to an obligatory ADH release will frequently result in a dilutional decrease in serum sodium to some degree. When hyponatremia is present, spurious causes must be excluded. This is accomplished most simply by evaluating the serum osmolality. Because osmolality measures the amount of solute per ‘‘mass’’ of water instead of volume, it is independent of the amount of water in a volume of serum. Normally, serum is 94% water. Increases in protein or lipid concentrations can, however, alter the amount of water and lead to an analytic error in sodium determination. Osmolality is not affected so. Consequently, hyponatremia in the setting of normal osmolality would raise the suspicion of paraproteinemia (e.g., multiple myeloma and macroglobulinemia) or hyperlipidemia. Alternatively, an increase in an extracellular solute such as glucose (or mannitol) causes a shift in water from the intracellular to extracellular space, to conserve osmotic equality. This results in a subsequent ‘‘dilution’’ of extracellular sodium, but a maintained osmolality because of the presence of another osmotically active substance, glucose. Identification of hyperglycemia in the setting of normal osmolality will explain a fall in sodium levels of approximately 1.6 mequiv/L for each 100 mg/100 mL rise in blood sugar above normal. When true hyponatremia does occur (low sodium plus low osmolality), evaluating how the kidney is behaving allows a rational interpretation of the probable pathophysiology. A careful assessment of overall fluid and electrolyte status (weight change, input and output summaries, presence or absence of edema, etc.) is essential (30). Characterizing the patient as ahead or behind in volume by clinical assessment allows appropriate interpretation of the renal response, which is best ascertained by measuring the urinary osmolality and sodium concentration. During active sodium conservation, urinary sodium is typically low (< 5 mequiv/L). This indicates either that there is a true deficit in sodium or that the kidney ‘‘thinks’’ there is one. The latter occurs when circulating substances (e.g., aldosterone) are inappropriately present in edematous states such as cirrhosis and chronic congestive heart failure (28,31). In each of these settings, however, the patient is edematous and probably has ascites. In the absence of either of these findings, true sodium depletion is probably present. Sodium administration should be guided by an estimate of the deficit, which must be considered independently of (added to) volume deficits that are isotonic (Na ¼ 140 mequiv/L). The sodium deficit approximates 0.6 mequiv/kg per milliequivalent fall in serum sodium. Unless neurologic symptoms are present, which would mandate urgent treatment, the sodium replacement should take 24 to 48 hours. After one-half of the deficit has been replaced, serum electrolyte levels should be rechecked. When urinary sodium is increased (>20 mequiv/L), simple sodium depletion can be excluded. Sodium conservation either will not or cannot take place. This occurs in the setting of abnormal ADH release, adrenal insufficiency, severe hyperthyroidism, recent use of diuretics, or intrinsic renal tubular dysfunction as in renal failure or interstitial nephritis. This can be clarified by measuring urinary osmolality.

Because the normal response to hyponatremia and hypo-osmolality is excretion of solute-free water (or dilute urine), the finding of a urine osmolality above that of serum, plus hyponatremia, indicates an abnormal ADH response (27,32). This implies a continued release of ADH in spite of a hypo-osmolar state. If this is present at the time of admission or in the absence of intravenous fluids, it implies a CNS abnormality (e.g., head trauma and intracranial tumor) or an ectopic site of production (e.g., bronchogenic carcinoma). In the patient receiving hypotonic fluids, this finding more commonly reflects the release of ADH associated with a central ‘‘acute phase response’’ or ‘‘systemic inflammatory response’’ to stress or illness. In either event, fluid balance will be ‘‘ahead’’ as is the total amount of sodium in the body. The hyponatremia means that water is ‘‘more ahead’’ than sodium. This paradoxic increased total sodium in the face of decreased serum sodium concentration results in a high urinary sodium excretion (>20 mequiv/L) and a high salt excretion fraction (>5%) (33). The finding of isosthenuria (urine osmolality equals serum osmolality) implies intrinsic renal dysfunction or pharmacologic dysfunction from diuretics. The finding of low urine osmolality and high urine sodium, however, suggests that ADH release is appropriately terminated, but that sodium is not being conserved. This situation is seen in mineralocorticoid insufficiency. Laboratory evaluation of the renal response to hyponatremia can be enlightening but does not replace clinical assessment. Because hyponatremia means either a decrease in sodium or an increase in water, appropriate interpretation of readily available data should allow discrimination. A decrease in sodium requires the presence of a route of loss and usually is associated with weight loss. Alternatively, an increase in water should be manifested by a gain in weight and excess fluid intake over output. In the patient who is not receiving intravenous fluids, a serum sodium below 135 mequiv/L constitutes hyponatremia and deserves investigation. In the patient who is receiving hypotonic fluids, however, mild hyponatremia is common enough to be expected. Although it is not normal, mild hyponatremia down to 130 mequiv/L can probably be ignored. A sodium level of 125 to 130 mequiv/L justifies vigorous diagnostic efforts to identify the cause. A serum sodium level of 120 to 125 mequiv/L necessitates vigorous institution of appropriate therapy. A sodium level of less than 120 mequiv/L is a medical emergency and frequently requires careful administration of hypertonic saline for correction. It is not the low sodium per se that is detrimental, but rather the increase in the intracellular fluid compartment, which accompanies this circumstance. This intracellular volume increase is especially detrimental to brain cells because the brain is contained within a fixed space with little or no room for expansion. When brain cells swell, an increase in intracranial pressure ultimately develops and presents clinically as a variety of manifestations, including apathy, confusion, weakness, nausea, and occasionally vomiting. If this water intoxication is not corrected, it will ultimately lead to convulsions, stupor, and possibly even death. When hyponatremia occurs in the setting of brain injury or intracranial surgery (particularly for cerebral aneurysms), the term ‘‘cerebral salt wasting’’ is often applied (34). The hypothesis is that humoral substances are released from the injured brain, either directly or as a result of the injury, which leads to an excessive loss of sodium in the kidney (35). More recent studies question whether this is actually a specific syndrome, or whether it is a manifestation

Chapter 2:

of increased ADH release in patients who have received very large amounts of isotonic crystalloid solutions. Although it is not clear which explanation is correct, it is certainly true that hyponatremia occurs in neurosurgical patients and that this hyponatremia may be multifactorial. Central to appropriate treatment is the assessment of actual fluid status (hypervolemia vs. hypovolemia) and a decision whether to administer fluids containing a high concentration of sodium or to restrict the amount of water being administered. In the absence of objective evidence of a sodium deficiency, it may be more appropriate to restrict water intake, as in the syndrome of increased ADH release, seen both in SIADH and in the acutely injured patient.

Pathophysiology of Fluid and Electrolyte Disorders

41

the very narrow range of normal potassium concentration in plasma and the relatively massive adjacent intracellular pool (5) (with exchange influenced by variables such as pH and drugs) make control of serum potassium both important and tenuous. This importance is underscored by the critical nature of severe potassium abnormalities on the cardiac, neural, and muscular systems and evidenced by the diligent monitoring of potassium concentrations, which occurs in operating suites and intensive care units. An understanding of the common circumstances leading to potassium abnormalities will facilitate maintenance of normal homeostasis and simplify treatment of abnormalities (16).

Hyperkalemia Hypernatremia The body rigorously defends itself against hyperosmolality. Even a small rise results in increased ADH release and subsequent free-water retention by the kidney, unless, of course, ADH release cannot occur (e.g., diabetes insipidus), ADH is ineffective (e.g., nephrogenic diabetes insipidus), or the patient is denied access to water (e.g., infants, geriatric patients, and unconscious or restrained patients). A simple review of the clinical situation will often identify the cause. Because hypernatremia implies either an increase in sodium or a decrease in water, both must be considered. An increase in sodium can result from aggressive administration of normal saline or sodium bicarbonate. A decrease in water can occur by renal or extrarenal mechanisms. Nonrenal causes should be clinically apparent and include such symptoms as diarrhea and excessive sweating. In addition to the forms of diabetes insipidus (pituitary dysfunction or nephrogenic), renal sources must include osmotic diuresis with obligatory ‘‘free-water’’ loss. In all cases, treatment is simple. Administration of hypotonic solutions will restore sodium concentration, and, when sodium excess is a component, judicious diuretic use will hasten sodium excretion. Care in treating hypernatremia must be exercised, however, because a rapid change in sodium concentration is of greater clinical importance than the actual sodium concentration (36). Too rapid a correction of sodium abnormalities can result in dramatic CNS effects, including seizures secondary to rapid changes in intracellular volume. Accordingly, treatment should be planned to correct one-half of the estimated deficit over 24 hours. In the case of hypernatremia, this usually translates into the administration of 2 mL of free water/kg over 24 hr for each milliequivalent of planned decrease in serum sodium concentration. In the patient with head injury or recent neurosurgical intervention with accompanying diabetes insipidus, unless actual hypophysectomy has been performed, early administration of vasopressin should be avoided. Careful replacement of losses will maintain fluid and electrolyte balance until the usually transient abnormality resolves. Accordingly, hypernatremia should be avoidable. If permanent diabetes insipidus is anticipated, however, administration of vasopressin or its synthetic analog, desmopressin acetate (DDAVP), should be instituted. Serious complications of diabetes insipidus (or inappropriate ADH secretion) are not a function of difficulty in treatment, but rather failure of recognition. Anticipation of such problems will greatly simplify their subsequent management.

Abnormalities of Potassium Because mammals usually ingest large amounts of potassium, it is not surprising that most abnormalities in potassium balance are the result not of intake, but of excretion. Further,

Because the kidney is the major site of potassium excretion, compromised renal function is almost always a factor contributing to hyperkalemia (9,11,13). Although increased potassium levels can occur from either excess intake or decreased excretion, the former is quite rare, is usually iatrogenic, and occurs in the setting of administering very large intravenous doses of potassium (or potassium-containing drugs such as penicillin) or giving potassium to a patient receiving a drug that inhibits potassium excretion (e.g., spironolactone, triamterene, and ACE inhibitors). Abnormalities of excretion are common and can be caused by intrinsic abnormalities of renal function (acute oliguric renal failure), disturbances in hormonal control of potassium exchange (hypoaldosteronism, adrenal insufficiency, etc.), or alterations in the potassium exchange mechanism per se (drugs, acidosis, etc.). Occasionally serum potassium is factitiously elevated (compared to plasma potassium) because of in vitro Kþ release from platelets during phlebotomy, in the setting of thrombocytosis (37). Hyperkalemia produces predictable clinical consequences that affect neuromuscular function (weakness, irritability, etc.) and cardiac conduction (peaking of T waves, prolongation of PR and QT intervals, widening of QRS complexes, and eventual heart block). Monitoring of the electrocardiogram provides a simple, noninvasive method of assessing change in the hyperkalemic or potentially hyperkalemic patient. Predictable changes, beginning with T-wave peaking, are the harbingers of subsequent complications, which are evidenced by alterations in the shape and duration of the major electrocardiogram complexes. Because hyperkalemia poses a life-threatening problem, urgent and definitive correction is imperative (17). A transient decrease in serum potassium can be accomplished by translocating extracellular potassium back into cells with glucose and insulin, and the membrane effects of hyperkalemia can be offset by the administration of calcium. In the rare patient with reasonable renal function and the need for a rapid decrease in serum potassium, a potassium diuresis can be induced by the use of loop-active diuretics. More commonly, potassium is removed from the body by the use of potassium-exchanging resins [sodium polystyrene sulfonate (Kayexalate)] that can be given by mouth or rectum and will predictably lower serum potassium. This is accomplished, however, through exchange with sodium and may alter fluid and sodium balance. Institution of peritoneal or hemodialysis is clearly the most effective, long-term approach to potassium control in the patient with renal compromise. It is important to remember, however, that continued administration of hypertonic glucose, as in parenteral nutrition, will result in a predictable and sustained fall in serum potassium because the egress of potassium associated with catabolism is prevented.

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Hypokalemia Hypokalemia is a common electrolyte abnormality in both hospitalized and ambulatory patients (16,38). Although it may be precipitated by an underlying disease (mineralocorticoid excess as in Cushing’s syndrome, potassium loss from a colonic villous adenoma, potassium wasting in renal disease, etc.) or by decreased potassium intake (low-potassium diet), most hypokalemia is iatrogenic in that it is induced pharmacologically by agents having potassium wasting as a side effect (36,39,40). Most notable are the diuretic agents currently used in the management of hypertension and fluid overload. The loop-active agents in particular are associated with a substantial potassium loss in the urine. Other classes of drugs, however, can also induce hypokalemia (21,41). The recent increased use of amphotericin B in the treatment of immune-compromised patients has uncovered a profound and difficult-to-manage hypokalemia (42). Use of newer synthetic penicillins in ultrahigh concentration is also associated with increased renal potassium loss and subsequent hypokalemia. Hypokalemia may manifest itself in various ways (e.g., muscle weakness and paralysis). More frequently, however, hypokalemia produces myocardial irritability and subsequent arrhythmias such as frequent premature contractions, sustained tachycardias, and potentiation of digitalis toxicity. Although these complications are more feared than common, they are serious complications and justify both respect and careful monitoring. Treatment is simple. Administration of potassium as the chloride salt will correct the usual associated chloride-dependent alkalosis, decrease renal potassium loss, and correct the hypokalemia. Beware, however, because the degree of hypokalemia is very poorly correlated to the magnitude of potassium deficit. Therefore, strict guidelines for replacement are dangerous. Because the hazards of hypokalemia appear to be virtually eliminated by institution of treatment rather than total correction, the identification of the abnormality and institution of treatment are more important than the rapidity of treatment. Frequently, discontinuation of the offending drug or control of the underlying disease will be necessary for long-term potassium control.

Abnormalities of Chloride Because chloride is the most abundant anion in plasma and extracellular water, it is not surprising that its range of variation is large and its mechanism of variation somewhat passive. The high concentration of chloride in gastric juice accounts for the hypochloremia that accompanies the metabolic alkalosis of conditions such as gastric outlet obstruction, repetitive vomiting, and pyloric stenosis. Chloride is absorbed in large quantities from the gut, and its level is controlled by urinary excretion. Most reabsorption of chloride occurs in the proximal tubule of the kidney, in association with sodium reabsorption. A considerable component is also actively reabsorbed in Henle’s limb (43). Alterations in proximal tubule absorption (osmotic diuretics) or Henle’s limb (loop-active diuretics) can lead to hypochloremia and metabolic alkalosis. In addition, in the presence of ‘‘hormone-dependent’’ (e.g., aldosterone) alkalosis, the fraction of chloride that is reabsorbed is decreased. This interaction of chloride and bicarbonate is important in the evaluation of the patient with metabolic alkalosis. Chloride is present in abundance in GI secretions. Substantial chloride loss can result from GI fluid losses from the upper tract (e.g., gastric outlet obstruction) or lower tract

(e.g., diarrhea). Assessment of the chloride deficit provides complementary information in the evaluation of volume depletion, secondary to GI losses.

Abnormalities of Calcium The most important factor in assessing calcium as an electrolyte is that clinically important abnormalities are very, very rare and easily categorized into hypocalcemia caused by hypoparathyroidism and hypercalcemia caused by hyperparathyroidism, tumors, systemic diseases, and medications. Hypocalcemia is rarely of clinical importance, except following parathyroid and thyroid surgery, and therefore infrequently requires treatment. Hypercalcemia, when the serum calcium is greater than 12, can be treated with aggressive saline administration, loop-active diuretics to minimize the fluid overload, and biphosphonates to reduce resorption of calcium from bone. Identification of the specific cause of the hypercalcemia (by measurement of ionized calcium, PTH, parathyroid hormone related peptide (PHRP), and 1,25-dihydroxyvitamin D3) is necessary to effect long-term control of the calcium level (18).

Isolated Acid/Base Abnormalities The availability of routine measurement of ‘‘arterial blood gases’’ has resulted in a much clearer understanding of the nature and treatment of acid/base disturbances in clinical practice. On the basis of the normal physiologic determinants previously described and the pathophysiologic mechanisms that will be enumerated, acid/base disturbances can be classified into acidosis or alkalosis. Both of the categories can be further subdivided into metabolic and respiratory, as well as combined metabolic/respiratory. Tables, nomograms, and algorithms have been developed for the assessment of the excess or deficit of acid (or base) to assist the clinician in management. Unfortunately, all of these aids are of only limited practical value because of the concurrent and efficient efforts of the compensatory mechanisms of the body to correct the acid/base disturbance. In other words, the onset of a disturbance in pH (by change in either HCO3 or pCO2) results in very rapid initiation of respiratory and/or metabolic compensatory mechanisms to partially correct the pH. It is essential in planning the treatment of acid/base disturbances to keep this compensation in mind, lest overaggressive management result in new, iatrogenic acid/base disturbances in the opposite direction.

Acidosis As described previously, acidosis can occur by the absolute gain in acid or loss of base from the body. Because the buffering system of the body is ‘‘open’’ in the sense that the CO2/HCO 3 system uses both respiratory and renal control systems, all but the most acute form of acidosis will be combined with compensatory adjustments. Consequently, all respiratory disturbances will have a metabolic compensatory component, and metabolic imbalances will stimulate respiratory compensation. Frequently, the pH is remarkably well corrected, and only a careful review of the historical facts associated with the illness will allow a clear analysis of the inciting cause. Occasionally, the cause is so elusive that only by disturbing the system with exogenous alkali and observing the respiratory response (or lack thereof) will the primary problem become apparent.

Respiratory Acidosis Pure respiratory acidosis is the simplest of the acid/base disturbances conceptually, because it results from a decrease

Chapter 2:

in effective alveolar ventilation (relative to carbon dioxide production). Normal carbon dioxide production from metabolic processes is approximately 450 L/day (20,000 mmol). Whenever carbon dioxide elimination lags behind production, respiratory acidosis ensues. The consequent laboratory abnormalities include a decrease in pH and an increase in pCO2. It is important to consider the pH first, because compensatory hypercapnea (increase in serum pCO2) in the setting of profound metabolic alkalosis is not uncommon, particularly in patients receiving mechanical ventilation by an intermittent (synchronized intermittent mandatory ventilation, pressure support ventilation, etc.) modality. Once respiratory acidosis is identified, a differential consideration of causes must include central respiratory depression (e.g., narcotics and intrinsic CNS disease), mechanical causes of decreased ventilation and/or increased dead space (e.g., tension pneumothorax, hemothorax, and massive pleural effusions), or pathophysiologic causes of increased carbon dioxide production. The common causes of acute or chronic respiratory acidosis are summarized as follows: & & & & & &

Airway obstruction Respiratory center depression Neuromuscular defects Restrictive lung diseases Smoke inhalation Inadequate mechanical ventilation

Appropriate treatment logically follows identification of the underlying mechanism and may include ventilatory assistance to enhance alveolar ventilation to eliminate the retained carbon dioxide and thereby correct the acidic pH. Occasionally, respiratory acidosis is produced by an increase in carbon dioxide production, because of either marked increase in oxygen consumption (active rewarming after heart surgery) or alteration in the respiratory quotient (excessive administration of carbohydrate-based parenteral nutrition). Because the pCO2 is proportional to the ratio of carbon dioxide production and minute alveolar ventilation, pCO2 ¼ kðV CO2 Þ=ðV AlvÞ; recognition of a high pCO2 and a high minute ventilation should assist in identifying patients with excessive carbon dioxide production.

Metabolic Acidosis In marked contradistinction to respiratory acidosis, metabolic acidosis can result from a variety of causes that include the gain of metabolic acids above excretion rates or the loss of bicarbonate greater than its rate of regeneration (44–47). Again, clinical evaluation and elucidation of the underlying mechanism greatly simplifies the task of evaluating the data. Also, it is important to avoid jumping to a conclusion of acidosis on the basis of only a decrease in the bicarbonate concentration of the serum, because this finding may also represent a normal metabolic compensation for a respiratory alkalosis. As is true with all biologic systems, electrical neutrality is maintained in all fluid compartments throughout the body by balancing the total number of cations with the total number of anions. In the extracellular compartment under normal conditions, the concentration of the cation sodium roughly equals the sum of the concentrations of the anions, chloride and bicarbonate, except for a small anion gap of

Pathophysiology of Fluid and Electrolyte Disorders

43

12 2 mequiv/L. An increase in this gap can give an important clue concerning the cause of acidosis. Thus it is helpful to subdivide metabolic acidosis into those categories that manifest an increase in unmeasured anions (increased anion gap) and those that do not and subsequently have a normal anion gap (nonanionic gap). As a generality, metabolic acidosis associated with the accumulation of organic acid will have an associated increased anion gap, whereas that caused by a loss of bicarbonate will have a normal or decreased anion gap. The distinction is important. Although both causes may require replacement of bicarbonate to correct the acidosis, the former group will require correction of an associated metabolic abnormality, and the latter will require attention to the site of bicarbonate loss (e.g., fistula).

Increased Gap Acidosis Any metabolic acidosis that is caused by the accumulation of organic acid (which is not measured by routine electrolyte analysis) will have a calculated anion gap that is greater than normal (uremia, diabetic ketoacidosis, lactic acidosis, and drug ingestion/poisoning). In some circumstances, e.g., diabetic ketoacidosis, combined mechanisms are operative, and the component caused by ketoacids is proportional to the anion gap. The presence of an acidosis with an anion gap greater than 14 mequiv/L implies either ingestion of an organic substance or an endogenous metabolic abnormality. Of the four commonly ingested ‘‘toxins’’ (methanol, ethylene glycol, ethanol, and isopropanol) only methanol (formic acid) and ethylene glycol (oxalic acid) produce a metabolic acidosis. Ethylene glycol also produces oxaluria, whereas methanol ingestion usually produces rapid retinal blindness. Acetylsalicylic acid (aspirin) and paraldehyde can also produce metabolic acidosis if ingested in large amounts, although paraldehyde is no longer available in the United States. Endogenous production of organic acid in excess of excretory capacity is seen in uremia, diabetic ketoacidosis, and lactic acidosis. Each is usually identifiable by the company it keeps: acidosis caused by renal failure is associated with the clinical and metabolic abnormalities of uremia; diabetic ketoacidosis is usually seen in known diabetics and is associated with hyperglycemia, dehydration, and ketosis in urine and serum (48); lactic acidosis can be caused by a variety of clinical states, but usually represents some form of hypoperfusion, such as sepsis or trauma (49). Lactic acidosis is associated with an increase in lactate in the blood (>4 mequiv/L) and an altered ratio of lactate to pyruvate (>30:1) and other oxidation–reduction pairs [e.g., acetoacetate/ betahydroxybutyrate and reduced nicotinamide adenine dinucleotide (NAD)/oxidized nicotinamide adenine dinucleotide (NADH)]. The actual cause of the increased lactic acid concentration is impairment of electron-to-oxygen transfer and subsequent accumulation of NADH, which shifts the equilibrium of pyruvate and lactate in the direction of lactate. Lactate serves as a reservoir or ‘‘cul de sac’’ during periods of deficient electron-to-oxygen transfer. Restoration of NADþ levels results in a prompt conversion of lactate back to pyruvate. It is often erroneously assumed that lactic acid is part of the pathophysiology of ‘‘lactic acidosis,’’ whereas it is actually only a secondary biochemical consequence of the underlying oxidation–reduction disorder. It is a quantitatively useful test to confirm the diagnosis and estimate the magnitude, however. Because it is often included in the battery of tests known as ‘‘blood gases,’’ it is important to confirm that a metabolic acidosis with an increased anion gap is

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present before suggesting a diagnosis of lactic acidosis, because lactate levels may be elevated in the absence of lactic acidosis. When lactic acidosis is present, attention needs to be paid to the underlying cause of the inadequate delivery of oxygen to tissues. It is also important to recognize that lactic acid may also be a component of the metabolic acidosis caused by diabetic ketoacidosis, methanol poisoning, and salicylate intoxication. Treatment of all forms of anion gap acidosis is both supportive (restore circulation, correct acidosis with bicarbonate, etc.) and specific (correct underlying abnormality). Bicarbonate should be given in adequate quantities to keep the pH above 7.2 and/or the bicarbonate level above 15 mequiv/L. Situations with ongoing tissue hypoxia may require substantial replacement, whereas transient problems such as postictal lactic acidosis usually correct themselves spontaneously. When bicarbonate is required, the bicarbonate deficit can be determined by a variety of formulas. An easy-toremember fact is that the bicarbonate space (apparent volume of distribution of the effective buffering system) corresponds roughly to one-half of total body water (approximately 30% of total body weight in kilograms). Thus: HCO 3 deficit ¼ 0:3 wt ð24 HCO3 Þ:

Usually one-half of the calculated deficit is replaced to avoid overcorrection, following which blood gases are repeated before infusing additional bicarbonate. In patients with compromised renal function and metabolic acidosis, it is important to remember that disorders associated with levels of potassium, magnesium, and phosphate are commonly found and should be sought. In lactic acidosis produced by hypovolemic shock, the treatment should focus on restoring circulation and perfusion, rather than on correcting the acidosis itself.

in conditions such as sepsis and hypovolemia) can be estimated by the following formula: pCO2 ¼ 1:5ðHCO 3 Þ þ D; where HCO3 is mequiv of HCO 3 and D is 8 2 mequiv/L. Recognition of this compensatory phenomenon will assist in preventing iatrogenic metabolic alkalosis from overtreatment with bicarbonate. It is also helpful to test whether the change in pCO2 is of an appropriate amount, because each increase or decrease of pCO2 of 10 torr should result in a change in pH in the opposite direction of 0.08.

Alkalosis As is the case with acidosis, alkalosis may be either respiratory or metabolic in origin. In contradistinction to acidosis, it is the metabolic component that is rather simple to define and correct, whereas respiratory alkalosis may have a myriad of causes.

Respiratory Alkalosis Respiratory alkalosis is probably the most common acid/ base disturbance in clinical medicine and can be induced by a multitude of underlying conditions. The pathophysiologic mechanism is excess alveolar ventilation above the requirements of carbon dioxide production. This abnormality typically represents some form of CNS overstimulation but may include as causes the factors summarized below: & & & & & &

Nonanion Gap Acidosis (Hyperchloremic) Causes of metabolic acidosis with a normal anion gap include bicarbonate loss, inability to excrete hydrogen ion, or administration of exogenous HCl or NH4Cl. Most commonly, there is either a recognized source of bicarbonate loss or an abnormality in the kidney (or adrenal). Recognized bicarbonate loss is usually from the GI tract distal to the pylorus and may include duodenal fistula, biliary drainage, pancreatic fistula, small intestinal fistula, ureterosigmoidostomy, and diarrhea. Intrinsic renal losses of bicarbonate include interstitial nephritis, renal tubular acidosis, adrenal insufficiency, hypoaldosteronism, and acetazolamide administration (50). Treatment of nonanion gap acidosis requires correction of the existing acidosis plus routine administration of a bicarbonate source on a regular basis. The dosage of bicarbonate required will depend on the underlying disease, its magnitude, and the need for dialysis. Guidelines for acute correction are the same as for anion gap acidosis: calculate the bicarbonate deficit (base deficit), estimate the bicarbonate space, and administer only as much as should correct one-half of the predicted deficit over a period of 24 hours. As indicated previously, compensation for metabolic acidosis is usual and is manifested as hyperpnea, tachypnea, nasal flaring, or even Kussmaul’s respiration. The degree of compensatory hyperventilation in a stable, chronic metabolic acidosis (as opposed to the acute changes seen

& & & &

Anxiety Fever Salicylate intoxication CNS disorders Intrathoracic processes Hypoxemia Hepatic insufficiency Gram-negative septicemia Pregnancy Mechanical hyperventilation

Treatment of respiratory alkalosis must be directed at correcting the underlying cause (e.g., correct fever with antipyretics). In patients requiring assisted ventilation, use of intermittent or partial ventilation systems will tend to minimize the overventilation that is often seen with controlled or assist/controlled ventilation. Other attempts at increasing pCO2, such as inspired carbon dioxide, rebreathing devices, and added mechanical dead space, are relatively ineffective.

Metabolic Alkalosis The fundamental abnormality in pure metabolic alkalosis is an absolute or relative excess of base (primarily bicarbonate) in extracellular fluid (44,51,52). This excess tends to be offset by a compensatory decrease in minute alveolar ventilation with subsequent hypercarbia. The mechanisms underlying metabolic alkalosis include the loss of hydrogen ion (with chloride), gain of exogenous base, or extracellular fluid–volume contraction. Identification of the latter two mechanisms should be straightforward by clinical evaluation, although gain of exogenous base may be masked as in blood or fresh frozen plasma transfusions (citrate is converted to bicarbonate) or excessive infusion of lactated Ringer’s solution.

Chapter 2:

The most common, clinically important forms of metabolic alkalosis are a result of loss of hydrogen ion and chloride from the stomach or hydrogen ion alone from the kidney. These two varieties can be separated by attention to the amount of chloride measured in the urine. Low urinary chloride (<10 mequiv/L) indicates a ‘‘chlorideresponsive’’ alkalosis. When there is insufficient chloride in the proximal nephron to maintain electroneutral reabsorption of sodium, bicarbonate is reabsorbed, leading to alkalosis, but with no chloride in the urine (53). When the abnormality occurs in the distal tubule (caused by hyperaldosteronism, increased plasma desoxycorticosterone, exogenous corticosteroids, licorice ingestion, or carbenoxolone therapy) the urine chloride will be greater than 20 mequiv/L, which confirms that chloride depletion is not the cause and therefore will not correct the abnormality by itself (54). More specifically, chloride-resistant alkalosis of renal origin can be thought of as excessive stimulation of the distal tubular, mineralocorticoid-mediated cation exchange mechanism resulting in increased excretion of Hþ and Kþ (55). This latter variety is perpetuated by (although not initiated by) hypokalemia, and correction of the potassium deficit is essential to restoration of acid/base balance (56,57). Active reabsorption of potassium (initiated by hypokalemia-induced hyperaldosteronism) in the distal renal tubule in ‘‘exchange’’ for Hþ, leads to the paradox of acid urine in the face of hypokalemic alkalosis (paradoxic aciduria). Occasionally, massive doses of potassium are required to achieve equilibrium; so the kidney does not have to work so hard reabsorbing it. When clinically significant hypoventilation develops as compensation for metabolic alkalosis, more rapid treatment may be needed (58). Two approaches are effective, including administration of acid and inducing renal tubular acidosis with acetazolamide, but neither will correct the underlying cause of the alkalosis. Recent experience with 0.1N HCl infusions (or as high as 0.2N) has shown this form of treatment to be quite safe if administered through a central venous line. The dose of Hþ should be calculated by determining base excess (or increase in bicarbonate above 28 mequiv/L), and multiplying it by one-half of estimated total body water (0.3 weight in kg). One-half of this calculated amount is then administered over 24 hours. Although repeated acetazolamide administration is effective in the treatment of severe metabolic alkalosis, correction of the excessive mineralocorticoid stimulation or maintenance of a high serum potassium is required to prevent recurrence.

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Table 6 Identification of Isolated Acid-Base Disturbances Disturbance Metabolic acidosis Metabolic alkalosis Respiratory acidosis (acute) Respiratory acidosis (chronic) Respiratory alkalosis (acute)

Respiratory alkalosis (chronic)

Effect pCO2 should fall by 1 to 1.5 times the fall in HCO3 PCO2 should rise by 0.5 to 1 times the rise in HCO3 Plasma HCO3 should be <30 mequiv/L Plasma HCO3 should rise by 4 mmol/10 mm Hg rise in pCO2 Plasma HCO3 should fall by 2.5 mmol/10 mm Hg fall in pCO2 (usually >18 mequiv/L) Plasma HCO3 should fall at least as much as acute but not <15 mequiv/L

Abbreviations: HCO3, Bicarbonate; pCO2, carbon dioxide partial pressure. Source: From Ref. 59.

clinical settings in which they are likely to occur are summarized below: Respiratory acidosis and metabolic alkalosis occur in patients with chronic respiratory acidosis with coexistent congestive heart failure, requiring diuretics. b. Respiratory acidosis and metabolic acidosis occur in patients with cardiopulmonary arrest, chronic lung disease in shock, or chronic renal failure with respiratory insufficiency. c. Respiratory alkalosis and metabolic acidosis may occur in hepatic failure with respiratory overstimulation or renal failure in sepsis. d. Respiratory alkalosis and metabolic alkalosis occur in hepatic cirrhosis with hyperventilation associated with vomiting or in chronic respiratory failure following institution of vigorous mechanical ventilation. e. Metabolic acidosis and metabolic alkalosis can be sequential rather than simultaneous abnormalities, although this may not be obvious. This situation is identified when the anion gap is numerically greater than the estimated HCO3 deficit and occurs with progressive contraction alkalosis caused by vomiting when hypovolemia becomes manifest. a.

PRINCIPLES OF FLUID THERAPY

Combined Acid/Base Disorders It is crucial to realize that acid/base disorders are in no way limited to a single pathophysiologic derangement at a time. Consequently, combinations of abnormalities are common. To distinguish single from combined disturbances, an objective approach is essential. Most standard texts contain graphs (e.g., Goldberg graphs) that indicate the 95% probability range for single acid/base disorders, i.e., the laboratory values that are 95% likely to represent a single pathophysiologic abnormality. In addition, numerous empiric equations have been developed to identify compensatory changes as opposed to coexistent abnormalities. Characteristics of isolated acid/base disturbances are listed in Table 6 (59). Certain combinations of acid/base disturbances are encountered commonly enough clinically to also justify listing. The components of these combinations and the

In any patient requiring intravenous fluid therapy, three categories of fluid loss must be taken into consideration. These include losses related to normal body maintenance (insensible plus urine), continuing losses of fluid and electrolytes, and preexisting fluid deficits resulting from the patient’s underlying disease. These should be thought of as a combination of maintenance and replacement fluids. As indicated in an earlier section of this chapter, all patients have normal maintenance requirements related to daily sensible and insensible losses. These losses will vary somewhat depending on the person’s age, body build, and sex, but generally average between 2000 and 2500 mL of fluid every 24 hrs (1500 mL/m2/day). The major sensible loss is urine, and the major insensible loss is related to evaporative losses from the skin and water losses through the lungs. All of these are primarily water losses, containing only small

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Table 7 Composition of Gastrointestinal Secretions

Stomach Pancreas Bile Intestine (primarily small)

Volume (mL/24 hr)

Na (mequiv/L)

K (mequiv/L)

Cl (mequiv/L)

HCO3 (mequiv/L)

1000–2000 300–800 300–600 2000–4000

60–100 135–145 135–145 120–140

10–20 5–10 5–10 5–10

100–130 70–90 90–130 90–140

– 95–120 30–40 30–40

amounts of electrolytes. If an individual patient requires intravenous therapy for only short periods of time, replacement of electrolyte losses is generally unnecessary, and maintenance volume losses can be replaced with solutions of 5% dextrose and water (Table 5) or 5% dextrose and half normal saline. If prolonged intravenous therapy is expected, the small but consistent daily electrolyte losses will need to be taken into account and can generally be managed by giving 2 mequiv/kg of body weight of sodium, chloride, and 1 mequiv/kg of potassium daily. Thus if a patient requires 2500 mL of fluid to meet normal maintenance needs, 1500 mL could be provided as dextrose and water, and the remaining 1000 mL could be given as half normal saline with the addition of appropriate amounts of potassium chloride (Table 5). Losses arising from the patient’s underlying disease must also be replaced. In surgical patients, these losses are almost always from some portion of the GI tract. As a general rule, intestinal secretions, bile, or pancreatic juice can be replaced on a volume-to-volume basis with a balanced salt solution such as Ringer’s lactate (although pancreatic replacement may require additional bicarbonate). Losses from an actively secreting stomach (emesis or nasogastric (NG) losses) require significant amounts of chloride (normal saline or half normal saline with potassium chloride added) (Table 5). The actual amount of potassium loss from GI secretions is small, although alkalosis can result in significant potassium loss from the kidney. Generally, vomiting or NG losses produce this abnormality (Table 7). To prevent the development of problems with hypokalemia, 20 to 40 mequiv of potassium for every liter of fluid loss from any of these sites should be administered. If long-term intravenous therapy is required, these requirements may increase and should be monitored by the determination of serum electrolyte values periodically. Another source of abnormal losses is an increased evaporative loss from the skin and respiratory tract in patients who are febrile. Loss of as much as 1000 mL daily may occur over and above normal insensible losses, if the body temperature is markedly elevated. In patients with persistent temperature elevations above 101 F (38.4 C), 500 to 1000 mL of additional fluid may be required daily (10% per C increase). Because such fluid loss is primarily water, it can generally be replaced with a ‘‘dextrose and water’’ intravenous solution or as water through a feeding tube. In critically ill patients receiving ventilatory support, respiratory losses (one-third of insensible loss) are no longer present because of the use of warm, humidified air, and fluid replacement should be reduced accordingly. Finally, preexisting solute or volume deficits must also be corrected. Again, such deficits generally arise from the GI tract and are the result of such problems as vomiting, diarrhea, or sequestration of fluid as may occur in patients with bowel obstruction. A correct estimate of these losses is often difficult, but careful attention to physical findings and important historical information from the patient, summarizing the duration of disease and the

frequency and amount of fluid losses (e.g., through vomiting or diarrhea), are usually helpful in deriving an appropriate estimate of the magnitude of underlying deficits. As emphasized previously, only one-half of this calculated estimate should be replaced in a given 24-hour period, and the patient reassessed clinically and with supporting laboratory data before attempting any further deficit replacement. If the principles just enumerated are followed, the large majority of patients requiring intravenous therapy can be managed without difficulty. Especially pertinent in such management is the frequent reassessment of the patient clinically and daily review of the patient’s input and output records to ensure that the proper amount and type of fluid is being given. Daily weight assessment are especially helpful in this regard and can give valuable clues as to whether abrupt or even subtle changes in fluid balance (either fluid overload or fluid deficit) are occurring. Further, attention to such detail helps prevent the development of any major imbalances that may occur insidiously before they become clinically obvious.

SUMMARY Plasma is a subset of the total fluid (extracellular fluid) that bathes the cells. As such when sampled, it allows an indirect look at the cellular events and the interaction between the cells and the extracellular fluid. Fluid and electrolyte disorders are legion and commonplace. The correct, critical interpretation of laboratory parameters to identify the pathophysiologic mechanisms underlying these abnormalities requires a willingness to go beyond the obvious. A logical analytic approach to the meaning of the observed laboratory disturbances should lead to an insight into the significance of the observation. A cookbook approach to ‘‘correct’’ the abnormal test result may actually worsen the situation. It is critical to appreciate that a concentration actually represents a ratio of the amount of a substance to the amount of fluid in which it is dissolved or suspended. Abnormalities in concentration can be caused by a change in either component. Attention to a variety of clinical factors (weight changes, vital signs, nursing notes, even history and physical examination, etc.) often provides the insight necessary to identify the true problem and chart its solution.

REFERENCES 1. Pitts R. Physiology of the Kidney and Body Fluids: Volume and Composition of the Body Fluids. Chicago: Year Book Medical Publishers, 1968. 2. Halperin ML, Bohn D. Clinical approach to disorders of salt and water balance. Emphasis on integrative physiology. Crit Care Clin 2002; 18:249–272.

Chapter 2: 3. Gruber UMA. Water and electrolyte balance. In: Diem K, ed. Documenta Geigy, Scientific Tables. Ardsley, NY: Geigy Pharmaceuticals, 1970. 4. Forbes R, Mitchell H, Cooper A. Further studies on the gross composition and mineral elements of the adult human body. J Biol chem 1956; 223:969. 5. Corsa L, et al. Measurement of exchangeable potassium in man by isotope dilution. J Clin Invest 1950; 29:1280. 6. Moore F. Determination of total body water and solids with isotopes. Science 1946; 104:157. 7. Moore F, et al. Body composition: total body water and electrolytes: intravascular and extravascular phase volumes. Metabolism 1956; 5:447. 8. Prough DS. Physiologic acid-base and electrolyte changes in acute and chronic renal failure patients. Anesthesiol Clin North Am 2000; 18:809–833, ix. 9. Beck LH. The aging kidney. Defending a delicate balance of fluid and electrolytes. Geriatrics 2000; 55:26–28, 31–32. 10. Miller M. Fluid and electrolyte homeostasis in the elderly: physiological changes of ageing and clinical consequences. Baillieres Clin Endocrinol Metab 1997; 11:367–387. 11. Jorgensen PL. Sodium and potassium ion pump in kidney tubules. Physiol Rev 1980; 60:864–917. 12. Kreimeier U. Pathophysiology of fluid imbalance. Crit Care 2000; 4:S3–S7. 13. Samson WK. Adrenomedullin and the control of fluid and electrolyte homeostasis. Annu Rev Physiol 1999; 61: 363–389. 14. Frazier SK. Neurohormonal responses during positive pressure mechanical ventilation. Heart Lung 1999; 28:149–165; quiz 166–167. 15. Cadnapaphornchai MA, Gurevich AK, Weinberger HD, Schrier RW. Pathophysiology of sodium and water retention in heart failure. Cardiology 2001; 96:122–131. 16. Gennari FJ. Disorders of potassium homeostasis. Hypokalemia and hyperkalemia. Crit Care Clin 2002; 18:273–288, vi. 17. Williams ME. Endocrine crises. Hyperkalemia. Crit Care Clin 1991; 7:155–174. 18. Kinder B, Stewart A. Hypercalcemia. Curr Probl Surg 2002; 39:349–448. 19. Fulop M. Algorithms for diagnosing some electrolyte disorders. Am J Emerg Med 1998; 16:76–84. 20. Ward P. Role of complement, chemokines, and regulatory cytokines in acute lung injury. Ann N Y Acad Sci 1996; 796:104–112. 21. Polderman KH, Girbes AR. Piperacillin-induced magnesium and potassium loss in intensive care unit patients. Intensive Care Med 2002; 28:520–522. 22. Madhusoodanan S, Bogunovic OJ, Moise D, et al. Hyponatraemia associated with psychotropic medications. A review of the literature and spontaneous reports. Adverse Drug React Toxicol Rev 2002; 21:17–29. 23. Liberopoulos EN, Alexandridis GH, Christidis DS, Elisaf MS. SIADH and hyponatremia with theophylline. Ann Pharmacother 2002; 36:1180–1182. 24. Fanestil DD, Park CS. Steroid hormones and the kidney. Annu Rev Physiol 1981; 43:637–649. 25. Goodenough RD, Burke JF. Fluid, electrolyte, and acid-base homeostasis in surgery. In: Burke JF, ed. Surgical Physiology. Philadelphia: WB Saunders, 1983. 26. Patel GP, Kasiar JB. Syndrome of inappropriate antidiuretic hormone-induced hyponatremia associated with amiodarone. Pharmacotherapy 2002; 22:649–651. 27. Diringer MN. Sodium disturbances frequently encountered in a neurologic intensive care unit. Neurol India 2001; 49(suppl 1): S19–S30. 28. Porcel A, Diaz F, Rendon P, et al. Dilutional hyponatremia in patients with cirrhosis and ascites. Arch Intern Med 2002; 162:323–328. 29. Oh MS, Carroll HJ. Disorders of sodium metabolism: hypernatremia and hyponatremia. Crit Care Med 1992; 20: 94–103.

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30. Milionis HJ, Liamis GL, Elisaf MS. The hyponatremic patient: a systematic approach to laboratory diagnosis. CMAJ 2002; 166:1056–1062. 31. Milionis HJ, Alexandrides GE, Liberopoulos EN, et al. Hypomagnesemia and concurrent acid-base and electrolyte abnormalities in patients with congestive heart failure. Eur J Heart Fail 2002; 4:167–173. 32. Wong LL, Verbalis JG. Systemic diseases associated with disorders of water homeostasis. Endocrinol Metab Clin North Am 2002; 31:121–140. 33. Espinel CH. The FENa test. Use in the differential diagnosis of acute renal failure. JAMA 1976; 236:579–581. 34. Qureshi AI, Suri MF, Sung GY, et al. Prognostic significance of hypernatremia and hyponatremia among patients with aneurysmal subarachnoid hemorrhage. Neurosurgery 2002; 50: 749–755; discussion 755–766. 35. Singh S, Bohn D, Carlotti AP, et al. Cerebral salt wasting: truths, fallacies, theories, and challenges. Crit Care Med 2002; 30:2575–2579. 36. Riggs JE. Neurologic manifestations of electrolyte disturbances. Neurol Clin 2002; 20:227–239, vii. 37. Johnson C, Hughes K. Pseudohyperkalemia secondary to postsplenectomy thrombocytosis. Am Surg 2001; 67:168–170. 38. Kapoor M, Chan GZ. Fluid and electrolyte abnormalities. Crit Care Clin 2001; 17:503–529. 39. Torpy DJ, Mullen N, Ilias I, Nieman LK. Association of hypertension and hypokalemia with Cushing’s syndrome caused by ectopic ACTH secretion: a series of 58 cases. Ann N Y Acad Sci 2002; 970:134–144. 40. Elisaf M, Liberopoulos E, Bairaktari E, Siamopoulos K. Hypokalaemia in alcoholic patients. Drug Alcohol Rev 2002; 21:73–76. 41. Preuss H, Burris J. Adverse metabolic effects of antihypertensive drugs. Implications for treatment. Drug Saf 1996; 14: 355–364. 42. Ural A, Avcu F, Cetin T, et al. Spironolactone: is it a novel drug for the prevention of amphotericin B-related hypokalemia in cancer patients? Eur J Clin Pharmacol 2002; 57:771–773. 43. Rocha AS, Kokko JP. Sodium chloride and water transport in the medullary thick ascending limb of Henle. Evidence for active chloride transport. J Clin Invest 1973; 52:612–623. 44. Sirker AA, Rhodes A, Grounds RM, Bennett ED. Acid-base physiology: the ’traditional’ and the ’modern’ approaches. Anaesthesia 2002; 57:348–356. 45. Story DA, Poustie S, Bellomo R. Estimating unmeasured anions in critically ill patients: anion-gap, base-deficit, and strong-iongap. Anaesthesia 2002; 57:1109–1114. 46. Hatherill M, Waggie Z, Purves L, et al. Correction of the anion gap for albumin in order to detect occult tissue anions in shock. Arch Dis Child 2002; 87:526–529. 47. Frangiosa A, De Santo LS, De Santo NG, et al. Acid-base state in patients after cardiac transplantation. Am J Nephrol 2002; 22:332–337. 48. Felig P. Diabetic ketoacidosis. N Engl J Med 1974; 290: 1360–1363. 49. Mizock B, Falk J. Lactic acidosis in critical illness. Crit Care Med 1992; 20:80–93. 50. Rothstein M, Obialo C, Hruska K. Renal tubular acidosis. Endocrinol Metab Clin North Am 1990; 19:869–887. 51. Wilkes NJ, Woolf R, Mutch M, et al. The effects of balanced versus saline-based hetastarch and crystalloid solutions on acid-base and electrolyte status and gastric mucosal perfusion in elderly surgical patients. Anesth Analg 2001; 93: 811–816. 52. Hodgkin JE, Soeprono FF, Chan DM. Incidence of metabolic alkalemia in hospitalized patients. Crit Care Med 1980; 8: 725–728. 53. Wesson D, Dolson G. Enhanced HCO3 secretion by distal tubule contributes to NaCl-induced correction of chronic alkalosis. Am J Physiol 1993; 264:F899–F906. 54. Hanna J, Scheinman J, Chan J. The kidney in acid-base balance. Pediatr Clin North Am 1995; 42:1365–1395.

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55. Gill JR Jr., Bartter FC. Evidence for a prostaglandin-independent defect in chloride reabsorption in the loop of Henle as a proximal cause of Bartter’s syndrome. Am J Med 1978; 65: 766–772. 56. Jacobson H, Seldin DW. On the generation, maintenance, and correction of metabolic alkalosis. Am J Physiol 1983; 245:F425–F432.

57. Khanna A, Kurtzman NA. Metabolic alkalosis. Respir Care 2001; 46:354–365. 58. Palmer B, Alpern R. Metabolic alkalosis. J Am Soc Nephrol 1997; 8:1462–1469. 59. Schrier RW, ed. Renal and Electrolyte Disorders. Boston: Little Brown, 1980.

3 Surgical Nutrition Rosemary A. Kozar, Margaret M. McQuiggan, and Frederick A. Moore

INTRODUCTION It has been said that outward appearances do not always convey things as they really are. This is certainly true with respect to surgical nutrition. In our society, where fast food diets have become the norm and a sedentary lifestyle is all too commonplace, protein-calorie malnutrition is being increasingly recognized in patients requiring hospitalization, despite how healthy a patient appears outwardly. Failure to identify this problem prior to embarking on a surgical procedure can have devastating effects on the outcome, not only from the standpoint of postoperative complications, but also, unfortunately and all too frequently, whether survival is impacted as well. Thus, it behooves the surgeon to have a thorough understanding of those factors that contribute to both good health and malnutrition and an understanding of how an operation or other stressful events can alter the adequacy of nutritional balance, and what strategies need to be employed to ensure that a healthy nutritional environment is maintained to minimize postoperative morbidity and mortality. The present chapter addresses these issues.

METABOLIC RESPONSE TO STRESS VS. STARVATION Endogenous supplies of carbohydrates, fat, and protein, are used to supply energy needs during starvation. In acute starvation, carbohydrates are initially utilized but stores are limited and quickly depleted. The body then depends heavily upon the breakdown of protein to provide amino acids as a primary energy source and as a source of new glucose for glucose-dependent tissues. These pathways provide about 85% of energy needs in this setting. Fat is a major fuel source available for gluconeogenesis and may also be oxidized to ketone bodies for use by the liver and other tissues. During brief starvation (approximately 72 hours), this process may be easily interrupted by providing exogenous substrate. After this, adaptive changes favor the mobilization of fat and reduce the breakdown of protein to a low level (30% of energy requirements). Fat, rather than protein, becomes the principal source of energy. With longer starvation, protein is further protected by a fall in total energy requirements. As with brief starvation, the process is quickly and easily reversed by providing exogenous glucose alone (which returns the patient to the brief starvation pattern) or exogenous glucose and amino acids. A variety of conditions, such as trauma, major elective surgery, burns, or sepsis, lead to a metabolic response called the ‘‘injury stress response.’’ This is an endocrinemediated response that increases circulating levels of

the counter-regulatory hormones, catecholamines, corticosteroids, and glucagon, and alters the metabolism of carbohydrates, protein, and fat. More recent evidence suggests that the systemic inflammatory response syndrome (SIRS) and its associated mediators [e.g., tumor necrosis factor (TNF)a, interleukin (IL)-1, IL-2, IL-6] also play an important role in the injury stress response. The best option to control the initiating insult is to provide high-risk patients exogenous substrates that support the metabolic environment. The injury stress response increases resting energy expenditure (REE). The increase in REE is dependent upon the type as well as the severity of the insult. It can increase as much as 100% after burns, 50% after sepsis, 40% after trauma, and 30% after major surgery (1–3). Similar to what occurs in starvation, endogenous substrates are mobilized. Glucose stores are quickly depleted and gluconeogenesis (principally in liver, but also in kidney) produces glucose that is shunted to glucose-dependent tissues (brain, erythrocytes, inflammatory cells, and wound tissue). Adipose tissue is stimulated to release free fatty acids and glycerol. The rate of lipolysis, however, exceeds lipid oxidation. As a result, plasma triglyceride levels increase and considerable reesterification occurs in the liver. This requires energy and creates futile cycles. The increase in protein catabolism is the most dramatic effect. Skeletal muscle proteins and later constitutive protein stores (such as albumin) are broken down and the released amino acids become the substrate for acute-phase protein synthesis, gluconeogenesis, and energy production. Metabolic differences between chronic starvation and the injury stress response are summarized in Table 1. Nutritional support needs to be tailored to the metabolic environment of the patient. The stressed intensive care unit (ICU) patient requires more nonprotein calories (25 kcal/kg/day) and more protein (1.3 g/kg/ day) compared to the starved patient (20 kcal/kg/day and 1.0 g/kg/day, respectively). As stress increases, patients become more catabolic and less tolerant to glucose. Therefore the amount of protein administered is increased (1.5–2.0 g/kg/day) and the percent of nonprotein calories delivered as glucose is decreased

Table 1 Metabolic Response of Starved vs. Stressed Patients Metabolic response

Prolonged starvation

Injury stress

Resting energy Primary fuel Proteolysis Gluconeogenesis Ketone production

Decreased Fat Increased (þ) Increased (þ) Increased (þþþ)

Increased Mixed Increased (þþþ) Increased (þþþ) Increased (þ)

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(80–70%). Thus the nonprotein calories to gram of nitrogen ratio is decreased from 120:1 to 80:1 with increasing metabolic stress.

RATIONALE FOR NUTRITIONAL SUPPORT The rationale for providing nutritional support is threefold: first, to prevent acute protein malnutrition, second, to modulate the immune response, and third, to promote vital gut function.

Prevention of Acute Protein Malnutrition Persistent hypercatabolism dominates the metabolic response to critical illness. Initially, amino acids demands are met by skeletal muscle proteolysis. However, in a short period of time, this depletes crucial constitutive structural elements as well as circulating proteins. The resulting acute protein malnutrition is associated with cardiac, pulmonary, hepatic, gastrointestinal, and immunologic dysfunction. Delayed infections then extend hypercatabolism with the progression to full-blown multiple-organ failure (MOF). A number of clinical studies have clearly demonstrated that early enteral nutrition improves outcome, as measured by improved nitrogen balance and constitutive protein levels, improved immune function, decreased incidence of infections, and decreased length of stay (4–7). However, these studies were performed in trauma and burn patients, an easily identified and cohesive group of patients who also tend to be young and free of comorbid disease. Whether these observations can be generalized to other ICU patient populations is not clear.

Modulation of Immune Response Despite tremendous advances in ICU care, nosocomial infections continue to be an unsolved problem. In large part, these late infections occur due to failure of local and systemic host defenses. While exact causes of late immunosuppression are not clear, it is now believed to occur in part as a result of dysfunctional regulation of inflammation (8). An initial insult, whether secondary to sepsis, trauma/burns, or major operation, can precipitate early systemic hyperinflammation (i.e., SIRS), which when intense can precipitate early MOF. Certain components of this early SIRS response are endogenously downregulated to prevent unnecessary, potentially auto-destructive inflammation. This is referred to as the compensatory anti-inflammatory response syndrome (i.e., CARS). The resulting delayed immunosuppression, however, sets the stage for secondary infection, which can either worsen early MOF or trigger late MOF. While no strategy can prevent or eliminate this dysfunctional inflammatory response, the delivery of specific immune-enhancing nutrients (generally via the gut) may prove beneficial.

Promotion of Vital Gut Function The dysfunctional gut is believed to be the ‘‘reservoir for pathogens’’ that cause late MOF-associated infections. The initial insult (via ischemia/reperfusion, sepsis, or inhibitory neuroendocrine reflexes) and emergency laparotomy (via anesthesia and bowel manipulation) cause an early ileus. Disuse (parenteral instead of enteral nutrition) and common ICU therapies (e.g., H2-antagonists, narcotics, and broadspectrum antibiotics) promote further gut dysfunction, characterized by progressive ileus, colonization of the upper gut, increased permeability, and decreased gut-associated lymphoid tissue function. Consequently, the upper gut becomes

a reservoir for pathogens, and local and systemic defense mechanisms that prevent the spread of these organisms become impaired; the primary route of dissemination (i.e., aspiration vs. translocation) is not clear. Although there is good epidemiologic evidence for this sequence of events, prospective randomized controlled trials (PRCT) of gutspecific therapies such as early enteral nutrition and, recently, immune-enhancing enteral formulas have consistently demonstrated a reduction in nosocomial infections and provide the most convincing evidence (7,9). Thus, the provision of early enteral nutrition to promote more normal gut function is believed to prevent the occurrence of this cascade of events.

INITIATION OF NUTRITIONAL SUPPORT Nutritional Assessment A nutritional assessment is completed early in the hospital stay. Physical assessment of the patient includes evaluation of body composition, edema, wound healing, nutrient composition of fluid losses through wounds or drains, and review of the laboratory and medication profile. A recording of the height and preadmission weight is obtained and information on chronic disease, activity level, medications, previous dietary restrictions, psychosocial data, and drug and tobacco patterns is elicited. A history of 10% weight loss over three months is considered significant. However, weight loss combined with physiologic impairment may be clinically more important, as this can be associated with more septic complications, pneumonia, and a longer hospital stay (10). Functional impairment may be assessed by crude measures such as physical assessment of muscle bellies and triceps skinfold, decreased prehospital activity, perception of fatigue, strength of handshake, strength of cough, and skin integrity. More sophisticated measures such as caliper measurements of skin folds, bioelectrical impedance analysis, indirect calorimetry, and dual-energy X-ray absorptiometry for bone mineral density, hand-grip dynamometry, and respiratory muscle strength may also be performed. Body protein stores as assessed by muscle mass and serum proteins correlate more closely with survival than adipose stores. The normal age-related loss of skeletal muscle is well recognized. Protein-energy malnutrition is a major predictor of mortality in elderly patients (11). Beginning in the second decade, there is a reduction in the total muscle fiber numbers that, with advancing age, produces a 40% reduction in muscle cross-sectional area (12). The malnourished, elderly patient undergoing a catabolic illness may reach a critical cardiac or respiratory threshold earlier than expected (13). Loss of lean body mass, predominately skeletal muscle, in the ICU after stress or sepsis may approach 1% daily (14). Thus, a healthy young person might approach a lethal decline in lean body mass (40%) in 40 days, while a 60-year-old would reach that level in 30 days, and a person over 70 years in 20 days. Reduced immunocompetence accompanies loss of lean body mass. Assessment of serum protein status is conducted upon admission and periodically throughout the hospital stay. Acute-phase proteins, such as C-reactive protein, rise markedly within 48 to 72 hours after sepsis, injury, or surgery, while the constitutive proteins—albumin, prealbumin, and transferrin—all decline (Table 2). Measuring both categories of proteins concurrently helps to determine the degree of physiologic stress and the return of anabolism.

Chapter 3: Surgical Nutrition

Table 2 Serum Proteins that Aid in Nutritional Assessment Protein

Site of synthesis

Half-life

Liver Liver Liver Liver

21 days 2–4 days 8–10 days 48–72 hours

Albumin (g/dL) Prealbumin (mg/dL) Transferrin (mg/dL) C-reactive protein

Table 4 Urinary Nitrogen Loss as a Function of Stress Type of protein Constitutive Constitutive Constitutive Acute-phase reactant

Once the acute-phase response has resolved, the constitutive proteins should rise on provision of adequate nutrition.

Energy Requirements The Harris–Benedict equation is a common method to estimate energy expenditure and thus determine caloric requirements. This equation was derived using healthy males and estimates basal energy expenditure (BEE) based on age, sex, height, and weight. The Harris–Benedict equations are as follows: Men : BEE ¼ 66 þ ð13:7 weightÞ þ ð5 heightÞ ð6:8 ageÞ Women : BEE ¼ 665 þ ð9:6 weightÞ þ ð1:9 heightÞ ð4:7 ageÞ where weight is actual or adjusted (kg), height is in cm, and age is in years. The BEE represents energy requirements in the fasted, resting, nonstressed state. In the presence of metabolic stress, the BEE must be multiplied by an empirically derived stress factor to obtain an estimate of the caloric requirement. The usual stress multiplication factors range from 1.2 to 1.6 times the BEE. Current recommendations for estimated caloric support are reviewed in Table 3. In general, most patients should receive 25 to 30 kcal/kg/day and 1.5 to 1.75 g/kg/day of protein. Needs may decrease as patients move farther from the initial surgery or trauma.

Monitoring Response to Therapy Urinary Urea Nitrogen Urinary urea nitrogen (UUN) is measured as an indicator of the protein catabolic rate (i.e., stress) and is used to determine the nitrogen balance. It represents 60% to 90% of the nitrogen excreted in the urine, and therefore, is a rough approximation of total urinary nitrogen. As the stress level increases, the concomitant increase in protein catabolism results in an increase in urinary nitrogen. Estimated urinary losses for various clinical conditions are reviewed in Table 4. However, in patients with compromised renal function (creatinine clearance <50 mL/min), measurement of UUN is not Table 3 Estimated Caloric Requirements in Surgical Patients by Body Weight Weight class Underweight Normal Overweight Obesitya Extreme obesitya a

BMI (kg/m2) < 18.5 18.5–24.9 25–29.9 30–34.9 35–39.9 40

Use adjusted body weight.

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Obesity class

Caloric intake (kcal/kg/day)

I II III

40 25–30 25–30 20–25 20–25 10–20

Level of stress Nonstressed Mild stress Moderate stress Severe stress

Clinical condition

Urinary nitrogen loss (g/day)

Starvation Elective surgery Trauma Sepsis

<8 8–12 13–18 > 18

valid and calculations of protein catabolism rates are necessary. These require serial measurements of blood urea nitrogen and weights, and calculations of interdialytic time period and urinary losses of nitrogen. Additionally, UUN is not useful in patients with spinal cord injury because obligatory losses due to disuse muscle wasting are generally extraordinary regardless of the level of support and persist for up to seven weeks postinjury (15). Nitrogen balance is the difference between nitrogen intake and nitrogen output. Nitrogen intake is determined from dietary intake per day, while nitrogen output per day is determined by measuring UUN in a 24-hour urine collection and adding 4 g to approximate nonurea nitrogen loss in the urine plus other insensible N2 losses. Ideally patients should be in positive nitrogen balance (3–5 g/day). Nitrogen balance is calculated as follows: Nitrogen balance ¼ ðg protein intake=6:25Þ ðg nitrogen in UUN þ 4Þ

Metabolic Gas Analysis Also known as indirect calorimetry, metabolic gas analysis is a tool that can measure REE. Oxygen consumption ðVO2 Þ and carbon dioxide production ðVCO2 Þ are measured, and the REE is then calculated from the Weir equation: REE ¼ 3:9 þ VO2 þ 1:1 VCO2 2:8 UUN Care, however, must be taken to ensure that a steady state (less than 10% change in VO2 and VCO2 during five consecutive minutes of the test) was achieved before relying on results (16). The REE obtained should then be used as the patient’s nutritional goal. No activity factor or thermogenesis factor (unless the patient is fasted or on intermittent feeds) needs to be added. Indirect calorimetry may be helpful when (i) overfeeding would be undesirable [as in diabetes, obesity, or chronic obstructive pulmonary disease (COPD)], (ii) underfeeding would be especially detrimental (renal failure, or large wounds), (iii) physical or clinical factors promote energy expenditure deviant from normal, (iv) drugs are used that may significantly alter energy expenditure (paralytic agents, beta-blockers, or corticosteroids), (v) patient response to calculated regimens is suboptimal, and (vi) body habitus makes energy expenditure predictions challenging (morbid obesity or quadriplegia). Although more commonly utilized in ventilated patients, indirect calorimetry can also be performed on spontaneously breathing patients. The second variable obtainable from indirect calorimetry is the respiratory quotient (RQ). This is calculated as follows: RQ ¼ VO2 =VCO2 ¼ CO2 production=O2 consumption RQ is a gross measurement of substrate utilization (Table 5) and should be used to verify the accuracy of the study (17).

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Table 5 Respiratory Quotient (RQ) for Various Substrates RQ 0.7 0.8 0.85 1.0 > 1.0

Substrate Fat Protein Mixed Carbohydrate Lipogenesis

Any test with an RQ value outside of the physiologic range (less than 0.67 or greater than 1.3) should be checked for accuracy. When an RQ value 1 is obtained and the patient is on total parenteral nutrition (TPN), CO2 production may be increased by one of two mechanisms: either a high proportion of nonprotein calories are being supplied as glucose (carbohydrates have RQ of 1) or the patient is being provided excess calories. With overfeeding, excess glucose is converted to fat and lipogenesis (RQ > 1) occurs.

Body Proteins that Aid in Nutritional Assessment Serum proteins that correlate with nutritional status should be assessed (Table 2). The liver normally produces a number of constitutive proteins that are helpful in nutritional assessment. Albumin is useful for evaluation of chronic malnutrition, but not in the acute setting as it has a long half-life. To determine if nutritional support is having an impact, proteins such as prealbumin and transferrin are more useful due to their shorter half-life. However, during the acutephase response, the liver undergoes reprioritization and downregulates the constitutive proteins and upregulates acute-phase proteins such as C-reactive protein. Measurement of proteins such as albumin and prealbumin are therefore not helpful as indicators of the adequacy of the nutrition support during the acute stress response. C-reactive protein is a sensitive acute-phase reactant that increases from a normal level near zero up to 20 to 30 mg/dL within 48 to 72 hours of injury. It can be used as an indicator of the severity of injury, inflammation, and sepsis. Only when this level begins to decline, can the liver begin to again synthesize constitutive proteins. Once C-reactive protein falls below 10 to 15 mg/dL, a prompt increase in prealbumin typically occurs (0.5–1.0 mg/dL daily). If not, then the adequacy of the support regimen should be reevaluated and other factors that are preventing anabolism should be investigated.

Impact of Nutritional Status on Surgical Outcome Clinical studies have clearly documented the predictive value of preoperative serum albumin levels on postoperative morbidity and mortality. In fact, albumin was the single best predictor of postoperative outcome in several large studies conducted by the Department of Veteran Affairs (18–20). Recently Kudsk et al. (21) demonstrated that albumin as well as the operative site was important in predicting postoperative complications. Patients undergoing colectomy and gastrectomy tolerated greater degrees of malnutrition than those patients undergoing pancreatectomy and esophagectomy. Interestingly, patients with the lowest preoperative albumin levels sustained the longest delays in resuming oral intake postoperatively, compounding the preexisting malnutrition. Despite these findings, preoperative malnutrition remains under-recognized and under-treated and suggests that additional prospective preoperative nutritional studies should be conducted with high-risk patients.

Special Considerations in the Obese Patient Overview of Obesity and Nutrition Obesity affects one-third of Americans and is associated with a number of comorbid conditions. Obesity and morbid obesity are increasingly encountered in the surgery patient and may increase risk for morbidity and mortality. Trauma patients with a body mass index (BMI) > 31 were found to have an eightfold higher rate of mortality following blunt trauma, frequently due to pulmonary complications (22). While controversial, hypocaloric feeding in the obese patient has also been suggested to lessen infectious complications secondary to hyperglycemia. Comparable nitrogen balance is achieved in this patient population when hypocaloric feeds are administered (23). Monitoring for clinical evidence of overfeeding (hypercapnea, hyperglycemia, insulin resistance, hypertriglyceridemia, diarrhea, and distention) is used to refine predictions. Obesity is defined as a BMI 30 (Table 3). Energy needs should be based on adjusted body weight (ABW) when actual body weight exceeds 120% of the ideal body weight (IBW) and is calculated as: ABW ¼ 0:25 ðactual body weight IBWÞ þ IBW: The ABW takes into account the increased lean body mass seen in obese patients and should be used in the Harris–Benedict equation or in caloric predictions (kcal/kg) of energy needs. Obese patients experience a similar metabolic response to critical illness but tend to be more resistant to insulin. Obese patients should be started on nutritional support as early as their nonobese counterparts. Hypocaloric feeding provides the obese individual with high protein (1.5–1.75 g/kg ABW) but low total calories (20–25 kcal/kg ABW).

Nutrition After Bariatric Surgery Nutritional treatment for uncomplicated bariatric procedures is somewhat standardized across the country, although little evidence-based research has been performed in this patient population. Immediately after surgery, most programs begin clear liquids for several days, which are consumed in 30 cc/hr increments. This is followed by a progressive diet (advanced every two weeks) of full liquids, then pureed, soft foods, and finally a regular diet as tolerated. Maintenance goals are set at 1000 to 1200 kcal daily. Protein goals are typically 60 to 80 g protein/day, which may deliver as little as 0.48 g protein/kg ABW, which is less than the recommended daily intake of 0.8 g/kg body weight of protein. Liquid multivitamins with mineral supplements, iron, vitamin C, vitamin B12, and calcium may also be added. The amount of nutritional follow-up and level of expertise varies greatly between programs. For the majority of patients, a standard bariatric diet can be followed. However, patients who develop postoperative complications necessitating admission to the ICU will generally require an alternative feeding regimen. If the complication is intra-abdominal, particularly if secondary to an anastomotic leak, nutrition will usually be provided by the parenteral route, as enteral access (to the small bowel) will generally require operative placement. An initial goal of adjusted weight at 10 to 20 kcal/kg and protein at 1.5 g/kg can be instituted, depending on the degree of insulin resistance. Indirect calorimetry is helpful and, if performed, feeds should be instituted at 60% to 75% REE. A UUN can then be used as a guide to achieve positive nitrogen balance. Type 2 diabetes is a

Chapter 3: Surgical Nutrition

frequent comorbidity and strict glycemic control (glucose < 110 mg/dL) should be employed in all patients. Intravenous lipids are not provided in the first one to two weeks, and then provided sparingly to prevent essential fatty acid deficiency. Enteral support typically requires a specialized combination of a high-protein polymeric formula with the addition of a protein modular product. A liquid multivitamin and mineral supplement may also be added. Bariatric patients who experience delayed complications or significant remote illnesses can pose a challenge. Many of these patients have been on the bariatric diet for a prolonged period of time and may have some degree of protein malnutrition from a low-calorie, low-protein diet. Due to malabsorption, micronutrient deficits may be present and the refeeding syndrome is likely to occur. Organ function may also be compromised through body cell mass depletion, and immunocompetence decreased via protein malnutrition. Kilocalorie and protein requirements can be extraordinary, especially with large wounds such as necrotizing fasciitis or decubiti. However, the approach is similar to that taken with early postoperative complications. Indirect calorimetry, 24-hour UUN, and sequential monitoring of C-reactive protein and prealbumin should be employed. If renal failure occurs, continuous dialysis is typically employed to facilitate adequate feeding. Depending on the length of time since the original surgery and the adequacy of postoperative nutritional follow-up, the patient may exhibit varying degrees of protein, iron, zinc, calcium, folate, and B12 deficiency.

ENTERAL NUTRITION Benefits of Enteral Nutrition Three prospective randomized controlled trials (PRCTs) performed in the 1980s had significant impact on clinical practice in surgical, and particularly trauma ICUs (4–6). These single-institution trials all randomized trauma patients to early enteral nutrition or TPN and all demonstrated that patients receiving early enteral nutrition had significantly fewer infectious complications. A meta-analysis that

53

combined data from eight PRCT (six published, two not published) was then conducted to assess the nutritional equivalence of enteral nutriton compared to TPN in highrisk trauma and/or postoperative patients (24). Similar to the single-institution trials, fewer infectious complications developed in patients receiving enteral nutrition. Even when patients with catheter-related sepsis were removed from the analysis, a significant difference in infections between groups remained. Taken together, these trials provide convincing evidence that enteral nutrition is preferred to TPN in patients sustaining major torso trauma. A recent meta-analysis evaluating the effect of early versus delayed enteral nutrition in acutely ill (medical and surgical) patients also confirmed a decrease in infectious complications in patients receiving early enteral nutrition (25).

Role of Immune-Enhancing Agents in Surgical Patients Recent basic and clinical research suggests that the beneficial effects of enteral nutrition can be amplified by supplementing specific nutrients that exert pharmacologic immune-enhancing effects beyond the prevention of acute protein malnutrition. There are at least 18 PRCT (Table 6) and three meta-analyses (24,26,27) where an immuneenhancing enteral diet is compared with a standard enteral diet or no diet, and where the patient outcome was a predetermined end point. Of the 18 PRCTs, 11 trials demonstrated improved outcome, four trials were highly suggestive of improved outcome, and three trials did not demonstrate any clinical outcome advantage. The majority of trials are in trauma and cancer patients, though a few trials include mixed ICU and septic ICU patients. The proposed immune-enhancing agents include glutamine, arginine, omega-3 polyunsaturated fatty acids (PUFA), and nucleotides, though the individual contributions of each have not been well investigated. Glutamine is actively absorbed across the intestinal epithelium and then metabolized in the small bowel to ammonia, citrulline, alanine, and proline, and serves as an energy source for the

Table 6 Patient Outcome in Prospective Randomized Controlled Trials Comparing IEDs vs. Standard Enteral Diets Author

Year, Journal

Patient type (Number)

Gottschlich Daly Brown Moore

1990, 1992, 1994, 1994,

JPEN Surgery Pharmacotherapy J Trauma

Burns (50) Cancer (77) Trauma (37) Trauma (98)

Bower Daly Kudsk Senkel Mendez Saffle Heslina Braga Atkinson Weimann Senkel Braga Snyderman Galban

1995, 1995, 1996, 1997, 1997, 1997, 1997, 1998, 1998, 1998, 1999, 1999, 1999, 2000,

Crit Care Med Ann Surg Ann Surg Crit Care Med J Trauma J Trauma Ann Surg Crit Care Med Crit Care Med Nutrition Arch Surg Arch Surg Laryngoscope JPEN

Mixed ICU (296) Cancer (60) Trauma (35) Cancer (154) Trauma (43) Burns (50) Cancer (154) Cancer (154) Mixed ICU (369) Trauma (32) Cancer (154) Cancer (206) Cancer (129) Septic ICU (181)

Results with IED (decrease in) Wound infection, LOS Wound complication, infections Infections Intra-abdominal infection, multiple organ failure Infections, LOS Wound complications, infections, LOS Antibiotics, infection, LOS Late infections * ARDS

Infections, LOS Ventilator days, LOS SIRS, multiple organ failure Late infections Infections Infections Late infections

a

Improved outcome Yes Yes Yes Yes ? Yes Yes Yes Yes No No No Yes ? Yes ? Yes Yes Yes Yes Yes

Compared IED to no diet. Abbreviations: ARDS, adult respiratory distress syndrome; ICU, intensive care unit; IED, immune-enhancing diet; SIRS, systemic inflammatory response syndrome; LOS, length of stay.

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enterocyte. Glutamine is therefore acknowledged to be the preferred fuel of the enterocyte, and stimulates lymphocyte and monocyte function. The demand for glutamine is increased during stressed states and supplementation at pharmacologic doses may be required. It also promotes protein synthesis, is a precursor for nucleotides as well as glutathione, and is thought to play a role in maintaining gut integrity. In a recent meta-analysis, glutamine (parenteral and enteral) administered to critically ill and surgical patients resulted in a lower mortality, less infectious complications, and shorter hospital stays (28). High-dose and parenteral glutamine had the greatest effect, though the study was not designed to examine these parameters. Additionally, a mixed patient population was included with limited (randomized) studies and clinical end points. A randomized trial of glutamine-enriched enteral nutrition in severely injured patients demonstrated a decrease in pneumonia, sepsis, and bacteremia (29). Arginine is a semiessential amino acid that is important for T-cell function and wound healing. Endogenous production is insufficient during periods of metabolic stress (such as illness) and exogenous supplementation is required for maximal function of the immune system. It is also a powerful secretagogue, increasing the production of growth hormone, prolactin, somatostatin, insulin, and glucagon. Additionally, arginine is the chief precursor of nitric oxide and has been shown to increase protein synthesis and improve wound healing (30). It is the association with nitric oxide production that has led to speculation that arginine may enhance the systemic inflammatory response and, therefore, be potentially harmful, particularly in septic patients (31). Sepsis increases levels of inducible nitric oxide synthetase (iNOS). Arginine is a substrate for iNOS, and in its presence arginine combines with molecular oxygen to produce citrulline and nitric oxide. The resulting nitric oxide could have numerous adverse effects in sepsis, including vasodilation, cardiac dysfunction, and direct cytotoxic injury by generating potent reactive oxygen species. Increased mortality has been demonstrated in some critically ill septic patients when receiving an immune-enhancing diet, and arginine has been implicated as the causative agent (32,33). Although traditional enteral products contain a high proportion of omega-6 PUFA, diets with a low omega-6 PUFA and high omega-3 PUFA content more favorably alter the fatty acid composition of membrane phospholipids toward reduced inflammation (34). Finally, nucleotides (purines and pyrimidines) are needed for DNA as well as RNA synthesis and may be necessary in stressed states to maintain rapid cell proliferation and responsiveness (35). In the setting of increased demand, most tissues can increase intracellular de novo synthesis of nucleotides. Lymphocytes, macrophages, and enterocytes, however, rely on increased salvage from the extracellular pool, which may be depleted during stress.

Contraindications to enteral nutrition can be divided into absolute and relative. Absolute contraindications would include functional complications such as bowel obstruction, peritonitis, progressive ileus, massive gastrointestinal hemorrhage, and gastrointestinal ischemia associated with shock and vasopressors. Relative contraindications include proven intolerance to enteral nutrition and intolerance associated with short-gut syndrome, high-output fistula, pancreatitis, and inflammatory bowel disease. A recent bowel anastomosis is not a contraindication. Laboratory data in fact suggests that healing may be hastened by early feeding (36).

Gastric Feeding Controversy exists as to the optimal site for delivery of enteral nutrients. Although there is a trend toward decreased ventilator-associated pneumonias with jejunal feeding, no significant differences have been demonstrated between postpyloric and gastric feeding (37–40). None of these studies, however, have been appropriately powered to demonstrate statistical significance. Additionally, a greater percentage of caloric goals are reached (37,41) and gastrointestinal complications are less frequent with postpyloric feeding. The stomach, but not the small bowel, commonly exhibits an ileus after surgery, major trauma, or other critical illnesses. Ritz et al. demonstrated that up to 45% of ventilated patients had delayed gastric emptying, impeding adequate delivery of gastric feeds (42). Several studies have shown that this can be modulated by the use of prokinetic agents (43,44). Erythromycin has a direct effect on the motilin receptors of the stomach and has been shown to improve the effectiveness of gastric feedings. Gastric feeding is appropriate for some surgical patients, particularly those farther out from their initial injury or insult. Prior to starting gastric feeds, however, patients should be screened for risk of aspiration (Table 7) (45). Aspiration is the second most common cause of nosocomial infection in hospitalized patients (46). The presence of an inflated endotracheal cuff does not preclude aspiration in intubated patients, as there is as high as a 50% to 75% incidence of aspiration in patients with endotracheal tubes and/or tracheostomies (47,48). Altered mental status, whether from dementia, sedation, or a closed head injury poses a risk as do neuromuscular disorders and stroke. Recent major abdominal surgery and the need for prolonged supine position can also increase the risk of aspiration. Persistently high gastric residual volumes have also been sited as a risk factor. What constitutes a high volume, however, has not been well defined. Approximately 1.5 L of saliva and 3 L of gastric secretions are produced daily and the stomach empties about a third to one-half of its contents hourly (49). Therefore, residual volumes approaching 500 cc/4 hr should be safe. To minimize the risk of aspiration, the head of the bed should remain elevated, oral care should be diligent,

Indications and Contraindications for Enteral Nutrition

Table 7 Factors Posing a Major Risk of Aspiration

Hospitalized patients who cannot adequately meet their nutritional goals by oral intake alone for a period of seven days should be considered for enteral supplementation. Additionally, early enteral nutrition should be considered for all acutely ill patients, such as patients with major torso trauma, chronically malnourished patients, and patients with limited physiologic reserve.

Endotracheal intubation Decreased level of consciousness Neuromuscular disorders or anatomical abnormalities of the upper gastrointestinal tract Recent stroke Recent major abdominal surgery Persistently high gastric residual volumes

Chapter 3: Surgical Nutrition

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tolerance should be regularly assessed, narcotics minimized, electrolyte abnormalities corrected, and glucose control optimized. Numerous strategies have been employed to identify occurrences of aspiration. Use of food, drug, and cosmetic (FD&C) blue No. 1 and methylene blue as means of visual detection for aspiration has been associated with clinically significant toxicity and this practice is discouraged (50). Furthermore, the presence of glucose in the tracheal aspirate has been found to be neither a reliable nor specific indicator of enteral feeding aspiration.

more partially digested macronutrients or combinations of nutrients and can be absorbed in patients with compromised gastrointestinal tracts), and modular formulas (which are composed of individual nutrients or combinations of nutrients, but are nutritionally incomplete and intended for use as supplements or in combination with other products). Unfortunately, with the exception of the immune-enhancing formulas, very little comparative data exists to guide clinicians in selecting the most appropriate formula. A recommended formula selection is reviewed in Table 9.

Obtaining Enteral Access

Administration of Feeds

Access can be divided into gastric (and duodenal) and jejunal with push, endoscopic, radiologic, and surgical options available (Table 8). For patients to be fed through an intragastric approach, a soft, nonsump nasogastric tube can be placed. For patients known to require long-term feeding access, a percutaneous endoscopic gastrostomy (PEG) can be obtained. For those patients identified as candidates for jejunal feeds, access can be obtained at the time of initial laparotomy, or at subsequent laparotomy if damage control is initially performed. The needle catheter jejunostomy (NCJ) is the preferred method of access and a commercially available kit containing a silastic 7 French catheter is available. Patients not undergoing laparotomy could have a ‘‘push’’ nasojejunal tube (Corpak Medsystems, Wheeling, Illinois) placed. If this is unsuccessful, an endoscopically placed nasojejunal tube can be placed through the biopsy channel of a flexible endoscope that has been advanced into the duodenum (51). Nasojejunal feeding may be done indefinitely, but if the need for long-term access becomes apparent, a PEG with a jejunal extension limb (PEG-J) can be placed.

Once enteral access has been obtained, feeds are begun at 15 cc/hr of full-strength formula and advanced by 15 cc/hr every 12 hours to a set goal of 60 cc/hr. To assure tolerance, this rate is maintained for 24 hours and then advanced by 15 cc/hr every 12 hours to a patient-specific targeted goal.

Formula Selection Many of the early clinical trials were performed using elemental formulas that were low in fat and surmised to be better tolerated. However, more recent studies suggest other formulas to be equally well tolerated. The numerous available formulas may be categorized into polymeric formulas (which contain nutrients in high-molecular-weight form, moderate fat loads, and require normal digestive and absorptive ability), predigested formulas (which contain one or

Complications of Enteral Nutrition Overview of Complications Technical complications that can be associated with the administration of enteral nutrition include aspiration, bowel perforation, clogged tubes, and tube malposition/dislodgement. Tube clogging can be prevented by routine saline irrigation of the tube and by administering only liquid medications through smaller bore tubes such as NCJs. Functional complications are the gastrointestinal manifestations frequently associated with intolerance and include emesis, nausea/ vomiting, cramping pain or distention, and diarrhea.

NCJ-Related Complications NCJs, although an ‘‘invasive’’ technique, are a safe and effective means to deliver jejunal feeds. Myers et al. reported only a 1% incidence of major complications and a 1.7% incidence of minor complications in a large study examining the safety of NCJs in patients undergoing major elective and emergency abdominal operations (52). The jejunostomyrelated complication rate in trauma patients has been reported to be slightly higher, with a major complications rate of 4% (53). However, the majority of these complications (10%) occurred in patients with a standard, open

Table 9 Formula Selection for Delivery of Enteral Nutrition Table 8 Options for Enteral Access Gastric tubes Manual Endoscopic Radiographic Surgical Jejunal tubes Manual Endoscopic

Radiographic

Nasogastric/nasoduodenal PEG Endoscopically placed nasoenteric (nasoduodenal tube) Fluoroscopy-guided nasoenteric tube PRG Open: Stamm, Witzel, or Janeway gastrostomy Laparoscopic gastrostomy ‘‘Push’’ nasojejunal tube PEJ PEG/JET Endoscopically placed nasojejunal tube PRG/PRJ PRG/JET

Abbreviations: JET, jejunal extension tube; PEG, percutaneous endoscopic gastrostomy; PRG, percutaneous radiographic gastrostomy; PEJ, percutaneous endoscopic jejunostomy; PRG/PRJ, percutaneous radiographic gastrostomy/jejunostomy.

Immune-enhancing diet Patients who have sustained major torso trauma, undergone major upper gastrointestinal surgery, or have limited physiologic reserve and who are at known risk for septic complications and multiple organ failure Polymeric high-protein formula Patients who do not meet the criteria for an immune-enhancing diet, have normal gut function, and are believed to have increased nitrogen requirements due to major injury, illness, or surgery. A modular protein component may be used in addition to the polymeric high-protein formula for use in the morbidly obese patient or in any patient that requires protein supplementation Elemental formula Patients who are intolerant to a polymeric formula or are acutely ill and who have not received enteral feeds for prolonged period of time Renal failure formula A concentrated, reduced electrolyte formulation is selected for use only in patients requiring intermittent hemodialysis. Often a modular protein component is used in addition to the commercially available renal formula to meet the increased nitrogen demands of the critically ill patient

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jejunostomy (typically a 14 French catheter), rather than with a NCJ (5–7 French, 2% incidence of complications). Major complications reported included volvuli with infarction, small bowel perforation, intraperitoneal leaks, and nonocclusive small bowel necrosis.

Nonocclusive Bowel Necrosis A rare but devastating complication of bowel necrosis has been associated with the administration of enteral feeds. The clinical presentation is similar to that of neonatal necrotizing enterocolitis, though the consistent association with enteral nutrition suggests that the inappropriate administration of nutrients into a dysfunctional gut plays a pathogenic role. The incidence is less than 1%, but the mortality frequently exceeds 50% (54,55). Most cases of nonocclusive bowel necrosis occur in a delayed fashion in critically ill patients with a complicated course (pneumonia, sepsis, renal failure, etc.) that requires progressively higher acuity care. Gastrointestinal signs and symptoms tend to occur late and, as a result, clinical monitoring fails to detect this entity early in its course. Computed tomography scan may reveal pneumatosis intestinalis or thickened bowel. Although most cases will require exploratory laparotomy, less advanced presentations may be managed with cessation of feeds, broadspectrum antibiotics, and aggressive fluid resuscitation.

Enteral Nutrition Protocol Rationale for an Enteral Nutrition Protocol An enteral feeding protocol can assist in providing a systematic, evidence-based approach to enteral nutrition and in minimizing complications. Assessing tolerance parameters are an important part of any enteral protocol. Frequently identified indicators of intolerance are vomiting, abdominal distention or cramping/tenderness, diarrhea, and high nasogastric tube output (56). Symptoms can be graded as mild, moderate, or severe. Mild symptoms of intolerance, such as mild abdominal distention or diarrhea, can just be monitored by repeat physical examinations with no change in the current rate of feeding. Moderate symptoms are managed based on the particular symptom. For distention, enteral feeds should be stopped and the patient assessed for evidence of a mechanical obstruction. If distention remains moderate, an elemental formula should be considered. Moderate diarrhea can be managed by maintaining but not increasing the current feeding rate and repeat examinations. Finally, for severe distention, enteral feeds should be stopped, intravenous hydration begun, and the possibility of nonocclusive bowel necrosis should be considered. For severe diarrhea, tube feeds can be reduced and the patient should be evaluated for possible Clostridium difficile infection. Vomiting is managed by ensuring adequate gastric decompression and either stopping gastric feeds or decreasing the tube feed infusion rate by half for jejunal feeds. Additionally, high nasogastric output in patients fed into the small bowel can be managed by verifying postpyloric placement of the feeding tube and checking the nasogastric aspirate for glucose. Any amount of glucose is considered abnormal and enteral feeds should be withheld. In general, enteral feeds should be discontinued if vasopressors are instituted.

Results of an Enteral Nutrition Protocol The incidence of enteral tolerance while using a protocol was analyzed in a prospective multi-institutional study

(57). Early tolerance (during advancement of enteral feeds to a goal of 60 cc/hr) was good in 84% (41/49) of patients and moderate in 16% (8/49). No patients experienced poor tolerance or complete intolerance. Late tolerance (after standard goal rate was met) was good in 80% (39/49), moderate in 16% (8/49), and poor in 4% (2/49) of patients. The site of feeding (gastric vs. jejunal) was not dictated by the protocol. Moderate intolerance was primarily due to high gastric output in patients fed via the stomach. All patients were successfully maintained on early enteral nutrition using this standardized protocol.

Specialized Enteral Diets Formulas with reduced carbohydrate and increased fat loads are available for use in patients with diabetes to potentially improve glycemic control. These products have not, however, undergone PRCT to demonstrate superior outcome in ICU patients. The use of standard high-protein formulas in an isocaloric or hypocaloric load, combined with aggressive insulin therapy, may be the most effective treatment for insulin resistance in the stressed, diabetic patient as opposed to carbohydrate restriction. Additionally, gastric feedings in the diabetic patient with gastroparesis may be associated with delayed gastric emptying and increased risk of aspiration, especially when high in fat content. There is one PRCT that demonstrated superior outcome, as demonstrated by reduced days on the ventilator, reduced ICU length of stay, and decreased incidence of organ failure in patients with adult respiratory distress syndrome (ARDS) when provided a high omega-3 fatty acid enteral product versus a high omega-6 ‘‘pulmonary’’ formula (58). However, the control diet was not the standard of care and may worsen ARDS. High omega-6 fatty acids increase inflammation and production of lipid mediators, which can worsen V/Q mismatch and thus worsen oxygenation in these patients. Although an enteral diet high in omega-3 fatty acids may be beneficial in early ARDS (59), prior to recommending such a diet, additional studies need to be performed.

TOTAL PARENTERAL NUTRITION Indications for TPN Whenever possible, the gastrointestinal track should be utilized for nutritional support. Table 10 reviews the basic indications for the administration of TPN. In general, TPN should not be started for at least seven days postoperatively if the patient is well nourished at baseline. Critically ill patients should be started on TPN if they are unable to achieve adequate caloric intake by postoperative day 6. Unlike with enteral feeding, there is no benefit to early TPN. Patients with persistent ileus, bowel obstruction, short gut, high-output fistulas, and malabsorption, may all benefit from TPN. Additionally, patients unable to tolerate enteral Table 10 Indications for TPN Persistent or progressive ileus Bowel obstruction Massive bowel resection refractory to enteral diet High-output fistula refractory to elemental diet Malabsorption High risk for nonocclusive bowel necrosis if fed enterally (shock resuscitation, a-agonists, persistent severe distention, or cramping) Documented intolerance to enteral nutrition Abbreviation: TPN, total parenteral nutrition.

Chapter 3: Surgical Nutrition

nutrition or who are at high risk for nonocclusive bowel necrosis (hypoperfusion or high vasopressor requirements) may benefit from TPN.

Table 11 Trace Element Deficiencies Trace element Iron Zinc

Preoperative and Perioperative TPN It is well documented that malnourished patients are at an increased risk for septic complications, problems with wound healing, longer hospital stays, and increased mortality (60). The unproved contention is that preoperative TPN can improve nutritional status and thereby reduce postoperative morbidity and mortality. However, studies evaluating preoperative TPN and outcome are variable. Results of randomized trials and a meta-analysis suggest that preoperative TPN for 7 to 10 days in severely malnourished patients may be beneficial in reducing the rates of infectious complications (61–65). For the mild to moderately malnourished patients, however, the risks of preoperative TPN (increase in septic morbidity) appear to outweigh the potential benefits. One of the confounding variables in preoperative TPN trials is that many of the patients were undergoing resection of a gastrointestinal cancer. Cancer patients in general, and particularly those with gastrointestinal malignancies, have cancer-associated weight loss, which can be very difficult to reverse. Prospective randomized trials of patients undergoing gastrointestinal surgery also failed to demonstrate any benefit, with the TPN group experiencing higher complication rates. Thus, routine postoperative TPN is not indicated and should be reserved for patients who cannot resume a diet by postoperative day 6 (66).

Initiation of TPN Components of TPN Components of TPN include (i) dextrose, (ii) fatty acids, (iii) amino acids, (iv) electrolytes, (v) vitamins, (vi) trace minerals, and (vii) fluids. Protein is provided based on grams per kilogram body weight, lipid is provided to deliver approximately 10% of total kilocalories needed, and dextrose provides the balance of kilocalories. Dextrose monohydrate (caloric density 3.4 kcal/g) is the carbohydrate. Fat emulsions (caloric density 2.0 kcal/cc of 20%) made from either soybean oil or a mixture of soybean oil and safflower oil provide fat calories and are the source of essential fatty acids (linoleic, linolenic, and arachidonic acids). Protein (caloric density 4 kcal/g) is provided as crystalline amino acids. Standard amino acid solutions contain a balance of essential and nonessential amino acids. The electrolyte cations, which include sodium, potassium, magnesium, phosphorus, and calcium, are admixed into the TPN solution using one of several anions. Acid–base status may be affected by the amount of chloride or acetate used in providing sodium and potassium. The concentrations of calcium and phosphorus are limited to avoid precipitation of a calcium phosphate salt. Multivitamin products that meet American Medical Association recommendations contain vitamins A, C, D, and E and the B vitamins, including folate, but not vitamin K, which must be added separately. A multi–trace mineral product is added to provide copper, chromium, manganese, zinc, and selenium. Symptoms of trace mineral deficiencies are reviewed in Table 11. TPN solutions are hyperosmolar and must be delivered through a large-lumen vein. When central access is unavailable or undesirable, peripheral parenteral nutrition (PPN) with a dilute solution (less than 800 mOsm/L) may be delivered through a peripheral vein for 7 to 10 days. Infusion sites must be rotated often to

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Copper Chromium Selenium Manganese Molybdenum

Symptom of deficiency Anemia Maculopapular rash, poor wound healing, cutaneous anergy, alopecia Anemia unresponsive to iron, pancytopenia Sudden glucose intolerance during TPN Proximal neuromuscular weakness and cardiac failure Weight loss, altered hair pigmentation, nausea Nausea/vomiting, tachycardia, central nervous system disturbances

Abbreviation: TPN, total parenteral nutrition.

reduce the incidence of thrombophlebitis. This therapy is best reserved for younger patients with easy peripheral access. PPN is seldom utilized. The combination of amino acids, dextrose, and lipids is referred to as total nutrient admixture (TNA) or a 3-in-1 solution. Typically an 8.5% or 10% amino acid solution is employed, although a 15% solution is available for patients requiring fluid restriction, such as those with congestive heart failure, renal failure, or hepatic failure. Dextrose 50% is the usual carbohydrate solution, although 70% solution may be used to restrict volume. Lipids are available in 10%, 20%, and 30% and are needed to prevent essential fatty acid deficiency. Ten percent of total kilocalories will accomplish this goal. Additional lipids can be given to provide an alternative calorie source in patients intolerant of high glucose loads. If lipids are administered separately, they must be infused for no greater than over 12 hours to prevent bacterial growth. Lipids are stable in TNA for 24 hours because the osmolarity and pH are less favorable to bacterial growth. The following is an example of TPN in an 80 kg male patient with significant injuries requiring 2400 kcal (30 kcal/kg) and 140 g protein (1.75 g/kg). A typical institutional TPN form is shown in Table 12.

Calculations (see Table 13): 1. 2.

Establish the kilocalories and protein desired: 2400 kcal, 140 g protein. Select the appropriate amino acid formula and quantity: using 10% amino acids 10 gm=100 mL 140 gm ¼ 1400 mL

3.

Calculate 10% of kcal as lipid emulsion: using 20% lipids 10% 2400 ¼ 240 kcal 240 kcal 1 mL=2 kcal ¼ 120 mL

4.

Add the kcal from amino acids and fat and subtract from goal:

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acids ¼ 140 g protein 4 cal=g ¼ 560 kcal kcal from fat ¼ 240 kcal goal kcal ðamino acid þ fatÞ kcal ¼ 2400 ð800 þ 240Þ ¼ 1600 kcal

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Part One: General Considerations

Table 12 Example of a Standard TPN Order Sheet

This is the amount of dextrose kcal needed. To determine the volume, 1600 kcal=3:4 kcal=g glucose ¼ 470 g glucose 470 g glucose=50 g=100 mL ¼ 940 mL The patient’s electrolyte profile is reviewed. If it is relatively normal, infusion is started with standard electrolyte requirements.

Complications Complications of TPN can be divided into technical, infectious, and metabolic. Administration of TPN requires central venous access due to its hypertonicity. Administration

through a large, central vein allows rapid dilution and therefore prevention of venous sclerosis and thrombophlebitis, which will occur with peripheral venous administration. The insertion of a central venous line, however, is not without risk. More than 15% of patients receiving central lines have some type of complication (67). Technical complications range from injury to major veins, arteries, or nerves, and violation of the pleural space. Complications can be minimized by strict observation of well-established guidelines for insertion, including adequate supervision. If a major artery is punctured, the needle should be withdrawn and pressure applied. If the vessel has been cannulated and it is unclear into which vessel (artery or vein) the catheter has been inserted, the catheter should be transduced. A venous tracing should be seen and the measured pressure should be approximately 4 to 10 mmHg.

Chapter 3: Surgical Nutrition

Table 13 Sample TPN Order Nonstandard TPN solution Base solution Standard amino acid (10 g/100 mL) Dextrose 50% (50 g/100 mL:3.4 kcal/g) Volume of base solution to be delivered in 24 hr Electrolytes Sodium chloride Sodium phosphate Potassium acetate Calcium gluconate Magnesium sulfate Multivitamins Trace elements Fat emulsion 20% (1 kcal/mL) Infusion rate

1400 mL 940 mL 2300 mL

35 mEq/L 8.3 mEq/L 35 mEq/L 4.16 mEq/L 3.3 mEq/L 10 mL 1 mL 120 mL 101 mL/hr

Abbreviation: TPN, total parenteral nutrition.

The pathogenesis of infectious complications most commonly stems from colonization of the skin and catheter hub. Short-term, noncuffed catheters (such as single- or triple-lumen catheters) become colonized from skin flora that migrate from the insertion site along the external surface of the catheter to the catheter tip and lead to catheterrelated bloodstream infections. Long-term catheters such as cuffed, tunneled, silicone catheters (Hickmans or Broviacs) or implanted catheters (ports) become colonized as bacteria migrate through the lumen of the hub, which becomes contaminated by skin flora. Other less frequent sources of contamination include infusion of contaminated solutions or hematogenous seeding from a distal site of infection. Preventative measures can be broken down into three categories: (i) catheter insertion, (ii) catheter care, and (iii) catheter removal. Important components of catheter insertion include skin preparation (chlorhexidine is more efficacious than alcohol or povidone iodine) and the use of

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maximal sterile barriers (68). Although it is commonly believed that multiple-lumen catheters have a higher rate of catheter-related sepsis compared to single-lumen catheters, randomized studies have not confirmed this practice (69). Recent randomized trials indicate that catheter-related sepsis can be reduced by the use of antibiotic- or antisepticbonded catheters (70). However, these catheters are considerably more expensive, can be associated with anaphylaxis, and have the potential to cause resistant organisms. Therefore, they should be used in select patients whose catheters are going to be in place for extended periods of time. With regard to catheter care, dressings may be either a transparent, semipermeable, polyurethane dressing or gauze. Insertion site antibiotic or antiseptic ointments are not recommended because of the potential to promote antibiotic resistance and fungal colonization. A meta-analysis of 12 randomized trials of catheter replacement did not support scheduled exchange of catheters over a guide wire or scheduled, routine replacement at a new site (71). Metabolic complications can include hyper- or hypoglycemia, electrolyte abnormalities, acid/base abnormalities, liver function abnormalities, and trace mineral deficiencies (Table 14) (72). Critical illness is accompanied by increased plasma counter-regulatory hormone levels, which have multiple effects on glucose homeostasis. The end result is hyperglycemia with resistance to insulin. Other factors that contribute include obesity, inflammatory cytokines associated with the SIRS (such as TNFa, IL-1, IL-2, and IL-6), advanced age, exogenous steroid or catecholamines, increased free fatty acids, and nutritional support (parenteral route greater than enteral route). The resulting hyperglycemia can adversely affect outcome through several mechanisms, including glycosuria with inappropriate diuresis, increase risk of infection by impairing neutrophil and immunoglobulin function, and exacerbation of cerebral edema. Van Den Berghe et al. (73) have recently demonstrated in a prospective randomized fashion a reduction of mortality in critically ill surgical patients, from 8% to 4.6%,

Table 14 Metabolic Complications Associated with TPN Abnormality Electrolyte Hyperkalemia Hypokalemia Hyperphosphatemia Hypophosphatemia Hypercalcemia Hypocalcemia Hypermagnesemia Hypomagnesemia Glucose Hyperglycemia Hypoglycemia Amino acids Hypercholeremia Metabolic acidosis Fatty acid deficiency Bleeding Abnormal liver functions

Etiology

Correction # " # " # "

Renal insufficiency/failure Inadequate intake, diuresis, diarrhea, refeeding Renal insufficiency/failure Inadequate administration, refeeding Excessive administration Inadequate administration, hypoalbuminemia, refeeding, rapid phosphorus repletion Excessive administration, renal failure Inadequate intake, diuresis, refeeding

# Administration " Administration

Excessive administration, sepsis, inadequate secretion of insulin Abrupt cessation of TPN, liver dysfunction

Strict glucose control with exogenous insulin, consider reducing glucose calories Taper TPN rate, administer 5% or 10% dextrose

Excessive chloride administration Excessive chloride administration Sepsis, inadequate perfusion, renal failure Inadequate administration Vitamin K deficiency Excessive fat or glucose administration, amino acid imbalance

# Administration Administer acetate Reassess patient " Administration " Administration Reassess patient

Abbreviation: TPN, total parenteral nutrition.

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Administration Administration Administration Administration Administration Administration

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Part One: General Considerations

when glucose levels were strictly controlled between 80 and 110 mg/dL versus conventional therapy (120–180 mg/dL). In a follow-up analysis, the same investigators demonstrated that the reduction in critical illness polyneuropathy, bacteremia, inflammation, and mortality was related to the lowering of blood glucose levels and not the amount of infused insulin per se (74). These data strongly support the maintenance of normoglycemia. The refeeding syndrome is a dramatic example of a metabolic complication that can be associated with TPN administration. It occurs with rapid and excessive feeding of patients with severe malnutrition due to conditions such as starvation, alcoholism, delayed nutritional support, anorexia nervosa, hyperemesis gravidarum, and massive weight loss after bariatric surgery (75). As a result of ion fluxes into the cell with refeeding, serum phosphate, magnesium, potassium, and calcium levels can drop rapidly. In the case of blunted basal insulin secretion, severe hyperglycemia may arise. Symptoms include cardiac arrhythmias, confusion, respiratory failure, and even death. This can be prevented by initiating TPN at a rate lower than the required goal, primarily by reducing dextrose calories. Exogenous insulin is frequently required.

patients have poor response to chemotherapy, radiation, and surgery as well as increased morbidity and mortality. When feasible, use of the gastrointestinal tract is the preferable mode of feeding. Immune-enhanced enteral diets in patients with gastrointestinal cancers have been shown to decrease postoperative infections (78,79). A recent metaanalysis that carried out a separate analysis of patients with gastrointestinal cancer, similarly demonstrated a reduction in major infectious complications but not death in the postoperative period (26). Traditionally, concern exists that perioperative TPN may promote tumor growth, i.e., feed the tumor not the patient. However, many studies supporting this theory were performed in animals whose life span is much shorter than humans (80). Thus, a 10-day course of TPN would represent a far greater segment of an animal’s life. Furthermore, the tumor size in these studies was large, representing up to 30% to 40% of the animal’s body weight. Nonetheless, the use of TPN in this patient population offers little benefit and may increase infection rates (81). Severely malnourished patients receiving a bone marrow transplant may be one subset of patients who can benefit from perioperative TPN.

Monitoring TPN

Short Gut

This is done to (i) determine the efficacy of the TPN therapy; (ii) determine changes in metabolic status (stress level); and (iii) detect complications associated with TPN. Measurements of efficacy in the acute-care setting include weight, constitutive protein status (e.g., albumin, transferrin, and prealbumin), nitrogen balance, and wound healing. Metabolic status should be viewed first from the clinical perspective. The presence of SIRS, infection, or high ventilation requirements all indicate a high metabolic rate. Metabolic status can be further assessed by laboratory variables that evaluate substrate tolerance (e.g., blood glucose and serum triglyceride concentrations) as well as protein catabolic rate (24-hour UUN).

Specialized Amino Acid TPN Solutions These formulas are designed to meet organ-failure specific requirements and include high-branched-chain, hepatic failure (low aromatic amino acid), and renal failure (high essential amino acids) formulations. The use of these specialty formulas remains controversial because of the expense and lack of outcome data. Studies comparing high-branchedchain amino acid solutions with standard formulas in stressed patients have shown improvements in nitrogen retention, constitutive protein levels, and immune function, but have failed to demonstrate reduced morbidity or mortality (76). One recent trial in Europe was able to demonstrate a reduced mortality in septic patients receiving a high-branched-chain formula (77). The use of hepatic and renal formulas has also not shown any proven benefit.

DISEASE-SPECIFIC NUTRITION Nutrition and Cancer Many forms of cancer are associated with weight loss and wasting or cachexia. Intake can be impaired due to the disease process, physical anomalies related to surgery or to the cancer itself, side effects of therapy, and psychosocial factors. Utilization of nutrients may be altered by malabsorption, cytokine activity, or the preponderance of inefficient cycles for energy metabolism. Malnourished cancer

Mesenteric infarction, Crohn’s disease, radiation enteritis, tumors, and trauma are the leading causes of short gut in adults (82). Factors that influence the management of short bowel syndrome include (i) the extent of resection; (ii) the site of resection; (iii) the presence or absence of the ileocecal valve; (iv) the residual function of the remaining small bowel; (v) the adaptive capacity of the intestinal remnant; (vi) the primary disease that precipitated the bowel loss; and (vii) the amount and the activity of the residual disease in the remnant (83). The average adult has 25 cm duodenum, 240 cm jejunum, and 360 cm ileum (84). In adults, the minimal length of small intestine (distal to the Ligament of Treitz) required for adequate digestive function is approximately 75 cm, although there are interindividual differences (85). For lesser amounts, patients will require long-term TPN. Additionally, patients requiring TPN three months after surgical resection can be considered as having short gut regardless of the extent of resection. The small bowel is the site of absorption for a variety of nutrients and electrolytes. Duodenectomy results in malabsorption of calcium, folate, and iron. Although the proximal jejunum is the usual site for protein, carbohydrate, and fat absorption, jejunectomy usually results in no major change in macronutrient and electrolyte absorption because the ileum can take over this role (86). Because the jejunum is the site of hormones that inhibit gastric secretion such as gastric inhibitory peptide and vasoactive intestinal polypeptide, gastric hypersecretion begins within 24 hours after jejunal resection. This produces a high sodium load in the stomach and reduces pH downstream, thus inactivating digestive enzymes and increasing diarrhea. The ileum plays an important role in slowing intestinal transit and concentrating luminal contents. Both vitamin B12 and bile salts are normally absorbed from the ileum. Following resection, the liver increases production of bile salts to compensate for the loss of absorption. However, this production can never be fully compensated for and bile salt depletion occurs, leading to steatorrhea and cholelithiasis. Cholestyramine is sometimes employed to treat the ensuing diarrhea and cholecystectomy is commonly required within two years after

Chapter 3: Surgical Nutrition

bowel resection. Steatorrhea promotes calcium binding to fatty acids rather than oxalate and allows more oxalate to be absorbed, leading to oxalate stones. Three stages of intestinal adaptation after resection have been identified (84): Stage I is characterized by fluid and electrolyte loss and lasts for approximately two weeks. It is treated with electrolyte replacement, an H2 blocker (to minimize hypersecretion), and parenteral nutrition. Hypersecretion of gastrin may result in peptic ulcer disease, gastroesophageal reflux disease, and proximal small bowel inflammation, so that H2 antagonists are a mainstay of therapy after intestinal resection. Stage II is a period of adaptation where oral intake begins and lasts from a few months up to one year. Feedings are advanced as stool and/or ostomy output becomes manageable. Stage III is maximal adaptation where normal home life may resume, although some patients may still require TPN. The residual small bowel as well as the colon undergo dilatation, lengthening, and mucosal proliferation along the course of adaptation. Experiments in rats have demonstrated superior adaptation in animals fed TPN and complex enteral diets as opposed to elemental diets (87). Aggressive use of enteral nutrition is thought to maximize gut adaptation (88). Although somatostatin may slow intestinal transit and reduce fluid and electrolyte loss acutely, it may also decrease enteroglucagon release, which exerts beneficial effects in bowel adaptation after resection. Octreotide is a synthetic analog of somatostatin with a longer half-life (three hours vs. three minutes). Octreotide reduces splanchnic blood flow and inhibits gastric, pancreatic, and small bowel secretions. These mechanisms may limit intestinal adaptation after bowel resection and reduce dietary fat digestion. Bowel output is reduced due to decreased secretions rather than improved absorption. Octreotide is generally recommended for use in patients with severe diarrhea/ostomy output (greater than 4 L daily) that is intractable to antidiarrheal agents. The use of antimotility agents may prove helpful in this circumstance. Although glutamine has been recommended, its role in morphological adaptation after bowel resection remains to be elucidated. Administering probiotics, the addition of live bacteria to the gastrointestinal tract, may also be beneficial in short gut. Healthy bacteria may be altered due to antibiotics, antimotility agents, H2 blockers, proton-pump inhibitors, and narcotics. Growth of healthy flora decreases growth of enteropathogens that may adhere to the mucosa and initiate inflammatory or immune sequelae (89). Structured programs have emerged that are geared toward decreasing TPN dependence after intestinal resection (90). Therapy consists of TPN (when necessary), peptide-based enteral nutrition, a low-fat, low-oxalate, high-protein, high–dietary fiber oral diet, and enteral glutamine in dipeptide form. Recombinant human growth hormone is provided for a specified period. Long-term follow-up of patients after intestinal resection should consist of monitoring sodium, potassium, chloride, zinc, copper, magnesium, iron, B12, folic acid, and fat-soluble vitamins. Zinc losses in diarrhea are substantial and may require supplementation above the quantity provided by intravenous multi–trace element products. Intramuscular vitamin B12 should be given if more than 60 cm of terminal ileum is resected. Chronic complications of short bowel syndrome include liver failure, gallstones, nutrient deficiencies, osteopenia, catheter-related sepsis, and small bowel bacterial overgrowth. Surgical approaches to increase functional bowel, including transplantation, remain an option.

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Enterocutaneous Fistula Abdominal operations account for over 80% of enterocutaneous fistulae. The remainder are due to Crohn’s, cancer, radiation, and infection. The nutritional management depends on the location of the fistula, the health of the bowel distal to the fistula, and the amount of fistula output. The advent of TPN in the 1960s greatly reduced mortality from fistulae. Spontaneous closure depends on location, comorbid factors, and nutritional state. Ninety percent of fistulae that are likely to close spontaneously will do so in the first 30 days (91). If fistulae are in the proximal bowel, full enteral feeds may be delivered distally.

Pancreatitis Acute pancreatitis induces severe hypercatabolism and without exogenous nutritional support, acute protein malnutrition can quickly occur. Due to concerns that enteral feeding would stimulate further secretion of pancreatic enzymes and worsen autolysis, TPN has been the mainstay of nutritional therapy. However, recent studies have indicated that enteral feeding into the jejunum in patients with acute pancreatitis is both safe and effective. Initial trials of enteral support recommended low-fat, elemental formulas (92–95), again due to concerns over pancreatic stimulation. However, Duerksen et al. demonstrated that, in patients who underwent partial pancreatectomy with exteriorization of pancreatic stents, there was no statistically significant difference in pancreatic secretion between polymeric and elemental formulas (96). When compared with TPN, enteral nutrition is associated with an attenuation of the acute-phase response (97), reduced septic complications (94), and markedly decreased cost.

SUMMARY Protein-calorie malnutrition has become increasingly recognized in hospitalized patients and has been shown to be present in as many as 50% of patients requiring surgical procedures. Understanding the metabolic response to starvation and surgical stress, and how a suboptimal nutritional environment can adversely affect this response is mandatory if postoperative morbidity and mortality are to be lessened. Fortunately, the last several decades have unraveled an enormous amount of new knowledge regarding the role that malnutrition plays in altering fundamental metabolic pathways that are essential to good patient care. Further, the ability to provide nutritional support by way of enteral or parenteral routes has enabled the provision of optimal nutritional therapy in even the patient with profound malnutrition requiring surgical intervention. Such advances have greatly minimized the attendant negative effects that suboptimal nutrition would otherwise have on patient outcome and survival following operation.

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Part One: General Considerations 23 days after the onset of peritonitis. Ann Surg 1998; 28: 146–158. Moore EE, Jones TN. Benefits of immediate jejunal feeding after major abdominal trauma – a prospective randomized study. J Trauma 1986; 26:874–881. Moore FA, Moore EE, Jones TN. TEN versus TPN following major abdominal trauma-reduced septic morbidity. J Trauma 1989; 29:916–922. Kudsk KA, Croce MA, Fabian TC, et al. Enteral versus parenteral feeding: effects on septic morbidity following blunt and penetrating abdominal trauma. Ann Surg 1992; 215:503–511. Moore FA, Feliciano DV, Andrassy RJ, et al. Early enteral feeding, compared with parenteral, reduces postoperative septic complications – the results of a meta-analysis. Ann Surg 1992; 216:172–183. Moore FA, Sauaia A, Moore EE, Haenel JB, Burch JM, Lezotte DC. Postinjury multiple organ failure: a bimodal phenomenon. J Trauma 1996; 40:501–512. Moore FA. Effects of immune-enhancing diets on infectious morbidity and multiple organ failure. J Parenter Enteral Nutr 2001; 25:S36–S42. Windsor JA, Hill GL. Weight loss with physiologic impairment: a basic indicator of surgical risk. Ann Surg 1988; 207(3):290–296. Heymsfield SB, McManus C, Stevens V, Smith J. Muscle mass: reliable indicator of protein-energy malnutrition severity and outcome. Am J Clin Nutr 1983; 35:1192–1199. Lexell J, Taylor CC, Sjostrom M. What is the cause of the aging atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateralis muscle from 15- to 83-year old men. J Neurol Sci 1988; 84:275–294. Griffiths RD. Muscle mass, survival and the elderly ICU patient. Nutrition 1996; 12:456–458. Finn PJ, Plan LD, Clark MA. Progressive cellular dehydration and proteolysis in critically ill patients. Lancet 1996; 347:654–656. Rodriguez DJ, Clevenger FW, Osler TM, Demarest GB, Fry DE. Obligatory negative nitrogen balance following spinal cord injury. J Parenter Enteral Nutr 1991; 15:319–322. McClave SA, Spain DA, Slolnick JL, et al. Achievement of steady state optimizes results when performing indirect calorimetry. J Parenter Enteral Nutr 2003; 27:16–20. Wooley JA, Sax HC. Indirect calorimetry: application to practice. Nutr Clin Pract 2003; 18:434–439. Gibbs J, Cull W, Henderson W, Daley J, Hur K, Khuri SF. Preoperative serum albumin level as a predictor of operative mortality and morbidity: results from the National Surgical Risk Study. Arch Surg 1999; 134:36–42. Daley J, Khuri SF, Henderson W, et al. Risk adjustments of the postoperative morbidity rate for the comparative assessment of the quality of surgical care. Results of the National Veterans Affairs Surgical Risk Study. J Am Coll Surg 1997; 185: 325–338. Khuri SF, Daley J, Henderson W, et al. The Department of Veterans Affairs NSQIP. The first national, validated, outcomebased risk-adjusted and peer-controlled program for the measurement and enhancement of the quality of surgical care. Ann Surg 1998; 228:491–507. Kudsk KA, Tolley EA, DeWitt RC, et al. Preoperative albumin and surgical site identify surgical risk for major postoperative complications. J Parenter Enteral Nutr 2003; 27:1–9. Smith-Choban P, Weireter LJ, Maynes C. Obesity and increased mortality in blunt trauma. J Trauma 1991; 31:1253–1257. Smith-Choban P, Burge JC, Scales D. Hypoenergetic nutrition support in hospitalized obese patients: a simplified method for clinical application. Am J Clin Nutr 1997; 66:546–550. Heyland DK, Novak F, Drover JW, Jain M, Su X Suchner U. Should immunonutrition become routine in the critically ill patient? JAMA 2001; 286:944. Marik PE, Zaloga P. Early enteral nutrition in acutely ill patients: a systematic review. Crit Care Med 2002; 29:2264–2270. Heys SD, Walker LG, Smith I, Eremin O. Enteral nutrition supplementation with key nutrients in patients with critical illness and cancer. Ann Surg 1999; 229:467–477.

27. Beale RJ, Bryg DJ, Bihari DJ. Immunonutrition in the critically ill: a systematic review of clinical outcome. Crit Care Med 1999; 27:2799. 28. Novak F, Heyland DK, Avenell A, Drover JW, Su X. Glutamine supplementation in serious illness: a systematic review of the evidence. Crit Care Med 2002; 30:2002–2029. 29. Houdijk APJ, Rijnsburger ER, Jansen J, et al. Randomized trial of glutamine-enriched enteral nutrition on infectious morbidity in patients with multiple trauma. Lancet 1998; 352: 772–776. 30. Barbul A, Lazarou SA, Efron DT. Arginine enhances wound healing and lymphocyte immune responses in humans. Surgery 1990; 108:331–336. 31. Suchner U, Heyland DK, Peter K. Immune-modulatory actions of arginine in the critically ill. Br J Nutr 2002; 87(suppl 1):S121. 32. Bower RH, Cerra FB, Bershadsky B, et al. Early administration of a formula (Impact) supplemented with arginine, nucleotides, and fish oil in intensive care patients: results of a mulitcenter, prospective, randomized, clinical trial. Crit Care Med 2001; 23:436–449. 33. Bruzzone R, Radrizzani D. Early enteral immunonutrition in patients with severe sepsis. Results of an interim analysis of a randomized multicenter clinical trial. Intensive Care Med 2003; 29:834–840. 34. Alexander JW, Saito H, Ogle CK, Trocki O. The importance of lipid type in the diet after burn injury. Ann Surg 1986; 204:1–8. 35. Van Buren CT, Kulkarni A, Fanslow WC, Rudoph FB. Dietary nucleotides, a requirement for helper/inducer T lymphocytes. Transplantation 1985; 40:694–697. 36. Khalili TM, Navarro A, Middleton Y, Margulies DR. Early postoperative enteral feeding increases anastomotic strength in a peritonitis model. Am J Surg 2001; 182:621–624. 37. Montecalvo MA, Steger KA, Farber HW, et al. Nutritional outcome and pneumonia in critical care patients randomized to gastric versus jejunal tube feedings. The Critical Care Research Team. Crit Care Med 1992; 20:1377–1387. 38. Montejo JC, Grau T, Acosta J, et al. Nutritional and Metabolic working group of the Spanish Society of Intensive Care Medicine and Coronary Units. Multicenter, prospective, randomized, single-blind study comparing the efficacy and gastrointestinal complications of early jejunal feeding with early gastric feeding in critically ill patients. Crit Care Med 2002; 30(4):796–800. 39. Kearns PJ, Chin D, Mueller L, Wallace K, Jensen WA, Kirsch CM. The incidence of ventilator-associated pneumonia and success in nutrient delivery with gastric versus small intestinal feeding: a randomized clinical trial. Crit Care Med 2000; 28(6):1742–1746. 40. Kortbeek JB, Haigh PI, Doig C. Duodenal versus gastric feeding in ventilated blunt trauma patients: a randomized controlled trial. J Trauma 1999; 46(6):992–996; discussion 996–998. 41. McClave SA, Sexton LK, Spain DA, et al. Enteral tube feeding in the intensive care unit: factors impeding adequate delivery. Crit Care Med 1999; 27(7):1252–1256. 42. Ritz MA, Fraser R, Edwards N, et al. Delayed gastric emptying in ventilated critically ill patients: measurement by 13 C-octanoic acid breath test. Crit Care Med 2001; 9(9):1744–1749. 43. Chapman MJ, Fraser RJ, Kluger MT, Buist MD, De Nichilo DJ. Erythromycin improves gastric emptying in critically ill patients intolerant of nasogastric feeding. Crit Care Med 2000; 28(7):2334–2337. 44. Boivin MA, Levy H. Gastric feeding with erythromycin is equivalent to transpyloric feeding in the critically ill. Crit Care Med 2001; 29(10):1916–1919. 45. McClave SA, DeMeo MT, DeLegge MH, et al. North American Summit on Aspiration in the Critically Ill Patient: consensus statement. J Parenter Enteral Nutr 2002; 26(suppl 6):S80–S85. 46. DeLegge MH. Aspiration pneumonia: incidence, mortality, and at-risk populations. J Parenter Enteral Nutr 2002; 26(suppl 6): S19–S24; discussion S24–S25.

Chapter 3: Surgical Nutrition 47. Elpern EH. Pulmonary aspiration in hospitalized adults. Nutr Clin Pract 1997; 12(1):5–13. 48. Seegobin RD, van Hasselt GL. Aspiration beyond endotracheal cuffs. Can Anaesth Soc J 1986; 33(3 Pt 1):273–279. 49. Lin HC, Van Citters GW. Stopping enteral feeding for arbitrary gastric residual volume may not be physiologically sound: results of a computer simulation model. J Parenter Enteral Nutr 1997; 21(5):286–289. 50. Maloney JP, Ryan TA. Detection of aspiration in enterally fed patients: a requirement for bedside monitors of aspiration. J Parenter Enteral Nutr 2002; 26(suppl 6):S34–S42. 51. Reed RL, Eachempati SR, Russell MK, Fahky C. Endoscopic placement of jejunal feeding catheters in critically ill patients by a ‘‘push’’ technique. J Trauma 1998; 45:388–393. 52. Myers JG, Page CP, Stewart RM, Schwesinger WH, Sirinek KR, Aust JB. Complications of needle catheter jejunostomy in 2002 consecutive applications. Am J Surg 1995; 170:547–551. 53. Holmes JH, Brundage SI, Hall RA, Yuen P, Maier RV, Jurkovich GJ. Complications of surgical jejunostomy in trauma patients. J Trauma 1999; 47:1009–1012. 54. Munshi I, Steingrub JS, Wolpert L. Small bowel necrosis associated with early post operative tube feeding in a trauma patient. J Trauma 2000; 49(1):163–165. 55. Marvin RG, McKinley BA, McQuiggan M, Cocanour CS, Moore FA. Nonocclusive bowel necrosis occurring in critically ill trauma patients receiving enteral nutrition manifests no reliable signs for early detection. Am J Surg 2000; 179:7–12. 56. McQuiggan MM, Marvin RG, McKinley BA, Moore FA. Enteral feeding following major torso trauma: from theory to practice. New Horizons 1999; 7:131–140. 57. Kozar RA, McQuiggan MM, Moore EE, Kudsk KA, Jurkovich GJ, Moore FA. Postinjury enteral tolerance is reliably achieved by a standardized protocol. J Surg Res 2002; 104:70–75. 58. Gadek JE, DeMichele SJ, Karlstad MD, et al. Effect of enteral feeding with eicosapentaenoic acid, gamma-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome. Crit Care Med 1999; 27(8):1409–1420. 59. American Society of Parenteral and Enteral Nutrition: Guidelines for the use of parenteral and enteral nutrition in adult and pediatric patients. J Parenter Enteral Nutr 2002; 17(suppl): 61SA–65SA. 60. Kudsk KA, Tolley EA, DeWitt RC, et al. Preoperative albumin and surgical site identify surgical risk for major postoperative complications. J Parenter Enteral Nutr 2003; 27:1–9. 61. Souba WW. Drug therapy: nutritional support. N Engl J Med 1997; 336:41–48. 62. Muller JM, Brenner U, Dienst C, Pichlmaier H. Preoperative parenteral feeding in patients with gastrointestinal cancer. Lancet 1982; 1:68–71. 63. The Veterans Affairs Total Parenteral Nutrition Cooperative Study Group. Perioperative total parenteral nutrition in surgical patients. N Engl J Med 1991; 325:525–532. 64. Bellantone R, Doglietto GB, Bossola M, et al. Preoperative parenteral nutrition in the high risk surgical patient. J Parenter Enteral Nutr 1988; 12:195–197. 65. Detsky AS, Baker JP, O’Rourke K, Goel V. Perioperative parenteral nutrition: a meta-analysis. Ann Intern Med 1987; 107: 195–203. 66. Sandstrom R, Drott C, Hyltander A, et al. The effect of postoperative intravenous feeding (TPN) on outcome following major surgery evaluated in a randomized study. Ann Surg 1993; 217:185–195. 67. McGee DC, Gould MK. Preventing complications of central venous catheterization. N Engl J Med 2003; 348:1123– 1233. 68. O’Grady NP, Alexander M, Pellinger EP, et al. Guidelines for the prevention of intravascular catheter-related infections. Clin Infect Dis 2002; 35:1281–1307. 69. Ma TY, Yoshinaka R, Banaag A, Jphnson B, Davis S, Berman SM. Total parenteral nutrition via multilumen catheters does not increase the risk of catheter-related sepsis: a randomized, prospective study. Clin Infect Dis 1998; 27:500–503.

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70. Raad I, Darouiche R, Dupuis J, et al. Central venous catheters coated with minocycline and rifampin for the prevention of catheter-related colonization and bloodstream infections: a randomized trial. Ann Intern Med 1997; 127:257–266. 71. Cook D, Randolph A, Kernerman P, et al. Central venous catheter replacement strategies: a systematic review of the literature. Crit Care Med 1997; 25:1417–1424. 72. Rombeau JL, Rolandelli RH, Wilmore DW, Daly JM. Nutritional support. In: Wilmore DW, Cheung LY, Harken AH, Holcroft JW, Meakins JL, eds. Care of the Surgical Patient. New York: Scientific American Surgery, 2001:15. 73. Van der Berghe G, Wouters P, Weekers F, et al. Intensive insulin therapy in critically ill patients. N Engl J Med 2001; 345: 1359–1367. 74. Van den Berghe G, Wouters PJ, Boullion R, et al. Outcome benefit of intensive insulin therapy in the critically ill: insulin dose versus glycemic control. Crit Care Med 2003; 31:359–366. 75. Crook MA, Hally V, Panteli JV. The importance of the refeeding syndrome. Nutrition 2001; 17:632–637. 76. Cerran FB, Mazuski JE, Chute E, et al. Branched chain metabolic support. A prospective, randomized, double-blind trial in surgical stress. Ann Surg 1984; 199:286–291. 77. Garcia-de-Lorenzio A, Ortiz-Leyba C, Planas M, et al. Parenteral administration of different amounts of branch-chain amino acids in septic patients: clinical and metabolic support. Crit Care Med 1997; 25:418–429. 78. Daly JM, Weintraub FN, Shou J, Rosato EF, Luci M. Enteral therapy in upper gastrointestinal cancer patients. Ann Surg 1995; 221:337–338. 79. Gianotti L, Braga M, Vignali A, et al. Effect of route of delivery and formulation of postoperative nutrition support in patients undergoing major operations for malignant neoplasms. Arch Surg 1997; 132:1222–1229. 80. Copeland EM. Historical perspective on nutritional support of cancer patients. CA Cancer J Clin 1998; 48:67–68. 81. Rivadeneira DE, Evoy D, Fahey TJ, Lieberman MD, Daly JM. Nutritional support of the cancer patient. CA Cancer J Clin 1998; 48:69–80. 82. Rombeau JL, Rolandelli RH. Enteral and parenteral nutrition in patients with enteric fistulas and short bowel syndrome. Surg Clin N Am 1987; 67:551–571. 83. Dudrick SJ, Latifi R, Fosnocht DE. Management of the short bowel syndrome. Surg Clin N Am 1997; 71(3):625–643. 84. Shanbhogue LKR, Molenar JC. Short bowel syndrome: metabolic and surgical management. Br J Surg 1994; 81:486–499. 85. Li JS. Short bowel syndrome. In: Shao JZ, Gu JF, Zhang SY, eds. Enteral Nutrition. Bejing: Military Medical Science Press, 1999:205–206. 86. Allard JP, Jeejeebhoy KN. Nutritional support and therapy in the short bowel syndrome. Gastroenterol Clin N Am 1989; 589–601. 87. Al-Jurf AS, Younasazi MK, Chapman-Furr F. Effect of nutritional method on adaptation of the intestinal remnant after massive small bowel resection. J Pediatr Gastroenterol Nutr 1985; 4:245–252. 88. Vanderhoof JA, Langnas AN. Short-bowel syndrome in children and adults. Gastroenterology 1997; 113:1767–1778. 89. Vanderhoof JA, Young RJ. The role of probiotics in the treatment of intestinal infections and inflammation. Curr Opin Gastroenterol 2001; 17:58–62. 90. Byrne TA, Persinger RL, Young LS, Ziegler TR, Wilmore DW. A new treatment for patients with short bowel syndrome. Ann Surg 1995; 222:243–255. 91. Pipkin WL, Gadacz TR. Nutritional considerations for dealing with intestinal diseases in the ICU. In: Shikora SA, Martindale RG, Schwaitzberg SD, eds. Nutritional Considerations in the ICU. Iowa: Kendall Hunt Publishing, 2002:279–285. 92. Olah A, Pardavi G, Belagyi T, Nagy A, Issekutz A, Mohamed GE. Early nasojejunal feeding in acute pancreatitis is associated with a lower complication rate. Nutrition 2002; 18:259–262. 93. Abu-Assi S, Craid K, O’Keefe SJ. Hypocaloric jejunal feeding is better than TPN in acute pancreatitis: results of a

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randomized comparative study. Am J Gastroenterol 2002; 97: 2255–2262. 94. Kalfarentzos F, Kehagias J, Mead N, Kokkinis K, Gogos CA. Enteral nutrition is superior to parenteral nutrition in severe acute pancreatitis: results of a randomized prospective trial. Br J Surg 1997; 84:1665–1669. 95. Braunschweig CL, Levy P, Sheean PM, Wang X. Enteral compared with parenteral nutrition: a meta-analysis. Am J Clin Nutr 2001; 74:534–542.

96. Duerksen DR, Bector S, Parry D, Yaffe C, Vajcner A, Lipschitz J. A comparison of the effect of elemental and immune-enhancing polymeric jejunal feeding on exocrine pancreatic function. J Parenter Enteral Nutr 2002; 26(3): 205–208. 97. Windsor AC, Kanwar S, Li AG, et al. Compared with parenteral nutrition, enteral feeding attenuates the acute phase response and improves disease severity in acute pancreatitis. Gut 1998; 42:431–435.

4 The Immune System and the Immunocompromised Patient Kathryn M. Verbanac, Lorita Rebellato, and Carl E. Haisch

INTRODUCTION The immune system functions to protect the body’s internal environment from a variety of potential insults such as foreign proteins, potentially adverse cells, microorganisms, and other noxious substances. Since immune competence plays such an important role in the insurance of good health and the response of a patient to surgical intervention, alterations in the immune system can greatly affect the outcome of an operation both in terms of morbidity and mortality. Immune competence reflects the highly integrated, carefully orchestrated homeostatic interplay of a number of physiologic processes whose primary function is to discriminate between substances that are part of the body’s own makeup (‘‘self’’) and those that are foreign to it (‘‘noself’’). The purpose of this chapter is to review the components of this immune system, how they interrelate, how this recognition of self and nonself occurs, and the impact that alterations in these processes can have on the surgical patient.

AN OVERVIEW OF THE IMMUNE SYSTEM In this section, we introduce the cellular components of the immune system, describe the initiation and effector stages of the humoral and cellular immune response, and discuss clinical assessment of immune function. This is to serve as a general introduction and review and to provide a basis for the clinical considerations that follow. There are several excellent textbooks on immunology, which should be referred to for a more comprehensive description of the immune system (1–5). In addition, we have cited key articles within each section that provide current reviews and references of the primary literature.

Cellular Components Leukocytes, the white blood cells that participate in the immune response, are derived from a common pluripotent stem cell in the bone marrow (Fig. 1). The mature cellular components of the immune system are distinguished on the basis of function and phenotype. Lymphocytes are distinguished from the other cells of the immune system because only lymphocytes exhibit diversity, specificity, memory, and self-/non–self-recognition. The cellular phenotype is principally defined by cell surface markers, the majority of which are designated cluster of differentiation (CD) antigens and are identified by monoclonal antibodies. Monoclonal antibodies serve as important immunological reagents for both the identification and quantitation of leukocyte and lymphocyte subpopulations that express these markers. There are currently 247 CD antigens that have been designated by the

International Workshops on Leukocyte Differentiation Antigens (6). Because the expression of cell surface antigens will vary qualitatively and quantitatively with both the maturation stage and activation state of the cell, monoclonal antibodies are also valuable for monitoring the phenotype of a specific leukocyte subpopulation (Table 1). Many of the cell surface antigens serve as specific receptors for ligands, thus ligand binding and receptor engagement have important immunological consequences. These ligands include immunoglobulins (Igs), complement, cytokines, viruses, and bacterial products.

B-Lymphocytes A schematic diagram of an Ig is presented in Figure 2. The development of the B-lymphocyte occurs predominantly in the bone marrow of mammals and is centered around the expression of membrane-bound Ig. Because the antigenbinding variable region of the membrane Ig is extracellular, it readily binds to soluble antigen. The expression of surface Ig will imbue the B-lymphocyte with both antigen specificity and memory. After antigen binding, signal transduction is mediated by another component of the B-cell receptor, a disulfide-bonded heterodimer that is noncovalently complexed with surface Ig and is designated as Ig-a/Ig-b or CD79a/CD79b (7). The DNA encoding antibody heavy chains, k light chains, and l light chains are located on different chromosomes. Within each chromosome, Ig sequences are present in germ line DNA as multigene families that contain numerous gene segments (Fig. 3). In progenitor and precursor B-cells, the variable region–D region–joining region (V-D-J) gene segments that comprise the variable region of the heavy chain undergo random gene rearrangement to form a functional Ig heavy chain (8). This is followed by light chain gene rearrangements that result in the expression of membrane-bound IgM. These rearrangements are regulated so that only one of the allelic forms of the Ig is expressed, that is, DNA from only one of the parental chromosomes is rearranged. This is referred to as allelic exclusion and insures that Ig with a single antigenic specificity is expressed by a given B-cell. B-cells at this developmental stage are released from the bone marrow into the blood. Most B-cells encounter antigen in regional lymph nodes or in the spleen, where they undergo antigen-dependent stages of differentiation. In the absence of antigen activation, peripheral B-cells will die within several days. If the membrane-bound antibody present on the surface of the B-cell encounters specific antigen, these cells undergo class switching by additional gene rearrangements of the constant region of the heavy chain and by changes in RNA processing. These classswitching events lead to different clones of B-cells that

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Pluripotent Stem Cell

Myeloid Progenitor

Lymphoid Progenitor THYMUS

B Lymphocyte

Erythroid Megakaryocyte Basophil Precursor Precursor

T Lymphocyte

Platelets

Basophils

Lymphoid/ Plasmacytoid Dendritic Cell DC2

Granulocyte-Monocyte Precursor

Eosinophil Precursor

Neutrophils

Erythrocytes

Natural Killer Cell

Monocytes

Eosinophils Macrophage

express different isotypes or classes of antibody with the same antigenic specificity. Different antibody isotypes have different effector functions, thus the range and type of immune response is broadened (Table 2). At this stage, some B-cells will clonally expand and differentiate into plasma cells that secrete various isotypes. Other B-cells will differentiate into memory B-cells that have a longer life span and express membrane-bound Ig of different isotypes. Memory B-cells are able to undergo somatic mutation to generate higher affinity antibodies, that is, antibodies with a greater binding strength or association constant, but of the same specificity. This process is termed affinity maturation. All of these mechanisms contribute to the generation of a high degree of antibody diversity; it is estimated that humans can produce antibodies that have 108 to 1011 different specificities.

T-Lymphocytes T-lymphocytes are termed ‘‘thymus dependent’’ because the lymphoid stem cell migrates from the bone marrow

Myeloid Dendritic Cell DC1

Figure 1 Maturation of blood cells from the pluripotent stem cell in bone marrow.

to the thymus, where it differentiates and matures during normal fetal development. It is in the thymus that T-cell receptor (TCR) rearrangement occurs to generate the diverse repertoire of antigen specificities exhibited by T-cell clones in the periphery. During thymic education, negative and positive selection processes result in the death of most high-affinity self-reactive T-cells (9). A large number of major histocompatibility complex (MHC)restricted T-lymphocyte clones survive that are tolerant to self-antigens and bear different antigen receptors, thus enabling adaptive immune responses to a large variety of pathogens. The TCR is the antigen-binding receptor on the surface of T-cells. It is expressed either as an a/b or a g/d heterodimer and is associated on the membrane with the multiple components of CD3 (Fig. 4). The invariant CD3 components (g, d, e, and z) are critical for the signal transduction events that occur after antigen binding (10). Interaction between an appropriate TCR and a peptide–MHC complex on antigen-presenting cells (APCs) or target cells induces

Table 1 Characteristic Leukocyte Cell Surface Markersa B-cell T-cell Th T cytotoxic NK cell Monocyte/macrophage Granulocytes a

CD19, CD20, CD21, CD22, slg, CD45RB, CD40, CD80/CD86 (B7-1/B7-2) CD2, CD3, CD5, TCR, CD28 CD4 CD8 CD2, CD16 (FcgRIII), CD56, CD11b (iC3bR), CD11c CD11b, CD11c, CD14, CD16, CD32 (FcgRII), CD64 (FcgRI), CD91 CD10 (CALLA), CD11b, CD11c, CD14, CD15 (Lewisx), CD16, CD32

This list is not comprehensive. The cell surface antigens unique to the cell type are in italics. The combination of coexpressed antigens is often most characteristic of a cell type. Abbreviations: CD, cluster of differentiation; Th, T helper; TCR, T-cell receptor.

Chapter 4: The Immune System and the Immunocompromised Patient

Figure 2 Schematic diagram of an immunoglobulin molecule. Each heavy (H) and light (L) chain contains an amino-terminal variable (V) region that is unique to each B-cell clone and which together form the antigen-binding site. The constant (C) domains exhibit limited variation and define the immunoglobulin isotype and effector functions. Intrachain and interchain disulfide bonds play a major role in the folding of the protein domains and in the formation of the polypeptide chain tetramer. The g, d, and a heavy chains contain a hinge region and the m and E heavy chains contain a fourth central CH domain.

the redistribution and/or catalytic activation of intracellular protein tyrosine kinases. These kinases then phosphorylate immunoreceptor tyrosine-based activation motifs (ITAM) within the cytoplasmic domains of the invariant chains of the CD3 subunits. This initial phosphorylation is followed

67

by a cascade of diverse intracellular signals, whose key outcome is to affect the expression of cytokine genes. Although the TCR is the unique distinguishing marker for the T-cells today, T-cells were historically distinguished from B-cells on the basis of the so-called E rosette receptor, now designated CD2. T-cells have been subdivided into two subtypes: those with helper function (primarily CD4þ) and those with cytotoxic function (primarily CD8þ). In the thymus, CD4CD8 bone marrow progenitors go through sequential developmental changes before the so-called single positive T-cells, expressing either CD4 or CD8, and the abTCR are produced. Although these latter cells comprise the vast majority of peripheral T-cells, it has recently been discovered that a small number of T-cells expressing the gdTCR are present in adult epithelial tissues and they can also be found in lymphoid organs. The majority of the abTCRþ T-cells in the periphery are naive or precursor T-cells that have not yet encountered antigen and are in the G0 stage of the cell cycle. Once antigen activated (as discussed below), these cells become effectors that perform helper, cytotoxic, or delayed-type hypersensitivity (DTH) functions. Memory T-cells are also generated during a primary response to antigen and recirculate in the blood and lymph as extremely long-lived resting cells with less stringent requirements for activation. Antigen-stimulated T helper (Th) cells have been classified into Th1 and Th2 cells on the basis of the distinct cytokines they secrete and on the functional effects of these cytokines. Originally discovered in mice, Th1 and Th2 patterns of cytokine exist in humans as well, but the cytokine patterns are less exclusive. In humans, interferon (IFN)-g is most consistently expressed by Th1 cells, and interleukin (IL)-4, IL-5, and IL-9 are most consistently produced by Th2 cells. IL-2 and tumor necrosis factor (TNF)-b (predominantly Th1 cytokines) and IL-6, IL-10, and IL-13 (predominantly Th2

Germ-line H-chain DNA (Chromosome 14) L VH1−L VH100

DH1−DH30 JH1−JH6

Cµ1 Cµ2

Cµ3

Cµ4

Cδ−Cγ−Cε−Cα

5'

3'

L Vκ1−L Vκ100 Germ-line κ-chain DNA (Chromosome 2)

Jκ1−Jκ100 Cκ

5'

3'

Germ-line λ-chain DNA (Chromosome 22) L Vλ1−L Vλ100

Jλ1 Cλ1

Jλ2 Cλ2 Jλ3 Cλ3 Jλ4 Cλ4 Jλ5 Cλ5 Jλ6 Cλ6

5'

3'

GENE REARRANGEMENT - DJ Joining, VDJ Joining, VJ Joining GENE TRANSCRIPTION - Polyadenylation and Splicing RNA TRANSLATION

Nascent Polypeptides VDJ Cµ1 Cµ2 Cµ3 Cµ4

VJ Cκ +

VHCµ

= VκCκ

Membrane lgM

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Figure 3 Immunoglobulin gene rearrangement for surface IgM expression in B-cells. Abbreviation: IgM, immunoglobulin M.

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Part One: General Considerations

Table 2 Properties and Biological Activitiesa of Serum Igs Property/activity c

Molecular weight (kDa) Heavy-chain component Normal serum level (mg/mL) In vivo serum half-life (days) Activates classical complement pathway Crosses placenta Present on membranes of mature B-cells Binds to phagocyte Fc receptors Mucosal transport Induces mast-cell degranulation

IgG1

IgG2

IgG3

IgG4

IgA1

IgA2

IgMb

IgE

IgD

150 1 9 23 þ

150 2 3 23 þ/

150 3 1 8 þþ

150 4 0.5 23 –

150–600 1 3 6 –

150–600 2 0.5 6 –

900 m 1.5 5 þþþ

190 E 0.0003 2.5 –

150 d 0.03 3 –

þ –

þ/ –

þ –

þ –

– –

– –

– þ

– –

– þ

þþ

þ/

þþ

þ

–

–

?

–

–

– –

– –

– –

– –

þþ –

þþ –

þ –

– þ

– –

a

Activity levels indicated as follows þþ, high; þ, moderate; þ/, minimal; ?, questionable; and , none. þ IgM is the first isotype produced by the neonate and during a primary immune response. c þ IgG, IgE, and IgD always exist as monomers; IgA can exist as a monomer, dimer, trimer, or tetramer. Membrane-bound IgM is a monomer, but secreted IgM in serum is a pentamer. Abbreviation: Ig, Immunoglobulins. Source: From Ref. 5. b

cytokines) tend to segregate less clearly among human subsets than in the mouse. Cytokines produced by APCs are the dominant factors guiding the development of Th1 and Th2 cells. Furthermore, specific transcription factors are required to be activated for each pathway: T-bet and signal transducer and activator of transcription (STAT)-4 for Th1 cells and GATA-3 and STAT-6 for Th2 cells (11,12). Reciprocal regulation occurs between the Th1 and Th2 subsets, mediated by Th cytokine effects on differentiation and on effector functions.

Figure 4 Schematic diagram of the TCR-CD3 complex. The polypeptide chains of CD3 noncovalently associate with the TCR a/b chains and with each other as heterodimers as well as ZZ homodimers. The solid rectangle represents an ITAM. Abbreviations: TCR, T-cell receptor; ITAM, immunoreceptors tyrosme-based activation motifs.

Antigens derived from pathogens stimulate the clonal proliferation of T-lymphocyte precursors that carry receptors specific for that antigen. Thus, in response to different pathogen-derived antigens, CD4 T-cells become either Th1 or Th2 cells. Th1 cells produce the cytokine IFN-g and are effective against intracellular bacteria, viruses, and protozoa, whereas Th2 cells produce IL-4, IL-5, and IL-13 and eliminate extracellular parasites. Like Th1 cells, CD8 T-cells are activated in response to intracellular pathogens and share some of the same effector mechanisms, particularly production of IFN-g. Upon antigenic stimulation, Th1 cells secrete IL-2, IFN-g, and TNF-b, which serve to channel the immune system toward cell-mediated immunity (CMI), including macrophage activation and DTH responses. Th1 cells are thus effective in the defense against intracellular pathogens (e.g., Leishmania) and are thought to be involved in the pathogenesis of acute allograft rejection, organ-specific autoimmune disorders, contact dermatitis, and certain chronic inflammatory diseases. In contrast, Th2 cells secrete IL-4, IL-5, IL-6, and IL-10, induce humoral and allergic responses, and are most effective against extracellular microorganisms and soluble toxins. Studies of Th2 cells have concentrated on IgE responses to helminth antigens and allergens. Th2 cytokines generally exert anti-inflammatory, immunosuppressive effects, and Th2 responses have been associated with transplantation tolerance, the immunopathology of chronic graft-versus-host disease, and systemic autoimmune disease. It is clear that reciprocal regulation occurs between the Th1 and Th2 subsets, mediated by specific cytokine effects on differentiation and on effector functions. Over the past five years, a great deal of interest has focused on regulatory T-cells (Treg) that appear to control the development of autoimmune disease and transplant rejection, and that might also play a critical role in controlling the expression of asthma and allergy. The term ‘‘regulatory T-cell’’ refers to cells that actively control or suppress the function of other cells, generally in an inhibitory fashion. CD4þCD25þ Treg cells have emerged as a unique population of suppressor T-cells that maintain peripheral immune

Chapter 4: The Immune System and the Immunocompromised Patient

tolerance (12,13). Transforming growth factor (TGF)-b has been implicated in the conversion of na€ve CD4þCD25 T-cells into CD4þCD25þ anergic/suppressor T-cells (14). The Treg subset has been isolated in humans, but it is still unclear if these regulatory cells can be used to inhibit ongoing T-cell responses in vivo or to reverse established pathology. There have not been similar functional divisions of CD8þ T-cell subpopulations. Although CD8 originally characterized suppressor and cytotoxic cells, a definitive CD8 suppressor cell has been elusive and has not been isolated or cloned. Therefore, most contemporary immunologists view the suppressor cell as a functional designation rather than the definition of a separate subpopulation. The classical CD8þ T-lymphocyte is the cytotoxic T-lymphocyte (CTL). CTL are MHC class I–restricted cells, which recognize and eliminate ‘‘altered self’’ target cells. Target cells include virally infected cells, malignant cells, and allogeneic cells. The primary mechanism of CTL-mediated killing, at least in vitro, involves granule exocytosis and release of a poreforming protein (perforin) and a battery of serine proteases (granzymes) that rapidly induce target cell lysis (15). CTL can also use the Fas (CD95) pathway to induce apoptosis, or programmed cell death, of target cells (16).

NK Cells Although their developmental pathway is not fully understood, natural killer (NK) cells, like T- and B-lymphocytes, arise from CD34þ lymphoid stem cells in the bone marrow. NK cells make up 5% to 10% of human peripheral blood mononuclear cells (PBMC). NK cells are termed null cells, because they do not express the distinct cell membrane markers that would characterize them as T- or B-cells, including the T-cell and B-cell antigen-binding receptors. NK cells and T-cells are both CD2þ and are thought to share a common thymic developmental precursor. Unlike CTL, NK cells typically express the phenotype CD16 and CD56 and do not typically express CD3 or CD8. Distinct subpopulations of NK cells with atypical phenotypes have been observed at very low levels in normal individuals, and at significant levels in recovering bone marrow–transplant recipients. NK cells were originally referred to as large granular lymphocytes, and discovered on the basis of their tumoricidal activity. The granules contain cytotoxic agents, including perforin and granzymes. The adjective ‘‘natural’’ refers to the important fact that NK cell–effector activity does not require antigen activation or sensitization and thus provides ‘‘natural immunity’’ or ‘‘innate’’ antiviral and antitumor protection. This immunity is critical in the early days of exposure, before CTL precursor cells can become activated and proliferate and differentiate into mature CTLs. Unlike CTLs, most NK cells do not express antigen receptors, hence they do not exhibit immunological specificity or memory. NK cells uniquely express inhibitory NK-cell receptors (NKR) that recognize polymorphic epitopes common to groups of human leukocyte antigen (HLA) alleles and transduce inhibitory signals, which suppress the NK-mediated cytolysis (17). NK cells thus recognize ‘‘absence of self,’’ and healthy cells avoid being killed via their expression of self-HLA. If HLA class I expression is downregulated by a viral infection or tumor transformation, no inhibitory signal is generated and the NK cells release their lytic mediators to kill these abnormal cells. In addition to inhibitory NKR, NK cells may also express activating NKR that are specific to MHC class I–related molecules expressed during infection, tumor transformation, or stress. The ligation of activating

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NKR will further enhance the NK-cell lytic activity. Each NK cell of an individual can express a unique receptor repertoire, with a spectrum of different numbers (1–19) and combinations (inhibitory and activating types) of killer immunoglobulin-like receptors (KIR) and NKG2 receptors with multiple class I specificities. NK cells also identify target cells and mediate antibody-dependent cell-mediated cytotoxicity (ADCC), which is described in detail later in this chapter. In short, NK cells express CD16 (the Fc gamma receptor IIIA) and can thus bind IgG that is bound to target cell membranes. CD16 engagement activates the NK cell, inducing the lytic mechanism and cytokine production. NK cells appear to kill target tumor cells and virally infected cells by mechanisms similar to those described for CTL, namely the release of lytic granules. NK cells have also been recently shown to induce apoptosis via direct target cell killing or in ADCC. These two mechanisms are probably not mutually exclusive and some studies suggest that perforin and granzymes may synergistically trigger apoptosis.

Monocytes/Macrophages The remaining cellular components to be described also develop from the pluripotent hematopoietic stem cell in the bone marrow, but from the myeloid lineage, rather than the lymphoid lineage. These cells differ from lymphocytes in several significant properties; they lack antigen specificity and they lack memory. The monocyte is a phagocytic mononuclear myeloid cell that circulates briefly before migrating into tissue where it differentiates into a macrophage. The macrophage can take up permanent residence in particular tissues or remain motile. The primary function of the monocyte/macrophage is to phagocytose and eliminate antigen (18). Their digestive granules contain lytic enzymes, reactive oxygen and nitrogen intermediates, and TNF. Macrophages can ingest and digest whole microorganisms as well as injured and dead cells, cellular debris, and activated clotting factors. The macrophage also digests internalized proteins and presents peptides in association with MHC class II on its surface. It plays a critical role in Th cell activation, both as an APC and as a source of IL-1. The Th cell, in turn, promotes macrophage activation by secretion of IFN-g. Activated macrophages have increased phagocytic and microbicidal activity and secrete many factors that promote the inflammatory response. Finally, monocytes and macrophages express all three forms of the FcgR (CD16, CD32, and CD64), and thus can participate in ADCC, which is discussed later.

Dendritic Cells The dendritic cell (DC) has achieved recent prominence as the most highly specialized APC due to its superior ability to activate na€ve T-cells to initiate immune responses (19). DCs appear to originate from bone marrow precursors of two different hematopoietic lineages. CD11cþ Type 1 DC (DC1) is myeloid derived, either directly from myeloid progenitors or from monocytes (20). CD11c Type 2 DC (DC2) appear to be lymphoid derived, and DC2 CD4þ precursors have been referred to as plasmacytoid T-cells, plasmacytoid monocytes, type 1 IFN-producing cells, and plasmacytoid DC (21). Although DC1 and DC2 were initially thought to secrete cytokines to induce respective Th1 and Th2 responses, it now appears that this functional correlation is not rigid. DCs are located throughout the body in different maturation states and they mature into potent APCs during

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conditions of infection or inflammation. DCs are characterized by branch-like membrane projections, motility in response to chemokine gradients, and surface expression of MHC class I and II and of the costimulatory molecules CD40, CD80, and CD86. These properties are responsible for their superior ability to capture and present antigen, to travel from the sites of infection to lymphoid organs where they activate na€ve T–cells, and initiate immune responses.

Granulocytes Granulocyte is a general term for a motile cell that contains cytoplasmic granules. These cells are categorized into basophils, eosinophils, and neutrophils on the basis of cell morphology and cytoplasmic staining properties. Granulocytes characteristically play a major role in the inflammatory response, which can be beneficial in host defenses and detrimental in inflammatory diseases. Neutrophils are also referred to as polymorphonuclear leukocytes (PMNs), and constitute 50% to 70% of the circulating white blood cells in humans. Neutrophils are active phagocytic scavengers of the immune system and are usually the first cells to arrive at an inflammatory site. Their granules contain lytic enzymes, bactericidal defensin peptides, and reactive oxygen and nitrogen intermediates; however, unlike macrophages, they do not contain TNF. The neutrophil is a critical effector cell in humoral and innate immunity and plays a key role in bacterial killing (22). Eosinophils are also phagocytic and function primarily against parasites via ADCC. They are the principal effector cell for the pathogenesis of allergic inflammation, caused by granular proteins that include eosinophil cationic protein, major basic protein, and eosinophil protein X (23). The nonphagocytic basophils play a major role in allergic responses when they release their granules of pharmacologic agents, including histamine.

The Innate Immune Response The innate immune response refers to the rapid response to microbial infection that relies on preexisting mechanisms (24). The innate immune system includes epithelial barriers, phagocytic cells, NK cells, the complement system, and cytokines. The innate immune response lacks both specificity and memory and is activated by a large variety of conserved molecular structures that are unique to microorganisms. These are primarily microbial metabolites that are referred to as pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), peptidoglycan, b-glucans and mannans, and double-stranded RNA. Families of Toll-like receptors (TLRs) on macrophages and DCs that recognize distinct PAMPs are now known to be the primary sensors of microbial infection. In addition to contributing to the signal transduction induced by many PAMPs, TLRs may also contribute to endogenous damage signals at inflammatory sites (25).

The Acquired Immune Response The interaction of a Th cell with an APC is the central event that initiates virtually all acquired immune responses, for generation of both CMI and antibody-mediated immunity (humoral). The T-cell and APC interact primarily in the secondary lymphoid organs—the lymph nodes and the spleen. Antigen can be transported to these organs as soluble antigen, as antigen-antibody complexes, or via mobile APC (including certain DCs and macrophages). After recognition of a peptide–MHC class II complex on an APC, Th cells are

activated and proliferate extensively. The Th cell progeny secrete cytokine ‘‘help’’ and play a central role in the activation of B-cells, T cytotoxic cells, and macrophages.

T-Cell–APC Interactions This association is based upon specific antigen recognition and is mediated by the requisite binding of two cell surface molecules, the TCR of the Th cell with the MHC–peptide complex of the APC. MHC molecules (termed ‘‘HLA’’ in humans) are cell surface molecules encoded by the MHC gene complex, which control the ability of an animal to respond immunologically to a given antigen. The multiple alleles (estimated at greater than 300) within a given HLA locus encode highly polymorphic gene products that vary in their individual ability to bind a specific peptide. The inherited HLA polymorphisms provide a large repertoire of specificities for antigen-derived peptides and thus provide the host with the ability to respond immunologically to a large range of different antigens. T-cells can also recognize microbial lipid and glycolipid antigens presented by CD1 proteins. CD1 belongs to MHC class I molecules that map outside of the MHC, have limited tissue distribution, and include H2M3 and MICA as members. CD1 proteins (CD1a, -b, and -c) can present foreign microbial lipid antigens, including several mycobacterial antigens. CD1d-restricted natural killer T-cells (NKT) play a role in immunity to bacteria, parasites, yeasts, and viruses (26). In particular, CD1d-restricted NKT-cells can activate innate and adaptive immune responses and appear to modulate immunity to infectious agents (27). Although difficult to characterize in humans, NKT-cells express a/b TCR and NKR1.1, a C-type lectin receptor. Many cell types can present antigen to T-cells, that is, they can degrade and present antigen-derived peptides via their MHC class I and class II molecules. The so-called ‘‘professional antigen presenting cells (APC)’’ are those cells that constitutively express MHC class II antigens, and, in humans, include DCs, macrophages, B-cells, and vascular endothelial cells. These cell types can vary in the effectiveness with which they present antigen and stimulate an immune response in vitro. The DC appears to be one of the most potent APC in vitro, expressing high levels of MHC class II and possessing long dendrite-like membrane processes that provide a large surface area for cell–cell interactions. The concentration of antigen, as well as other variables, may determine which cell type is the operative APC in vivo. Because a B-cell recognizes an antigen specifically via its surface Ig, it can present antigen effectively even at very low antigen concentrations. After intracellular degradation of foreign proteins, the MHC class I and class II molecules of the APC bind certain processed peptides within a groove formed by the polymorphic residues of the MHC. The TCR thus recognizes the specific amino acid sequence of a peptide (processed antigen) in association with self-MHC. This interaction is termed self-MHC restriction or associative recognition. APC generally ‘‘present’’ processed foreign antigen that are in association with MHC class II to CD4 T-cells and in association with MHC class I to CD8 T-cells (Fig. 5). CD4 and CD8 specifically recognize residues within the monomorphic conserved domains of MHC class II and class I antigens, respectively; thus CD4 and CD8 cells are termed MHC class II– and MHC class I–restricted, respectively. There are additional accessory cell surface proteins that play important roles in the T-cell–APC interaction. Some act as adhesion molecules to stabilize or increase the strength of T-cell–APC interactions and redistribute within the membrane to contribute to the formation of the immunological synapse

Chapter 4: The Immune System and the Immunocompromised Patient

Figure 5 Antigen presentation and recognition.

between the two cell types. Examples of such T-cell–APC ligand pairs include CD2–leukocyte function antigen (LFA)-3; LFA-1– ICAM-1 or ICAM-2; CD4–MHC class II; and CD8– MHC class I. Some ligand pair interactions provide signals to enhance T-cell activation—many ligands have cytoplasmic domains possessing enzymatic activity, usually kinase activity (phosphorylating enzymes). Such ligands are often called signal transducers because they send a signal from outside the cell to the inside. CD4 and CD8 molecules are associated with intracellular T-cell protein tyrosine kinases that appear to be activated by binding to the monomorphic domains of MHC molecules. There is compelling evidence that certain ligand pair interactions provide critical ‘‘second signals’’ required for T-cell activation after the antigenic ‘‘signal 1’’ is delivered via TCR ligation to the MHC peptide. In addition to the TCR, T-cells have multiple so-called ‘‘coreceptors’’ with both shared and unique properties that regulate the T-cell response. The most significant activating ligand pairs that have been identified on T-cells and APC, respectively, are CD40L–CD40 and CD28–CD80 (28,29). In the current working model of Th cell activation (Fig. 6), CD40L expression is upregulated by T-cells that have received signal 1 (ligation

Figure 6 T-cell activation.

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of the TCR with MHC antigen). CD40L can then engage CD40, its cognate ligand on the APC, which in turn triggers the APC to upregulate the expression of CD80 and CD86 (originally named B7.1 and B7.2). CD80 binding to CD28, which is constitutively expressed on T-cells, results in full T-cell activation. Inducible costimulatory molecule is a coreceptor that is induced in na€ve and memory T-cells, binds to B7H on APC, and apparently provides signals that can substitute for CD28. Finally, CTL antigen-4 is a T-cell coreceptor that is homologous to CD28, and is a negative regulator of T-cell responses by inhibiting proliferation. It is important to note that APC activation is usually a corollary to these interactions and is also dependent on CD40–CD40L engagement. Activated B-cells, macrophages, and DCs are key players in the effector phase of an immune response. Soluble cytokines are also involved in mediating this ‘‘cross-talk,’’ and include T-cell–derived IL-4 as well as IL-1, IL-6, and IL12, which are secreted by specific APC cell types. Once the Th cell is activated by productive engagement of the appropriate receptor ligand pairs, and after receipt of the appropriate signals, it produces IL-2 ‘‘help,’’ which functions in an autocrine manner to induce proliferation and clonal expansion. These Th progeny can now produce cytokines that ‘‘help’’ B-cells, macrophages, and CTLs differentiate and perform their full effector functions. The central role of the Th cell in the effector mechanisms of the immune response is presented in Figure 7.

The Humoral Immune Response It is clear that B-cells, T–cells, and APC are required for the generation of most humoral immune responses. As described above, the B-cell antigen receptor is a multicomponent receptor consisting of surface Ig noncovalently associated with two other distinct transmembrane proteins. The surface antibody mediates the internalization of specific antigen via receptor-mediated endocytosis. In the B-cell endosome, newly synthesized MHC class II antigens encounter peptides derived from antigen proteolysis and present certain peptides on the surface of the B-cell. These ‘‘antigen primed’’ B-cells act as APC to interact with activated Th cells (which are antigen specific on the basis of their TCR) and form specific B–Th cell conjugates. Antigen-specific conjugate formation appears to actually cause redistribution of the TCR, LFA-1, and CD4 molecules within the T-cell at the interface of the cell-to-cell contact. Conjugate formation induces T-cell expression of CD40L, which then engages CD40 on the B-cell surface, as described above. The B-cell receptor itself is associated with cytoplasmic src-family tyrosine protein kinases that are activated upon antigen binding and initiate an intracellular signaling cascade that leads to many cellular responses, including cytokine production. B-cell activation ensues, triggered by a combination of membrane events and cytokine signaling. The Th cytokines IL-1 and IL-4 play predominant roles as the costimulatory signals in B-cell activation. The activated B-cell is driven into the S (or DNA synthesis) phase of the cell cycle by IL-2, IL-4, and IL-5. As discussed earlier, IgM is the first isotype expressed by plasma cells during a primary immune response. Numerous Th1 and/ or Th2 cytokines (including IL-2, IL-4, IL-5, IFN-g, and TGF-b) induce differentiation into plasma cells, producing different Ig isotypes. The changes in the activation and maturation stage of the cells are caused as well as marked by changes in gene expression.

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APC

T helper cell

IL-2, IL-4, IL-5, IL-6 IL-2

TDTH

Activated macrophage

IL-2

+

+

CD8 TC

CD4 TC

B cell

IFN-γ TNF-β

Cytotoxicity MHC expression

CD8+ CTL

Lytic enzymes Class I MHC alloantigen

Membrane damage Class II MHC alloantigen

CD4+ CTL

NK cell or macrophage C

Lysis

Target tissue

It must be noted that ‘‘T-independent’’ antigens exist that can directly stimulate B-cells and elicit antibody responses without the assistance of Th cells (30). This type of antigen, usually carbohydrate in nature and comprised of repeating epitopes, is best exemplified by bacterial polysaccharides. Complement-Mediated Cytolysis It is the major effector of the humoral immune response. Complement refers to a group of at least 20 distinct serum proteins that participate in a highly regulated enzymatic cascade (31,32). There are two pathways of complement activation, the classical and the alternative pathways. Both pathways generate a membrane attack complex that forms pores in cell membranes and causes cell lysis. A conformational change occurs when an antibody binds to its antigen on a cell surface, such that a binding site on the Fc portion of the molecule is exposed. Binding of the C1 component of the complement system to the Fc sites exposed on two adjacent antibody molecules activates the classical pathway. In humans, IgM and certain subclasses of IgG (IgG-1, -2, and -3) are capable of ‘‘fixing’’ complement in this manner. The alternative pathway is usually initiated by bacteria, yeast, and certain viruses in an antibody-independent fashion. Complement reaction products cause vasodilation and chemotaxis to promote a localized inflammatory response and also act as opsonins to promote phagocytosis of antigen. Anaphylaxis It is an immediate hypersensitivity reaction that is triggered by IgE-mediated mast cell or basophil degranulation and

ADCC

Fc receptor

Figure 7 Schematic of the central role of the Th cell in the effector mechanisms of the immune response. Abbreviations: C, complement; ADCC, antibody-dependent cell-mediated cytotoxicity; Th cell, T helper cell. Source: From Ref. 5.

is initiated within 2 to 30 minutes of antigen exposure. Anaphylaxis can be localized to a specific target tissue or can occur throughout the organism, inducing systemic effects that can be fatal. Upon primary exposure to parasitic antigens or allergens, B-cells are activated to become IgEsecreting plasma cells. IgE binds to the cell surface of mast cells and basophils via the high-affinity FceRI, thus increasing the half-life of IgE from days to weeks. Mast cells are bone marrow–derived cells that differentiate in connective tissues where they reside. Upon secondary exposure to antigen, the IgE on these sensitized cells is cross-linked, which triggers degranulation and the release of pharmacologically active mediators. These mediators include histamine and serotonin, which cause increased vascular permeability and smooth muscle contraction, as well as granulocyte chemotactic factors and proteases that mediate tissue destruction. Additional secondary mediators such as leukotrienes are generated by the breakdown of the mast cell membrane phospholipids after degranulation. Eosinophils are also attracted to the site and can bind directly to antibody-coated antigen. They in turn are activated, degranulate, and release inflammatory mediators. Opsonization It refers to the deposition of molecules on an antigen (including a microorganism) that promotes contact with an appropriate phagocytic cell. Such molecules, or opsonins, are usually antibodies or components of complement. Because macrophages and granulocytes express receptors for both antibody and complement on their cell surfaces, opsonization increases antigen binding by these phagocytes.

Chapter 4: The Immune System and the Immunocompromised Patient

Opsonins thus serve as a bridge between antigens and effector cells. The enhanced antigen binding results in a significant increase in the rate of phagocytosis.

Antibody-Dependent Cell-Mediated Cytotoxicity Antibodies can also affect cell-mediated responses to foreign cells in a complement-independent process termed ADCC (22). In this scenario, antibody serves as a bridge connecting a foreign target cell to an immune effector cell, and thus provides antigen specificity to a nonspecific cytotoxic cell. NK cells, neutrophils, macrophages, and eosinophils express Fc receptors on their cell surfaces. These receptors can thus recognize the carboxy terminal region of an Ig molecule that is bound to foreign cells via its amino-terminal antigen-binding hypervariable domains. This binding stimulates the cytotoxic cell to increase the lytic components in their cytoplasmic granules (including enzymes, TNF, and perforin) and then to release the granule contents, culminating in destruction of the target cell.

CTL-Mediated Cytotoxicity The CTL-mediated immune response is initiated by the activation and differentiation of precursor CTL (CTLp) into functional effectors. The interaction of the TCR of a resting CTLp with the MHC class I–peptide complex of its target cell induces the expression of T-cell IL-2 receptors. As described earlier, IL-2 ‘‘help’’ is produced as a result of a similar interaction between CD4þTh and MHC class IIþ APC. This IL-2 provides the principal ‘‘second signal’’ required by CTLp for activation, triggering its clonal proliferation and differentiation into a mature CTL effector. As discussed above, other ligand interactions also appear to supply strong costimulatory signals. The B7/CD28 interaction has been reported to induce CTLp to express IL-2 by themselves, bypassing the requirement for Th cells. The CTL progeny, mature effectors, are now able to form conjugates with target cells that bear the appropriate MHC class I–peptide complexes. Exocytosis of cytoplasmic granules at the interface of cell–cell contact releases lytic molecules that damage the membrane of the target cell and cause death, as described above. Secondary to antigen recognition, the LFA-1/ICAM interaction between the T-cell and target cell appears to be critical for both conjugate formation and subsequent dissociation of the CTL from the target cell.

DTH Reactions DTH responses refer to an increased reactivity to a specific antigen, which is T-cell–mediated. The increased reactivity is a function of previous antigen sensitization (via APC–T-cell interactions described above) that has induced T-cells to proliferate and differentiate into a subset of T-cells (usually CD4þ Th1), designated TDTH cells. Following the second exposure to an antigen, TDTH cells secrete a myriad of cytokines, which induce a localized inflammatory reaction (DTH). Macrophages are attracted by these cytokines and activated. The DTH response usually peaks 48 to 72 hours after secondary contact. This response is important and generally very effective in the defense against intracellular pathogens and contact antigens.

Mediators of Immune Function As previously discussed, cytokines play a major role in immune function and regulation (33). Cytokines are regulatory proteins that have pleiotropic effects on cells that participate

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in host defense and repair processes via control of cell differentiation, activation, and proliferation. Originally termed ‘‘lymphokines,’’ the cytokine terminology evolved to indicate that these factors can be non–lymphocyte derived and can also have nonlymphoid targets. The current ‘‘interleukin’’ terminology was born at the 1978 Second International Lymphocyte Workshop, and incorporates the concept of bidirectional, intercellular signaling between somatic, myeloid, and lymphoid cells. At this writing, IL-30, a member of the IL-2 subfamily, is the most recent cytokine named by the IUIS/WHO Standing Committee on Interleukin Designation (34). Cytokines typically are very potent—they act at picogram-nanogram levels and exert paracrine and autocrine effects. The expression of cytokines is inducible, triggered by diverse stimuli from diverse cellular sources. Cytokines operate in a network that can be overlapping, redundant, synergistic, additive, and/or antagonistic. Cytokines are grouped into families based on structural homologies and are also classified by function (see Table 3 for representative examples). Finally, they act by binding to high-affinity receptors that are transmembrane proteins, many possessing kinase activity or enzymatic activities, which activate the kinases that effect signal transduction. Further comprehensive descriptions of the cytokines are beyond the scope of this chapter and can be found elsewhere (35,36).

Clinical Assessment of Immune Function Clinical assessment of human immune function can help define defects in local and systemic defense mechanisms. This is true for both patients with primary hereditary immunodeficiencies and for those with acquired immunodeficiencies. Although some of the tests described below are not routinely conducted clinically, they are available at most tertiary care centers and can be valuable for the identification of the immunocompromised patient at increased risk for local and systemic infections. A summary of laboratory tests used to detect immunodeficiencies is found in Figure 8. An obvious key to the successful treatment of infection in the immunocompromised host is early diagnosis of infection. Discussion of these detection techniques is beyond the scope of this review; however, it should be noted that advances in molecular biology have brought innovations to these analyses. Molecular techniques include the use of chemiluminescent-labeled DNA probes that hybridize to DNA of specific bacteria and viruses as well as virus- and bacteria-specific DNA primers that have been developed for DNA amplification in the polymerase chain reaction. In this section, we will briefly discuss some of the clinical predictors and laboratory tests that are available.

Leukocyte Enumeration and Subsets Quantitative determinations of circulating leukocytes are routinely determined by critical blood count (CBC), including platelet size and number and differential analysis (Table 4). This test will detect gross changes in cells that might occur in conditions such as leukemia or in certain immunodeficiency diseases. CBC is the first step to evaluating all suspected immune defects and the absolute counts must be compared to age-appropriate normative values. Small-sized platelets are a characteristic finding in Wiskott– Aldrich syndrome. The absolute neutrophil count is very important in cancer patients; less than 1000 cells/mL strongly correlates with an increased incidence of sepsis and less than

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Table 3 Cytokine Classification by General Function Hematopoietic B-cell Growth T-Cell Growth Antiviral Inflammatory Chemotactic Antimitotic Immunosuppressive

IL-3, IL-11, IL-17, colony-stimulating factors, stem cell factor, erythropoeitin IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-14, IL-15, IL-21, TGF-b IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, IL-16, IL-18, IL-21, IL-23, IL-27 Interferon family (a/b/c/x, s), IL-28, IL-29 TNF family (TNFa/b, Lymphotoxin), IL-1 family (IL-1a/b, IL-1RA), MIF, IL-6, IL-8, IL-17 IL-8, MCP-1-MCP-5, MIP-1, MGSA/GRO, RANTES, IL-16 TNF family IL-4, IL-10, IL-13, TGF-b

Abbreviations: IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor; IL-1 RA, IL-1 receptor antagonist; MCP, macrophage chemotactic protein; MIF, migration inhibitory factor; MGSA, melanocyte growth stimulatory activity; GRO, growth-related oncogenes.

500 cells/mL, if not corrected, is associated with an increased incidence of death. The CBC may be normal, however, in the most common immune deficiency, asymptomatic selective IgA (SIGA) deficiency. If lymphopenia is observed, screening for T- and B-cell disorders should be initiated. Enumeration of lymphocyte subsets can be performed by flow cytometric analysis, and characteristic markers have been described in the previous sections. Flow cytometry has become an important part of the workup of patients suspected of having primary immunodeficiency diseases (38). For example, there is a marked reduction of both T- and B-cell counts in severe combined immunodeficiency disease (SCID). Although lymphocytes comprise about 25% of the mononuclear cells in the human circulation, the blood actually contains a small proportion (about 2%) of the body’s total T- and B-lymphocyte pool (39). Lymphocyte trafficking is regulated largely by differential expression of adhesion molecules and chemokine receptors on T-cells (40). The

majority of the lymphocytes are concentrated in the lymph nodes and the spleen, however, it is estimated that about 500 109 lymphocytes travel through the blood compartment each day. The CD4:CD8 ratio in peripheral blood cells is approximately 2:1 in healthy individuals, and deviations from this ratio can be prognostic indicators of immune dysfunction, especially of immunodeficiency and autoimmune diseases. This ratio is monitored closely clinically in conditions such as AIDS. However, the blood does not always mirror lymphocyte alterations in other tissues, and inferences about pathological changes in other organs must be made judiciously. As a further caution, CD4 and CD8 are not absolute indicators of cell function. Bone marrow and lymph nodes may also be examined in addition to blood, to determine if the immune cell distributions are normal. Flow cytometry can also analyze cell surface antigens that are unique to cells at different stages of differentiation,

Figure 8 A diagnostic testing algorithm for primary immunodeficiency diseases. Source: From Ref. 37.

Chapter 4: The Immune System and the Immunocompromised Patient

Table 4 Normal Adult Circulating White Blood Cell Populations Cell type White blood cells Lymphocytes T-cells B-cells NK cells Monocytes Granulocytes Neutrophils Eosinophils Basophils

Absolute counta 4.5–11.0 1–4.8

0–0.8 1.8–8.4 1.8–7.7 0–0.5 0–0.2

Percentage 25–30% of white blood cells ~70% of lymphocytes 15–25% of lymphocytes 5–15% of lymphocytes

50–70% of white blood cells

(103 cells mm3) Abbreviation: NK, natural killer. a

as described earlier for surface Ig isotypes in B-cell development. Flow cytometry has basically replaced previous tests such as E-rosette formation for T-cell detection and fluorescent microscopy detection of surface Igs for B-cells. Flow cytometry can be used to enumerate a specific cell type, evaluate function, or detect a specific gene product. Most major immunodeficiency diseases can be diagnosed by flow cytometry, with the exception of Che´diak–Higashi syndrome, ataxia telangiectasia, and complement deficiencies (38). The enzyme-linked immunospot (ELISPOT) was developed to detect and quantitate plasma (antibody-secreting) cells and provided a rapid and versatile alternative to conventional plaque-forming assays. The ELISPOT is very sensitive and detects the high concentration of antibody that surrounds each plasma cell. The ELISPOT has more recently been used to enumerate antigen-specific T-cells via detection of cell-associated cytokine production in patients with viral infections, cancer patients, and transplant recipients. Peripheral blood cells can be measured directly or after ex vivo stimulation with antigen. The frequency of antigen-specific CTL, Th cells, and their precursors can be measured in peripheral blood by in vitro limiting dilution analysis (LDA) of PBMC. LDA is not routinely available in clinical laboratories. Although LDA is a standard immunological method in many research laboratories, it is cumbersome, labor intensive, and operator dependent.

Serum Components Serum Ig levels can be determined electrophoretically and by enzyme-linked immunoabsorbent assay (ELISA). In humoral immunodeficiency diseases, serum Igs may be totally absent, as occurs in X-linked agammaglobulinemia, or decreased, as occurs in common variable hypergammaglobulinema. There may also be selective increases or decreases in certain classes of Igs in the immunocompromised patient. Complement components can also be accurately measured in serum (41), most typically by the CH50 test, which simply measures the amount of serum required to lyse 50% of antibody-coated sheep red cells. If abnormal, this test is followed by analysis of individual complement components, usually C3 and C4. Although not commonly occurring in the homozygous state in humans, genetic deficiencies have been identified in each of the complement components and are associated with increased susceptibility to bacterial infections and/or immune complex diseases, including systemic lupus erythematosus and glomerulonephritis (31). Interestingly, an inherited deficiency in C1 inhibitor, a complement regula-

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tory protein, occurs more frequently and results in hereditary angioedema. The functional activity of lymphoid cells can be evaluated on the basis of the cytokines they secrete. As described above, the different populations and subpopulations of leukocytes secrete distinct cytokines and all of the major human cytokines can be measured by ELISA or bioassay and more recently by flow cytometry. However, accurate methods are still lacking for the standard quantitation of many immune mediators in biological fluids and cells (42). For many cytokines, serum levels have not proven to be consistent indicators of immune status. This is partly due to their short half-lives and the fact that many cytokines are associated with protein carriers in the serum or are predominantly present in latent, inactive forms. In addition, cytokines typically act locally in an autocrine or paracrine manner and are not usually systemic effectors. Cytokine production is thus often measured after the isolation of leukocytes from an individual and subsequent in vitro culture with antigen. In bacterial septic shock, high levels of both pro- and anti-inflammatory cytokines are detectable within the bloodstream (43). In this condition, bacterial cell wall endotoxins stimulate macrophages to overproduce IL-1 and TNF-a, and high blood levels of these cytokines have been associated with poor survival. These and other proinflammatory cytokines mediate drastic systemic effects that can be fatal, including a drop in blood pressure, fever, diarrhea, and extensive blood clotting. However, therapies targeting these cytokines have been clinically ineffective (44). Although not routinely analyzed clinically, other regulatory molecules circulate in the serum in soluble form and may modulate the immune response. Many of these are cell surface antigens that can be synthesized in a soluble form by alternative mRNA splicing or can be ‘‘shed’’ or generated via proteolytic cleavage from the cell surface. Soluble forms of certain antigens are frequently produced by activated cells, thus elevations may indicate an ongoing immune response. For example, many cytokine receptors exist as secreted/soluble proteins (45) and are involved in the endogenous regulation of cytokine activity. Certain soluble cytokine receptors appear to serve as markers of immune suppression (IL-2R) and sepsis (TNFR). There is substantial interest in the potential application of soluble cytokine receptors in biological therapy (46). Serum components can also be assayed indirectly by measuring their mRNA levels. Quantitative molecular methods are being implemented, such as real time reverse transcriptase–polymerase chain reaction, and offer the advantages of speed and specificity.

Leukocyte Function Analyses can also be conducted to determine if the leukocytes that are present in a patient exhibit normal functional activities. PBMC are generally isolated by density gradient centrifugation on Ficoll-Hypaque, prior to analysis in one of the following tests. Mitogenic Proliferation Mitogens are substances that induce DNA synthesis and cell division. Mitogens are often termed polyclonal activators, because they activate cells without regard to their clonal antigenic specificities. Many mitogens are plant proteins termed ‘‘lectins,’’ which have affinities for specific carbohydrate moieties on cell surface glycoproteins and glycolipids. Some

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mitogens are T-cell specific [such as concanavalin A (ConA) and phytohemagglutinin (PHA)], some are B-cell specific (such as the LPS of gram-negative bacteria), and some mitogens activate both T- and B-cells (such as pokeweed mitogen). Proliferation is generally measured in the laboratory by incorporation of 3H-thymidine into DNA. Finally, many superantigens are potent activators of T-cells (e.g., staphylococcal enterotoxin A) and B-cells (e.g., staphylococcal protein A). Mixed Lymphocyte Reaction The mixed lymphocyte reaction (MLR) is an assay in which lymphocytes from different individuals are cultured together, and proliferate in response to allogeneic antigens. In the typical one-way MLR, cells from the individual to be tested (the responder cells) are mixed with different allogeneic stimulator cells that have been irradiated to prevent proliferation. This assay can also be used to test the ability of the stimulator lymphocytes (the APC) to present antigen. The amount of 3H-thymidine incorporated into DNA is determined after four days of culture and is proportional to the proliferative response. The MLR primarily measures the proliferation of Th cells and thus is a strong indicator of differences in the MHC class II antigens of responder and stimulator cells. However, MHC class I antigens as well as minor transplantation antigens have also been reported to contribute to this response. Nowadays, this assay is no longer used to measure compatibility at the MHC class II region. Molecular methods using polymerase chain reaction–based methods are being used to determine MHC alleles. Cell-Mediated Lympholysis It is an analogous assay to the MLR, but analyzes the activity of CD8þ CTL instead of CD4þ Th cells. This assay can evaluate in vivo–generated CTL (isolated from the circulation) or CTL that are induced in vitro by MLR. In either case, effector function is measured by the ability of these cells to lyse target cells. Target cells are generally allogeneic or virally infected syngeneic cells. Target cells are genetically identical to the cells injected or transplanted in vivo or identical to the stimulator cells for in vitro MLR-induced cell-mediated lympholysis (CML) assays. Target cells are prelabeled intracellularly with 51chromium or a dye that, upon lysis, is released from the cell into solution at a level proportional to the level of cell-mediated cytotoxicity. When conducted as an MLR-induced CML, this assay can also be used to evaluate the functional ability of Th cells to induce CTL effectors.

In Vivo Tests DTH is the most common in vivo assay of cell-mediated immune status in humans. The presence of a DTH reaction can be measured in vivo by injecting the antigen intradermally. A characteristic skin lesion caused by erythema at the injection site indicates a positive reaction and is evident 48 to 72 hours after antigen application. DTH can be used to assess cellular immune function by evaluating the response of an individual to recall antigens—antigens to which the patient presumably had been previously exposed. The CMI multitest includes eight recall antigens. Dinitrochlorobenzene (DNCB) has also been used as an antigen to test for cellular immune function, and patient response to de novo antigen challenge is measured two weeks after the initial DNCB skin contact. DTH has been used in a variety of clinical settings, including patients in intensive care units, surgical populations,

and transplant and HIV patients. This assay has been used to predict prognosis, overall survival, and response to therapies. The prototype of this test in humans is the administration of the purified protein derivative (PPD) extracted from the cell wall of Mycobacterium tuberculosis; however, other test antigens from the organisms causing histoplasmosis, candidiasis, and nocardiosis are also used. This test is usually given to determine if an individual has been previously exposed to the bacteria (either through infection or prior immunization). A more sensitive ELISPOT has been developed to detect T-cells specific for M. tuberculosis using antigens that are absent from Mycobacterium vaccines and most environmental mycobacteria (47). The status of humoral immune function can be tested in vivo by testing antibody titers to recall antigens such as diptheria and tetanus toxins. If titers are low, antibody responses to these antigens can also be tested two weeks after antigen boost or after active vaccination. Pneumococcal vaccine has been used in this manner to test human antibody responses to carbohydrate antigens. B-cell function can also be tested by analyzing serum for appropriate isohemagglutinin titers (antibodies against the A or B blood group antigens). In addition, if the patient has experienced a documented infection, the current antibody titer against the specific organism can be informative. Finally, the function of circulating neutrophils and monocytes can be evaluated by the nitroblue tetrazolium (NBT) test. NBT is reduced during the normal respiratory oxidative burst and can provide both qualitative and quantitative data on freshly isolated cells.

THE IMMUNOCOMPROMISED SURGICAL PATIENT Immunodeficiency Clinical States Primary Immunodeficiency There are more than 95 primary immunodeficiency diseases (Table 5). Some are manifest from birth while others are not identified until much later in life. The World Health Organization has categorized primary immunodeficiencies into five basic groups: (i) combined deficiencies, (ii) predominantly antibody deficiencies, (iii) cellular deficiencies, (iv) complement deficiencies, and (v) defects of phagocytic function (49). The major complication of primary immunodeficiencies is an increased susceptibility to viral or bacterial infection, although there are increases in certain types of cancer. Patients with severe T- or B-cell immune defects have an increased incidence of cancer and autoimmune diseases. The type of immunodeficiency can determine the type of cancer that develops. In those patients with combined immunodeficiencies, non-Hodgkin’s lymphomas represent the major type of tumor reported. This was the case even though almost half of the patients had received some therapy aimed at immunoreconstitution (50). In patients with SCID, the major cause of death is infection. Those patients with primary antibody deficiencies have an increased propensity to recurrent and chronic pyogenic infections, usually involving the respiratory tract. The usual tumor is a lymphoma, with gastrointestinal carcinomas following. Compared to other immunodeficiency states, central nervous system lymphomas are less common. The incidence of gastric carcinoma may be related to the gastric atrophy, achlorhydria, and decreased acid secretions. Many of these patients have low IgA levels and may be at increased risk of Helicobacter or related infections (51).

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Table 5 Examples of Primary Immunodeficiency Diseases Designation

Gene

Description/pathogenesis

Antibody deficiencies XLA

BTK

Mutations in the gene encoding BTK, a regulator of B-cell development; absence of mature circulating B-cells and undetectable or substantially low serum Ig levels lead to recurrent bacterial infections during the first year of life. Absent or marked reduction of serum IgA; majority of patients are asymptomatic; others have recurring respiratory infections, chronic diarrhea, allergies, or autoimmune disease. Defect in the AICDA required for Ig isotope switching and somatic hypermutation in Bcells; low IgG and IgA, normal or increased IgM. Low IgG, IgA; normal or increased IgM; bacterial and opportunistic infections.

IgA deficiency

Hyper-IgM syndrome type 2 (AID deficiency) Hyper-IgM syndrome type 3

IGAD1

AID (AICDA) CD40

Combined B- and T-cell deficiencies X-linked SCID

IL2RG

Jak3 deficiency

JAK3

RAG1 deficiency RAG2 deficiency ADA deficiency

RAG1 RAG2 ADA

X-linked hyper-IgM syndrome

CD40L

WAS

WASP

Ataxia-telangiectasia X-linked lymphoproliferative syndrome

ATM SH2D1A (SAP)

Phagocytic defects LAD1

ITGB2

LAD2

FUCT1

Most common form of SCID; caused by a mutation in the IL-2 receptor gene on the X chromosome needed for the normal growth and function of T-cells and B-cells; lymphopenia occurs primarily from the absence or near absence of T-cells and natural killer cells; B-cells are immature and defective. Mutation in the gene that encodes JAK3 needed for differentiation of hematopoietic cells; lymphopenia occurs primarily from the absence or near absence of T-cells and natural killer cells; B-cells are present but defective. Mutations in RAG1 lead absence of mature B- and T-cells. Mutations in RAG2 lead absence of mature B- and T-cells. Mutation in a gene encoding the enzyme ADA; lymphopenia occurs from the death of B-cells because of accumulation of toxic metabolite and functional antibodies are decreased or absent. Mutations in the CD40 ligand gene lead to impairment of T-cell/B-cell interaction, lack of Ig isotope switching; recurrent and opportunistic infections. Defect in cytoskeletal WASP, affecting platelets and T-cells, leads to thrombocytopenia, small defective platelets, eczema, lymphomas, autoimmune disease, and infections. Progressive multisystem disorder characterized by neurologic impairment with ataxia, telangiectasia of the conjunctiva and skin, malignancy, and radiation sensitivity. Uncontrolled lymphoproliferation induced by severe EBV infections, B-cell lymphoma.

Disorder of neutrophil adhesion caused by lack of CD18 characterized by recurrent or progressive necrotic soft-tissue infection, periodontitis, poor wound healing, leukocytosis, and delayed umbilical cord detachment. Defect in GDP fucose transporter 1; associated with mental retardation, soft-tissue infection, and delayed healing.

Complement deficiencies Deficiency of individual complement CIQA, C1QB, C1QG, Absence of complement components; results in increased infections and lupus-like components C1q, C1r, C1s, C2, C3, C4, C5, C1R, C15, C2, C3, diseases; C1, C2, C3, and C4 associated with autoimmunity and pyogenic C6, C7, C8, C9 C4A, C4B, C5, C6, C7, infections. C8A, C8B, C8G, C9 Factor B, Factor H1 BF, HF1 C5–C9 and properdin deficiencies associated with neisserial infections. Abbreviations: Ig, immunoglobulin; SCID, severe combined immunodeficiency disease; WAS, Wiskott–Aldrich syndrome; BTK, Bruton’s tyrosine kinase; AICDA, activation-induced cytidine deaminase; XLA, X-linked agammaglobulinemia; JAK3, Janus-associated kinase 3; RAG, recombinase activity gene; ADA, adenosine deaminae; WASP, WAS protein; EBV, Epstein–Barr virus; GDP, guanosine diphosphate. Source: From Ref. 48.

Those patients with well-characterized immunodeficiencies such as Wiskott–Aldrich syndrome also have an increased risk of developing malignant tumors. This risk is almost 100% by the age of 30 in patients with Wiskott–Aldrich syndrome, with the most common site of involvement being the brain (52). Respiratory infections are common in ataxia telangiectasia. Non-Hodgkin’s lymphomas account for almost half of the malignancies in this group of patients, however, almost a quarter of the patients develop leukemia. Bone marrow transplantation has been used successfully in the treatment of patients with immunodeficiencies. Gene therapy has also been used but with limited success. The patients who have a primary immunodeficiency and those who develop a secondary immunodeficiency develop a high proportion of lymphoproliferative disorders.

The incidence of cancer in patients with primary immunodeficiencies is approximately 1% to 4% (47). It must be remembered that this apparently low figure is for all patients with immunodeficiencies and that there are specific immunodeficiencies that have a very high incidence of cancer. This incidence is comparable to the incidence of cancer in organ transplant patients, which is approximately 1% to 5% (53). In cardiac transplant patients, the risk of posttransplant B-cell lymphoproliferative disease is 5% to 6% of patients. This is believed to be due to the very high immunosuppressive drug doses that cardiac recipients receive (54). The increased use of powerful immunosuppressants as induction agents for many solid organ transplants has increased the incidence of post-transplant lymphoproliferative disease. This has been shown to be associated with the

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use of OKT3 or antithymocyte globulin, but not with antiCD25 antibody (55). Together, these data would lead to the conclusion that there may be a common mechanism to explain the incidence of cancer in patients who have been immunosuppressed to maintain an organ transplant and in patients suffering from primary immunodeficiencies.

Transplant Recipients as Secondary Immunodeficiency Patients Transplant recipients can be viewed as secondary immunodeficiency patients due to the chronic use of immunosuppressive agents (Table 6). The interval between transplantation and the onset of a cancer after transplant (approximately 32 months) is much shorter than that in the general population that has been exposed to a carcinogen (5–20 years) (56). The time of appearance of the cancer after transplant is dependent on the type of cancer that occurs. Kaposi’s sarcoma appears an average of 21 months after transplant compared with anogenital carcinomas, which occur an average of 115 months after transplant. The incidence of most common cancers such as lung, breast, prostate, colon, and invasive uterine cancer observed in the general population is not changed in transplant recipients. However, squamous cell carcinomas of the skin are markedly increased. A variety of tumors in the transplant recipients compared to the general population are as follows: lymphomas, 23% versus 5%; lip cancers, 7% versus 0.3%; Kaposi’s sarcoma, 6% versus minimal; carcinoma of the kidney, 5% versus 2%; and sarcomas, 1.7% versus 0.5%, respectively (57). A population-based study from Sweden shows the same patterns: an excess of nonmelanomatous skin cancers with a standardized incidence ratio (SIR) of 56, lip cancer (SIR 53), and non-Hodgkin’s lymphoma (SIR 6). There was also a 10-fold increased incidence of anal cancer and a 20-fold increase in vulvar and vaginal cancer compared to the general population. Those patients with a nonrenal transplant had a higher risk of cancer compared to those with a renal transplant (58). Thus in certain tumors the incidence is markedly different from the general population. Viral infections have been postulated to play a role in the etiology of cancer development in these immuno-incompetent patients. Epstein–Barr virus (EBV) has been associated with Burkitt’s lymphoma and Hodgkin’s disease in the general population. Approximately 30% to 60% of patients with primary disorders are EBV positive. The postulated mechanism is that the virus infects B-cells and stimulates a polyclonal B-cell proliferation (59). B-cell proliferation is abated in normal patients by cytotoxic T-lymphocytes, but in immunocompromised individuals the proliferation continues

unchecked. This proliferation of multiple B-cells is occasionally interrupted by an unknown event, which results in the emergence of a monoclonal tumor, thus explaining the incidence of EBV-DNA–specific sequences in tumor specimens of patients who have received a transplant, who are congenitally immunodeficient, or who have AIDS (60,61). The reasons why patients with primary immunodeficiency or transplants develop disturbances in immunity that result in cancer are not clear. There are several theories that are open to discussion. The first theory is that the patients have a defective surveillance system. If this were the case, then one would expect a higher percentage of cancers of all types and not an increase in certain cancers that have been described. Indeed in those patients who develop a non-Hodgkin’s lymphoma or a Kaposi’s sarcoma, when immunosuppression is withdrawn, the patients will frequently have a regression of their tumor. Thus the innate immune response can be effective. Another popular theory is that patients have chronic antigenic stimulation. Animal models have shown that continuous stimulation with a foreign antigen can give a high yield of lymphomas (57). AIDS patients have chronic infections, which could result in continued immunologic stimulation. Transplant patients have a chronic source of antigenic stimulation from the transplant, which could thereby result in the development of lymphomas. Transplant patients may also develop cancer because of the carcinogenic effects of the immunosuppressive agents. Azathioprine, cyclophosphamide, and cyclosporin may directly damage DNA and cause malignant changes (57). However, most animal models require these drugs in combination with another chemical or physical agent to develop cancer. There is also an established increased incidence of skin cancer in patients who reside in climates with sun exposure, such as the Southwestern United States or Australia. The most fascinating question regarding cancer development in immunocompromised patients is why such a small percentage of these patients develop cancer. There may be a genetic predisposition in a small number of patients for an increased susceptibility to viral infections or malignancies. There are several studies that link HLA types to susceptibility or resistance to Kaposi’s sarcoma.

Malnutrition General Malnutrition is an important problem in surgical patients. In fact it is the most common cause of acquired immunodeficiency in the world. Surgical patients who present with cancer, fistulas, burns, and trauma are predisposed to the

Table 6 Effects of Major Immunosuppressive Agents

Myelosuppression Cell specificity Delayed wound healing Impaired microbial defense Organotoxicity Tumorigenesis Diabetogenicity Therapeutic monitoring possible a

Calcineurin inhibitors

Sirolimus (Rapamycin)

Mycophenolate mofetil

– – þ þa Yes

þ þ þ – – Yes

þ þ – – – Yes

Significant for Tacrolimus (FK506), not cyclosporin. Abbreviations: , No effect; þ, small, variable effect; , significant effect.

Corticosteroids

Anti-CD3 monoclonal antibody

Anti-CD25 monoclonal antibody

Polyclonal antilymphocyte antibody

– – – – þ No

– – þ – – No

– – – – – – No

þ – – – – No

Chapter 4: The Immune System and the Immunocompromised Patient

development of sepsis and infection. The relationship between host nutrition and wound healing has been recently reviewed (62). A consistent finding in malnutrition is the lack of maturation of T-lymphocytes in the thymic environment. This is caused by decreased amounts of thymic hormones and a defect in the thymic epithelium, and results in the release of immature cells to the periphery (63). The decrease in mature T-lymphocytes is manifested by in vivo abnormalities such as lack of impaired DTH responses. In vitro, this is detected as a decrease in mitogen and antigen lymphocyte proliferative responses to mitogens and antigens (64). Malnourished patients exhibit many abnormal T-cell responses that lead to additional host abnormalities in those processes that are dependent on the T-cell response. These include macrophage activation, T-cell–dependent antibody production, and T-cell–mediated cytotoxic responses to viruses. The patient who is malnourished as well as vitamin A deficient has depressed CMI. These individuals also exhibit decreases in antibody response, and there can be defects in B-cell clonal expansion. One can show that Ig levels are normal or even high in these malnourished patients (65). Even with the above changes, the use of live vaccines in malnourished children is generally safe. The complement cascade is very important in the defense against infection. Malnutrition results in decreased serum levels of most complement components, particularly C3. In addition, the normal increase in complement levels seen in stress situations is blunted. In malnourished children, complement activation by both pathways appears to be intact but diminished (particularly the alternative pathway). This may be a major predisposing factor to the increased susceptibility of malnourished patients to gramnegative sepsis (66). Clinically malnourished patients tend to fare poorly and are found to have prolonged infections or infections of greater severity than patients with normal nutritional levels. Malnutrition and infection can become part of a vicious cycle, with infection causing increased malnutrition, which in turn causes prolonged or exacerbated infection. Malnutrition is a major predisposing factor for complications in postoperative patients. It is associated with decreased wound healing, an increased complication rate, and an increased death rate (67). The route of nutritional support has also been determined to be of importance in the posttraumatic care of patients. Moore et al., have shown that among patients with abdominal trauma, those who received early enteral feedings had significantly fewer intra-abdominal abscesses and lower rates of pneumonia, compared to patients who received delayed total parenteral nutrition (TPN) (68). These findings were confirmed by Kudsk et al., who found a decreased incidence of intra-abdominal abscesses, pneumonia, and number of infections in patients who received enteral feeding (69). Interestingly, these findings are opposite to those found by authors who reviewed enteral and parenteral nutrition in patients with severe head injuries, who show that parenteral feedings were more favorable (70). Thus one must evaluate a patient carefully and make sure that there are no contraindications for a particular form of nutrition. A recent review of data published in 2001 and 2002 confirmed the benefits of preoperative administration of immunonutrition in surgical patients (71). Decreased infectious complications were observed in critically ill patients receiving immune-enhancing diets. However, postoperative

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administration offered no advantages to surgical, critically injured, or critically ill patients in this data review.

Nutritional Supplements The number of nutritional supplements that have been identified in nutritional support has continued to increase. There are some agents that have been shown to have clinical utility and others that are still experimental. These agents include vitamins, prostaglandins, lipids, and amino acids. Vitamins that have been studied include A, D, E, and K. Vitamin A deficiency has been shown to have a markedly favorable influence on the survival of children who have measles and in patients with bacterial, protozoal, and viral infections. Vitamin A levels are markedly decreased in postoperative patients or in those who have suffered a major burn. In these groups of patients, vitamin A supplementation is crucial and can reverse the postinjury immunosuppression (72). Other fat-soluble vitamins such as D, E, and K are important in immune function. Vitamin E has a major influence on immune function and its deficiency results in a decrease in T-cell–mediated antibody response. Fatty acids also have an influence on immune function. Linoleic acid can prolong graft survival in mice, whereas a deficiency in the same fatty acid decreases allograft survival in the same model. These results indicate the importance of linoleic acid in immunomodulation (73). Diets high in soybean, corn, sunflower, and safflower oil suppress the response of splenocytes and T-cells to mitogens, in mice and guinea pigs. These same sources of fatty acids also result in decreased immune competence. Large amounts of prostaglandin E2 (PGE2) are produced by suppressor cells in chronic inflammatory conditions such as multiple sclerosis, rheumatoid arthritis, and infections (74). PGE2 is produced by monocytes and macrophages and can modulate the responses of immune cells. The importance of PGE2 has been studied in patients with chronic infections, who have abnormally high circulating levels of PGE2. Postoperative patients who experience a suppressed immune function have a marked increase in PGE2 synthesis (75). Tumor cells can also stimulate large amounts of PGE2 to be released by macrophages. These macrophages seem to produce PGE2 in an uncontrolled fashion and are not subject to the normal feedback mechanism. PGE2 can suppress NK cells, T-cell proliferation, and cytotoxic T-cells and cause an overall decrease in immune surveillance, and may be one mechanism by which tumor cells escape normal immune surveillance (76). Patients with Hodgkin’s disease produce four times the normal amount of PGE2. This suppresses the immune response by inhibiting the production of lymphokines necessary for lymphocyte function. Prostaglandin synthetase inhibitors have been shown in some models to improve immune function and inhibit tumor growth. An amino acid that has been shown to be of major importance in stress situations is arginine. L-Arginine is the substrate for the synthesis of nitric oxide as well as other biologically active molecules. It is now viewed as an essential amino acid for young mammals (including parentally fed human infants), and as a conditionally essential amino acid for adults under conditions such as trauma and burn injury (77). Arginine has also been shown to be of importance in wound healing (62). Through both nitric oxide– dependent and nitric oxide–independent effects, it has been shown to reverse endothelial dysfunction and improve many common cardiovascular disorders (78).

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Finally, arginine has documented effects on immune functions. However, both animal and human studies have shown that enteral arginine supplementation as a single variable does not show clear immunologic benefit (79). Inconsistent results to date may be due to differences in the amount and timing of arginine administration, the animal species, and model. A recent study suggests that expression of the arginine-metabolizing enzyme arginase may be essential in regulating the cellular immune response and the inflammatory process during a critical illness (80). In human studies, oral arginine significantly increases peripheral blood lymphocyte blastogenesis in response to ConA and PHA. This change begins within three days of supplementation. Supplementation has also been studied in the clinical setting of patients receiving TPN with and without arginine. Those patients who received arginine showed an increased response of both PHA and ConA stimulation at days 3 and 7 after surgery, compared to those patients who did not receive arginine (81). The same author completed a study of HIV patients with a CD4/CD8 ratio of 0.8 or less (normal ¼ 2.0). These patients were supplemented with 20 g of arginine daily for at least two weeks. There were no changes in the CD4/CD8 ratio but there was a significant increase in the mitogenic responses to ConA and PHA in the patients tested. Arginine has also been used in postoperative surgical patients and has been shown to give an increased T-cell response to ConA and PHA as well as increased Th cell numbers. There was no change in other T-cell subsets (82). As a note of caution, Sucher et al. indicate that arginine in larger amounts may increase systemic inflammation and result in poorer clinical outcomes (83). Glutamine, a nonessential amino acid that has large body stores, is depleted following major insults such as infection and injury. Supplementation with additional glutamine in adult trauma patients resulted in a decrease in soluble tumor necrosis receptors and a lower incidence of sepsis and pneumonia (84). Similar improved outcomes were shown in severely burned patients (total body surface area greater than 25%). Intravenous glutamine was given to isonitrogenous controls or treatment patients. C-reactive protein levels and the incidence of gram-negative bacteremia were significantly decreased in treated patients. Nutritional parameters such as transferrin and prealbumin were improved compared to controls at 14 days. These studies suggest the benefits of glutamine, but the mechanisms and eventual place in surgical therapy remain to be defined (85). Cerra et al. reported the use of arginine, RNA precursor purines and pyrimidines, and n-3 polyunsaturated fatty acids, a major component of the cell membrane, in a group of intensive care unit (ICU) patients (86). These three agents in combination were associated with an improvement in the in vitro responses to ConA, PHA, and tetanus antigen. This study showed that nutritional supplementation must not only supply calories but must also supply elements that are essential to the improvement of immune parameters.

Trauma, Burns, and Surgery Trauma and Surgery The importance of immune changes during trauma, burns, or surgery is becoming better understood. Much of the data that have been discovered are now being used as a basis for intervention and treatment to prevent further morbidity after the traumatic event. These immune changes may occur secondary to blood transfusions, tissue injury, or the neuroendocrine changes that have occurred (Table 7).

Even in the individual who has undergone uncomplicated surgery there are postoperative changes in the immune system, which include a depression of both T- and B-lymphocytes. There is also a decrease in the response to a number of agents that cause a blastogenic response, including PPD, Staphylococcus aureus, and Escherichia coli, pokeweed mitogen, and phytohemagglutinin. Even with uncomplicated surgery there can be major depression in these blastogenic responses. The major change occurs on day 3 and normality is restored by day 9 (88). In patients who were judged to have moderate to severe injuries, creatine phosphokinase (CPK), cortisol, and white blood cell and T- and B-cell counts were performed. There were depressions in the total number of T- and B-lymphocytes as well as in the WBC by injury day 1, which returned to normal within approximately five days. During these changes in lymphocyte levels, the serum cortisol and CPK levels also increased markedly and returned to normal within five to seven days (89). The realization that major trauma caused a change in immune status provided impetus to examine other measures of immune reactivity. Keane et al. studied 31 injured patients and their lymphocyte responsiveness for up to 20 days after injury, comparing patients who became septic with those who did not. When compared to normals, the immune responses in trauma patients were depressed for over 20 days. In addition, the immune responses studied were significantly lower in the septic compared to the nonseptic patients. Afterinjury responses to streptokinase, streptodornase, mumps antigen, ConA, and PHA were all depressed and did not return to normal levels even after 20 days. In addition, the mixed lymphocyte culture was also abnormally depressed. Those patients were severely injured as indicated by the number of units of blood required and by the number of organ systems injured. Blood transfusions may also have contributed to the observed immune depression (90). Another study reviewed cellular immune depression after multiple trauma in patients who did not become septic during their hospital course. Significant suppression of lymphocyte responses to mitogens was noted, and there was a reversal of the Th–suppressor ratio (normal 2.0:1 vs. patients 0.96:1) suppressor cell functional activity was also detected early after trauma. This suppressor cell activity decreased, but persisted in three patients who developed sepsis (one of whom died). These data indicate that lymphocyte abnormalities exist after major trauma and some of these depressions can be associated with ultimate sepsis (91). This depression in cellular immunity is also reflected in antibody production in surgical patients. Nohr et al. have shown that abnormalities of both in vivo and in vitro antibody production are decreased in all surgical patients and antibody production is most depressed in those patients who have demonstrated reduced DTH responses (92). The studies outlined above have basically examined lymphocyte function. There are also abnormalities in the PMN in these seriously injured patients. Defects in the adherence and killing ability of PMNs have been reported along with migration abnormalities (93). These latter patients showed marked depression of both serum and cell-mediated migration that was proportional to the degree of trauma suffered. The macrophage is a pivotal cell in the immune response exerting both helper and suppressive effects, and may play a major role in the immunosuppressive effects of trauma. In one study, macrophage function was measured in a group of traumatized patients with a mean injury

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Table 7 Effect of Illness, Injury, or Treatment on Host Defense Mechanisms Condition Primary immunodeficiencies Secondary (acquired) immunodeficiencies Age Malnutrition Malignancy Burns Infection Trauma or surgery Anesthesia Blood transfusion Diabetes mellitus Renal failure Liver disease Splenectomy Radiation Protein-losing diseases Foreign bodies Drugs

Local immunity

T-cell function

B-cell function

Phagocytic function

Complement

#

#

#

#

#

NC NC NC # # # NC NC # NC NC NC # NC # NC

# # # # # # # # # # # NC ? # NC #

# " or # # # ? ? ? ? ? NC or # # # ? # NC " or #

NC # NC # # # # ? # # ? # ? NC NC #

NC # NC # ? ? ? ? ? ? ? ? ? ? ? ?

Abbreviations: NC, no change; ", increased; #, decreased; ?, unknown. Source: From Ref. 87.

severity score (ISS) of 38 (ISS of 25–40 gives approximately 50% mortality). The number of macrophages increased almost threefold from day 3 to 14 and the number of Th cells markedly decreased. During this same period, the level of PGE2 synthesis in vitro of isolated monocytes was also found to be highly elevated and, in fact, correlated with the level of injury reflected by the ISS. The level of IL-2 generated in an in vitro assay was also decreased, which would lead to decreased clonal expansion of T-cells (94). Changes in the surface expression of MHC antigens on monocytes and T-cells can be detected in postoperative patients. Because these molecules are critical for antigen presentation, their expression is central to the specific immune response that an individual can mount to a unique antigen. Wakefield et al. detected changes in MHC class II expression in a group of patients after surgery. HLA-DR expression on monocytes declined in all postoperative patients, with a more marked decline in those who developed sepsis. All postoperative patients had a marked increase in the number of T-cells that expressed HLA-DR, with a larger increase in those patients who did not develop sepsis (95). The exact mechanism of these changes is not known, but it appears that the patients who developed sepsis were unable to mount an appropriate immune response and had major defects in antigen presentation and T-cell activation. IFN-g production has been reported to be decreased after trauma, and it has been suggested that exogenous administration of IFN-g may be effective in reducing infection in trauma patients. This effect may be related to effects on HLA-DR expression (96). Polk et al. have tested the hypothesis that increased HLA class II expression may make a difference in the survival of patients after use of recombinant IFN-g. In a randomized, prospective trial, the authors were able to show that those trauma patients who received IFN-g for 10 days had an increased level of HLA-DR expression on their monocytes, compared to those patients who received placebo only. Although there was a trend for a decreased death rate among IFN-g–treated patients, the changes were not statistically significant. There were also fewer severe infections in the treatment group that required reoperation or computerized axial tomography (CAT) scan– guided drainage. The authors believed that a larger trial or a

longer treatment period would be useful to determine the utility of IFN-g in these patients (97). Other cytokines also appear to act in part by this mechanism; granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to increase the HLA-DR expression of monocytes and thus their antigen presenting capacity (98). It may be possible to predict the potential for infection in patients based on postoperative levels of HLA-DR expression on monocytes or T-cells. Peripheral blood monocytes have also been examined for the expression of CD4, CD14, and CD16. Kampalath et al. have shown a pattern in trauma patients, which is similar to that of cord blood from immunologically naive newborns. Thus monocytes may be central to both antigen presentation as well as to the clearance of bacteria in the trauma patients (99). IL-4 and IL-10, two cytokines associated with the Th2 lymphocyte subset, have also been implicated in the occurrence of sepsis after surgery. These cytokines are generally considered immunosuppressive. In a posthemorrhage mouse model, splenocytes and T-cells showed increased levels of IL-10 release compared to macrophages. PGE2 was shown to stimulate the release of IL-10, suggesting that IL-10 may be the ultimate effector of posttrauma PGE2 (100). Mack et al. have used a mouse model in which the animals received a femur fracture and hemorrhage to show that the cytokine pattern in splenocytes after injury was consistent with Th2. This pattern may explain some of the changes in the cellular immune response after injury (101). IL-4 activity has been studied in a group of trauma patients with an ISS > 25. Plasma IL-4 was found to be higher in those with greater severity of injury, lower patient age (age 30 years or younger), and hypotension, when admitted to the hospital. Those patients who had high levels of IL-4 on admission had a lower incidence of nosocomial infection compared to those patients with a lower IL-4 level. However, if the IL-4 increased during the ICU stay, the patients had a greater incidence of sepsis, pneumonia, or renal dysfunction even if they had shown low IL-4 levels on admission (102). IL-6 in septic patients who died was elevated compared to nonseptic patients. Those patients who had a poor cytokine response to a septic challenge had a poor clinical outcome (103).

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In studying the interrelationship between Th1 and Th2 cells in trauma and burns, Goebel et al. studied the occurrence of IL-2 and IFN-g (Th1 cytokines) and IL-4 (Th2 cytokine). Following trauma (ISS > 25) in human subjects, the authors showed a markedly decreased level of IL-2 when compared to normal controls. However, the production of IL-4 was markedly increased. The same pattern was observed in a murine burn model. IL-12 was found to be significantly decreased in injured humans. In the concurrent murine model in the study, when IL-12 was given to the animals after cecal puncture and ligation, a significant decrease in death from sepsis resulted. This study suggests that there may be a shift away from Th1 cells toward Th2 cells, and this may result in an increased susceptibility to sepsis after trauma but replacement may improve survival (104). The cause of the shift from Th1 to Th2 cytokine pattern is still being investigated. The change may be due to increased levels of glucocorticoids and epinephrine, both of which inhibit Th1 but not Th2 cytokine production. The changes in cytokine levels in the traumatized and septic patient have yielded information that may be helpful in treating the patient. However, understanding of the recruitment of active cells to the site of injury has been shown to be an important aspect of medicine. Chemokines, a class of chemoattractant cytokines, are being studied in trauma, transplantation, atherosclerosis, and infectious disease. There are four main families of chemokines, represented as CC, CXC, C, and CX3C based on the cysteine motif at the amino terminal of the protein. These families are specific chemoattractants for different leukocyte subgroups. CC chemokines attract macrophages and lymphocytes, whereas CXC attract leukocytes. These proteins are major players in augmenting the ability of leukocytes to adhere to endothelial cells and then to move through the vessel wall into tissues. Chemokines have been identified in patients after trauma. Adams et al. found growth-related oncogenes (GRO)-alpha (a chemokine with a new designation CXCL1) in laparotomy pads of patients who had undergone a laparotomy and had damage control laparotomy performed (105). A subsequent study by the same authors showed the presence of IL-8, a proinflammatory chemokine, in the plasma of patients who had undergone exploration of the abdomen after damage-control laparotomy (106). Thus the homing of leukocyte subclasses is dependent on chemokines. Understanding of the role of these proteins in the surgical patient may allow manipulation to improve patient outcomes by increasing the number of cells at the damaged site.

Burns Those patients who have undergone a severe burn injury also have major changes in immune status. In addition to nonspecific loss of the skin barrier, they have other immune defects. The total number of lymphocytes is reduced, as in severe nonburn trauma, and there is a decrease in the number of Th cells as well as T suppressor cells. Lymphocytes from severely burned patients exhibit decreased expression of HLA-DR and IL-2 receptor, both markers of T-cell activation. Serum from burned mice has been shown to inhibit lymphocyte proliferation and surface antigen expression in vitro. This may be due to PGE2 effects. Patients who have experienced large burns exhibited a 5- to 10-fold increase in PGE2, which results in a concomitant suppression of the T-lymphocyte response. The lymphocyte response can be restored when PGE2 synthesis is blocked (107). Burn patients have a decreased level of IL-2, which may also contribute to the immunosuppression evident

in them. IL-2 plays a central role in immunoregulation— supporting lymphocyte proliferation, increasing Th cell proliferation, and causing CTL precursors to differentiate into cytotoxic T-cells (108). Surgery and other trauma can result in marked changes in the immune system. These changes are being studied and defined and various measures are being designed for intervention. It is hoped that these interventions will result in decreased morbidity and mortality.

Aging Surgery in the aged patient is fraught with many physiological concerns such as cardiac and pulmonary disease. The mortality rate increases with age after major surgery and after major trauma. In the aged patient, the natural barriers against infection are impaired. The skin becomes thinner and there is a loss of Langerhans cells. There is also a decrease in blood supply to the skin. There is loss of bacteriostatic effects of urine and decline of renal function leading to less acidification of the urine, and a propensity for urinary tract infection. The pulmonary tree is also compromised by decreased ventilation and decreased saliva. There are also immunological changes that occur with aging, which influence the susceptibility to infection in these patients. A study by Charpentier et al. found that there were no significant differences in T-cell function between young patients and those over 70 years of age, when T-cell levels, PHA stimulation, and allogeneic responses were compared. However, if low levels of mitogens were used, lower levels of stimulation were observed in the older patients (109). Age-related changes have also been detected in B-cell function. A Japanese group showed that there was a decrease in IgG antibody synthesis after administration of tetanus toxoid in aged patients (110). Most T- and B-cells decrease in numbers with age, however, the changes in NK cells are less straightforward. NK cells with high activity actually increase with age in humans, which is in contrast to NK cells with low activity, which may be a compensatory mechanism to cope with a decreased T-cell number (111). The activity of NK cells in different compartments of the body may provide insights into the reason for increased occurrence of neoplasms in elderly. NK cells isolated from murine spleen and lymph nodes exhibited markedly decreased NK activity. This is in contrast to those NK cells isolated from human peripheral blood cells that showed normal activity. Thus, in solid organs, cells that could prevent neoplasm may be lacking effective activity. Specific cytokines and lipid mediators are also decreased during aging. As mentioned above, there is an in vivo decrease of delayed hypersensitivity to common antigen skin tests. This decrease may also be due to decreased production of IL-2 by stimulated cells, and therefore a decrease in the number of Th cells and T suppressor cells. This decrease may be due to a decrease in the level of thymic hormones. During the aging process, there are markedly decreased levels of natural antibodies and a decrease in the primary immune response to an antigen. It has been postulated that the immunosuppression of old age is due to the development of a greater sensitivity to PGE2, which would cause increased activation of suppressor T-cells by PGE2. Newer evidence indicates an increase in IL-10 production, indicating a shift toward the anti-inflammatory Th2 cytokine response (112). These changes in delayed cellular immunity increase the susceptibility to all infectious agents. This can result in an increase in colonization and frank infection and increase the incidence of bacteremia, septicemia, and infections that

Chapter 4: The Immune System and the Immunocompromised Patient

are spread via the hematogenous route. Because of these changes, care must be taken to make certain the aged patient is as well physiologically as possible prior to elective surgery. Adequate nutrition is required. Underlying diseases must be controlled prior to any invasive procedures. Vaccinations also decrease morbidity and mortality in these patients.

Splenectomy and Blood Transfusion Splenectomy The incidence of overwhelming sepsis after splenectomy is increased compared to patients with an intact spleen. The important phagocytic and antibody production capabilities of the spleen are lost after splenectomy. The greatest risk for sepsis is from encapsulated organisms and appears to be the greatest within the early postsplenectomy period, however, this risk remains lifelong. In a study in adult patients who had either underlying malignancy or trauma as the reason for splenectomy, lethal sepsis occurred in 2.7% of adult patients approximately two years after splenectomy (113). The highest risk was in those patients who had an underlying malignancy. In this study, the most common organism involved was Streptococcus pneumoniae. Splenectomy is known to be associated with gram-negative rods, Neisseria menigitidis, E. coli, and H. influenza. Less common problems include babesiosis, histoplasmosis, and malaria. Splenectomy decreases a major protective mechanism in the body. Splenectomy in animals has been shown to decrease the removal of bacteria from the bloodstream. The liver can remove organisms that are well opsonized, but the spleen can remove those organisms that are not well opsonized. The spleen is a major site of antigen presentation to B-cells; splenic removal of an organism not only removes the organism but also promotes an antibody response in which the spleen plays a pivotal role. The spleen is the first site in which antibody to an organism is detected after exposure to the offending agent. Consistent with this observation, splenectomy also causes IgM antibody levels to decrease. Strategies for prophylaxis include prophylactic antibiotics or starting prescribed antibiotics at the first sign of infection. Pneumococcal vaccine is ideally given to patients after splenectomy. Those patients who have had splenectomy for malignancy show a poor antibody response to the vaccine. Those who have received it for trauma apparently have a better response, but this is less than in normal controls in terms of absolute titer, relative rise, and rate of rise. Those patients who receive meningococcal and H. influenza vaccine show similar titers to those obtained with pneumococcal vaccine. Clearly patients must be educated about the risks, have a supply of antibiotics, and be given the vaccines. Some physicians recommend repeating pneumococcal vaccination at 5- to 10-year intervals or in the face of a falling titer.

Hemorrhage and Blood Transfusion Hemorrhage itself can have an effect on the immune response. Experimentally, there are many changes that are documented in animal models. In a rat model, there is a marked decline in the mitogen-induced response of lymphocytes and a decrease in the production of IL-2. However, this response did return to normal within 48 hours after hemorrhage without resuscitation (114). The serum from these animals will also depress the mitogen response of lymphocytes from normal animals. The above results were confirmed using a mouse model. In addition, although there was no change in the

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relative numbers of T- and B-cells, there was still the depression of splenocyte mitogenic responses. An additional murine study added resuscitation after the hemorrhage and showed the same impairment in mitogen responses, which were evident up to day 10 when they returned to normal. The production of IL-2 was also depressed during this time course (115). Additional studies confirmed the finding that murine splenocyte IL-2 levels were depressed after hemorrhage, but also showed that IL-3, IL-6, and IFN-g were also depressed. The depression of IFN-g might result in the marked decline of macrophage function, depression of NK cell function, and decreased activation of cytotoxic T-cells. The lack of IL-6 production may result in the depressed humoral function because this cytokine is heavily involved in B-cell activation (116). Humoral immunity can also be shown to be depressed after hemorrhage in a murine model. The cause of nonspecific immune dysfunction after hemorrhage is unknown. There is data to indicate that this depression may be due to macrophage dysfunction. The macrophage is also key to antigen presentation in a shock state. In a murine model, Stephan et al. demonstrated that antigen presentation was markedly depressed after hemorrhage and resuscitation one hour later (117). The length of this depression is at least 120 hours, with an onset of as early as 15 minutes with a blood pressure of 35 mmHg. Thus this change in antigen presentation in a murine model can be early and prolonged even in the face of adequate resuscitation. Additional studies using Kupffer cells have shown similar findings. The cause of the changes in immune function after hemorrhage is yet to be clearly elucidated. The agent that is most widely implicated is endotoxin, believed to come from the gastrointestinal tract. The poor O2 and nutrient delivery during severe hemorrhage may also cause immune suppression. Hemorrhage is also a major stimulator of catecholamine release. Epinephrine has been shown in humans to result in altered mitogen-induced proliferation of lymphocytes and changes in the ratios of different subsets of lymphocytes. Catecholamines can also suppress phagocyte function and B-cell function. These changes may be addressed by various specific pharmacologic interventions. Hemorrhage plus injury has an additive detrimental effect on immune function. Wichmann et al. have shown in a murine model that hemorrhage caused a depression in splenic and macrophage function; however, the addition of a femur fracture caused an even deeper depression in these parameters (118). Thus trauma patients may be experiencing an immune depression secondary to the blood loss and shock as well as the injury. Blood transfusions are given with little thought to the potential immunological consequences. Blood transfusions are believed to have some deleterious effects on the immune system in those patients who have cancer. These changes have been studied in a number of types of cancers. In those patients with colorectal cancer, cancer recurrence was increased in patients who received blood. The results of this retrospective study took into account the complexity of the surgery. Foster et al. confirmed these findings showing that those patients who did not receive any blood during their hospitalization did significantly better than those who received blood transfusions (119). In evaluating those patients who underwent surgery for lung cancer, similar findings have been discovered. Hyman et al. reported that a group of patients who underwent resection for non–small cell carcinoma of the lung and

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were transfused showed a significantly worse five-year survival rate than those patients who had not been transfused (120). The same sort of association between blood transfusion and long-term survival has been identified in patients suffering from soft tissue sarcomas. Those patients who received blood transfusions had a 20% decrease in five-year survival. The three major cancer groups examined above are in contrast to breast cancer, renal cell carcinoma, and head and neck cancers. Breast cancer patients who had invasive breast cancer and had either a complete or partial mastectomy showed no effect of blood transfusion on long-term survival (121). Likewise, no effects of blood transfusion can be found in those patients with renal cell carcinoma and those with head and neck cancer in long-term survival. It is unclear why transfusion effects in cancer patients appear to be tissue specific. This immunosuppressive effect is clearly seen in those patients who have received a renal transplant. Early in the history of transplantation, blood transfusions to the recipient were largely avoided; however, in 1973, Opelz et al. reported a beneficial effect of transfusions on graft survival. The major reason for this effect was believed to be the immunosuppressive action of blood (122). This finding could not be replicated in those patients who received frozen packed red cells. Fischer et al. showed that transfusion with one to three units of blood caused suppression of cellular immunity, defined by a mixed lymphocyte response. When he compared the effects to autologous units, there was no depression in cellular immunity (123). Matsumoto et al. studied MLR in patients who had undergone donor-specific transfusions while being readied for transplant. He found both suppressor cells and antibodies, which he thought were anti-idiotypic antibodies. This latter finding was confirmed by Singal who showed that sera from transfused patients resulted in depression of MLR, while sera from nontransfused patients did not give the same depression (124,125). Animal studies suggested that prostaglandin E may be a mediator of these changes. There is no doubt that blood transfusion has a major influence on the immune system. Some of these immunosuppressive effects maybe desirable, as in the case of a transplant in which transfusion improves graft survival. Transfusion effects are also undesirable in the case of patients with certain types of cancer. The use of transfusions is always worth careful evaluation for the individual patient.

Renal Failure Infections are common in patients with renal failure and are a major cause of death in patients with end-stage renal disease. One would expect major changes in the immune system of these patients and, in fact, they show defects in specific as well as nonspecific immunity. Abnormal cellular immunity has been confirmed clinically in various ways. These patients also have a markedly decreased response to cutaneous injection of various antigens (126). Hepatitis B vaccination in chronic renal failure patients on dialysis resulted in a very low rate of seroconversion after vaccination compared to the general population (127). This was in the face of the normal levels of Ig in the dialysis patients. Lymphocyte counts have also been shown to be decreased in these patients; however, the CD4/CD8 ratios are reported to be normal (128). In vitro studies have shown that uremic patients have impaired responses to mitogens and allogeneic lymphocytes. Because these findings are marked in the presence

of uremic serum, there may be a soluble factor that causes this depression (129). Because these changes were seen in patients who are being dialyzed, the substance is not dialyzable. There were indications that the TCR/CD3 antigen complex in the uremic patient may be downregulated. This finding may be due to a combination of uremia, hyperparathyroidism, and dialysis itself (130). Other cells that are affected in uremia are B-cells and NK cells. Uremic patients show normal to supranormal levels of IgG and IgM, although the actual mechanism for this finding is not well delineated. There is recent evidence that monocytes may be defective in uremic patients (131). This defect also influences how the monocytes present antigens. Thus, the defects that are seen may be a combination of both T-cell and APC deficiencies. The disturbances outlined above undoubtedly have an influence on the decreased ability of these patients to handle both bacterial and viral infectious challenges. Vigilance for infection is necessary in these patients. This concern is heightened for the large number of dialysis patients who are diabetic. Patients who are diabetic and on dialysis appear to be at an especially high risk of developing infections.

Diabetes Mellitus Most of the deaths in these patients can be attributed to cardiovascular disease; however, infection is a major cause of morbidity in these patients. Early studies indicated that diabetics had a greater number of infections than did control subjects. For example, one early study reported a 2.4% incidence of limb infection in diabetic patients compared to 0.5% in nondiabetic subjects (132). In addition, those patients with diabetes for longer than 20 years were found to have a prevalence of bacteriuria (19% compared to controls with a prevalence of 8%) (133). Most clinicians agree that diabetics will have more severe infections and a more protracted clinical course than nondiabetics. The World Health Organization classifies diabetes as a secondary immunodeficiency disease. The ability of patients to fight infection is altered in diabetics. PMN granulocytes have several functional abnormalities, with decreased migration characteristics, especially in those patients with poorly controlled serum glucose levels (134). Phagocytosis is defective in diabetics, especially when the glucose level is elevated (135). However, following normalization of glucose levels, these abnormalities disappear. Lymphocyte subsets can be altered in diabetics. A study of type 1 diabetics reported a decrease in circulating CD4 cells, which results in a decrease of the CD4/CD8 ratio (136). This change may be due to decreased levels of insulin or decreased insulin activity. As was found with neutrophil function, normalization of insulin levels results in normal lymphocyte transformation upon stimulation, as well as normalization of lymphocyte levels (137). However, the report that approximately 50% of the diabetic patients who received hepatitis B vaccine responded poorly indicates that there may be additional immunosuppressive factors operative in these patients. Patients who have a well-controlled glucose level do not have a higher susceptibility to Staphyloccus infections. However, in older patients, the risk of bacteremia is higher. Specific infections can be more fatal in diabetics compared to normal patients, and those patients who are diabetic and develop bacteremia have a higher mortality rate than those without diabetes (138). Candida infections are more prevalent

Chapter 4: The Immune System and the Immunocompromised Patient

in diabetics compared to the normal population and are particularly more common in those patients whose glucose levels are poorly controlled. However, this was not the case with other fungal infections. The treatment of fungal and bacterial infections requires the initiation of good glucose control and appropriate antibiotic or antifungal therapy. Major factors that lead to increased infections in diabetics in addition to poor glucose control include underlying vascular disease and nerve damage. Poor blood supply results in inadequate oxygenation, which leads to anaerobic infection and also limits host-defense mechanisms. If a patient has a peripheral neuropathy, the result can be an ulcer, which can become secondarily infected. The key to prevention of infection in the diabetic includes control of hyperglycemia; if one reduces the glucose to normal levels the incidence of infections decreases. Complications that lead to increased levels of insulin must be avoided. One must also aggressively look for infection and treat those conditions that can result in the infection becoming widespread or locally uncontrollable.

Prevention and Therapy Clinical Guidelines Prevention of infection in the immunocompromised patient calls for an awareness of the possible infections these patients can develop and an approach to deal with the problem. One must also be aware that patients with certain immune defects can develop cancer. When these patients are examined, one must check for the presence of palpable lymph nodes and also carefully examine the skin to look for suspicious lesions. The risk of infection in these patients is dependent on both the immunosuppressive status of the patient and environmental factors. The amount of immunosuppression is dependent on the level of nutrition, the state of the various portions of the immune system, defects in the skin or mucus membranes, and any invasive lines or procedures that have been undertaken. The importance of environmental factors is illustrated by the realization that even a normal healthy individual can be infected if in contact with a large enough inoculum of an infectious agent. Thus a patient who is immunosuppressed can become infected on exposure to a proportionately smaller number of infectious agents, depending upon his overall immune status. In caring for these patients, one must apply the principles outlined in Table 8. Hand washing is particularly critical in these patients to prevent patient-to-patient transmission of infection. Deep lines such as subclavian and internal jugular lines, and Swan–Ganz catheters must be watched particularly carefully. In immunosuppressed patients, there will be no evidence of infection, and therefore changing the lines or making sure they are absolutely necessary is imperative. Nutritional support will bolster the immune system and prevent a catabolic state in the immunocompromised patient. One must also be certain that the integument is intact and that the skin does not become a site for entrance of bacteria. The Guidelines for Prevention of Surgical Site Infection from the Centers for Disease Control should be rigorously followed in this group of patients. Immunocompromised patients can develop infections in a number of places. These infections can include skin, respiratory, gastrointestinal or central nervous systems. Suspicious skin lesions should be biopsied, aspirated, or cultured. The neutropenic patient who develops a fever requires careful evaluation. Approximately 40% of patients who are

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neutropenic and develop a fever will have a documented infection (140). Only 20% of fevers in these patients are due to the underlying disease or to other noninfectious causes. The remaining 40% are due to possible but nonproven infections. The classical signs of infection in these patients frequently are missing. They will not exhibit fluctuance, calor, rubor, or lymphadenopathy as commonly as patients with a normal neutrophil count do. The physical examination should focus on skin, mucosa, and perianal areas. The therapy early on should include broad-spectrum antibiotics after adequate cultures have been obtained. The optimal drug choice is still debated. Antibiotics are useful in these patients but other methods of stimulating the immune response are being sought.

Biologic Response Modifiers Biologic response modifiers (BRM) are being explored in hopes of increasing the activity of specific groups of cells in the body. Commonly used biological response modifiers include cytokines, colony-stimulating factors, IFNs, and TNFs, but can include microorganisms, polysaccharides, polypeptides, thymic factors, synthetic compounds, polyribonucleotides, and vaccines. Most were named based upon a primary property, but these agents have a large amount of overlap in their individual effects and have many diverse effects. IL-1 is a product of monocytes and has a myriad of effects. These include activation of T, B, and NK cells and activation of vascular endothelium, fibroblast proliferation, and activity against human tumors. It has also been shown to prevent infection and protect against IL-2 toxicity. It has been shown to protect against bacterial infections in mice (141). IL-1 has also been used in patients with cancers and has been shown to increase the white blood cell count, increase the cellularity in bone marrow, and give rise to major hypotension after use (142). IL-2 is produced primarily by Th1 cells. Treatment of NK cells with IL-2 results in their differentiation into lymphokineactivated killer cells, which appear to home to the site of viral infection. Because of these promising results it was the first BRM used in clinical trials. The cytokine has been shown to be effective in a number of animal models, yet its use in clinical trials has been disappointing. Surprisingly, when used in AIDS patients, it was shown to decrease some immune functions such as that of NK cells. Because of its general effectiveness in increasing the activity of NK cells, it is still used in cancer patients. The precise dose and combination of additional chemotherapeutic agents are being evaluated in metastatic melanoma, acute myelogenous leukemia, and metastatic renal cell carcinoma. It is used at the maximal tolerated dose with the major side effects being cardiovascular. Colony-stimulating factors are commonly used in cancer chemotherapy. The major interest in these agents is because they can reverse the severe neutropenia after chemotherapy, which can lead to severe bacterial infections. Granulocyte colony-stimulating factor (G-CSF) is produced by a number of cells in the body including endothelial cells, fibroblasts, and macrophages. The agent has been used in cancer chemotherapy to prevent the periods of severe neutropenia and the use of broad-spectrum antibiotics (143). G-CSF has minimal toxicities, which include mild bone pain and cellulitis at the injection site. G-CSF is not useful in patients who have an established infection, but is only useful in preventing the neutropenia associated with cancer chemotherapy (144).

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Table 8 Principles of Infection Prevention in the Compromised Host Reduce acquisition of ambient organisms from hospital environment Avoid hospitalization Segregate patients with potentially communicable diseases Avoid overcrowding in wards and ICU Enforce strict hygiene, particularly hand washing, among physicians, nurses, and aides Avoid invasive procedures whenever possible Employ indwelling intravenous and urinary catheters only if absolutely necessary Change scalp vein needles, tubing, and intravenous bottles daily Use respiratory assistance devices, particularly those requiring intubation, with great care and with close monitoring to ensure that they are adequately decontaminated Reduce number of colonizing organisms Remove or reduce sites of prior infection Consider the use of isoniazid prophylaxis in patients with histories, positive skin tests, or chest X-ray findings of old tuberculosis Closely monitor serologic tests and clinical course in patients with evidence of old coccidioidomycosis or histoplasmosis who have now become immunosuppressed Bolster host defense mechanisms Successfully treat the underlying disease Prescribe adequate nutrition and exercise Improve respiratory toilet Control diabetes mellitus, congestive heart failure, or respiratory insufficiency Reduce local obstruction caused by tumor Protect the integrity of oral and anorectal mucosae When appropriate, prescribe immunotherapy with vaccines and/or immunoglobulin preparation Abbreviation: ICU, intensive care unit. Source: From Ref. 139.

GM-CSF factor has an effect on both stem cells and megakaryocytes and stimulates the production and antibacterial function of neutrophils and monocytes. Like G-CSF, it is produced by endothelial cells, fibroblasts, and T-lymphocytes. GM-CSF has been used in patients with aplastic anemia and those who have undergone bone marrow transplantation. It has also found clinical applications in patients with acute and chronic leukemia, Hodgkin’s and non-Hodgkin’s lymphomas, treatment-related cytopenias from chemotherapy in solid tumors (145,146), and AIDSrelated neutropenia (147). The functional capabilities of the induced cells are normal. The therapy results in increased numbers of leukocytes in peripheral blood. The toxicity is mild when used in small doses; however, there is marked toxicity in large doses, including fever, capillary leak syndrome, and pericarditis. Data from animal experiments and ex vivo human experiments suggested that administration of GM-CSF might provide clinical benefit in the treatment or prevention of neonatal infections. Although a significant number of clinical studies have administered GM-CSF to neonates, there is insufficient evidence of clear efficacy or benefit to support their routine use. Several recent clinical reviews agree that further adequately powered, randomized, placebo-controlled clinical trials of this promising immunotherapy are warranted (148). The IFNs were among the first BRM discovered to have clinical application. IFN-a has shown clinical use in hairy cell leukemia, condyloma acuminatum, AIDSassociated Kaposi’s sarcoma, melanoma, lymphoma, and chronic myelogenous leukemia (149–151). The side effects are primarily a flu-like syndrome including, fever, headache, nausea, vomiting, anorexia, and occasionally impaired cognitive function. In most patients, these symptoms abate and therapy is not limited. IFN-g is important in macrophage activation and in B- and T-cell responses (152,153). IFN-a has been used to treat hepatitis B. It has been shown to give a serocoversion rate of approximately 25% to 40%

(154). Hepatitis C has been especially prominent in the dialysis population and is associated with a chronic carrier state. Treatment with IFN-a for approximately six months has been shown to give a response rate of approximately 50%, but about half of those who respond will relapse (155). The response rate has been shown to increase with the addition of ribavirin to the IFN-a. The last group of BRM to be discussed is TNFs. This group of agents was discovered when it was shown that certain bacterial products could reduce the size of tumors (156). TNF-a is released by macrophages. It has many biological effects including: proliferation of T-cells and NK cells; adherence of neutrophils to endothelium; increased cytolytic activity of T- and NK cells; and stimulation of other BRM such as IL-1 and IL-6. It has been proved useful in hairy cell leukemia and in patients with Kaposi’s sarcoma (149,157). During clinical use the side effects of TNF-a are quite mild, including fever, headache, nausea, and vomiting. TNF-b carries some similarity to TNF-a and is produced by T-, B-, and NK-cells. It mediates the biological response to microbial infections. The agent has been used in an attempt to improve outcomes in infection in an animal model (158). Antibodies to TNF have been used in septic patients, however, there was no survival benefit noted in this group of patients (159). The use of these agents in the treatment of human disease is still being explored. The importance of BRM is now being realized, as is the complexity of their interactions. Much additional work will need to be completed before their potential for therapy can be realized.

SUMMARY The causes of immunosuppression in surgical patients have been outlined. These include malnutrition, trauma, blood transfusions, sepsis, diabetes mellitus, and cancer. Each of these causes can be evaluated and the individual problem

Chapter 4: The Immune System and the Immunocompromised Patient

corrected if possible. The individual patient with severe neutropenia must be evaluated very carefully and steps taken to prevent infection. The place of BRMs has been defined in neutropenia in certain disease states but additional uses still are to be defined.

ACKNOWLEDGMENTS The authors wish to thank Mrs. Valerie Brookins and Mr. Dorian Araneda for their assistance in the preparation of Figures and Tables.

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5 Physiologic Basis of Transplantation Yuan Zhai and Rafik M. Ghobrial

INTRODUCTION Successful clinical transplantation is limited not by technical pitfalls but rather by the immune process that mediates rejection of the transplanted tissue or organ (1). Thus, the physiologic basis of transplantation rests on understanding the immunologic rejection process. The goal of the transplant surgeon is to manipulate either the host (i.e., the recipient of the transplanted organ or tissue) or the allograft (i.e., the organ or tissue being transplanted from a donor, within the same species) to avert, minimize, or reduce this physiologic process. Immunologic events leading to allograft rejection can be divided into afferent, central, and efferent limbs. The afferent limb is the initiation of immune responses, which includes presentation of foreign histocompatibility antigens to T-lymphocytes by antigen-presenting cell (APC). The activation of antigen-nonspecific innate immunity is critical for this process, which by itself is also able to directly cause damage to allografts. The central limb is the activation of alloantigen-specific T- and B-lymphocytes, including their proliferation and differentiation. The efferent limb is execution of effector functions of activated lymphocytes, including both cellular and soluble cytotoxicities to destroy grafts. In the following discussion, we will use alloreactive T-cells as an example, and provide a mechanistic overview of various components of alloimmune responses and how alloimmune responses proceed.

ALLOGRAFT REJECTION Afferent Limb This section aims to delineate the molecular and cellular components of the immune system, which provide the structural basis of T-cell recognition of allogeneic antigens. As we know now, organ transplantation presents both physical and biological stresses to the host immune system. The first response to these stresses is inflammation, which is mediated by the host innate immune system and provides the initial milieu for subsequent activation of the host’s antigen-specific adaptive immune system.

Innate Immunity The innate immune system consists of both cellular and soluble components. Dendritic cells, macrophages, neutrophils, eosinophils, mast cells, natural killer (NK) cells, as well as intraepithelial lymphocytes at the mucosal surface, form the first line of cellular defence against infectious agents in mammalian tissues. Soluble components include complements, C-reactive protein, mannan-binding lectin, cytokines, and chemokines. Activation of the innate immune system is mediated by receptor–ligand interactions. Infectious agents

carry distinctive pathogen-associated molecular patterns, which are often lipid and carbohydrate moieties, constituents of microbial cell walls (2). They can be recognized by various cell–surface pattern recognition receptors, including toll-like receptors (TLRs), scavenger receptors (sialic acid), macrophage mannose receptor, b-glycan receptor, and galactose-specific lectin. Phagocytosis is one of the classical outcomes of the innate immune reaction. Recently, it has been recognized that the activation of innate immunity is in fact a prerequirement for effective adaptive immunity involving antigen-specific T- and B-cell responses. Despite its sentinel role in host immune responses, innate immunity has long been ignored in allograft rejection because of a common but misleading observation, in animal models, that allografts survive long term in T- and B-cell–deficient recipients. In fact, the first wave of graft-infiltrating lymphocytes are macrophages and neutrophils, but not T- or B-cells, in both allo- and xenograft transplantation (3). While it is true that innate immune responses alone may not be sufficient to acutely reject allografts, it was recently demonstrated that inhibition of innate responses could disrupt the development of alloantigen-specific T-cell responses and prolong allograft survival. An elegant example is the observation that the absence of local synthesis of the complement component C3 can promote major histocompatibility complex (MHC)–fully mismatched renal allograft survival in a murine model (4). On the other hand, with the effective control of acute allograft rejection by current immunosuppressive agents, innate immune responses, possibly spared from T-cell or B-cell targeted therapies, may show critical impact on chronic allograft rejection. In the solid organ transplantation setting, the surgical procedure and ischemia–reperfusion injury are two major antigen-independent stimulants that trigger innate immune responses in the early post-transplantation phase. Cell death is a key event in initiating the inflammation cascade, which is enhanced during reperfusion by reactive oxygen species, cellular adenosine triphosphate depletion, expression of death ligands [tumor necrosis factor (TNF)-a, FasL], and complement activation. Although, the molecular mechanism activating the innate immune system in transplantation has not been determined, the finding of endogenous ligand for TLR implied the potential role for this particular innate receptor system (5). TLRs were initially identified as homologues of the Drosophila Toll proteins, and developed in mammalians to a highly conserved receptor system that recognizes functionally essential molecules shared by microbial pathogens (6). TLRs are expressed on different cell types, particularly APCs, such as macrophages and dendritic cells (DCs). TLR engagement by microbial products initiates the process of DC maturation, resulting in the upregulation of surface expression of MHC and costimulatory

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molecules, and the production of immunostimulatory cytokines. These matured DCs are the key leading to T-cell activation and the development of adaptive immunity. Molecules generated during tissue damage and inflammation including heat shock proteins (HSPs), hyaluronan, heparin sulfate, and fibronectin have been shown to act as endogenous TLR ligands and effectively stimulate DCs via TLR system (5). Upregulation of chemokine and cytokine production as a result of intragraft DC and endothelial activation leads to the recruitment and activation of infiltrating lymphocytes, including macrophages, NK cells, and T-cells. Clearly, there are bidirectional interactions between innate and adaptive immune responses during this progressive graft rejection process: the initial innate immune activation probably starts the cellular cascade in an antigen-nonspecific fashion to trigger T-cells, whose activation then not only elicits antigen-specific adaptive immune responses, but also feeds back and amplifies the innate immune responses. Chemokines and Their Receptors As one of the major products from innate immune system activation, chemokines play critical roles in intragraft recruitment and activation of not only antigen-nonspecific leukocytes of innate immunity, but also T- and B-cells of adaptive immunity. Chemokines are a large family of low-molecular-weight (8–11 kDa), heparin-binding proteins initially recognized with chemoattractant activity for leukocytes and later with other important developmental functions, e.g., hematopoiesis and organogenesis (7). Currently, there are approximately 60 chemokines divided into four subfamilies, based on cysteine motifs near the amino-terminal end of the molecules: C, CC, CXC, and Cx3C. Functionally, however, chemokines within the same subfamily vary significantly in their biological targets and effects. Two major functional groups of chemokines involve either inflammation or homeostasis, which also differ in their expression regulation (inducible by proinflammatory or constitutive stimuli, respectively). Chemokine receptors are members of the serpentine, heterotrimeric G-protein–coupled, seven-transmembrane– spanning receptor superfamily. Eighteen chemokine receptors have been identified so far, with each binding a distinct set (more than one) of chemokines (7). The complexity and specificity of the chemokine system stem from not only the multitude of receptor–ligand pairing but also the regulated expression of ligands and receptors, which differ according to cell subsets and state of cell activation. The array of chemokine receptors displayed on a leukocyte surface regulate its homing and chemotactic potentials. Thus, each chemokine recruits only specific cell types at a particular activation or differentiation state. Table 1 lists some of the common chemokines and their receptors that have been implicated in transplant immune responses (8,9).

Alloantigen Presentation and Recognition Alloantigen-specific immune responses involve three types of lymphocytes: T-cells, B-cells, and APCs (particularly DCs). As in the innate immune response, the ‘‘adaptive immune response’’ is also mediated by receptor–ligand interactions, but in a much more complicated fashion. Multiple pairs of receptor–ligand interactions are required for effective T (or B) -cell activation. In this section, we will review the molecular structure of such receptors or ligands, including the MHC, minor histocompatibility antigens, T- or B-cell receptors, and costimulation molecules.

Table 1 Chemokine Cascades Following Organ Transplantation Chemokine cascade

Receptor

Early (3–72 hr) Gro-a/MIP-2

CXCR2

Neutrophils

IL-8 MCP-1/JE

CXCR1/2 CCR2

Neutrophils Macrophages, T

MIP-1a

CCR1/5

MIP-1b

CCR1/5

IP-10

CXCR3

Fractalkine

CX3CR1

Late (48–72þ hr) RANTES

CCR1/3/5

Target lymphocyte

Murine cardiac allograft outcome after treatment Delayed rejection (<20 days) by anti-Gro-a

Delayed rejection in CCR2 KO Macrophages, NK, No effect in MIPT 1aKO; delay rejection in CCR5 KO Macrophages, NK, T Activated T Prolonged survival ( < 20 days) in IP-10 donor KO, CXCR3 KO, or anti-CXCR3; delayed rejection by anti-IP-10 T, NK No effect in CX3CR1KO, prolong survival by anti-CX3CR1

MIG

CXCR3

T, NK, macrophages Activated T

IP-10 I-TAC

CXCR3 CXCR3

Activated T Activated T

No effect in RANTES KO Delayed rejection by anti-MIG

Abbreviations: IL, interleukin; MCP, monocyte chemotactic protein; NK, natural killer.

Major Histocompatibility Antigens Major Histocompatibility Complex. The antigen (Ag) disparity between the host and the graft initiates the immune response and ultimately the rejection process. Early experiments in clinical transplantation revealed the presence of a single, strong, antigenic system controlling the outcome of transplantation in every species. The human MHC encodes the human leukocyte antigens (HLA), including class I, class II, and class III, and many other genes that encode proteins of unknown function. The MHC spans 3.5 million DNA base pairs, approximately 2% of the genetic material in autosomal chromosome 6 (Fig. 1). The class II genes are located closest to the centromere in the order DP, DQ, and DR. The class III region encodes components of the complement system (C2, C4A, C4B, and Factor B) as well as the enzyme steroid 21-hydroxylase. The genes for HSP, TNF, lymphotoxin (LT)-a, and LT-b lie between the complement and class I genes. The most telomeric portion of the MHC contains the class I-ab chain genes in the sequence B, C, and A. Nonpolymorphic class I–like genes and pseudogenes, located in the large space between the C and A genes and telomeric to HLA-A, occupy 11 centimorgans. The function of the class I–like genes remains largely unknown. However, these genes may serve as a repository for alternative nucleic acid sequences used for generating polymorphism in the true class I molecules by the process

Chapter 5:

Figure 1 Chromosomal map of the human major histocompatibility complex (MHC). The MHC locus encodes HLA class I (A, B, and C), class II (DP, DQ, and DR), and class III, including the complement (C0 ) locus. Proteasome and transporter (TAP-1 and TAP-2) genes are located in the class II region. Genes encoding HSP and TNF/LT family of cytokines (LT-b, LT, and TNF) sit in a telomeric position to class III loci. Class I–like genes and multiple pseudogenes are located between the C and A loci. Abbreviations: HSP, heat shock proteins; HLA, human leukocyte antigen; LT, lymphotoxin; TAP, transporter in antigen processing; TNF, tumor necrosis factor.

of gene conversion (incorporation of variant sequences in the class I or class II genes without reciprocal crossing over). Located within the class II region are the genes encoding multiple subunits of a cytosolic protease termed proteasome, which is involved in the generation of peptide fragments from intracellular endogenous cytosolic proteins. The transporter in antigen processing (TAP) 1 and 2 genes are located next to the proteasome complex in the class II region. Such TAP genes encode subunits of a heterodimeric pump protein that transports the endogenous peptide fragments into the endoplasmic reticulum (ER) where such peptides bind to class I, but not to class II, molecules. Class I and class II glycoproteins serve as scaffolds for the presentation of processed antigenic peptides to T-cells: Class I molecules present peptides from endogenous proteins to CD8þ T-cells, while class II molecules present peptides from exogenous antigens to CD4þ T-cells. The polymorphism, and therefore antigenicity, of the MHC class I and class II transplantation antigens is the result of amino acid (a.a.) sequence differences between donor and recipient MHC molecules. Characteristics of Class I MHC Antigens Structure. The class I MHC antigens are expressed as complexes of a membrane-bound glycoprotein, heavy-chain polypeptide (45 kDa) that is noncovalently associated with a non-MHC encoded b2-microglobulin (b2m; 12 kDa) light chain (Fig. 2A). Crystallographic analysis of human HLA-A2, HLA-B27, and HLA-Aw68, and mouse H-2Db class I molecules revealed similar molecular configurations. The membrane-distal polymorphic a1 (90 a.a.) and a2 (92 a.a.) domains bear eight antiparallel b-pleated strands bordered by two a-helical regions (the first from the a1 and the second from the a2 domain), which jointly form the peptide-binding cleft (Fig. 2B). The a domain is attached to a less-polymorphic, immunoglobulin (Ig)-like, membrane-proximal (92 a.a.) domain, which is followed by conserved transmembrane (25 a.a.), and intracytoplasmic (30 a.a.) segments. The a3 domain contains the binding sites for the CD8 molecule. The b2m forms an Ig-like domain, which is noncovalently associated with the extracellular

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Figure 2 Major histocompatibility complex class I antigen. (A) Schematic illustration of the heavy chain including a1, a2, and a3, transmembrane and intracytoplasmic domains noncovalently associated with the light chain (b2m). (B) Schematic illustration of the peptide-binding cleft formed by the a1 and the a2 domains, each contributing an a-helix and four b-strands. Both domains are in continuity with one another (180 a.a.) to provide a peptide-binding groove with closed ends. A–F denote peptide-binding pockets formed by polymorphic a.a. residues in the cleft. NH2 and COOH are the amino and carboxy-terminal ends of the protein.

portion of the heavy chain and appears to be critical in maintaining the native confirmation of class I molecules. Antigen Presentation. Class I molecules present peptides derived from proteins in the cytosol to CD8þ-bearing T-cells. T-cells recognize both the MHC molecule and the associated peptide presented in the peptide-binding groove. The six pockets (A, B, C, D, E, and F) present within the groove (Fig. 2B) provide specificity for interactions with the peptide side chains. The closed ends of the groove result in a strong bias for the stable binding of short peptides in the range of 8 to 10 residues (10). A broad peptide-binding capacity arises from hydrogen bonding between the NH2 and COOH groups of the peptide and the residues at the end of the groove (A and F pockets). A second, more specific bonding links the polymorphic residues in the pockets B to E to specific peptide side chains (anchor residues). Such anchor peptide residues that fit optimally into the pockets of the peptide-binding groove stipulate the repertoire of peptides that associate with a particular class I molecule. For example, HLA-A0201 binds peptides exhibiting leucine at position 2 and valine at position 9. Such peptide motifs have been defined for HLA-A0205 and HLA-B27 as well as many mouse class I molecules. Thus each class I allelic product selects its bound peptides according to ‘‘specific motifs’’ characterized by an allele-specific length and positions of anchor peptide residues. Assembly and Expression. Short nanomeric peptides loaded onto MHC class I molecules are generated in the cytosol by proteasome degradation of newly synthesized proteins. Targeting of proteins for proteasomal degradation requires their ubiquitination. The peptides are transferred to ER by specialized peptide transporters, TAP, and loaded on new MHC class I molecules (consisting of class I heavy chain and b2m) under the control of a loading complex composed of several ER resident chaperons (including tapasin,

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calnexin, and calreticulin), which retard the intracellular transport of peptide-free class I molecules (11). Once associated to peptides, the trimolecular complexes are rapidly transferred through the golgi apparatus to the plasma membrane via the constitutive secretory pathway, and expressed as membrane-bound glycoproteins (Fig. 3). Class I antigens are expressed on almost all cells of the body, although levels of expression may vary and are typically highest on hematopoietic elements. Recent studies have confirmed the existence of ‘‘cross presentation’’ by MHC class I molecules, in which internalized exogenous antigens are loaded onto MHC class I molecules (12). Macropinocytosis allows receptor-independent cross presentation of soluble antigens by DCs. Physiologically, phagocytosis is the major route for antigen uptake and cross presentation. Phagocytosis of bacteria and apoptotic cells, Fc receptor (FcR)–mediated uptake of immune complex, opsonized liposomes, or dead cells all result in efficient cross presentation. Peptide bound to HSPs, which are internalized through receptors, such as CD91, also induce cross presentation. Two main intracellular pathways for cross presentation have been identified. TAP-independent endocytic cross presentation utilizes recycling MHC class I molecules in endosomes and lysosomes, where exogenous peptides exchange with endogenous peptides at low pH. TAP-dependent cross presentation utilizes a membrane transport pathway linking the lumen

of endocytic compartment and the cytosol, which allows selective delivery of internalized antigens to the cytosol, leading to ER loading of the exogenous antigens. In vivo cross presentation was mostly dependent on TAP, which may play an important role in peripheral tolerance of cytotoxic T-lymphocytes (CTLs) to tissue-specific antigens. Characteristics of Class II MHC Antigens Structure and Antigen Presentation. Class II MHC antigens are composed of a heterodimer of two transmembrane a (33 kDa) and b (29 kDa) molecular chains, each bearing one peptide-binding domain (a1 or b1) and one Ig domain (a2 or b2; Fig. 4A). Crystallographic analysis of the class II HLA-DR1 revealed a three-dimensional structure similar to that of class I molecules (13). Similar to class I molecules, class II polymorphic residues are concentrated in the a-helical sides or the b-strands of the peptide-binding cleft, which bear allele-specific motifs that interact with the peptide anchor residues at five binding pockets. In contrast to class I molecules, the peptide-binding site of class II molecules includes two noncovalently associated halves (a1 or b1), with one a-helix and four bb-strands (90 a.a.) contributed by each chain (Fig. 4B) to form an open peptide-binding groove (Fig. 5). In addition, class II molecules form conserved bonds with the main chain atoms of the peptide, not the terminal groups, thus allowing the ends of the peptides to extend out of the binding groove. Thus, class II–associated peptides exhibit a broader range of lengths, typically 12 to 24 residues (Fig. 5). Class II molecules may even bind whole proteins. Assembly and Expression. Whereas class I molecules present peptides derived primarily from endogenous proteins synthesized within the cell, class II molecules bind to foreign peptides introduced into the cell through the endocytic pathway (exogenous antigen). These differences result from distinct intracellular trafficking and assembly pathways of class I and class II molecules. In contradistinction to the endogenous pathway utilized by class I molecules,

Figure 3 Schematic illustration of antigen processing and presentation by class I. Endogenously generated proteins are degraded in the cytoplasm by the action of a macromolecular proteasome complex. Endogenous peptides are transported into the endoplasmic reticulum (ER) by specialized peptide transportors, TAP, and loaded on to new MHC class I molecules (consisting of class I heavy chain and b2m). The loading of peptides is under the control of a loading complex composed of several ER resident chaperons (including tapasin, calnexin, and calrecticulin), which retard the intracellular transport of peptide-free class I molecules. The assembled trimolecular complex (peptide, heavy, and b2m chains) is transported through the golgi complex to the plasma membranes via the constitutive secretory pathway for surface expression. Abbreviations: MHC, major histocompatibility complex; TAP, transporter in antigen processing.

Figure 4 Class II major histocompatibilty complex class antigen. (A) Schematic illustration of the a and b chains, each contains the membrane-distal a1 and b1domains that form the peptide-binding cleft, and the membrane-proximal a2 and b2 domains. Each is followed by a transmembrane and an intracytoplasmic domain. (B) Schematic illustration of the peptide-binding cleft formed by two separate a1 and b1 domains, each containing approximately 90 a.a. One a-helix and four b-strands are contributed by each domain. NH2 and COOH, amino- and carboxy-terminal ends.

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class II molecules followed by the association of the peptides with the a/b heterodimers (15,16). Vesicularly transported class II a/b-peptide complexes are then expressed on the cell surface for antigen presentation (Fig. 6). Class II molecules exhibit limited distribution to vascular endothelium and cells of lymphoreticular origin, specifically B-lymphocytes, monocytes, APCs (including macrophages, B-cells, DCs, and langerhans epidermal cells), and immune-activated T-lymphocytes.

Figure 5 Schematic illustration of peptides binding to class I or class II molecules. The continuity between a1 and a2 domains of the class I molecules provides a closed peptide-binding groove that limits binding to peptides that are 8 to 10 a.a. long. Class II molecules exhibit an open peptide-binding groove formed by two separate a1 and b1 domains, thus allowing longer peptides (12–24 a.a.) to bind and protrude beyond the peptide-binding groove. Abbreviation: a.a., amino acids.

an exogenous assembly pathway links class II molecules to the antigenic peptides generated after endocytosis and endosomal digestion of antigens by proteases including cathepsins and asparaginyl endopeptidases (14). The class II chains synthesized intracellularly associate in the ER with a nonpolymorphic, non-MHC–encoded chain, termed ‘‘invariant chain’’ (li, 30 kDa). Three invariant chains combine to form a homotrimer, with each subunit binding to a newly synthesized class II a/b heterodimer to form a nine-chain complex. The invariant chain serves to make the class II a/b heterodimers more compact, and reduces the binding of the class II molecules to endogenous peptides in the ER. The invariant chain then guides the class II complexes to the lysosomal compartments of the exogenous pathway, which contain the proteolytically cleaved, exogenous antigenic peptides. The acidic environment of the endosomes allows the dissociation of the invariant chain from

Blood Group Antigens: A Minor Histocompatibility Antigen Complex Red blood cell carbohydrate antigens, which are considered as minor histocompatibility antigens, vary in their tissue distribution and degree of expression. Rh antigens are found exclusively on red cells, while ABO antigens are widely distributed on other tissues and body fluids. The ABO system of blood typing is the only non-HLA histocompatibility system that has been identified to mediate rejection in humans (17). Renal transplantation across an ABO incompatibility is almost always unsuccessful (18). The recipient responds as a presensitized host, because he bears circulating isoantibodies toward foreign blood group antigens, thereby resulting in hyperacute rejection (see below). Renal function ceases within minutes to hours. Recently, the development of techniques for removal of anti–A and anti–B isoantibodies by plasmapheresis renewed some interest in ABO incompatible kidney transplants. Alexandre et al. reported a series of successful ABO incompatible, living, related and unrelated kidney transplants (19). Interestingly, after treatment, the isoagglutinin titers returned to near preoperative levels, even in successful grafts. A successful outcome of cadaveric renal transplantation from donors with the blood group A2 to recipients with blood group O has been reported, a success attributed to the lower density of the A2 form of antigen expressed on the donor kidney. Lewis (Le) Ags, which are blood group–related complex carbohydrate antigens consisting of 1,3 fucosylated N-acetylglucosamine groups within repeating lactosamine subunits, may also influence renal allograft survival. They result from a complex interaction between gene products of the ABO, Rh, Secretor, and Le loci. The Le antigens exist in a soluble form in the serum (Lea and Leb) and body fluids but not on erythrocytes. Oriol et al. implicated incompatible

Figure 6 Schematic illustration of antigen processing and presentation by class II molecules. The class II chains synthesized intracellularly associate in the ER with a nonpolymorphic, non-major histocompatibility complex–encoded chain, termed as the invariant chain (li). Three invariant chains combine to form a homotrimer, with each subunit binding to a newly synthesized class II a/b heterodimer to form a nine-chain complex. The invariant chain then guides the class II complexes to the lysosomal compartments of the exogenous pathway, which contain the proteolytically cleaved, exogenous antigenic peptides. The acidic environment of the endosomes allows the dissociation of the invariant chain from class II molecules followed by the association of the peptides with the a/b heterodimers. Vesicularly transported class II a/b peptide complexes are then expressed on the cell surface for antigen presentation. Abbreviation: ER, endoplasmic reticulum.

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Le Ags as the cause of renal allograft failure in presensitized hosts bearing anti-Le antibodies following multiple blood transfusions (20). Although Le and HLA disparities display negative additive effects on transplant survival, HLA identity cannot override the deleterious influence of a Le Ag mismatch. Le Ags are expressed on lymphocytes and on epithelial cells of both the distal convoluted tubules and collecting ducts. Spitalnik et al. reported that eight Le-negative individuals with circulating anti-Le antibodies, who received Le-incompatible grafts, all displayed allograft rejection (21). In contrast, Rh incompatibility in renal transplantation plays a minor role in allograft survival, except possibly in a presensitized Rh(-) recipient (17). Currently, ABO incompatibility between donor and recipient is routinely avoided by blood grouping, but Le mismatches are only sought if there has been an extensive recipient transfusion history. T-Lymphocytes T-Cell Receptor. There are two forms of T-cell receptor (TcR): the a/b heterodimers, which are expressed on the majority of mature peripheral blood T-cells (Fig. 7), and the g/d heterodimers, which are expressed on 10% of intestinal T-cells and 5% of peripheral blood T-cells. The a/b TcR recognizes processed peptides presented by classical polymorphic class I or class II MHC molecules in order to activate a wide variety of T-cell–mediated immune mechanisms, including the alloimmune response. Activation of a/b T-cells is thus ‘‘restricted’’ by the recognition of the appropriate MHC antigens. T-cells expressing the g/d TcR are not restricted by the conventional polymorphic self-MHC,

but may recognize the nonpolymorphic MHC-like molecules. The function of the g/d T-cells is largely unknown; they may play a role in the initiation of the immune response to some commonly encountered microbial antigens at the epithelial boundaries. The a/b TcR consists of two polypeptide chains (Fig. 7), the a chain (40–50 kDa) and the b chain (40–45 kDa), which bear striking similarities to Igs. Both chains have variable (V) and constant (C) domains with appropriately spaced cysteine residues, to allow the formation of interchain disulfide bonded loops similar to Ig V and C domains. Thus the TcR and many other membrane proteins, structurally homologous to Ig domains, are referred to as members of the Ig superfamily (IgSF), and the genes encoding these molecules are termed the Ig gene superfamily (Table 2). The C-terminal end of the V region in both chains (the junction between the V and C domains) is encoded by a joining (J) segment gene, and in case of the b chain only, by an additional diversity (D) segment gene. The presence of V, D, J, and C regions is also characteristic of Igs. The similarity between the TcR and Ig may reflect the common function of both structures to detect foreign antigens. The Ig domain of the C region of both chains is followed by a hinge region that forms a disulfide linkage between the two chains. The transmembrane segments exhibit positively charged side chains that interact with the negatively charged residues in the CD3 polypeptides. The carboxy terminus of the C regions of both the a and b chains forms a 5 to 12 a.a. intracytoplasmic tail, which is too short for signal transduction, rather this function is performed by the TcR-associated CD3 complex. TcR-Associated Proteins. The CD3 complex contains three (g, d, e) peptide products. The g, d, and e chains, encoded on chromosome 11, are nonpolymorphic, highly homologous peptides of 20 to 26 kDa (Fig. 6). Each chain exhibits an Ig-likeextracellular domain (79–104 a.a.), a transmembrane segment containing a negatively charged aspartic acid residue important for association with the TcR, and a cytoplasmic domain ranging from 43 to 55 a.a. The TcR x chain encoded on chromosome 1 is a 16 kDa peptide that exists primarily as a disulfide-linked homodimer, and is structurally and genetically distinct from the CD3 subunits. x chain has a short extracellular domain of only 9 a.a. and long cytoplasmic tail of 113 to 155 a.a.; its dimerization occurs through the single cysteine residue located at the extracellular boundary of the plasma membrane. The TcR, currently

Table 2 Immunoglobulin Superfamily Members

Figure 7 Schematic illustration of the T-cell receptor (TcR) complex. The TcR consists of two chains a and b, each containing an immunoglobulin-like variable domain (V) and a constant domain (C). Each chain has a transmembrane and a short intracytoplasmic segment. The TcR-associated proteins consist of the CD3 complex d, g and e chains associated with z chain homodimer. The transmembrane segments of the TcR a and b chains exhibit positively charged side chains (þ) that interact with the negatively charged residues (–) in the transmembrane portions of the g, d, e, and x chains. Antigen recognition activation motifs containing sites for tyrosine phosphorylation (P) are present in the cytoplasmic tails of the g, d, e, and x chains.

IgG Major histocompatibility antigens (class I, class II) TcR CD3 (g,d,e) Invariant chain (li) CD4, CD8 Thy-1 CD2 (LFA-2) ICAM-1, ICAM-2 VCAM-1 CD28, CTLA-4, B7/BB1, B7.2 IL-1R Abbreviations: ICAM, intercellular adhesion molecule; IgG, immunoglobulin; IL-1R, interleukin-1 receptor; LFA-2, lymphocyte function-associated antigen-2; TcR, T-cell receptor; VCAM-1, vascular cell adhesion molecule-1.

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viewed as an octomer model of receptor structure, consists of four relatively stable dimmers: ab, CD3-de, CD3-ge, and zz (Fig. 7). The cytoplasmic tails of the CD3-g, CD3-d, CD3-e, and TcR z chains, essential for the TcR signal transduction, contain a critical signaling motif known as the immunoreceptor tyrosine-based activation motif (ITAM). The concensus sequence for an ITAM is YxxL(x)6–8YxxL, (single-letter code for a.a. with x denoting any a.a.). The tyrosine residues undergo phosphorylation during TcR activation. This motif occurs once in the tails of CD3-g, CD3-d, and CD3-e chains, and thrice on the tail of the TcR z chain. Rearrangement and Expression of TcR a and b Genes. Genomic sequences encoding the b chain are located on human chromosome 7 (Fig. 8). Each Cb gene is associated with a 50 cluster of six Jb genes and one Db gene. Vb segments (75–100) are located at the 50 of the D J C clusters. The a-chain sequences on human chromosome 14 contain a single Ca gene associated with at least 60 different Ja genes and 75 Va gene segments, grouped in 12 different families (Fig. 8). The assembly of the multiple units of the V, D, and J segments (somatic recombination) is mediated by the products of the recombination activator genes RAG -1 and RAG-2 (22), found only in the immature T- and B-cells. The multiple combinatorial associations of different V, D, and J segments during TcR rearrangement allows a broad TcR diversity. Further, the ‘‘N-region diversification,’’ involving the random addition of nucleotides that are not a part of the genomic sequences at the VJ, VD, and DJ junctions, and the pairing of aa/b chains serve to multiply the diversity of the TcR. Thus, the potential size of the generated T-cell repertoire may exceed 1015 specificities. T-Cell Maturation and MHC Restriction. After migration from the bone marrow, T-cells mature in the thymus where rearrangement and expression of the TcR genes occur. The CD4 and CD8 molecules expressed on mature T-cell subsets are commonly used as markers for thymocyte maturation. Most immature cells in the thymic cortex

Figure 8 Genomic sequences encoding the TcR a and b chains. Vb segments are located 50 of the D J C clusters, while each Cb gene is associated with a 50 cluster of six Jb genes and one 30 Db gene. The a chain sequences contain a single Ca gene associated with multiple Ja and Va gene segments. The assembly of different V, D, and J segments with the C gene segments results in multiple genomic sequences encoding a and b chains with different specificities. Abbreviation: TcR, T-cell receptor.

are the rapidly dividing thymocytes, which lack CD4 and CD8 molecules (CD4CD8, double negative), but are actively rearranging the a- and b TcR genes (Fig. 9). The majority of the double-negative cells (80%) beginning to express CD4 and CD8, thus develop into CD4þCD8þ (double positive). The vast majority of double-negative cells die in vivo, and only 20% of such cells develop into CD4þ TcR a/b or CD8þ TcR a/b MHC–restricted (single positive) mature T-cells, which are predominantly detected in the medulla. These single positive T-cells constitute the vast majority of the T-cells in the peripheral circulation. A small proportion of double-negative T-cells will never express CD4 or CD8 and rearrange the g/d but not the a/b TcR genes, giving rise to the g/d TcR–expressing T-cells, which are not restricted by the conventional polymorphic self-MHC. T-cells maturing in the thymus exhibit a broad range of specificities, including clones potentially reactive to

Figure 9 T-cell maturation in the thymus. In the a/b T-cell lineage, double-negative (CD4CD8) thymocytes develop into CD4þCD8þ (double positive). The majority of double-positive thymocytes die in vivo and the remaining minority ultimately gives rise to the CD4þ TcR ab or CD8þ TCR ab self-major histocompatibility complex (MHC)– restricted (single positive) mature T-cells that recognize foreign antigen in association with self-MHC. Positive selection allows thymocytes with TcRs that recognize self-MHC molecules to survive and eliminates all non–self-MHC–reactive cells. Negative selection eliminates all T-cells that recognize self-antigens in association with selfMHC. A small population of double-negative thymocytes expresses the g/d TcR and develops into CD4CD8 non–MHC-restricted T-cells. Abbreviation: TcR, T-cell receptor.

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Figure 10 Interaction of TcR complex and accessory molecules with target ligands. (A) Interaction of CD4þ T-helper cell with APC. (B) Interaction of CD8þ T cytotoxic cell with target cell. LFA-1 binds to ICAM-1, CD2 to LFA-3, and CD28 to B7. The antigen-binding site of the TcR complex interacts with both the peptide and the peptide-binding groove of the MHC molecule. CD4 and CD8 molecules bind to MHC class II or class I, respectively. Abbreviations: APC, antigen presenting cell; ICAM, intercellular adhesion molecules; LFA, leukocyte function–associated antigen; MHC, major histocompatibility complex; TcR, T-cell receptor.

self-antigens. Thus, the second important event in the thymic T-cell maturation is the selection of T-cells that are tolerant to self-antigens, yet reactive to foreign antigens. On the one hand, there is selection of T-cells with the receptors recognizing foreign antigens in association with self-MHC molecules (positive selection) (23). On the other hand, there is selective elimination of T-cells (negative selection) that bind with high affinity to self-antigens, in association with self-MHC (24,25). Such self-reacting T-cell clones undergo clonal deletion by a Ca2þdependent process of cell death or apoptosis (26). Thus, thymic selection results in a T-cell repertoire recognizing foreign antigens, including alloantigens, in the context of self-MHC.

of the Src oncogene family p56lck. Rapid phosphorylation of the serine residues in the cytoplasmic tail upon antigenspecific stimulation of the T-cell (MHC–peptide complex interaction with TcR) underlines the role of CD4 in signal transduction. Binding of the CD4 coreceptor to the lateral surface of the MHC molecule augments signaling via the TcR up to 100-fold. However, ligation of the CD4 molecules alone, without TcR activation, inhibits T-cell activation (27). This dual opposing behavior presumably reflects an interplay between p56lck and the cytoplasmic tails of the CD3 z chains. The major function of CD4þ T-cells (65% of the a/

Accessory Molecules. The binding of the peptidehistocompatibility antigen assembly to the TcR is complemented by at least three sets of independent, accessory receptor–ligand interactions: coreceptor, CD4 to class II MHC molecules or CD8 markers to class I (Fig. 10); costimulatory molecules such as B7 to CD28; and adhesion molecules such as leukocyte function–associated antigen-1 (LFA-1) of the integrin gene family to intercellular adhesion molecules (ICAM-1 or ICAM-2). Although the TcR–CD3 complex are the key molecules in the antigen-specific recognition of the MHC–peptide complex and T-cell activation, binding of the accessory molecules is necessary for modulation of signal transduction, as well as to increase the affinity of cellular interactions with T-cells, as evidenced by the capacity of blocking antibodies directed against accessory molecules to inhibit T-cell activation. Coreceptor Molecules, CD4 and CD8. These nonpolymorphic transmembrane glycoprotein members of the Ig gene superfamily are expressed in a mutually exclusive fashion on mature T-cell subsets, which are either CD4þ or CD8þ. The CD4 (T4, L3T4) molecule (55 kDa), encoded on chromosome 12, contains four extracellular Ig-like domains, a transmembrane domain, and an intracytoplasmic tail (Fig. 11). The two N-terminal Ig-like domains only bind to the b2 domain of class II MHC, thus imposing a restriction of CD4þ T-cell interactions to class II MHC–bearing cells such as APCs. The intracytoplasmic tail is physically associated with a lymphocyte-specific protein tyrosine kinase

Figure 11 Schematic illustration of coreceptor molecules. The CD4 molecule (55 kDa) is expressed on cellular membranes as a monomer, which consists of four extracellular immunoglobulin-like domains, a transmembrane domain, and an intracytoplasmic tail. The two membrane-distal domains contain the binding sites for class II major histocompatibility complex (MHC) b2 domain, while the intracytoplasmic domain is associated with p56lck src protein kinase. The CD8 molecule is mostly expressed as a heterodimer of a (34 kDa) and b (34 kDa) chains that contain the class I MHC–binding domain in the extracellular domains. Similar to CD4, the CD8 intracytoplasmic domain is associated with p56lck.

Chapter 5:

b mature T-cells) following antigen-specific stimulation is the secretion of cytokines that act on other T-cells, B-cells, macrophages, neutrophils, and vascular endothelium. These cells are thus designated helper–inducer T-cells (Th). The CD8 (T8, Lyt-2) molecule exists in two forms: either as a homodimer of CD8a chain (34 kDa) or as a heterodimer of CD8a and CD8b (34 kDa) chains (Fig. 11) encoded by genes located on chromosome 2. The extracellular domains contain the binding sites to MHC class I a3 domain, resulting in restricted interaction of CD8þ T-cells (35% of the a/b T-cells) with class I MHC–bearing cells. Similar to the CD4 molecule, the intracytoplasmic domains of CD8 undergo serine phosphorylation following antigenspecific stimulation of CD8þ T-cells due to the physical association of p56lck. The CD8 marker is expressed on killer or cytotoxic T-lymphocytes (CTL; Tc), which constitute the effector T-cell population that ultimately contribute to allograft destruction. Cytokines involved in the differentiation and induction of CTLs include interleukin (IL)-2, interferon (IFN)-g, and IL-6. The division of T-cells into Th or CTL populations based on the expression of CD4 or CD8 molecules, while convenient, is not exclusive. Cytotoxic activity has been shown to be mediated by CD4þ T-cells, while helper activity can be provided by CD8 cells. Animal studies utilizing antibodies directed against CD8 or CD4 have demonstrated that rejection of class I allo-disparate grafts is primarily mediated by CD8þ cells, while CD4þ cells are responsible for rejection of class II–disparate allografts (28). However, CD4 and CD8 cells cooperate in nearly all episodes of clinical graft rejection. Costimulation Molecules. As we will discuss next, T-cell activation requires two essential signals to fully drive their proliferation and differentiation. TcR and its associated

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molecules (CD3, CD4, or CD8) interacting with MHC– peptide complex provides the first and antigen-specific signal. At the same time, a secondary and non–antigen-specific signal is required, which acts synergistically by either delivering a qualitatively distinctive intracellular signal or simply enhancing the endogenously weak TcR engagement. The second signal is provided by costimulation molecules on T-cell side interacting with their ligand or receptor counterparts on APCs. Currently, there are two major families of well-characterized costimulation molecule pairs: IgSF (29), such as CD28-B7, and TNF-receptor (TNFR) superfamily, such as CD40L–CD40 (30) (Fig. 12). With the identification of a multitude of costimulation molecules in each of these two families, and the discovery of the regulated expression of costimulation molecules depending on the stages of T-cell differentiation and the types of inflammatory environment, the current hypothesis of costimulatory mechanism of T-cell activation acknowledges the necessity of multiple costimulations and the variation of costimulation, despite redundancy. The existence of these many costimulation pathways may meet the need of at least three in vivo scenario, which are: (i) requirement of several signals simultaneously at any one time in responding T-cells, (ii) requirement of different signals at different times and/or stages of T-cell responses, or (iii) promotion of different effector functions (Th, Tc, Treg, Tmemory, etc.). (a) IgSF. Members of this family can be categorized into CD28-like molecules expressed on T-cells and B7-like molecules expressed on APCs. CD28 and its relatives including inducible costimulator (ICOS), CTLA-4, PD-1, CD7, and TIM1 are characterized by a single V-like Ig domain extended by a stalk from the T-cell surface (Fig. 12). They form homodimers and, except PD-1 (in 2q37.2), have their genes coded in the 2q33. On the other

Figure 12 Two superfamilies of T-cell costimulatory molecules. Costimulation molecules on T-cell side are interacting with their ligand– receptor counterparts on APCs. Two major families of well-characterized costimulation molecule pairs are: IgSF, such as CD28-B7, and TNFR superfamily, such as CD40L–CD40. Abbreviations: APCs, antigen-presenting cells; IgSF, immunoglobulin superfamily; TNF, tumor necrosis factor; TNFR, TNF-receptor.

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hand, B7-like molecules, B7-1, B7-2, B7H, and PD-L1/2, are made of a distal V-like and a proximal C-like Ig domain and probably also form homodimers. They are coded in the 2q23 region. CD28 is the first, well-characterized costimulatory molecule expressed constitutively on the surface of both na€ve and activated T-cells. Currently, there are two known ligands, B7-1(CD80) and B7-2(CD86), which have different expression pattern and induction kinetics on APCs. Ligation of CD28 can reduce the number of TcRs that must be engaged for a response, lower the activation threshold, and allow a response to low-affinity peptide. CD28 signaling can also alter the balance between Th1 and Th2 cell differentiation and prevent the induction of nonresponsiveness (anergy) and cell death (apoptosis). Although it has been shown that na€ve or primary T-cell responses are dependent on CD28 costimulation for optimal production of cytokines and proliferation, it is now also clear that secondary or memory T-cell responses are less dependent or independent of CD28 molecule. Other costimulatory molecules, such as ICOS, can substitute CD28 to provide necessary costimulation for memory T-cell activations. CTLA-4 was identified soon after CD28 as one of its close relatives, and binds to the same ligands but with much higher affinity. In contrast to CD28, CTLA-4 was not expressed constitutively on T-cells: resting T-cells express little surface CTLA-4, but its surface level is induced upon T-cell activation (particularly by CD28 costimulation) via both redistribution of an intracellular pool and increased synthesis. Most importantly, ligation of CTLA-4 delivers a negative signal to T-cells and inhibits T-cell proliferation and IL-2 synthesis in response to stimulation with anti-CD3 and anti-CD28 antibodies in vitro. Interference of CTLA-4–B7 interaction enhances T-cell responses, both in vitro and in vivo. Deficiency of CTLA-4 in mice leads to the development of massive lymphoadenopathy, autoimmunity, and early death. Thus, it has been hypothesized that CTLA-4 sets the threshold of T-cell activation and is the master switch of self-limiting T-cell activation: na€ve T-cells without CTLA-4 are activated by TcR and costimulatory signals, which accompany the induction of CTLA-4 to prevent overreaction of T-cells and eventually shut down T-cell response. The existence of CD28-independent costimulation pathways is indicated by the facts that mice lacking CD28 are still able to mount effective responses to some virus, acutely reject various kinds of allografts, and produce near normal levels of cytokines by their T-cells, particularly memory type, upon activation. ICOS was first identified in a screen for unique molecules expressed on human peripheral blood T-cells following activation. Thus, unlike CD28, ICOS is expressed on activated but not resting na€ve T-cells. Memory T-cells express ICOS constitutively, and its level is further increased following activation. Although ICOS costimulation might support activation of na€ve T-cells, it seems to be more important for enhancing ongoing and recallproliferative responses. ICOS costimulation does not induce IL-2 production but increases secretion of IL-4, IL-5, IL-10, IFN-g, and TNF-a, suggesting its primary role in inducing T-cell effector function. ICOS is structurally related to CD28 but does not bind to B7-1 and B7-2 molecules. Instead, it binds to a novel B7 family member, B7H, which is regulated differentially from B7-1 or B7-2 on APCs. Importantly, B7H expression can be induced in fibroblasts in culture, and in nonlymphoid tissues in mice, by treatment with the inflammatory agents LPS and TNF-a, whereas B7-1 or B7-2 are not induced in these same tissues by similar

treatment. Thus, inflamed peripheral tissues would be able to stimulate memory, but not na€ve, T-cells, due to the differential regulation of the ligands for CD28 and ICOS. PD-1 was initially cloned due to its involvement in apoptosis. Unlike other members of the CD28 family, PD-1 is widely expressed on hematopoietic-derived tissues. It expresses on T-cells, B-cells, monocytes, and myeloid cells following activation. The functional role of PD-1 is not completely understood. Although, like CTLA-4, it may deliver an inhibitory signal to T (and B)-cells, it works only under certain conditions and in certain aspects of immune responses. PD-1–deficient mice become ill owing to the inappropriate activation of lymphocyte, particularly production of autoreactive IgG3 (C57Bl/6) or IgG1 (Balb/c). In marked contrast to CTLA-4–deficient mice, all of which died within three to four weeks after birth, severe autoimmune symptoms are only observed in half of the PD-1 deficient mice and occur late after birth (up to 14 month). All of these suggest that PD-1 is not the primary inhibitory signal for T-cells, and is less potent compared with CTLA-4. The ligand for PD-1 was recently cloned (B7H1) and shares structural homolog with B7-1 or B7-2. It is constitutively expressed on both nonlymphoid tissues, including heart, lung, kidney, skeletal muscle, and placenta, and some hematopoietic tissues, such as macrophage and T-cells. Its expression is upregulated on DCs by LPS and IFN-g, as well as on nonlymphoid tissues such as keratinocytes. The expression pattern of B7H1 suggests that PD-1 might function to inhibit lymphocyte responses in nonlymphoid tissues. (b) TNF/TNFR Superfamily. The first member of this large family of costimulatory molecules is CD40–CD40L pair. The TNFR-like CD40 has been known for long-time presence on B-cells and other APCs, including DCs, monocytes, macrophages, mast cells, fibroblasts, and endothelial cells. Its engagement is critical for B-cell clonal expansion, as well as APC activation that enables them to activate na€ve T-cells. Its ligand, TNF-like CD40L, is induced on na€ve T-cells within a few hours of their activation by TcR-mediated signal 1 alone, and this induction is enhanced by CD28-mediated signal 2. The ligation of CD40L on T-cells leads to the upregulation of cell surface CD28 expression and later to induce ICOS. The importance of this costimulatory pathway is manifested by the disorders observed in the human hyperIgM syndrome, an X-linked immunodeficiency where the CD40L molecules is nonfunctional. Using CD40 or CD40L KO animals or blocking antibodies, it has been shown that CD40–CD40L interactions are critical for the development of the CD4 T-cell–dependent, cell-mediated immune responses at multiple levels. Lack of these interactions results in greatly reduced activation of CD4 T-cells resulting from inefficient priming and expansion of antigen-specific T-cells, due to a lack of induction of costimulatory activity on APCs and a failure in the activation of APC to produce cytokines such as IL-12. This will lead to the impairment of many CD4 T-cell effector functions, including help for B-cells and activation of monocytes, macrophages, and DCs, which in turn affect the activation of CD8 T-cells. Additionally, CD40–CD40L interaction upregulates expressions of adhesion molecules on endothelia, and its blockade in vivo will inhibit the migration of T-cells to secondary lymphoid organs and inflamed tissues. At the effector stage, CD40– CD40L interactions could amplify immune responses by upregulating costimulation and promoting secretion of inflammatory cytokines. Thus, CD40–CD40L interaction is mandatory for primary APC–T-cell cooperation, and plays

Chapter 5: Physiologic Basis of Transplantation

a central role in CD4 T-cell help to B-cells, as well as cytotoxic CD8 T-cells. This family of costimulatory molecules is expanded rapidly in recent years, and now it includes at least five other pairs (TNFR–TNF) (30): OX40–OX40L, CD27–CD70, CD30– CD30L, 4-1BB–4-1BBL, and HVEM–LIGHT (Fig. 12). A simplified pattern of the cellular expression of these new, paired molecules on lymphocytes is that TNFR family members are expressed by T-cells, and their TNF-family ligands are expressed by APCs. However, each ligand can also be expressed by T-cells. Additionally, there are two patterns of expression kinetics of these TNFR family members by na€ve T-cells: constitutive low-to-medium level expression, such as CD27 and HVEM, and inducible expression, which occurs several hours after signal 1 engagement and often peaks for two to four days into the responses, such as OX40, 4-1BB, and CD30, depending on antigens and the environment during T-cell responses. None of the TNF-family ligands is expressed constitutively on T-cells. TcR-mediated signal 1 is sufficient to induce the inducible TNFR-family molecules, however, CD28 signaling augments their kinetics and levels. Corresponding to the receptor-like molecules on T-cells, their ligands on APCs also have two expression patterns. OX40L, 4-1BB, CD70, and CD30L are not constitutively expressed by resting or immature APCs, but are inducible 24 hours to several days after activation by CD40, LPS (on DCs), and BCR (on B-cells), coinciding with the peak level of expression of their receptors by T-cells. LIGHT is expressed by immature DCs, and unlike others, it is downregulated during the process of maturation. Thus, functionally, the constitutive expression of HVEM–LIGHT pair indicates their possible role in the initial activation of T-cells and APCs, while other inducible pairs involve more likely either in clonal expansion phase or to sustain the ongoing responses of T-cells. Adhesion Molecules. The ICAM-1 (114 kDa) contains five Ig-like extracellular domains, and is expressed on T- and Bcells, fibroblasts, and endothelial cells (Fig. 13). ICAM-1 contains the binding sites for the adhesion molecules LFA-1 and macrophage antigen-1 (Mac-1), both members of the b2 integrin family. The binding of LFA-1 to ICAM-1 may deliver a costimulatory signal to T-cells, thus enhancing the TcR-mediated signal for T-cell activation. ICAM-2 bears only two Ig-like extracellular domains, and can only bind to LFA-1. CD2 [T11, LFA-2, Tp50, and sheep red blood cell receptor (SRBC); 50 kDa] is expressed on mature T-cells, on 50% of thymocytes, and on NK. CD2 binds to a molecule that is structurally similar to itself: leukocyte function–associated antigen-3 (LFA-3, CD58; 55 kDa), which is expressed on a wide variety of hematopoietic cells. CD2 binding to LFA-3 is important for the binding of Th cells to APCs or CTLs to their targets. In addition to its adhesive function, CD2 plays an important role in T-cell activation, as evidenced by the ability of some anti-CD2 antibodies to stimulate T-cell proliferation and cytokine production. This led to the hypothesis that CD2 may mediate an alternate pathway for T-cell activation, independent of the TcR. The vascular cell adhesion molecule-1 (VCAM-1), expressed on activated endothelial cells, contains seven Ig-like extracellular domains. VCAM-1 is the ligand for very late activation (VLA)-4 (b1 integrin) expressed on T-cells. The VCAM-1–VLA-4 interaction is critical for T-cell migration from the blood stream into the sites of inflammation, and may further provide a costimulatory signal for T-cell activation.

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Figure 13 Adhesion molecules of the immunoglobulin (Ig) gene superfamily. These are characterized by the presence of the Ig homology unit, demonstrated schematically as a loop that is stabilized by a disulfide bond (–S–S–), contributed by conserved cysteine residues. Members of this family include ICAM-1, ICAM-2, CD2, VCAM-1, CD28, and B7 molecules. Each molecule contains multiple Ig-like domains, a transmembrane region, and an intracytoplasmic segment. NH2 and COOH, amino- and carboxyterminal ends. Abbreviations: ICAM, intercellular adhesion molecules; VCAM-1, vascular cell adhesion molecule-1.

Selectins (lectin adhesion molecules), a family of three closely related proteins L-, E-, and P-selectin, provide a calcium-dependent specific recognition signals upon interaction with carbohydrate (31), but not with protein, leading primarily to the adhesion of leukocytes to endothelial cells. All three selectins are single chain, transmembrane glycoproteins that share similar structural features (Fig. 14A): first, a lectin-binding domain at the N-terminus, then an epidermal growth factor–like domain and a number of tandemly repeated domains homologous to the complement regulatory proteins, followed by a transmembrane domain and a short intracytoplasmic tail. The L-selectin (CD62L, MEL-14, LAM-1; 90 kDa) expressed on leukocytes serves as a homing receptor for lymphocytes to attach to lymph node– high endothelial venules. It also binds leukocytes to cytokine (TNF, IL-1, and IFN-g)-activated endothelial cells at the site of inflammation. E-selectin (CD62E, ELAM-1; 110 kDa) is exclusively expressed by cytokine-activated endothelial cells. Its ligand is a complex sialylated carbohydrate group related to Lex antigens. P-selectin [CD62P, PADGEM, guanosine monophosphate (GMP) -140; 140 kDa] is present in the secretory granules of platelets and endothelial cells. Upon activation of endothelial cells, P-selectins are rapidly distributed to the cell surface in order to bind circulating monocytes and neutrophils. P- and E-selectins recognize similar ligands. Integrin superfamily members are heterodimeric cell surface proteins composed of two noncovalently associated a (120–200 kDa) and b chains (90–110 kDa; Fig. 14B) (32). The integrin b chains contain tandem repeats of a cysteine-rich domain. The globular heads of the a chains contain a series of divalent cation–binding regions important for receptor function, because many integrins have been shown to

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Figure 14 Schematic illustration of selectins and integrins. (A) The extracellular regions of L-, E-, and P-selectin consist of the characteristic lectin-binding domain and an epidermal growth factor–like domain, followed by a variable number of short consensus repeats similar to the complement-regulatory proteins. Each molecule contains transmembrane and short intracytoplasmic domains. (B) Integrins are heterodimeric complexes of noncovalently associated a and b chains, with characteristic globular heads that contribute to interchain linkage and ligand interaction. Stalks extend from the heads to the cellular membrane, followed by transmembranous and cytoplasmic segments.

require divalent cations for ligand binding. The extracellular domains of the two chains bind to a wide variety of ligands including extracellular matrix glycoproteins, including fibronectin and vitronectin, other cell surface proteins, and complement components. The three original integrin subfamilies were defined based on the b subunits, which were believed to bind several distinct nonoverlapping sets of a chains. Recently, at least eight forms of the b-chain have been identified; furthermore, several a chains have been shown to pair with more than one b subunit. The VLA molecules or b1 integrins (CD49a–f CD29) display six different a chains (CD49a–f) linked to the same b1 chain (CD29). VLA-4 (a4b1, CD49d CD29) mediates adhesion of T-cells to endothelial cells by binding to the VCAM-1. In addition, VLA-4 (a4b1, CD49d CD29) and VLA-5 (a5b1, CD49e CD29) bind to fibronectin, and VLA-6 (a6b1, CD49f CD29) binds to laminin in the extracellular matrix. Such interactions may be important for the retention of T-cells in the tissues following extravasation from the intravascular compartment. Further, the binding of VLA-4 to VCAM-1 and extracellular matrix proteins can provide costimulatory signals for T-cell activation. The three b2 integrins—LFA-1 (CD11a CD18), Mac-1 (CR3; CD11b CD18), and p150, 95 (CR4; CD11c CD18)— share the same b2 subunit (CD18) that combines with three different a chains (CD11a, CD11b, and CD11c). These molecules play an important role in the adhesion of lymphocytes to other cells, such as the APCs and vascular endothelium. Ligands for LFA-1 include ICAM-1 and ICAM-2 (members of the Ig gene superfamily). Mac-1 also binds to ICAM-1. Anti–LFA-1 antibodies inhibit a wide variety of lymphocyte functions including APC induced Th cell activation and CTL target cell lysis. This is presumably due to inhibition of the conjugate formation between the involved cells. TcR-specific stimulation results in functional modulation of integrins, resulting in an increase in their binding affinity to their corresponding ligands. The increased affinity may be caused by phosphorylation of the integrin intracytoplasmic tails. In addition, following ligand binding, integrins deliver stimulatory signals to cells. The critical role of the integrin function is demonstrated in the familial disease leukocyte deficiency-type 1, which occurs as a result of genetic

mutation of the b2 chain (CD18). Afflicted individuals display a deficiency in all b2 integrins, leading to recurrent bacterial and fungal infections due to lack of polymorphonuclear leukocyte accumulation and adhesion-dependent lymphocyte function. Adhesion molecules play an important role in the migration of cells from the vascular compartment to the sites of inflammation (33). Inflammatory cytokines (mainly TNF) induce this complex process leading to the activation of endothelial cells, which regulate the extravasation of neutrophils, monocytes, and T-cells. Endothelial cells release vasodilators such as prostacyclin (PGI2) and nitric oxide (NO), which increase blood flow at the inflammatory site. TNF production enhances PGI2 synthesis, increases NO production, and induces endothelial cell surface E-selectin expression. E-selectin–mediated interaction with neutrophils results in slowing and rolling of cells (Fig. 15). Endothelial cell expression of VCAM-1 and ICAM-1, which occurs approximately four to six hours after the initiation of the inflammatory response, is critical for the attachment of leukocytes. The T-cell ligand for ICAM-1 is LFA-1, and VLA-4 for VCAM-1, while neutrophils engage ICAM-1 through Mac-1. Chemokines, secreted by the endothelial cells under the effect of TNF-a such as IL-8 and monocyte chemotactic protein-1, induce leukocyte flattening and cell locomotion. TNF-a and IFN-g cause endothelial basement membrane changes that allow migration and extravasation of cells as well as other macromolecules such as fibrinogen. Once leukocytes enter the tissues, VLA-4, VLA-5, and VLA-6 encourage cell binding to the extracellular matrix proteins. The extravasation of neutrophils and monocytes accompanied by the deposition of fibrin result in induration at the site of inflammation or graft rejection. Such induration is characteristic of a delayed type hypersensitivity (DTH) reaction, which is apparent 18 hours after the deposition of antigen eliciting the inflammatory response, and reaches a maximum at 24 to 48 hours. Antigen-activated CD8þ or CD4þ T-cells can adoptively transfer DTH-like reactions. Monocytes deposited in the tissues differentiate into macrophages, which are the main effector cells in DTH. IFN-g is the main cytokine in DTH due to its macrophage activating capacity. IFN-g augments the phagocytic

Chapter 5: Physiologic Basis of Transplantation

Figure 15 Transendothelial migration of leukocytes. 1. Rolling: inflammatory cytokines (TNF) induce the expression of L- and E-selectins on endothelial cells, which initiate rolling of leukocytes. 2. Attachment: ICAM-1 expressed on endothelial cell surfaces binds to MAC-1 on neutrophils or LFA-1 on T-cells. VCAM-1 engages VLA-4 on T-cells. IL-8 and MCP-1 induce leukocyte conformational changes. 3. Migration: attached leukocytes migrate into the interstitial space. Abbreviations: ICAM, intercellular adhesion molecule; IL, interleukin; LFA, leukocyte function–associated antigen; MCP, monocyte chemotactic protein; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule; VLA, very late activation.

activity of macrophages and increases the expression of the high-affinity receptors for the Fc portion of Ig molecules, thereby enhancing phagocytosis of opsonized bacteria. Activated macrophages display more efficient APC function, express higher levels of class II, ICAM-1, and LFA-3, and release platelet-activating factor, prostaglandins, thrombin, transforming growth factor (TGF) -b, fibroblast growth factor (FGF), tissue factors that initiate the extrinsic clotting cascade, and other inflammatory mediators. DTH results in tissue destruction (necrosis) followed by angiogenesis and fibrosis when the antigen is eliminated. Dendritic Cells Dendritic cells are bone marrow–derived APC that are found both in lymphoid and nonlymphoid tissues. They are the most critical cellular component bridging innate and adaptive immunity (34). DCs at immature stage reside in most peripheral tissue, particularly at sites of interface with the environment, where they take up 1% to 2% of the total cell numbers. In the absence of ongoing inflammation and immune responses, DCs patrol constantly through the blood, peripheral tissues, lymph, and secondary lymphoid organs. Although they are capable to take up self- and non–self-antigens via endocytosis or phagocytosis, immature DCs are quite inefficient in presenting antigens and are not able to fully activate T-cells. They have to go through a maturation process, which is induced during infection or inflammation by bacterial or viral products, inflammatory cytokines, as well as some self-molecules, identified in recent literatures as endogenous danger signals. Via specific cell surface receptors (e.g., TLRs, CD40, TNFR, FcR), stimulated DCs dramatically increase their surface expression of T-cell costimulation molecules, and the efficiency of antigen presentation (by enhancing antigen uptake, intracellular transport and degradation, and the intracellular traffic of MHC molecules, leading to increased peptide loading half-life and delivery to the cell surface of MHC molecules). Matured DCs are the

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most potent APCs and the only ones capable of activating na€ve T-cells and initiating adaptive immune response. Concomitant with the modification of their antigen presentation abilities, maturation also induces massive migration of DCs out of peripheral tissues. Alterations in the expression of chemokine receptors and adhesion molecules, as well as changes of the cytoskeleton organization, contribute this migration of DCs through afferent lymph to secondary lymphoid organs where they interact with T-cells. DCs represent a heterogenous cell population. Improved techniques for the isolation of DCs from various tissues have led to the identification of at least five types of DCs, based on their phenotypes and functions (35). They are myeloid or monocytoid DC (CD11cþCD1aþCD123–), plasmacytoid DC (CD11c–CD1a–CD123þ), Langerhans cell– derived DC (CD11cþCD1a–CD123–), B-cell–like DC, and follicular DC in humans, and CD8a- (myeloid) and CD8aþ DC, B-cell–like DC, plasmacytoid DC, and Langerhans cell–derived DC in murines. Although they all express MHC class II and CD40 (and CD11c in mice), different DC subsets and DCs at different maturation stages express distinct surface molecules and secrete different cytokines. Human plasmacytoid DC are the principal IFN-producing cells of the immune system, and monocytoid DC produce IL-12. In mice, although both CD8a– and CD8aþDC mature into potent immunostimulatory APCs, CD8aþCD11b-DC secrete IL-12 and prime na€ve CD4 T-cell secrete Th1 cytokines and promote antiviral immunity by inducing CTL via secretion of IFN-g; CD8-CD11bþDC, on the other hand, stimulate Th2 responses and secrete neither IL-12 nor IFN-g. In transplantation models, alloantigen presentation by immature DC has been shown to promote graft tolerance. Thus, during antigen presentation, DCs can determine selectively the type of induced immune responses.

Central Limb: Proliferation and Differentiation Although multiple effectors, both cells and molecules, contribute to allograft rejection, the most critical one is the T-cell. It is not only able to directly reject grafts by itself, but also influences other effectors in reacting to grafts. In this section, we will discuss both biochemical and structural mechanisms of alloreactive T-cell activation, in particular CD4þ T-helper subset.

T-Cell Activation Two Signal Hypothesis for T-Cell Activation The two-signal model was originally proposed by Bretscher and Cohn in 1970 as a hypothesis to account for self-tolerance in the peripheral. The current form of this model assumes that T-cells require two signals to be activated by APCs. The first signal is delivered via TcR upon antigen presentation. The second signal has two important features: it has no cognition of the antigen, and it can be produced by distinctive molecular interactions between an APC and a T-cell. Lymphocytes stimulated through the antigen receptor alone fail to produce cytokines, are unable to sustain proliferation, and often undergo apoptosis or become nonresponsive to subsequent stimulation. Early studies found soluble factors, such as cytokines (IL-1, IL-2, and IL-4), could enhance the activation of both T- and B-cells. Now, it is clear that interactions between receptor–ligand pairs of cell surface molecules on the responder lymphocyte and an APC represent the critical event, costimulation, in the activation process. The binding of TcR with a peptide-loaded MHC molecule is very brief. To fully activate a primary T-cell with signal 1 alone, it

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has been calculated that the engagement of 8000 TcR were required. However, with the costimulation of CD28, the number of TcR engagements required is lowered to 1000 (36). Biochemically, the secondary costimulation may deliver a qualitatively distinct activation signal, or act synergistically with the first signal (see below for details). As discussed above, amultitude of costimulation molecules have been identified. Although hierarchy and redundancy exist, these different molecules may function in different types of T-cells (e.g., CD4, CD8, Th, Treg, and Tc) and/or at different stages of T-cell differentiation (na€ve, effector, and memory). The Immunological Synapse and Lipid Raft Effective TcR interactions with MHC–peptide complex occur in the nanometer-scale gap between T-cells and APCs, referred to as an immunological synapse (IS) (37). T-cell activation requires sustained interactions with APCs with signaling duration on the order of hours, while individual receptor–ligand pair between these cells has half-lives only on the order of seconds. IS provides a molecular organization that helps make the longevity and specificity of the T-cell–activation signaling possible. The mature IS has been defined by the bull’s eye arrangement of supramolecular activation clusters (SMACs) that form within a few minutes of T-cell–APC contact. SMACs are detected by fluorescence microscopy and appear as increased densities of specific molecules. The center of the bull’s eye or cSMAC is enriched with TcR (including CD3 and CD4/8) and MHC–peptide complexes. The ring of the bull’s eye or pSMAC contains the integrin LFA-1, its major counter-receptor ICAM-1, and CD2. The cSMAC also includes the signaling molecules on the T-cell cytoplasmic side of the IS, including protein kinase C y and the src family kinase lck (see below for detail). Interestingly, costimulatory molecule, CD28, is also localized in the cSMAC. The structural basis to form IS is the preexisting membrane domain, lipid raft, which segregates proteins and lipids into liquid-ordered domains with distinct biophysical properties. Experimentally, these membrane domains are relatively insoluble in Triton X-100 detergent at low temperature, which are also referred to as detergent-insoluble, glycolipid-enriched domains, or glycosphingolipid-enriched membranes. Crucial signaling proteins are enriched in these lipid rafts. They include palmitoylated or myristoylated src kinase, ras-related proteins, such as RAC and CDC42, and adaptor proteins, such as palmitoylated linker for activation of T-cells (LAT). TcR is only weakly associated with lipid rafts in unstimulated state. However, engagement of TcR with antigen promotes the entry of receptors into the lipid rafts, and early TcR-induced tyrosine phosphorylation is restricted within the lipid raft. Coreceptor CD4 and CD8 are strongly associated with lipid rafts and may play an important role in the initial alternations in the lipid and signaling environment of the TcR. Costimulation modulates lipid raft accumulation in IS such that CD28 coligation with TcR enhances the process, while CTLA-4 potently inhibits TcR and TcR–CD28–mediated raft expression. Biochemical Basis of T-Cell Activation As previously discussed, TcR is a multisubunit complex consisting of ligand recognition component of polymorphic ab chains noncovalently associated with the signal transduction component of invariant CD3-g, CD3-d, and CD3-e and TcRz chain dimer. All of these molecules are integral TcR components and as such are required for TcR expression. The nonpolymorphic signal transduction chains are

members of a family of immunoreceptor signal transduction subunits that contain signaling motif ITAM, as described above. Each of the CD3-g, CD3-d, and CD3-e subunits contain one ITAM, and TcRz contains three ITAM. The tyrosine residues within each ITAM become rapidly phosphorylated upon optimal TcR engagement (Fig. 7), which is mediated by two members of the Src family of protein tyrosine kinases (PTKs) found in T-cells. Lck is the predominant enzyme involved in ITAM phosphorylation, while Fyn also has this capacity. The phosphorylated ITAM becomes subject to binding by additional molecules, most importantly zetaassociated protein (ZAP)-70. ZAP-70 is a cytoplasmic PTK exclusively expressed in T-cells and NK cells. It contains two SH2 domains in its N-terminal, both of which are essential for its binding to two phosphorylated tyrosines in a signal ITAM. Once bound to the TcR, ZAP-70 is activated by phosphorylation at multiple sites mediated by the Src PTKs, which then acts as both kinase and binding sites for other proteins. The activated TcR is thus characterized by phosphorylated ITAMs associated with phosphorylated, activated ZAP-70 (Fig. 16) (38). One of the most critical substrates of ZAP-70 is LAT (39), which is a type-III membrane protein without a signal sequence. There are two Cys residues, which undergo palmitoylation and localize this protein to the plasma membrane. LAT contains a very short extracellular domain and a long cytosolic region with nine tyrosine residues. Tyrosine phosphorylations of LAT lead to recruitment of a number of key, downstream signaling molecules, including Grb2 family proteins, SLP-76, and phospholipase C-g1. Both Grb2 family proteins and SLP-76 are adapter molecules, which themselves do not have any enzyme activities but contain SH3 domain to recruit other effector molecules, including SOS, Cbl. Additionally, SLP-76 contains multiple tyrosine residues at its amino-terminals, which can be phosphorylated by ZAP-70 and become binding sites for other proteins with SH2 domains, noticeably Vav, Nck, and Itk. It was shown recently that Itk plays a critical role in the activation of PLCg1 recruited to LAT. PLCg1 catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol-1,4,5-triphosphate and diacylglycerol (DAG). Phosphatidylinositol 3 (PI3) induces release of Ca2þ from ER via its receptors. The deprivation of intracellular Ca2þ storage triggers the opening of the cytoplasmic Ca2þ release– activated Ca2þ channels and leads to influx of Ca2þ from the extracellular environment. Elevated intracellular Ca2þ activates calcineurin, a Ca2þ/calmodulin (CaM) -dependent phosphotase, which dephosphorylates NFAT family of transcription factors. Increase of DAG activates RasGRP and protein kinase y. Activated RasGRP directly interacts with Ras and induces guanosine diphosphate–guanosine triphosphate exchange, which may be involved in the initiation of extracellular signal–regulated kinase activation, leading to c-fos induction, a AP-1 component. Protein kinase y was shown to play an essential role in NF-kB activation via activation of IkB kinase (IKK)b that phosphorylates IkB proteins and induces their degradation. NFAT, AP-1, and NF-kB are all essential transcription factors for IL-2 production (Fig. 16), a hallmark of complete T-cell activation. Costimulation contributes to T-cell activation. The signaling pathways downstream of costimulation molecules have been difficult to characterize and have only recently begun to be elucidated. CD28 is the primary costimulation molecule for na€ve CD4 T-cells. Despite being short, its cytoplasmic domain contains multiple binding motifs for various signaling molecules, including PI3-kinase (PI3K,

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Figure 16 Biochemical basis of T-cell activation. Following T-cell antigen recognition, the tyrosine residues within each immunoreceptor tyrosine-based activation (ITAM) become rapidly phosphorylated by Lck and Fyn. The phosphorylated ITAM become subject to binding by zeta-associated protein (ZAP-70). Once bound to the TCR, ZAP-70 is activated by phosphorylation at multiple sites mediated by the Src PTKs, which then act as both kinase and binding sites for other proteins. One of the most critical substrates of ZAP-70 is LAT, whose tyrosine phosphorylations lead to recruitment of a numbers of key downstream signaling molecules, including Grb2 family proteins, SLP-76, and phospholipase C-g1. SLP-76 contains multiple tyrosine residues at its amino-terminals, which can be phosphorylated by ZAP-70, and become binding sites for other proteins with SH-2 domains, including Vav, Nck, and Itk. Itk plays a critical role in the activation of PLCg1 recruited to LAT. PLCg1 catalyzes hydrolysis of PIP2 into IP3 and DAG. PI3 induces release of Ca2þ from ER via its receptors. The deprivation of intracellular Ca2þ storage triggers the opening of the CRAC and leads to influx of Ca2þ from the extracellular environment. Elevated intracellular Ca2þ activates calcineurin, a Ca2þ/calmodulin–dependent phosphotase, which dephosphorylates NFAT family of transcription factors. Increase of DAG activates RasGRP and protein kinase y. Activated RasGRP directly interacts with Ras and induces GDP–guanosine triphosphate (GTP) exchange, which may be involved in the initiation of ERK activation leading to c-fos induction, a AP-1 component. Protein kinase y plays an essential role in NF-kB activation via activation of IKKb that phosphorylates IkB proteins, and induces their degradation. NFAT, AP-1, and NF-kB are all essential transcription factors for IL-2 production, a hallmark of complete T-cell activation. Abbreviations: CRAC, cytoplasmic Ca2þ release–activated Ca2þ channels; DAG, diacylglycerol; ER, endoplasmic reticulum; GDP, guanosine diphosphate; IL, interleukin; IP3, inositol-1,4,5-triphosphate; PIP2, phosphatidyl inositol 4,5-bisphosphate.

via its adaptor subnit p85, SH2), ITK (SH3), LCK (SH3), and GRB2/GADS (SH2 and SH3). CD28 phosphorylation subsequent to TcR activation is required for the recruitment of these proteins. Three intracellular signal transduction pathways have been attributed biochemically to the costimulatory effects of CD28 (29). Central to all three pathways are the D3 lipid products of PI3K, phosphatidylinositol (3,4)bisphosphate and phosphatidylinositol (3–5) trisphosphate, which anchor to the inner face of the plasma membrane where they bind and recruit various proteins with pleckstrin-homology domains. (i) Phosphoinositide-dependent kinase–protein kinase B (PKB)–glycogen synthase kinase-3 pathway prevents T-cell apoptosis, increases cellular glycolysis, and enhances IL-2 gene transcription [via nuclear factor of activated T-cells (NFAT)]. (ii) VAV-MEKK1-JNK pathway also upregulates IL-2 transcription [via activating protein-1 (AP-1)], and additionally induces cytoskeleton remodeling. (iii) PKB–MEKK1–MAGUK–mediated NF-kB pathway, as the third avenue, also contributes to the induced transcription of IL-2 and some antiapoptosis genes. Enhanced degradation of inhibitor of NF-kB and increased activity of IKK promotes translocation of NF-kB to the nucleus after CD28 ligation (Fig. 16). T-Helper Cell Activation and Differentiation Central to host adaptive immunity, Th cell activation and differentiation defines the nature of developing immune responses. Although oversimplified, the paradigm that

activated CD4 T-cells differentiate into distinctive subsets producing different arrays of cytokines provides us vital insights into the pathogenesis of immune-mediated diseases (40). As we discussed above, T-cell activation requires two essential signals to drive their proliferation. At the same time, the environment of T-cell activation provides a third signal, which will determine the characters of the descendent T-cells. It is now clear that Th differentiation is regulated at multiple levels. Although the natures of antigens and costimulation molecules, as well as the key transcription factors all play important roles, cytokine condition is the most important regulator in initiating the differentiation processes and determining the types of Th cells generated (41,42). The classical Th subsets include Th1 and Th2 (Fig. 17). Th1 effector cells produce IFN-g and mediate macrophage activation and DTH, which are collectively termed proinflammatory or cell-mediated immune responses, critical to control intracellular pathogens. In contrast, Th2 effector cells produce IL-4 or IL-5, promote secretion of IgG1 and IgE from B-cells, and mediate immediate-type hypersensitivity reactions (allergy), which are collectively termed humoral immunity. Recently, a third type of Th cell has been defined as Treg, which exerts immune regulatory functions. IL-12 is the classical cytokine used for in vitro differentiation of Th1 cells from naive precursors. It is a heterodimeric cytokine composed of two subunits, p35 and p40. The p35 subunit is expressed ubiquitously and

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Figure 17 Cytokine induced differentiation of T-helper cells. The classical Th subsets include Th1 and Th2. Th1 effector cells produce IFN-g and mediate macrophage activation and DTH, which are collectively termed proinflammatory or cell-mediated immune responses. In contrast, Th2 effector cells produce IL-4 or IL-5, promote secretion of IgG1 and IgE from B-cells, and mediate immediate-type hypersensitivity reactions (allergy), which are collectively termed humoral immunity. IL-12 is the classical cytokine used for in vitro differentiation of Th1 cells from niave precursors. The transcriptional factor Stat4 is critical to IL-12R signaling, which upon activation by phosphorylation may directly interact with IFN-g promoter and induce its production. T-bet is another important transcriptional factor that mediates the positive feedback loop of IFN-g production. IL-4 is the major known factor regulating Th2 differentiation. TSLP in human can activate DCs to prime na€ve CD4 T-cells to produce IL-4 and other Th2 cytokines. IL-4 binding to their receptors activates Stat6, which is the central transcriptional factor driving Th2 differentiation. The activated Stat6 influences the remodeling of the IL-4 or IL-13 locus and induces another transcription factor GATA-3. GATA-3 can directly bind to the promoters of IL-5 and IL-13 genes and activate their transcriptions. Abbreviations: DTH, delayed type hypersensitivity; IFN, interferon; TSLP, hymic stromal lymphopoietin; IL, interleukin; MHC, major histocompatibility complex; TcR, T-cell receptor; TNF, tumor necrosis factor; CTL, cytotoxic T-lymphocytes; Ig, immunoglobulin.

constitutively at low levels, while the p49 production is highly regulated and induced by products of infectious microorganisms in monocytes, neutrophils, macrophages, and DCs, via TLRs. IL-12 is also induced by the engagement of CD40 on APCs. The IL-12 receptor expressed by T-cells consists of two subunits, b1 and b2, both of which are not expressed in resting T-cells but induced upon T-cell activation. Critical to IL-12R signaling is the transcriptional factor Stat4, which upon activation by phosphorylation may directly interact with IFN-g promoter and induce IFN-g production (Fig. 17). T-bet is another important transcriptional factor, which mediates the positive feedback loop of IFN-g production. Synergized with TcR signaling, IFN-g activates Stat1 and upregulates expression of T-bet, which alters the chromatin structure of IFN-g locus and stabilizes expression of IL-12Rb2. This permits the cells’ continued responsiveness to IL-12, which further induces IFN-g production and Th1 differentiation (43). Other cytokines produced by APCs in response to pathogens and capable of inducing Th1 differentiation include type-1 IFNs, IL-18, and two newly identified IL-12 homolog IL-23 and IL-27. In contrast to the multiplicity of cytokines produced by DC to promote Th1 differentiation, there are few clear initiators produced by APCs in response to pathogens to drive Th2 differentiation. In human, it appears that thymic stromal lymphopoietin, which is highly expressed by epithelial cells, can activate DCs to prime na€ve CD4 T-cells to produce IL-4 and other Th2 cytokines (41). The dose of antigen is another determinant that may initiate Th2 differentiation. Low doses of antigen induce Th2 polarization. Currently, IL-4 is the major known factor regulating Th2 differentiation, both in vitro and in vivo. However, it is unclear as to which sources, including eosinophils, mast cells, and NK1.1 T-cells, and CD4 T-cells polarizing towards Th2, produce the initiating IL-4. Na€ve CD4 T-cells stimulated through TcRs in the presence of IL-4 develop into Th2 effector cells capable of producing IL-4, IL-5, and IL-13. IL-4 is also a potent suppressor of IFN-g–producing Th1 cells. Two types of IL-4 receptors exit, both using IL-4Ra chain.

The type I receptor comprises the IL-4Ra and the common g chain, and the type II receptor comprises IL-4Ra and IL-13R. IL-4 binding to either of these receptors activates Stat6, which is the central transcriptional factor driving Th2 differentiation (Fig. 17). The activated Stat6 influences the remodeling of the IL-4 or IL-13 loci, and induces another transcription factor GATA-3 (43). GATA-3 can directly bind to the promoters of IL-5 and IL-13 genes and activate their transcriptions. GATA-3 does not affect IL-4 gene expression as a cis-acting transcriptional regulator, but rather by influencing chromatin remodeling, as T-bet to IFN-g gene. Another transcription factor preferentially expressed in Th2 cells is the proto-oncogen c-Maf, which strongly transactivates the IL-4 promoter but not the IL-5 or IL-13 promoters. Other cytokines capable of inducing Th2 differentiation include IL-13 and a newly described cytokine IL-21, related to IL-2/-4/-15 family.

Alloimmune Recognition Allorecognition refers to T-cell recognition of genetically encoded polymorphisms between individuals of the same species. The principal targets of the allorecognition are MHC molecules. Additionally, minor histocompatibility antigens, peptides derived from allelically polymorphic host proteins, presented in the groove of MHC class I and II are also recognized by host T-cells. Vigorous T-cell responses initiated by multiple T-cell clones expressing a wide variety of receptor genes in response to alloantigen are the essential components of graft rejection. In contrast to the low frequency of T-cells (< 0.1%) responding to nominal antigens, as much as 10% of host T-cells can respond to alloantigen. Molecular Basis of Allorecognition The a.a. sequence variation between donor and recipient polymorphic class I or class II MHC alloantigens induces rejection. This variability is mainly confined to a.a. present on the top, sides, and floor of the peptide-binding groove of MHC molecules (Fig. 18A) (44). Polymorphic a.a. residues

Chapter 5: Physiologic Basis of Transplantation

within the peptide-binding groove serve as sites for peptide binding and/or TcR recognition, with the TcR recognizing a complex formed of a peptide anchored in the peptidebinding groove (Fig. 18A) (45). Normally, thymic selection allows for a T-cell repertoire that is tolerant to self-peptides presented on self-MHC (Fig. 18B, Panel 1), but is activated by foreignc peptides presented on self-MHC molecules (Fig. 18B, Panel 2). Thus, MHC–peptide complexes that exhibit a.a. differences from self-peptide–MHC complexes trigger the alloimmune response (46). T-cells recognize alloantigens either as intact molecules expressed on the graft (direct recognition) or as processed allopeptides presented by self-MHC (indirect recognition) (47–49). Acute allograft rejection is conventionally thought to be mediated largely by direct recognition of allo-MHC– peptide complexes, which is a unique type of immune

Figure 18 Molecular basis of allorecognition. (A) TcR recognition sites. Polymorphic amino acid (a.a.) present in the MHC peptide-binding groove serve as sites for peptide and TcR binding. The TcR binds to a.a. present in both the peptide and the MHC-binding groove. (B) Direct recognition. Host TcRs are tolerant to self-MHC molecules presenting self-peptides (Panel 1), but are reactive to self-MHC molecules presenting foreign peptides (Panel 2) or allo-MHC–peptide complexes that resemble foreign peptide self-MHC complexes (Panel 3). (C) Indirect recognition. MHC alloantigens are processed and presented as allopeptides on self-MHC molecules to stimulate host T-cells. Abbreviations: APC, antigen-presenting cell; TcR, T-cell receptor; MHC, major histocompatibility complex.

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response occurring only in allogeneic transplantation settings. The vigorous response observed when MHC-incompatible lymphocytes are cocultured (mixed lymphocyte reaction, MLR) is the result of this direct allorecognition. In the model of direct recognition (Fig. 18B), it is believed that the three-dimensional surface determinant formed by alloMHC–peptide complex (Fig. 15B, Panel 3) resembles that of a foreign peptide bound to self-MHC (Fig. 15B, Panel 2), which optimally binds to and activates host TcR, but differs from the self-MHC–peptide complex to which the T-cell is tolerant (Fig. 18B, Panel 1). Thus, direct recognition of alloantigens represents cross-reactivity for a TcR selected to recognize foreign antigen in the context of self-MHC. The presence of multiple a.a. differences between allo-MHC– peptide and self-MHC–peptide complexes generates numerous determinants that trigger multiple, cross-reactive T-cell clones. An important concept arising from the direct recognition model is that alloreactive T-cells are the same cells involved in foreign antigen recognition. Thus, the allospecific and antigen-specific T-cell–repertoires are one and the same. There are two major hypotheses to account for the unusually high precursor frequency of alloreactive T-cells involved in this particular antigen recognition pathway (49,50). The first one (high determinant density) proposes that the ligand of the alloreactive T-cells is the allogeneic MHC molecule itself, independent of what peptides are bound. Thus, every foreign MHC molecule of a given type could act as a ligand for the alloreactive T-cells, creating a very high ligand density. As a consequence, the affinity of the alloreactive T cell’s receptor could afford to be substantially lower than that required for a peptide– self-MHC complex, thereby calling into play the alloreactive T-cell–repertoire T-cells with low, medium, and high affinity, and creating a high precursor frequency. The second hypothesis (multiple binary complex) suggests that alloreactive T-cells are specific for individual peptide–allo-MHC complexes. Given that MHC molecules at the cell surface are known to be occupied by a diverse array of peptides, a single allogeneic MHC molecule could stimulate a large range of alloreactive T-cells, each specific for an individual peptide–MHC complex. Despite these hypotheses, the role of direct allorecognition in vivo has not been definitively proven until recently. It is now also realized that the relevance of na€ve direct alloreactive T-cells in rejecting allografts may be limited to the very early stage of transplantation due to the availability of donor-derived DCs. On the other hand, recent evidence suggests that indirect recognition plays an important role in both the initiation and amplification of the rejection process (47,51). In the indirect recognition model, class I or class II MHC alloantigens undergo cellular digestion, processing, and presentation as allopeptides by host APCs (Fig. 18C), in a similar fashion to foreign antigens. Thus, peptides derived from MHC alloantigens trigger the alloimmune response when presented on self–class II. A single alloantigen may produce multiple peptides exhibiting variable lengths and a.a. composition. Each cell can display up to 104 copies of an individual peptide, and up to 102 to 103 different peptides bound to the same MHC molecule (46). Furthermore, each peptide may specifically bind to different MHC molecules. It is estimated that a heterozygous individual can express 10 to 20 different class II and six different class I molecules per cell. Thus, binding of numerous selfpeptides to allo-MHC molecules, or conversely, processing and presentation of multiple allo-peptides derived from allo-MHC molecules on self-MHC molecules result in a vast

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diversity of MHC–peptide complexes that differ from selfMHC–peptide complexes. This diversity accounts for the large number of T-cell clones responding to a single alloantigen. Nevertheless, several reports have observed that one or several Vb segments of the responding TcRs recur with a high frequency (52), suggesting the dominance of some epitopes displayed by the MHC–peptide complexes in triggering the alloimmune reaction. The model of indirect alloantigen recognition is currently under intense investigation because recent evidence suggests that modified MHC-derived allopeptides may deliver an inhibitory (53) rather than an immunogenic signal to the TcR that results in tolerance, rather than rejection of allografts (54,55). Cellular Basis of Allorecognition A unifying perspective in the alloimmune response is that rejection is initiated by disparate histocompatibility antigens (class I, class II, or both), which activate cytokine-producing Th cells, and is executed by cytokine-dependent cytolytic lymphocytes (CTL), although many phenotypes of cells may participate. The mixed lymphocyte culture (MLC) reaction (Fig. 19) seems to provide an in vitro correlate of the in vivo rejection response to dissect the contribution of individual elements. MLC Reaction. A ‘‘two-way’’ allogeneic MLC is induced by culturing mononuclear leukocytes (T-cells, B-cells, mononuclear phagocytes, and DCs) obtained from peripheral blood of one individual (donor cells) with a similar population of cells obtained from another individual (recipient cells). These cultures display proliferation of both cell populations, if there are allelic differences at MHC loci. In a ‘‘one-way’’ MLC, donor lymphocytes are gamma irradiated to inhibit their proliferation, thereby allowing one to selectively measure the proliferative response of responder host cells (measured by 3H-thymidine incorporation) toward stimulator donor cells. Two major populations of alloreactive

Figure 19 Mixed lymphocyte culture (MLC) reaction. The MLC is an in vivo determination of histoincompatibility. Proliferative responses of responder lymphocytes toward donor stimulators are determined by 3H-thymidine incorporation.

T-cells emerge during the MLC. The first is IL-2–producing CD4þ T-cells specific for allogeneic class II molecules (HLADR, DP, and DQ), which undergo the proliferative response. In practice, HLA-D disparity is detected by the proliferative response of recipient to donor cells. The magnitude of the proliferative response seems to correlate well with the length of graft survival: living, related donor–recipient combinations producing high MLC stimulation indicative of a significant class II Ag mismatch have a lower one year survival than low MLC responders (56). However, treatment with cyclosporine (CsA) and prednisone resulted in excellent graft survival in MLC high-responder, living, related recipients (56). The second cell type CD8þ T-cells function as cytolytic T-cells–recognizing foreign class I MHC product namely HLA-A, B, or C. Each individual CTL is specific for only one class I MHC gene product. However, the bulk population contains CTLs directed against most class I differences between the stimulators and responders. CTLs produce IL-2, IFN-g, TNF-a, and LT. It is the cooperation between these two distinct cell populations that ultimately leads to allograft destruction. It is emphasized that the functional subdivision of alloreactive T-cells into CD4þ helper and CD8þ CTLs is not absolute; both CD4þ CTLs and CD8þ helper cells have been identified to participate in MLR response.

Cytokine Network Cytokines are a diverse group of proteins that regulate both the specific and nonspecific phases of the immune response. They are produced by a variety of cells and act on target cells in an autocrine (same secreting cell), paracrine (nearby cell), or endocrine (distant cell) fashion. Their short-lived effect is mediated by their binding to high affinity receptors on a wide variety of cells. Most cytokines are pleiotropic, where a single cytokine exhibits a wide range of biological effects on various tissues and cells. Conversely, a single cell has different receptors for multiple cytokines and can interact with more than one cytokine. Cytokines also function in a redundant fashion; different cytokines act on the same cell to produce similar effects. This complex, intercellular cytokine network allows T, B, and/or hemopoietic cells to be differentially activated and to produce varied responses. Based on their origin, sequence of discovery, and chromosome location, cytokines have been subdivided as ILs, IFNs, colony-stimulating factors (CSFs), TNFs, TGFs chemokines, and other growth factors (Table 3) (57). Structurally, cytokines may be grouped into large families that share conserved motifs or sequence homologies. For example hematopoietins comprise the set of molecules IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, and IL-12, granulocyte macrophage–colony-stimulating factor (GM–CSFq), granulocyte– colony-stimulating factor, and erythropoietin. These molecules contain a four a-helix bundle motif, which is also shared by growth hormone prolactin, and a ciliary neurotrophic factor (58). Similarly, IL-1a, IL-1b, FGF, and keratinocyte growth factor exhibit a b-trefoil motif, whereas NGF, TGF-b, and platelet-derived growth factor assume a cysteine knot motif (two pairs of antiparallel b-strands anchored by three cysteine bridges) (59). Table 4 illustrates the most important cytokines and their prominent features. Functionally, cytokines may be organized into three broad overlapping categories: cytokines that influence T- and B-cell activation, cytokines that mediate nonspecific immunity and inflammation, and cytokines that stimulate hematopoiesis (60).

Chapter 5: Physiologic Basis of Transplantation

Table 3 Cytokine Families Interleukins IL-1a, b IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-9 IL-10 IL-11 IL-12

CSFs and TNFs

Interferons IFN-a IFN-b IFN-g

Chemokines

TGF-b

GM–CSF G–CSF M–CSF EPO TNF-a LT (TNF-b) LT-b

TGF-b1 TGF-b2 TGF-b3 TGF-b4 TGF-b5 Activins Inhibins BMPs

IL-8 MCP-1

Growth factors EGF TGF-a FGF KGF PDGF NGF

Abbreviations: BMP, bone morphogenic protein; CSF, colony-stimulating factor; EPO, erythropoietin; EGF, epidermal GF; FGF, fibroblast GF; GF, growth factor; G–CSF, granulocyte-CSF; GM–CSF, granulocyte macrophage–CSF; IL, interleukin; IFN, interferon; KGF, keratinocyte GF; LT, lymphotoxin; M–CSF, macrophage–CSF; MCP, monocyte chemotactic protein; NGF, nerve GF; TGF, transforming growth factor; TNF, tumor necrosis factor; PDGF, platelet derived GF.

Efferent Limb: Alloreactive Effectors and Graft Destruction The rejection process (Table 5), which is initiated by Ag recognition and lymphocyte activation, is ultimately effected by: (a) antigen-specific cytotoxic T cells reacting specifically

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with donor class I (CD8 CTL) or class II (CD4 CTL) Agbearing cells; (b) antigen-nonspecific effector cells activated by a lymphokine mechanism, e.g., macrophage (61); and/or (c) humoral antibodies, including a variety of complementdependent and lymphocyte-dependent moieties. As we discussed above, the outcome of T-cell recognition of antigens depends on the nature of cellular interactions and the surrounding milieu. In vivo, a third dimension of T-cell activation, i.e., location, also determines the outcome of this antigen encounter, as shown recently that peripheral secondary lymphoid organs, i.e., LN and spleen, are critical for eliciting alloimmune responses after transplantation (62). Clinically, graft rejection is classified on the basis of its timing as hyperacute, accelerated, acute, or chronic.

Multiple Pathways for Allograft Rejection Central to the allograft rejection cascade is the activation of alloreactive T-cells. After organ transplantation, there are two-way traffics of lymphocytes in and out of the graft, directed by proinflammatory chemokines or cytokines. Critical to the acute graft rejection, donor DCs migrate to recipient’s draining LNs or spleen to initiate the activation of na€ve alloreactive T-cell, directly recognizing alloantigens. At the same time, recipient’s DCs travel through the graft, pick up and process donor antigens, and present to na€ve alloreactive T-cell, indirectly recognizing alloantigens in the recipient’s draining LNs or spleen. While alloreactive CD4 T-cells are activated by DCs only, the alloreactive CD8 T-cell

Table 4 Major Cytokines and Their Function Cytokine

Gene number

IL-1 (17 kDa) Monomer

2(IL-1a IL-lb)

IL-2 (17 kDa) Monomer

1

IL-3 (26 kDa) Monomer IL-4 (20 kDa) Monomer EL-5(20kDa) Homodimer IL-6 (26 kDa) Monomer IL-7 (25 kDa) Monomer IL-10 (18kDa) Homodimer

1 1 1 1 1 1

IL-12 (40 kDa) Heterodimer

2

Type I IFN (a, b)(18 kDa) Monomer

20 IFN-a 1 IFN-b

Type II (IFN-g) (24 kDa) Homodimer

1

TGF-b (14 kDa) Homodimer Chemokines (8–10 kDa) Monomer

Multiple Multiple

TNF (17 kDa) Homotrimer

1

LT (TNF- b)(24 Kda) Monomer GM-CSF (22kDa) Homodimer

1 1

M-CSF (40 kDa)

1

G-CSF (19 kDa)

1

Cell source

Function

Macrophages, APCs

Facilitates activation of Th cells, acute phase reactants CD4 þ T cells Autocrine and paracrine T cell growth and differentiation, activation of NK and B cells CD4 þ T cells Growth and differentiation of hematopoietic stem cells CD4 þ T cells B-cell differentiation, IgE production, mast cell growth T cells B-cell and eosinophil differentiation, IgA production Macrophages, Th cells B-cell differentiation, acute phase reactants Bone marrow, stromal cells Growth and differentiation of B-cell progenitors T cells Inhibit TNF, IL-1 production by macrophages, B cell activation Macrophages Activation of NK cells, CTL activation, Th1 differentiation Macrophages a fibroblasts b Inhibit viral replication, cell proliferation, increase class I expression and NK activity T cells Activation of macrophages, T, B, NK, and endothelial cells. Increases class I and class II expression, anti-viral activity Macrophages, T cells Inhibition of macrophages and T cells Endothelial cells, T cells, phagocytes, Leukocyte chemotaxis, activation fibroblasts Macrophages, T cells Activation of neutrophils, endothelial cells, fever acute phase reactants, cachexia T and B-cell stimulator Activated T cells Similar to TNF Endothelial cells, T cells, phagocytes, Acts on marrow progenitors for growth and fibroblasts differentiation of granulocytes and monoclear phagocytes. Granulocyte activation Phagocytes, fibroblasts, endothelial Differentiation to mononuclear phagocytes cells Phagocytes, fibroblasts, endothelial Differentiation granulocytes cells

Notes: Mononuclear phagocytes include monocytes and macrophages; granulocytes include esinophils, basophils and neutrophils. Abbreviations: CSF, colony stimulating factors; CTL, cytotoxic T-lymphocytes; Ig, immunoglobulin; IL, interleukin; kDa, kilodalton; NK, natural killer cells; TNF, tumor necrosis factor.

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Table 5 Clinical Types of Rejection Rejection

Time course

Clinical signs and symptoms

Mechanism

Pathology

Hyperacute

Sudden onset, min/hr after vascularization

Abrupt loss of function; no perfusion

Humoral with preformed anti-HLA antibodies

Accelerated

Rapid loss of function, 72–100 hr after transplantation

Abrupt loss of function; tender, swollen graft

Acute

First 3 mo after transplantation

Graft swelling, fever, and oliguria

Cellular presensitization: destruction by specific Tc cells or ADCC Cellular; Tc cells and DTH mechanism

Fibrin thrombi within arterioles. Endothelial sloughing, platelet aggregation Interstitial hemorrhage. Fibrinoid necrosis of small arterial vessels

Chronic

Gradual decline in graft function

Progressive loss of function, hypertension, and glomerulopathy

Humoral

Interstitial and perivascular mononuclear infiltration, tubulitis, and interstitial edema Obliterative vascular changes, interstitial fibrosis

Response to therapy () Irreversible

()

(þ) Reversible

() Irreverisble

Abbreviations: ADCC, antibody-dependent cell-mediated cytotoxicity; DTH, delayed-type hypersensitivity; HLA, human leucocyte antigen.

activation may require additional CD4 help, which is either cytokine, such as IL-2, or costimulatory interaction with DCs, such as CD154–CD40. The activation of alloreactive B-cells is mediated by activated CD4 T-cells via direct CD154–CD40 interactions. Activated T-cells are now able to infiltrate into the graft and exert their effector functions (63). Cytotoxic CD8 T-cells recognize donor MHC class I molecules expressed on the graft, and kill the cells preferentially by the perforin/granzyme pathway; cytotoxic CD4 T-cells, on the other hand, recognize donor class II molecules, which can be induced by inflammatory cytokines on certain types of cells, e.g., endothelia, and kill the cells by the Fas/FasL pathway. Additionally, activated T-cells, in both direct as well as indirect allorecognition pathways, produce proinflammatory cytokines, such as IFN-g, which induce the delay-type hypersensitivity reaction where macrophages release toxic molecules, e.g., NO, TNF-a, and oxygen species. Antibodies reactive to donor MHC molecules will mediate complement activation, such C6, or antibodydependent cell-mediated cytotoxicity, e.g., FcR-mediated NK cytotoxicity. While the cytotoxic T-cell–mediated allograft rejection is the dominant effector mechanism of Th1-type alloimmune response, Th2-type immune response is also able to reject allografts. In addition to alloreactive antibodies, IL-4, IL-5, or IL-13 mediate eosinophil recruitment and activation within grafts, and the release of cytotoxic granules from activated eosinophils can directly damage allografts (64).

Hyperacute Rejection This is characterized by the cessation of graft function minutes to hours after graft revascularization. This form of rejection is thought to be secondary to host presensitization, i.e., to the presence of host preformed antidonor antibodies. Furthermore, it is not amenable to any form of pharmacologic immunosuppressive agents. A classic setting for hyperacute rejection is ABO incompatibility wherein circulating antidonor hemagglutinins rapidly thrombose the graft after binding to endothelial cells, and through their Fc tails activate complement. Damaged endothelial cells secrete high-molecular-weight forms of von willebrand factor that mediates platelet adhesion and aggregation and granulocyte activation. Although, anti-ABO antibodies do not present a clinical problem due to pretransplant ABO

typing, preformed antibodies toward HLA or endothelial E–M antigens are the more usual causes of hyperacute rejection. These antibodies may be induced by prior transplants, blood transfusion, or pregnancy. Correlation between a positive pretransplant microcytotoxicity cross-match (an assay for detection of recipient antidonor antibodies) and the occurrence of hyperacute rejection is well documented (65). Pretransplant testing of recipients for antidonor antibodies has markedly reduced the incidence of hyperacute rejection in clinical allotransplantation. On the one hand, renal and heart transplants are particularly susceptible to hyperacute rejection. Thus, a negative T-cell cross-match and ABO compatibility are usual prerequisites. On the other hand, the liver is less susceptible to hyperacute rejection, and liver transplants have been performed in the face of ABO incompatibility and positive cross-matches. Microscopically, hyperacutely rejected transplants show fibrin thrombi within the small arteries and arterioles, capillary dilation, endothelial cell sloughing, platelet aggregation, and adhesion of polymorphonuclear leukocytes. Both animal and human renal allograft studies suggest that rejection is initiated by a primary immune injury to the vascular endothelium. Circulating antibodies bind specifically to the class I histocompatibility Ags exposed on the vascular endothelial cell surface and activate complement. Platelet aggregation and subsequent release of platelet factors and vasoactive substances occur at the site of injury. The initial vasoconstrictive response progresses to thrombosis after aggregation of platelets, red blood cells, and polymorphonuclear leukocytes, followed by fibrin deposition. The ischemic cascade obviates perfusion to the transplanted organ. Sometimes the reaction is not immediate but rather occurs 24 to 48 hours after transplantation, in which case it is denoted as ‘‘delayed hyperacute’’ rejection. Presumably, these cases reflect initial low titers of antibodies with a prompt secondary response. Thus, delayed hyperacute rejection appears to be the result of a qualitatively weaker immune response than that which causes hyperacute rejection.

Accelerated Rejection Accelerated rejection frequently produces rapid loss of graft function; in many cases, it is refractory to antirejection

Chapter 5: Physiologic Basis of Transplantation

therapy. The graft usually functions satisfactorily for 72 to 100 hours, before graft function declines precipitously. Histologically, there are vascular lesions of capillary disruption with interstitial hemorrhage, hemorrhagic infarcts, and fibrinoid necrosis of small arterial vessels. Two pathogenic mechanisms appear to mediate the response: cellular presensitization with rapid graft destruction by specific T-cytotoxic cells, or a secondary antibody production of a moiety that depends on lymphocytes to execute cytotoxicity (antibody-dependent cell-mediated cytotoxicity). In such a reaction, antibody acts as a bridge between donor tissue and cytotoxic cells, an interaction that results in activation of the lytic mechanism of the cytotoxic cell with subsequent tissue destruction.

Acute Rejection Acute rejection, unlike the accelerated or hyperacute mechanisms, tends to be reversible with therapy. Acute rejection episodes are the most common type, affecting 30% of renal allografts and usually occurring during the first three months following surgery with a peak incidence at five weeks. Acute rejection is uncommon after the first year unless there is noncompliance to the drug regimen by the patient or an inappropriate reduction of immunotherapy by the physician. The clinical features of cell-mediated renal allograft rejection including graft swelling and tenderness, fever, oliguria, and hypertension are rarely obscured in the CsA era. Laboratory assessment reveals an increased serum creatinine, and the diagnosis may be confirmed by percutaneous needle biopsy. However, attenuation of the clinical picture by CsA therapy has led to greater reliance on laboratory data and imaging studies. The greatest dilemma is the differentiation between acute rejection and CsA-induced nephrotoxicity. Furthermore, anuria secondary to acute tubular necrosis (ATN) may occur immediately following operation or within the first few weeks after transplantation. In general, a 25% rise in serum creatinine demands immediate evaluation (66). Imaging studies include the evaluation of the physiologic status of the allograft by nuclear renograms or ultrasound examination enhanced by color flow Doppler (CFD). Nuclear renograms include three phases that utilize two radiolabeled compounds (99mTc-DTPA and 99m Tc-MAG3). The first phase evaluates renal perfusion, which should be evident within four seconds of the aortic blush following the administration of 99mTc-DTPA. The second phase quantitates graft flow by the interval between injection and the appearance of parenchymal activity. The third phase evaluates the excretory function by comparing the amount of the isotope in the bladder relative to the graft. In acute rejection, there is decreased perfusion, poor parenchyma uptake, and excretion. Good perfusion with impaired parenchymal uptake is common in ATN. The findings in early CsA toxicity are similar to those of ATN. In severe CsA toxicity, altered perfusion may be present due to the alpha-agonist effect of CsA. Thus, the diagnosis of CsA toxicity is tentative until rejection and ATN are ruled out. Ultrasound examination with CFD accurately detects allograft swelling common to both ATN and rejection, but it is less helpful than nuclear scans in differentiating both entities. It also demonstrates ureteric or renal pelvic dilatation secondary to obstruction or accumulation of perinephric fluid collections that contribute to the declining renal allograft function. CFD sensitively detects the increase in vascular resistance, which is associated with

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rejection, but is not an early feature of CsA toxicity. However, CFD is operator dependent, and the evaluation of acute rejection often demands histopathologic examination. Histologic features of acute rejection include prominent interstitial and perivascular mononuclear infiltrates with interstitial edema and tubulitis (Fig. 20A). Macrophages and T-lymphocytes, specifically cytotoxic T-cells, are the predominant infiltrating cell types. In addition, CD4 helper cells mediating delayed-type hypersensitivity mechanisms are important contributors. Significant interstitial cellular infiltration with moderate tubulitis (greater than four mononuclear cells/tubular cross section) constitute grade I ‘‘mild’’ acute rejection. Grade II ‘‘moderate’’ rejection demonstrates significant interstitial infiltration, severe tubulitis, and/or intimal arteritis, while ‘‘severe’’ grade III rejection involves transmural arteritis, medial smooth muscle necrosis, and/or focal infarction and interstitial hemorrhage. In mild-to-moderate cellular rejection, two cell types predominate within the interstitial cellular infiltrates (67): macrophages, comprising 40% to 50%, and T-lymphocytes, 30% to 40%. Microscopically, infiltrating T-cells include cytotoxic T-cells (Tc) bearing the CD8 cell surface marker and helper CD4 cells. In severe cellular rejection, the cellular infiltrate is composed of 60% macrophages, 20% to 30% polymorphonuclear leukocytes, and 15%

Figure 20 Renal allograft rejection. (A) Acute rejection demonstrating prominent interstitial and perivascular mononuclear infiltrates with interstitial edema. (B) Chronic rejection characterized by interstitial fibrosis and obliterative vascular changes.

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T-lymphocytes, the majority of which are Tc cells. Although there are no classical histological changes of acute CsA toxicity, inclusion bodies, peritubular capillary congestion, tubular vacuolization, microcalcifications and thrombotic microangiopathy have been described. ATN is a difficult histologic diagnosis, although swelling of tubular cells and disrupted tubules in the absence of signs of rejection or drug toxicity is helpful information. After activation of CD4 and CD8 lymphocytes, circulating immune cells recognize the allograft at the periphery. The CD4 cells play a critical role by releasing lymphokines that recruit and activate immunologically incompetent lymphocytes and macrophages toward the allograft through a delayed-type hypersensitivity mechanism. This mechanism is specific, although the target cytotoxic cell activated by lymphokines is not. On the one hand, the delayed-type hypersensitivity effector CD4 cell does not directly mediate allograft damage but secretes lymphocytotoxins responsible for cellular destruction and lymphokines that attract and activate macrophages, lymphocytes, and NK cells to produce cellular necrosis. On the other hand, cytotoxic CD8 lymphocyte displays specific activity against class I alloantigen on the graft, thereby participating in the tissue destruction. Although the CD8 Tc cell is the predominant specific lymphocyte involved in cellular rejection, the CD4 helper cell is an important component of the process because of its ability to magnify the cellular response by recruitment of other immune elements. In summary, cytotoxic T-cells specifically react with the class I alloantigen disparity, whereas the delayed-type hypersensitivity mechanism of CD4 helper cells causes a nonspecific response in recognition of the class II alloantigen disparity. Acute rejection episodes may be precipitated by viral syndromes that are caused by the following (68):

low serum complement levels suggest that the humoral antibodies mediate a glomerular lesion. The Ig deposits appear to produce obliterative vascular changes (Fig. 20B) eventuating in interstitial fibrosis (65). Chronic rejection tends to be a separate pathologic and clinical entity reflecting an alloantibody response frequently triggered by but not due to recurrent acute rejection episodes. In recent years, the role of T-cells recognizing alloantigens indirectly has been implied (69–71). At least three lines of evidence support the case: first, by manipulating animal models (e.g., class I KO recipients of MHC class II KO graft), it was shown that indirect allorecognition alone was able to mediate graft rejection; second, by analyzing lymphocytes from those animal models, it was confirmed that there was a small population of self-MHC–restricted T-cells responsive to donor-derived peptides; third, in clinical patients, it has been demonstrated that there is a positive correlation between T-cell reactivity to synthetic allopeptides derived from the donor MHC and chronic graft rejection. It is now clear that the nature of antigenic stimulations from an allograft changes over time. At the late stage of transplantation, processed alloantigen becomes the dominant type, because the donor-derived APCs residing in and emigrating from the graft diminish, and all of the parenchymal cells of the graft can serve as a substrate for host APC processing. Due to the fact that the T-cell precursor frequency in indirect allorecognition pathway is several orders of magnitude less than that in the direct allorecognition pathway, the indirect alloimmune response represents a more slow-paced immune reaction, consistent with the natural history of chronic rejection.

IMMUNOSUPPRESSIVE THERAPY

From the opposite standpoint, allograft rejection sometimes produces activation of a latent viral infection, caused either by viral transformation of lymphocytes or by release of virus from damaged donor kidney cells.

The ultimate goal in organ transplantation is to achieve immunologic tolerance of the host to the graft, thereby obviating any rejection process. Although there have been important experimental leads toward the goal of decreasing graft immunogenicity, only the recipient immunosuppressive approach has proven clinical efficacy. The ideal immunosuppressive regimen would specifically dampen host responses toward donor, but not toward third-party transplants. Currently, the immunosuppressive modalities used for organ transplantation are nonspecific, altering host responses not only toward histocompatibility but also toward an array of other antigens. In general, two types of agents are used in clinical practice: pharmacologic agents exemplified by corticosteroids, CsA, and antiproliferative agents, and biologic agents such as antilymphocyte sera (ALS) and monoclonal antibodies. Irradiation, although utilized in the early days of clinical transplantation, has no current applications.

Chronic Rejection

Mechanisms of Pharmacologic Agents

1. 2.

3.

Direct viral damage, e.g., as in mumps or influenza viral syndromes, releasing increased amounts of histocompatibility Ags from the graft. Viral Ags expressed during a clinical infection may cross-react with donor histocompatibility Ags. Thus the immune response directed against the virus can potentially result in graft rejection. The virus may augment the host’s immune system by acting as an adjuvant, making him more susceptible to an acute rejection episode, although in most cases, particularly with cytomegalovirus, the infection tends to induce immunodepression.

Chronic rejection is characterized by a gradual decline in graft function and tends to be unresponsive to steroid antirejection therapy and to eventuate in transplant failure. Although CsA has reduced the incidence of acute renal allograft rejection, it has failed to reduce the previously observed 10% incidence prior to its use (66). The clinical syndrome of progressive deterioration of graft function following renal transplantation includes hypertension and glomerulopathy with hematuria and/or proteinuria. Graft damage has been ascribed to a humoral process based on detection of donorspecific circulating antibodies in recipient sera. Deposition of Igs and complement on glomerular capillary walls and

Current clinical and experimental immunosuppressive agents (72) seek to inhibit the recognition of alloantigens, destroy alloreactive T-cells, or alter the regulatory events leading to maturation and differentiation of mature alloreactive elements (Fig. 21).

Inhibition of Antigen Recognition, Processing, and Presentation Corticosteroids The first step in the development of transplant rejection is the generation of costimulatory activity by APCs, an event necessary for T-cell activation. Corticosteroids impair transcription

Chapter 5: Physiologic Basis of Transplantation

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evidence suggests that the activated ligand–receptor complex impedes the activation, but not the binding, effects of AP-1 and NFAT DNA–binding proteins on the IL-2 gene promoter region. Because of their tendency to inhibit nonspecific resistance more intensively than specific resistance (low immunosuppressive index), and their pleiotropic toxicities due to widespread utilization of GRE in other gene transcription units (an unfavorable therapeutic index), steroids are the weakest link in present clinical regimens. Steroid therapy may produce multiple, severe side effects such as hypertension, cushingoid syndrome, steroid-induced diabetes, osteoporosis, avascular necrosis of bones, weight gain, growth retardation, poor wound healing, cataracts, pancreatitis, peptic ulceration, colonic perforation, gastrointestinal bleeding, and psychosis.

Lymphokine Synthesis Inhibitors (Calcineurin Inhibitors) Figure 21 Sites of action of immunosuppressive drugs in relation to the cell cycle. Steroids and 15-DSG block initial activation events at the G0 phase. CsA and FK506 (Tacrolimus) inhibit G0 to G1 progression, by inhibiting IL-2 transcription. RAPA acts during the late G1 phase by blocking cytokine signal transduction. Aza, MM, MTX, and MZB inhibit purine, while BQR inhibits pyrimidine synthesis at the S phase. Abbreviations: 15-DSG, 15-deoxyspergualin; Aza, Azathioprine; BQR, Brequinar; CsA, cyclosporine; MM, Mycophenolate Mofetil; MTX, Methotrexate; MZB, Mizorbine; RAPA, Rapamycin.

of the IL-1b gene, cause rapid degradation of IL-1 mRNA, as well as reduce IL-6 transcription, thereby inhibiting the generation of the costimulatory signal (73). Moreover, steroids also inhibit the gene transcription and expression of IL-2, TNF, and IFN-g. These compounds diffuse intracellularly and bind to cytoplasmic receptors that exist in association with the 90 kDa HSP. Steroid binding induces the dissociation of the HSP from the complex and translocation of the ligand– receptor complex into the nucleus. The activated complex reversibly binds to DNA sites, collectively called glucocorticoid response elements (GRE), which exhibit the TGTTCT DNA motif and are present on multiple promoter regions of several cytokine genes including the IL-2 promoter. Recent

Cyclosporine CsA (1202 Da; 74) is a cyclic endecapeptide, extracted from the soil fungus Tolypocladium Inflatum Gams, which exhibits a hydrophobic exterior and a hydrophilic center shielded in the interior of the structure. CsA binds to a cytoplasmic protein, cyclophilin (CYP; Fig. 22), which displays cis– trans peptidyl-propyl-isomerase activity, which everts the hydrophilic CsA site from the interior to the exterior of the molecule, presumably converting the prodrug into the active moiety. CsA inhibits processes dependent upon an increased cytoplasmic calcium burst. CsA binding to CYP changes the substrate affinity of the isomerase such that it complexes with and inhibits calcineurins (CaN) A and B, serine–threonine phosphatases associated with Ca2þ, and CaM. The formation of the complex CaN A–CaN B–Ca2þ– CaM–CsA–CYP prevents the dephosphorylation, activation, and nuclear translocation of NFATc, the first regulatory protein controlling the enhancer region of the IL-2 gene (75). CsA inhibits T-cell synthesis of not only IL-2, but also IL-3, IFN-g, IL-6, and IL-7. IL-1 synthesis and IL-2-receptor (IL-2R) generation appear to be calcium independent and therefore CsA resistant. T-helper and T-cytotoxic cell functions are downregulated by CsA in addition to the generation of regulatory T-cells that appear to enhance allograft survival in animal models (74). The lack of inhibitory effects of CsA on the generation, proliferation, or function of

Figure 22 Proposed mechanism of action of calcineurin inhibitors on the regulatory sequences of the IL-2 gene. The promoter region of the IL-2 gene contains binding sites for the Oct-1, AP-1, OAP, and NF-kB DNA–regulatory proteins. NFIL-2A and NFIL-2E sites bind Oct-1/OAP and NFAT proteins, respectively. CD28RE, CD28 responsive element. The pentameric complexes CaN A– CaN B–Ca2þ–CaM–CsA–CYP or CaN A–CaN B–Ca2þ–CaM– FK506–CYP prevent activation and nuclear migration of NFATc, which normally binds to NFATn to induce activation of the IL-2 promoter at the NFIL-2E site. Abbreviations: Ca2þ, calcium; CaM, calmodulin; CaN, calcineurin; CsA, cyclosporine; CYP, cyclophilin; FKBP, FK506 binding protein; NFAT, nuclear factor of activated Tcells; IL, interleukin.

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nonspecific host resistance elements produces a high immunosuppressive index. CsA exhibits a variety of side effects that include nephrotoxicity, hypertension, hyperkalemia, hyperuricemia, hypercholestrolemia, hypertriglyceridemia, hyperglycemia, hemolytic uremic syndrome, hepatotoxicity, and mild neurologic symptoms such as insomnia, hyperactivity tremors, and depression. Further, the concentration of CsA is affected by several drugs that enhance or inhibit CsA metabolism through the cytochrome P-4503A enzyme system. Rifampin, phenobarbital, phenytoin, and valproic acid increase CsA metabolism, whereas erythromycin, verapamil, and ketoconazole reduce CsA metabolism. It has been difficult to optimize CsA therapy because of the agent’s marked interindividual variations in pharmacokinetics and in pharmacodynamics. Among renal transplant recipients, there is a sixfold span of drug clearance rates and a ninefold span of relative oral bioavailability. Thus, CsA doses are based upon measured drug levels or calculated pharmacokinetic parameters, rather than on fixed weight- or surface area–adjusted amounts. CsA whole blood levels are more reproducible than plasma levels. CsA levels are measured by either fluorescence or radioimmunoassays that measure primarily the parent compound and about 15% of its metabolites, or by high-performance liquid chromatography, which measures only the parent compound. Some centers tailor CsA therapy based on trough levels (175–225 ng/mL for renal and 350–450 ng/mL for hepatic transplants). However, clinically obtained trough levels do not show a good correlation with areas under the concentration curve (AUC; 76). Nevertheless, in clinical practice, AUCs are often difficult to obtain and trough levels are widely utilized to guide dosing protocols. The use of the new glycol-furan microemulsion formulation of CsA (Neoral) has facilitated dose prediction. Neoral absorption appears to be independent of bile and food composition. Tacrolimus (FK506, Prograf) Tacrolimus is a polycyclic macrolide antibiotic produced by Streptomyces tsukubaensis that inhibits T-cell production of IL-2, IL-3, IL-4, and IFN-g, 10- to 100-fold more potently than CsA. Furthermore, it blocks the expression of IL-2R and inhibits the synthesis of TNF-a GM–CSF, and c-myc. Tacrolimus, like CsA, inhibits Ca2þ-dependent intracellular pathways mediated by interactions with an intracellular binding protein [FK506 binding protein (FKBP); Fig. 22], which, like CYP, is a member of the cis–trans propyl isomerase ‘‘immunophilin’’ family. This interaction ultimately results in inhibition of IL-2 production (75). Tacrolimus and CsA exhibit in vitro and in vivo antagonism, which may be explained by the fact that they share a common intracellular pathway. The two drugs, however, may not act by precisely the same mechanism, because, in experimental models, Tacrolimus inhibits, whereas CsA facilitates the induction of regulatory cells. Tacrolimus absorption, following oral administration, is less bile dependent than CsA. This may be beneficial during the biliary diversion with T-tube drainage following hepatic transplantation. This may explain, in part, the wider utilization of Tacrolimus as the primary immunosuppressive agent in liver transplantation when compared with CsA (77). In the recent years, Tacrolimus has been widely utilized in clinical pancreatic and renal transplantation. Similar to CsA, Tacrolimus dosing is guided by whole blood trough concentrations.

Side effects of Tacrolimus therapy include nephrotoxicity caused by decreased renal cortical blood flow and reduced glomerular filtration rates and drug-induced hyperkalemia and hypertension, findings previously reported with CsA therapy. Excessibely high doses of Tacrolimus may be associated with neurotoxicity, which includes expressive aphasia, coma, and seizures, nephrotoxicity, new onset diabetes mellitus, and, rarely, lymphoproliferative disorders. Thus, Tacrolimus appears to have an equivalent immunotherapeutic effect to CsA. Although immunosuppressive protocols that avoid calcineurin inhibitors may be utilized in the future, most clinicians currently utilize either agent as the primary immunotherapeutic medication. The choice between CsA and Tacrolimus is mostly based on clinical preference and the experience of the center with each drug.

Inhibitors of Late T-Cell Function (TOR Inhibitors) The inhibitors of mammalian target of rapamycin (mTOR) are a relatively new class of immunosuppressants (78). This class includes two drugs: sirolimus and everolimus. Sirolimus (Rapamycin, RAPA, Rapamune1; Wyeth Pharmaceuticals) Sirolimus (79) a macrolide antibiotic produced by the actinomycete Streptomyces hygroscopicus is structurally related to FK506. Unlike CsA and Tacrolimus, which influence the G0 to G1 progression, Sirolimus does not affect lymphokine synthesis, but rather inhibits their effects on the G1 build-up phase to the S phase (Fig. 21). Thus, RAPA inhibits a broad array of calcium-independent activation events mediated via CD28, protein kinase C, and lymphokine stimulation. Although Sirolimus binds to FKBP to form RAPA–FKBP complex, the target of its inhibitory effects on isolated lymphocytes is unknown. Various authors report retarded endocytosis of receptor complexes or inhibited PI3-kinase activity necessary to generate the 3-phosphorylated phosphoinositides (PI-3-P, PI-3,4-P2, and PI-3,4,5-P3), which function as growth and division signals. RAPA also inhibits the IL-2 stimulated T-cell S-phase entry (80) by blocking p70S6 or the p34cdc2 kinases, thereby blocking the proliferative but not the differentiation events induced by IL-2. The clinical utilization of RAPA has been based, in part, upon its synergistic immunosuppressive effect with CsA. Thus, the combination of RAPA and CsA may have allowed the utilization of both drugs at extremely low doses to produce a high immunosuppressive index with a low toxic profile. In renal transplantation, RAPA is widely used in conjunction with CsA in ‘‘steroid-sparing’’ immunosuppressive protocols. Unfortunately, a black-box warning has been placed on RAPA in liver transplantation after the initial clinical trials resulted in a high rate of side effects that included hepatic artery thrombosis and delayed wound healing. Side effects most commonly documented with sirolimus include nausea, vomiting, and elevated liver enzymes, cholesterol, and triglycerides. Everolimus (Certican1, RAD-001; Novartis) Everolimus has a mechanism of action identical to that of sirolimus. Once inside the cell, it binds to FKBP-12. The complex then binds mTOR, inhibiting activation of p70S6 kinase and arresting the cell cycle at the G1 to S phase. This results in a blockade of growth factor–mediated, IL-2 and IL-5–driven proliferation of T- and B-lymphocytes and vascular smooth muscle cells.

Chapter 5: Physiologic Basis of Transplantation

Drug–drug interactions must be considered with the mTOR inhibitors. Coadministration with CsA increases exposure to everolimus, but this interaction does not appear to be as severe as that seen with CsA and sirolimus. Nevertheless, withdrawal of CsA will require retitration of the everolimus dose. Everolimus is metabolized by the cytochrome P450 3A4 enzyme system, resulting in a potential for important interactions with drugs such as macrolide antibiotics, azole antifungals, and phenobarbital or phenytoin. Currently, this drug is in clinical trial with very promising results (81).

Nucleoside Synthesis Inhibitors Azathioprine Aza, a purine analog, is a nitroimidazole derivative 6-mercaptopurine (6-MP). It was first used at the Peter Bent Brigham Hospital in Boston and rapidly became the cornerstone of chemical immunosuppression in the 1960s (72). Aza must be metabolized in the liver via conversion to 6-MP and then to its active metabolite 6-thioinosinic acid. It exerts its immunosuppressive effect by broadly inhibiting DNA and RNA synthesis by preventing the synthesis of adenosine monophosphate and GMP from inosinic acid (de novo purine synthesis) and blocking the interconversion among the precursors of purine nucleotides (salvage pathway). However, Aza exhibits multiple limitations. Because Aza can be incorporated into DNA as a fraudulent base, it carries an increased proclivity to malignancy and papillomatosis. Aza also produces a hepatotoxicity that can potentiate the CsA injury. Because Aza acts as a competitive enzyme inhibitor of both de novo and salvage pathways, it must be used at high concentrations, resulting in bone marrow depression to a greater degree than its immunosuppressive effect. The limitations of Aza as an immunosuppressive drug led to an urgent need to identify new nucleoside synthesis inhibitors that (a) acted noncompetitively to inhibit synthesis of either purines [Mizorbine (MZB) and Mycophenolate Mofetil (MMF)] or pyrimidines [Brequinar (BQR)], (b) were not incorporated into DNA, and (c) showed reduced myelotoxicity. In the early days of transplantation, Aza–steroid regimens displayed a low therapeutic index, with 90% of renal allograft recipients experiencing rejection episodes and only 50% one-year graft survival. Following the advent of CsA, Aza was utilized as a third agent with the CsA–steroid regimen. Currently, the clinical use of Aza has been largely replaced by MMF. MMF (RS-61443, Cellcept1; Hoffmann–La Roche) MMF is a semisynthetic morpholinoethyl ester of mycophenolic acid (MPA), which is produced by the fungus Penicillin glaucum. It was introduced in the mid-1990s after a series of clinical trials showed its superiority to Aza in preventing allograft rejection in kidney transplant recipients (82). It is now commonly used for maintenance immunosuppression in various regimens containing calcineurin inhibitors, steroids, and/or sirolimus (83). It is rapidly absorbed following oral administration and hydrolyzed to form MPA, its active metabolite. MPA inhibits the de novo pathway of guanosine nucleotide synthesis through reversible inhibition of inosine monophosphate dehydrogenase (IMPDH) and possibly also GMP-synthetase, two crucial enzymes in the de novo purine synthesis pathway, thereby depleting phosphoribosyl pyrophosphate (PRPP). Depletion of PRPP inhibits generation of hypoxanthine guanine phosphoryl ribosyl transferase, an important intermediate in the major purine salvage

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pathway. The result is a relatively selective antiproliferative effect on lymphocytes, because they rely heavily upon this de novo pathway for purine synthesis. Initial reports suggested that MMF does not produce significant hepato- or nephrotoxicity. The most problematic adverse events associated with this agent include gastrointestinal effects (diarrhea, abdominal pain, nausea, vomiting, and dyspepsia) and hematologic disorders (leukopenia, anemia, and thrombocytopenia). In most cases, these effects respond promptly to dose reductions. The drug is now widely utilized in all solid organ transplants, as a third agent in conjunction with steroids in CsA- or Tacrolimus-based triple immunosuppressive regimens. Moreover, MMF has been successfully utilized with either CsA or Tacrolimus in steroid-sparing immunosuppressive protocols. MPA Sodium (Myfortic1, ERL-080; Novartis) Mycophenolate acid sodium is an enteric-coated formulation of the sodium salt of MPA. It is considered a second generation IMPDH, with a mechanism of action that is the same as that described above for MMF. Because it is enteric coated, it allows a delayed release of MPA into the small intestine. This is hoped to ameliorate some of the upper gastrointestinal side effects associated with MMF’s immediate release of MPA into the stomach. A dose of 720 mg of mycophenolate acid sodium offers systemic exposure equivalent to 1000 mg of MMF. Two pivotal, Phase III, multicenter trials were conducted in renal transplant recipients to assess tolerability and efficacy (84). In each of these trials, mycophenolate acid sodium (ERL-080) 720 mg, twice daily, was compared to MMF 1000 mg, twice daily, in combination with CsA and corticosteroids. Both groups demonstrated equivalent rates of biopsy-proven acute rejection, graft loss, and patient death. The ERL-080 group, however, seemed to have fewer moderate or severe acute rejection episodes. No significant difference in the incidence or frequency of GI adverse events was noted at three or six months. The group receiving ERL080, however, had a lower incidence of severe neutropenia, fewer serious adverse events, and fewer serious infections. Myfortic has been recently approved for clinical utilization. MZB; Bredinin MZB is an imidazole nucleoside antibiotic isolated from Eupenicillum brefeldianum, and is converted to its active monophosphate form by adenosine kinase (72). It has been used experimentally and, to a limited extent, clinically in Japan as an immunosuppressive agent. MZB noncompetitively inhibits IMPDH, which converts IMP to xanthine monophosphate, an intermediate compound in the synthesis of GMP. Thus, like Aza and MMF (MM), MZB progressively depletes intracellular GMP stores. Its immunosuppressive capacity is estimated to be comparable to that of Aza, whereas associated marrow depression is less prominent. The drug is not metabolized in the liver and demonstrates no significant hepatotoxicity. Its use has been associated with significant anorexia and subsequent weight loss in the canine model. As yet no clinical trials have been published outside of Japan, and worldwide distribution of the drug is not anticipated. Leflunomide (Arava1; Aventis) Leflunomide is currently approved for use in rheumatoid arthritis and previous data suggested a promising role for

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it in organ transplantation. Leflunomide is rapidly and extensively metabolized to A77 1726, an active metabolite that is responsible for most of the drug’s activity (85). It inhibits de novo pyrimidine synthesis by reversibly blocking dihydro-orotate dehydrogenase (DHODH). This inhibition blocks T- and B-cell proliferation and strongly suppresses the production of IgM and IgG antibodies. Doses required for use in kidney and liver transplantation are significantly higher than those used for rheumatoid arthritis. Leflunomide carries a black box warning against use in pregnancy and is not suggested for use in patients with significant hepatic dysfunction or positive hepatitis B or C serologies. Within the field of organ transplantation, leflunomide has been studied in a variety of different capacities. It has been shown to control and reverse acute rejection, control the aggressive antibody responses in xeno-rejection, reverse an established chronic rejection response in rodents, have synergistic or additive effects with the calcineurin phosphatase inhibitors, and have the ability to halt replication of herpes viruses including cytomegalovirus (CMV) and herpes simplex virus (HSV). Due to its adverse event profile, however, it may not be widely used in transplantation. Toxicity, including anemia and elevations in liver function tests, correlates with serum concentration. ‘‘Public Citizen,’’ a consumer watchdog group, has petitioned the FDA to remove leflunomide from the market, citing 130 cases of severe liver dysfunction and up to 22 deaths associated with its use. FK778 (Fujisawa) FK778 is an analogue of A77 1726—leflunomide’s active metabolite. It is a novel molecule with a mechanism of action similar to leflunomide. Like leflunomide, it appears to have antiviral effects and has been effective in preventing acute allograft rejection in animal models (86). Compared to the parent compound, FK778 has demonstrated an equivalent or higher immunosuppressive effect with a shorter half-life in vitro and in vivo. FK778 is currently in Phase II clinical trials in Europe. Brequinar BQR is a synthetic difluoroquinoline carboxylic acid derivative, which was originally developed as an antineoplastic drug. It reversibly inhibits the mitochondrial enzyme DHODH, the fourth enzyme in the de novo pyrimidine biosynthesis pathway. The greater potency of BQR than that of MM, MZB, or Aza is probably due to the 100fold lower concentration of pyrimidine than of purine precursors (72). Oral drug administration prolongs the survival of heart, kidney, and liver transplants in rat models. BQR exhibits synergistic interaction with CsA, both in vitro and in vivo. Although high-dose BQR therapy in cancer patients produced gastric toxicity, Phase I study only showed thrombocytopenia, which can be avoided by the use of lower drug doses. Unfortunately, Phase II trials to assess its efficacy in combination with CsA and steroids were suspended. Its clinical future is therefore doubtful. Methotrexate MTX is an antimetabolite that antagonizes folic acid metabolism by binding dihydrofolate reductase, thereby inhibiting DNA synthesis (72). The immunosuppressive effects have been attributed to inhibition of macrophage migration due

to decreased IL-1 production and to reduced accumulation of inflammatory cells and/or immune mediators. Combined low-dose treatment with MTX and CsA downregulates the transcription of some gene products that mediate cytotoxicity, including TNF-a, LT, and some serine proteases, thereby prolonging transplant survival. However, utilization in clinical practice is limited.

Other Agents FTY720 (Novartis) is a novel agent with a structure that is completely different from conventional immunosuppressants and a mechanism of action that is equally unique. After oral administration, it is metabolized by sphingosine kinase to FTY720-phosphate, the active metabolite. This metabolite acts as an agonist at four of five receptors for sphingosine 1-phosphate, where it increases the intrinsic mobility of cells. This results in a reversible redistribution of lymphocytes from the periphery to lymphoid tissues (87). It does not impair T-cell activation or expansion, or memory to systemic viral infection. Rather, its immunosuppressive activity is a result of altered chemokine response and resultant removal of T- and B-cells from circulation while sequestering them in lymph nodes and Peyer’s patches. Administration of FTY720 results in a rapid, dosedependent decrease in peripheral blood lymphocyte counts. This occurs within hours and is reversible upon discontinuation of the drug. Doses of FTY720 in clinical studies have ranged from 0.125 to 5 mg, administered orally, once daily. FTY720 exhibits low interpatient variability compared with other immunosuppressants, and it is believed that therapeutic drug monitoring will not be necessary. It is extensively and completely metabolized by the liver with an elimination half-life of approximately seven days (88). Dose adjustments may only be necessary in patients with severe hepatic insufficiency. To date, clinical studies with FTY720 have been conducted solely in renal transplant recipients. A multicenter, randomized, open-label, dose-finding study compared FTY720 to MMF in combination with CsA and corticosteroids. FTY720 doses of 2.5 mg reduced the rate of biopsyproven acute rejection below that of MMF 2 g/day (89). Another study showed that FTY720 doses of 5 mg/day may allow for reduced-dose CsA with equivalent immunosuppressive efficacy. The major adverse event noted was bradycardia (12%), occurring within hours of the first dose. It is suspected that this is caused by a direct effect of FTY720 on the sinus node, and the toxicity is enhanced by concomitant b–blocker therapy. Phase III trials are underway.

Biologic Agents Polyclonal Antibodies Potent, nonspecific, immunosuppressive antibodies can be produced by immunizing a xenogeneic host, i.e., a speciesdivergent host, with human lymphocytes. These sera contain polyclonal antibodies, which opsonize the corresponding peripheral blood cellular elements, including T-cells, leading to their autodestruction in the liver and spleen. In addition, antibody binding onto lymphocytes may ‘‘blind’’ them to antigenic stimuli. The resulting in vivo depletion or inactivation of T-lymphocytes interferes with Tcell–mediated reactions, including allograft rejection, delayed hypersensitivity (DTH), and graft versus host reactions. Immunosuppression with ALS or purified globulin fractions (ALGs) has been demonstrated in experimental animal trials to prevent skin allograft rejection in mice, rats, and

Chapter 5: Physiologic Basis of Transplantation

primates and to prevent rejection of vascularized allografts in experimental canine renal and hepatic transplantation. Polyclonal antilymphocyte preparations have been conventionally used for prophylactic therapy during the first week after transplantation, to reduce the frequency and severity of early rejection episodes. However, clinical studies have not yielded unequivocal evidence supporting the use of ALS prophylactically. Some centers demonstrated improved graft survival and fewer rejection episodes, with a relative paucity of the reported side effects of serum sickness, anaphylactoid reactions, vascular thrombosis, or infection. On the other hand, other well-controlled studies suggest no therapeutic benefit from prophylactic ALG, but rather a significant risk of fatal infections. Alternatively, polyclonal antibodies can be used for therapy of acute renal allograft rejection episodes. The use of ALG in conjunction with high-dose steroids to reverse acute renal allograft rejection episodes produces a 13% increased incidence of viral sepsis and a 13% incidence of serum sickness, but an improved one-year allograft survival. Therefore, sequential rather than simultaneous use of ALG and high-dose steroids is recommended for rejection therapy. In clinical utilization, ALS and ALG preparations have been replaced by thymoglobulin, which is obtained by immunization of rabbits with human thymocytes. Like the previously used polyclonal antibodies, thymoglobulin requires central venous access for administration. Adverse events of thymoglobulin include fever, chills, and leukopenia and viral, bacterial, and fungal infections. The incidence of malignancies (less than 3%) is also comparable to other polyclonal antibodies.

Monoclonal Antibodies A second class of biologic agents that disrupt early activation events are monoclonal antibodies (MAb) directed toward surface receptor (abTcR, CD3) or coreceptor (CD4) epitopes present on T-cells or on APCs, such as B7, LFA-1, or ICAM-1. Immunosuppressive therapy with MAb reagents inactivates cell surface epitopes that mediate alloreactivity. Hybridoma technology, which was initially reported by Kohler and Milstein (90), yields readily standardized, specific xenogeneic murine MAb of greater purity and molecular specificity than polyclonal reagents. Unfortunately, clinical utilization of MAb produced in the mouse may result in the production of human antimouse antibodies (HAMA) that preclude retreament. Two innovations have been adopted to avoid the production of HAMA following administration of mouse MAbs. Chimeric antibodies combine the variable regions of mouse antibodies with human antibody constant regions and, therefore, present fewer foreign a.a. sequences to the host. However, one-third of the structure is still of mouse origin, which may stimulate the production of anti-idiotypic antibodies. Humanized antibodies combine only the smallest part of a mouse antibody that is required for target recognition, the distinctive complementarity-determining regions, with human variable and constant region frameworks (Fig. 23). However, humanized antibodies may exhibit reduced affinity to antigen epitopes and their beneficial effects can be clarified only by randomized clinical trials. Anti-CD3 and TcR MAbs OKT3 is a murine IgG2a MAb that dampens alloantigen recognition and cytolytic function of T-cells, by modulating

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Figure 23 Schematic illustration of chimeric and humanized antibodies. The murine portions of the antibody is shown in black and the human portions in white. Abbreviations: CDR, V (variable); C (constant) regions; CDR, complementarity-determining region.

or ‘‘blinding’’ cell surface CD3 epitopes, thereby reversing allorejection episodes. However, OKT3 therapy may evoke the production of HAMA including anti-idiotypic and antiisotypic varieties (91) that preclude retreatment with mouse antibodies. Further, the administration of OKT3 may be associated with fever, chills, diarrhea, headache, pulmonary edema, wheezing, hypotension, aseptic meningitis, seizures, or even coma due to the release of IFN-g, IL-1, and TNF. In addition to the acute cytokine release reactions, excessive OKT3 therapy predisposes to cytomegalovirus infections and post-transplant lymphoproliferative diseases (PTLD). Second generation MAbs seek to avoid the severe systemic reactions due to the cytokine release that follows OKT3 administration, and the proclivity toward lymphoma development. Visilizumab (NuvionTM, HuM291; Protein Design Labs) and huOKT3g1 (R. W. Johnson Pharmaceutical Research Institute) are two new humanized anti-CD3 monoclonal antibodies. These modified agents may exhibit less mitogenic and less cytokine-releasing activity than muromonab-CD3. Visilizumab, in a Phase I study in de novo live donor renal transplant recipients, induced a rapid, dose-dependent, marked depletion of peripheral T-cells within two hours. At higher doses, this effect lasted for approximately one week. Most adverse events were mild to moderate, occurred within the first few hours after administration, and were likely related to cytokine release (92). In a separate phase I study, huOKT3g1 showed the ability to reverse acute rejection episodes in kidney transplant and kidney–pancreas transplant patients. Minimal first-dose reactions occurred and anti-OKT3 antibodies were not detected (93). T10B9 and BMA 031 are two MAbs that are putatively directed against the ab TcR, which represents the initial site of T-cell recognition. Both antibodies are currently under clinical evaluation, the former for use in acute rejection episodes and the latter for prophylaxis of rejection. A major limitation of treatment with anti-TcR antibodies is the rapid development of HAMA. MAbs Against Accessory Molecules Alternate approaches seek to utilize MAb directed against accessory T-cell molecules. OKT4A IgG2a MAb, which reacts with the CD4 coreceptor on Th cells, appears to provoke fewer side effects than OKT3 (94). Unfortunately, OKT4A also generated strong HAMA. The lack of efficacy of the original murine antibody seems to be overcome with the humanized variant of OKT4A.

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A second target is the molecular pair LFA-1 on T-cells and ICAM-1 (CD54) on monocytes. A phase I clinical trial of anti-CD54 MAb induction therapy in ‘‘high risk recipients’’ demonstrated a 78% renal allograft survival, in contrast to 56% with conventional immunosuppression (95). AntiLFA-1 MAb successfully reversed steroid-resistant acute graft versus host reactions in man. These promising results using MAbs directed against T-cell/monocyte coreceptors await confirmation in controlled clinical trials. The CD28–B7 interaction is perhaps one of the best understood of the costimulatory pathways. CTLA-4-Ig and LEA29Y (Bristol-Myers Squibb and Novartis) are fusion proteins and homologues of CD28. They bind the B7 molecule, thereby blocking the interaction with CD28. LEA29Y is considered a second-generation CTLA-4-Ig and has shown to be more effective for inhibiting T-cell effector functions in vitro than CTLA-4-Ig. Early trials are not yet published, and Phase II and III clinical trials are currently in progress. IL-2R Antagonists The newest monoclonal antibodies for induction in solid organ transplantation are the IL-2 receptor blockers daclizumab and basiliximab. These agents share a similar pharmacologic mechanism. The differences between these agents lie in the amount of murine antibody sequences, with basiliximab being a chimeric molecule and daclizumab being a humanized molecule that contains fewer murine antibody sequences. Also, these agents have different dosing guidelines based on the study designs from the currently available literature. Both of these agents have demonstrated a very low occurrence of adverse reactions found to be essentially equal to placebo-treated patients without the cytokine release syndrome commonly seen with the traditional induction agents, mainly OKT3. These agents have proven efficacy in reducing the incidence of acute rejection episodes in renal transplantation, when used in combination with maintenance immunosuppression. Emerging Anti–B-Cell MAbs Alemtuzumab (Campath1; Schering AG) is an anti-CD52 monoclonal antibody currently approved for treatment of B-cell chronic lymphocytic leukemia. It targets T- and B-lymphocytes as well as monocytes. After intravenous (IV) administration, alemtuzumab rapidly and completely depletes lymphocytes from circulation and from the periphery. The most significant adverse event is a profound, prolonged lymphopenia and the subsequent increased risk of opportunistic infections. However, excessive adverse events associated with immunosuppression were not seen despite extremely low CD4 counts (96). Preliminary data suggest a trend toward a reduced incidence and severity of rejection episodes in renal, liver, and multivisceral transplant recipients. Rituximab (Rituxan1; Genentech) is a murine/human chimeric monoclonal antibody approved for use in treating non-Hodgkin lymphomas. It is specific for the CD20 surface marker on B-cells. Within the field of transplantation, it has been used primarily in cases of PTLD. It is currently under investigation for utilization in antibody-mediated rejection episodes as well as in B-cell induced disorders in transplantation.

Irradiation The immunosuppressive effect of irradiation is caused by the inhibition of DNA synthesis and cellular replication. Total

body irradiation of renal transplant recipients has been employed in the early days of clinical transplantation with limited but unequivocal success. However, total body irradiation was abandoned in favor of local graft radiotherapy, because of its associated toxicity including severe bone marrow depression and fatal infections. Subsequent clinical trials demonstrated that local graft irradiation increased the rate of reversal of allograft rejection but adversely affected the ultimate fate of the graft when used in combination with steroids. These findings, coupled with the superiority of other emerging immunologic strategies in managing graft rejection, resulted in very limited clinical application of local graft irradiation, which was ultimately abandoned. Because of the potent immunodepressive effect of radiotherapy and the need to minimize other organ toxicity, a regimen called total lymphoid irradiation (TLI) that focuses on cervical, axillary, mediastinal, para-aortic, splenic, and ilioinguinal nodes was devised and first applied for treatment of Hodgkin’s disease. After therapy, hosts displayed a state of immunosuppression characterized by T-lymphocytopenia, decreased alloreactivity, and induction of nonspecific T-suppressor cells. A decreased number and function of T-cells were documented for years after therapy. Although TLI decreases cell-mediated immunity and prolongs allograft survival in mice, success in large mammal models has not been consistent. Even if the logistic issue that TLI must be performed before transplantation is ignored, the complications associated with its administration (i.e., nausea, vomiting, possible increased incidence of lymphoma, and opportunistic infections) have limited its application in the clinical arena.

Risks of Immunosuppression Neoplasia Immunodeficiency states predispose to the development of de novo cancers since the first report of this association in 1969. The incidence of neoplasms in renal allograft recipients varies between 1% and 16%, far greater than the general population matched for age (97). Nonmelanotic skin and lip cancers, lymphoproliferative diseases, and cervical carcinomas account for 60% of the total cancers (98). Conversely, the malignancies that are most commonly encountered in the general population, namely carcinoma of the breast, lung, prostate, and colon are relatively uncommon in transplant recipients. There is an increased incidence of de novo skin and lip cancers in transplant recipients, 39% of which are squamous cell and basal cell carcinomas. Melanomas, which comprise only about 5% of skin cancers, behave in a similar fashion to the general population. OKT3 treatment poses a particularly high risk for PTLD, which encompasses a wide morphologic spectrum of Epstein–Barr virus (EBV)– induced B-cell diseases ranging from reactive, polyclonal hyperplasia, to true monoclonal malignant lymphoma. A common picture in renal allograft recipients (97) shows non-Hodgkin’s lymphomas containing TcR-b gene rearrangements consistent with monoclonal T-cell proliferation. Treatment with Aza, prednisone, and/or MTX, as well as the high frequency of herpes virus infections predispose recipients to cervical dysplasia and carcinoma in 21% of women who develop post-transplant cancers. Treatment is based on surgical extirpation of the tumors, if possible, together with reduction of immunosuppressive therapy. Conflicting data suggest that immunosuppression potentiates the growth of existing cancers. Based on recurrence rates of neoplasms posttransplantation, a two-year

Chapter 5: Physiologic Basis of Transplantation

waiting period between treatment of cancers and transplantation is justified for most neoplasms, except incidentally discovered renal carcinomas, in situ carcinomas, low-grade bladder cancers, and basal cell skin cancers.

Infections Over 80% of transplant recipients develop at least one infection after transplantation, and 40% of deaths are due to infectious complications occurring alone or following rejection and treatment. About 55% of posttransplant infections are caused by bacterial agents, 30% are by viruses, and 15% are by fungi (99). Most bacterial infections arise in the urinary tract, plastic venous catheters, the surgical wound, or intra-abdominal sites due to a spectrum of gram-negative and gram-positive agents akin to those occurring in other surgical patients. The most common viral infections after transplantation are caused by DNA viruses of the herpes virus family including CMV, EBV, HSV, and varicella zoster virus (HSV). Viral infections tend to be latent but are potentially oncogenic. Symptomatic CMV is associated with intense immunosuppression, particularly the use of ATG or OKT3, exhibiting a peak incidence at six weeks posttransplantation. In addition, CMV constitutes a risk factor for subsequent rejection episodes and bacterial, fungal, and parasitic (particularly Pneumocystis carinii) super-infections. The reduced incidence of such infections under CsA rather than Aza therapy may reflect both the less frequent rejection episodes requiring monoclonal or polyclonal intervention (31) as well as its capacity to spare nonspecific immune elements. Candida albicans is the most common fungal infection, although Candida globrata is seen with increasing frequency. Aspergillus fumigates and A. flavus are the second most common causes of fungal infections and present as pulmonary infiltrates. Other less common agents include Coccidioides immitis, Histoplasma capsulatum, and Nocardia asteroides. The protozoan parasite P. carinii has become an important cause of pneumonia in the immunocompromised patient. However, prophylaxis with trimethoprim–sulfamethoxazole has nearly eliminated these infections in transplant patients.

Clinical Application Unfortunately, current clinical practices are based upon empiric rather than scientific foundations. In spite of the widely disparate intrinsic immunogenicity of various organs, the various degrees of donor-to-recipient incompatibility, and the well-recognized interindividual variations in host immunosuppressiveness, transplant centers tend to utilize uniform initial induction and early maintenance immunosuppressive regimens.

Induction Immunosuppression The first two weeks post-transplant pose the greatest challenge, due to the requirement to promptly establish adequate immunosuppression in order to avert an acute allograft rejection episode. Many clinicians initiate CsA therapy either prior to or immediately after the transplantation procedure because experimental data suggest that CsA exerts maximal benefit at the time of antigen presentation. Others cite the proclivity of CsA to produce renal and/or hepatic dysfunction as the basis for delayed CsA administration, preferring sequential initiation protocols with antilymphocyte antibodies that provide rapid T-cell depletion with significant effects on granulocytes and platelets. Selective effects on T-cells may be

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achieved by OKT3, although its early administration carries the risk of development of HAMA that precludes its subsequent administration. Most workers, therefore, prefer the polyclonal reagents for the initial induction and reserve the use of the MAb for subsequent acute rejection episodes. Antilymphocyte induction protocols carry several penalties: delayed hospital discharge due to postponing optimization of CsA regimen, delayed onset (but not reduced incidence) of acute rejection episodes, and proclivity for viral infections and post-transplant lymphoproliferative disease.

Short-Term Maintenance Immunosuppression The short-term immunosuppressive regimen utilizes the dual drug combination of calcineurin inhibitors (CsA/ Tacrolimus) and steroids to avert allograft rejection during the first 90 days posttransplant. The most popular strategy starts with an 8 to 10 mg/kg CsA divided as a twice a day (BID) regimen. The dose is adjusted based upon measured trough levels. Most clinician’s strive for whole blood parent CsA compound concentration of 175 to 350 ng/mL. When Tacrolimus is utilized, trough levels of 10 to 15 ng/mL are usually targeted in the early postoperative period. Tripple immunosuppression regimens combine either MMF or RAPA in addition to calcineurin inhibitors and steroids. The combination of corticosteroids with calcineurin inhibitors is based upon both clinical and experimental data that suggest a synergistic effect. Almost all steroid regimens include a massive (250–1000 mg) intraoperative dose of methylprednisolone to dampen graft tissue injury and reduce T-cell activation. During the immediate postoperative period, steroid doses are rapidly tapered from 200 mg to 20–30 mg by day 7, followed by a more gradual taper to 15 mg between 30 and 90 days.

Antirejection Immunosuppression The principles of treatment for rejection episodes are relatively uniform across organs and among transplant centers. Most centers demand histopathological evidence of rejection, due to the significant penalties of excessive treatment of an adequately immunosuppressed patient. Steroid regimens combine large IV doses of methyl prednisolone with an oral prednisone taper. Delivery of IV steroids achieves high plasma concentrations and rapid reversal of symptoms. The risks of therapy include exacerbating or inducing diabetes mellitus, gastrointestinal irritation/ perforation, and triggering psychosis. Administration of more than 7 g of methyl prednisolone within the first 30 days posttransplantation constitutes a risk factor for survival, due to the pronounced depression of the nonspecific immune resistance of the host. It is important to reduce the level of steroids to the pretherapy dose within one month, as long-term administration of high doses predisposes to gastrointestinal bleeding, myopathy, osteoporosis, bacterial/fugal infections, and hypertension. The advantages of steroid treatment are the low medication costs and the 90% efficacy rate. After three to five days of steroid treatment, most centers assess the success based upon remission of symptoms and improved graft function. However, this assessment is not clinically easy. On the one hand, blood chemistry values, which reflect tissue damage end points, slowly reverse even after control of the immune process; on the other hand, delay in the delivery of adequate immunosuppression may result in graft failure or severe permanent impairment. If the

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response to steroids is deemed inadequate, and if the transplant biopsy documents persistent rejection, the physician proceeds with the administration of an antilymphocyte preparation. Most workers use OKT3 rather than polyclonal sera. To minimize the toxicity of OKT3 due to cytokine release, our regimen employs a split dose approach, delivering 1, 2, and 2 mg IV Q eight hours for the first day, followed by 2.5 mg Q 12 hours on the second day, and 5 mg/day thereafter. Each dose is administered with an anti-inflammatory pretreatment cocktail of 100 mg hydrocortisone, 50 mg benadryl, and 120 mg acetaminophen. Additionally, administration of monoclonal antibodies is always combined with a CMV prophylactic protocol. An intermediate point for the efficacy of OKT3 therapy is the reduction of T-cells bearing the CD3 marker from about 65% to < 10%, determined by flow cytometry analysis of whole blood samples with a fluoresceinated OKT3 reagent. Unfortunately, there are no secure algorithms for the length of OKT3 therapy, because there is a delay in the therapeutic effect by 7 to 10 days and in the toxic effects by 14 to 30 days.

Long-Term Maintenance Immunosuppression Although CsA reduced the incidence and severity of graft rejection episodes, it has had little impact on the 10% incidence of chronic rejection observed under Aza–Pred therapy. While it is possible that the failure represents inadequate CsA therapy due to suboptimal dose adjustments, it is more likely that host B-cell responses are less CsA sensitive than T-cell responses. Long-term management schemes show more variation than short-term regimens—there are advocates for CsA-only, CsA–Pred, CsA–Aza, and Aza–Pred strategies. Although no data suggest a difference in outcome, there have not been truly randomized trials. More recently, tacrolimus have been substituted for CsA in renal, liver, pancreas, and mutlivisceral organ transplant recipients. Furthermore, AZA use has been replaced by either MMF or RAPA.

CLINICAL TRANSPLANT OUTCOMES The modern era of organ transplantation began with the first successful grafting of a kidney between identical twins in 1954 (46). Subsequent advances in knowledge concerning the pathogenesis of rejection and the ability to manipulate this immune response by various therapeutic strategies have allowed transplantation to proceed to a point where many diseased organs can now be replaced with a minimum of difficulty. Kidney transplantation is relatively commonplace throughout the United States and many other countries, and organs such as the liver and heart are now being transplanted with increasing frequency. Although allograft rejection continues to be an important problem, CsA has done much to improve graft survival rates. Thus, for many organs, the availability of donors has posed more of a problem than the potential difficulties surrounding the rejection process. To gain an insight into the advances that have been made in organ transplantation over the last three decades, this section will briefly review the current status of organs that are transplantable. For purposes of discussion, three types of donor grafts should be recognized. These include: (i) grafting between twins in which the immunologic match is identical between host and donor, (ii) living, related grafts in which there is considerable immunologic similarity between the host and donor, but not to the same

degree as with twins, and (iii) cadaveric grafts obtained from unrelated individuals (i.e., subjects who are brain dead, but in whom function of the organ to be potentially transplanted is normal) in which immunologic matching between host and donor may be quite dissimilar, even though every attempt is made before transplantation to optimize this match as much as possible. For renal transplantation, all three types of grafts have been used. For transplantation of organs such as the heart, it is obvious that only cadaver grafts can be used.

Renal Transplantation Approximately 6000 to 10,000 new patients annually develop end-stage renal disease in the United States, srequiring treatment with either chronic dialysis or renal transplantation. For patients aged 70 years or lesser, renal transplantation has emerged as the treatment of choice. This relates to the recent advances in immunosuppression, particularly the discovery of CsA, which has drastically improved both renal allograft and patient survival (66). Before the availability of CsA, survival rates for patients receiving cadaveric allografts approached 90%, whereas graft survivals approximated 50%. With the advent of CsA, graft and patient one-year survival rates of 80% and 95%, respectively, have been routinely achieved in cadaveric transplantation. Similar results have been obtained in patients receiving living, related donor kidneys. With HLA-identical grafts, graft survival has approached 90% to 95%, and for all other living, related grafts, 75% to 85% graft survival has been obtained. Historically, patient survival following renal transplantation with a kidney from a living, related donor always exceeded that achieved with hemodialysis. In contrast, patient survival after cadaveric transplantation generally paralleled that associated with hemodialysis. With the use of CsA, however, a clear improvement in patient survival following cadaveric transplantation has been observed, when compared to that in hemodialysis. In addition, renal transplant patients demonstrated a significantly improved rehabilitation rate. Without question, both the longevity and quality of life have been clearly improved with this therapeutic modality. The renal transplanted kidney is usually placed in the retroperitonium of the iliac fossa through a lower abdominal incision (Fig. 24). Generally, the blood supply is assured by anastomosing the renal artery to the external iliac artery in an end-to-side or to the hypogastric artery in an end-toend fashion. The renal vein is anastomosed to the iliac vein end-to-side. Urinary tract continuity is usually established by a ureteroneocystostomy by placing the ureter either through a submucosal tunnel or by a direct implant that prevents the subsequent development of reflux. The success of this surgical approach has been clearly demonstrated in the thousands of kidneys that have been transplanted over the last 30 years.

Cardiac Transplantation Since the performance of the first human cardiac transplant by Dr. Christian Barnard in December 1967, thousands of heart transplants have been performed to prolong the life expectancy of patients with end-stage cardiac disease. Although enthusiasm for cardiac transplantation has waxed and waned over the last two decades, the use of CsA to treat rejection clearly improved the clinical outcome and rejuvenated interest in this procedure. The impact of CsA was

Chapter 5: Physiologic Basis of Transplantation

Figure 24 Renal transplantation. The donor kidney is placed in the retroperitonium of the iliac fossa. The donor renal artery and vein are anastomosed to the recipient iliac artery and vein, respectively in an end-to-side fashion. Ureteroneocystostomy establishes urinary continuity.

evidenced by the worldwide increase in cardiac transplant centers. Currently, cardiac transplantation is routinely performed in many centers in the United States. Patients considered as suitable candidates for cardiac transplantation are those with end-stage heart disease, provided that they have no other disease condition that potentially affects long-term survival. Generally, candidates for cardiac transplantation should be under the age of 65, and without an underlying malignant disease, insulindependent diabetes mellitus, severe peripheral vascular disease, morbid obesity, or an active infectious process. In addition, a high fixed pulmonary vascular resistance is usually considered a contraindication for this procedure. Cardiac allotransplantation (100) is usually performed orthotopically; the recipient heart is removed, leaving a remnant of the right and left atria posteriorly. The atria, pulmonary artery, and aorta from the donor heart are then anastomosed primarily. Heterotopic cardiac transplantation has also been performed in which the allograft is connected in parallel with the native heart. The theoretic advantage of this latter procedure is the maintenance of the native myocardium to sustain the recipient for reimplantation, if the organ comes from a suboptimal donor or if it undergoes acute irreversible rejection. The current overall patient survival rate exceeds 85% and 70%, with graft survival rates of 85% and 70% at one and five years, respectively. Regularly scheduled right endocardial biopsies in conjunction with cardiac catheterization and coronary angiography are used to evaluate the status of the transplanted heart. Although fever, hemodynamic instability, signs of congestive heart failure, arrhythmias, and electrocardiographic alterations are clinical indices suggesting rejection, histologic evaluation of the endocardium is usually necessary to rule out a rejection episode. Although rejection may occur at any time following transplantation, late graft failure is often associated with atherosclerotic coronary artery disease and chronic rejection of the transplanted heart.

Hepatic Transplantation Liver transplantation is now considered an accepted therapeutic modality for a number of hepatic disorders, and, in

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certain conditions, the treatment of choice. As with heart transplantation, the general acceptance of this procedure has paralleled the significant improvement in survival resulting from the development of the surgical procedure and the use of CsA. Before CsA, Starzl and coworkers, pioneers in clinical hepatic transplantation, reported a one- and five-year survival rate of 32.9% and 20%, respectively, between 1963 and 1980. Immunosuppression with CsA has drastically improved these results, with the actuarial survival rates at one and five years now being 75% and 70%, respectively. When liver transplantation is carried to improve the quality of life in noncritically ill patients, the one-year survival is in excess of 90% (101). Indications for liver transplantation are now clearly defined. In the pediatric population, biliary atresia is the most common clinical condition in which this procedure is used. Because clinical success with hepatic transplantation has exceeded that of the Kasai procedure for biliary atresia, it is now considered by some researchers to be the treatment of choice for this pediatric disease. Other pediatric conditions in which hepatic transplantation offers a therapeutic alternative include various inborne errors of metabolism such as Wilson’s disease, tyrosinemia, glycogen storage disease, and the alpha1-antitrypsin deficiency. However, the severe shortage of pediatric liver donors has limited the expansion of liver transplantation in the pediatric population. The development of new surgical approaches including reduced-size liver transplant, split-liver transplantation, and living, related donors has increased the supply of organs for the pediatric population. End-stage liver disease (ESLD) caused by hepatitis C virus infection has become the most common indication for transplantation of the liver. Other indications include ESLD caused by cryptogenic and primary biliary cirrhosis, sclerosing cholangitis, alcohol related liver disease (Lannec’s cirrhosis), fulminant hepatic failure, hepatitis B, primary hepatocellular carcinoma, and hemangioendothelioma. Although the literature is replete with examples of transplantation in patients with cholangiocarcinoma, the relatively high recurrence rate in patients with this neoplastic disease has questioned the value of this procedure. As a general rule, patients with hepatic malignancy secondary to an extrahepatic source are no longer considered acceptable candidates for liver transplantation. Individuals with significant cardiopulmonary disease, advanced alcoholic disease, sepsis, or those deemed psychologically unfit for transplantation because of potential difficulty with immunologic drug compliance are also eliminated as acceptable transplant candidates. Age greater than 70 years currently constitutes a relative contraindication for liver transplantation. Liver transplantation (Fig. 25) is a formidable procedure conducted in three phases. The first phase involves dissection and removal of the recipient liver through a bilateral subcostal incision with an upper midline extension. During the second anhepatic phase, the venovenous bypass employed by most centers, decompresses the splanchnic bed, lower extremities, and kidneys by establishing venous flow from the portal vein and inferior vena cava to the axillary vein via a centrifugal pump. The third phase of hepatic implantation involves four vascular anastomoses including the inferior vena cava above and below the liver, the portal vein, and the hepatic artery. The most common biliary reconstruction is a choledochocholedochostomy followed by a choledochoenteric anastomosis via a standard Roux-en-Y choledochojejunostomy. Reported complications following liver transplantation ranged from 14% to 55%. These include

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have been patients with end-stage pulmonary vascular disease such as primary pulmonary hypertension, and those with severe parenchymal disease, including chronic obstructive pulmonary disease, respiratory burns, pulmonary fibrosis, toxic pneumonitis, and bronchiectasis.

Pancreas Transplantation

Figure 25 Hepatic transplantation. Vascular continuity is established through four anastomoses that include the IVC above and below the liver, the portal vein, and the hepatic artery. Biliary tract reconstruction is obtained by a choledochocholedochostomy performed over a T-tube. Abbreviation: IVC, inferior vena cava.

postoperative hemorrhage, vascular thrombosis, biliary leak and obstruction, bowel obstruction and perforation, gastrointestinal bleeding, and wound complications (101). In deciding whether a patient is a suitable candidate for hepatic transplantation, the most important determinants are clearly the extent to which the patient’s underlying disease is interfering with quality of life and how transplantation will ultimately influence the long-term prognosis of this disease.

Lung Transplantation Transplantation of the lung can be performed either by itself or as an en bloc heart–lung combination. Clinical experience with solitary lung transplants has been fraught with considerable problems, including infection, hemorrhagic consolidation, and severe rejection, although this latter problem appears to be less noteworthy with the availability of CsA. Most centers in which solitary transplants have been performed have had few survivors beyond one month. For this reason, the heart–lung combination transplant has gained in popularity. Not only does the combined transplant ensure a better blood supply to the transplant bronchus, which has historically been a major problem with solitary pulmonary transplants in which anastomotic dehiscence of the bronchus has frequently occurred, but the operation is also technically more feasible than the solitary transplant and provides a maximum amount of lung parenchyma to optimize function of the allograft. However, in the last decade, lung transplantation outcomes have dramatically improved. Currently, lung grafting boasts one- and three-year survival rates approaching 78% and 60%, respectively. Survival following combined heart–lung procedures is approximately 70% and 40%, at one and three years, respectively. Although clinical experience with lung transplantation has been limited when compared with kidney, heart, or liver transplantation, potential recipients include those individuals with irreversible and terminal pulmonary failure. Among individuals who have received lung transplants

More than one million patients in the United States currently suffer from insulin-dependent diabetes mellitus. An additional 10,000 to 15,000 new cases can be expected each year. Despite exacting control of the serum glucose by exogenously administered insulin, serious complications are associated with this disease. The microangioplastic lesions resulting from aberrant diabetic carbohydrate metabolism produce severe retinopathy that may ultimately lead to blindness, nephropathy eventuating in renal failure, and peripheral vascular disease that is commonly associated with neuropathy and limb loss. Pancreatic transplantation in rodent models has successfully prevented and, in some cases, halted the progression of these microangiopathic lesions; thus it is not surprising that enormous efforts have been made to develop successful means of transplanting pancreatic tissue in humans. The first pancreatic transplant was performed by Kelly at the University of Minnesota in 1960. The one-year survival for all pancreas transplants between 1966 and 1980 was 21%, the outcome being influenced greatly by the magnitude of the rejection process. The advent of CsA, has renewed interest in this endeavor. Currently, approximately 1200 pancreatic transplantation are performed yearly in the United States in type 1 diabetic (T1D) patients. The most common procedure is simultaneous pancreas and kidney transplant in T1D recipients with renal failure. Pancreas transplantation alone is generally reserved for T1D patients who exhibit severe diabetes-related complications, but no evidence of renal failure. Rarely, the procedure is employed for type 2 diabetes, which is considered a contraindication for transplantation by most researchers. Presently, one-year patient and graft survival rates approximate 95% and 85%, respectively. Whole organ pancreatic transplants (102) include en bloc placement of the pancreas and a portion of duodenum surrounding the papilla of Vater to the urinary bladder in order to provide a means of exocrine drainage (Fig. 26). More recently, enteric drainage of the duodenal portion of the transplanted pancreas has been widely adopted because of the high complication rates and electrolyte imbalance associated with bladder drainage. The splenic artery and vein are then anastomosed to the hypogastric artery and external iliac vein, respectively, to reestablish blood supply. Pancreas transplantation is associated with higher morbidity than that encountered in renal transplant recipients. Such complications include vascular thrombosis and duodenal leaks that frequently necessitates allograft removal. In addition to the vascularized pancreatic allograft, treatment of diabetes has also been attempted with transplantation of isolated pancreatic islet cells. It is estimated that approximately 500,000 islets are required for normal carbohydrate metabolism. For this procedure, the pancreas is mechanically and enzymatically disrupted to harvest the islet cells. These cells are then transferred to the donor. A variety of donor sites have been used, including the testes, peritoneal cavity, beneath the renal capsule, spleen, and portal vein, the latter being the most often used. Although this technique has been quite successful experimentally in preventing or halting the metabolic consequences of diabetes in various animal models, clinical trials to date in diabetic

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have recently been successful in preventing the rejection process and GVHD in various experimental models include CsA or FK506 administration, donor organ irradiation, and microsurgical excision of mesenteric lymph nodes. In the 1960s, several attempts have been made clinically to transplant human intestine. However, all patients died because of technical difficulties associated with the operation, sepsis, graft rejection, or a combination of these factors. Interest in SBT in the 1970s faded due to the advent of total parenteral nutrition (TPN). In the 1980s, interest in SBT was renewed due the realization of TPN limitations and the success of CsA in prolonging SBT survival in experimental animals. Current attempts of isolated SBT or combined liver/small bowel transplants have utilized high doses of FK506 and prostaglandin E1 with a 70% actuarial graft and patient survival (103). However, this is associated with a 90% incidence of bacterial infections, 43% viral infections, and an increased rate of lymphoproliferative disorders.

Bone-Marrow Transplantation Figure 26 Pancreas transplantation. Donor portal vein is anastomosed to the recipient iliac vein. Donor iliac ‘‘Y’’ graft connects donor SMA and donor splenic artery to recipient iliac artery. The duodenal segment is anastomosed to the recipient urinary bladder. Abbreviations: SMA, superior mesenteric artery; SMV, superior mesenteric vein.

human subjects have failed to abrogate the need for insulin, although some groups reported total insulin independence for one year. Thus, despite its potential advantage, islet transplant is still in its infancy.

Small Bowel Transplantation Annually, thousands of patients become victims of the short gut syndrome, usually secondary to thrombosis or embolization of the superior mesenteric artery, necessitating massive intestinal resection. The minimal digestive absorptive capacity with which these patients are left is often inadequate to maintain a normal nutritional status, requiring the use of long-term parenteral alimentation for survival. Obviously, replacement of this missing intestine with a small bowel transplant would be highly desirable. Experimentally, small bowel transplantation (SBT) has been successfully performed in animals since 1959. Lillehei, Goott, and Miller were the first to describe this technique with a canine model. Continuing experimental refinements have made the technique of SBT quite feasible from a technical standpoint. As with other organ transplants, the major problem in making SBT a clinically useful modality concerns the control of rejection. The magnitude of this problem directly relates to the length of intestine transplanted. Large segments of small intestine have resulted experimentally in a graft-versus-host disease (GVHD) reaction, because of the large amount of lymphoid tissue contained in intestinal mesenteric lymph nodes, peyer’s patches, and lamina propria that are capable of mounting an immune response against the host. In humans, the typical erythematous rash of GVHD develops concurrently with the appearance of donor lymphocytes in the recipient’s peripheral circulation. When small segments of intestine are transplanted, this is less of a problem, but the allograft still undergoes the standard rejection process. Immunosuppression regimens that

The indications for bone-marrow transplantation include severe inherited immune deficiency disorders, bone marrow failure, and certain malignancies. The potentially fatal inherited diseases for which bone marrow transplantation has been used include various hemoglobinopathies such as thalassemia and sickle cell disease, immunodeficiency states, and certain enzymatic disorders such as mucopolysaccharoidosis, osteopetrosis, chronic granulomatous disease, adenosine deaminase deficiency, and Diamond–Blackfan anemia. Acquired disorders of marrow failure responding to bone-marrow transplantation have included aplastic anemia and various malignancies such as acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, Hodgkin’s disease, non-Hodgkin’s lymphoma, neuroblastoma, multiple myeloma, and acute myelofibrosis. Marrow transplantation has also been used in selected cases of marrow failure secondary to toxic chemotherapeutic agents. Human bone-marrow transplantation have stressed the use of HLA-histocompatible marrow between donor and recipient, as determined by serologic typing for the alleles of HLA-A, HLA-B, HLA-DR, and HLA-D loci and nonreactive MLC. Bone marrow for transplantation has been obtained from syngeneic, allogeneic, and autologous sources. Whereas syngeneic marrow transplantation refers to a transfer of bone marrow between genetically identical twins, allogeneic marrow transplantation is usually accomplished between HLA-identical siblings who inherited the same haplotype of chromosome 6 from each parent. The chance of HLA identity between siblings is one in four. Recipients of allogeneic or syngeneic marrow are pretreated with immunosuppressive agents and myeloablative drugs, if the underlying cause of marrow failure is a malignancy. In autologous transplantation, marrow is harvested from the patient during remission and cryopreserved. The marrow is then replanted following myeloablative antineoplastic therapy to eliminate residual malignant cells. Of interest, autologous transplantation has been as effective as the allogeneic or syngeneic marrow transplantation, in treating acute nonlymphocytic and chronic myelogenous leukemia. The major complications associated with bonemarrow transplantation have included rejection of the grafted marrow, GVHD, and potentially lethal infections. If the transplanted marrow functions only briefly and a marrow biopsy reveals the absence of marrow elements, this

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indicates that the recipient has rejected the donor marrow because of presensitization, usually resulting from prior blood transfusions. GVHD results from the transplanted T-cells reacting to the genetically different host cells. The lymphohematopoietic system, including the skin, liver, and gastrointestinal tract, are primarily affected by the graft versus host response, which is manifest clinically as dermatitis, diarrhea, alterations in liver function, weight loss, and a high susceptibility to infection. GVHD occurs in 30% to 70% of allogeneic marrow transplant recipients and results in death of 20% to 40% of those individuals affected. The infectious complications following bone-marrow transplantation usually occur within the first several weeks before the production of granulocytes by the grafted marrow. The recipient is susceptible to all types of infections, including those caused by bacteria, fungi, and herpes simplex. After the first month following grafting, the increase in the peripheral white blood cell count suggests the engraftment of the donor marrow. Subsequent infections include cytomegalovirus and P. carinii infections, both of which are usually manifested as interstitial pneumonias. The use of prophylactic trimethoprim–sulfamethoxazole has greatly reduced the incidence of interstitial pneumonia secondary to Pneumocystis. Those patients having cytomegalovirus as the offending organism are much less fortunate, and many of these individuals will die because no current effective treatment exists. Thus, to prevent cytomegalovirus infections, hyperimmune globulin is usually administered prophylactically, early after transplantation.

Corneal Transplantation Corneal transplantation, also referred to as corneal grafting or penetrating keratoplasty, is performed in the United States at a rate of approximately 10,000 operations per year. The success of this procedure is limited only by the technical skills of the surgeon and not by the immune response. This is because the cornea is an immunologically privileged structure in which oxygen is derived from the oxygen content of the internal aqueous humor rather than from specific blood vessels. Consequently, circulating antibodies are isolated from the transplanted cornea. When they occur, rejection episodes are usually mild and develop at a rate of only 10% to 12%. If rejection becomes a problem, it is usually ablated by the use of topical steroids. In over 90% of operations performed, corneal transplantation proves to be successful. Currently, corneal transplantation is the procedure of choice for all patients suffering from opaque corneas. It is limited only by the availability of donor corneas. The persistence of the immunologically privileged status of the transplanted cornea depends on the vascularity of the graft. Scarring of the cornea results in an increased vascularity and consequently an increased risk of rejection. Under this circumstance, the cornea has a 25% incidence of irreversible rejection. Rejection occurs primarily through a cellular immune response. Previous corneal grafting resulting in corneal scarring and increased vascularity has been associated with graft failure. Histocompatibility matching is not routinely necessary, but is beneficial in the presence of heavily vascularized corneas.

SUMMARY Successful transplantation of organs or tissues rests on an understanding of the immunologic rejection process. The events leading to allograft rejection consist of afferent,

central, and efferent limbs. The presentation and recognition of foreign antigens are manifestations of the afferent limb. The central limb encompasses the proliferation, differentiation, and production of lymphokines by T-cells and of humoral antibody by B-cells, whereas the efferent limb represents the activation of various cell populations that ultimately lead to the rejection of the transplanted tissue or organ. Clinically, allograft rejection is classified as hyperacute, accelerated, acute, or chronic. A variety of immunologic strategies have been used to avert, minimize, or reduce the rejection response of the host or the immunogenicity of the allograft. The latter approach has only had limited success experimentally with nonvascularized grafts. Host immunosuppression by means of chemical substances (i.e., corticosteroids, CsA, and Aza) nonspecifically diminishes the immune response. More recently, new potent immunosuppressive medications (Tacrolimus, MMF, and RAPA) have gained wide application in transplantation. Biologic agents (i.e., ALS and monoclonal antibodies) provide both generalized and specific immunosuppressive effects. ALS nonspecifically depletes the host of lymphoid cells, whereas monoclonal antibodies deplete a specific T-cell subset. To date, immunologic manipulation to achieve transplantation tolerance has not progressed to a level satisfactory for clinical use. Continued research in basic immunology provides foundations for further advances in immunosuppressive manipulations. The next era of transplantation will witness progress from the nonspecific immunosuppressive strategy that is associated with a high morbidity to a strategy that seeks to alter regulation of the immune response, in order to selectively downregulate antidonor responses to achieve immunological tolerance, the ideal state of immunosuppression (104).

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6 Surgical Infection: Principles of Management and Antibiotic Usage Christina Paylan and Rodney Durham

INTRODUCTION The prevention and treatment of microbial infections are important components of the surgeon’s clinical practice. Patients may first present to a surgeon because of an infection requiring drainage or some other form of surgical treatment. In addition, postoperative infections are among the most common complications of surgical therapy, even in patients whose original problem was noninfectious in nature. For these reasons, it is important for the surgeon to have an understanding of the basic pathophysiology of infection, as well as the fundamental principles of management, including antibiotic therapy.

PATHOPHYSIOLOGY OF SURGICAL INFECTIONS Infections result when the balance between pathogenic organisms and the host’s defense mechanisms is disrupted. Large numbers of bacteria and other microorganisms colonize many of the epithelial surfaces of the body, including the skin, most portions of the upper aerodigestive tract, the gastrointestinal (GI) tract below the duodenum, the vagina, and the lower genitourinary tract. Overall, approximately 1014 bacteria colonize the human body, or about 10 bacteria for every eucaryotic cell (1). Anaerobic bacteria predominate over aerobic bacteria by a 10:1 ratio, and this ratio may be even higher in the lower GI tract. These endogenous microorganisms prevent colonization of epithelial surfaces by more pathogenic organisms, but can themselves be pathogenic under appropriate circumstances. Furthermore, endogenous flora is responsible for many of the infections observed in surgical patients. The virulence of a particular microbe is related to many different properties. Specific molecules on the microbial surface may allow an organism to adhere to an epithelial surface, thereby initiating an infection. For instance, Escherichia coli strains bearing specific types of fimbriae can attach to the urinary epithelium; these strains are much more likely to produce clinical urinary tract infections than strains not bearing those structures. Other microbial products may facilitate invasion of the host tissues. Streptokinase, hyaluronidase, and collagenase are enzymes secreted by some types of bacteria that allow spreading infections to occur. Finally, many microorganisms have mechanisms that allow them to escape the host defenses. Thus microbial proteins that disrupt chemotaxis by white blood cells, interfere with phagocytosis, or prevent intracellular killing have been identified. Thus, certain intracellular pathogens, such as Listeria, actually survive within the phagocyte.

Various exotoxins and endotoxins may be responsible for many of the deleterious effects of microbial infections. Many times, the actual infection itself is of little consequence, but the potent toxins released lead to fulminant disease. Examples of such infections are those produced by diphtheria, cholera, and tetanus bacteria. Microbial toxins produce a variety of effects on the host. Some exotoxins are cytotoxic for eucaryotic cells, such as the exotoxins produced by Clostridium difficile. Other toxins trigger an overwhelming host response, and it is the host response rather than the actual infection that results in damage to distant tissues. For instance, many of the pathogenic effects of lipopolysaccharide, the complex endotoxin of gram-negative bacteria, are thought to be mediated by this overwhelming host response. There are numerous mechanisms by which the host prevents these pathogenic microorganisms from producing frank infection (2). The intact epithelium of the skin or mucosal surface presents the initial barrier against microbial invasion. Both physical and chemical components contribute to this barrier function. Tight junctions and other components of the intercellular adhesion complex prevent microorganisms from gaining access to internal tissues. Mucosal surfaces are protected by a surface layer of mucus, which prevents bacterial attachment, and the dry, somewhat acidic, keratinized surface of the skin inhibits bacterial proliferation. In addition, epithelial cells secrete a number of substances, such as lysozyme, which are also inhibitory for microbial growth. Finally, some epithelial surfaces have mechanical mechanisms, by which to directly remove microorganisms, as is seen with the mucociliary apparatus of the respiratory epithelium. Microbial organisms that breach the epithelial barrier or otherwise gain access to interior tissues have to face both nonspecific and specific humoral and cellular defense mechanisms. Nonspecific humoral components of the inflammatory response, such as the complement cascade, the kallikrein–kinin system, and the coagulation system, are activated. This activation helps to localize the invading microorganisms and also signals for the recruitment of phagocytic cells, such as neutrophils and macrophages, to the site of invasion. The nonspecific inflammatory response is accompanied by the development of the specific immune response. The humoral arm of the immune response is represented by the various immunoglobulin molecules produced by B-lymphocytes and plasma cells, and the cellular arm by specific cytotoxic T-lymphocytes and natural killer cells, among others. Many aspects of surgical treatment interfere with these host-defense mechanisms. The most obvious of these is the surgical incision through intact skin or mucosa.

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The placement of intravenous and other vascular access catheters also breaks the intact skin barrier. Other devices, such as endotracheal tubes and indwelling urinary catheters, provide microorganisms with direct access from sites of heavy colonization to normally sterile areas. Ischemic and devitalized tissues produced as a result of a surgical procedure provide a haven for the growth of bacteria, because they may be inaccessible to phagocytic cells. In addition, the presence of blood in areas of damage provides essential nutrients for bacterial growth. The use of prophylatic antibiotics for many operative procedures, while undeniably reducing the risk of wound infections, may also change the resident microbial flora, allowing a pathogen to proliferate. This has been well demonstrated in the development of antibiotic-associated colitis (AAC). The patient’s preexisting medical condition can also interfere with host-defense mechanisms. For instance, the presence of diabetes mellitus or the prior use of corticosteroid or immunosuppressive medications may impair the development of an adequate inflammatory or immunologic response. Severe malnutrition is also associated with an increased risk of postoperative infections. Finally, the pathologic process that has necessitated the surgical procedure may increase the risk of a significant infection. There is a high likelihood of developing an intraabdominal abscess or surgical wound infection when massive peritoneal contamination, for instance, from a free colonic perforation, has occurred. Shock and hypothermia also result in a generalized impairment in the function of phagocytic cells, as well as in the cellular immune responses, and predispose patients to postoperative infections.

SYSTEMIC INFLAMMATORY RESPONSE The local inflammatory response generated as a result of an infection is usually accompanied by a systemic response of the organism. This systemic response to a proinflammatory stimulus, whether infectious or noninfectious, is now referred to as the systemic inflammatory response syndrome (SIRS) (3). SIRS is considered to be present if a patient has two or more abnormalities in temperature, heart rate, respiratory function, or white blood cell count (Table 1). When an infection is the cause of SIRS, sepsis is considered to be present. Although alterations in these four parameters suffice to define the clinical syndrome of SIRS, the systemic response to infection involves changes in many more physiologic parameters. In general, this systemic response includes the development of a hyperdynamic cardiovascular state characterized by increased cardiac output and decreased systemic vascular resistance and a hypermetabolic state characterized by increased oxygen consumption and carbon dioxide production, production and utilization of glucose, mobilization of free fatty acids, and breakdown of the body’s muscle protein stores (4). This overall systemic response is important in ensuring the organism’s survival during an infectious episode. Indeed, in experimental settings, it has been shown that the development of fever is associated with increased survival following a normally lethal bacterial challenge (5). It has also been observed in clinical studies that the inability to develop an adequate cardiovascular or metabolic response has an adverse impact on patient survival during sepsis (6,7). The mechanisms by which the systemic inflammatory response is generated have been extensively investigated in recent years. It is apparent that a large number of endogenous

Table 1 Definitions Infection: Microbial phenomenon characterized by an inflammatory response to the presence of microorganisms or the invasion of normally sterile host tissue by those organism Bacteremia: The presence of viable bacteria in the blood SIRS: The systemic inflammatory response to a variety of severe clinical insults. The response is manifested by two or more of the following conditions: Temperature >38 C or <36 C Heart rate >90 beats/min Respiratory rate >20 breaths/min or PaCO2 <32 Torr (<4.3 kPa) WBC >12,000 cells/mm3, <4000 cells/mm3, or >10% immature (band) forms Sepsis: The systemic response to infection. This systemic response is manifested by two or more of the following conditions as a result of infection: Temperature >38 C or <36 C Heart rate >90 beats/min Respiratory rate >20 breaths/min or PaCO2 <3 Torr (< 4.3 kPa) WBC >12,000 cells/mm3, <4000 cells/mm3, or >10% immature (band) forms Severe sepsis: Sepsis associated with organ dysfunction, hypoperfusion, or hypotension. Hypoperfusion and perfusion abnormalities may include, but are not limited to lactic acidosis, oliguria, or an acute alteration in mental status Septic shock: Sepsis with hypotension, despite adequate fluid resuscitation, along with the presence of perfusion abnormalities that may include, but are not limited to, lactic acidosis, oliguria, or an acute alteration in mental status. Patients who are receiving inotropic or vasopressor agents may not be hypotensive at the time that perfusion abnormalities are measured Hypotension: A systolic blood pressure of <90 mmHg or a reduction of <40 mmHg from baseline in the absence of other causes for hypotension MODS: Presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention Abbreviations: MODS, multiple-organ dysfunction syndrome; SIRS, systemic inflammatory response syndrome. Source: From Ref. 3.

mediators are involved in this response. Such mediators include neurotransmitters and hormones (such as catecholamines, glucagon, and glucocorticoids), various proinflammatory mediators (including lipid-derived mediators such as eicosanoids and platelet-activating factor), and polypeptide mediators (such as activated components of the complement system). Of particular importance in triggering the systemic response to infections are certain peptide regulatory factors referred to as cytokines. Cytokines that may be especially important in the development of this response include tumor necrosis factor (TNF), lymphotoxin, colonystimulating factors, and various interleukins [particularly interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-8 (IL-8), and, interferon-a, interferon-b, and interferon-g] (8). The importance of these agents has been demonstrated in experimental studies in which endotoxin was infused into laboratory animals or human volunteers. Endotoxin infusion was found to be followed by the sequential release of TNF, IL-1, and IL-6 (9). Inhibition of TNF using a neutralizing antibody abrogated the release of the other cytokines and also suppressed many of the physiologic changes typical of the systemic inflammatory response (10). In clinical studies, however, circulating levels of IL-6 rather than TNF have been found to correlate most closely with an adverse clinical outcome, but the combined inflammatory score based on circulating concentrations of

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endotoxin, TNF, IL-1, and IL-6 appeared to be the best predictor of outcome in patients with the sepsis syndrome (11). Given the importance of these proinflammatory mediators in the development of the clinical manifestations of infections, it is not surprising that antagonists of these cytokines have been developed and subjected to clinical trials. In general, however, the use of antiendotoxin antibodies, anti-TNF antibodies, and an IL-1 receptor antagonist has not proved beneficial. There are several potential reasons why these agents have not been useful clinically. First of all, although the experimental infusion of endotoxin produces an orderly release of cytokines with TNF playing a primary role, most clinical infections, even those due to gram-negative organisms, are likely to involve a much more complicated pattern of mediator release. Second, the therapeutic use of cytokine antagonists may occur too late, because much of the initial wave of cytokine release occurs within a few hours of the infusion of bacteria or endotoxin. It is only under very special circumstances, in which an antagonist can be given prior to the expected release of the mediators, that anticytokine therapy has proved beneficial (12). Finally, from the biologic viewpoint, it should be noted that the systemic inflammatory response has evolved in vertebrates as a mechanism to counteract infectious and other damaging stimuli. Accordingly, this reaction should ultimately be advantageous for survival, even though acutely it increases the physiologic demands placed on the organism. Thus it is certainly possible that counteracting this response may not benefit the patient.

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OVERALL APPROACH TO SEPSIS In March of 2004, an international panel of critical care and infectious disease specialists published guidelines for the management of patients with severe sepsis and septic shock under the auspices of Surviving Sepsis Campaign (13). The multimodality approach to sepsis outlined by Surviving Sepsis Campaign is as follows:

m. n.

a.

Goals at initial resuscitation within the first six hours include the following:

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c.

d. e.

CVP of 8–12 mmHg Mean arterial pressure of 65 mmHg Urine output greater than 0.5 cc/kg/hr Mixed venous saturation greater than 70%

If mixed venous saturation of more than 70% is not achieved within the first six hours with fluid resuscitation to a central venous pressure of 8 to 12 mmHg, then transfusion with one unit of packed red blood cells to achieve a hemotocrit of more than 30% is recommended. This goal can also be attempted by administration of dobutamine if necessary. Goals at diagnosis include drawing appropriate cultures before any antimicrobial therapy is begun. At least two types of blood cultures, one drawn percutaneously and one drawn through each vascular access device, are recommended. Intravenous antibiotic therapy should be started within the first hour of recognition of severe sepsis after appropriate cultures are obtained. Initial empirical anti-infective therapy should be directed against the likely bacterial or fungal organisms. Antimicrobial regimen should always be reassessed after 48 to 72 hours with the aim of streamlining to

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prevent development of resistance and to reduce toxicity as well as costs. Source control should be evaluated in every patient presenting with sepsis. In this regard, drainage of an abscess, debridement of infected necrotic tissue, and the removal of a potentially infected device should not be delayed. Fluid resuscitation is important and may consist of natural or artifical colloids or crystalloids. There is no evidence-based support for one type of fluid over another. Vasopressor therapy is important when fluid resuscitation becomes inadequate to control blood pressure. Either norepinephrine or dopamine is the first choice as a vasopressor agent to correct hypotension in septic shock. However low-dose dopamine should not be used for renal protection as part of treatment for severe sepsis. All patients requiring vasopressors should have an arterial catheter placed as soon as practical. Vasopressin may be considered in patients with refractory shock despite adequate fluid resuscitation. Inotropic therapy is recommended in severe sepsis when the cardiac output is low. Dobutamine is the agent of choice, and if used in the presence of low blood pressure, it should be combined with vasopressor therapy. Intravenous corticosteroids (200–3000 mg/day for seven days in three divided doses or by continuous infusion) are recommended in patients with septic shock, who despite adequate fluid replacement require vasopressor therapy to maintain adequate blood pressure. Some experts use 250 mc adrenocorticotropic hormone (ACTH)–stimulation test to identify responders (9 mg/dL increase in cortisol 30–60 minutes post–ACTH administration). Human-activated protein C, known as Xigris, is recommended in patients at high risk of death. Blood product administration should only occur when red blood cell concentration falls below 7 g/dL. Erythropoietin is not recommended as a specific treatment of anemia associated with severe sepsis, but may be used when septic patients have other accepted reasons for administration, such as renal failure–induced compromise of red blood cell production. In addition, routine use of fresh frozen plasma to correct laboratory clotting abnormalities in the absence of bleeding or planned invasive procedures is not recommended. High tidal volumes coupled with high plateau pressures should be avoided. Hypercapnia can be tolerated in patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Unless contraindicated, mechanically ventilated patients should be maintained semirecumbent with the head of bed at 45 to prevent the development of ventilator-associated pneumonia (VAP). Neuromuscular blockers should be avoided due to the risk of prolonged neuromuscular blockade following discontinuation. Blood glucose levels should be maintained at less than 150 mg/dL by using continuous infusion of insulin. In acute renal failure, and in the absence of hemodynamic instability, continuous venovenous hemofiltration and intermittent hemodialysis are considered equivalent.

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u. Bicarbonate therapy for the purpose of improving hemodynamics or reducing vasopressor requirements is not recommended. v. Deep vein thrombosis therapy is recommended with unfractioned or low-molecular-weight heparin. w. Stress ulcer prophylaxis should be given to all patients with severe sepsis. H2 receptor inhibitors are more efficacious than sucralfate and are the preferred agents.

DEFINITIONS OF SURGICAL INFECTIONS In 1992, the Centers for Disease Control and Prevention revised its definition of surgical wound infections. At this time, it was proposed to change the terminology from wound infection to surgical-site infections (SSI) (14). This latter terminology was designed to take into account both incisional (superficial and deep) infections and infections of organs or body spaces (e.g., subphrenic abscesses). More distant infections (e.g., postoperative pneumonia), excluding blood infections directly related to SSI, are classified as postoperative complications. Superficial incisional infections involve only the skin and subcutaneous tissues. Deep incisional infections involve the fascial and muscle layers, and organ or space infections involve any organs or body cavities that were manipulated during the surgical procedure. To be considered an SSI, the infection must occur within 30 days of the procedure, unless an implant is in place, in which case the time period is lengthened to one year. Characteristics of the different types of infection are listed in Table 2. Infections that arise in tissues begin as a cellulitis. This is manifested clinically by local tenderness, heat, swelling, redness, and pain. Because this type of infection has no localized areas of pus formation, it does not lend itself to drainage and can often be managed by rest, application of local heat, and antibiotic therapy, and elevation if an extremity is involved. If left untreated, cellulitis may spread through adjacent tissue planes (as occurs with b-hemolytic Streptococcus) or remain localized to form an abscess (as

may occur with Staphylococcus). In contrast to cellulitis, an abscess denotes a local collection of necrotic tissue, white blood cells, and bacteria, designated clinically as pus. The increased osmotic pressure that exists within a walled-off abscess can lead to movement of water into the area from adjacent tissues, resulting in pressure that is seen clinically as severe pain and tenderness. All abscesses should be drained to prevent the risk of bacterial spread along adjacent tissue planes or through the blood or lymphatic vessels and to provide symptomatic relief. Adjunctive antibiotic therapy is indicated to treat associated areas of cellulitis and to control any bacteria that may have entered the lymph or blood during the drainage process. Not uncommonly, products of infection from areas of cellulitis or abscess formation gain access to the blood and lymph systems. Occasionally, during the spread of infection, lymph channels may become infected and are seen clinically as hyperemic streaks. This condition is called acute lymphangitis, colloquially referred to as blood poisoning. If this infection is not controlled, the involved lymph nodes may later abscess and progress to a more severe state of infection known as suppurative lymphangitis. Invasion of vascular channels by bacteria is referred to as bacteremia. Because of the direct consequences that may result from bacterial spread, both lymphatically and through the vascular system, aggressive treatment including appropriate antibiotic therapy and local management of the source of infection with drainage of any abscesses is indicated. Approximately 30% of surgical patients either enter the hospital with an infection or develop one subsequently during the course of their postoperative care. Operative intervention is usually required in patients who are admitted to surgical services with evidence of infection. Infections of this nature include such diseases as appendicitis, cholecystitis, diverticular abscess, and gangrene of the leg. Although antibiotics are used as an adjunct to surgery in these patients, antibiotics by themselves are usually not efficacious in controlling the infectious process. The most frequent types of infection encountered in surgical patients are postoperative infections. In general, this classification

Table 2 Characteristics of Surgical Site Infections Superficial incisional SSIs Infection occurs within 30 days after the operation and infection involves only skin and subcutaneous tissue of the incision and at least one of the following: (i) Purulent drainage, with or without laboratory confirmation, from the superficial incision (ii) At least one of the following signs or symptoms of infection: Pain or tenderness, localized swelling, and redness, or heat, and superficial incision is deliberately opened by surgeon, unless culture result of incision is negative Deep incisional SSIs Infection occurs within 30 days after the operation if no implant is left in place or within 1 yr if implant is in place and the infection, appears to be related to the operation and infection involves deep soft tissues (e.g., fascial and muscle layers) of the incision (i) A deep incision spontaneously dehisces or is deliberately opened by a surgeon when the patient has at least one of the following signs or symptoms: Fever (higher than 38 C), localized pain, or tenderness, unless culture result of the incision is negative Diagnosis of a deep incisional SSI by a surgeon or attending physician Organ or space SSIs Infection occurs within 30 days after the operation if no implant is left in place or within 1 yr if implant is in place and the infection, appears to be related to the operation and infection involves any part of the anatomy (e.g., organs or spaces) other than the incision, which was opened or manipulated during an operation and at least one of the following: (i) Purulent drainage from a drain placed through a stab wound into an organ/space (ii) Organisms isolated from an aseptically obtained culture or fluid or tissue in the organ or space (iii) An abscess or other evidence of infection involving the organ or space on direct examination, during reoperation, or by histopathologic or radiologic examination (iv) Diagnosis of an organ or space SSI by a surgeon or attending physician Abbreviations: NNISS, National Nosocomial Infection Surveillance System; SSI, surgical site infection. Source: From Ref. 14.

Chapter 6:

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implies a hospital-acquired infection, commonly referred to as a nosocomial infection. Of all nosocomial infections, as many as 70% occur in surgical patients (16). Approximately 40% of these infections occur at the surgical site, 42% originate in the urinary tract, 14% are respiratory infections, and 4% are blood stream infections. As discussed in the preceding section, nosocomial infections are most commonly caused by the patient’s normal endogenous flora and are a consequence of breaches in the patient’s host defenses from the surgical procedure itself or secondary to other invasive procedures such as the placement of intravenous catheters. However, if the patient has been hospitalized for a period of time, colonization of the patient with hospital-acquired organisms occurs and may result in infections that are resistant to multiple antibiotics (17). Nosocomial infections are less frequently caused by organisms acquired directly from the hospital environment. Examples include infections derived from contaminated respiratory equipment resulting in pneumonia, intravenous fluid contamination resulting in sepsis, and the infrequent situation in which hospital personnel may be the source of infection.

EVALUATION OF SUSPECTED INFECTION IN THE SURGICAL PATIENT An understanding of the clinical manifestations and types of infection that develop postoperatively assists greatly in expediting the diagnosis and in instituting appropriate therapy for the infectious process. Normal body temperature is maintained by the thermoregulatory center in the hypothalamus within narrow limits (98.6 0.9 F) and varies only slightly throughout a given 24-hour period. An elevation in temperature above normal daily fluctuations represents the presence of fever. Although fever in the postoperative period may be due to numerous causes (Table 3), the presence of fever should be assumed to be due to infection until proved otherwise. The timing of the onset of fever is a classic aid in determining the possible cause of these infections. A temperature elevation in the first day or two following surgery is usually related to atelectasis involving one or both lungs. In the next 24 to 48 hours, urinary tract manifestations may become apparent. Under most conditions, wound infections do not cause fever until four to six days postoperatively, although streptococcal and clostridial wound infections can be seen as early as 24 hours after surgery. Thrombophlebitis almost never occurs before the third postoperative day and usually does not become apparent until a week to 10 days following surgery. Venous disorders involving the lower extremities as a cause of postoperative fever are generally less commonly encountered than those associated with phlebitis secondary to upper-extremity venous catheter placement (i.e., intravenous lines). A mnemonic to assist in remembering this sequence of fever onset is wind (pulmonary), water (urinary), wound, and walking (phlebitis). Despite the convenience of these guidelines, they should not preclude careful examination of other potential sources for infection. Furthermore, exceptions to the timing of these guidelines and fever patterns are legion so that the rational assessment of postoperative fever should begin with an organized approach to diagnosis (Fig. 1). In addition to elicitation of the patient’s history, a careful physical examination focusing on likely sources of infection is mandatory. Wounds and drain sites must be

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Table 3 Causes of Postoperative Fever Respiratory tract infections Atelectasis Pneumonia Urinary tract infections Wound infections Early wound infections Streptococcus species Clostridium species Late wound infections Staphylococcus species Mixed aerobic–anaerobic organisms Thrombophlebitis Upper extremity (almost always catheter-related infections) Lower extremity (usually related to venous stasis and not infected) Intra-abdominal causes Peritonitis Abscess Miscellaneous causes (considerably less common) GI infections Blood transfusion reaction Allergies Dehydration Undrained sterile fluids (e.g., pleural fluid and hematoma) Pancreatitis Blood transfusion–related infections (e.g., non-A hepatitis, non-B hepatitis, and cytomegalovirus infection) Abbreviation: GI, gastrointestinal.

inspected carefully. Patients with urinary bladder catheters should be examined for signs of urinary tract infection (e.g., flank pain and cloudy urine). Intravenous catheter sites may be a source of serious infection even though the evidence for inflammation at the catheter site may be subtle. Swelling in the arm distal to a subclavian catheter might indicate septic thrombophlebitis. The skin should be carefully examined for rashes, which may be allergic or infectious in origin. Infection lesions should be uncapped, and a Gram stain and culture of the pustular contents should be performed. Careful assessment of the lung and chest tube sites, if present, is mandatory to exclude pneumonitis and other pulmonary infections such as empyema. Evaluation of the abdomen may disclose peritonitis or a suspected subphrenic or other deep abscess. The rectal and pelvic examinations must not be excluded; otherwise, perirectal and pelvic abscesses might be missed. Appropriate laboratory and radiologic studies are obtained based on the clinical findings after completion of elicitation of the history and physical examination.

ROLE OF THE LABORATORY IN INFECTION DIAGNOSIS Stress caused by trauma, burns, or infections result in a nonspecific host response that has been referred to as the acute phase response (18,19). This response occurs within hours to days after tissue injury and may last indefinitely, depending on the persistence of the disease. This response results in metabolic changes in numerous organ systems (hepatic, hematologic, endocrine, and neurologic systems), some of which may be detected by laboratory tests. One of the more sensitive indicators of the acute phase response is an increase in the number and a decrease in the maturity of circulating neutrophils. Many surgical infections are

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Figure 1 Approach to the clinical management of the surgical patient with postoperative fever.

characterized by leukocytosis with a left shift in the differential count. However, white blood cell counts by themselves can be misleading. As noted in the definitions of sepsis discussed in Table 1, a low white blood cell count, especially in the elderly or compromised host, may be associated with particularly severe episodes of sepsis. A number of other nonspecific laboratory tests may also be indicative of infection. An increase in hepatic protein synthesis results in elevated levels of C-reactive protein and an increase in the erythrocyte sedimentation rate during the acute phase response. In addition, abnormalities of glucose metabolism during sepsis can lead to both hypoglycemia and hyperglycemia (20,21). Laboratory tests may also support the presence of specific disease processes. Liver function studies may reveal elevated levels of bilirubin and alkaline phosphatase secondary to infections of the liver such as ascending cholangitis, cholecystitis, liver abscess, or hepatitis. However, the range of overlap in liver function derangements in fundamentally different disease states is so high that these tests may be difficult to interpret clinically. For example, exceedingly high aminotransferase levels (i.e., in the thousands) are virtually diagnostic of hepatitis (usually A, B, and

non-A/non-B), lower levels (i.e., in the hundreds) can be seen in hepatitis caused by cytomegalovirus infections, Epstein–Barr viral disease, syphilis, and toxoplasmosis. These lower levels can be seen in alcoholic hepatitis, liver abscess, and, occasionally, gallbladder disease, among other disease states. If hepatitis is considered a possibility, more specific serum tests, such as hepatitis B surface antigen and cytomegalovirus titers, should be obtained. Although the history, physical examination, radiographic tests, and nonspecific laboratory tests are valuable in establishing the presence and defining the focus of infection, identifying the offending organism depends on obtaining stains and cultures of relevant material. Care must be taken to ensure appropriate collection of these specimens (22). Many surgical infections occur adjacent to mucous membranes or other areas where colonizing flora may be confused with causal flora. Classic examples of inappropriate and irrelevant cultures are as follows: culturing sputum for anaerobes in necrotizing pneumonia, culturing chest tube contents rather than percutaneously obtained pleural space material when empyema is suspected, swabbing wounds for culture rather than aspirating underlying abscesses through intact skin or cellulitic areas, and

Chapter 6:

Surgical Infection: Principles of Management and Antibiotic Usage

culturing sinus tracts rather than obtaining bone biopsies without contamination from the tract when osteomyelitis is suspected. Direct examination of a specimen either as a wet mount or as a stained specimen is often useful in the diagnosis of infection. Wet mounts examined with an ordinary light microscope are useful in the identification of protozoa and fungi (23). Gram’s stain is the most widely used procedure for the direct examination of a specimen. Stained smears can demonstrate the presence of inflammatory cells and bacteria. The presence of inflammatory cells may be useful in determining the adequacy of a specimen, particularly in Gram’s stains of sputum in which the presence of inflammatory cells suggests pneumonia, whereas a stain demonstrating squamous epithelial cells and a wide variety of organisms suggests that the specimen has been contaminated with saliva (23). With experience, many causative agents can be predicted, and the Gram’s stain result can guide empiric therapy. For instance, clumps of staphylococci are clearly different from chains of streptococci. Clostridial species have a characteristic appearance of plump, gram-positive rods. Among surgical infections, mixed anaerobic–aerobic infections are common; the Gram’s stain result may be the only clue to the presence of an anaerobe that may fail to grow in culture. Traditionally, urine, sputum, and blood cultures in addition to cultures of any infected material obtained from debridement or aspiration have been used to evaluate patients suspected of having a postoperative infection. However, if negative urinalysis results are obtained and the patient has no symptoms relating to the urinary tract, urine cultures may be omitted. In addition, routine culturing of the patient need not include a sputum sample if there is no realistic concern for pulmonary infection (i.e., no cough, no rales during pulmonary auscultation, and a normal chest radiogram). Finally, the utility of routine culturing of the blood in patients with postoperative fevers in the absence of clinical signs of sepsis or neutropenia has been questioned. Theuer et al. (24) found the yield of positive blood cultures per patient febrile event was only 5% when routine blood cultures were obtained. Of interest, bacterial cultures in patients receiving broad-spectrum antibiotics were uniformly negative. Because many patients were begun on antibiotic coverage after obtaining one set of cultures, reculturing the patient was not productive if initial cultures did not reveal the source of fever. As a result, appropriateness of antibiotic therapy could not be evaluated in these patients. Therefore, it has been suggested that antibiotic therapy be withheld until there is a defined bacterial source of infection in patients who are not neutropenic or septic. Empiric antibiotic therapy can and should be initiated in these patients if clinical deterioration occurs. The approach outlined above is designed to have an organism available in the laboratory so that tests can be performed on it to verify that the correct antibiotics have been chosen and to allow for appropriate changes in antibiotic coverage if the organism proves to be resistant. To predict antibiotic susceptibility of an organism, most microbiology laboratories use the disk diffusion test in which an antibiotic impregnated in a paper disk diffuses into the Mueller– Hinton agar plate on which bacteria are plated (25,26). The utility of this test is limited to organisms that grow rapidly on this agar (staphylococci, Enterobacteriaceae, pseudomonads, and Haemophilus influenzae). This method is not reliable for determining susceptibility of anaerobic

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bacteria. In 24 hours, a sizable clear zone (no growth) around the disk indicates that the organism is susceptible to that antibiotic. It must be noted, however, that the amount of antibiotic in a disk has been chosen so that the result of the test predicts that an organism in the blood stream can be killed or its growth inhibited by concentrations of antibiotic achievable in the blood stream. The minimum inhibitory concentration (MIC) must be exceeded in the blood stream to penetrate an abscess and kill susceptible organisms. Conversely, a urinary tract infection caused by an ‘‘antibiotic-resistant’’ organism may actually be cured by that antibiotic because the antibiotic is excreted, and thereby concentrated, in the urine. The apparent susceptibilities of certain organisms are also subject to the exact number of organisms placed on the agar plate (i.e., inoculum effect). Susceptibilities of organisms such as Pseudomonas aeruginosa are very dependent on control of the cationic content of the media. Finally, a slow-growing organism may appear falsely susceptible to antibiotic therapy by disk diffusion testing. Additional information about antibiotic susceptibility can be obtained by dilution susceptibility testing, which may be performed in agar or broth. A highly controlled inoculum (often in serum to control the effects of protein binding) is exposed to specific antibiotics in twofold dilutions. The MIC and minimum bactericidal concentration of the antibiotic can then be defined and related to the measured or anticipated concentration of antibiotic achieved or achievable in target tissues (27,28). This kind of data is useful for testing of slow-growing bacteria, when the duration of antibiotic therapy is long (i.e., osteomyelitis and endocarditis), or if another antibiotic must be substituted because of the development of adverse reactions. Automated systems using both the disk diffusion and the dilution method can produce susceptibility results in three to seven hours. However, results for important isolates should be confirmed by conventional methods, which require 24 to 48 hours to perform. Most surgical infections can be well managed by using standard antibiotic susceptibility data. One very important exception to this rule is the apparent susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) to cephalosporins when tested by the disk diffusion method (29). MRSA may appear to be susceptible, but when checked by dilution susceptibility testing, the MICs are found to be high. Consequently, clinical failures can occur when MRSA infection is treated with even higher doses of cephalosporins. For this reason, many laboratories no longer report the cephalosporin susceptibility of MRSA.

TYPES OF POSTOPERATIVE INFECTIONS Respiratory Infections Pneumonia is the second most common nosocomial infection after urinary tract infections but is the leading infectious cause of morbidity and mortality in hospitals in the United States. Nosocomial pneumonias related to surgery have been noted to occur in 5.4 to 11.2 cases per 1000 discharges, depending on the type of operation performed (30). Nosocomial pneumonia is now characterized to include the spectrum of (i) hospital acquired-pneumonia (HAP), (ii) VAP, and (iii) healthcare associated pneumonia (HCAP). Since the initial 1996 American Thoracic Society guidelines, a number of new developments have appeared requiring a new evidence-based guideline for HAP, VAP, and HCAP.

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Some of the key recommendations and principles in this new-evidence based guideline are as follows: &

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&

&

HCAP is included in the spectrum of HAP and VAP, and patients with HCAP need therapy for multidrugresistant (MDR) pathogens. A lower respiratory tract culture needs to be collected from all patients before antibiotic therapy, but the collection of cultures should not delay the initiation of therapy in critically ill patients. Either semiquantitative or ‘‘quantitative’’ culture data can be used for the management of patients with HAP. Lower respiratory cultures can be obtained bronchoscopically and nonbronchoscopically and can be cultured quantitatively or semiquantitatively. Quantitative cultures increase specificity of the diagnosis of HAP without deleterious consequences. Negative lower respiratory tract cultures can be used to stop antibiotic therapy in a patient who has had cultures obtained in the absence of an antibiotic change in the past 72 hours. Early, appropriate, broad-spectrum antibiotic therapy should be prescribed with adequate doses to optimize antimicrobial efficacy. An empiric therapy regimen should include agents that are from a different antibiotic class than that the patient has recently received. Combination therapy for a specific pathogen should be used judiciously in the treatment for HAP, and consideration should be given to short-duration (5 days) aminoglycoside therapy, when used in combination with a b-lactam to treat P. aeruginosa pneumonia. Linezolid is an alternative to vancomycin, and unconfirmed, preliminary data suggest that it may have an advantage for proven VAP due to MRSA. Colistin should be considered as therapy for patients with VAP due to a cabapenem-resistant Acinetobacter species. Aerosolized antibiotics may have value as adjunctive therapy in patients with VAP due to some MDR pathogens. De-escalation of antibiotics should be considered once data are available on the results of lower respiratory tract cultures and the patient’s clinical response. A shorter duration of antibiotic therapy (7–8 days) is recommended for patients with uncomplicated HAP, VAP, or HCAP, who have received initially appropriate therapy and have had a good clinical response, with no evidence of infection with nonfermenting gramnegative bacilli.

A brief discussion of the incidence, pathogenesis, and the modifiable risk factors for each of the three types of nosocomial respiratory infections is warranted to appreciate the extent of the morbidity and mortality that is caused by these infections. HAP is usually caused by bacteria (and currently is the second most common nosocomial infection in the United States) and is associated with high mortality and morbidity (31). HAP increases hospital stay by an average of seven to nine days per patient and has been reported to produce an excess cost of more than $40,000 per patient. Although HAP is not a reportable illness, available data suggest that it occurs at a rate between 5 and 10 cases per 1000 hospital admissions, with the incidence increasing by as much as 6- to 20-fold in mechanically ventilated patients. Time of

onset of pneumonia is an important epidemiologic variable and risk factor for specific pathogens and outcomes in patients with HAP and VAP. Early onset HAP and VAP carries a better prognosis and is defined as pneumonia occurring within the first four days of hospitalization. These are more likely to be caused by antibiotic sensitive bacteria. Pneumonia occurring after five days or more of hospitalization tends to carry a worse prognosis, because they are more likely to be caused by MDR (31). All three types of nosocomial pneumonia may be caused by a wide spectrum of bacterial pathogens, may be polymicrobial, and, rarely are due to viral or fungal pathogens, in immunocompetent hosts. Common pathogens include gram-negative bacilli such as P. aeruginosa, E. coli, Klebsiella pneumoniae, and Acinetobacter species. Infections due to gram-positive cocci, such as Staphylococcus aureus, particularly MRSA, have been rapidly emerging in the United States. Pneumonia due to S. aureus is more common in patients with diabetes mellitus, head trauma, and those hospitalized in intensive care units. The management of nosocomial pneumonia is especially challenging when the pneumonia is caused by multidrug-resistant bacteria. Rates of HAP due to MDR pathogens have increased dramatically in hospitalized patients, especially in intensive care and transplant patients. In dealing with respiratory infections, it is also important to keep in mind that a number of factors predispose surgical patients to the development of respiratory infections. These include suppression of the immune system, which occurs after surgical or accidental trauma, infections occurring at surgical sites remote from the lung, which may lead to ARDS, and mechanical interference with lung function secondary to operative procedures. Thoracic and upper abdominal incisions are particularly likely to impair respiratory function. Pain and splinting from these incisions lead to postoperative hypoventilation and bronchiolar constriction, resulting in atelectasis and a reduction in the functional residual capacity (FRC) of the lung. The extent of the decrease in FRC depends on the type of incision used. Thoracic incisions have been shown to decrease FRC by as much as 65%. Upper abdominal incisions decrease FRC by about 35%, whereas lower abdominal incisions decrease FRC by as little as 7% (32). Atelectasis, defined as areas of alveolar collapse, result in the retention of bronchial secretions and the entrapment of bacteria. Because alveolar macrophages and systemically recruited neutrophils attempt to eradicate these bacteria, fever ensues. Because atelectasis is a common, inciting event leading to the development of postoperative pneumonia, every effort should be directed at preventing or reexpanding collapsed alveoli. This goal may be accomplished through early ambulation, frequent coughing and deep breathing, frequent changes in position when in bed, and the use of incentive spirometry to improve pulmonary expansion. Patient-related risk factors for the development of postoperative pulmonary complications include advanced age, comorbid illnesses (e.g., chronic obstructive lung disease, diabetes, and alcoholism), malnutrition, decreased mental status, and cigarette smoking (33,34). In addition, microaspiration of small volumes of gastric and oropharyngeal secretions colonized with pathogenic bacteria is a common precursor of pneumonia. Whereas microaspiration may occur in up to 45% of normal volunteers, it does not usually lead to pneumonia in the absence of colonization by pathogenic bacteria. As many as 35% of moderately ill patients and about 75% of critically ill patients develop

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oropharyngeal colonization with pathogenic bacteria, primarily gram-negative enteric organisms, during the course of hospitalization (35).Colonization of gastric secretions is particularly likely in patients receiving acid-reducing therapy for the prophylaxis of stress gastritis. Aspiration of oropharyngeal pathogens or leakage of bacteria around the endotracheal tube cuff is the primary route of bacterial entry into the trachea and may lead to pneumonia. Endotracheal intubation and mechanical ventilation, as mentioned above, in association with discussions of HAP, HCAP, and VAP, is one of the leading factors that predispose the patient to develop pneumonia by interfering with lung defenses and by allowing direct access of bacteria to the bronchial tree. Efforts to minimize the likelihood of aspiration of oropharyngeal and gastric contents are equally important in the prevention of postoperative pneumonia. Thus, removal of tubes that alter the normal function of the gastroesophageal (i.e., a nasogastric tube) and pharyngoesophageal sphincters (i.e., an endotracheal tube) and discontinuation of drugs that depress the central nervous system (CNS) should be effected as early as is safely possible. Clinically, the diagnosis of pneumonia is made by the demonstration of an infiltrate on a chest x-ray examination and the presence of purulent sputum, fever, and leucocytosis (36). If a pleural effusion is identified on chest x-ray examination, a diagnostic thoracentesis should be performed. Computed tomography (CT) scans of the chest are not used routinely but may be useful to identify empyemas, lung abscesses, and other pulmonary pathology in patients who are not responding to therapy. Blood cultures should be obtained and have been shown to isolate the etiologic pathogen in 8% to 20% of cases (37). Gram’s stains and cultures of purulent sputum should be obtained. However, it should be recognized that the utility of sputum cultures in patients with hospital-acquired pneumonia is limited and that the interpretation of culture results is based on how the specimen was collected. Cultures of expectorated sputum are neither sensitive nor specific for identifying the infecting pathogen and are primarily useful to establish sensitivity patterns when resistant organisms are suspected (35). In patients who are intubated, routine nonquantitative cultures are a sensitive but not specific method of identifying possible pathogens, because critically ill patients often have colonization of the lower respiratory tract with pathogenic organisms. The simple recovery of a pathogenic organism in these patients does not establish that organism as the cause of infection. However, these culture results have a significant negative predictive value so that antibiotic regimens may be modified once culture results are available. Quantitative and semiquantitative cultures obtained using fiber-optic bronchoscopy may be useful in some intubated patients. Organisms for culture are obtained by use of a protected specimen brash (PSB) or by bronchoalveolar lavage (BAL). The etiologic pathogen is defined by the recovery of bacteria above a predetermined threshold (PSB > 103 and BAL >104 or 105 colony-forming units per milliliter). However, the role of quantitative culture techniques in the diagnosis of hospital-acquired pneumonia remains controversial, and the techniques for obtaining the cultures and concentration of bacteria that establish the presence of infection have not been standardized. The choice of antibiotics for the treatment of nosocomial pneumonias is based on likely pathogens and the spectrum of activity, pharmacodynamic profile, and adverse reactions associated with individual drugs (38). Likely pathogens may be deduced from the timing of the onset

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and the severity of the pneumonia, as well as from the presence of patient-associated risk factors. The choice of antibiotic is also influenced by the concentrations of the agent attainable in respiratory secretions. Quinolones attain concentrations in respiratory secretions close to those attained in serum. On the other hand, aminoglycosides penetrate respiratory secretions poorly and should not be used alone to treat gram-negative respiratory infections (39). Clinical improvement should be used as the ultimate end point of therapy. In general, clinical improvement does not occur before 48 to 72 hours after the initiation of therapy. Changes in antibiotics during this period should not be undertaken unless progressive deterioration of the patient’s clinical status is apparent. Every effort should be made to determine the reason for failure in patients who are unresponsive to therapy after this time period (40). The optimal duration of treatment for nosocomial pneumonias has not been established. For gram-negative pneumonias, which are associated with high rates of treatment failure, relapse, and death, 14 to 21 days of treatment has been recommended. Other less virulent organisms may be adequately treated with a 7- to 10-day course of antibiotics.

Urinary Tract Infections The largest percentage of infections that occur on surgery services arise in the urinary tract (approximately 13.3 cases per 1000 hospital discharges) (41). This circumstance is a simple consequence of the placement of urinary bladder catheters. Although serious infectious problems may ensue from the catheterization of the elderly male with an unrecognized prostate infection or obstruction, most urinary tract problems occur because closed urinary drainage systems eventually become colonized by enteric bacteria such as Escherichia from which the patient may become infected. No measure is particularly effective in preventing these catheter-related infections, although a number of approaches have been tried including the administration of antibiotics (both systemic and local), the use of special tubes with irrigants, and various regimens of catheter care (42). Use of prophylactic antibiotics encourages infection by resistant bacteria or the fungus Candida (especially in diabetic patients). Frequent catheter care with cleansing of the urethral meatus leads to an increased frequency of urinary tract infections so that only daily (rather than more frequent) catheter care has become standard practice. The best prevention of urinary tract infections is to avoid prolonged catheterization and to use alternatives such as intermittent catheterization or, in the case of men, condom catheters whenever possible. Regardless of the technique of drainage, patients should be kept well hydrated so that the bladder is constantly flushed, thereby decreasing the number of potentially infecting bacteria. Many physicians overreact to the presence of more than 105 organisms in the patient’s urine without first obtaining a urinalysis to prove the presence (or absence) of pyuria. Colonization without pyuria can usually be resolved without the use of antibiotics, simply by removing the patient’s catheter and maintaining adequate hydration. In the patient with significant bacteriuria and pyuria in combination and a high temperature elevation in the range of 103 to 104 F, a source of infection other than the bladder should be sought. Pyelonephritis with its attendant high frequency of bacteremia may be present under these circumstances and require aggressive antibiotic therapy; simple cystitis rarely causes high fever (43). The majority

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of catheterized patients with bacteriuria and pyuria have a low-grade fever secondary to a simple catheter-related cystitis. The principles of management in this latter circumstance include removal of the catheter, if possible, and in most instances a single dose of an antimicrobial agent that is excreted in the urine. For those patients in whom catheter placement must remain in the presence of pyuria, the use of a brief course of an antibiotic is warranted to prevent the bladder from becoming a nidus for systemic infection. This latter condition is most likely to occur if the catheter becomes obstructed. Although most postoperative urinary tract infections occur in association with indwelling urethral catheters, occasionally such infections develop in the early postoperative period secondary to urinary stasis and necessitate catheter placement as a therapeutic measure. Patients at risk for this problem include those with prostatic hypertrophy and various neuromuscular disorders such as multiple sclerosis, those who had undergone previous anorectal procedures that have resulted in ineffective postoperative voiding, and those individuals in whom a spinal anesthetic was used. Under most circumstances, a urinary catheter should be prophylactically placed in such patients before surgery to prevent these stasis problems and should be removed as soon as normal voiding can be ensured. If a catheter-associated infection develops, the principles of treatment outlined previously should be followed.

Table 4 NRC Wound Classification Criteria Classificationa

Criteria

Clean (< 2%)

Elective (not urgent or emergency), primarily closed; no acute inflammation or transection of GI oropharyngeal, genitourinary, biliary, or tracheobronchial tracts; no technique break (e.g., elective inguinal herniorrhaphy) Clean contaminated Urgent or emergency case that is otherwise ‘‘clean’’; (< 10%) elective, controlled opening of GI, oropharyngeal, biliary, or tracheobronchial tracts; minimal spillage and/or minor technique break; reoperation via ‘‘clean’’ incision within 7 days: blunt trauma, intact skin, negative exploration (e.g., vagotomy and pyloroplasty) Contaminated (20%) Acute, nonpurulent inflammation (note absence of purulence); major technique break or major spill from hollow organ; penetrating trauma <4 hr old; chronic open wounds to be grated or covered (e.g., acute, nonperforated, nongangrenous appendicitis) Dirty (40%) Purulence or abscess, e.g., preoperative perforation of GI, oropharyngeal, biliary, or tracheobronchial tracts; penetrating trauma >4 hr old (e.g., perforated appendicitis with abscess) a

Wound infection rate after Ref. 44. Abbreviations: NRC, National Research Council; GI, gastrointestinal. Source: From Ref. 45.

Wound and Soft Tissue Infections The development of SSI, usually in the skin and subcutaneous tissues of the operative side, is a common postoperative problem. Overall, it is estimated that 4% to 7% of surgical patients develop these infections postoperatively. These infections contribute to postoperative morbidity and increased length of stay, with attendant increased costs of medical care. The prevention of SSIs relies on correct surgical technique, modification of host risk factors, and adequate antimicrobial prophylaxis. Both local factors, such as the degree of contamination of the wound, and systemic factors, such as the overall condition of the patient, influence the rates of wound infections. Systemic factors, such as hypoxic lung disease, should not be underestimated when evaluating SSI. The degree of contamination of the surgical wound has traditionally been assessed according to the National Research Council (NRC) wound classification scheme. In this system, wounds are classified as clean, clean contaminated, contaminated, or dirty (Table 4). The NRC wound classification does not, however, take into consideration any other risk factors for wound infection (44,45). Additional risk factors for wound infection have been defined. In one prospective study involving nearly 24,000 patients, advanced age, the presence of diabetes, obesity, or malnutrition, and a perioperative hospital stay of more than two weeks were identified as risk factors (46). In other studies, malignancy, alcoholism, recent cigarette smoking, hypoxemia, remote infection, chronic inflammation, prior surgical site irradiation, recent operation, prior use of antibiotics, and recent use of corticosteroids or cytotoxic agents have also been associated with an increased risk of postoperative infection (45,47). The interaction between these and other factors and the NRC wound classification was investigated in two very large epidemiologic evaluations. One of these took place during the Study on the Efficacy of Nosocomial Infection

Control (SENIC) project (48). This analysis of nearly 60,000 patients undergoing surgical procedures identified four factors of nearly equal weight in predicting postoperative wound infection: an abdominal operation, an operation that took longer than two hours, a wound classified as either contaminated or dirty, and the presence of three or more diagnoses at the time of discharge. The surgical wound infection rates in patients ranged from 1.0% in patients with no risk factors to 27.0% in patients with four risk factors (Table 5). Even in patients with clean wounds, the postoperative wound infection rate ranged from 1.1% to 15.8% as the number of risk factors increased from zero to three (49). The second large study involved patients evaluated as part of the National Nosocomial Infection Surveillance System (NNISS), sponsored by the Centers for Disease Control and Prevention (50). In this survey of nearly 85,000 operations, three risk factors were identified: a patient having an American Society of Anesthesiologists (ASA) score of 3, 4, or 5, an operation considered contaminated or dirty-infected, and an operation that took longer than T hours, where the value of T depended on the actual operative procedure and ranged from one hour for a simple procedure such as appendectomy to four hours for complex procedures on the liver, bile ducts, or pancreas. The risk of a surgical wound infection varied from 1.5% to 13% as the number of risk factors increased from zero to three (Fig. 2). In patients with clean wounds, the infection rates varied from 1% to 5.4% as the number of risk factors increased from zero to two. The NNISS data also showed that there was an increased risk of other nosocomial infections, including pneumonias, blood stream infections, and urinary tract infections as the number of risk factors increased. Thus the inclusion of additional factors along with the NRC wound classification predicted the risk of surgical

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Table 5 Distribution of Patients and Infections by the SENIC Risk Factors and NRC Wound Classification % Infection by NRC wound classification SENIC risk factors 0 1 2 3 4 NRC, % of all patients NRC, % of patients with infection NRC, % of group with infection

SENIC, % of patients with infection

SENIC, % of group with infection

Clean contaminated

Contaminated

Dirty

SENIC, % of all patients

1.1 3.9 8.4 15.8 N/A 55d

0.6 2.8 8.4 17.7 N/A 36

N/A 4.5 8.3 11.0 23.9 2

N/A 6.7 10.9 18.8 27.4 7

46a 32 16 5 1 100

10b 29 35 20 6 –

1c 3.6 8.9 17.2 26.7 –

39e

35

4

22

–

100

–

8.5

12.6

–

–

Clean

2.9f

3.9

4.1

Note: SENIC risk factors include abdominal operations, operations longer than two hours, three or more associated diagnoses, and dirty or contaminated wounds, as defined by NRC wound classification. Patients with contaminated or dirty wounds cannot have zero SENIC risk factors. Patterns with clean or clean-contaminated wounds cannot have four SENIC risk factors. Example statements indicating how to read this table: a Patients with zero SENIC risk factors accounted for 46% of all patients. b Patients with zero SENIC risk factors accounted for 10% of all infected patients. c The infection rate for patients with zero SENIC risk factors was 1%. d The NRC clean wounds accounted for 55% of all patients. e The NRC clean wounds accounted for 39% of all infected patients. f The infection rate for patients with NRC clean wounds was 2.9%. Abbreviations: NRC, National Research Council; SENIC, Study on the Efficacy of Nosocomial Infection Control. Source: From Ref. 49.

wound infection better than did the NRC wound classification scheme alone. Although the variables identified in these two large prospective studies were different, it is apparent that the length of the procedure and the patient’s premorbid condition, as assessed in the SENIC study by the number of discharge diagnoses and in the NNISS study by the ASA classification, have as much bearing on wound infection rates as the NRC wound classification category. Rational interventions designed to decrease the risk of surgical wound infection can be developed on the basis of this risk factor data. Currently, one of the most commonly used interventions is the use of perioperative prophylactic antibiotics. Because of the low rate of infections (approximately 1–3%) usually observed in patients classified as having clean wounds, it had been argued that prophylactic antibiotics were not needed in that group of patients.

Figure 2 SWI rates within categories of the surgical patient risk index. Abbreviations: G, Goodman–Kruskal correlation coefficient; s.e., standard error; SWI, surgical wound infection. Source: From Ref. 44.

However, both the SENIC and the NNISS studies indicate that there are patients with clean wounds who have a much higher risk of wound infection. Thus, selected patients having clean operations might warrant prophylaxis. The use of perioperative antibiotics is discussed in more detail later in this chapter. Other factors might also be subject to modification. The length of the surgical procedure was identified in both studies as a risk factor for the development of surgical wound infection. This might suggest that efforts made by the surgeon to increase the speed of the operation would result in decreased infectious complications. However, it is unclear from these studies if the length of the surgical procedure was actually a surrogate end point for surgical skill or was rather related to the difficulty of the particular procedure as determined by the complexity of the patient’s pathologic condition. Thus if the latter were true, it is unlikely that efforts designed to increase the rapidity with which an operative procedure is carried out would have a significant effect on postoperative infections. From additional studies, it does appear that the prevention of intraoperative hypothermia reduces the likelihood of surgical wound infections. In a recent prospective, randomized study, the maintenance of normothermia during elective colorectal surgery decreased the rate of wound infections threefold (51). It was speculated that hypothermia interfered with blood flow to the surgical wound by triggering vasoconstriction or, in some other way, impaired the immunologic response in those areas. Many of the risk factors related to the patient’s pathologic process and underlying physiologic reserves cannot be readily controlled. However, several factors are related directly or indirectly to nutritional status. Thus interventions designed to improve the patient’s nutritional status, particularly the use of parenteral nutrition, have been

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proposed as a means of reducing postoperative infectious morbidity. However, preoperative parenteral nutritional support has not been found to benefit the majority of patients and may even contribute to postoperative infections. In the Veterans Affairs Total Parenteral Nutrition Cooperative Study, only the patients with severe malnutrition had a slight benefit from preoperative nutritional support (52). In the remainder of the patients, who had mild-to-moderate malnutrition, preoperative therapy may actually have been detrimental. This investigation did not examine the potential role of oral or enteral nutrition preoperatively, and there is no reason to discourage adequate nutrition during the preoperative period if it does not unnecessarily delay a needed procedure. However, exhaustive efforts to improve nutritional intake, particularly using parenteral nutrition, are unlikely to be useful except in the most severely malnourished patients. One final intervention that appears useful in reducing global rates of surgical wound infections is an effective infection control program (53). This intervention has been estimated to provide reductions in wound infection rates of 20% to 38%. Effective programs should include wound surveillance, both in the inpatient and outpatient setting, to detect the presence of surgical wound infections. However, of greater importance is feedback to individual practitioners with regard to their own wound infection rates and an effective program for policy development and teaching that allows individual practitioners to adapt effective techniques in controlling wound infection rates.

Phlebitis Although phlebitis of the lower extremities may develop in any patient following surgery and result in postoperative fever, it seldom has a bacterial cause. In contrast, catheterrelated phlebitis is a true infection. Bacteremias secondary to intravascular devices occur in as many as 25,000 to 50,000 patients per year in the United States (54). An intravenous catheter breaches local skin defenses and allows several portals of entry for bacteria. The most common portal of entry is along the skin tract on the outside of the catheter. The predominant organisms that gain entry by this route are skin flora (e.g., S. aureus, but also, with increasing recognition, Staphylococcus epidermidis). Contamination of the catheter hub infusion junction has also been implicated as a cause of bacteremia in patients with central venous lines. Again, organisms of the skin flora are the most common ones. Rarely, bacteremias may occur from the infusion of contaminated solutions. These infections are most commonly due to gram-negative organisms (e.g., Pseudomonas and Enterobacteriaceae). Definitive diagnosis of a catheter-related infection can only be made if there is purulence or inflammation at the insertion site with recovery of a pathogen on culture or if blood culture results and culture results from of the catheter tip are positive for the same organism and there is no other identifiable source of bacteremia. The diagnosis is considered probable if (i) there is purulence at the insertion site but no pathogen is recovered, (ii) blood and catheter-tip culture results are positive for the same organism and a separate identifiable source of bacteremia is present, or (iii) bacteremia is present without an identifiable source and the catheter tip and insertion site were not cultured. Catheter-tip cultures are obtained when a blood stream infection is thought to be related to contamination of the catheter. Under these circumstances, the tip of the catheter is cut off with sterile scissors and placed in a sterile

container so that semiquantitative cultures can be obtained. Maki et al. (55) reported that these cultures have a 76% to 96% specificity and a positive predictive value of 16% to 31% for identifying the presence of a catheter-related infection. Prevention of infection requires careful use of sterile technique for the placement of all intravascular catheters and particularly for central venous lines. In addition, the insertion site must be dressed appropriately. A metaanalysis (56) found a significantly increased risk of catheter colonization when transparent semipermeable dressings were used compared with gauze dressings. This study also found a trend toward an increase in bacteremia with the use of transparent dressings, but this difference was not statistically significant. Frequent catheter changes (every 72–96 hours) may be necessary in patients with clinical signs of infection. This is especially true if the catheters are being frequently manipulated to administer medications or to draw blood samples. In the absence of clinical signs of infection and, particularly, if the catheter is dedicated for a single purpose (i.e., total parenteral nutrition administration), the catheters may be left in place indefinitely. Exchange over a guide wire should not be used if a device infection is suspected (57). Three therapeutic decisions involving intravenous catheters are particularly controversial. The first is bacteremia caused by S. aureus, which may be secondary to infected intravenous catheters. S. aureus is notorious for persisting within leukocytes and disseminating widely throughout the body (58). A body of data has suggested that if the focus of infection is removable (e.g., an intravenous catheter that can be removed or a furuncle that can be rapidly drained), the patient can be treated with a relatively short course of antibiotics, specifically, two weeks. Conflicting evidence reveals the occasional case of endocarditis that develops despite adherence to appropriate principles of short-course antistaphylococcal therapy (59). This has led some infectious disease experts to recommend a long (4–6 weeks) course of antibiotics, particularly if the patient is immunocompromised or if the patient has persistent staphylococcal bacteremia. A second and relatively common therapeutic dilemma is the approach to C. albicans fungemia related to an infected intravenous catheter site. Until recently, the usual approach was to remove the catheter and, because C. albicans is an avirulent pathogen, to rely on host defenses to clear the infection. With the introduction of less toxic antifungal agents, therapy is commonly administered to almost all patients with fungemia. Compelling data have documented that fluconazole can replace amphotericin B for the treatment of susceptible fungi in patients without neutropenia (60). Fluconazole should not be used to treat Candida krusei, Torulopsis glabrata, and Candida lusitaniae because of the high incidence of resistance of these organisms to triazoles. In patients with obvious host deficits (e.g., neutropenia and immunosuppressive drugs), the current recommendation is removal of the catheter followed by a short course (7–10 days) of amphotericin B. The third difficulty in the treatment of infected intravenous catheter sites arises when thrombophlebitis supervenes. If a central vein is thrombosed and infected, high-dose antimicrobial therapy often suffices. The incidence of pulmonary embolism in this setting is probably higher than previously thought, and the use of heparin may be appropriate for selected patients. When a peripheral vein is thrombosed, as evidenced by a palpable venous cord, and is believed to be a source of continuing infection, efforts

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to aspirate the vein are usually unrevealing but worth attempting. In general, treatment consists of a conventional course of antibiotics for approximately 10 days. If the patient does not exhibit a clinical response to antibiotic therapy, excision of the vein may be necessary. It should be recognized that veins that appear grossly normal at the time of surgery may in fact contain multiple microabscesses in their walls when they are examined microscopically.

Intra-Abdominal Infections Intra-abdominal infection may occur with or without contamination of the peritoneal cavity. Infections (e.g., appendicitis and simple cholecystitis) confined to a diseased organ that is resectable are usually easily cured surgically, may not require prolonged antibiotic therapy, and are not associated with diffuse peritonitis. Peritonitis is defined as inflammation of the peritoneum from any cause, with the most common cause being intra-abdominal infection secondary to bacteria or other microorganisms. Bacterial peritonitis has been classified into primary, secondary, and tertiary peritonitis. Primary peritonitis occurs because of seeding of the peritoneum with bacteria from the blood stream or the lymphatic system and occurs most commonly in cirrhotic patients with ascites. It accounts for less than 1% of peritonitis and treatment is primarily medical with antibiotic therapy (61). Tertiary peritonitis is a peritonitis-like syndrome occurring after a patient has had persistent intra-abdominal infection. Organisms causing tertiary peritonitis are generally nosocomial pathogens such as multiply resistant bacteria or fungi. Most of these patients have been on multiple antibiotics and have had numerous abdominal procedures performed. Secondary peritonitis is the most common form of peritonitis encountered by the surgeon and occurs because of a loss of integrity of the GI tract with resultant contamination of the peritoneal cavity by GI contents (62). The natural history of secondary peritonitis has been demonstrated in animal experiments. In these studies, a two-stage process was noted to occur (63,64). The first stage, lasting approximately five days, was a generalized peritonitis in which gram-negative enteric aerobes were the predominate organism. E. coli bacteremia was common, and a mortality rate of 43% occurred during this stage without treatment. The second stage occurred after five days as the peritoneal defenses attempted to wall off the infection. This resulted in multiple intra-abdominal abscesses with grossly purulent material contained within a collagen wall. The predominant organism within these abscesses was Bacteroides fragilis. Antibiotic therapy (e.g., aminoglycosides) directed toward aerobic enteric organisms decreased early mortality and the incidence of E. coli bacteremia, but did not prevent abscess formation. Conversely, treatment directed against anaerobic organisms (metronidazole and clindamycin) did not affect early mortality but prevented the formation of abscesses. Other studies have demonstrated that mortality rates are directly correlated with the E. coli inoculum size. In addition, large inoculums of bacteria within an abscess cavity inactivate antimicrobial agents at a rate proportional to bacterial density and to the individual inactivating ability. Dead bacteria and debris within the abscess cavity can bind to and inactivate antimicrobial agents. Finally inactivation of antibiotics, particularly clindamycin and aminoglycosides, occurs because of the acidic conditions within the abscess. The diagnosis of secondary peritonitis in an alert patient may be made by physical examination, which characteristically demonstrates diffuse tenderness, rebound

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tenderness, involuntary muscle guarding, and abdominal wall rigidity. The presence of these signs of generalized peritonitis should lead to prompt operation without further diagnostic studies. However, the physical examination may be difficult to interpret in critically ill patients, patients with altered mental status, postoperative patients, and patients with intra-abdominal abscesses. In addition, elderly patients and patients who are immunosuppressed may not exhibit signs of peritonitis even in the face of generalized infection. Ancillary diagnostic tests may be necessary in these patients (65). Plain films may be of use in detecting a perforated viscus if free peritoneal air is demonstrated. In the absence of significant findings on plain films, ultrasonography has been used as the initial procedure to diagnose intra-abdominal infection in some studies. It offers the advantage of the ability to perform the test at the bedside in critically ill patients, low cost, and rapid results. However, interpretation of the scans may be limited by wounds, dressings, ostomies, obesity, and air-filled bowel loops. Ultrasound may be most useful for detecting infections originating in the liver or biliary tree. Overall sensitivity of ultrasound for the detection of intra-abdominal infection is between 75% and 82%. For the majority of patients, CT scan is the most appropriate initial study. Although CT scan is more costly than ultrasound, it has a sensitivity of 78% to 100% for the detection of intra-abdominal infection. Suspicious fluid collections identified on CT scan should be aspirated for gram’s stain and culture. When aspiration is used in conjunction with CT scans, specificity for the identification of intra-abdominal abscesses is 98%. Optimal management of intra-abdominal infection requires physiologic support of the patient, resection or repair of the source of infection, drainage of any established abscesses, and elimination of residual contamination with antimicrobial therapy (66). Patients with diffuse peritonitis exhibit marked sequestration of extracellular fluid within the inflamed peritoneal cavity, and the intestine and may require large amounts of volume resuscitation. In addition, these patients may develop multiple organ dysfunction syndrome (MODS) secondary to intra-abdominal sepsis and require support of other organ systems (i.e., mechanical ventilation and dialysis) while the primary focus of sepsis is being addressed. Operation is usually necessary to control the source of infection. When frank perforation of the GI tract is the source of infection, the operation may involve resection or repair of the intestine with or without anastomoses or exteriorization of the intestine. Established abscesses should be drained to reduce the bacterial inoculum. This may be accomplished by CT-guided percutaneous drainage or by operative drainage (67–69). A safe percutaneous drainage route that avoids puncturing solid or hollow viscera can be identified in 85% to 90% of patients. After successful drainage, there should be prompt clinical improvement within 48 to 72 hours. Failure to improve within this time frame requires a repeat CT scanning. If residual fluid cannot be evacuated with the placement of additional drains, surgical drainage should be performed. The success of percutaneous drainage of well-defined unilocular abscesses ranges from 80% to 90% and CT-guided drainage has become the procedure of choice for these abscesses (63,70). Percutaneous drainage of complex abscesses (i.e., loculated, pancreatic, interloop, or multiple, or abscesses associated with enteric fistula) has been less successful and may require surgery. Based on the experimental data just discussed, antibiotic therapy should be directed toward both aerobic and

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anaerobic enteric organisms to prevent early mortality and subsequent abscess formation. This may be accomplished with combination therapy or with broad-spectrum single-agent therapy. The requirement for treatment of enterococcus and Candida recovered from intra-abdominal infections continues to be an area of controversy. In animal studies and in past clinical experience, successful treatment of intra-abdominal infection has been accomplished with agents that have no activity against these organisms. However, both organisms are commonly found in the GI tract and have become major nosocomial pathogens over the last 15 to 20 years. Currently, recommendations are that these organisms do not require antibiotic coverage if they are a part of a polymicrobial infection. However, if these organisms are isolated in pure culture or are recovered from the blood stream or, in the case of Candida, if disseminated infection is present, treatment of these organisms should be initiated. A complete discussion of antibiotic therapy in the treatment of intra-abdominal infection is beyond the scope of this chapter but may be found in a consensus statement by the Surgical Infection Society (71).

Miscellaneous Infections Although the foregoing discussion has considered the usual causes of postoperative infection, in reality many other causes can exist. For this reason, the surgeon must always maintain an open mind and frequently reexamine the patient to look for the underlying source of infection if the patient continues to exhibit clinical signs of infection. An unexplained heart murmur may lead to a diagnosis of endocarditis on echocardiogram, whereas unusual obtundation may be secondary to undiagnosed meningitis or brain abscess. In traumatized patients with facial fractures, sinusitis may occur, particularly in patients who are nasotracheally intubated. Parotitis from an obstructed salivary gland duct may also be an incipient source of infection. Conversely, in some patients, fever may not be related to infection at all, but may be secondary to a drug reaction or may be an allergic reaction to a blood transfusion. Finally, an additional type of infectious process is the intrinsic infections of the GI tract that may occasionally occur following surgery. The most common manifestations of these infections are fever and diarrhea. However, these are nonspecific findings, and in some patients, diarrhea may be related to enteral feedings and have nothing to do with the fever. Of more concern to the surgeon is the patient who has received antibiotic therapy and then has developed diarrhea. In many instances, this diarrhea is caused by an overgrowth of C. difficile, which produces a cytotoxin (72,73). Antibiotics that are notably linked with AAC include ampicillin, cephalosporins, and clindamycin, but the syndrome may occur with the administration of almost any antibiotic. AAC may be accompanied by fever and toxicity and is frequently overlooked as the potential source of postoperative fever. Diagnosis includes an assessment of stool for the presence of C. difficile toxin. The fecal leukocyte examination results may be positive, but negative results do not exclude AAC. The diagnosis may also be made by sigmoidoscopy if the typical pseudomembranous lesions associated with this entity are seen. After diagnosis, therapy is oral vancomycin—125 mg four times daily for 10 days. Parenteral vancomycin is not recommended because the object of therapy is to obtain intraluminal levels of antibiotic, which intravenous vancomycin does not provide. An alternative therapeutic option is oral (250 mg four times

daily) or intravenous metronidazole, because both routes of administration of metronidazole are effective. Because Clostridium spores may survive a course of vancomycin or metronidazole, relapses of AAC may occur. These are treated by an additional course of the same antimicrobial agent.

PATHOGENS RESPONSIBLE FOR SURGICAL INFECTION A common approach to characterizing surgical infection is to list the microbial agents that cause such infections. Considering the phenomenal number of organisms that can infect compromised hosts, a list of ‘‘surgical microbes’’ becomes merely a weak attempt at a review of the entire field of microbiology. Equally inappropriate are lists of microbes and the preferred antimicrobial agents to treat them. Memorizing such lists is a relatively fruitless exercise because many surgical infections are polymicrobic; thus the surgeon must more realistically design an empiric therapy with a broad-spectrum agent or a combination of agents The rational approach to surgical microbiology is to learn those microbes that can singly cause important infections and that may have classic clinical presentations. Also the surgeon must learn the combinations of microbes that are frequently encountered in surgical situations so that empiric therapy can be instituted, until the results of appropriate Gram’s stains and cultures are available. Table 6 lists the most common pathogens identified by the NNISS from 1986 to 1989 (74). Among the fungi, C. albicans is the most important and is not infrequently involved in intravenous catheter-related fungemia. A recent multicenter study has documented the efficacy of fluconazole in the treatment of candidemia in non-neutropenic patients (60). In immunocompromised hosts, however, amphotericin B should be used until further data are available. Two new lipophilic preparations of amphotericin B may reduce the toxicity of this agent (75). The easiest way to control Candida infections in routine surgical practice is to prevent their occurrence by choosing the most narrow spectrum antibiotic or combination of antibiotics and using them for the briefest duration consistent with guaranteed clinical success (76). Other fungal pathogens, including Aspergillus and Mucor, are still occasionally problems for the surgeon and may cause systemic infections in debilitated patients The source of these infections (e.g., the burn wound or diabetic abscess) requires aggressive surgical excision in conjunction with high-dose (as much as 1 mg/ kg/day for the 1st week) amphotericin B if effective treatment is to be rendered (77). The role of viruses in surgical infection is poorly studied except for the blood-borne hepatitides such as hepatitis B and C and cytomegalovirus. Cytomegalovirus is a common problem in transplantation recipients. Presently, no effective therapy exists for cytomegalovirus, but new agents are being tested. HIV is an uncommon cause of postoperative infection in surgical patients; however, most surgeons will be called on to treat patients with HIV infections. Although occupational risks for the infection of health care workers is low, all surgeons should be aware of preventive strategies to limit transmission of this disease (78,79). In addition, a number of parasites that are not commonly seen, including Pneumocystis carinii and Toxoplasma gondii, are encountered routinely in AIDS patients, obliging all physicians, including surgeons, to become reacquainted with their clinical presentations.

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Table 6 Pathogen Distribution for Major Sites of Nosocomial Infection, NNISS, 1986–1989 Pathogen E. coli Enterococci P. aeruginosa S. aureus Coagulase-negative staphylococci Enterobacter sp. K. pneumoniae C. albicans P. mirabilis Streptococcal species Citrobacter sp. Candida sp. S. marcescens

Urinary tract infection 11,135 6720 5127 823 1634 2339 2664 2978 2312 207 812 853 367

(26) (16) (12) (2) (4) (6) (6) (7) (5) (0) (2) (2) (1)

a

Wound

Pneumonia

Blood stream

1951 2645 1668 3439 2472

(10) (13] (8) (17) (12)

946 342 2598 2401 293

(6) (2) (17) (16) (2)

733 1037 543 1984 3384

(6) (8) (4) (16) (27)

1529 618 481 712 539 321 81 271

(8) (3) (2) (4) (3) (2) (0) (1)

1625 1042 615 503 231 226 109 579

(11) (7) (4) (3) (1) (1) (1) (4)

610 548 617 105 465 82 330 152

(5) (4) (5) (1) (4) (1) (3) (1)

Total 14,765 10,744 9936 8647 7783 6103 4872 4691 3632 1442 1441 1373 1369

(16) (12) (11) (10) (9) (7) (5) (5) (4) (2) (2) (2) (2)

Note: A site may have up to four pathogens. a Number (%). Abbreviation: NNISS, National Nosocomial Infection Surveillance System. Source: From Ref. 74.

Knowledge of other parasitic infections may also be relevant to surgical practice. The classic example is the huge solitary liver abscess in the right lobe of the liver. In such cases, amebic disease must be suspected and confirmed by an ameba serology test. Metronidazole is the antibiotic of choice for these infections. Unless such abscesses threaten to rupture, they are usually managed with antimicrobial agents alone, and surgery or percutaneous drainage is generally not necessary (80). When hydatid disease is suspected, it should be confirmed serologically, and serious consideration should be given to treating the patient with benzimidazole compounds (mebendazole and albendazole) before surgery (81).

of a particular antibiotic. It is for these reasons that an antibiotic, although once effective therapeutically against a specific pathogen, may lose its efficacy. A discussion of the various classes of antibiotics is warranted in order to appreciate the understanding of the mechanism by which they are designed to overcome the numerous pathogens causing postoperative infectious complications.

Table 7 Mechanisms of Action Responsible for Efficacy of Antibiotics Against Different Microorganisms

ANTIBIOTICS IN THE MANAGEMENT OF INFECTION

Antibiotic

It is beyond the scope of this chapter to discuss the use of antibiotics in the treatment of infection in a comprehensive fashion. Therefore a brief discussion of the mechanisms of antibiotic action, antibiotic prophylaxis, and the therapeutic use of antibiotics is presented.

Aminoglycoside Inhibit protein synthesis by binding to 30S ribosome b-Lactams Inhibit cell wall synthesis by interleukin with the production or cross-linking of peptidoglycans Clindamycin Binds to 50S ribosome subunits and inhibits peptide bond formation Macrolides Binds to 50S ribosome subunit and inhibits protein synthesis Metronidazole Redox reaction produces toxic metabolites that damage bacterial DNA, inhibits DNA gyrase Quinolones Interfering with DNA replication and repair Rifampin Blocks RNA synthesis by inhibiting DNA-dependent RNA polymerase Sulfonamides/ Blocks folic-acid synthesis; binds to trimethoprim 30S ribosomal subunit and inhibits protein synthesis Tetracyclines Interfering with production of purine and pyrimidine, blocks dihydrofolate reductase Vancomycin Inhibits cell wall synthesis by interfering with peptidoglycan production at a different site from penicillins; also may alter membrane permeability and inhibit RNA synthesis

Mechanisms of Action Differing modes of action are responsible for the efficacy of individual antibiotics against various microorganisms (Table 7). The extent to which these antibiotic effects prevent bacterial growth often influences the efficacy of an antibiotic under a given set of clinical conditions. A bactericidal antibiotic is often preferable to a bacteriostatic antibiotic, which inhibits growth or multiplication of bacteria and allows normal host defenses to actually effect bacterial destruction. In an immunocompromised patient in whom host defenses may be severely depleted, use of a bactericidal drug is even more important. In the same way that antibiotics differ in their modes of action, microorganisms develop bacterial resistance in a variety of ways. These ways may include mutation to a resistant strain or the production of an enzyme such as penicillinase or b-lactamase that can destroy the antibiotic effects of penicillins and cephalosporins, respectively. In addition, a given pathogen may activate a latent biochemical process or acquire, by transfer, a chromosome-like factor, known as a resistance factor that may block the mode of action

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Mechanism of action

Bacteriostatic/ bactericidal Bactericidal Bactericidal

Bactericidal Bacteriostatic Bactericidal

Bactericidal Bactericidal Bacteriostatic

Bacteriostatic

Bactericidal

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b-Lactam Antibiotics b-lactam antibiotics are among the most commonly prescribed drugs, grouped together because of their shared structural feature, the b-lactam ring. They include: & & & & &

Penicillins Cephalosporins Carbapenems Monobactams b-lactamase inhibitors

b-lactam antibiotics cause the inhibition of synthesis of the bacterial cell wall, leaving bacteria without a protective outer shield. Without this protective outer shelf, water from isotonic body fluids move in freely into bacteria, creating a bursting effect. Bacteria with cell wall susceptible to b-lactam antibiotics are typically known as gram-positive organisms. The mechanism of action involves prevention of the normal cross linkage of peptidoglycan, an essential structural component of bacterial cell wall, or by interference of the biosynthesis of peptidoglycans (82). b-lactam inhibition of cell wall synthesis leads to activation of the autolytic system through a twocomponent system, VncR/S, which initiates a cell death program. An understanding of the generational differences among the b-lactams remains essential for the surgeon who is often challenged with the decision to choose from a variety of antimicrobial agents, with the precaution that resistant strains may emerge. Thus, while better and stronger agents have been synthesized and are available to the surgeon today, the surgeon must still remain cognizant of treating simpler infections with the simpler, narrower spectrum antibiotics in order to combat the fight against the emergence of resistant strains. Penicillins Penicillins can be classified into three groups: & &

&

Penicillin G Antistaphylococcal penicillins (nafcillin, oxacillin, cloxacillin, and dicloxacillin) Broad spectrum penicillins

Blood spectrum penicillins are further classified into: &

& &

Second generation (ampicillin, amoxicillin, and related agents) Third generation (carbenicillin and ticarcillin) Fourth generation (mezlocillin and piperacillin)

Spectrum of Activity. Penicillin G is effective against &

& &

&

Gram-positive cocci (except penicillinase-producing staphylococci, penicillin-resistant pneumococci and enterococci, and oxacillin-resistant staphylococci) Gram-positive rods such as Listeria Gram-negative cocci such as Neisseria sp. (except penicillinase-producing Neisseria gonorrhoeae) Most anaerobes (with certain exceptions such as Bacteroides)

Antistaphylococcal penicillins inhibit penicillinaseproducing staphylococci but are inactive against oxacillinresistant staphylococci. Broad-Spectrum Penicillins. The broad-spectrum penicillins are distinguished by their-activity against gram-negative

bacilli. These agents have been stratified into the secondgeneration penicillins (ampicillin and amoxicillin), the third-generation penicillins (carbanecillin and ticarcillin), and the fourth-generation penicillins (mezlocillin and piperacillin). None of the broad-spectrum penicillins are effective against penicillinase-producing staphylococci. Second-Generation Penicillins. Ampicillin, amoxicillin, and closely related antibiotics are able to penetrate the porin channel of gram-negative bacteria but are not stable to b-lactamases. These antibiotics are active against the majority of strains of E. coli, Proteus mirabilis, Salmonella, Shigella, and H. influenzae. Whereas a large percentage of encapsulated H. influenzae type b from the blood and cerebrospinal fluid of children are b-lactamase positive (and ampicillin resistant), only 15% of the non–type b isolates from adult patients with community-acquired pneumonia are b-lactamase positive (83). Amoxicillin and ampicillin have an identical spectrum of activity, but amoxicillin is better absorbed from the intestine when administered orally and yields higher blood and urine levels. Amoxicillin is available generically and is preferable to ampicillin for oral use except in the therapy of Shigella infections sensitive to ampicillin. Third-Generation Penicillins. Carbenicillin and ticarcillin also can penetrate the porin channel of gram-negative bacteria in high doses, but they are less active than ampicillin on a weight basis. However, the carboxy group on the side chain of these antibiotics expands the spectrum of activity by rendering them more resistant to the chromosomal b-lactamases of certain organisms, such as indole-positive Proteus species, Enterobacter species, and P. aeruginosa. Third and fourth generation penicillins are most useful in infections caused by these organisms. Ticarcillin has the same spectrum of activity as carbenicillin but is two to four times more active on a weight basis against P. aeruginosa; the normal maximum parenteral dose is 18 g/day. Fourth-Generation Penicillins. Mezlocillin and piperacillin are derivatives of ampicillin. They cover much the same spectrum as carbenicillin and ticarcillin but are more active in vitro on a weight basis. In addition, they have some activity against strains of Klebsiella, although cephalosporins remain the preferred agents. They are more active than carbenicillin or ticarcillin against enterococci and B. fragilis, but other agents are preferred for the treatment of these organisms as well. Mezlocillin is comparable to ticarcillin against P. aeruginosa and somewhat more active against Enterobacteriaceae than carbenicillin or ticarcillin. Piperacillin is as active as mezlocillin against Enterobacteriaceae and more active than mezlocillin or ticarcillin against P. aeruginosa. As with ticarcillin, clinical failures have occurred when these newer penicillins are used as single agents to treat serious Pseudomonas infections. The third- and fourth-generation penicillins are generally considered together as antipseudomonal penicillins and only a single representative employed as standard therapy at a given hospital. Several factors are considered in deciding which of these agents should be chosen: &

The fourth-generation penicillins are more active in vitro on a weight basis in inhibiting bacterial growth but not in bacterial killing; for P. aeruginosa, ticarcillin is more rapidly bactericidal.

Chapter 6: &

&

Surgical Infection: Principles of Management and Antibiotic Usage

The fourth-generation penicillins have little enhanced stability to b-lactamases compared with the thirdgeneration agents. The fourth-generation penicillins, especially mezlocillin, have less effect than ticarcillin on platelet function.

Cephalosporins Cephalosporins include the closely related cephamycin compounds. The parenteral agents are commonly classified into the following categories: & & & &

First generation Second generation Third generation Fourth generation (cefepime)

Spectrum of Activity. Most of the available cephalosporins are semisynthetic derivatives of cephalosporin C, a compound with antibacterial activity produced by the fungus Cephalosporium. The closely related cephamycin compounds (derived from Streptomyces spp.) are regarded as members of the cephalosporin class. In clinical practice, these antibiotics have frequently been grouped into four ‘‘generations’’ based upon their spectrum of activity against aerobic and facultative gram-negative bacilli. First-Generation Cephalosporins. Cephalothin is the oldest of the first-generation cephalosporins and was previously used as the prototype of this group. Cephalothin is active against most gram-positive cocci (including penicillinase-producing staphylococci), but does not have clinically useful activity against enterococci, Listeria, oxacillin-resistant staphylococci, or penicillin-resistant pneumococci. Cephalothin is active against most strains of E. coli, P. mirabilis, and K. pneumoniae, but has little activity against indole-positive Proteus, Enterobacter, Serratia, and the nonenteric gram-negative bacilli such as Acinetobacter and P. aeruginosa. Gram-negative cocci (such as the gonococcus and meningococcus) and H. influenzae are generally resistant. Cephalothin is active against most of the common anaerobic pathogens, with certain exceptions such as Bacteroides species, particularly B. fragilis. Second-Generation Cephalosporins. The second-generation cephalosporins are somewhat less active against grampositive cocci than the first-generation agents, but are more active against certain gram-negative bacilli. This generation of compounds can be divided into two subgroups, one with activity against H. influenzae and the other, the cephamycins, with activity against Bacteroides. Cephamycin Subgroup (Active Against Bacteroides). The cephamycin subgroup of the second-generation cephalosporins includes cefoxitin and cefotetan. This subgroup is active against most strains of E. coli, P. mirabilis, and Klebsiella, like the first generation cephalosporins. Unlike the first-generation cephalosporins, the cephamycins are active against many strains of Bacteroides. The combination of activity against common aerobic and facultative gram-negative bacilli plus Bacteroides has led to the use of the cephamycins in the prophylaxis and therapy of infections in the abdominal and pelvic cavities (where these organisms predominate). The cephamycins have no clear advantages over the first-generation cephalosporins for infections outside of the abdominal and pelvic areas.

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Cefotetan has a substantially longer serum half-life than cefoxitin, allowing for less frequent dosing for therapy and single-dose prophylaxis. Cefotetan is more active than cefoxitin against aerobic and facultative gram-negative bacilli although not comparable to third-generation cephalosporins, but less active against Bacteroides. Third-Generation Cephalosporins. The third-generation cephalosporin class is marked by stability to the common blactamases of gram-negative bacilli, and these compounds are highly active against Enterobacteriaceae (E. coli, P. mirabilis, indole-positive Proteus, Klebsiella, Enterobacter, Serratia, and Citrobacter), Neisseria, and H. influenzae. Mutants of Enterobacter, indole-positive Proteus, Serratia, and Citrobacter, with stable derepression of the chromosomal b-lactamase, however, are resistant to these antibiotics. The third-generation cephalosporins are less active against gram-positive organisms than the first-generation cephalosporins and are inactive against enterococci, Listeria, oxacillin-resistant staphylococci, and Acinetobacter. Cefotaxime and ceftriaxone are usually active against pneumococci with intermediate susceptibility to penicillin, but strains fully resistant to penicillin are often resistant to the third generation cephalosporins as well. In the late 1990s, for example, 25% of strains of Streptococcus pneumoniae in the United States were intermediately or fully resistant to penicillin and 14% were resistant to third generation cephalosporins (84,85). Fourth-Generation Cephalosporins. Cefepime is a member of a new class of cephalosporins. It has a positively charged quaternary ammonium attached to the dihydrothiazone ring, which results in better penetration through the outer membrane of gram-negative bacteria and a lower affinity than the third-generation cephalosporins for certain chromosomal b-lactamases of gram-negative bacilli. Cefepime has similar activity to cefotaxime and ceftriaxone against pneumococci (including penicillin-intermediate strains) and oxacillin-sensitive S. aureus. Like the earlier third-generation agents, it is active against the Enterobacteriaceae, Neisseria, and H. influenzae, but has greater activity against the gram-negative enterics that have a broad-spectrum, inducible, chromosomal b-lactamase (Enterobacter, indole-positive Proteus, Citrobacter, and Serratia). The role of cefepime in therapy of infections due to stably derepressed mutants of these organisms, however, has not yet been fully defined. Cefepime is as active as ceftazidime for P. aeruginosa, and is active against some ceftazidime-resistant isolates. However, cefepime is not yet approved for the therapy of meningitis. Summary of Treatment Indications for Third- or Fourth-Generation Drugs & &

& &

&

Gram-negative meningitis caused by Enterobacteriaceae. Penicillin-resistant gonococcal infections—Ceftriaoxone is therapy of choice, which is also the recommended therapy for Lyme disease involving the CNS or joints. H. influenza that are ampicillin resistant. In renal dysfunction, later-generation cephalosporins are useful alternatives to the aminoglycosides in treating gram-negative infections, particularly, resistant to other b-lactams. Caution should be used, however, in using a latergeneration cephalosporin as a single agent for the

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&

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Part One: General Considerations

treatment of serious infections with Enterobacter, indolepositive Proteus, Serratia, and Citrobacter, because of the possibility of resistance emerging during therapy. Ceftazidime and cefepime are effective therapies for serious infections due to P. aeruginosa, when the organism is resistant to the antipseudomonal penicillins or the patient is penicillin allergic; as with the antipseudomonal penicillins, however, ceftazidime and cefepime should generally be given in combination with an aminoglycoside for treatment of serious P. aeruginosa infection. Ceftazidime is effective therapy for meningitis caused by P. aeruginosa. Later-generation cephalosporins are not particularly useful in treating infections due to gram-positive cocci (except penicillin-resistant pneumococci) or Acinetobacter. They are not currently recommended for prophylactic use in surgery.

Carbapenems Imipenem and meropenem are carbapenems that are exceedingly resistant to cleavage by most plasmid and chromosomal b-lactamase (86). They have a very broad spectrum of activity encompassing: &

&

&

Gram-positive organisms (including Enterococcus faecalis and Listeria) Gram-negative organisms (including b-lactamase– producing H. influenzae and N. gonorrhoeae, the Enterobacteriaceae, and P. aeruginosa) Anaerobes (including B. fragilis)

Neither drug is generally active against Stenotrophomonas maltophilia (which has a carbapenem-hydrolyzing chromosomal b-lactamase) Although initial isolates of P. aeruginosa are usually susceptible to the carbapenems, resistance may emerge during therapy when these drugs are used as a single agent. Evidence suggests that carbapenems do not traverse the outer membrane of P. aeruginosa through the normal porin channel used by the other b-lactams but rather through a different channel (86). Carbapenem-resistant strains of P. aeruginosa arising on therapy generally have altered permeability to these drugs and specific changes in their outer membrane proteins; such strains are generally not crossresistant to other b-lactams nor do they produce increased or novel b-lactamase activity. Imipenem is inactivated in the proximal renal tubule by the normal human enzyme dehydropeptidase I, with resultant low urinary levels of active drug and necrosis of the proximal tubule in the rabbit model. Such cleavage of imipenem is prevented by coadministration of cilastatin, a specific inhibitor of this dehydropeptidase. Imipenem–cilastatin therapy has been associated with CNS toxicity, including change in mental state, myoclonus, and, particularly, seizures (86). These effects are especially evident in patients with underlying CNS disease or impaired renal function. Imipenem should not be used for the therapy of meningitis. The dosage of imipenem administered should be carefully titrated; patients with glomerular filtration rates of less than 5 mL/min should generally not receive imipenem unless hemodialysis is ongoing or will start within 48 hours. Meropenem is another carbapenem approved for use in the United States; its spectrum of activity is quite similar to imipenem. Meropenem is stable to human renal dehydropeptidase I, and the compound is administered without

cilastatin. Meropenem appears to have a lower risk of producing seizures than imipenem–cilastatin, and it is approved for the treatment of bacterial meningitis. Ertapenem, a new carbapenem, has recently been approved by the U.S. Food and Drug Administration (FDA). This drug has a narrower spectrum of activity than imipenem or meropenem. It is active against most Enterobacteriaceae and anaerobes but less active than the other carbapenems meant for the treatment of infection caused by P. aeruginosa, Acinetobacter, and gram-positive bacteria, particularly enterococci and penicillin-resistant pneumococci. Clinical trials of this new drug have mainly been published as abstracts thus far; its major benefit over other carbapenems is that it has a long half-life and can be administered once daily. Unlike meropenem, ertapenem is not approved for the therapy of meningitis. b-Lactamase Inhibitors Clavulanate, sulbactam, and tazobactam are b-lactamase inhibitors, which have little intrinsic antibacterial activity but inhibit the activity of a number of plasmid-mediated b-lactamases (87). They generally do not inhibit chromosomally mediated b-lactamases. Combination of these agents with ampicillin, amoxicillin, ticarcillin, or piperacillin results in antibiotics with an enhanced spectrum of activity against many, but not all, organisms containing plasmid-mediated b-lactamases. In addition, these compounds inhibit the chromosomal b-lactamase of many Bacteroides species, extending the spectrum of coverage for these organisms as well. Amoxicillin–clavulanate, Unasyn, will inhibit most strains of oxacillin-sensitive S. aureus and b-lactamase–producing H. influenzae in addition to the usual organisms inhibited by amoxicillin alone. At the high drug concentrations achieved in urine, the combination is also active against certain b-lactamase–producing Enterobacteriaceae. Unasyn is a parenteral formulation that expands the spectrum of ampicillin to include most strains of S. aureus and b-lactamase–producing H. influenzae, some Enterobacteriaceae, and anaerobes (including B. fragilis). This combination has been used for prophylaxis and therapy of intra-abdominal and pelvic infections instead of cefoxitin or cefotetan. Randomized, double-blind trials showed ampicillin–sulbactam to be equivalent to cefoxitin in the prophylaxis for abdominal surgery and in the treatment of intra-abdominal and pelvic infections. It has also been used to treat patients with diabetic foot ulcers. Ticarcillin–clavulanate (Timentin) and piperacillin– tazobactam (Zosyn) expand the spectrum of the respective penicillins to include b-lactamase–producing S. aureus, H. influenzae, N. gonorrhoeae, some Enterobacteriaceae, and anaerobes (including B. fragilis) (88). These combinations are generally not effective against ticarcillin- or piperacillinresistant strains of P. aeruginosa. In addition, piperacillin– tazobactam, dosed at 3.375 g every six hours, may not be effective for the treatment of P. aeruginosa infections. Thus, the spectrum and clinical utility of these two agents are similar to ampicillin–sulbactam. Monobactams Aztreonam is a monocyclic b-lactam antibiotic with good activity against the majority of gram-negative aerobic and facultative bacteria, including the Enterobacteriaceae and P. aeruginosa. It has virtually no activity against grampositive organisms or anaerobes: the majority of strains of Acinetobacter and S. maltophilia are resistant, and resistant strains of P. aeruginosa frequently emerge during therapy with aztreonam alone. The spectrum of activity of

Chapter 6:

Surgical Infection: Principles of Management and Antibiotic Usage

aztreonam is similar to that of the aminoglycosides. However, it is less reliable therapy than aminoglycosides for the nonenteric gram-negative bacilli such as Acinetobacter, P. aeruginosa, and S. maltophilia. Data support the absence of cross allergenicity between aztreonam and other b-lactam antibiotic (86). However, patients with ceftazidime allergy may be allergic to aztreonam. The clinical situation in which aztreonam is most useful is in place of an extended spectrum penicillin or cephalosporin, in situations when these compounds are indicated but cannot be used because of allergy. Aztreonam is the only monobactam currently marketed. The dosing of these novel b-lactams and dose modifications in patients with renal dysfunction are important considerations in prescribing these drugs. A number of these drugs have unique properties compared to other b-lactams, such as inhibition of b-lactamases or inherent resistance to cleavage by b-lactamases, which extend the spectrum of the combination agents and carbapenems. The monobactams have a narrower spectrum of activity but less cross allergenicity. These agents should not be used widely, to avoid expense and the development of drug resistance. Resistance to b-Lactam Antibiotics Mechanisms of Bacterial Resistance. Three general mechanisms of bacterial resistance to antibiotics, including the b-lactams, have been well characterized: decreased penetration to the target site, alteration of the target site, and inactivation of the antibiotic by a bacterial enzyme (86,87). Decreased Penetration to the Target Site. The outer membrane of gram-negative bacilli provides an efficient barrier to the penetration of b-lactam antibiotics to their target pencillin binding proteins (PBPs) in the bacterial plasma membrane. b-lactams usually must pass through the hydrophilic porin protein channels in the outer membrane of gram-negative bacilli to reach the periplasmic space and plasma membrane. The permeability barrier of the outer membrane is a major factor in the resistance of P. aeruginosa to many b-lactam antibiotics. Alteration of the Target Site. The target sites for the b-lactams are the PBPs in the cytoplasmic membrane. Alterations in PBPs may influence their binding affinity for b-lactam antibiotics and, therefore, the sensitivity of the altered bacterial cell to inhibition by these antibiotics. Such a mechanism is responsible for penicillin resistance in pneumococci, methicillin (oxacillin) resistance in staphylococci, and for an increasing number of bacteria with intrinsic resistance to b-lactams, such as gonococci, enterococci, and H. influenzae (86). Inactivation by a Bacterial Enzyme. Production of b-lactamase is the major mechanism of resistance to the b-lactam antibiotics in clinical isolates. Such bacterial enzymes may cleave predominantly penicillins (penicillinases), cephalosporins (cephalosporinases), or both (b-lactamases). Their production may be encoded within the bacterial chromosome (and hence be characteristic of an entire species), or the genes may be acquired on a plasmid or transposon (and hence be characteristic of an individual strain rather than the species). Bacteria may synthesize the b-lactamase constitutively (as occurring in the case of many plasmid-mediated enzymes) or synthesis may be inducible in the presence of antibiotic (as occurring in the case of many chromosomal enzymes). Inducible b-lactamases may not be reliably detected by initial susceptibility testing, particularly with the newer rapid methods.

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Chromosomal b-Lactamases. Although virtually all gramnegative bacilli possess a chromosomal b-lactamase gene, certain species produce insignificant amounts of this enzyme, and their susceptibility to b-lactams is largely determined by plasmid-mediated b-lactamases and antibiotic permeability. These include E. coli, P. mirabilis, Salmonella, Shigella, and H. influenzae. K. pneumoniae produces a chromosomal b-lactamase that is primarily a penicillinase; thus, these strains are frequently susceptible to the cephalosporins. The last group of species within the Enterobacteriaceae, including Enterobacter, indole-positive Proteus, Serratia, and Citrobacter, produce an inducible chromosomal b-lactamase that may be difficult to detect on initial susceptibility testing but that can mediate resistance to all currently available b-lactams with the exception of the carbapenems. In addition to inducible production of this chromosomal enzyme, these species may give rise to regulatory mutants that are ‘‘derepressed’’ and produce high levels of this broadspectrum chromosomal enzyme constitutively. Plasmid-Mediated b-Lactamases. The most common plasmidmediated b-lactamases of gram-negative bacteria (such as TEM-1, TEM-2, and SHV-1) mediate resistance to the penicillins and first- and second-generation cephalosporins, but not cefuroxime, cephamycins, third- and fourth-generation cephalosporins, or the novel b-lactam compounds such as the carbapenems or aztreonam. More recently, extended-spectrum plasmid-mediated b-lactamases (derived from the common TEM and SHV enzymes) have arisen, which are capable of cleaving later-generation cephalosporins and aztreonam (86). Originally described in strains of Klebsiella from Europe, these b-lactamases have now been found in a variety of gramnegative bacilli (i.e., E. coli and K. pneumoniae) in several areas of the United States, and the spread of these organisms between patients in intensive care units has been documented. It has also been documented that nursing home patients may be an important reservoir for strains of Enterobacteriaceae producing extended-spectrum plasmid-mediated b-lactamases (89). Although the strains of resistant E. coli and K. pneumoniae usually differ, most harbor a common plasmid encoding the extended-spectrum b-lactamase, suggesting intragenic and intraspecies transfer of the plasmid between strains, rather than transfer of a single strain between patients. These strains are almost always resistant to ceftazidime, gentamicin, and tobramycin, and frequently are also resistant to trimethoprim–sulfamethoxazole and ciprofloxacin. These enzymes mediate high-level resistance to the third- and fourth-generation cephalosporins and aztreonam, but not to the cephamycins (cefoxitin and cefotetan) or the carbapenems. However, use of the cephamycins against strains containing these new enzymes is limited by the development of permeability mutants in the porin protein, OmpF. The b-lactamase inhibitors, clavulanate, sulbactam, and tazobactam, have generally retained the ability to inhibit these newer plasmid-mediated b-lactamases. Another plasmid-mediated b-lactamase has been described in Klebsiella, which is homologous to the chromosomal cephalosporinase of Enterobacter cloacae (89). This plasmid-mediated b-lactamase is capable of cleaving all of the currently available b-lactams (with the exception of the carbapenems), and its activity is not inhibited by clavulanate, sulbactam, or tazobactam. This plasmidmediated b-lactamase confers a broad resistance pattern similar to stably derepressed mutants of Enterobacter.

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Part One: General Considerations

Resistance to b-lactam antibiotics occurs through one or a combination of three mechanisms: production of a b-lactamase, which inactivates the b-lactam ring, alteration of the target penicillin-binding protein of the cell surface, or interference with the antibiotic’s ability to reach the protein target. The problem with resistance to b-lactam antibiotics has been partially addressed by the introduction of a number of b-lactamase inhibitors. These inhibitors are most effective against plasmid-encoded b-lactamases. Enterobacteriaceae produce chromosomal b-lactamases that are only weakly inhibited. Currently, three b-lactamase inhibitors are commercially available: clavulanate, sulbactam, and tazobactam. Although there are some minor pharmacologic differences in these compounds, therapeutic efficacy is similar. These products are only available in fixed combinations with certain b-lactam antibiotics in which the antibacterial activity of the combination is determined by the spectrum of the antibiotic.

Aminoglycosides The aminoglycosides cause a misreading of the mRNA by irreversibly binding to the 30S subunit of bacterial ribosomes (90). This causes a subsequent interference with amino acid replication. Without amino acid replication, bacteria lose their capacity to sustain or proliferate. Nine aminoglycosides (gentamicin, tobramycin, amikacin, streptomycin, neomycin, kanamycin, paromomycin, netilmicin, and spectinomycin) are approved by the FDA for clinical use in the United States. Of these, gentamicin, tobramycin, and amikacin are the most frequently prescribed, although netilmicin possesses comparable efficacy for select indications. The most common clinical application (either alone or as part of combination therapy) of the aminoglycosides is in the treatment of serious infections caused by aerobic gramnegative bacilli (86,91). In addition, selected aminoglycosides have demonstrated clinically relevant activity against protozoa (paromomycin), N. gonorrhoeae (spectinomycin), and mycobacterial infections (amikacin). The aminoglycosides have demonstrated relative stability against resistance, and the emergence of resistance during therapy (especially when used in combination with other agents) is rare. The most frequently encountered toxicity resulting from aminoglycoside administration is nephrotoxicity. Ototoxicity can also result from aminoglycoside administration, but its prevalence and clinical detection are more problematic to determine. These side effects may be reduced by identification and avoidance in patients at risk, and by appropriate dosing and monitoring of serum drug concentration. Neurotoxicity is rarely encountered, but may be severe and life threatening. Newer strategies for dosing include consolidated administration (also known as ‘‘oncedaily’’ administration) in selected patients to take advantage of the postantibiotic effect (PAE), concentration-dependent killing, and the potential for reduced toxicity. This overview will focus on the use of gentamicin, tobramycin, amikacin, and netilmicin against aerobic gram-negative bacilli and for synergy in the treatment of aerobic gram-positive cocci. Mechanism of Action The aminoglycosides are generally considered bactericidal antibiotics. They impair bacterial protein synthesis (90). These drugs work primarily by binding to the 30S ribosomal subunit, leading to misreading of the genetic code and

inhibition of translocation (90). The initial steps required for peptide synthesis are uninterrupted, such as binding of mRNA and the association of the 50S ribosomal subunit, but elongation fails to occur due to disruption of the mechanisms for ensuring translational accuracy (86,90). The ensuing antimicrobial activity is usually bactericidal against susceptible aerobic gram-negative bacilli. Aminoglycosides initially penetrate the organism by disrupting the magnesium bridges between lipopolysaccharide moieties. They are transported across the cytoplasmic membrane in an energy-dependent manner. This step can be inhibited in vitro by divalent cations, increased osmolality, acidic pH, and an anaerobic environment. The microbiologic activity of aminoglycosides is pH dependent. As a result, the antimicrobial effect may be reduced at the low pH found in lung and bronchial secretions. As an example, the MIC of aminoglycosides was increased almost fivefold at pH < 6.5 in one in vitro study. Two important pharmacodynamic properties of aminoglycosides are the PAE and concentration-dependent killing: &

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The PAE refers to the persistent suppression of bacterial growth that occurs after the drug has been removed in vitro or cleared by drug metabolism and elimination in vivo. Initially described for gram-negative bacilli, aminoglycosides also exhibit PAE against S. aureus but not against other gram-positive cocci. The duration of the PAE [approximately 3 hours (range 1–7.5 hours)] depends upon the method of evaluation and the organism studied. The duration of the PAE is reduced in the absence of polymorphonuclear leukocytes. Concentration-dependent killing refers to the ability of higher concentrations of aminoglycosides (relative to the organism’s MIC) to induce more rapid, and complete, killing of the pathogen. Aminoglycosides exhibit concentration-dependent microbiologic activity in both in vivo and in vitro models. Achieving optimal peak concentrations of aminoglycosides with standard dosing regimens can be difficult, because efforts must be made to avoid sustained elevated trough concentrations (which are believed to be important in avoiding nephrotoxicity). Relative to traditional dosing methods, the consolidated dosing approach is more likely to achieve optimal peak concentrations that result in concentrationdependent killing.

A synergistic effect has been demonstrated in vitro for selected organisms when aminoglycosides are used in combination with other antibiotics, most consistently with cell wall–active agents (e.g., b-lactam antibiotics). Spectrum of Activity Aminoglycosides exhibit potent in vitro activity against a wide range of aerobic gram-negative pathogens, including Enterobacteriaceae, Pseudomonas spp., and H. influenzae. However, in vitro activity against Burkholderia cepacia, S. maltophilia, and anaerobic bacteria is usually poor or absent. Aminoglycosides also demonstrate activity against gram-positive organisms such as methicillin-susceptible S. aureus. However, most authorities believe these drugs are not adequate therapy for serious infections caused by MRSA and S. pneumoniae (see ‘‘Treatment and prevention of methicillin-resistant S. aureus infection’’).

Chapter 6:

Surgical Infection: Principles of Management and Antibiotic Usage

Minor differences exist among the in vitro potencies of the various aminoglycosides. Gentamicin usually demonstrates superior in vitro activity to tobramycin against Serratia spp., whereas tobramycin is usually more potent than gentamicin against P. aeruginosa. Clinical Uses. The most frequent clinical use of aminoglycosides (most commonly in combination with other antibacterial agents) is empiric therapy of serious infections such as septicemia, respiratory tract infections, complicated urinary tract infections, intra-abdominal infections, and osteomyelitis caused by aerobic gram-negative bacilli. Once an organism has been identified and susceptibilities determined, aminoglycosides are usually discontinued in favor of less toxic antibiotics to complete a treatment course. Aminoglycosides are also employed frequently in combination (usually with a b-lactam antibiotic to which the organism is susceptible in vitro) for serious infections caused by Serratia spp., Pseudomonas spp., indole-positive Proteus, Citrobacter spp., Acinetobacter spp, and Enterobacter spp., due to the pathogen’s potential to exhibit inducible resistance to the b-lactam. For infections caused by Enterobacter spp., the addition of an aminoglycoside to a third-generation cephalosporin (most frequently associated with ceftazidime) has not been shown to decrease the development of resistance that the latter drug promotes. Combination therapy with gentamicin is frequently used for the treatment of invasive enterococcal infections not exhibiting high-level aminoglycoside resistance (such as bacter