Handbook of Mediators in Septic Shock [1 ed.] 9781315892023, 9781351071123, 9781351088022, 9781351096478, 9781351079570

Table of contents :

1. Toxins 2. Biogenic Amines 3. Oligo- and Polypeptides 4. Proteins 5. Fatty Acid Derivatives 6. Varia 7. Future Perspectives

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HANDBOOK MEDIATORS /„ SEPTIC SHOCK

HANDBOOK MEDIATORS * SEPTIC SHOCK Edited by

Edmund A. Neugebauer, Ph.D. Associate Professor and Head Biochemical and Experimental Division II. Department of Surgery University of Cologne Cologne, Germany

John W. Holaday, Ph.D. Professor Chief Executive Officer EntreMed, Inc. Rockville, Maryland

Press C CRC Taylor & Francis Group

CR

Boca Raton London New York

CRC Press Boca Raton Ann Arbor London Tokyo CRC Press is an imprint of the Taylor & Francis Group, an informa business

First published 1993 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 Reissued 2018 by CRC Press © 1993 by CRC Press, Inc. CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright. com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Handbook of mediators in septic shock/edited by Edmund A. Neugebauer, John W. Holaday. p. cm. Includes bibliographical references and index. ISBN 0-8493-3548-5 1.  Septic Shock—Handbooks, manuals, etc.  2.  Inflammation -Mediators—Handbooks, manuals, etc. I. Neugebauer, Edmund A. II. Holaday, John W. [DNLM:  1.  Endotoxins.  2.  Shock, Septic. QZ 140 H2355] RC182.S4H26 1993 616.9’44—dc20 DNLM/DLC for Library of Congress 

92-48305

A Library of Congress record exists under LC control number: 92048305 Publisher’s Note The publisher has gone to great lengths to ensure the quality of this reprint but points out that some imperfections in the original copies may be apparent. Disclaimer The publisher has made every effort to trace copyright holders and welcomes correspondence from those they have been unable to contact. ISBN 13: 978-1-315-89202-3 (hbk) ISBN 13: 978-1-351-07112-3 (ebk) Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

DEDICATION This handbook is dedicated to our beloved wives Marlene and Dorinda. Thank you for always being understanding and supportive.

Science is facts. Just as houses are made of stones, so is science made of facts. But a pile of stones is not a house and a collection of facts is not necessarily science.* Jules Henri Poincare 1854-1912

* We thank Professor Kirkpatrick from RWTH Aachen, Germany for bringing our attention to this appropriate citation.

PREFACE Septic shock remains as a major killer despite decades of research directed at uncovering its cause and potential cure. According to statistics reported in the U.S., at least one patient out of every two hundred hospital admissions develops septic shock; of these, 25 to 40 percent die. Despite the promise of ever more sophisticated technology, representing an exponential growth in biomedical knowledge, the clinical application of this technology has, as of yet, failed to significantly affect the morbidity and mortality of septic shock. Until recently, the term "septic shock" was used synonomously with "septicemia", "sepsis syndrome", "endotoxemia" and various other descriptive nomenclature, each endorsed by vocal advocates. In order to clarify this confusion, a concensus committee of experts recently redefined these terms as examples of a generalized "systemic inflammatory response syndrome'' (SIRS) in recognition of the often similar clinical presentation of septic shock, pancreatitis, trauma, and other generalized inflammatory disorders.1 Although the pathogenesis of septic shock is distinguished from these other syndromes by the presence of infection, with the exception of bacterial toxins, SIRS shares many, if not all, of the mediators of septic shock reviewed in this book. Septic shock resulting from bacterial infection can result from the proliferation of either Gram-positive or Gram-negative organisms; however, Gram-negative sepsis predominates. In septic shock, the spectrum of signs and symptoms are brought about by the severe disruption of normal cell, tissue, and organ function. The complexity of septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria and, in turn, from the infected host. As more mediators become progressively involved, the dyshomeostatic state resulting from septic shock may ultimately become irreversible. An obvious way of treating septic shock is to kill the offending organisms, and the prophylactic and therapeutic use of antibiotics has represented one key milestone in the management of septic shock. Paradoxically, however, the use of antibiotics to kill the offending organisms may even exacerbate the shock syndrome through the release of exotoxins and endotoxins from the dying Gram-positive and Gram-negative bacteria (respectively). Although exotoxins are byproducts of Gram-positive organisms, their pathophysiological actions may involve a sensitization to the pathogenic effects of endotoxins derived from gut translocation or concomitant infection. Chemically referred to as "lipopolysaccharides", endotoxins are known to activate the broad spectrum of mediators that contribute to septic shock. There are thousands of books and scientific reports directed at uncovering the cause and treatment of septic and endotoxic shock. Over the last century, many hundreds of mediators of septic shock have been elucidated, each resulting in the development of different potential therapeutic strategies. In reviewing this massive literature, the putative importance of individual mediators of sepsis may be well documented by extensive data or poorly supported by limited information. Nonetheless, the relative "importance" ascribed to each of these mediators based upon the number of publications does not necessarily reflect the strength of the supportive evidence. In this book, we are attempting to provide a systematic evaluation of the various putative mediators of septic shock. Instead of a series of separate chapters reviewing the creative research efforts of individual laboratories, we have asked the authors, as experts in their individual mediators, to objectively evaluate the collective literature by the use of metaanalysis. After sorting out data and references according to a series of exclusion criteria using decision trees, the authors were asked to employ the KOCH-DALE criteria to evaluate the causality of their individual mediators of septic shock.

An introductory chapter is provided by Dr. Bone and colleagues that defines septic shock and provides a brief overview of the subsequent chapters. We have divided "mediators" into seven general chapter categories, including toxins, biogenic amines, oligo- and polypeptides, proteins, fatty acid derivatives and varia. The concluding chapters will provide a collective evaluation of the individual mediators, pointing to the sufficiency of existing data or to the need for further experimentation. Through the editorial process, we have learned that the rigidity of meta-analysis and the KOCH-DALE criteria may not provide the flexibility required for evaluating the complexities of all septic shock mediators. For this reason, additional methods of objective analysis have been proposed. Finally, a heuristic look at other ways of designing experiments and interpreting data will be provided, with a brief discussion of chaos theory as it may apply to the cascade of events that characterize septic shock. It is perhaps unfortunate that the process of scientific research emphasizes new discoveries, often prematurely dismissing the old. Today, state-of-the-art research in circulatory shock emphasizes cytokines, monoclonal antibodies, eicosanoids and other "sexy" mediators. However fickle the scientists, the changing tides of scientific enthusiasm do not dictate biological relevance; the "true" relevance of mediators is dictated by biology itself. Although many promising new therapeutic strategies for the treatment of septic shock, such as the use of passive immunization against endotoxins or specific cytokines, have recently caught the attention of the biomedical community, the problems of septic shock are far from being resolved. By the time of diagnosis, the sequence of early mediator events that initiate the septic shock cascade may be well on the way. If this is the case, the proverbial "cat is out of the bag", and treatment with monoclonal antibodies as described above may be of little use. Ultimately, therapeutic strategies will reflect the evolution of mediator interactions over time, perhaps requiring different "cocktails" of drugs at different stages of septic shock. Even using this approach, with present technology it would be impossible to treat the adverse effects of all of the mediators at times when their roles become primary. There is obviously a need for evaluation of the ' 'state of the art", and this book provides an attempt objectively to evaluate the existing data and to point the way towards new research strategies to address the confounding problems of septic shock. Edmund A. Neugebauer John W. Holaday

REFERENCE 1. Members of the American College of Chest Physicians/Society of Critical Care Medicine consensus conference committee: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis, Crit. Care Med., 20, 864, 1992.

INTRODUCTION An uncontrolled, systemic inflammatory response is the underlying cause of the clinical condition known as septic shock. It is a leading cause of death in the intensive care unit and a dilemma for the physicians whose patients' lives are in danger. This inflammatory response is effected by a complicated system of interacting, endogenous mediators. The true number of these mediators is not known; more are being described each year. The tremendous growth of knowledge in this relatively new field has left a gap where detailed textbooks covering the subject should be; the need for a work such as this is quite apparent. I feel that this publication will become a much-used reference on the subject of mediators. The chapters in this book have been written by experts and well-known authors, all of whom have conducted original investigations in the field. In order to summarize the large quantity of data available on mediators, these authors were asked to apply the method of meta-analysis to relevant, published data on the mediators with which they have become familiar. The technique of meta-analysis, first presented and published in 1987 at the "First International Conference on Shock" in Montreal (Canada) by Drs. Edmund A. Neugebauer and Wilfried Lorenz, consists of a systematic and quantitative pooling of all relevant data from studies in the field to construct a consensus picture. Meta-analysis utilizes a "decision tree" — a series of yes-no, decision-making questions that branch from a central course leading toward the final decision of relevancy. For instance, the results of a study may be excluded from further consideration because an animal model inappropriate to the clinical setting was used. When data from a study are found to be relevant through the use of the decision tree, it is used in an analysis that may ultimately suggest cause and effect relationship. The questions and criteria that form the branches of the decision tree, termed test nodes, are determined before the start of the meta-analysis. The first mediator analyzed with this methodology was histamine. The original paper, published in a 1987 issue of Theoretical Surgery by Neugebauer et al., served as a forerunner to this text on mediators. This new approach also started a controversial discussion on the limitations and relevance of applying meta-analysis to the question of mediator involvement in sepsis. A discussion forum was therefore initiated by Drs. Edmund A. Neugebauer and Wilfried Lorenz to examine the effects of histamine in sepsis from the point of view of specialists in the fields of surgery, anaesthesiology, physiology, pharmacology, statistics, biostatistics, and philosophy. This highly interesting discussion is included as an appendix to this book. The relevance and limitations of applying meta-analysis to the question of the various mediators involved in sepsis is discussed. Also discussed are different methods of causality assessment: reasons why the KOCH-DALE methodology of causality alone is inappropriate for this subject, in which multiple mediators and effects are involved in causing sepsis; in addition, promising and alternative approaches are outlined. The first effector analyzed in terms of meta-analysis is endotoxin, appropriate since it is probably involved in some of the first reactions that take place during the cascade of events that culminate in Gram-negative sepsis and its sequelae. This exogenous agent, which has a remarkably complicated and somewhat variable structure, is found in the cell walls of Gram-negative bacteria. The authors, Drs. Carolyn S. Cody and David L. Dunn, describe the characteristics and effects of endotoxin, then report on various methods and problems that may be encountered in raising antibodies against portions of the endotoxin moiety, and on their use for the relief of sepsis. Their meta-analysis of the significance of endotoxin in sepsis and on the protective capacity of these antibodies, at the end of the chapter, is accompanied by the test node questions used in the analysis. A summary of their accumulated data is presented in a series of tables.

The next chapter, by Dr. Sucharit Bhakdi, contains an analysis of the role of exotoxin in sepsis. Exotoxins released by Gram-positive bacteria have been associated with toxic shock syndrome, a response that is similar to the systemic inflammatory response seen in Gram-negative sepsis. The application of meta-analysis to the effects of exotoxin is difficult, since only a small amount of data exists on the subject. Another potential mediator, serotonin (5-HT), is the subject of the chapter, written by Drs. James R. Parratt and Brian L. Furman. They outline the known distribution, action, and receptor locations for 5-HT. They include a discussion on the actions of 5-HT that are similar to events seen in sepsis especially those that affect the circulatory system, including the endothelium, platelet function, and cellular metabolism. This brief review includes a discussion on the use of receptor antagonists to relieve symptoms of sepsis induced in various animals models. A full meta-analysis were not performed, due to a lack of data on the subject. A meta-analysis of the role of catecholamines in sepsis is included in the chapter by Drs. Reuven Rabinovici and Giora Feuerstein. Their methods section details the test node questions used in the analysis. They found that most of the studies available were not suitable for use in determining the effects of catecholamines in sepsis. Their conclusions were limited to a confirmation of a catecholaminergic causative factor in sepsis models using the rat. Obviously, more research is needed on this mediator, particularly with human subjects and, too, they noted that many of the studies were not applicable due to a lack of suitable controls. Doctor Arthur Revhaug's chapter on the effects of vasoactive intestinal polypeptide (VIP) in sepsis follows. Their meta-analysis included seven original investigations published since 1975. Although acknowledging that the data was sparse on this little-studied peptide, they concluded that the suggestion of a causal relationship between VIP and sepsis does exist, largely due to its effects on hemodynamics. They also cited the problem of specificity in quantitative RIA testing of serum VIP. The chapter on the role of kallikrein-kinin system in sepsis was written by Gerd Bonner. A thorough review of this very complicated subject precedes the conclusion that the system is active in sepsis. This chapter is of value simply as an excellent review of the subject. Due to the large number of studies reviewed in this analysis, the reliability of the conclusion is likely to be quite high. A review of the literature and a meta-analysis of data compiled on the role of vasopressin in sepsis was performed by Drs. Michael F. Wilson and Daniel J. Brackett. Their discussion ranged from studies of the possible effects of vasopressin that could mimic the symptoms of shock, to the effects of antagonists in preventing septic disease. In their summary, the authors state that altered vasopressin levels can be associated with the presence of endotoxemia, but the data are not sufficient to determine causality. Dr. Nelson Gurll wrote the chapter on the role of the endogenous opiates in sepsis. His fine analysis included the results of fifty-two studies. The conclusion indicates that endorphins play a role in septic shock, although with the caveat that most of the studies reviewed utilized the opiate receptor blocker, naloxone. The chapter written by Drs. Anders Bengtsson, Heinz Redl, Mats Heideman, and Giinther Schlag examined the effects of complement in sepsis. The complement system, a known mediator of inflammatory responses, can be activated by a variety of causes, including various types of bacteria, tissue injury, and the presence of immune complexes. Thus, complement is a likely candidate as a powerful mediator in clinical sepsis. Because good techniques have been developed to measure serum levels of complement, and because there have been many clinical studies of complement in sepsis, it seems likely that meta-analysis should yield positive results. However, in concluding that complement is activated in sepsis, the authors declined to estimate its relative importance. This was due to the development of new "truly specific" immunoassays, which may put previously accepted studies into

doubt, the lack of antagonists to complement, and the use of animals (particularly rodents) which are complement deficient, rather than primates, which have complement systems similar to that found in man. Hemodynamic changes are important in sepsis; both vasodilation and vasoconstriction occur at various times during the course of sepsis. The next chapter, written by Drs. Michel Burnier, Ernst Biirki, Bernard Waeber, and Hans-Rudolph Brunner, includes a review of the renin-angiotensin system, which can cause a potent vasoconstriction. Their application of meta-analysis to this question is hampered by the failure of studies to pass questions on the decision tree. However, they note that, using the KOCH-DALE criteria that blockade of a mediators effects should alter the course of disease, the renin-angiotensin system does have effects in sepsis. The chapter written by Doctors Ernst Biirki, Michel Burnier, Dominique Evequoz, Bernard Waeber, and Hans-Rudolph Brunner deals with the role of neuropeptide Y in sepsis. After a thorough review on the effects of endotoxemia on serum levels of neuropeptide Y, he discusses the effect of exogenous neuropeptide Y on septic shock-like conditions. However, the authors could not conclude that the peptide plays a definite role in sepsis: the cited studies contained much conflicting information. The role of the interleukins in sepsis has long been assumed to be an important one. This group of mediators is near the top of the list when sepsis is discussed, and Dr. Judy Spitzer does the subject justice in this chapter. However, the difficulty in measuring these molecules was strongly implicated in the author's unwillingness to state the causal nature of these cytokines. Also cited was the significance of interactions among the presumed mediators of sepsis: while not necessarily causative, a mediator may be contributory to sepsis. The involvement of cachectin, or tumor necrosis factor (TNF), in sepsis is the subject of the next chapter, written by Drs. Kevin J. Tracey and Anthony Cerami. TNF has a relatively long-standing reputation as a primary mediator in the development of septic shock, and these author's review of the literature supports that premise. In covering this potentially deleterious cytokine, the authors discuss in detail its regulation, binding, and actions, both good and bad, along with the protective effects of anti-TNF antibodies. An analysis of the role of fibronectin (FN) in sepsis is found in the chapter written by Drs. Manfred Nagelschmidt and Andreas Rink. From their review of the role played by FN, it is clear that serum levels of the protein decrease during sepsis. Further, preliminary results indicate that exogenous FN is beneficial to patients with sepsis. However, the authors felt that the studies were compromised by poor design. In sum, it was concluded that FN has no role as mediator in sepsis, but the changes that occur in its concentration during sepsis indicate that it may be protective in sepsis, rather than harmful; more work needs to be done with this peptide. The release of proteinases by polymorphonuclear leukocytes and macrophages has been shown to occur in the systemic inflammatory response; as such, they may be considered as mediators of the damage caused by sepsis. However, they can have another role in sepsis, activating true mediators through proteolysis of precursor molecules. The chapter by Drs. Marianne Jochum, Werner Machleidt, and Hans Fritz examines these issues. A rather complicated picture is drawn, in which the specific inhibitors of a certain proteinase may be destroyed by yet another proteinase. Despite the complexity, this review also explores the possibility that exogenous proteinase inhibitors can be used therapeutically. Elastase is a proteinase of note; it has the capability of damaging vascular tissue. Circulating levels of elastase (and other proteases) may have a predictive value in determining patient outcome in sepsis. These authors summarized their review by stating that proteinases have a "prominent role" in acute inflammation, based on published research. However, as with many

other authors in this book, a strict use of the decision tree would have resulted in very little data to analyze. Doctors Markus Biichler, Waldemar Uhl, and Timo J. Nevalainen next present a brief treatise on the effects of phospholipase A2 in sepsis. This enzyme is one of several that are capable of hydrolyzing the phospholipids found in the cell membrane, and are thus an important part of the digestive process. However, it may also be a part of the mediator cascade in sepsis since it also liberates arachidonic acid from membranes. This catabolite is a building block for several potent mediators in sepsis. It appears that certain other mediators in sepsis, such as TNF and the interleukins, can affect the production of phospholipase A2. The authors conclude that this mediator is involved in certain inflammatory diseases, and has an important influence on the septic condition. Endothelium-derived relaxing factor and endothelin are the subject of a chapter by Drs. James R. Parratt, James C. Stoclet, and Brian L. Furman. These highly vasoactive peptides are released by endothelial cells, and are believed to be important in sepsis, since elements of both vasoconstriction and vasodilation may be observed in sepsis. The authors conclude by saying that these factors may potentially be involved in sepsis, but that the evidence is far from conclusive at this point. The enzymes involved in the cyclo-oxygenase pathway, the subject of the chapter by Drs. T. Robin Wagner, James A. Cook, and Perry V. Halushka, are important in producing prostaglandins and thromboxane from arachidonic acid, a product released during metabolic break down of the cell membrane. These products, and arachidonic acid itself, are probable mediators that have been associated with inflammation. This includes their effects on smooth muscle, blood cell aggregation, and vascular resistance. The activity of these enzymes depends on the level of endotoxin present and any tolerance to endotoxin that may have been acquired. Analysis of the significance of these mediators, termed eicosanoids, in sepsis is made more difficult by their very brief half-life in circulation. The metabolism and production of these arachidonic acid derivatives are influenced by the cytokines in complicated ways; the authors do a thorough job of summarizing a very complicated subject, one which will require much research to fully understand. The leukotrienes have been much cited in the literature on sepsis, and are the subject of the chapter, written by Drs. Edward F. Smith, III and Claudia R. Turner. These too, are products of arachidonic acid breakdown, through an enzyme system termed the 5-lipoxygenase pathway. These mediators are capable of eliciting responses characteristic of septic shock, particularly hemodynamic changes, fluid compartment distribution perturbations, and changes in lung function. Again, accurate measure of leukotriene levels in the blood stream, hence analysis of their role in sepsis, is made more difficult because of their very short half life. Work with receptor antagonists has, however, helped to elucidate the role of leukotrienes in sepsis. In their meta-analysis, the authors found that most of the studies performed to date do not conform to the strict criteria of the decision tree; it was thus inconclusive with respect to the significance of leukotrienes in clinical sepsis. The chapter concerned with the effects of platelet-activating factor (PAF) was written by Drs. David Hosford, Matyas Koltai, Monique Paubert Braquet, and Pierre Braquet. This chapter includes a brief critique of the meta-analysis method, followed by an analysis of PAF's role in sepsis using a "probability matrix" in place of the meta-analysis. This method allows more flexibility in the inclusion of data, since a numerical scale is used instead of the yes/no answers demanded by the decision tree. There is evidence that PAF facilitates the release of TNF and interleukins from macrophages, and superoxide release from polymorphonuclear leukocytes, both events that probably occur in sepsis. PAF is also believed to have a direct effect on capillary permeability and coagulation. These effects must often be prompted by the synergistic effects of other suspected mediators. The condition of sepsis has been associated with increased serum levels of PAF, and changes in the number of PAF

receptors on platelets. There is considerable evidence that PAF antagonists ameliorate many of the symptoms of sepsis. These facts allow the authors to conclude that there is a high likelihood that PAF is involved in sepsis. The release of oxygen free radicals during the inflammatory response may also be important as a cause of damage in sepsis. These reactive molecules were examined in the next chapter by Drs. Bengt Gerdin and Ulf Haglund. They discuss the location, production, and effects of radicals in the body. Due to their transient nature, it is impossible to measure their concentrations in the body; even documenting their presence is very difficult. Estimates can be obtained, however, through measures of the weak chemiluminescent emission that results from radical formation, through observation of the "electron spin trapping" characteristics of the tissue, or through the amount of damage avoided by the presence of substances that reduce the damaging effects of radicals. The authors conclude that few of the studies would pass the criteria set down by the decision tree, and that more research is necessary before the clinical use of various agents that decrease the damage caused by free radicals can be instituted. Calcium is found in relatively high concentrations throughout the body and is responsible for mediating numerous events that range from photoreceptor response to muscle contraction. However, calcium may also be responsible for cell damage during sepsis through its activation of intracellular degradative enzymes. The possibility that calcium may mediate sepsis is analyzed in a chapter by Drs. Gary P. Zaloga and Diana Malcolm. In their summary, they state that sepsis alters intra- and extracellular calcium levels and that such increased levels of calcium can exacerbate the damage in animal models of sepsis. However, in clinical sepsis, there is a deficit of data on the subject which prohibits the use of agents that could therapeutically modify serum calcium levels. In the last chapter to discuss specific mediators, Dr. Lerner B. Hinshaw discusses the potential use of glucocorticoids as an adjunctive therapy in the treatment of septic shock. Although animal studies had shown that this may be beneficial, it has not been confirmed in humans. The theoretical basis for the use of corticosteroids lies in their ability to powerfully "dampen" the production, release, and effect of various mediators involved in sepsis, particularly TNF. However, in vitro studies and animal research have indicated that it may be necessary to apply the steroid before the genetic induction of these mediators take place. This would make appropriate application of these agents in therapy very difficult. This book is a superb summary of the information available on the various molecular species that mediate the clinical condition of sepsis. The editors, Drs. Edmund A. Neugebauer and John W. Holaday, have done an outstanding job in putting this text together. This text is singular in its field and will doubtless remain as a standard for years to come. Roger C. Bone, M.D. Rush Medical College Vice President for Medical Affairs The Ralph C. Brown, M.D. Professor of Internal Medicine Chief, Pulmonary Disease Rush-Presbyterian-St. Luke's Medical Center Chicago, Illinois

THE EDITORS Edmund, Alwin, Martin Neugebauer, Ph.D., is Associate Professor of Theoretical Surgery and Head of the Biochemical and Experimental Division, II. Department of Surgery, University of Cologne, Germany. Dr. Neugebauer obtained his basic training first in chemistry at the Max-Planck-Institute for Experimental Medicine in Gottingen and received a first degree as Chemical Engineer in 1974 at the University of Aachen. He obtained his Ph.D. degree in Chemistry at the University of Marburg in June 1982. While doing postdoctoral work at the University of Marburg at the Department of Theoretical Surgery, he studied medicine and was appointed Assistant Professor of Theoretical Surgery in 1988 and elected as Deputy Chief of the Department of Theoretical Surgery at the Philipps-University of Marburg. It was in March 1989 that he assumed his present position. Dr. Neugebauer is a member of ten scientific societies: The Shock Society, The European Shock Society, The European Society for Surgical Research, The European Society for Critical Care, The European Histamine Research Society (EHRS), The German Surgical Society — Section Experimental Surgery, The Surgical Society for Clinical Trials (CAS) of the German Surgical Society, The Austrian Surgical Society, The Society for Intensive and Emergency Care (CAIN), The European Society for Intensive Care Medicine, The European Association for Endoscopic Surgery and other Interventional Techniques (EAES), Society of Critical Care Medicine (SCCM), and The Surgical Infection Society Europe (SIS-E). Dr. Neugebauer is also treasurer of the EHRS and Vice President of the Surgical Society for Clinical Trials of the German Surgical Society and member of the working group Surgical Forum of the Section of Experimental Surgery of the German Surgical Society. In 1992 he received the award for special achievements in surgical intensive care from the German Surgical Society. Dr. Neugebauer is the author of more than 140 publications and has been the author and co-author of 26 book chapters. He is on the editorial board of Agents & Actions and Unfallchirurg as well as guest editor of Theoretical Surgery and Clinical Investigator. His current major research interests relate to pathomechanisms of shock, trauma, and multiple organ failure as well as technology assessment of surgical procedures, including quality of life and acute pain research. John W. Holaday, Ph.D., is the President and Chief Executive Officer of EntreMed, Inc., a biopharmaceutical company developing vaccines and immunological products located in Rockville, Maryland. He also serves as an Associate Professor of Medicine at The Johns Hopkins University School of Medicine, Baltimore, Maryland and as President of the Shock Society, an organization of clinical and preclinical researchers dedicated to understanding the causes and treatments of circulatory shock. Dr. Holaday obtained his training at the University of Alabama, receiving the B.S. degree, with a double major in Biology and Chemistry, in 1966, and an M.S. degree in Molecular Biology in 1969. After serving four years as a Captain in the Medical Service Corps at the Walter Reed Army Institute of Research, Washington, D.C., he obtained a Ph.D. degree (with honors) in Pharmacology from the University of California, San Francisco, School of Medicine in 1977. While on active duty in the U.S. Army Reserves, Dr. Holaday served as Chief Biochemist, Department of Psychiatry (1968-1972), and as Senior Research Neuropharmacologist, Department of Medical Neurosciences, Division of Neuropsychiatry (1976-1980), Walter Reed Army Institute of Research where he served as Chief (1980-1989). His academic

positions include an appointment as Adjunct Professor of Pharmacology and Psychiatry at the Uniformed Services University School of Medicine, Bethesda, MD (1981 to the present) and as the Clinical Assistant Professor of Surgery at the University of Connecticut Health Center, Farmington, Connecticut (1991 to the present). He is the co-founder of publicly and privately owned pharmaceutical corporations, including Medicis Pharmaceutical Corporation, New York (1988-1992) and EntreMed, Incorporated, Rockville, Maryland (1992 to the present). Dr. Holaday has been a member of the Shock Society since 1978, where he has served as Treasurer, Secretary, Program Chair and President. He is also a member of the American Society for Pharmacology and Experimental Therapeutics, the Society for Critical Care Medicine (program committee; Fellow, 1992), The American College of Neuropsychopharmacology (Fellow, 1989), International Narcotics Research Conference (program chair, 1990), Society for Neuroscience, Neurotrauma Society, N.Y. Academy of Sciences, Sigma Xi and the American Association for the Advancement of Science. He had received numerous honors and awards, including the Dean Calvert Award and the U.S. Army Research and Development Achievement Award. Dr. Holaday has edited or co-edited five books and has authored more than 200 papers and book chapters and 300 scientific abstracts. He serves as Associate Editor, Circulatory Shock, and is on the editorial board of six additional journals. He continues to serve on peer-review committees evaluating grant applications for the NIH and the NIDA. He holds over 20 U.S. and foreign patents, and his research interests include the definition of mediators and mechanisms in circulatory shock and psychoneuroimmunology.

CONTRIBUTORS Anders Bengtsson, M.D., Ph.D. Department of Anesthesiology Sahlgren's Hospital Gothenburg University Gothenburg, Sweden

Markus Biichler, M.D. Associate Professor Department of General Surgery University of Ulm — Safranberg Ulm, Germany

Sucharit Bhakdi, M.D. Professor Medical Microbiology Johannes-Gutenberg-University Mainz Mainz, Germany

Ernst Burki, Ph.D. Division D'Hypertension Centre Hospitalier Universitaire Vaudois Lausanne, Switzerland

Gerd Bonner, M.D. Associate Professor Department of Medicine University of Cologne Cologne, Germany

Michel Burnier, M.D. Policlinique Medicale Universitaire Lausanne, Switzerland

Roger C. Bone, M.D. Dean, Rush Medical College Vice President for Medical Affairs The Ralph C. Brown, M.D. Professor of Internal Medicine Chief, Pulmonary Disease Rush-Presbyterian-St. Luke's Medical Center Chicago, Illinois Daniel J. Brackett, M.D. Assistant Professor Departments of Surgery and Anesthesiology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma Pierre Braquet, Ph.D. Professor Institut Henri Beaufour Le Plessis Robinson, France Hans-Rudolph Brunner, M.D. Professor Department of Medicine CHUV Lausanne, Switzerland

Daniel B. Carr, M.D. Associate Professor Harvard University Departments of Anesthesia Massachusetts General Hospital Boston, Massachusetts Anthony Cerami, Ph.D. Professor Picower Institute for Medical Research Manhasset, New York Carolyn S. Cody, M.D. Department of Surgery Medical School University of Minnesota Minneapolis, Minnesota James A. Cook, Ph.D. Professor of Physiology Department of Physiology Medical University of South Carolina Charleston, South Carolina David L. Dunn, M.D., Ph.D. Associate Professor of Surgery Head, Surgical Infectious Diseases Department of Surgery Medical School University of Minnesota Minneapolis, Minnesota

Dominique Evequoz Policlinique Medicale Universitaire Lausanne, Switzerland Giora Z. Feuerstein, M.Sc., M.D. Cardiovascular Pharmacology SmithKline Beecham Pharmaceuticals King of Prussia, Pennsylvania Hans Fritz, Ph.D. Professor Department of Clinical Chemistry and Clinical Biochemistry Surgical Clinic City University of Munich Munich, Germany Brian L. Furman, Ph.D. Department of Physiology and Pharmacology University of Strathclyde Royal College Glasgow, United Kingdom Bengt Gerdin, M.D., Ph.D. Associate Professor Department of Surgery University Hospital Uppsala, Sweden Nelson J. Gurll, M.D. Professor Department of Surgery The University of Iowa Hospitals and Clinics Iowa City, Iowa Ulf Haglund, M.D., Ph.D. Professor Department of Surgery Uppsala University Hospital Uppsala, Sweden Perry V. Halushka, M.D., Ph.D. Professor and Director Clinical Pharmacology Department of Pharmacology Medical University of South Carolina Charleston, South Carolina

Matz Heideman, M.D. Department of Surgery Boras Hospital Boras, Sweden Lerner B. Hinshaw, Ph.D. Professor George Lynn Cross Research Professor Department of Molecular Toxicology Oklahoma Medical Research Foundation University of Oklahoma Oklahoma City, Oklahoma John W. Holaday, Ph.D. Professor and Chief Executive Officer EntreMed, Inc. Rockville, Maryland David J. Hosford, Ph.D., M.I. Biol., C. Biol. Director of Communications Institut Henri Beaufour Le Plessis Robinson, France Marianne Jochum, Ph.D. Professor Department of Clinical Chemistry and Clinical Biochemistry Surgical Clinic City University of Munich Miinchen, Germany Hans-Jorg Klotter, M.D. Surgical Clinic Philipps-University Marburg Marburg, Germany Matyas Koltai, Ph.D. Institut Henri Beaufour Le Plessis Robinson, France Helmut Lill, M.D. Surgical Clinic Philipps-University Marburg Marburg, Germany Wilfried Lorenz, M.D. Professor Institute of Theoretical Surgery Marburg, Germany

Werner Machleidt, M.D. Professor Institute of Physiological Chemistry, Physical Biochemistry, and Cell Biology University of Munich Munich, Germany Diana Malcolm, Ph.D. Associate Professor Uniformed Services University of the Health Sciences F. Edward Hebert School of Medicine Bethesda, Maryland Manfred Nagelschmidt, Ph.D. Biochemical and Experimental Division II. Department of Surgery University of Cologne Cologne, Germany Edmund A. Neugebauer, Ph.D. Associate Professor and Head Biochemical and Experimental Division II. Department of Surgery University of Cologne Cologne, Germany Timo J. Nevalainen, M.D. Department of Pathology University of Turku Turku, Finland James R. Parratt, Ph.D., D.Sc., M.Dhc., M.R.C. Path. Professor, Chair of Cardiovascular Pharmacology Department of Physiology and Pharmacology University of Strathclyde Glasgow, United Kingdom

Heinz Redl, Ph.D. Associate Professor Ludwig-Boltzmann-Institute for Experimental and Clinical Traumatology Wien, Austria Arthur Revhaug, M.D., Ph.D. Professor Department of Surgery University Hospital of Tromso Tromso, Norway Andreas Rink, M.D. Biochemical and Experimental Division II. Department of Surgery University of Cologne Cologne, Germany Dieter Rixen, M.D. Surgical Clinic Merheim Department of Surgery University of Cologne Cologne, Germany Gunther Schlag, M.D. Professor and Chair Ludwig-Boltzmann-Institut for Experimental and Clinical Traumatology Wien, Austria Helmut Sitter, Ph.D. Institute of Theoretical Surgery Philipps-University Marburg Marburg, Germany Edward F. Smith, III, Ph.D. Mallinckrodt Medical, Inc. St. Louis, Missouri

Monique Paubert-Braquet Bio-Inova Plaisir, France

Judy A. Spitzer, Ph.D. Professor of Physiology and Medicine Louisiana State University Medical Center New Orleans, Louisiana

Reuven Rabinovici, M.D. Trauma Division Department of Surgery Jefferson Medical School Philadelphia, Pennsylvania

James C. Stoclet, Ph.D. Laboratoire de Pharmacologie Universite Louis Pasteur Strasbourg, France

Kevin J. Tracey, M.D. Division of Neurosurgery North Shore University Hospital Cornell University Medical Center Manhasset, New York Claudia R. Turner, Ph.D. Senior Research Scientist Pfizer Central Research Groton, Connecticut Waldemar Uhl, M.D. Department of General Surgery University of Ulm Ulm, Germany

Bernard Waeber, M.D. Division D' Hypertension Centre Hospitalier Universitaire Vaudois Lausanne, Switzerland T. Robin Wagner, D.V.M. Department of Physiology Medical University of South Carolina Charleston, South Carolina Michael F. Wilson, M.D. Professor Department of Medicine/Cardiology Millard Fillmore Hospitals State University of New York at Buffalo Buffalo, New York

Gary Paul Zaloga, M.A., M.D. Professor of Anesthesia and Medicine Department of Anesthesia Bowman Gray School of Medicine Winston Salem, North Carolina

ACKNOWLEDGMENTS As editors, we owe a special debt of thanks to the authors of this handbook. They have given us extra effort in sharing their expertise in this novel format, despite the extreme limitations of their professional time, our demands for revisions, and the demands of the CRC Press for timely submissions. We are also indebted to our many colleagues and collaborators whose tireless efforts have allowed us to complete this handbook. We especially want to acknowledge the grace, skill, and decision of Mrs. Karin Nasskau. In addition to managing the preparations of the manuscripts, dealing with many authors around the globe, and responding to our editorial demands, Mrs. Nasskau diplomatically dealt with authors who were late with their submissions. She was primarily responsible for the final organization of the chapters, and this handbook would not have been possible without her outstanding assistance. We would also like to thank Mrs. Vera Sorg for her tireless work, good spirit, and her dedication to the completion of this handbook.

TABLE OF CONTENTS PART I: TOXINS 1.

Endotoxins in Septic Shock C. S. Cody and D. L. Dunn

2.

Possible Relevance of Bacterial Exotoxins in the Pathogenesis of Septic Shock S. Bhakdi

1

39

PART II: BIOGENIC AMINES 3.

Histamine in Septic/Endotoxic Shock E. Neugebauer, D. Rixen, and W. Lorenz

51

4.

5-Hydroxytryptamine as a Chemical Mediator of Shock J. R. Parratt and B. L. Furman

127

5.

Catecholamines in Septic Shock: A Meta-Analysis Evaluation R. Rabinovici and G. Feuerstein

141

PART III: OLIGO- AND POLYPEPTIDES 6.

Vasoactive Intestinal Polypeptide (VIP) in Septic Shock A. Revhaug

159

7.

Kallikrein-Kinin Systems in Shock G. Bonner

167

8.

Vasopressin as a Potential Mediator during Sepsis and Endotoxemia M. F. Wilson and D. J. Bracket

193

9.

Endogenous Opiates in Septic/Endotoxic Shock N. Gurll

205

10.

Complement in Septic Shock A. Bengtsson, H. Redl, M. Heideman, and G. Schlag

231

11.

Mediators in Septic Shock: The Renin-Angiotensin System M. Burnier, E. Biirki, B. Waeber, and H. R. Brunner

259

12.

Role of Neuropeptide Y in Endotoxemia E. Biirki, M. Burnier, D. Evequoz, B. Waeber, and H. R. Brunner

271

13.

Interleukins in Sepsis J. A. Spitzer

279

14.

Cachetin/Tumor Necrosis Factor in Septic/Endotoxic Shock K. J. Tracey and A. Cerami

291

PART IV: PROTEINS 15.

Fibronectin and Its Possible Role as a Mediator in Septic Shock M. Nagelschmidt and A. Rink

16.

Phagocyte Proteinases in Multiple Trauma and Sepsis: Pathomechanisms and Related Therapeutic Approaches M. Jochum, W. Machleidt, and H. Fritz

17.

Phospholipase A2 M. Biichler, W. Uhl, and T. J. Nevalainen

18.

Substances Mainly Derived from Vascular Endothelium (EndotheliumDerived Relaxing Factor, or Nitric Oxide, and Endothelin) as Chemical Mediators in Sepsis and Endotoxemia J. R. Parratt, J.-C. Stoclet, and B. L. Furman

309

335 363

381

PART V: FATTY ACID DERIVATIVES 19.

Cyclo-Oxygenase Products in Septic and Endotoxic Shock T. R. Wagner, P. V. Halushka, and J. A. Cook

395

20.

Leukotrienes in Endotoxic and Septic Shock E. F. Smith III and C. R. Turner

419

21.

Analysis of Platelet-Activating Factor in Endotoxic Shock and Sepsis Using a Probability Matrix: A Critique of Meta-Analysis D. Hosford, M. Koltai, M. Paubert-Braquet, and P. Braquet

439

PART VI: VARIA 22.

Possible Involvement of Oxygen Free Radicals (OFR) in Shock and Shock-Related States B. Gerdin and U. Haglund

457

23.

Calcium as a Mediator in Septic Shock G. P. Zaloga and D. Malcolm

475

24.

Cortisol in Septic Shock L. B. Hinshaw

487

PART VII: FUTURE PERSPECTIVES 25.

Models for Causality Assessment H. Sitter, W. Lorenz, H. J. Klotter, and H. Lill

499

26.

Meta2: An Analysis of Meta-Analyses of Mediators in Septic Shock J. W. Holaday, E. Neugebauer, and D. B. Carr

523

Appendix (Discussion Forum)

535

Index

563

Part I. Toxins

Chapter 1

ENDOTOXINS IN SEPTIC SHOCK Carolyn S. Cody and David L. Dunn TABLE OF CONTENTS I.

Introduction

2

II.

Physicochemical Characteristics of Endotoxins A. Introduction B. Structure of Endotoxin 1. Effects of Extraction Methods 2. Polysaccharide Regions 3. Lipid A

4 4 4 4 4 5

III.

Biologic Effects of Endotoxins A. Introduction B. Cytokine Response 1. Introduction 2. TNF-ct 3. Interleukins C. Effects on Organ Systems 1. Cardiovascular Effects 2. Pulmonary Effects 3. Gastrointestinal Effects

6 6 6 6 7 9 10 10 10 11

IV.

Antibodies Directed Against Endotoxins A. Introduction B. Polyclonal Antibodies 1. Serotype-Specific Antibodies 2. Cross-Reactive Antibodies a. Antibodies Directed Against Common GramNegative Bacterial Antigens b. Antibodies Directed Against Core LPS c. Antibodies Directed Against Lipid A

12 12 12 12 13

C.

21 21 21 21 23 23

D.

Monoclonal Antibodies 1. Serotype-Specific Antibodies 2. Cross-Reactive Antibodies a. In Vitro Evaluation of Cross-Reactivity b. Protection Studies in Animal Models c. Protection Studies in Human Subjects Meta-Analysis of the Protective Capacity of Antiendotoxin Antibodies 1. Decision Tree: Test Nodes a. Is the Study Design Appropriate? b. Is the Shock Model Clinically Relevant?

0-8493-3548-5/93/$0.00 + $.50 C 1993 by CRC Press, Inc.

13 13 20

24 24 24 24

1

2

Handbook of Mediators in Septic Shock

2.

V.

c. Are Other Causes of Shock Excluded? d. Are Antibody Controls Appropriate? e. Is Antibody Dosage Appropriate? f. Is Response Caused by Antibody Itself? Decision Tree 2: Test Nodes a. Has the Antibody Demonstrated Protective Capacity? b. Are Monoclonal or Polyclonal Antibodies more Protective? c. Are Serotype-Specific Antibodies more Protective than Anti-Deep-Core/Lipid A Antibodies? d. Does Antibody Class or Subclass Affect Protective Capacity? e. Does Whole Cell or Endotoxin Immunization Produce more Protective Antibodies? f. Does Antibody Reactivity In Vitro Correlate with Protective Capacity? g. Are Human Antibodies more Protective than Those Made in Other Species? h. Do Protective Antiendotoxin Antibodies Affect the Activity of Second Mediators?

Summary

25 25 25 25 28 28 28 28 29 30 30 30 30 30

Acknowledgments

31

References

31

I. INTRODUCTION Despite antibiotic therapy, aggressive hemodynamic monitoring, fluid resuscitation, and metabolic support, Gram-negative bacterial sepsis remains a lethal disease in normal and immunocompromised patients. A large body of data has accumulated, providing evidence that the mechanisms which underlie the lethality of this disease are derived from the complex interactions of the host with both the intact organism and Gram-negative bacterial endotoxins. Nearly 100 years ago, Richard Pfeiffer, working with lysates of heat-killed Vibrio cholerae, found that exposure of animals to these lysates resulted in shock and death.1 He termed the heat-stable component of these cultures endotoxin, and distinguished its effects from those of bacterial exotoxins. Bacterial endotoxins were initially defined by virtue of their pyrogenicity, and were subsequently found to be: (1) unique components of Gram-negative bacteria, (2) ubiquitous among Gram-negative organisms, and (3) localized to the Gramnegative bacterial cell wall. 2 Extraction procedures first developed by Boivin and Mesrobeanu

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3

permitted the characterization of endotoxins as lipopolysaccharide (LPS)-protein-phospholipid complexes. Subsequent preparation of protein-free LPS by Westphal and others, identified LPS as the active component of endotoxin.2 4 Further chemical studies demonstrated that LPS consisted of a nontoxic polysaccharide portion, some components of which were shared among bacterial genera, and a toxic, highly conserved lipid moiety (lipid A), which has been associated with the majority of the deleterious effects of endotoxin. LPS exerts diverse physiologic, biochemical, and immunologic effects that act in concert upon host tissues to produce what has been characterized as the "septic state or response" or "sepsis". Shwartzman documented the localized tissue necrosis that occurs after initial local injection of endotoxin is followed by systemic endotoxemia, and subsequently demonstrated that disseminated intravascular coagulation was a sequela of endotoxemia.5-6 Other investigators have characterized the host response to endotoxin as consisting of a variety of components that include fever, systemic acidosis, disordered substrate and oxygen utilization, abnormal metabolism, hyperkalemia, hyperglycemia, decreased systemic vascular resistance, elevated cardiac output, and hypotension. It is now apparent that the majority of these responses do not result from the direct effects of endotoxin upon host cells and tissues. Rather, they are the end results of complex interactions of endotoxin with humoral and cellular components of host defenses that culminate in the release of secondary mediators of sepsis. Several groups of investigators have developed experimental models which make it clear that endotoxin initiates and perpetuates the release of cellular mediators that act to further augment host mediator responses and, via excessive mediator secretion, cause target organ damage, organ failure, and death. The development of antibodies (Abs) against endotoxins has provided a new therapeutic modality with which to intervene in the cascade of events leading to Gram-negative bacterial septic shock and death. Initial studies demonstrated the protective capacity of polyclonal, serotype-specific antiendotoxin Abs during experimental Gram-negative bacterial sepsis. Since that time, polyclonal and monoclonal antiendotoxin Abs with reactivity against conserved LPS saccharide moieties and against lipid A have been developed and tested in experimental models of sepsis and in clinical trials. Major controversies exist regarding the ability of antiendotoxin Abs to exert protective capacity during Gram-negative bacterial sepsis, despite in vitro and in vivo evidence of functional capacity of cross-reactive antiendotoxin Abs that includes: (1) neutralization of endotoxicity, (2) enhancement of bacterial clearance, and (3) abrogation of LPS-stimulated production of secondary mediators of sepsis. The current controversy may be summarized with the following questions: 1. 2.

Do clinical studies and experimental models of sepsis clearly demonstrate a reduction in mortality associated with the administration of antiendotoxin Abs? What are the characteristics of antiendotoxin Abs that contribute to protective capacity?

In response to these questions, we will utilize the methodology of meta-analysis to evaluate the ability of antiendotoxin Abs to exert protective capacity and to identify those characteristics that are required to produce protective capacity. In this chapter, we will first review the structure and biochemistry of endotoxin, with an emphasis upon those portions of the molecule that confer antigenicity and toxicity. Second, we will present an overview of the physiologic and biologic effects of endotoxin at both the systemic and cellular levels. In the third section, we will discuss the development and testing of antiendotoxin Abs, and last will be presented a meta-analysis of the protective capacity of antiendotoxin Abs with an attempt to define characteristics that produce protective capacity.

4

Handbook of Mediators in Septic Shock

II. PHYSICOCHEMICAL CHARACTERISTICS OF ENDOTOXINS A. INTRODUCTION Endotoxins (LPS) are a heterogeneous group of macromolecules with an approximate molecular weight of 200,000 that reside in the outer membrane of the cell wall of Gramnegative bacteria. Endotoxin is composed of protein and LPS molecules that are shed from bacteria during active growth and are released in copious amounts when cell lysis occurs. LPS is an integral membrane component composed of three distinct structural regions that include: 1. 2. 3.

An outer polysaccharide structure [O-antigen (O-Ag)] that is unique and comprises the major antigenic determinants for each bacterial strain A core polysaccharide that links outer polysaccharide to lipid A and is structurally similar among many bacterial genera Lipid A, the toxic moiety of LPS that is extremely homogeneous among Gram-negative microorganisms.

A series of rough mutant organisms (so-named due to their characteristic colonial morphology) that do not express the O-Ag region on their cell surface due to mutations that produce enzymic deletions has been derived from Salmonella Minnesota (vide infra). Analogous Escherichia coli mutants have been isolated as well. Each mutant expresses only a portion of the core region of LPS on its outer membrane. The use of these rough mutants has fostered understanding of the biological activities of each structural region of LPS and promoted the discovery and development of therapeutic cross-reactive antiendotoxin Abs against shared bacterial antigens. B. STRUCTURE OF ENDOTOXIN 1. Effects of Extraction Methods Understanding of the relationship of endotoxin structure and function has been furthered by structural analysis of endotoxins extracted by different methods from bacterial membranes. The Boivin method extraction of bacterial cell wall with cold tricarboxylic acid results in endotoxin consisting of LPS, lipid, and cell wall proteins that are mitogenic for lymphocytes from endotoxin-resistant C3H/HeJ mice.6-7 Removal of the protein component by hot aqueous phenol (Westphal extraction) results in loss of mitogenicity but retention of all toxic properties.8 Many other extraction methods have been developed which yield LPS, polysaccharides, or free lipid A. Antigenicity is retained in intact polysaccharides, and antigenicity and toxicity can both be demonstrated in those preparations that contain lipid A. Free lipid A is released by hydrolysis from LPS and is poorly soluble, exhibiting minimal biologic activity, although this substance exhibits toxicity when it is coupled to a protein carrier. While it is not unexpected that different fractions of the endotoxin molecule may possess varying degrees of biological activities, it is apparent that profound alterations in primary and secondary molecular structure may be imposed by the method of extraction and may affect biologic function. 2. Polysaccharide Regions The polysaccharide component of LPS is comprised of two distinct regions: (1) an outer hydrophilic O-Ag region, consisting of repeating saccharide units that are unique for each strain of bacteria; and (2) an acidic core region that links the outer polysaccharide to lipid A and that may be identical for many divergent species and genera of Gram-negative bacteria. The composition of O-Ag is highly variable: analysis via gel electrophoresis of smooth

Cody and Dunn

5

strains of E. coli and Salmonella sp. (wild-type strains that display smooth colonial morphology), has demonstrated that the average O-Ag polysaccharide chain length is between 30 and 100 saccharide residues, with high variability among species and among organisms of the same species.4-9JO Composition analysis of these O-Ag saccharide residues reveals hexosamines, hexoses, and deoxyhexoses to be predominant, linked together in 1-4 and 1-6 configurations. Delineation of the composition of O-Ags has permitted the classification of species of Salmonella, Escherichia, Pseudomonas, and other genera into chemotypes that are common among small groups of bacteria within a species and serve to confer diverse serologic reactivity even within a specific genus. The core polysaccharide region of LPS is a series of 10 to 12 saccharide residues that links O-Ag to lipid A and is itself composed of three regions: outer, intermediate, and inner (deep) core. Core LPS is best defined by a series of rough mutant organisms of Salmonella minnesota and E. coli, each blocked at a more proximal step in the synthesis of core LPS. This series consists of S. minnesota Ra (outer, lipid A + 11 sugars), S. minnesota Rb (outer, lipid A + 10 sugars), E. coli J5 (intermediate, lipid A + 7 sugars), S. minnesota Re (intermediate, lipid A + 6 sugars), S. minnesota Rd (intermediate, lipid A + 5 sugars), and S. minnesota Re (deep, lipid A + 3 sugars). "-12 All core oligosaccharides have a common proximal basal structure that includes glucose or galactose bound to heptose that is in turn bound to 2-keto-3-deoxy-D-mannoctulosonic acid (KDO). However, five different core types have been identified based primarily upon variations or substitutions in the saccharide moieties linked to KDO or glucose. 13 ~ 15 These alterations are exhibited principally in the outer and intermediate regions of core LPS, while the deep core region is less variable and represents a relatively homogeneous portion of LPS. In addition to sugar substitutions, core oligosaccharides are multiply substituted with phosphates that cause the core region to be highly charged, and that may contribute to the limited antigenic heterogeneity of the region.11-13 The core region of LPS appears to represent an ideal antigenic candidate for the formation of Abs against Gram-negative bacteria because it is biochemically and immunologically highly conserved among a wide variety of Gram-negative microorganisms as well as being coupled to the toxic lipid-A moiety. Abs against deep-core epitopes expressed on the surface of mutant E. coli and S. minnesota have been developed and have been shown to be broadly cross-reactive against many Gram-negative microorganisms. 3. Lipid A Lipid A is the toxic moiety of endotoxin and is structurally the most highly conserved portion of the LPS macromolecule.16 It is linked to KDO through an ester linkage and is composed of diphosphorylated diglucosamine residues containing ester and amide-linked fatty acids.4 Ester-linked fatty acids may be even or odd-numbered in chain length and are generally C14-16. All amide-linked fatty acids are even-numbered and are p-hydroxyl substituted, conditions that are unique to fatty acids associated with lipid A. 3 - 6 Also of note is the observation that fatty-acid chain length and composition are influenced by the medium in which the organism is grown. Therefore, the structure of lipid A is fully defined only for a population of bacteria sharing the same cultural conditions.6 Although diverse groups of Gram-negative organisms exhibit similar lipid-A composition, variants of lipid A have been isolated from mutant organisms.17'18 Analysis of the biologic activities of variants and of synthetic lipid A has permitted correlation of specific structures within the lipid-A molecule with toxicity and immunologic reactivity.19 Toxicity appears to be associated with diphosphoryl lipid A, a supposition supported by the finding that injection of endotoxin-susceptible species with monophosphoryl lipid A or monophosphoryl monosaccharide lipid-A precursors (lipid X or lipid Y, respectively) fails to produce fever, Shwartzman reaction, or shock although these preparations show endotoxic reactivity

6

Handbook of Mediators in Septic Shock

in the limulus amoebocyte assay. In contrast, all lipid-A preparations are antigenic, activate macrophages, and are active as B-cell mitogens. While phosphate moieties appear to confer mitogenicity, the antigenic capacity of lipid X appears to require specific amide-linked hydroxy myristoyl groups.18 This observation has been extended to natural lipid A, in that reduced structures which consist of disaccharide and a single amide-linked 3-hydroxyl myristic acid retain full antigenic activity.20 In summary, structural requirements of lipid A for expression of full endotoxic activity appear to include: (1) glucosamine disaccharide, (2) two phosphoryl residues, and (3) 3-hydroxyl fatty acid. Despite the definition of structural requirements for full expression of endotoxin activity, there is evidence that toxicity and antigenicity of lipid A are determined not only by primary structure, but also by the physicochemical state and environment of lipid A.21 Free lipid A in its insoluble form is poorly antigenic and displays weak toxicity. Coupling of lipid A to human or bovine serum albumin markedly increases lipid A toxicity and improves antigenicity, leading to the hypothesis that the saccharide moieties of LPS serve to solubilize lipid A in the host milieu.22-23 Discovery that the immunoreactivity of lipid A is also increased when the molecule is embedded within lipid suggests that conformational changes may occur upon binding of solubilized lipid A to lipoprotein or cellular targets, resulting in the exposure of cryptic lipid A antigens.21 The exposure of cryptic lipid A antigenic determinants only upon binding may help to explain the failure of Abs directed against soluble, intact lipid A to completely abrogate the biologic responses associated with lipid A activity and supports the concept of the existence of an endotoxic conformation for lipid A that is dependent not only on structural composition of the molecule, but on the physicochemical environment.

III. BIOLOGIC EFFECTS OF ENDOTOXINS A. INTRODUCTION The physiologic derangements which accompany septic shock represent a cascade of metabolic, hemodynamic, and immunologic alterations of the normal host homeostatic mechanisms. Host septic responses to endotoxin are legion, but generally consist of fever, systemic acidosis, disordered substrate and oxygen utilization, abnormal metabolism, arterial hypotension, decreased systemic vascular resistance, and elevated cardiac output. The cellular and molecular effects of endotoxin which underlie the systemic responses are now being described, and result from multiple mechanisms of toxicity, including: (1) activation of host defense mechanisms; (2) endotoxin-stimulated release of secondary mediators; and (3) derangement of cellular metabolism. The precise relationship, however, between the cellular and molecular mechanisms of endotoxicity and end-organ responses has yet to be defined. There is evidence that LPS induction and perpetuation of host-mediator secretion produces many if not all of the alterations of septic shock as well as multiple system organ failure. For that reason, early interventions designed to abrogate the interaction of endotoxin with host effector cells and to neutralize endotoxin, combined with aggressive resuscitation after the advent of cardiovascular collapse, and metabolic support may serve to decrease the early mortality associated with endotoxemia and to prevent late morbidity and mortality resulting from multiple organ failure. B. CYTOKINE RESPONSE 1. Introduction It is now apparent that many of the biological effects of LPS are mediated by cytokines. Activation of macrophages by LPS initiates a cascade of endogenous mediators that includes tumor necrosis factor-a (TNF), interleukin-1 (IL-1) and interleukin 6 (IL-6). Although the precise roles of these mediators in the development of septic shock are yet to be delineated,

Cody and Dunn

1

TNF and IL-1 appear to be primary mediators of host defense within the local milieu, exerting deleterious effects on the host after large amounts are secreted and enter the systemic circulation.24'27 Elevated levels of IL-6 have been demonstrated in the serum of patients with septic shock and at the site of infections; administration of high doses of IL-6, however, does not appear to be correlated with mortality.28 The temporal relationships of TNF, IL-1, and IL-6 have been examined after bacterial or endotoxin challenge in man and in nonhuman primates and have been characterized by: (1) peak appearance of TNF at 60 to 90 min; (2) elevation of IL-1 levels at 3 to 4 h; and (3) a rise in IL-6 levels at 4 to 8 h.25'27-29-30 This sequence suggests a pivotal role for TNF in the cascade of cytokine release and thus it has been shown that Abs to TNF abrogate the appearance of IL-1 and IL-6.31 However, unequivocal support for the role of TNF as the primary and sole mediator of the release of other cytokines is lacking. For example, LPS appears to be a direct stimulus of IL-1 production In vivo and induces the expression of IL-6 from a variety of cell types in vitro.27-32 Anti-LPS Abs abrogate LPS-induced IL-1 and IL-6 expression, and have been shown to decrease TNF production and enhance survival in murine models of sepsis.32'35 Understanding of the temporal and causal relationships of LPS and endogenous mediators of sepsis has permitted both the development of protective antiLPS Abs that prevent release of cytokines and the development of protective anticytokine Abs. In addition, selective intervention within the cytokine cascade may interrupt progression beyond the level of proximal mediators and permit beneficial activity within the local host milieu while abrogating the overexuberant host response associated with end-organ damage and mortality. 2. TNF-a Cachectin was first isolated from the blood of septic, hyperlipemic rabbits infected with Trypanosoma sp., and was found to be identical in sequence to TNF, a 17-kDa protein released by tumor-bearing hosts in response in endotoxin injection.36-37 TNF has been detected in the serum of patients with acute meningococcemia, infected burns, at onset of shock after E. coli infusion in baboons, and after experimental endotoxin challenge in humans and in animal models.38'41 Definition, however, of the precise role of TNF as a causal agent of endotoxic/septic shock is difficult and is dependent on: (1) characterization of the kinetics of endotoxin-stimulated TNF release; (2) replication of the shock state after exogenous TNF administration; and (3) correlation of occurrence and degree of septic shock with elevated endogenous TNF production. Lack of consistent demonstration of elevated TNF levels after bacterial or LPS challenge has prevented the unequivocal establishment of TNF as the pivotal mediator of the sepsis state.25-30-38 40 Failure to demonstrate elevated TNF levels in clinical and experimental studies may relate to the proximal position of TNF in the cytokine sequence and to the rapid clearance of TNF from the circulation. In support of this explanation, Michie et al.25 established that TNF was detectable in serum 90 min after bolus injection of LPS into healthy human volunteers and remained elevated only for approximately 3 h. Studies of clearance of recombinant TNF (rTNF) administered to cancer patients also have established that the halflife is less than 20 min.42 Similar kinetics have been displayed in baboon, rabbit, and mouse models of endotoxemia and Gram-negative bacterial infection.24-30-43-44 A wide spectrum of physiologic abnormalities is observed in conjunction with elevated levels of TNF in both clinical and experimental investigations of the effects of TNF. Administration of large doses of rTNF to dogs and rodents is associated with a shock-like state and death, while chronic infusion of rTNF in rodents and in humans results in fever, fluid retention, and anorexia.45"50 In contrast, rats treated with lethal doses of TNF previously shown to contain a concentration of endotoxin of less than 5 U/mg protein did not develop

8

Handbook of Mediators in Septic Shock

manifestations of shock.51 In this and other studies, simultaneous infusion of endotoxin and TNF resulted in shock and death, leading to uncertainty regarding the independent role of TNF as a mediator of septic shock.51-52 Important information regarding the role of TNF in the production of LPS-induced septic shock has been provided by studies of the C3H/HeJ mouse strain. This endotoxin-resistant mouse line displays a mutational event which prevents TNF (as well as IL-1, interferon 7, and colony-stimulating factor) release in response to an endotoxin challenge.53 Bone marrow transplantation with marrow from the endotoxin-sensitive C3H/HeN strain restores TNF production and endotoxin sensitivity.54 Endotoxin resistance associated with decreased production of TNF has been documented in normal animals and has been implicated as the mechanism that confers protection against lethal endotoxemia.55 Measure of TNF levels after administration of sublethal doses of endotoxin to mice, revealed peak production of TNF 2 h after initial LPS injection; thereafter, TNF levels declined and subject animals were refractory to further LPS stimulation for 3 to 5 d. During this same period, animals were resistant to subsequent lethal doses of both LPS and TNF. Similarly, pretreatment of rats with sublethal doses of rTNF has been associated with tolerance to subsequent lethal doses of rTNF and endotoxin.56 The cellular mechanisms which underlie this lack of responsiveness are not clear, but may include: (1) down-regulation of LPS and/or TNF receptors; (2) blockade of endogenous TNF production; and (3) activation or deactivation of genetic modulators of TNF production. Additional studies may serve to precisely define the "set point" of the host response that may either provide tolerance or provoke lethality. Protection against the lethal effects of endotoxin has also been demonstrated after administration of anti-TNF Abs.57 Treatment of mice or rabbits with polyclonal antisera against TNF prior to endotoxin injection has conferred protection against lethal challenge.57-58 Similar protection has been afforded by monoclonal Abs (mAbs) against TNF in murine and primate models of E. coli bacteremia, and in a single clinical study of patients in septic shock.57'59 Although it appears that anti-TNF mAbs offer protection against many of the deleterious effects of endotoxin-stimulated TNF production, there is evidence that this protection is incomplete: (1) the febrile response to LPS remains intact despite Ab efficacy;57-59 (2) LPSinduced adrenocortical hormone production and hypoglycemic responses are unaffected;60 and (3) neutrophil activation is not reduced by the presence of anti-TNF mAbs.59'61 The differential effects of endotoxin and TNF suggest that although TNF is a significant, proximal mediator of septic shock, prevention of the interaction of endotoxin with effector cells is required to eliminate the full spectrum of deleterious host responses. The interrelationship of endotoxin and TNF has been explored via the use of Abs to both TNF and endotoxin. In vitro studies utilizing macrophages, the primary cellular source of TNF, have revealed that endotoxin is the most potent known stimulus of TNF secretion, inducing TNF-DNA transcription and enhancing translation of cellular pools of TNFmRNA.36-53 Compounds which abrogate the toxicity of LPS, including polymyxin B and anti-LPS Abs have been examined for their abilities to alter the production of TNF in vivo and in vitro. Baumgartner et al. determined that protection afforded by polyclonal O-specific anti-LPS mAbs in a murine model of bacteremia and endotoxemia was associated with decreased TNF levels, while administration of human anti-lipid A mAb was associated with neither decreased TNF levels nor protection.32 Murine mAbs against core LPS have been found to protect mice against a live bacterial challenge, but did not decrease serum TNF levels compared to those in animals that died.62 In contrast, treatment of mice with mAbs against the O-Ag portion of LPS has demonstrated both protection and decreased TNF levels.34 In vitro examinations of the capacity of anti-LPS mAbs to decrease LPS-induced TNF production by macrophages have yielded conflicting results. Chia et al.,63 using anti-O-Ag

Cody and Dunn

9

specific, anti-core LPS, and anti-lipid A mAbs failed to demonstrate as much as 50% inhibition of TNF after preincubation of LPS or lipid A with mAbs. Mayoral et al.35 and Priest et al.64 have reported significant decreases in TNF production by peritoneal macrophages after LPS was bound by anti-O-Ag-specific and cross-reactive mAbs. Incubation of LPS with, or treatment of macrophages with polymyxin B prior to stimulation with LPS or lipid A abolishes LPS-induced TNF production.65 These studies suggest that decreased TNF production by macrophages may be due to binding to lipid A epitopes and neutralization of lipid A toxicity, or to the prevention of receptor-ligand interactions through direct effects on the mammalian cell LPS receptor. The ability of some anti-O-Ag-specific mAbs to decrease TNF production may be explained by the capacity of the mAbs to block (perhaps through nonspecific mechanisms such as steric hindrance) binding of the lipid A portion of LPS to the macrophage LPS receptor. Thus, production of effective modulators of LPSstimulated TNF production awaits understanding of the ligand specificity of the LPS receptor and of the cellular signaling mechanisms which convert LPS-macrophage interactions into cytokine release. 3. Interleukins IL-1 and IL-6 are part of a family of monokines whose production is stimulated by the interaction of endotoxin with macrophages, and which exert diverse effects upon the mammalian host.26-66 These effects include: fever; induction of synthesis of acute-phase proteins; reduction of plasma iron stores; and immunomodulation. Secretion of IL-1 follows the appearance of TNF in serum after endotoxin or bacterial challenge, after which elevated levels of IL-6 appear. Induction of IL-1 production upon TNF stimulation has not been conclusively shown. Abs directed against LPS or TNF, however, negate the rise in IL-1 levels and prevent secretion of IL-6.28 Stimulation of IL-1 appears to be associated with many of the systemic alterations associated with inflammation, as well as tissue-specific effects such as inflammation within pathologic joint effusions and within the central nervous system.67 Prominent among the systemic effects of IL-1 is fever, resulting from the action of IL-1 on the thermoregulatory center of the brain. IL-1 is also an effective immunomodulator: administration of low doses of recombinant IL-1 to granulocytopenic mice was associated with accelerated improvement in neutrophil counts and with increased survival in experimental E. coli infection.68"70 In normal animals and in C3H/HeJ mice, IL-1 induced profound neutrophilia.71'72 In composite, these findings indicate that IL-1 may exert some protective effects during sepsis. However, stimulation by IL-1 of the many components of the acute phase response during Gramnegative bacterial infection may culminate in overall deleterious effects upon the host, including septic shock and organ failure. The role of IL-6 in host defense is less clear, despite the observation of high levels of IL-6 in body fluids during acute bacterial infection and within infected wounds.66 Elevated serum levels of IL-6 occur during septic shock and in thermally injured patients; administration of large doses of IL-6, however, was not associated with increased mortality in a baboon model of sepsis.28-66 Conversely, administration of anti-IL-6 mAbs was associated with decreased mortality in a mouse model.73 Thus, the roles that IL-1 and IL-6 play in host defense during sepsis have not been completed elucidated, and these mediators in fact, may exert opposing effects. This poses a therapeutic dilemma in that treatment of septic shock with antiendotoxin or anti-TNF Abs may abolish both beneficial and deleterious effects of interleukin. The significance of IL release during sepsis to the maintenance of host homeo&tasis needs to be established, and the composite effects of anti-LPS Abs or Abs directed against proximal mediators of sepsis (e.g., TNF) will have to be carefully examined in this regard.

10

Handbook of Mediators in Septic Shock

C. EFFECTS ON ORGAN SYSTEMS 1. Cardiovascular Effects Although the cardiovascular effects of endotoxin have been examined in a variety of studies, few animal models have been developed that appear to closely simulate human disease. Initial studies in dogs given bolus injections of endotoxin showed a decrease in cardiac output associated with venous pooling, and no intrinsic cardiac dysfunction was found. Subsequently, other canine endotoxin models were developed, and alterations such as low cardiac index (CI), high systemic vascular resistance (SVR), and both increased and decreased myocardial contractility after bolus endotoxin injection were observed.74-75 In an awake canine model using viable or heat-killed E. coli contained in a fibrin clot implanted into the peritoneum, Natanson et al. induced a time course of cardiovascular changes characterized by stable or increased CI, low SVR, decreased ejection fraction (EF), and a dilated left ventricle (LV).75-77 These abnormalities are similar to those observed in human septic shock, which is characterized by high CI, low SVR, low mean arterial pressure, and reversible LV dysfunction. Studies of cardiovascular dysfunction in human septic shock have, however, been hampered by the need for immediate therapy which precludes observation of isolated effects of Gram-negative bacterial sepsis. Suffredini et al.78 examined the effects of endotoxin administration on cardiovascular performance in a group of healthy volunteers. Changes in LV parameters measured after intravenous endotoxin injection were qualitatively similar to those observed during septic shock: subjects developed elevated CI, decreased SVR and dilated LV. Decreased EF, increased left ventricular ejection fraction (LVEF), and depressed LV performance were observed by gated radionuclide scanning. Two mechanisms have been postulated to cause decreased cardiac performance in endotoxemia and septic shock: global cardiac ischemia, and the presence of circulating myocardial depressant factor.79"83 Evaluation of coronary artery blood flow in patients with septic shock using thermodilution catheters placed within the coronary sinus revealed normal or increased coronary artery blood flow. Although there was no evidence of ischemia, abnormalities of coronary oxygen transport have been observed, suggesting arteriovenous shunting within the myocardium, similar to that which occurs in other organs during septic shock.84 The presence of abnormal ventricular function despite adequate volume loading has led to a search for a myocardial depressant factor active during septic and traumatic shock.83'85-86 Because identical patterns of hemodynamic alteration in dogs treated with Gram-negative or Gram-positive bacteria or Candida have been observed, the hypothesis that infection may cause induction of a final common pathway of injury by structurally unrelated stimuli seems attractive. In support of this contention is the observation that administration of TNF reproduces the cardiovascular profile initially demonstrated after endotoxemia in animal models, and in vitro studies of the effect of TNF on isolated myocardial fiber shortening have demonstrated depressant activity.84 No effects were observed with the administration of IL-1, IL-2, or endotoxin alone. Sera from patients with septic shock and undetectable levels of serum TNF have been shown to decrease myocardial contractility in vitro.85 Taken together these studies suggest that TNF is one of several circulating cardiac depressants present during septic shock. 2. Pulmonary Effects Sublethal doses of endotoxin administered to unanesthetized sheep have provided a model of endotoxin-induced lung injury that may be relevant to the physiologic changes observed in the lungs of humans.87 Initial events in the ovine model include pulmonary hypertension with decreased compliance and resistance to air flow, followed by increased vascular permeability with loss of vasoconstrictor response to hypoxia, increased alveolararterial oxygen gradient (A-aO2) and decreased systemic partial pressure of oxygen

Cody and Dunn

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(pO2).88'91 Several hours after administration of low doses of endotoxin, a trend toward normalization of lung mechanics occurs, but the animals display increased airway reactivity which contributes to persistent oxygenation abnormalities.92 The effects of endotoxin appear to increase in severity with increasing endotoxin dose, in that sheep given large doses of endotoxin display similar initial vascular, ventilatory, and oxygenation abnormalities, but sequentially develop fulminant pulmonary edema, severe hypoxemia, and respiratory collapse.93 Endotoxin-induced injury to the lungs appears to be neutrophil-dependent, a hypothesis supported by studies in which neutrophil depletion in the whole animal reduced endotoxininduced alterations in lung mechanics and may decrease pulmonary hypertension.87-94-95 Even severe depletion of circulating neutrophils, however, does not prevent lung injury and recent evidence indicates that a resident population of neutrophils in the lung may be stimulated by exposure to endotoxin.96 The role of resident populations of phagocytic cells in the production of lung injury has been emphasized by the demonstration that severe and prolonged lung injury may be initiated by endotoxin stimulation of alveolar macrophages via production of toxic metabolites of oxygen, release of proteolytic enzymes, and generation of IL-1 and TNF.68'97 In addition to leukocyte release of deleterious mediators, endotoxin directly stimulates the release of toxic metabolites of oxygen and arachidonic acid from parenchymal cells and platelets.87-98 Of these mediators, thromboxane and various leukotrienes have been demonstrated to induce bronchoconstriction and may increase vascular permeability, thereby amplifying endotoxin-induced lung injury.99"102 Intravenous endotoxin administration in human volunteers results in an increased A-aO2 gradient, minimal arterial hypoxemia, and increased alveolar permeability, paralleling the alterations in pulmonary function observed in the ovine model. Bronchoalveolar lavage showed no evidence of increase in number of neutrophils present after endotoxin administration.103 In summary, endotoxin administration in humans and animal models results in lung injury, alterations in pulmonary mechanics, hypoxemia, and increased airway reactivity that may culminate in severe respiratory compromise and failure. 3. Gastrointestinal Effects Investigation of the role of endotoxin in alteration of gastrointestinal function has centered on the role of the gastrointestinal tract as a reservoir of Gram-negative pathogens.104"108 In animals, small amounts of labeled LPS have been shown to enter the circulation from the intestine in inverted gut-sac models, but no significant transport of LPS through the intact bowel wall has been demonstrated.109-110 In contrast, small amounts of endotoxin have been determined in the portal blood of laparotomized rats in which the lumen of the bowel was not entered.111 Translocation of both bacteria and endotoxin through the gut has been shown to be facilitated bowel ischemia, hypoxia, and chemical peritonitis. In addition to direct bowel injury, vasoactive amines generated during disseminated intravascular coagulation and shock increase the permeability of the bowel to endotoxins.112 Both portal vein and lymphatic transport of endotoxin have been demonstrated to occur in animals, followed by clearance of endotoxin via the liver and components of the reticuloendothelial system. n1 -" 2 Similar to the findings in animals studies, portal endotoxin has not been demonstrable in humans without the occurrence of concomitant bowel disease.113-114 Both portal and systemic endotoxemia have been found to be present in conditions characterized by mucosal disruption, including necrotizing enterocolitis in immature infants, Crohn's disease, ulcerative colitis, and after colonoscopy.115"117 Liver injury secondary to obstructive disease and acute pancreatitis is associated with systemic endotoxemia and, in addition, liver injury may be mediated by endotoxin passage through the portal circulation.114 The release of toxic metabolites and monokines by Kupffer cells, with subsequent damage to parenchymal cells

12

Handbook of Mediators in Septic Shock

is stimulated by endotoxin exposure in in vitro models, and Mazuski et al. 118 have demonstrated direct effects of endotoxin on hepatic parenchymal cell protein synthesis.

IV. ANTIBODIES DIRECTED AGAINST ENDOTOXINS A. INTRODUCTION The current medical armamentarium used to treat Gram-negative bacterial sepsis and shock has reduced, but not eliminated the morbidity and mortality of this lethal disease process. Mortality remains substantial, approaching 20% overall, and reaching 50% in immunocompromised patients. Augmentation of the host humoral immune system with antiendotoxin Abs represents a therapeutic modality that may serve to reduce mortality both through direct neutralization of LPS toxicity, and through intervention in the host effector response to endotoxin. Early investigators observed that active immunization of animals with Gram-negative bacteria protected against septic shock, and that passive transfer of serum from immunized animals provided protection during Gram-negative infections.119-120 Subsequent studies using purified polyclonal Ab established that this was the protective component in serum and revealed the therapeutic potential of antiendotoxin Abs.121 In this section, the authors will review and compare the immunologic and immunobiologic basis of the ability of Abs against different structural regions of LPS to provide protection during experimental Gram-negative bacterial sepsis and examine their use as therapeutic agents in Gram-negative bacterial sepsis and shock. (See Table 1.) B. POLYCLONAL ANTIBODIES 1. Serotype-Specific Antibodies The host humoral response to Gram-negative bacterial infection is primarily serotypespecific. Thus, immunization of animals with heat-killed bacteria yields polyclonal IgG and IgM (immunoglobulin G and M, respectively) Abs directed largely against O-Ag determinants with little Ab against other LPS determinants produced.1'9"123 In rabbit and murine models of bacteremia and endotoxemia, passive transfer of these Abs protected against the development of the dermal Shwartzman phenomenon and against death from bacterial or endotoxic challenge using the homologous organism.124 Initial studies of Gram-negative bacterial infection in man, revealed that liter of O-Ab against homologous organisms did not correlate with degree of protection against Gram-negative shock and death except when the liter of anti-O-Ag Abs was greater than 1:640.l25 However, there was a trend toward correlation of elevated anti-Salmonella minnesota Re Ab liters with protection against shock or death due lo heterologous Gram-negative bacteria. Despite the lack of a clear correlation of anti-LPS Ab levels with protection, clinical efficacy of polyclonal anti-LPS Abs was suggested by the studies of Lachman et al.,126 who utilized freeze-dried human plasma containing high tilers of naturally-occurring anli-LPS IgG in Ihe Irealment of septic shock. A decrease in mortality from 47 lo 7% was seen in Ihe group of palienls receiving hightiler plasma compared lo Ihe group receiving plasma in which the Ab liter was not determined. However, Ihese results must be interpreted wilh caution, since Ihe sludy was nol blinded, prelrealment endogenous tilers of anli-LPS Ig were nol determined in eilher experimenlal or conlrol groups, and patients in the experimental group received various doses of hightiler plasma. Further examination of the effects of passive transfer of anti-LPS Abs in human plasma was performed in a randomized, double-blind trial of therapy in patienls wilh septic shock of surgical origin.127 Inlramuscular administration of hyperimmune globulin containing antiLPS Abs resulted in no diminution of endotoxemia or mortality. No increase in serum Ab tilers could be demonstrated after Irealmenl, and there was no correlation of pretreatment anti-LPS Ab tiler wilh survival in Ihe Irealmenl or control group. In neither of these two

Cody and Dunn

13

studies was the Ab preparation characterized, although previous studies of anti-LPS IgG in this population revealed Abs to multiple Gram-negative organisms, and it is possible that anti-O specific Abs predominated in the polyclonal antisera. Although anti-O Abs appear to afford protection in animal models and may be protective in man, the clinical utility of passive transfer using serotype-specific Abs is limited by (1) the need for conclusive identification of the specific bacterial O-Ag structure prior to Ab administration, and (2) lack of Ab availability during the critical early phase of infection where Ab might serve to limit bacterial replication and endotoxin dissemination. 2. Cross-Reactive Antibodies a. Antibodies Directed Against Common Gram-Negative Bacterial Antigens The Gram-negative bacterial cell wall contains at least three distinct structural regions that are structurally highly conserved among most species and genera of Gram-negative bacteria: enterobacterial common antigen (EGA), core LPS, and lipid A. 128 - 129 Because of the findings that (1) naturally occurring Ab against EGA was found to have no effect on the course of bacteremia in humans, and (2) passive transfer of Ab against EGA provided no consistent protection in animal models, the majority of recent efforts have been directed toward the development of cross-reactive Abs directed against the homogeneous deep-core LPS and lipid A regions of LPS. The association of anti-core Abs with protection against heterologous bacterial infection in humans, first documented by McCabe et al.' 25 and Pollack et al.,130 suggested that naturally-occurring cross-reactive Abs constitute an important facet of host defense. Chedid had earlier proposed a mechanism by which Abs might be formed in vivo against core LPS determinants.123 He proposed that smooth bacterial strains were modified by serum or digestive enzymes, thereby unmasking core LPS and exposing deep-core/lipid A epitopes. It was postulated that these Abs provided nonspecific protection against a wide variety of Gramnegative microorganisms, delaying the consequences of infection until activation of specific immune mechanisms. These findings served to bolster efforts to develop polyclonal Abs against broadly shared bacterial antigens in order to provide a means of early immunologic intervention during the inciting events of Gram-negative bacterial sepsis. b. Antibodies Directed Against Core LPS i. Anti-S. minnesota Re Abs S. minnesota Re structural components, but not lipid A, have been demonstrated on the surface of many types of bacteria.11-12 As previously mentioned, these rough mutants express deep core LPS epitopes extensively upon their cell surface, and represent ideal immunogens for the developrhent of cross-reactive Abs. In early studies, active immunization with the S. minnesota Re mutant afforded protection in mice and in leukopenic rabbits against lethal heterologous bacterial challenge.131'133 Passive transfer of rabbit Ab directed against the S. minnesota rough mutant chemotypes Rd and Re afforded similar protection in mice against a lethal challenge of either Klebsiella pneumoniae or E. coli 107. Naturally occurring Ab against the S. minnesota Re mutant has been found in patients with Gram-negative bacteremia, and the presence of this Ab was highly correlated with decreased morbidity and mortality during Gram-negative bacterial infection.125 This correlation has encouraged the effort to develop anti-51. minnesota Re Abs, and initial trials of human and rabbit anti-S. minnesota Re Abs in murine models of sepsis have demonstrated cross-protective capacj t y_ 134,135

ii. Anti-E. coli J5 Abs

(a) Protection Studies in Animal Models

The presence of naturally occurring Abs against the E. coli mutant J5 derived from E. coli 0111:B4 (wild-type) has been shown to be positively correlated with survival during

+ Murine Live bacteria iv

+ "experimental = '7 "conn-oi = 21

+ "experimental = 40 "control = 40

+ "experimental > 300 "control > 150

+ Murine a. Live bacleria ip b. Live bacleria iv

+

Agranulocylic rabbil fed live bacteria, reclal manipulation + Guinea pig Live bacleria iv

+ "experimental = 78 "control = 97

Murine LPS iv

+ Murine Live bacteria iv

+ "experimental > 100 "control > 100

+ "experimental > 200 "control > 200

+ Murine LPS + actinomycin D iv

Shock model relevant

+ "experimental > 300 "control > 300

Study design appropriate

Other causes excluded Ab Dosage appropriate

Rabbit Polyclonal Serum: 1 . Serolype-specific 2. Cross-reactive

Rabbil Polyclonal Serum: 1 . Serolype-specific 2. Cross-reaclive

Equine Polyclonal F(ab')2 Cross-reaclive

Rabbil Polyclonal Serum Cross-reaclive

Rabbit Polyclonal Serum: 1 . Serolype-specific 2. Cross-reactive

Rabbit Polyclonal: 1 . Cross-reactive Serum 2. Cross-reactive IgG fraction 3. Cross-reactive IgM fraclion

Rabbit Polyclonal Serum: 1 . Serotype-specif ic 2. Cross-reactive antideep core 3. Cross-reactive antilipid A

-

-

-

-

-

-

Single dose, known tiler

+ Range of doses, known liler

Single dose, known liler

Single dose, known liter

Single dose, known liter

Single dose, known liter

+ Range of doses, known liter

ANIMAL POLYCLONAL Ab STUDIES

Monoclonal or polyclonal Ab

+ Saline and preimmune serum

+ Saline, preimmune serum, or unrelated anliserum

+ Saline or preimmune F(ab')2

+ Saline or normal rabbil serum

+ Nonimmune serum

+ Saline or preimmune serum

+ Normal serum Ab tested for LPS

Ab Controls appropriate

TABLE 1 Meta-Analysis of Anti-LPS Antibody Protection Studies

+* -

+* —

-1- ***

+***

+* +***

+ ** -** +**

+* -I- ** -

Ab > C1

— +***

—

-

_

-

-

-

Ab = C2

Protection

-

—

—

-

-

-

+ **

— Ab — C3

140

1 39

138

137

136

135

133

Ref .

14 Handbook of Mediators in Septic Shock

"experimental = 90

-f

+ "experimental > 30

Murine

+ Neutropenic mouse a. Live bacteria iv b. LPS + aclinomycin D iv c. Live bacteria + mucin ip

+ "experimental = 50 "control = 50

a. LPS iv b. Live bacteria + mucin ip

Murine

-f"experimental = 30 = "control 18 +

"ot reported

+ Murine a. Live bacteria iv b. Live bacteria + hgb ip c. Actinomycin D + LPS iv + Rabbil (Dermal Shwartzman phenomenon) +

+ "experimental = 50 "control = 50

=

-fMurine a. Live bacteria iv b. LPS iv + actinomycin D

-f"experimental > 100 "control > 100

"control

+ Murine Live bacteria iv

ip

+ Murine Live bacteria + hgb

+ "experimental ~ 20 "control = 20

+ "experimental = 50 "control = 50

— + Range of doses, known liter

Murine Monoclonal Cross-reactive:

Murine Monoclonal Serotype-specific: 1 . IgG3 2. IgM

Murine Monoclonal Serolype-specific IgG3

Murine monoclonal: 1 . Serotype-specif ic IgG2l 2. Cross-reactive IgG[

Murine Monoclonal Serotype-specific IgG2a

—

—

—

-

—

-

— Single dose of Ab,

+ Single dose of Ab, Ab liter known

— Range of doses, known Ab liter

+ Range of doses, known Ab liter

— Range of doses given, known Ab liter

+ Range of doses, known Ab liters

ANIMAL MONOCLONAL Ab STUDIES Murine Monoclonal Four Cross-reactive (combined administration)

IgG,

Cross-reactive 1 . Equine Polyclonal Ig 2. Murine Monoclonal —

-

Saline

Saline

—

—

— Normal rabbit serum

Saline

+ Unrelated IgGia mAb

Unrelated IgG mAb

Saline

+* +*

+* +*

+*

+* + ***

+*

+ **

+*** +***

—

— -

-

-

~

—

_ _

—

— -

-

-

—

—

_

159

154

153

152

151

150

162

Cody and Dunn 15

Other causes excluded Ab Dosage appropriate

+ Murine Live bacteria + mucin ip

+ Murine a. Live bacteria ip b. Live bacteria + mucin ip

+ Obstetrical and gynecologic palienls wilh sep-

+ "experimental > 12 ncontroi > 12

+ "experimental ~ 24 "control = 24

+ Nonrandomized, controlled

—

-

-

-

Range of doses, Ab liter known

+ Range of doses, Ab liter known

— Single dose of Ab, Ab liter known

+ Range of doses

Ab liter known

Human Polyclonal Plasma Cross-reactive vs. Multiple anligen specific!-

+

One of more doses, Ab tiler variable

-

Medium or cullure supernalanls

Not recorded

Saline

— Rabbit serum or ascites

Ab Controls appropriate

+ Normal human plasma

HUMAN POLYCLONAL AND MONOCLONAL Ab STUDIES

Human Monoclonal Cross-reactive IgM antilipid A

Murine Monoclonal: 1 . Serotype-specif ic IgG3 (two) 2. Cross-reactive: a. Limited (antiouter core LPS) b. Broad (anti-deep core/lipid A)

Murine Monoclonal Cross-reactive (antideep core/lipid A)

+ Murine a. Live bacteria iv b. LPS + actinomycin D iv

+ "experimental = 40 "control = 40

Two anti-core Five anti-lipid A

ANIMAL MONOCLONAL Ab STUDIES

— Murine Monoclonal Murine Six Cross-reactive LPS iv + P. acnes or mAbs actinomycin D

a. Live or killed bacteria iv b. LPS + actinomycin D ip

Shock model relevant

Monoclonal or polyclonal Ab

+ "experimental > 100 "control > 100

icomroi > 30

Study design appropriate

TABLE 1 (continued) Meta- Analysis of Anti-LPS Antibody Protection Studies

+

—

—

—

±** + **

+ **

-

-

-

— —

Ab = C2

Protection

+*

+**

-

+* +*

Ab > C

1

-

—

—

—

-

+ **

—

— Ab — C3

126

165

164

163

160

Ref.

16 Handbook of Mediators in Septic Shock

=

2

+

-t-

+

+

+

— Single dose of mAb, Ab tiler known

— Single dose of mAb, Ab liter known

Single dose on mg/ kg basis, Ab liter known

Single dose on mg/ kg basis, Ab liter known

Single dose only on mg/kg basis, Ab titer known

— Human serum albumin

— D5 Normal Saline

-tMatched preimmune human plasma

+ Matched preimmune human plasma

+ Matched preimmune human plasma

+

+

-

+

+

-

-

-

-

—

-

-

+

-

—

169

168

148

147

146

Ab — antibody; ac D — actinomycin D; hgb = hemoglobin; LPS = lipopolysaccharide; mAb = monoclonal antibody; ncontroi = number of subjects in control group; ne)[perLmenta] = number of subjects in experimental group; n.r. — not reported; P. acnes = Propionibacterium acnes.

Murine Monoclonal IgM Cross-reaclive, amilipid A

Murine Monoclonal IgM Cross-reactive, antilipid A

Human Polyclonal Plasma Cross-reactive

Human Polyclonal Plasma Cross-reactive

Human Polyclonal Plasma Cross-reactive

ties

Protection by anti-LPS antibodies Similar protective capacity of anti-LPS Abs and controls 3 Protection by neither Abs nor controls * Challenge using homologous {immunogen) species ** Challenge using homologous (immunogen) and heterologous species *** Challenge using heterologous species

1

Note:

"control = 281

+ Randomized, controlled, double-blind "experimental = 262

-tSepsis defined by specific criteria and proven gram-negative infection

-f Sepsis defined by specific criteria and proven gram-negative infeclion

+ Randomized, controlled, double-blind "experimental ~ 164

152

+ Prophylaxis during prolonged ncutropenia

+ Randomized, controlled "experimental ~ 47 "control = 53

"control =

+ Prophylaxis of surgical patients undergoing high-risk operative procedures

+ Randomized, controlled, double-blind "experiment.] = '26 "control = 136

^c Shock

+ Patients with evidence of sepsis, gram-negative infection or condition predisposing to gramnegative infection

1^

+ Randomized, controlled, double-blind "experimental = 103 "control = '09

"control ~ 19

"experimental

Cody and Dunn

18

Handbook of Mediators in Septic Shock

Pseudomonas sepsis, suggesting a role for anli-E. coli J5 Ab as a cross-protective agent in Gram-negative bacterial sepsis.130 In studies examining the cross-protective capacity of passively transferred equine and rabbit anti-J5 Abs, polyclonal Ab has shown utility against E. coli, K. pneumoniae, and Pseudomonas sp. In a rabbit model of sepsis, agranulocytic animals were fed live wild-type E. coli, and after the onset of sepsis a single dose of anliE. coli J5 polyclonal antiserum was given by intravenous bolus injection. Survival after E. coli challenge was enhanced after antiserum administration, while nonimmune serum was without effect. In this study, antiserum appeared to function both as an opsonin and as an antitoxin and as such was postulated to combine with endotoxin at the surface of bacteria enzymatically denuded of O-Ags.136 Using a guinea pig model of intravenous Gram-negative bacterial infection, Dunn and Ferguson137 demonstrated that rabbit anli-E. coli J5 antiserum administered prior to lethal challenge was protective against either E. coli 0111:B4 or Pseudomonas aeruginosa challenge.137 Normal rabbit serum did not enhance survival. In an in vitro correlate, the anli-E. coli J5 antiserum was found to have high liter IgG Ab that bound to heat-killed E. coli J5 and P. aeruginosa. Concurrent examination of the mechanism of cross-protection revealed enhanced phagocytosis of the challenge organisms and of three other Gram-negative organisms. To examine further the role of the Fc portion of Ig in protection, horse anti-E. coli J5 F(ab')2 polyclonal Ab fragments were used in a murine model of sepsis.138 Passive transfer of equine F(ab')2 fragments of IgG prior to intravenous bacterial challenge resulted in protection of mice given two times LD50 of E. coli J5, £. coli 0111:B4, P. aeruginosa, or K. pneumoniae. A phagocytosis assay using F(ab')2 or intact IgG demonstrated enhanced bacterial uptake with the latter only, although in vivo protection was equivalent. Via ELISA determinations, Ab binding of IgG or F(ab')2 to LPS derived from Gram-negative organisms was greater than that to whole bacteria. These findings suggest that polyclonal Ab directed against E. coli J5 is broadly cross-protective and may function in vivo primarily as an antitoxin serving to neutralize endotoxin. In contrast, however, Greisman and Johnston found that passive transfer of high-liter rabbit anlisera lo E. coli J5 or S. minnesota Re mulanl failed lo prolecl mice against endotoxin challenge (E. coli 0111:B4, E. coli 0127:B8, Salmonella typhimurium, S. minnesota, or Citrobacterfreundii).139-140 Control mice were given matched preimmune sera or saline, but preimmune Ab tilers were not determined. Failure of rabbit anti-E. coli J5 antiserum to provide protection in mice against S. typhimurium was also reported by Sakulramrung when a large bacterial inoculum was administered.141 Of note, however, was Ihe observalion lhal a reduction in the size of the inoculum was associated with a modicum of protective capacity. Thus the ability of anti-core LPS polyclonal Abs lo provide cross-protection during experimental Gram-negative bacterial infection has not been an invarianl finding. Experimental studies can be broadly grouped inlo Ihree categories, that consist of those in which: (1) anticore LPS Abs protected when compared lo conlrol antiserum; (2) similar protective capacity of bolh anti-core LPS Abs and control antiserum occurred; and (3) neither preparation provided protective capacity. Many of Ihese sludies may have been hampered by: lack of reagent specificity; failure lo use appropriate conlrols and lo control for antiserum and subjecl animal nalural Ab; and Ihe effecl of inoculum size. Of additional concern both in early studies of protective capacity of cross-reaclive polyclonal Abs, and in currenl evaluations of mAb protective capacity is contamination of Ab preparations by endotoxin. Although endoloxin-free as determined by limulus amoebocyte assay, Ab preparations may conlain minute quantities of endotoxin which serve as nonspecific stimulators of Ihe immune response. Chong and Huston142 have evaluated Ihe effecl of endotoxin contamination on Ihe protective capacity of core-reactive Abs and have determined lhal: (1) the addition of picogram quantities of endotoxin to

Cody and Dunn

19

nonprotective Abs rendered them protective in a murine model of bacteremia; (2) endotoxininduced protection was maximal when contaminated core-reactive Ab preparations were administered prior to and by the same route of injection as the bacterial challenge (in contrast to the protection afforded by serotype-specific Abs administered either before or after bacterial challenge); and (3) both rough and smooth endotoxins induced nonspecific host protection. Postulated reasons for the failure of polyclonal anti-core LPS Abs to confer protection against homologous wild-type or heterologous LPS involves consideration of the homogeneity of both the antigen itself and the subsequent challenge organisms or LPS. Primarily, several authors including ourselves have hypothesized that core LPS epitopes may be unavailable for binding in some circumstances due to cloaking by O-Ag polysaccharide sidechains.123'138-143 However, the presence of naturally occurring anti-S. Minnesota Re and anti-£. coll J5 Abs calls into question the in vivo relevance of this postulated effect. Chedid, in 1968, proposed that serum proteases may cleave overlying O side chains, exposing deepcore antigens.123 These antigens may also be differentially exposed in rapid phases of bacterial growth, when O-polysaccharide substitution is incomplete. Such exposure may also result in an antigen that may provoke the formation of natural anticore LPS Ab. Thus, the protective capacity of anticore Ab against a given preparation of LPS may depend upon duration of bacterial culture and culture conditions prior to LPS extraction that may give rise to the presence of varying amounts of only smooth (lipid A + core + O-Ag), or smooth and rough (lipid A + core) LPS. The end result of such postulated variations in challenge LPS preparations might be significant variability in anticore LPS protective capacity. Other potential confounding variables that may have precluded demonstration of a significant effect of anticore LPS Abs upon survival during experimental Gram-negative bacterial sepsis involve the inadvertent production of anti-O-Ag Abs via: (1) endotoxin-induced polyclonal activation of B cells, with increase in endogenous anti-O-specific Ab liters; and (2) induction of serotype-specific Ab against the parental smooth LPS through specific Ab production against wild-type O-polysaccharide chains contained within the mutant cell but that are not translocated to the bacterial cell surface.144 These chains are postulated to become accessible to Ab formation during the process of heat killing.145 Unfortunately, at the time of these initial studies, monospecific Ab reagents were not available, and thus many of the above mentioned nonspecific effects could not be excluded. Despite these drawbacks, the data obtained from these experimental studies provided enough cogent information that human trials were implemented. (b) Protection Studies in Human Subjects

Ziegler et al.146 developed polyclonal human antisera directed against E. coli J5 by immunizing normal human volunteers with heat-killed E. coll J5, and in a randomized, double-blind study, a single unit of human immune or preimmune control serum was administered to critically ill patients with presumed Gram-negative bacterial sepsis. Outcome was assessed in relation to death from Gram-negative bacteremia or from causes directly attributable to bacteremia. Mortality associated with Gram-negative bacteremia occurred less frequently in those patients given human anti-£. coli J5 antiserum than in those given preimmune serum (22 vs. 39%) and the degree of protection was greater in those patients with clinical evidence of shock, with or without positive blood cultures (44 vs. 77%). Preand postimmune levels of naturally occurring Abs against E. coli J5 were not measured, nor were levels of naturally occurring O-specific Abs measured. The possibility, therefore, exists that a portion of the protective effect of antisera was due to O-specific Ab active against the bacteriologic strain present in the patient, but not in the control material. The same authors extended their studies of anti-£". coli J5 antiserum to prophylaxis.147 Anti-E. coli J5 antiserum or control serum was administered to a group of patients identified

20

Handbook of Mediators in Septic Shock

as being at high risk for, but without clinical signs of bacteremia. Anti-E. coli J5 antiserum failed to prevent focal Gram-negative infections, although the infections appeared less severe than those experienced by controls. The incidence of shock and death was reduced in those patients receiving anti-£. coli J5 antiserum, providing support for the findings of the initial clinical trial. In a second human trial of anti-E. coli J5-antiserum prophylaxis, however, the administration of pre- and postimmune anti-E. coli J5 antisera to neutropenic patients resulted in no reduction in bacteremia, febrile episodes, or mortality. The authors attributed the failure of prophylaxis to the small number of cases of Gram-negative bacteremia observed precluding demonstration of a small protective effect or to the need for administration of additional doses of the Ab.148 c. Antibodies Directed Against Lipid A Lipid A is the most highly conserved structural region of LPS and is responsible for many if not all of the pathological effects associated with septic shock, including fever, hypotension, shock disseminated intravascular coagulation, and organ failure.19 Interestingly, although Abs directed against lipid A have been shown to occur in as many as 73% of healthy humans, 149 the induction of anti-lipid A Ab synthesis in animals has proved somewhat difficult. One explanation of this observation may be that lipid A is not exposed on the intact bacterium or on smooth LPS, while degradation in vivo of whole bacteria or of LPS may yield lipid A in an immunogenic form.20 Supporting evidence for this hypothesis has been provided from studies that demonstrated Ab with low levels of anti-lipid A reactivity has been obtained via immunization of rabbits with S. minnesota R595 and E. coli mutants. Increased liters of anti-lipid A Abs were obtained after these rough mutant organisms were treated with mild acid hydrolysis to expose free lipid A on their outer membranes. Brade et al.21 evaluated the immunogenicity and antigenicity of natural lipid A and of a variety of synthetic whole and partial lipid A compounds derived from E. coli and S. minnesota. The antigens were presented in soluble and insoluble forms, as well as complexed to a variety of carriers. Analysis of polyclonal rabbit antisera using a passive hemolysis assay revealed diversity of rabbit Abs against natural and synthetic lipid A. At least five specificities were identified, all of which recognized epitopes in the phosphorylated diglucosamine region of lipid A. These studies also demonstrated that immunoreactivity of lipid A was dependent on the physicochemical environment. Immunogenicity of lipid A was greatest when those compounds were coated onto sheep red blood cells (SRBCs) or contained within liposomes. When lipid A was used as an antigen in passive hemolysis inhibition assays, however, epitopes within the hydrophilic backbone of lipid A were not exposed in aqueous solutions, but were reactive with anti-lipid A Ab when it was incorporated into SRBCs or within liposomes. Based on these studies, it would appear that the amphipathic nature of lipid A must be preserved in order for the molecule to effectively function as an antigen in vivo and in vitro. While many investigators have attempted to develop anti-lipid A Abs and to test their in vitro and in vivo reactivity, the results obtained frequently are difficult to interpret. For example, polyclonal Ab directed against lipid A has exhibited broad cross-reactivity to LPS derived from both smooth and rough bacteria when examined via passive hemolysis, implying that outer membrane associated lipid A is accessible to Ab in these preparations. When rabbit anti-E. coli or S. minnesota 595 lipid A serum was examined via enzyme-linked immunosorbent assay (ELISA) using formalin-fixed smooth and rough whole organisms, however, no reactivity was observed. When these same sera were tested against LPS released from the same organisms after heating, only reactivity against LPS released by rough strains could be demonstrated.16 Therefore, although broad cross-reactivity of anti-lipid A Abs can be demonstrated in vitro, the degree of exposure of lipid A on intact bacteria may limit the clinical utility of these Abs.

Cody and Dunn

21

C. MONOCLONAL ANTIBODIES 1. Serotype-Specific Antibodies The need for widely available, standardized preparations of Abs for effective immunotherapy has led to the development of cross-reactive mAbs with defined epitope binding specificities in the deep-core/lipid A region of LPS. That mAbs could protect against endotoxemic and bacterial challenge was initially demonstrated in the studies of Dunn et 153 using a serotype-specific mAb to LPS from E. coli al 150-152 am j Kirkland and Ziegler 0111:B4. Immunization with mAb prior to intravenous bolus injection of LPS or live E. coli 0111:B4 mAb raised the LD50 for both LPS and intact organisms. Type-specific protection against endotoxemia and bacteremia was therefore shown to be achieved with Ab to a single epitope of E. coli 0111:B4. Additional studies revealed that type-specific protection was not dependent on Ab class or subclass.154 MAbs of IgG3 and IgM class and subclass with specificity against the O-Ag portion of E. coli 0111:B4 LPS were demonstrated to afford equal protection in an immunosuppressed mouse model of bacterial and endotoxin sepsis. Despite these demonstrations of efficacy, empiric therapy of septic shock or sepsis syndrome with serotype-specific mAbs is impractical. Therefore, the development of protective mAbs has focused on mAbs against broadly shared bacterial antigens. 2. Cross-Reactive Antibodies a. In Vitro Evaluation of Cross-Reactivity Efforts to develop cross-reactive mAbs have yielded many anti-deep-core/lipid A mAbs.150'152'155"169 Examination of these murine or human anti-E. coli J5 and anti-5. minnesota mAbs raised against whole cell or LPS antigens by radioimmunoassay (RIA), ELISA, or hemagglutination techniques has revealed cross-reactivity to LPS isolated from E. coli J5, 5. typhimurium, S. minnesota Re595, Agrobacterium tumefaciens, and Pseudomonas aeruginosa, and to whole cells of S. minnesota Re595, E. coli 0111:B4, P. aeruginosa, and K. pneumoniae.l45't50J5JA5S Several authors have localized the reactivity of broadly crossreactive mAbs against S. minnesota R595 and E. coli J5 to the lipid A fraction of LPS via Western immunotransblot analysis using LPS from rough and smooth bacterial strains. 155 - 162164 Both ELISA and RIA binding to intact and heat-killed cells have been examined, and cross-reactive binding of these mAbs have been examined, and were directly correlated with lipid A reactivity.153'163 Despite the above examples, the majority of mAbs generated against rough mutant organisms have lacked reactivity against homologous wild-type smooth organisms or LPS derived from these organisms, heterologous organisms or LPS, or lipid A.156"160 There are several possible explanations for the apparent lack of cross-reactivity of mAbs that include: (1) choice of binding target; (2) physical alteration of binding target by fixation; (3) inaccessibility of deep-core/lipid A epitopes on intact bacteria and on LPS; and (4) occurrence and antigenicity of unique binding determinants within the deep-core/lipid A region. Each of these points will be discussed in turn. i. Choice of Binding Target MAbs directed against whole intact or heat-killed organisms were evaluated for binding to chromatographically purified LPS or lipid A by Miner et al.160 who have shown that mAbs generated against whole bacteria demonstrate cross-reactivity in direct-binding RIA against only whole bacteria. The authors suggest that the lack of binding in corresponding preparations of LPS may be explained by alteration of antigenic determinants during LPS processing, by the presence of other common surface antigens on the bacterial cell wall against which mAb are made, but which are absent in LPS preparations, or by the presence of small quantities of incomplete LPS moieties on the whole organisms which are antigenic but which are absent from preparations of LPS.145-160

22

Handbook of Mediators in Septic Shock

ii. Physical Alteration of Binding Target by Fixation Alteration of mAb binding pattern in ELISA to fixed and intact organisms was examined by Aydintug et al.145 Extensive cross-reactivity of murine mAbs generated against heatkilled E. coli J5 could be demonstrated using heat-killed targets, but not when live or formalin-fixed organisms were used. Heating may alter the configuration of epitopes on the bacterial surface, increase bacterial permeability, or expose cryptic antigens which are not accessible to Ab binding on the intact organism. Formalin fixation causes extensive alkylation of hydroxyl and amide moieties of carbohydrate on the bacterial surface, thus altering epitope characteristics and decreasing binding of mAb to these epitopes. The recent work of Cody et al.,161 using serotype-specific anticore/lipid A mAbs in a fluorescence microscopy study of mAb binding to heat-killed and live bacterial targets revealed; (1) increased binding of deep-core/lipid A mAbs to heat-killed E. coli 0111:B4 compared to intact organisms; (2) increased binding of serotype-specific mAbs to heat-killed E. coli J5 compared to intact organisms; and (3) no difference in binding of serotype-specific mAbs to intact or heatkilled E. coli 0111:B4. These results indicate that heat-killing of organisms may result in the emergence or alteration of antigenic sites. The effect is pronounced for deep-core/lipid A Ag sites, and may lead to both over- and underestimates of binding of cross-reactive mAbs to bacteria in vivo. Hi. Inaccessibility of Deep-CorelLipid A Epitopes on Intact Bacteria and on LPS Although deep-core/lipid A Ag on mutant organisms represent ideal targets for the creation of broadly cross-reactive mAbs, the degree to which deep-core/lipid A antigens are exposed on intact or heat-killed bacteria or on LPS is not clear. Pollack et al.157 have shown anti-lipid A mAb binding to free lipid A and to lipid A in LPS from Re mutant bacteria, but not to LPS from mutant bacteria containing other core sugars or O saccharides. This indicated that mAb binding to lipid A was significantly affected not only by O sidechains but by sugars which form part of the deep-core/lipid A structural region. In contrast, Dunn et al.151-163 and Board et al.155 have demonstrated binding of mAbs against deep-core/lipid A and lipid A epitopes to heat-killed wild type whole organisms and to LPS derived from these organisms. In further investigations from this laboratory, mAbs, assessing binding specificity defined by immunodotblot analysis to be within the deep-core (Re) region of LPS, were found to be reactive against wild type E. coli, S. typhimurium, and S. minnesota, on Western immunotransblot analysis.164 Cross-protective capacity of these mAbs was demonstrated in a murine model, and was compared to protection afforded by serotype-specific mAbs against homologous bacteria. Protective capacity after mAb pretreatment was achieved with both serotype-specific and cross-reactive mAbs, although higher doses of cross-reactive mAbs were required for protection. These studies demonstrated that deep-core/lipid A structures on wild type bacteria and LPS are accessible to mAbs with specificity for inner core and lipid A epitopes. iv. Occurrence and Antigenicity of Unique Binding Determinants within the Deep-Core/ Lipid A Region Although the deep-core/lipid A region is broadly conserved among bacterial genera, Pollack et al.157 and Priest et al.164 have reported pronounced specificity of core and lipid A-reactive mAbs which reflect inter- and intraspecies differences in covalent core structure. Pollack et al.157 state that the apparent cross-reactive capacity of many deep-core/lipid A mAbs may be explained by: (1) nonspecific binding of proteins or Abs present in highly concentrated ascites fluid or tissue culture supernatant; (2) poor controls and lack of confirmatory assays of mAb binding; (3) failure to evaluate reactivity against a variety of rough mutant LPS types differing in covalent structure; and (4) use of whole bacteria rather than

Cody and Dunn

23

isolated LPS as an antigen in solid-phase assays, increasing the likelihood of nonspecific binding. b. Protection Studies in Animal Models Although large numbers of cross-reactive mAbs have been generated, the protective capacity of these mAbs has been variable in studies using a lethal endotoxin or bacterial challenge. For example, Miner in 1988 found that cross-reactive mAbs failed to protect in animal models although binding to whole heat-killed bacteria in vitro was observed by ELISA.160 In previously published studies, Dunn et al.150 had demonstrated in a murine model that protection was afforded by four broadly cross-reactive anti-E. coli J5 mAbs against lethal doses of mild-type E. coli 0111:B4, E. coli J5, and Klebsiella pneumoniae. Extension of these studies utilizing a broadly cross-reactive IgG mAb revealed: (1) broad cross-protective capacity in murine models of LPS endotoxemia and Gram-negative bacteremia;152-1KM64 (2) enhancement of phagocytosis in vitro by human polymorphonuclear cells of opsonized live bacteria;163 and (3) protective capacity in mice equal to that afforded by polyclonal anti-deep-core/lipid A Abs with similar specificity.162 Further correlations of cross-reactivity and cross-protective capacity were made by Priest et al.164 who demonstrated that a panel of mAbs exhibited cross-reactivity binding in vitro and provided protection in vivo against a large number of different types of Gram-negative bacteria or their derived outer membrane LPS. Taken together, these studies confirmed the protective capacity of cross-reactive mAbs and suggested the therapeutic value of these mAbs in the treatment of human Gram-negative bacterial infection. While murine mAbs have been the focus of much study, human monoclonal anticore/ lipid A Abs also have been isolated.165'169 Teng et al.165 first developed a human monoclonal IgM Ab against deep-core/lipid A determinants. The extensive cross-reactive and crossprotective capacities of these Ab preparations were demonstrated by: (1) broad cross-reactivity of mAbs a variety of Gram-negative bacterial species isolated from the blood of patients; (2) abrogation of the dermal Shwartzman reaction in mice; and (3) protection of mice against lethal bacteremia induced by a broad range of Gram-negative organisms. The highly crossreactive Ab appeared to bind avidly to its lipid A target despite the presence of O polysaccharides, since the Ab was protective at low doses. c. Protection Studies in Human Subjects Since the first demonstration of the efficacy of human mAbs against Gram-negative bacterial infection, clinical trials of human anti-deep-core/lipid A mAbs have been instituted. Clinical trials of E5, a cross-reactive murine deep-core/lipid A mAb have been initiated.167-168 The IgM mAb preparation has been determined to be safe and without significant side effects or allergic reactions in Phase I study, and initial findings from studies of efficacy against Gram-negative bacterial infection in humans demonstrate protection against mortality in those patients who were not in septic shock.168 Ziegler et al.169 have demonstrated protection afforded by HA-1A, a cross-reactive human IgM mAb in a population of bacteremic patients in septic shock. In this multicenter trial, patients with sepsis and suspected Gram-negative bacterial infection were randomly assigned to receive HA-1A or placebo at entry into the study. Usual resuscitative and supportive measures including fluid, pressors, and appropriate antibiotics were continued, and patients were followed for 28 d or until death. In 196 patients with Gram-negative bacteremia, 63% of patients receiving HA-1A survived 28 d, compared to 48% of controls. A 42% reduction was observed when mortality was examined in those patients with septic shock. The results of follow-up studies using either E5 or HA-IA, however, have not demonstrated efficacy, and the ability of these mAbs to effectively bind to LPS must be questioned. Further research should serve to reveal those Ab characteristics

24

Handbook of Mediators in Septic Shock

that define cross-protective capacity and will assist in the design and production of highly effective Abs for the therapy of Gram-negative bacterial infection and septic shock. D. META-ANALYSIS OF THE PROTECTIVE CAPACITY OF ANTIENDOTOXIN ANTIBODIES Meta-analysis provides a means by which cross-study comparison of Abs developed by a large number of laboratories may be compared with regard to protective capacity. A large body of literature exists regarding mAb-mediated protection, but the failure to establish criteria which define valid models of protection has prevented the recognition of those Ab characteristics which are common to protective Abs. We have designed two decision trees, the first to address those issues pertinent to design and execution of valid protection studies and the second to summarize and compare the characteristics of protective and nonprotective Abs in decision tree format. Studies were grouped by type of model and by type of Ab employed, enabling systematic comparisons of: (1) protective capacity within or across animal species; (2) protective capacity of polyclonal vs. monoclonal Ab preparations; and (3) protective capacity of serotype-specific vs. anti-deep-core/lipid A Abs. (See Table 2.) None of the studies examined satisfied all test nodes of the first decision tree, but results of this analysis should nonetheless prove useful. Systematic assessment of the literature by meta-analysis will: (1) aid in the design of valid animal models or clinical trials protection; (2) assist in the development of therapeutic Abs by identifying those Ab characteristics which are common to protective Abs; and (3) decrease the cost of development and increase the availability of protective Ab preparations. 1. Decision Tree 1: Test Nodes (Figure 1) a. Is the Study Design Appropriate? Examples of study design included: randomized controlled, controlled, and open. Few studies qualified as randomized, controlled studies; most were controlled studies utilizing similar, concurrently treated patient or animal populations. Of significance in randomized controlled and controlled animal and human studies was the size of the control group compared to the size of the treatment group. Both treatment and control groups should have had equal numbers of subjects, particularly if small numbers of subjects were used. In patient trials, the study population should have been clearly defined with respect to underlying disease and demonstration of universally accepted signs and symptoms of septic shock before entry into the study. b. Is the Shock Model Clinically Relevant? The choice of an animal model of septic shock for use in protection studies presupposes relevance to the human disease. Animal species vary widely in their susceptibility to endotoxin, with humans and rabbits being among the most highly susceptible and rodents the least susceptible. Also, relevant conditions may be employed to produce endotoxic shock in poorly susceptible species, including abrogation of natural defenses with actinomycin D, and large, rapid intravenous bolus of organisms or purified endotoxin. Of consideration also should be the type of endotoxin or bacterium employed. The use of nonpathogenic bacteria or LPS forms that are not clinically relevant in order to demonstrate binding or protection should be avoided. Many models fail to mimic the human disease, in which endotoxic shock appears to evolve gradually after Gram-negative bacterial infection. We believe that the preferred model should employ a range of doses of intact organisms, injected intravenously or instilled into the peritoneal cavity. Confirmation of bacterial inoculum numbers should be reported by precise bacterial enumeration.

Cody and Dunn

25

c. Are Other Causes of Shock Excluded? Cardiac or hypovolemic shock or shock due to infection with other microorganisms may create an appearance of shock indistinguishable from that of endotoxic shock. Appropriate cardiovascular monitoring, vigorous fluid resuscitation, and obtaining blood cultures to rule out infection with other organisms should be performed in human studies. Animals should receive appropriate fluid resuscitation and blood cultures should be performed at study termination to determine which microorganisms are present. d. Are Antibody Controls Appropriate? Results of Ab treatment in the experimental groups were frequently compared to the results of treatment with saline in the control group. More appropriate controls consist of unrelated Ab of the same subclass, in the case of mAb, or preimmune sera, in the case of polyclonal Abs. Both preimmune sera and unrelated Ab may have nonspecific effects on host resistance or on endotoxin clearance, which may lead to underestimation of specific anticore/LPS Ab activity. In addition, preimmune sera may contain significant tilers of serotype-specific Ab or cross-reactive Ab, resulting in a similar misinterpretation of specific Ab activity. e. Is Antibody Dosage Appropriate? Many factors determine the success of Ab therapy, including amount of Ab given on the basis of test subject weight, single or multiple dosing of Ab, and timing of Ab administration. Ab dose should be calculated on a per weight basis, and should be consistent for all subjects. The use of very high dosages on a per weight basis should have been avoided in animal models, as it is not possible to reproduce high Ab concentrations in clinical trials. In addition, effectiveness of Ab against endotoxin may not be shown if Ab is given at a single dosage. For example, it has been demonstrated that cross-reactive Abs require higher Ab dosages than do serotype-specific Abs to demonstrate protection. Therefore, a protective effect of Ab may be missed if a range of dosages is not employed in protection studies. All study participants should have received the same number of doses of Ab, and at the same intervals after onset of endotoxemia and thereafter. /. Is Response Caused by Antibody Itself? The need for precise definition of all Abs present in polyclonal antiserum in order to avoid the misinterpretation of unrelated Ab effects as those of specific protection has already been alluded to. An additional cause of confusion regarding the activity of Ab preparations resides in the presence of endotoxin in Ab preparations. Although endotoxin is a nearly ubiquitous contaminant of laboratory preparations and may contaminate both polyclonal and monoclonal Abs, Abs are seldom analyzed for the presence of endotoxin. Minute amounts of endotoxin in Ab preparations may stimulate host defenses and confer the appearance of protection where no specific protective capacity exists, or bind to the Ab preparation and decrease protective effect. Anti-LPS Abs may function as LPS scavengers, binding large quantities of endotoxin. Bound endotoxin may cause sepsis-like syndrome when present in large amounts, or stimulate the host response, mimicking a protective effect when bound in small quantities. A further consideration (of particular significance in patient trials) is the use of antibiotics concurrent with Ab administration. The appropriateness of antibiotic choice for the infecting organism and the duration of antibiotic therapy may affect mortality and morbidity independent of Ab administration. Although ethical considerations preclude withholding antibiotic therapy, patient groups should be evaluated for major differences in antibiotic therapies between subject and control groups as well as differences among individual subjects. Other concurrent therapies, such as percutaneous abscess drainage and

Polyclonal Polyclonal Polyclonal

Polyclonal

Polyclonal

Polyclonal

Polyclonal

Polyclonal Polycional

Polyclonal Polyclonal

Polyclonal

Polyclonal

Yes Yes No

Yes IgM > IgG

Yes Yes

Yes

Yes

Yes No

Yes No

Yes

Yes

Protective?

Antibody character

Cross-reactive

Cross-reactive

Serotype-specific Cross-reactive

Serotype-specific Cross-reactive

Cross-reactive

Cross-reactive

Serotype-specific Cross-reactive

Cross-reactive

Serotype-specific Cross-reactive (anti-deep core) Cross-reactive (anti-lipid A)

Reactivity

Not determined

Not determined

Not determined Not determined

Not determined

IgG F(ab') 2

Not determined

Not determined

IgM IgG

Not determined Not determined Not determined

Antibody class or subclass

Heat-killed whole cells

Heat-killed whole cells

Boiled whole cells Boiled whole cells

Boiled, acetone, or ether fixed whole cells, or LPS

Heat-killed whole cells

Heat-killed whole cells

Boiled whole cells

Acetone-fixed whole cells

Acetone-fixed whole cells

Immunization scheme

Hemagglutination

Hemagglutination

Not determined Not determined

Passive hemagglutination

ELISA

ELISA

Hemagglutination

ELISA

Indirect hemagglutination

In Vitro characterization

Human

Human

Rabbit Rabbit

Rabbit

Horse

Rabbit

Rabbit

Rabbit

Rabbit

Animal species of Ab

TABLE 2 Meta- Analysis of Characteristics of Protective Anti-LPS Antibodies

Not determined

Not determined

Not determined Not determined

Not determined

Not determined

Not determined

Not determined

Not determined

Not determined

Effect on secondary mediators

147

146

140

139

138

137

136

135

133

Ref.

26 Handbook of Mediators in Septic Shock

Monoclonal

Monoclonal Monoclonal

Monoclonal

Monoclonal

Monoclonal

Yes

Yes Yes

Yes

Yes; IgG = IgM

Yes

Monoclonal

Monoclonal

Monoclonal

Yes

Yes

Yes

Cross-reactive

Cross-reactive

Serotype-specific Cross-reactive

Cross-reactive

Cross-reactive Cross-reactive

Cross-reactive anti-core anti-lipid A Not determined

Serotype-specific

Serotype-specific

Serotype-specific Cross-reactive

Serotype-specific

Cross-reactive

IgM

IgM

IgG2a, IgG3

IgGi

Ig IgGi

Not determined

IgG3, IgM

IgG,

IgG2a IgG,

IgG2a

IgG4

Heat-killed whole cells

Heat-killed whole cells

Heat-killed whole cells and LPS

Heat-killed whole cells and LPS

Heat-killed whole cells and LPS

Heat-killed whole cells and LPS

Heat-killed whole cells and LPS

Heat-killed whole cells

Heat-killed whole cells and LPS

Heat-killed whole cells

Heat-killed whole cells

Ab = antibody; ELISA = enzyme-linked immunosorbent assay; LPS = lipopolysaccharide.

Monoclonal

Yes

Note:

Polyclonal Monoclonal

Yes; Polyclonal = Monoclonal

Polyclonal

Monoclonal

Yes

Not recorded

ELISA, dot blot

ELISA immunodot blot, Western immunotransblot

ELISA

ELISA, Western immunotransblot

RIA

ELISA, immunodot blot, Western immunotransblot

ELISA

ELISA

ELISA

ELISA

Human

Human

Mouse

Mouse

Equine Mouse

Rabbit

Mouse

Mouse

Mouse

Mouse

Mouse

Mouse

Not determined

Not determined

Not determined

Not determined

Not determined

Not determined

Not determined

Not determined

Not determined

Not determined

Not determined

150

169

165

164

163

162

159

154

153

152

151

Cody and Dunn 27

28

Handbook of Mediators in Septic Shock ,

Study design appropriate?

\

+ \

Shock model or patient group well-defined?

\

+ -X

Other causes of shock excluded?

\

+ \

Antibody controls appropriate?

\

+ \

Antibody dosage appropriate? -f

\

X

Response caused by Ab itself?

\

+

FIGURE 1. Meta-analysis of protection studies of anti-LPS antibodies. If all nodes are satisfied in this decision tree, then the study shows a real association between antibody administration and decreased mortality from endotoxemia.

surgical abscess drainage or debridement should be examined for their independent contributions to decreased patient morbidity and mortality, so that appropriate stratification occurs. 2. Decision Tree 2: Test Nodes (Figure 2) a. Has the Antibody Demonstrated Protective Capacity? As previously discussed, no protection study fulfilled all test nodes for the demonstration of protection by an Ab preparation. Therefore, in this decision tree, both protective and nonprotective Abs will be examined for those characteristics that contribute to reactivity. b. Are Monoclonal or Polyclonal Antibodies more Protective? Passive transfer of both polyclonal and monoclonal O-specific and core-reactive Ab appears to provide protection. In addition, both polyclonal and monoclonal anti-deep-core/ lipid A Abs have been shown to mitigate the effects of septic shock or to reduce lethality in animal and human trials. However, the superiority of one preparation over the other in providing protection has not been established. Only one study provides data regarding specific comparisons, and no differences in protective capacity were noted.162 c. Are Serotype-Specific Antibodies more Protective than Anti-Deep-Core/Lipid A Antibodies? Priest et al.164 and McCabe et al.135 directly compared O-specific and core-reactive mAbs and found O-specific mAbs to be more protective. No analysis of the specific core epitopes to which binding of Ab affords the greatest degree of protection has been performed. Thus, it is not known whether the use of Abs directed against these epitopes that reside in the deep-core/lipid A region will invariably serve to maximize both cross-reactivity and crossprotective capacity. Systematic examination of the reactivity of anti-deep-core/lipid A mAbs

29

Cody and Dunn v Has the Ab demonstrated protective capacity?

—\

+

\ Is the Ab monoclonal or polyclonal? + -\ monoclonal \ polyclonal \ Serotype-specific or cross-reactive? +-X serotype-specific \ cross-reactive \ Does antibody class or subclass affect protective capacity? + -\ IgM •A IgG \ other \ Does whole cell or endotoxin immunization \ produce more protective Abs? + —X whole cell + -\ LPS \ Does Ab reactivity in vitro correlate with \ protective capacity? +-X ELJSA + -\ Immunodot blot \ Western immunotransblot \ Are human Abs more protective than \ others? + —\ human + —\ other species \ Is activity of secondary mediators affected? \ + A. TNF-a +A

-

IL-1

FIGURE 2. Meta-analysis of characteristics of protective anti-LPS antibodies. This figure represents an adaptation of the decision-tree format that was applied in order to summarize those characteristics that may be relevant to the in vivo protective capacity of anti-LPS antibodies.

directed against rough mutant bacteria, rough LPS, and against monophosphoryl and diphosphoryl lipid A using Western immunotransblot analysis and ELISA may serve to delineate those epitopes that serve to simultaneously confer cross-reactivity and cross-protective Ab capacity. Rapid development of both monoclonal and polyclonal reagents directed against these selected antigenic sites could then occur. d. Does Antibody Class or Subclass Affect Protective Capacity? Previous studies indicate that anti-O Abs and anti-deep-core/lipid A Abs function in similar fashions. However, the relationship of Ab class and subclass to protective capacity has not been completely evaluated. Correlation of protective capacity with Ab class and subclass would aid in the development of screening assays for protective Abs. In addition, production of protective mAbs may be increased through the development of class switch clones derived from highly cross-reactive mAb-secreting hybridoma cell lines.

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Handbook of Mediators in Septic Shock

e. Does Whole Cell or Endotoxin Immunization Produce more Protective Antibodies? Highly protective serotype-specific Abs result from immunization of animals with either whole bacteria or LPS from wild-type organisms. The high density of O saccharides on the bacterial surface and the relative stability of O saccharides to chemical manipulation during LPS purification contributes to the efficacy of these immunogens in the production of protective Abs. More marked effects of the physical association of immunogens upon protective capacity may be found when anti-deep-core/lipid A Abs are analyzed. It is unclear if: (1) immunization with rough LPS or rough mutant bacteria maximizes Ab production to deep-core/lipid A epitopes; (2) the use of mutants or purified LPS immunogens results in the production of Abs that are protective against wild-type organisms; or (3) there is a selective advantage in the use of one form of immunogen over the other in the production of cross-protective Abs. /. Does Antibody Reactivity In Vitro Correlate with Protective Capacity? A variety of in vitro methods have been developed to select cross-reactive Abs for use in protection studies, yet few studies have correlated protective capacity to binding observed in vitro. Correlation may aid in the selection of in vitro targets which best reflect binding. Of particular significance may be the in vitro reactivity of anti-deep-core/lipid Abs to wild type organisms as a predictor of protective capacity. g. Are Human Antibodies more Protective than Those Made in Other Species? Polyclonal Abs from human, murine and equine sources have been utilized in human and animal studies of protection. Other Ab sources, including equine and lapine, have been used in animal models. MAb studies have employed primarily murine mAbs. The advent, however, of human and chimeric myeloma fusion partners has increased the availability of human mAbs. No comparisons of the efficacy of human mAbs to those of other species has been attempted. h. Do Protective Antiendotoxin Antibodies Affect the Activity of Second Mediators? The production of IL-1, IL-6, and TNF by macrophages in vitro after endotoxin stimulation has been shown to correlate with in vivo lethality. The administration of protective serotype-specific anti-LPS mAbs appears to reduce systemic TNF levels during experimental Gram-negative bacterial infection in animal models and inhibits TNF production by macrophages in vitro. Abs to lipid A have been shown to inhibit in vitro human macrophage production of IL-1, IL-6, and TNF resulting from stimulation by lipid A but not after stimulation by LPS. Reduction in release of deleterious cytokines as a result of the protective capacity of anti-deep-core monoclonal or polyclonal Ab preparations remains to be demonstrated in vivo.

V. SUMMARY Intensive investigative efforts over the past decade have led to the characterization of the biologic effects of endotoxin in the mammalian host. It has become increasingly clear that this microbial cell-wall component is responsible for virtually all of the toxic effects that occur during Gram-negative bacterial sepsis. Endotoxin appears to exert its effects upon the host by provoking the release of macrophage monokines such as TNF, IL-1, IL-6, and others, that act upon the host to produce toxicity. Therapy of Gram-negative bacterial sepsis and shock has, for many years, centered around the use of antimicrobial agents, fluid resuscitation, hemodynamic monitoring, and metabolic support. Despite such therapy, Gramnegative bacterial sepsis remains a highly lethal disease process. For this reason, polyclonal

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and monoclonal antibody preparations directed against endotoxin have been developed and studied in vitro and in vivo in experimental models of infection, and tested in septic patients. Meta-analysis of these studies indicates that this form of therapy holds great promise, and that these antibodies may serve as important tools to dissect the mechanisms by which endotoxin exerts deleterious effects and causes host lethality, concurrently acting to stimulate further clinical and experimental research in the hopes of establishing the optimum means of interdicting endotoxin toxicity.

ACKNOWLEDGMENTS The authors would like to thank Ms. Laci Aase and Ms. Lora Esch for their expert assistance in the preparation of the manuscript.

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Surg., 125, 24, 1990. 35. Mayoral, J. and Dunn, D., Cross-reactive murine monoclonal antibodies directed against the core/lipid A region of endotoxin inhibit production of tumor necrosis factor, J. Surg. Res., 49, 287, 1990. 36. Beutler, B. and Cerami, A., Cachectin: more than a tumor necrosis factor, N. Engl. J. Med., 316, 379, 1987. 37. Carswell, E., Old, L., Kassel, R., Green, S., Fiore, N., and Williamson, B., An endotoxin-induced serum factor that causes necrosis of tumors, Proc. Natl. Acad. Sci. U.S.A., 72, 3666, 1975. 38. Waage, A., Halstensen, A., and Espevik, T., Association between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease, Lancet, i, 355, 1987. 39. Marano, M., Fong, Y., Moldawer, L., Wei, H., Calvano, S., Tracey, K., Barie. P., Manogue, K., Cerami, A., Shires, G., and Lowry, S., Serum cachectin/TNF in critically ill burn patients correlates with infection and mortality, Surg. Gynecol. Obstet., 170, 32, 1990. 40. 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42. Blick, M., Sherwin, S., Rosenblum, M., and Gutterman, J., Phase I study of recombinant tumor necrosis factor in cancer patients, Cancer Res., 47, 2986, 1987. 43. Mathison, J., Wolfson, E., and Ulevitch, R., Participation of tumor necrosis factor in the mediation of gram-negative bacterial lipopolysaccharide-induced injury in rabbits, J. Clin. Invest., 81, 1925, 1988. 44. Beutler, B., Milsark, I., and Cerami, A., Cachectin/tumor necrosis factor: production, distribution, and metabolic fate in vivo, J. Immunol., 135, 3972, 1985. 45. Tracey, K., Beutler, B., Lowry, S., Merryweather, J., Wolpe, S., Milsark, I., Hariri, R., Fahey, T., Zentilla, A., and Albert, J., Shock and tissue injury induced by recombinant human cachectin, Science, 234, 470, 1986. 46. Tracey, K., Lowry, S., Fahey, T., Albert, J., Fong, Y., Hesse, D., Beutler, B., Manogue, K., and Calvano, S., Cachectin/tumor necrosis factor induce lethal shock and stress hormone responses in the dog, Surg. Gynecol. 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Handbook of Mediators in Septic Shock 68. Dinarello, C., Interleukin-1 and the pathogenesis of the acute-phase response, N. Engl. J. Med., 311, 1413, 1984. 69. Cross, A., Sadoff, J., Kelly, N., Bern ton, E., and Gemski, P., Pretreatment with recombinant murine tumor necrosis factor-a/cachectin and murine interleukin la protects mice from lethal bacterial infection, J. Exp. Med., 169, 2021, 1989. 70. Van der Meer, J., Barza, M., Wolff, S., and Dinarello, C., A low dose of recombinant interleukin 1 protects granulocytopenic mice from lethal gram-negative infection, Proc. Natl. Acad. Sci. U.S.A., 85, 1620, 1988. 7 1 . Mclntyre, K., Unowsky, J., DeLorenzo, W., and Benjamin W., Enhancement of antibacterial resistance of neutropenic, bone marrow-suppressed mice by interleukin la, Infect. Immun., 57, 48, 1989. 72. Kampschmidt, R., Pulliam, L., and Upchurch, H., The activity of partially purified leukocytic endogenous mediator in endotoxin-resistant C3H/HeJ mice, J. Lab. Clin. 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95. Heflin, A. and Brigham, K., Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia, J. Clin. Invest., 68, 1253, 1981. 96. Staub, N., Schultz, E., and Albertine, K., Leukocytes and pulmonary vascular injury, Ann. N.Y. Acad. Sci., 384, 332, 1982. 97. Smith, R., Bowman, B., and Speziale, S., Interleukin-1 stimulates granulocyte exocytosis from human neutrophils, J. Leukoc. Biol, 39, 746, 1985. 98. Brigham, K., Metabolites of arachidonic acid in experimental lung vascular injury, Fed. Proc., 44, 43, 1985. 99. Bowers, R., Ellis, E., Brigham, K., and Bates, J., Effects of prostaglandin cyclic endoperoxides on the lung circulation of sheep, J. Clin. Invest., 63, 131, 1979. 100. Brigham, K. and Ogletree, M., Effects of prostaglandins and related compounds on lung vascular permeability, Bull. Eur. Pathophysiol. Resp., 17, 705, 1981. 101. Winn, R., Harlan, J., Nadir, B., Marker, J., and Hildebrandt, J., Thromboxane A2 mediates lung vasoconstriction but not permeability after endotoxin, /. Clin. Invest., 72, 911, 1983. 102. Ogletree, M., Snapper, J., and Brigham, K., Immediate pulmonary vascular and airway responses after intravenous leukotriene (LT) D4 injections in awake sheep, Physiologist, 28, 275, 1982. 103. Suffredini, A., Shelhammer, J., and Neumann, R., Intravenous endotoxin administration to normal humans causes gas exchange abnormalities and increased alveolar permeability (abst.), Clin. Res., 36, 511A, 1988. 104. Ravin, H., Rowley, D., Jenkins, C., and Fine, J., On the absorption of bacterial endotoxin from the gastrointestinal tract of the normal and shocked animal, /. Exp. Med., 112, 783, 1960. 105. Tamakuma, S., Rojas-Corona, R., Cuevas, P., and Fine, J., Demonstration of a lethal endotoxemia of intestinal origin in refractory non-septic shock, Ann. Surg., 17, 219, 1971. 106. Cuevas, P. and Fine, J., Role of intraintestinal endotoxin in death from peritonitis, Surg. Gynecol. Obstet., 134, 953, 1972. 107. Marshall, J., Christou, N., Horn, R., and Meakins, J., The microbiology of multiple organ failure. The proximal gastrointestinal tract as an occult reservoir of pathogens, Arch. Surg., 123, 309, 1988. 108. Cans, H. and Matsumoto, K., The escape of endotoxin from the intestine, Surg. Gynecol. Obstet., 139, 395, 1974. 109. Nolan, J., Hare, D., McDevitt, J., and Vilagat, A., In vitro studies of intestinal endotoxin absorption. I. Kinetics of absorption in the isolated gut sac, Gastroenterology, 72, 434, 1977. 110. Olafson. P., Nylander, G., and Olson, P., Endotoxin: routes of transport in experimental peritonitis, Am. J. Surg., 151, 443, 1986. 1 1 1 . Van Deventer, S., ten Gate, J., and Tygat, G., Intestinal endotoxemia: clinical significance, Gastroenterology, 94, 825, 1988. 112. Nolan, J., Endotoxin, reticuloendothelial function and liver injury, Hepatology, 1, 458, 1981. 113. Oleay, I., Kitaham, A., Miller, R., Drapanas, T., Trejo, R., and DiLuzio, W., Reticuloendothelial dysfunction and endotoxemia following portal vein occlusion, Surgery, 75, 64, 1974. 114. Brearly, S., Harris, R., Stone, P., and Keighley, M., Endotoxin in portal and systemic blood, Digest. Surg., 2, 70, 1985. 115. Palmer, K., Duderen, B., and Holsworth, C., Bacteriological and endotoxin studies in cases of ulcerative colitis submitted to surgery, Gut, 21, 851, 1980. 116. Kelley, C., Ingoldby, C., Blenkhorn, J., and Wood, C., Colonoscopy related endotoxemia, Surg. Gynecol. Obstet., 161, 332, 1985. 117. Scheifele, D., Olson, E., and Pendray, M., Endotoxemia and thrombocytopenia during neonatal necrotizing enterocolitis, Am. J. Clin. Pathol, 83, 227, 1985. 118. Mazuski, J., Platt, J., West, M., Simmons, R., Towle, H., and Cerra, F., Direct effects of endotoxin on hepatocytes, Arch. Surg., 123, 340, 1988. 119. Tate, W., Douglas, H., Braude, A., and Wills, W., Protection against lethality of E. coli endotoxin with O antiserum, Ann. N.Y. Acad. Sci., 133, 746, 1966. 120. Watson, D. and Kim, Y., Modification of host responses to bacterial endotoxins. 1. Specificity of pyrogen tolerance and role of hypersensitivity in pyrogenicity, lethality and skin reactivity, J. Exp. Med., 118, 425, 1963. 121. Kim, Y. and Watson, D., Modification of host responses to bacterial endotoxins. II, Passive transfer of immunity to bacterial endotoxin with fractions containing 19S antibodies, J. Exp. Med., 121, 751, 1965. 122. Greisman, S., Young, E., and DuBuy, B., Mechanisms of endotoxin tolerance. VIII. Specificity of serum transfer, J. Immunol., 111, 1349, 1973. 123. Chedid, L., Parant, M., Parant, F., and Boyer, F., A proposed mechanism for natural immunity to enterobacterial pathogens, J. Immunol., 100, 292, 1968. 124. Ziegler, E., Douglas, H., and Braude, A., Human antiserum for prevention of the local Shwartzman reaction and death from bacterial lipopolysaccharide, J. Clin. Invest., 52, 3236, 1973.

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125. McCabe, W., Kreger, B., and Johns, M., Type-specific and cross-reactive antibodies in gram-negative bacteremia, N. Engl. J. Med., 287, 261, 1972. 126. Lachman, E., Pitsoe, S., andGaffin, S., Anti-lipopolysaccharideimmunotherapy in management of septic shock of obstetric and gynaecological origin, Lancet, 1, 981, 1984. 127. Aitchison, J. and Arbuckle, D., Anti-endotoxin in the treatment of severe surgical septic shock. Results of a randomized, double-blind trial, S. Afr. Med. J., 68, 787, 1985. 128. Kunin, C., Separation, characterization, and biological significance of a common antigen in enterobacteriaciae, /. Exp. Med., 118, 565, 1963. 129. McCabe, W. and Greely, A., Common enterobacterial antigen. II. Effect of immunization on challenge with heterologous bacilli, Infect. Immun., 1, 386, 1973. 130. Pollack, M., Huang, A., Prescott, R., Young, L., Hunter, K., Cruess, D., and Tsai, C., Enhanced survival in Pseudomonas aeruginosa septicemia associated with high levels of circulating antibody to Escherichia coli endotoxin core, /. Clin. Invest., 72, 1874, 1983. 131. McCabe, W., Immunization with R mutants of S. Minnesota. I. Protection against challenge with heterologous Gram-negative bacilli, J. Immunol., 108, 601, 1972. 132. Bruins, S., Stumacher, R., Johns, M., and McCabe, W., Immunization with R mutants of Salmonella minnesota. III. Comparison of the protective effect of immunization with lipid A and the Re mutant, Infect. Immun., 17, 12, 1977. 133. Johns, M., Skehill, A., and McCabe, W., Immunization with rough mutants of Salmonella minnesota. IV. Protection by antisera to O and rough antigens against endotoxin, J. Infect. Dis., 147, 57, 1983. 134. DeMaria, A., Johns, M., Berberich, H., and McCabe, W., Immunization with rough mutants of Salmonella minnesota: initial studies in human subjects, /. Infect. Dis., 158, 301, 1988. 135. McCabe, W., DeMaria, A., Berberich, H., and Johns, M., Immunization with rough mutants of Salmonella minnesota: protective activity of IgM and IgG antibody to R595 (Re chemotype) mutant, J. Infect. Dis., 158, 291, 1988. 136. Ziegler, E., Douglas, H., Sherman, J., Davis, C., and Braude, A., Treatment of E. coli and Klebsiella bacteremia in agranulocytic animals with antiserum to a UDP-gal epimerase-deficient mutant, J. Immunol., 111,433, 1973. 137. Dunn, D. and Ferguson, R., Immunotherapy of gram-negative bacterial sepsis: enhanced survival in a guinea pig model by use of rabbit antiserum to Escherichia coli J5, Surgery, 92, 212, 1982. 138. Dunn, D., Mach, P., Condie, R., and Cerra, F., Anticore endotoxin F(ab')2 equine immunoglobulin fragments protect against lethal effects of Gram-negative bacterial sepsis, Surgery, 96, 440, 1984. 139. Greisman, S. and Johnston, C., Failure of antisera to J5 and R595 rough mutants to reduce endotoxemic lethality, J. Infect. Dis., 157, 54, 1988. 140. Greisman, S., DuBuy, B., and Woodward, C., Experimental Gram-negative bacterial sepsis: re-evaluation of the ability of rough mutant antisera to protect mice, Proc. Soc. Exp. Biol., 15, 482, 1978. 141. Sakulramrung, R. and Domingue, G., Cross-reactive immunoprotective antibodies to Escherichia coli O l l l rough mutant J5, J. Infect. Dis., 151, 995, 1985. 142. Chong, K. and Huston, M., Implications of endotoxin contamination in the evaluation of antibodies to lipopolysaccharides in a murine model of Gram-negative sepsis, J. Infect. Dis., 156, 713, 1987. 143. Gigliotti, F. and Shenep, J., Failure of monoclonal antibodies to core glycolipid to bind intact smooth strains of Escherichia coli, J. Infect. Dis., 151, 1005, 1985. 144. McCallus, D. and Norcross, N., Antibody specific for Escherichia coli J5 cross-reacts to various degrees with an Escherichia coli clinical isolate grown for different lengths of time, Infect. Immun., 55, 1042, 1987. 145. Aydintug, M., Inzana, T., Letonja, T., Davis, W., and Corbeil, L., Cross-reactivity of monoclonal antibodies to Escherichia coli J5 with heterologous Gram-negative bacteria and extracted lipopolysaccharides, J. Infect. Dis., 160, 846, 1989. 146. Ziegler, E., McCutchan, J., Fierer, J., Glauser, M., Sadoff, J., Douglas, H., and Braude, A., Treatment of Gram-negative bacteremia and shock with human antiserum to a mutant Escherichia coli, N. Engl. J. Med., 307, 1225, 1982. 147. Baumgartner, J., Glauser, M., McCutchan, J., Ziegler, E., van Melle, G., Kouber, M., Vogt, M., Meuhlen, E., and Luethy, R., Prevention of Gram-negative shock and death in surgical patients by antibody to endotoxin core glycolipid, Lancet, 2, 59, 1985. 148. McCutchan, J., Wolf, J., Ziegler, E., and Braude, A., Ineffectiveness of single-dose human antiserum to core glycolipid (E. coli J5) for prophylaxis of bacteremic, Gram-negative infections in patients with prolonged neutropenia, Schweiz. Med. Wochenschr., 113, 40, 1983. 149. Mattsby-Baltzer, I. and Alving, C., Antibodies to lipid A: occurrence in humans, Rev. Infect. Dis., 6, 553, 1984.

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150. Dunn, D., Mach, P., and Cerra, F., Monoclonal antibodies protect against lethal effects of Gram-negative sepsis, Surg. Forum, 14, 142, 1983. 151. Dunn, D., Bogard, W., and Cerra, F., Enhanced survival during murine Gram-negative sepsis by use of a murine monoclonal antibody, Arch. Surg., 120, 50, 1985. 152. Dunn, D., Bogard, W., and Cerra, F., Efficacy of type-specific and cross-reactive murine monoclonal antibodies directed against endotoxin during experimental sepsis, Surgery, 98, 283, 1985. 153. Kirkland, L. and Ziegler, E., An immunoprotective monoclonal antibody to lipopolysaccharide, J. Immunol., 132, 2590, 1984. 154. Dunn, D., Antibody immunotherapy of Gram-negative bacterial sepsis in an immunosuppressed animal model, Transplantation, 45, 424, 1988. 155. Bogard, W., Dunn, D., Abernethy, K., Kilgarriff, C., and Kung, P., Isolation and characterization of murine monoclonal antibodies specific for Gram-negative bacterial lipopolysaccharide: association of crossgenus reactivity with lipid A specificity, Infect. Immun., 55, 899, 1987. 156. Nelles, M. and Niswander, C., Mouse monoclonal antibodies reactive with J5 lipopolysaccharide exhibit serological cross-reactivity with a variety of Gram-negative bacteria, Infect. Immun., 46, 677, 1984. 157. Pollack, M., Chia, J., Koles, N., Miller, M., and Guelde, S., Specificity and cross-reactivity of monoclonal antibodies reactive with the core and lipid A regions of bacterial lipopolysaccharides, J. Infect. Dis., 159, 168, 1989. 158. Mutharia, L., Crockford, G., Bogard, W., and Hancock, R., Monoclonal antibodies specific for Escherichia coli J5 lipopolysaccharide: cross-reaction with other Gram-negative bacterial species, Infect. Immun., 45, 631, 1984. 159. Ward, M., Michalek, S. and McGhee, J., Monoclonal antibodies to Salmonella lipopolysaccharides: functional analysis of anti-lipid A antibodies, Clin. Exp. Immunol., 12, 157, 1988. 160. Miner, K., Manyak, C., Williams, E., Jackson, J., Jewell, M., Gammon, M., Ehrenfreund, C., Hayes, E., Callahan, L., Zweerink, H., and Sigal, N., Characterization of murine monoclonal antibodies to Escherichia coli J5, Infect. Immun., 52, 56, 1986. 161. Cody, C., Raymond, C., and Dunn, D., Fluorescence microscopic examination of monoclonal antibody binding to deep core/lipid A epitopes on live and heat-killed bacteria, submitted. 162. Dunn, D., Priest, B., and Condie, R., Protective capacity of polyclonal and monoclonal antibodies directed against endotoxin during experimental sepsis, Arch. Surg., 123, 1389, 1988. 163. Dunn, D., Ewald, D., Chandan, N., and Cerra, F., Immunotherapy of Gram-negative bacterial sepsis. A single murine monoclonal antibody provides cross-genera protection, Arch. Surg., 121, 58, 1986. 164. Priest, B., Brinson, D., Schroeder, D., and Dunn, D., Treatment of experimental Gram-negative bacterial sepsis with murine monoclonal antibodies directed against lipopolysaccharide, Surgery, 106, 147, 1989. 165. Teng, N., Kaplan, H., Herbert, J., Moore, C., Douglas, H., Wunderlich, A., and Braude, A., Protection against gram-negative bacteremia and endotoxemia with human monoclonal IgM antibodies, Proc. Natl. Acad. Sci. U.S.A., 82, 1790, 1985. 166. Pollack, M., Raubitschek, A., and Larrick, J., Human monoclonal antibodies that recognize conserved epitopes in the core-lipid region of lipopolysaccharide, J. Clin. Invest., 79, 1421, 1987. 167. Harkonen, S., Scannon, P., Mischak, R., Spiter, L., Foxall, C., Kennedy, D., and Greenberg, R., Phase I study of a murine monoclonal anti-lipid A antibody in bacteremic and nonbacteremic patients, Antimicrob. Agents Chemother., 32, 710, 1988. 168. Greenman, R., Schein, R., Martin, M., Wenzel, R., Maclntyre, N., Emmanuel, G., Chmel, H., Kohler, R., McCarthy, M., Plouffe, J., and Russell, J., Xoma Sepsis Study Group, A controlled clinical trial of E5 murine monoclonal IgM antibody to endotoxin in the treatment of Gram-negative sepsis, JAMA, 266, 1097, 1991. 169. Ziegler, E., Fisher, C., Sprung, C., Straube, R., Sadoff, J., and Foulke, G., Treatment of Gramnegative bacteremia and septic shock with HA-IA human monoclonal antibody against endotoxin, N. Engl. J. Med., 324, 429, 1991.

Chapter 2

POSSIBLE RELEVANCE OF BACTERIAL EXOTOXINS IN THE PATHOGENESIS OF SEPTIC SHOCK Sucharit Bhakdi

TABLE OF CONTENTS I.

Introduction

40

II.

General Features of Transmembrane Pore Formation

40

III.

Factors Influencing the Overall Susceptibility of Cells A. Cellular Factors B. Humoral Factors

41 41 43

IV.

Cell Biological and Pathophysiological Consequences of Membrane Damage by Pore-Forming Cytolysins A. Cytocidal Effects B. Secondary Cellular Reactions 1. Secretion 2. Stimulation of Arachidonic-Acid Metabolism 3. Derangement of Cytoskeleton Organization and Function 4. Other Processes

43 43 45 45 45 45 46

Conclusion and Perspectives

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V.

References

0-8493-3548-5/93/$0.00 + $.50 e 1993 by CRC Press, Inc.

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I. INTRODUCTION The objective of other assays contained in this volume is to evaluate the evidence for a causative role of various mediator substances in the development of septic shock using meta-analysis. The discussion of exotoxins in this context is, however, not yet possible because methods for quantifying potentially relevant toxins are not available. On the other hand, in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. Since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made in this chapter to provide a brief overview of this generally neglected topic. Protein toxins act intracellularly or they damage the integrity and function of the plasma membrane. Major representatives of the former group are the adenosine diphosphate (ADP)ribosylating toxins (e.g., cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. Most medically relevant toxins of this category have been studied in great detail. Although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. In contrast, experimental evidence is accumulating for a role of membrane-damaging toxins in the pathogenesis of inflammatory lesions. Formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur,1'2 the present discussion will be restricted to this category of toxins. The author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. Thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage, so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. Major obstacles that currently block the path towards meta-analysis will be alluded to in the concluding section.

II. GENERAL FEATURES OF TRANSMEMBRANE PORE FORMATION All pore-forming toxins are produced and released by bacteria initially as water-soluble proteins, but are able to undergo a unique and possibly always irreversible transition from a hydrophilic to an amphiphilic state upon interaction with a target lipid bilayer.1'2 Primary binding to a target membrane may require the presence of specific binder molecules. A well documented example for this type of interaction is represented by the sulfhydryl-activated cytolysins (e.g., streptolysin-O [SLO]), which probably initially bind to membrane cholesterol. Often, however, cytolysins will be of the "receptorless" type that binds indiscriminately to lipid bilayers. Escherichia coll hemolysin and related toxins are considered representatives of this second group. Staphylococcal a-toxin occupies an intermediate position, since it can interact both with specific binding sites and via nonspecific absorption to lipid bilayers (unpublished data). We will briefly consider the interactions of a-toxin with target cells, since they have been characterized in detail. Only certain cell types express the highaffinity binding sites for a-toxin on their surface. When present at low concentrations, the toxin will bind exclusively to such sites, so that cells lacking the toxin-binder will be exempted from attack. At high concentrations, a-toxin will additionally absorb in a nonspecific fashion to lipid bilayers, causing damage also to cells that are devoid of the specific binding site. When present, the high-affinity binding sites are usually only expressed in low numbers; therefore, overall consumption of toxin is low. The attack process is very effective, requiring only a small number of toxin molecules to be deposited on each cell target in order for membrane permeabilization to take place.

Bhakdi

41

Insertion into the lipid bilayer follows as a second and sometimes temporally dissociated step after toxin molecules have bound to the membrane surface. Generally, membrane insertion can take place at 0°C, albeit at a slower rate. In some instances, pores are probably generated by insertion of protein monomers into the bilayer (e.g., E. coli hemolysin3). Alternatively, some pore-formers need to aggregate with each other to form oligomers before pores can be generated; this is the case with a-toxin4"6 and SLO.7 Monomeric pores are too small to be visualized by electron microscopy, whereas several oligomeric pores have been amenable to ultrastructural characterization. In the latter cases, the pores are generally seen as partially or fully circularized structures. Oligomeric pores are sometimes heterogeneous in size, due to variations in the number of protomers that constitute the individual channels.7 Staphylococcus aureus a-toxin oligomers tend to be more homogeneous (Figure 1). The effective functional diameters of pores span a very large range, from approximately 1.5 nm (a-toxin) to over 35 nm (SLO). Even the smallest pores are, however, large enough to permit rapid transmembrane flux of ions and small molecules. Generally, oligomeric pores have been found to display little ion selectivity and no dependence on membrane potential.8 In contrast, monomeric pores created by E. coli hemolysin require a correct transmembrane potential and display a strong selectivity for cations over anions.9 Although the primary structures of a-toxin,10 E. coli hemolysin," and several sulfhydrylactivated toxins12-13 are known, the domains that are membrane-embedded have yet to be identified. No details are available on the polypeptide conformation and three-dimensional structure of any protein pore. One surface of the membrane-embedded protein must necessarily be hydrophobic to permit its stable interaction with the lipid domain of the bilayer. The other must be hydrophilic to permit the passage of ions and small hydrophilic molecules across the membrane.1'2 It is not known whether the pores always span the bilayer, or whether partial penetration might suffice to generate a functional lesion. The extent to which pores are lined by protein is also unknown. In the case of the atoxin hexamer as well as with fully circularized SLO pores, the channels appear to indeed traverse the interior of the protein cylinders. However, incompletely circularized SLO pores may be lined by an edge of free lipid arising through lateral repellent of apolar lipid domains from the inserted, hydrophilic faces of the protein.7 It is conceivable that monomerically inserted proteins such as hemolysin of E. coli similarly do not require circularized protein structures to generate transmembrane channels. The fate of pores after their formation in membranes of nucleated cells has not been analyzed in any detail. No study on the deposition of any pore-forming bacterial cytolysin in human tissues has been performed to date.

III. FACTORS INFLUENCING THE OVERALL SUSCEPTIBILITY OF CELLS In order to assess the relevance of any cytolysin, it is essential to determine which cells represent vulnerable targets under physiological conditions. Two main categories of factors, cellular and humoral, influence the overall susceptibility of cells towards a given toxin. A. CELLULAR FACTORS Specific high-affinity binding sites — When present, such sites a toxin to the respective cell target. Nonspecific cell-surface characteristics — Nonspecific factors organization of charged molecules can probably affect the efficiency diffusion of a toxin to a membrane bilayer occurs. For reasons discussed may imply that a toxin will not be able to efficiently attack a cell conditions.

will direct attack of such as the surface and speed at which below, slow binding under physiological

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Handbook of Mediators in Septic Shock

FIGURE 1. (A) Negatively stained fragment of rabbit erythrocyte lysed with staphylococcal a-toxin. Numerous 10-nm ring-shaped structures are seen over the membrane (arrows). (B) Isolated toxin hexamer in detergent solution. (C) Lecithin liposomes carrying reincorporated a-toxin hexamers. The hexamers are seen as stubs along the edge of the liposomal membrane and as rings over the membrane (arrows). Characteristically, liposomes that escape incorporation of the toxin are impermeable to the stain. (D) Negatively stained erythrocyte membrane lysed by streptolysin-0 (SLO) showing numerous 25 to 100 nm long and approximately 7.5 nm broad, curved rods of 13 to 16 nm inner radius of curvature. Most rods are approximately semicircular, often joined in pairs at their ends. Dense accumulation of stain are seen at the concave side of the rods. When these do not form closed profiles, the stain deposit is partly bordered by a "free" edge of the erythrocyte membrane (arrows). (E) Negative staining of isolated SLO oligomers, showing numerous curved rod structures identical to those found on toxin-treated membranes. (F) purified SLO complexes reincorporated into cholesterol-free lecithin liposomes. The toxin oligomers form holes in the liposomes (unlabeled arrows); (p) indicates a lesion seen in profile. (Bars = 100 nm.) (Uranylacetate was used as negative stain in A. Sodium silicotungstate was used as negative stain in B through F.)

43

Bhakdi

Intrinsic resistance — Intrinsic resistance towards a toxin is exemplified by human leukocytes and their resistance to Staphylococcus aureus a-toxin (unpublished data). The term implies that a toxin can bind to a cell without inflicting functional membrane damage. The mechanisms underlying such intrinsic resistance have not been delineated. Repair mechanisms — Theoretically, it is conceivable that nucleated cells can repair a limited number of damaged membrane sites. However, membrane repair has not been clearly documented for any bacterial pore-forming to date. In those cases studied (e.g., attack of leukocytes by E. coli hemolysin), repair mechanisms could not be detected even following attack by low toxin doses.14 B. HUMORAL FACTORS Pore-forming bacterial toxins are generally potent immunogens, and respective antibodies are therefore normally present in healthy individuals. Moreover, many pore-formers are bound and inactivated by plasma lipoproteins.15 When liberated into the host environment, the attack of a given toxin will thus be counteracted by several inhibitory humoral factors. The net outcome will be determined by the affinity, binding kinetics, and the concentration of the reaction partners. The speed of binding of a toxin to a target cell may vary considerably. For example, E. coli binds extremely rapidly to leukocytes but relatively slowly to erythrocytes (unpublished data). Whenever present, humoral components hence will be able to prevent E. coli hemolysin from attacking erythrocytes, but their action may often be too slow to similarly protect leukocytes. As a consequence, addition of low amounts of E. coli hemolysin to whole human blood results in its selective attack on leukocytes'4 and monocytes.16 Another example for selective attack of a pore-forming cytolysin is the action of a-toxin on human blood platelets. Upon addition of this toxin to whole human blood, the cytolysin binds rapidly to specific sites on blood platelets and leukocytes, less efficiently to lymphocytes, and not at all to erythrocytes. Leukocytes can intrinsically withstand toxin attack, but platelets and monocytes are highly susceptible. As a consequence, selective damage to the latter cells ensues.17'18 Theoretically, it should be possible to augment the neutralizing capacity of antibodies by increasing antibody liters. In a model study, high-titered human hyperimmune globulins against S. aureus a-toxin were prepared by immunization of volunteers, and tested for their neutralizing capacity in vitro and in vivo. Experimental protocols omitted a preincubation step in order to more closely mimic the situation arising during bacterial infections. It was found that these hyperimmune globulins, but not any of the commercially available intravenous immunoglobulin preparations, were able to totally suppress the deleterious action of a-toxin.19 Clinical trials with these hyperimmune globulins are currently being planned.

IV. CELL BIOLOGICAL AND PATHOPHYSIOLOGICAL CONSEQUENCES OF MEMBRANE DAMAGE BY POREFORMING CYTOLYSINS A. CYTOCIDAL EFFECTS Cell death is the most obvious and inevitable consequence of transmembrane poreformation if a lesion cannot be removed or repaired. Death ensues because the cell is rapidly depleted of adenosine triphosphate (via efflux through the pores; Figure 2) and because it

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Handbook of Mediators in Septic Shock

FIGURE 2. Release of ATP from PMN (106 cells in 400 p.1) suspended in PBS (A) or in a solution mix containing physiological concentrations of human serum albumin, high-density lipids, low density lipids, and IgG (B) induced by E. coli hemolysin (ECH). ATP was measured continuously using the firefly assay. The final concentrations of ECH applied are given in HU/ml. The assays were calibrated with ATP as depicted. Note the relatively poor protection of PMN against toxin action afforded by the plasma protein components. (Reproduced from Bhakdi et al., J. Exp. Med., 169, 737-754, 1989. With copyright permission of the Rockefeller University Press.)

Bhakdi

45

is unable to counteract the deleterious effects of ionic disequilibrium and loss of its "milieu interieur" that is essential for sustainment of metabolic processes. Loss of cellular function can have direct consequences. For example, killing of leukocytes and monocytes by E. coli hemolysin cripples the local phagocytic defense system and probably fosters the invasion of other pathogens that colonize the lesions. Damage to endothelial cells by Staphylococcus aureus a-toxin causes massive derangements in lung vasculature, and loss of endothelial cell lining leads to rapid development of pulmonary edema.20'21 Renal tubular epithelia are highly susceptible to E. coli hemolysin22 and this may partially explain disturbances in tubular function that commonly develop during renal infections with toxin-producing bacterial strains. B. SECONDARY CELLULAR REACTIONS Until recently, the awareness was generally lacking that perturbation of the plasma membrane permeability barrier might trigger various cellular reactions. Today, it is apparent that such reactions are inevitable and they may contribute significantly to the progression of tissue lesions. Passive flux of extracellular calcium ions into the damaged cells represents a major trigger for such secondary reactions.1-2 Depending on the cell target, a variety of responses may ensue, of which the following have been studied in some detail. 1. Secretion Exocytoxic liberation of vesicular components has been demonstrated in leukocytes,14 platelets,17'18 and neurological cells.23 Leukocytes succumbing to attack by E. coli hemolysin release large amounts of vesicular components.14 The release of elastase is noteworthy since this enzyme has been discussed as a factor that may contribute to the development of pulmonary lesions. Platelets attacked by a-toxin secrete large quantities of granule constituents including platelet-factor 4 and Factor V.17'18 Release of the latter leads to assembly of platelet-bound, prothrombinase complexes that generate thrombin. S. aureus a-toxin thus activates human platelets and promotes coagulation (Figure 3), processes that bear high potential relevance in staphylococcal infections. 2. Stimulation of Arachidonic Acid Metabolism Since elevations in intracellular calcium concentrations trigger arachidonic-acid metabolism, it is not surprising that formation of transmembrane pores by bacterial toxins leads to generation of eicosanoids in nucleated cells. Endothelial cells treated with very low doses of a-toxin orE. coli hemolysin produce large amounts of prostaglandins24 and leukotrienes.25 Such processes could obviously contribute to the development of septic shock. Perfusion of isolated, blood-free rabbit lungs with a-toxin,20'21 E. coli hemolysin,26 or with viable hemolytic E. coli21 leads to massive lung vascular injury. A steep rise in pulmonary arterial pressure is observed that probably derives from thromboxane overload. In addition, vascular leakage ensues, due possibly to damage of the endothelial cell layer. These findings collectively point to the hitherto neglected possibility that bacterial cytolysins can produce pulmonary lesions leading to conditions akin to those seen in adult respiratory-distress syndrome. Uncontrolled production and liberation of eicosanoids may be a significant factor contributing to systemic inflammatory reactions that are the hallmark of septic shock. 3. Derangement of Cytoskeleton Organization and Function Steep rises in intracellular calcium levels could cause functional derangements of contractile microfilaments. In a model study, endothelial cells were found to respond to attack by very low doses of a-toxin with formation of intercellular gaps that permitted free passage of macrornolecules across the originally intact monolayer.28 Evidence was obtained that this derived from contraction and rounding up of the adherent cells, probably because of cyto-

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Handbook of Mediators in Septic Shock

FIGURE 3. Reduction in clot times of recalcified human plasma by a-toxin. Citrated plasma samples containing the depicted numbers of platelets (per nanoliter) were given 12 mM Ca2+ and a-toxin at the given final concentrations. Marked reductions of clot times were noted at toxin concentrations of 1 to 1.5 fig/ml, dependent on the presence of platelets in the plasma sample. No clot indicates lack of clot formation within 900 s. (Reproduced from Bhakdi et al., J. Exp. Med., 168, 527-542, 1988. With copyright permission of the Rockefeller University Press.)

skeletal dysfunction. If such processes occurred in vivo, they would be expected to create leaks across the vascular endothelial lining, and promote edema formation. 4. Other Processes Leukocytes attacked by very low doses of E. coli hemolysin produce enhanced amounts of oxygen radicals (unpublished data). These cells also become hyperresponsive with respect to superoxide production when exposed to conventional stimuli such as phorbol esters. The cause of these findings is not known but may be related to a calcium-dependent stimulation of phospholipase C with subsequent production of diacylglycerol which in turn activates surface-associated oxidase. Production of oxygen radicals has been implicated as an important cause of pulmonary damage during septic shock. Finally, attack on monocytes by 5. aureus a-toxin29 and E. coli hemolysin16 can cause liberation of large amounts of interleukin (IL)-lp from these cells. A prerequisite for this appears to be the prior intracellular accumulation of IL-lfi precursor. Thus, unstimulated monocytes do not release IL-1(3 in response to toxin attack, but large amounts of cytokine are liberated from cells that have been in short-term culture or that have been co-stimulated with very low amounts of lipopolysaccharide (LPS).16'29 Pore-forming toxins may therefore synergize with LPS and with other substances that induce IL-lp synthesis. This concept is

47

Bhakdl

FIGURE 4. Effects of incubation of viable E. coli with freshly isolated human monocytes. Six strains were used with the given characteristics and at the given, approximate bacteria to cell ratios. Measurements of cellular ATP (A) and of IL-lfJ (B) and tumor necrosis factor (TNF)-a (C) in the cell supernatants were performed after 70 min incubation at 37°C. All strains of toxin-producers evoked ATP-depletion and IL-lji production, but depressed TNFproduction presumably due to their cytocidal action. The 100% ATP-content corresponded to 140 pmol ATP per well. (Reproduced from Bhakdi et al., J. Clin. Invest., 85, 1746-1753, 1990. With copyright permission of the Rockefeller University Press.)

supported by the finding that incubation of freshly isolated monocytes with toxin-producing E. coli at a cellular ratio of only 1:1 causes production and release of IL-1J3 within a few hours (Figure 4).16 Such a dramatic and rapid effect was not observed when cells were incubated with genetically related, nonhemolytic E. coli. The possible relevance of IL-lp in the pathogenesis of septic shock is currently being studied in several laboratories.

V. CONCLUSION AND PERSPECTIVES In contrast to endotoxins, membrane-damaging bacterial exotoxins have hitherto received little attention as possible contributors to the pathogenesis of septic shock. Despite the fact that they are produced by many important bacterial pathogens, the very existence of these toxins has escaped the attention of many clinicians. Attack of pore formers on nucleated cells and platelets will evoke complex secondary reactions including exocytosis, stimulation of eicosanoid production, release of oxygen radicals, and liberation of cytokines. These toxins can therefore influence hemostasis and trigger production and release of many potent mediators whose possible roles in the pathogenesis of septic shock are currently in the forefront of discussion.

48

Handbook of Mediators in Septic Shock

Because of their unusual properties, pore-forming toxins are unfortunately very difficult to quantify in biological fluids and tissues. An appreciable fraction will become bound to blood or tissue cells, and another fraction will become bound to antibodies and lipoproteins. Quantitation of the proteins is only feasible if methods can be devised to release them from their bound state. At present, no satisfactory solution to the problem is available. Since the primary dilemma of constructing sensitive immunoassays has not been solved, meta-analysis is impossible. Furthermore, an additional circumstance warrants consideration. Pore-forming toxins need not necessarily be released in measurable quantities into body fluids or tissues in order to exert their action. Very few molecules are generally required to generate a membrane lesion, and attack on a susceptible cell may occur within a very small, circumscribed area when viable bacteria gain intimate contact with a target. This will probably suffice to evoke the various cytotoxic effects discussed above. Indeed, seeding toxin-producing E. coli onto monocytes at a cellular ratio of only 1:1 led to release of IL-1 (3 followed by cell death, in the absence of detectable toxin in the cell supernatants.16 Similarly, perfusion of isolated, blood-free rabbit lungs with hemolytic E. coli also caused massive derangements in the pulmonary vasculature in the absence of detectable circulating toxin.26 Thus, even if measurements of toxin concentrations in body tissues and fluids become possible, the feasibility of conducting meaningful meta-analyses could still remain questionable.

REFERENCES 1. Bhakdi, S. and Tranum-Jensen, J., Damage to mammalian cells by proteins that form transmembrane pores, Rev. Physioi. Biochem. Pharmacol., 107, 147, 1987. 2. Bhakdi, S. and Tranum-Jensen, J., Damage to cell membranes by pore-forming bacterial cytolysins, Prog. Allergy, 40, 1, 1988. 3. Bhakdi, S., Mackman, N., Nicaud, H. M., and Holland, I. B., E. coli hemolysin may damage target cell membranes by generating transmembrane pores, Infect. Immun., 52, 634, 1986. 4. Fiissle, R., Bhakdi, S., Sziegoleit, A., Tranum-Jensen, J., Kranz, T., and Wellensiek, H. J., On the mechanism of membrane damage by S. aureus a-toxin, J. Cell. Biol., 91, 83, 1981. 5. Bhakdi, S., Muhly, M., and Fiissle, R., Correlation between toxin binding and hemolytic activity in membrane damage by staphylococcal a-toxin, Infect. Immun., 46, 318, 1984. 6. Reichwein, J., Hugo, F., Roth, M., Sinner, A., and Bhakdi, S., Quantitative analysis of the binding and oligomerisation of staphylococcal a-toxin in target erythrocyte membranes, Infect. Immun., 55, 2940, 1987. 7. Bhakdi, S., Tranum-Jensen, J., and Sziegoleit, A., Mechanism of membrane damage by streptolysinO, Infect. Immun., 47, 52, 1985. 8. Menestrina, G., Ionic channels formed by Staphylococcus aureus a-toxin: voltage-dependent inhibition by divalent cations, J. Membr. Biol., 90, 177, 1986. 9. Menestrina, G., Mackman, N., Holland, I. B., and Bhakdi, S., E. coli hemolysin forms voltagedependent ion channels in lipid membranes, Biochim. Biophys. Ada, 905, 109, 1987. 10. Gray, G. S. and Kehoe, M., Primary sequence of the a-toxin gene from Staphylococcus aureus Wood 46, Infect. Immun., 46, 615, 1984. 11. Felmlee, T., Pellet, S., and Welch, R., Nucleotide sequence of an Escherichia coli chromosomal hemolysin, J. Bact., 163, 94, 1985. 12. Kehoe, M. A., Miller, L., Walker, J. A., and Boulnois, G., Nucleotide sequence of the streptolysin-O (SLO) gene: structural homologies between SLO and other membrane-damaging, thiol-activated toxins, Infect. Immun., 55, 3228, 1987. 13. Tweten, R., Nucleotide sequence of the gene for perfringolysin O from Clostridium perfringens: significant homology with the genes for streptolysin O and pneumolysin, Infect. Immun., 56, 3235, 1988. 14. Bhakdi, S., Greulich, S., Muhly, M., Eberspacher, F., Becker, H., Thiele, A., and Hugo, F., Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes, /. Exp. Med., 169, 737, 1989. 15. Bhakdi, S., Fiissle, R., Utermann, G., and Tranum-Jensen, J., Binding and partial inactivation of S. aureus a-toxin by human plasma low density lipoprotein, J. Biol. Chem., 258, 5899, 1983.

Bhakdi

49

16. Bhakdi, S., Muhly, M., Korom, S., and Schmidt, G., Effects of Escherichia coli hemolysin on human monocytes. Cytocidal action and stimulation of interleukin 1 release, /. Clin. Invest., 85, 1746, 1990. 17. Bhakdi, S., Muhly, M., Mannhardt, U., Hugo, F., Klapettek, K., Mueller-Eckhardt, C., and Roka, L., Staphylococcal a-toxin promotes blood coagulation via activation of human platelets, /. Exp. Med., 168, 527, 1988. 18. Arvand, M., Bhakdi, S., Dahlback, B., and Preissner, K. T., Staphylococcus aureus a-toxin attack on human platelets promotes assembly of the prothrombinase complex, J. Biol. Chem., 265, 14,377, 1990. 19. Bhakdi, S., Mannhardt, H., Ronneberger, R., and Hungerer, K. D., Human hyperimmune globulin protects against the cytotoxic action of Staphylococcal a-toxin in vitro and in vivo, Infect. Immun., 57, 3214, 1989. 20. Seeger, W., Bauer, M., and Bhakdi, S., Staphylococcal a-toxin elicits hypertension in isolated rabbit lungs due to stimulation of the arachidonic acid cascade, J. Clin. Invest., 74, 849, 1984. 21. Seeger, W., Birkemeyer, R. G., Ermert, L., Suttorp, N., Bhakdi, S., and Duncker, H. R., Staphylococcal a-toxin-induced vascular leakage in isolated perfused rabbit lungs, Lab. Invest., in press, 1990. 22. Keane, W. F., Welch, R., Gekker, G., and Peterson, P. K., Mechanism of Escherichia coli alphahemolysin induced injury to isolated renal tubular cells, Am. J. Pathol., 126, 350, 1987. 23. Ahnert-Hilger, G., Bhakdi, S., and Gratzl, M., Minimal requirements for exocytosis: a study using PC 12 cells permeabilized with Staphylococcal a-toxin, /. Biol. Chem., 260, 12,730, 1985. 24. Suttorp, N., Seeger, W., Dewein, E., Bhakdi, S., and Roka, L., Staphylococcal a-toxin stimulates synthesis of prostacyclin by cultured endothelial cells from pig pulmonary arteries, Am. J. Physiol., 248, C127, 1985. 25. Grimminger, F., Walmrath, D., Birkemeyer, R. G., Bhakdi, S., and Seeger, W., Leukotriene- and hete-generation elicited by low doses of Escherichia coli hemolysin in rabbit lungs, Infect. Immun., 58, 2659, 1990. 26. Seeger, W., Walter, H., Suttorp, N., and Bhakdi, S., Thromboxane-mediated hypertension and vascular leakage evoked by low doses of Escherichia coli hemolysin in rabbit lungs, J. Clin. Invest., 84, 220, 1988. 27. Grimminger, F., Thomas, M., Obernitz, R., Walmrath, D., Bhakdi, S., and Seeger, W., Inflammatory lipid mediator generation elicited by viable hemolysin-forming Escherichia coli in lung vasculature, /. Exp. Med., 172, 1115, 1990. 28. Suttorp, N., Hessz, T., Seeger, W., Wilke, A., Koob, R., Lutz, F., and Drenckhahn, D., Bacterial exotoxins and endothelial permeability for water and albumin in vitro, Am. J. Physiol., 2555, C368, 1988. 29. Bhakdi, S., Muhly, M., Korom, S., and Hugo, F., Release of interleukin-l(Ja associated with potent cytocidal action of Staphylococcal a-toxin on human monocytes, Infect. Immun., 51, 2512, 1989.

Part II. Biogenic Amines

Chapter 3 HISTAMINE IN SEPTIC/ENDOTOXIC SHOCK E. Neugebauer, D. Rixen, W. Lorenz

TABLE OF CONTENTS I.

Introduction

52

II.

Database: Histamine in Septic/Endotoxic Shock

54

III.

Koch-Dale Criteria 1 and 2: Presence in Disease and Absence in Health A. Conducting the Search and Selection of Studies for Inclusion in the Meta-Analysis B. Method of the Meta-Analysis with the Decision Tree: The Criteria and Methodological Standards of the Test Nodes in the Decision Tree 1. Assay for Histamine Determination Reliable? 2. Sample Preparation Appropriate? 3. Sampling in the Relevant Body Fluid or Organ System? 4. Sampling at the Right Time? 5. Study Design Appropriate? 6. Shock Model Clinically Relevant? 7. Species Clinically Relevant? 8. Histamine-Release Response Caused by the Shock Itself? 9. Evaluation of Shortcomings in the Individual Studies and Their Interpretation C. Results of the Meta-Analysis with the Decision-Tree 1. Primates (Man and Monkey) Dog and Cat 2. 3. Rabbit, Rat, and Mouse D. Descriptive Statistics on Methodological Shortcomings and Their Evaluation

54

IV.

Koch-Dale Criterion 3: Eliciting the Disease by Exogenous Administration, Endogenous Release, or Formation Pro — Histamine is a "Shock Toxin'' A. B. Contra — Histamine is not a "Shock Toxin'' C. Conclusion

V.

Koch-Dale Criterion 4: Blockade of Histamine Effects by Antagonists and Preventing or Ameliorating the Disease A. Conducting the Search and Selection of Studies for Inclusion in the Meta-Analysis B. Method of the Meta-Analysis with the Decision Tree: The Criteria and Methodological Standards of the Test Nodes in the Decision Tree 1. Are Studied Antagonists Efficient and Specific for Histamine?

0-8493-3548-5/93«0.00 + $.50 C 1993 by CRC Press, Inc.

54 55 56 61 62 64 65 65 66 67 67 68 68 69 75 79 79 89 93 96 96 97 97 99

51

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Handbook of Mediators in Septic Shock

C.

D.

a. Efficacy of Antihistamines b. Specificity of Antihistamines 2. Correct Dose-Response Relationship? Correct Temporal Relationship? 3. 4. Study Design Appropriate and Shock Model Clinically Relevant? 5. Species Clinically Relevant? 6. Measured Hemodynamic, Respiratory, or Metabolic Parameters Adequate? 7. Development of Shock or Outcome Affected? 8. Evaluation of Shortcomings in the Individual Studies and Their Interpretation Results of the Meta-Analysis with the Decision Tree 1. Man 2. Dog, Sheep, Pig, and Cat Rat and Mouse 3. a. Rat b. Mouse Descriptive Statistics on Methodological Shortcomings and Their Evaluation ,

99 99 100 101 101 101 101 102 103 103 103 103 108 108 113 113

VI.

Discussion

115

VII.

Summary

116

References

118

I. INTRODUCTION Over the years, histamine, like other mediators, has been assigned a greater or less important role in the pathogenesis of different types of shock. This amine is the mediator with the longest history of all active substances currently discussed in shock. It has been considered as a shock toxin since 1920, when it was discovered that this amine dilates small blood vessels, increases capillary permeability and produces a lethal shock syndrome when given in large doses.21 About 20 years later, Moon, primarily on the basis of evidence of histological changes noted in the shocked subject, proposed that histamine was the single etiologic and toxic substance in all kinds of shock.96 In 1960, Hinshaw's group introduced histamine specifically into the pathogenetic concept of septic shock.48 They recognized and emphasized the similarities between the vascular actions of histamine and those of endotoxin. Consequently, they interpreted their findings as support for the hypothesis that histamine plays a crucial role in the progressive development of hypotension following endotoxin administration. Moreover, the simultaneous rise of another hypothesis, which postulated a continuous action of histamine during progressive shock (Schayer's115 induced histamine

Neugebauer, Rixen, and Lorenz

53

concept), catapulted histamine, especially after the World Congress of Physiology in Ley den 1962, to the star of the show in septic shock. With only a few mediators to account for most of the biological effects and clinical features of shock, histamine has held the leading role. There were, however, some inconsistent findings in the experiments. Histamine administration could increase the survival rate in endotoxic shock, released histamine failed to alter vascular and metabolic responses to endotoxin, and long-term infusion of histamine did not elicit pulmonary edema.31-56'100 A more important loss of credit was produced by the discovery of other substances, which in bioassays were more vasoactive. Immediately, biologically plausible arguments were constructed for kinins, prostanoids, and endorphins. Finally, the keenness of fickle scientists for this year's novelty on the market (leucotrienes, platelet activating factor, oxygen free radicals, cytokins, etc.) pushed histamine back from being the cause in septic shock into the group of other possible factors associated with septic shock. Now, histamine has become an "ugly" mediator, no longer interesting to pharmacologists and physiologists in shock research and thus no longer, or only rarely, included in flow diagrams or other graphic presentations of shock pathogenesis. Recent review articles on mediators in septic shock are examples of this type of general assessment.27'55'102-132-133 Despite its well-known subjectivity, its questionable validity and its inefficiency, the traditional narrative approach to reviewing and evaluating the literature is still the custom in life sciences and clinical medicine.130 However, in the last 15 years new methods such as meta-analysis have been developed, especially in the social sciences, to overcome the drawbacks of the traditional procedure.37-38-121-130 Meta-analysis uses existing data, as does the narrative approach, but provides a more scientific alternative by focusing on the qualitative and quantitative evaluation of findings and methods in groups of independent studies.130 It is a prerequisite for meta-analysis that the original studies must report findings in sufficient numerical details for quantitative analysis. To establish a cause and effect relationship of a mediator in a given disease, i.e., septic/ endotoxic shock, it is generally accepted to use the four classical Koch-Dale criteria.91 These criteria include: 1. 2. 3. 4.

The presence of the mediator in the disease (plasma or target organs) The absence of the mediator in health The possibility to elicit the disease by exogenous administration or endogenous formation/release To block its effect(s) by inhibitors of synthesis/release or by specific antagonists and preventing or ameliorating the disease

Unfortunately, much confusion exists due to the misinterpretation of published findings as cause and effect. To increase empirical evidence for a causal relationship, several other types of association — mainly bias, role of chance, and confounding bias — must be excluded. This can be achieved — thus coming to a more valid evaluation of the current status of single mediators in shock — by using the model of a decision tree. This model ("thinking-aloud technique") has proved useful in analyzing the process of medical diagnosis and was first applied in pharmacology to define an intravenous agent as a chemical liberator of histamine in several clinical states.60-88-93 The decision trees constructed in this paper will be used to evaluate the evidence for the single-mediator histamine as a causal chemical factor in clinical and experimental septic/ endotoxic shock. The results of all available in vivo studies on histamine in this field for all four Koch-Dale criteria will be analyzed and evaluated according to the criteria and methodological standards, defined in the "Methods" section of each study, for each question node in the decision trees (meta-analysis). The analysis and evaluation for the first two Koch-Dale criteria has already been published with all studies reported until December

54

Handbook of Mediators in Septic Shock

1987.134 This paper is an updated version for the first two Koch-Dale criteria but moreover includes all findings for the Koch-Dale criteria 3 and 4 as well. It must be stated that this process is considered highly useful to establish a cause-effect relationship, but not to assess the relative importance of the mediator histamine in the presence of all other causal factors in septic shock.135 However, this type of evaluation is a prerequisite and basis for causality analysis of a single mediator in the presence of many other causal factors with multicausal models, causal networks based on group theory or chaos theory (see Chapter 25 in this book).

II. DATABASE: HISTAMINE IN SEPTIC/ENDOTOXIC SHOCK The primary database for retrieval of studies published on histamine in septic/endotoxic shock was the manual research library system of the Institute of Theoretical Surgery in Marburg. There were 45,352 publications on mediators in shock until December 31, 1987, on secretory procedures and pathophysiology of peptic ulcer disease, and, in particular, on every aspect of histamine research when we started the first meta-analysis.134 The research library system is organized as a transient, consecutive series of reprints and photocopies covering a collection period of 26 years (reprints from 1910 to today). Using a typewriter, every paper was registered in the Vancouver reference style and indexed alphabetically by the name of the first author. The special research library system was built over the years by continuous ordering and registration of the current literature, using two bibliographic services — the Institute of Scientific Information, Philadelphia, PA, U.S.A., Current Contents-Life Sciences, and Ascatopics — and the key word Shock. The primary database was completed by a further literature search with the DIMDI database (Deutsches Institut fur Medizinische Dokumentation und Information, Cologne, Germany) which mainly covers MEDLINE and EMBASE starting with the year 1987. The secondary database consisted of literature cited in original papers and review articles and completed the special database system on this topic.

III. KOCH-DALE CRITERIA 1 AND 2: PRESENCE IN DISEASE AND ABSENCE IN HEALTH A. CONDUCTING THE SEARCH AND SELECTION OF STUDIES FOR INCLUSION IN THE META-ANALYSIS The first retrieval of articles on the more general topic of' 'histamine in shock conditions" was performed by hand. After 3 weeks of work by two of the authors sitting together, 1222 papers were selected from the primary database. Careful reading of all these papers, including a handwritten brief summary by the first author, took another 4 months and ended with only 25 publications for the Koch-Dale criteria 1 and 2 until December 31, 1987. Another four publications were added from the years 1988 until December 1990. All of these had investigated the target topic of "histamine release (HR) in septic/endotoxic shock conditions in vivo,'' thus fulfilling the admission criteria for the evaluation of the Koch-Dale criteria 1 and 2. From the 29 studies, 27 were finally included in the meta-analysis. Two papers were excluded because they provided insufficient numerical details on several questions of the decision tree. The 27 studies listed in Table 1 were divided by the evaluation of their results: histamine release was (yes) or was not (no) demonstrated. Unfortunately, the definition of histamine release was sometimes equivocal. Histamine release was defined as either an increase or a decrease of the histamine concentration in a body fluid and/or a tissue. The definition of histamine release used in this meta-analysis is provided in the description of the question nodes in the decision tree.

55

Neugebauer, Rixen, and Lorenz TABLE 1 Histamine Release Demonstrated in Septic/Endotoxic Shock Conditions In Vivo: A Complete Survey of the Literature (1940-1990) Species

Yes (n = 21)

No (n = 8)

Man

(l) a Panse and Dutta (1963)101 (2) Kobold et 1. (1963)62 (3) Griffiths (1972)40 (4) Cho et al.16 (1985)" (5) Neugebauer et al. (1988)136

(6) Rose and Browne (1940)"2 (7) Jacobs et al. (1989)137 (8) Casale et al. (1990)138

Monkey

(9) Hinshaw et al. (1961)49

Dog

(10) (11) (12) (13) (14) (15) (16) (17)

Cat

(20) Naik et al. (1978)98

Weil and Spink (1957)129 Hinshaw et al. (I960)48 Hinshaw et al. (1961)50 Spink et al. (1964)"9 Kobold et al. (1964)63 Reddin et al. (1966)107 Vick et al. (1971)127 Caldwell et al. (1975)14

(21) Parratt et al. (1986)'03

65

Rabbit

(22) (23) (19) (24)

Rat

(25) Schumer (1972)116 (26) Bracket! et al. (1990)139

Mouse

(18) Jacobson et al. (1964)56 (19) Corrado et al. (1964)20 (15) Reddin et al.107 (1966)c

Kun (1947) Davis et al. (1963)22 Corrado et al. (1964)20 Rampart et al. (1982)106

(27) Markley et al. (1974)94

Note: Yes and no represent the opinion of the authors on their own work of whether histamine release is or is not demonstrated. The criteria for staging histamine release response are not uniform between the various authors. Our own definitions are given in the text. Only clinical or experimental in vivo studies are included in the table; experiments with isolated systems (cells, perfused organs, etc.) are not included. ' b 0

Study number in parentheses; these numers are used throughout chapter and tables. Infants = 35 weeks. Puppies = 1-6 months.

From Neugebauer, E., Lorenz, W., Maroske, D., Barthien, W., and Ennis, M., Theor. Surg., 2, 1-28, 1987. With permission of Springer-Verlag, Heidelberg.

B. METHOD OF THE META-ANALYSIS WITH THE DECISION TREE: THE CRITERIA AND METHODOLOGICAL STANDARDS OF THE TEST NODES IN THE DECISION TREE In addition to the authors' own assessment of their results, every study listed in Table 1 was re-analyzed by the decision-tree model (Figure 1). This type of decision tree is made up of a sequence of hierarchical questions at test nodes with binary branches (yes or no answers). Beginning with the first question, each study was analyzed. If the answer to the question of one of the test nodes was no (—), the study should have been — strictly speaking — excluded from further analysis. However, because it became obvious that the demand

56

Handbook of Mediators in Septic Shock

/

\+ yO. Q

Publication on histamine release response in septic/endotoxic shock conditions in vivo? Assay for histamine determination reliable? n

D

Sample preparation appropriate? JO.

D

Sampling in the relevant bodyfluid or organ system?

i).

Sampling at the right time? p

Q D

Study design appropriate? JO.

D

Shock model clinically relevant?

JO. Species clinically relevant?

/ Vn

Q

G

Response caused by the shock itself? O

Yes

. study shows a real association between histamine release and septic/ endotoxic shock in vivo

FIGURE 1. Decision tree for the investigation of a real existing association between histamine release and septic/ endotoxic shock under in vivo conditions. Koch-Dale criteria 1 and 2. The results of each published study listed in Table 1 were analyzed and evaluated following the logistics of the tree (meta-analysis). The structure includes ten test nodes and nine binary branches, of which nine were positive. If the answer to one question is no ( — ) , the study must be excluded from further analysis. For further explanation and a definition of standards implicated by the test nodes, see the text. The tree was constructed according to Lusted.93 (From Neugebauer, E. et al., Theor. Surg., 2, 1, 1987. With permission of Springer-Verlag, Heidelberg.)

on absolute reliability of the reported data was impracticable, shortcomings were estimated in a further step of the analysis (see below). If the question was answered yes ( + ), including a positive judgment of the shortcomings at this test node, the examination proceeded to the next test node. Only a positive answer to the last question of the tree classified the study as suitable for demonstrating a real association between histamine release and septic/endotoxic shock in vivo. Real associations indicate a high probability for cause-effect relationships. Calculation of conditional probabilities can quantitate the degree of certainty of a causal relationship.91-135 The criteria and methodological standards at each operation node (Figure 1) were tabulated in detail. A 3-d consensus conference of the whole research group was organized to assess, describe, and explain all these criteria with tables and figures (see Tables 12 through 17).95 1. Assay for Histamine Determination Reliable? Every publication was first scrutinized for the method of estimating histamine release. Of the methods available today, only the three classical and direct methods — the bioassay, the fluorometric assay, and the radioenzymatic assay in their varous modifications (Table 2) — were used in all studies.9>92>I4° Changes in the histamine content of whole blood, plasma, or serum, were always considered as an indicator for histamine release. The reliability of all these assays is controversial.u-88-140 We consider that the original fluorometric assay of Shore et al.118 has the most and the bioassay the second-most limitations. The fluorometricfluoroenzymatic assay and the enzymatic double-isotope dilution assay are equally suitable for measuring histamine in many conditions. 10'77>81 However, in certain circumstances, for

57

Neugebauer, Rlxen, and Lorenz

TABLE 2 Methods Used for Determining Histamine in Body Fluids in Studies on Septic and Endotoxic Shock Principle

First publication

Optimum development regarding reliability*

Bioassay on the terminal, isolated guinea-pig ileum

Guggenheim and Loffler (1916)

Adam et al. (1957)

Fluorometric assay following condensation with o-phthaldialdehyde

Shore et al. (1959)

Lorenz et al. (1970, 1972) Lorenz and Doenicke (1978)

Enzymatic double isotype dilution assay using histamine methyltransferase

Snyder et al. (1966)

Beaven et al. (1972) Taylor and Snyder (1972) Beaven and Horakova (1978) Dyeretal. (1982)

Note: The fluorometric assay of Lorenz et al. is denoted in the text as fluorometric-fluoroenzymatic since the identification of the substance measured in the assay among other procedures is carried out with purified histamine methyltransferase. The distinction between fluorometric and fluoroenzymatic has become necessary because of the repeatedly proposed argument of the low sensitivity and specificity against the fluorometric assay (Beaven et al., Domschke et al., Taylor et al.) which is true for the Shore procedure, but definitely not for the assay of Lorenz et al. For references not included in the list of references in this publication.88'140 1

Reliability: sensitivity, specificity, precision, and accuracy.

From Lorenz, W., Doenicke, A., Schoning, B., and Neugebauer, E., in Adverse Reactions of Anaesthetic Drugs, Vol. 8, Thornton, J. A., Ed., Elsevier/North-Holland Biomedical Press, Amsterdam, 1981, 169. With permission.

example, after administration of intravenous agents and plasma substitutes, both these assays can be unreliable.88'140 For this reason, it is essential to test the reliability of any histamine assay used in the investigation of the effects of a new drug, operation, or disease.72'73'77'81 -88-92 Unfortunately, however, not all the assays used in the single studies under investigation have been sufficiently tested. It is irrational to develop theories about any pathomechanism of histamine release before this possible error has been carefully excluded. The reliability of the assay for measuring histamine concentrations in body fluids cannot be transferred from one species to another, but must always be re-evaluated. Humans — In human whole blood there is no general problem in determining histamine levels with any of the methods listed in Table 2, since the histamine concentration of about 50 ng/ml in normal subjects is relatively high.9-72-141 There is good agreement between all methods and all authors regarding this value. However, in the fluorometric assay of Anton and Sayre,4 isoamyl alcohol is used to extract of histamine from samples; this can interfere markedly with histamine-induced fluorescence readings.92 For human plasma histamine concentrations, only partial agreement is found between the results of serial assays (Table 3). Of the older biological, spectrophotometrical, and fluorometric methods, only the bioassay of Adam et al.1 and the fluorometric test of Graham et al.39 produced plasma histamine levels which are in the same order of magnitude as those measured with the fluorometricfluoroenzymatic assay of Lorenz et al.77'81 and with the isotope techniques.9 The widely used method of Shore et al.118 is unsuitable for the assay of histamine in plasma since it lacks both sensitivity and specificity (interference with many amines, amino acids, and polyamines). At present, the modified fluorometric-fluoroenzymatic assay,85'142 the gas chromatographic-mass spectrometric procedure,58-59 and the modified radioenzymatic assays are

58

Handbook of Mediators in Septic Shock n TABLE 3 Comparison of the Plasma Histamine Concentrations of Several Mammalian Species Obtained with Biological, Spectrophotometric, Fluorometric, and Isotopic Methods Species Man

Monkey Dog

Cat Guinea-pig Rat

Histamine (ng/ml)' 88 This question was also constructed to exclude single studies in which release of histamine was demonstrated, but where the histamine levels measured did not correspond to the degree of biological effects and clinical reactions and to their time course (Tables 9 and 11). Finally, if a particular study could not be evaluated because of ambiguous answers to preceding questions, then this last test node required a consensus regarding its retention in this analysis. 9. Evaluation of Shortcomings in the Individual Studies and Their Interpretation Because only few, if any, studies finished the decision-tree process without failures, their shortcomings were further evaluated in a "second look", in which the main shortcomings of each were listed and their relevance for a yes or no decision was evaluated during

68

Handbook of Mediators in Septic Shock

the consensus conference. In studies with minor shortcomings compensated by major histamine release, we evaluated whether they met the criteria of histamine release listed in Table 11. Definitions of histamine release using whole blood levels (increase or decrease) were not considered reliable except in dogs and provided the assay and sample preparation were reliable. Descriptive statistics were used to evaluate all of the individual studies. C. RESULTS OF THE META-ANALYSIS WITH THE DECISION TREE This section provides an analysis and evaluation of all clinical and experimental in vivo studies listed in Table 1 on histamine release in septic/endotoxic shock (Koch-Dale criteria 1 and 2) with the aid of the decision tree. 1. Primates (Man and Monkey) Eight studies have been published on histamine release in patients with septic shock, three of them within the recent 2 years (Table 1). One study revealed these conditions in experimentally induced endotoxic shock in monkeys.49 All of these studies were analyzed by means of the questions and answers in the decision tree (Figure 1); the decisions and their explanations are outlined in Table 12. Details for understanding these explanations were given in the extended methodological section (see Section II.B). As a result, none of the five older studies in man (Studies 1 through 4, Study 6; and also Study 9, the single study in monkeys) completed the decision tree without shortcomings! Our study (Study 5,136) was conceived and conducted after the first publication of this metaanalysis and took care of all the objections received in the question nodes of the decision tree.134 The two other studies from the well-respected group of Kaliner (Studies 7,137 and 8138) had only a few, but significant, shortcomings in their study designs for demonstration of histamine release. However, to evaluate whether, despite the failures, any of the studies might be suitable for calculating conditional probabilities and defining histamine, at least as a contributory determinant in septic shock,91-144 each study was examined for the degree or effect of histamine release (Table 13). Following this two-step procedure none of the primate studies, except Study 5 in our opinion, could be considered as valid to demonstrate histamine release in septic shock. The main shortcomings judged not to be remediable were (1) estimation of histamine release in whole blood (wrong body fluid in this species; four of nine studies) and (2) inappropriate study design (seven of nine studies). In contrast to the author's estimation about their demonstration of histamine release (yes/ no = 6/3, Table 1), their interpretation was not confirmed in any but one of the studies (Table 13). Our study (Study 5136) was performed with the knowledge of the shortcomings of all previous studies published through 1987. The study was designed as a prospective crosssectional study with a test group of 20 patients with confirmed sepsis (inclusion criteria of the Veterans Administration Cooperative Study Group) and of a concomitant control group of 20 postoperative patients with peripheral trauma but without signs of local or systemic infection.145 The patients were well matched and the groups were not significantly different in all criteria with known influence on histamine release.149 A regression analysis was performed to exclude and to determine confounding factors which could also explain histamine release (drugs, underlying diseases, etc.). As a result, histamine release was shown to be causally associated with sepsis and septic shock. Figure 3 summarizes the results of the study of both groups. Comparison of the median values of each group on days one through five revealed significantly higher values in the test group on days one through four, but not on day five. Whereas only 1 of 29 patients in the control group showed histamine values >1 ng/ml during the study period (maximum level = 2.5 ng/ml), 9 of 20 patients

Neugebauer, Rixen, and Lorenz

69

in the test group exceeded the pathological cut-off point of 1 ng/ml with extreme values up to 48 ng/ml. Our findings are in contrast to the most recent studies of Kaliner's group (Studies 7137 and 8138). In Study 7, nearly all subjects had unmeasurable histamine values due to the presence of an "inhibitor" in the plasma, which interferes with the assay. After removal of the inhibitor septic shock, plasma histamine levels were normal. The main points of criticism of that study are (1) that the degree of the removal of the inhibitory factors is uncertain and that other confounding factors have not been considered, and (2) that the probability to detect elevated plasma histamine levels by only one sample per patient is near zero.150 Many hours may have elapsed following an early release of histamine, because the patients were in an advanced stage of shock. The normal t1/2 of histamine in plasma is only 2 to 2.5 min. The second argument is also true for Study 8.'38 They obtained blood samples every 2 h and, as a result, may have missed an increase in histamine between sampling periods. In conclusion, further controlled clinical studies with careful selection of the appropriate control groups are needed for confirmation of our study results and for a better definition of the phases of histamine release under clinical conditions.

2. Dog and Cat The preferred model for studying histamine release in experimental conditions has been the endotoxin shock model in the dog (n = 10, Studies 10 through 19) and the cat (n = 2, Studies 20 and 21) (Table 1). These results were analyzed as if for primates (Table 14). The most striking shortcomings in all of these studies was the use of unreliable histamine assays — a finding which can easily be understood after reading the section on methods. In only one of the dog studies (Study 10) were the results considered reliable, and this was ^ased on the old bioassay method of Guggenheim and Loffler.41'129 It must be mentioned, lowever, that the specificity of the assay was not definitely shown (no antihistamines tested) nd the sensitivity was not very high.151 Few (two of ten studies) had any relevant control group. Histamine release was investigated under extreme (nonclinical) conditions by intravenous administration of high doses of endotoxin. This disappointing result demonstrates a tremendous gap between the experimental work in animals and the clinical situation. Answers on histamine involvement in septic shock could at best be suspected in extreme clinical situations. Nevertheless, the high endotoxin doses were judged as ambiguous because they may serve as a model for the initial histamine release response after pulsatile endotoxin inflow. None of the studies on cats or dogs was considered faultless. However, an evaluation of the shortcomings of all studies in Table 15 revealed two that were suitable, in general, for demonstrating histamine release as having a real association with endotoxic shock.127-129 Unfortunately, in Study 10, only one case was demonstrated in which histamine levels were correlated with the shock-related systemic hypotension, and this was not sufficient for calculating conditional probabilities. Such a calculation was, however, possible in a study by Vick et al.127 (Study 16) in which the effects of varying doses of endotoxin on plasma histamine levels (P) and fatality were investigated in a series of 22 adult beagle dogs. There were 13 deaths (L) in 22 dogs (P/L = 0.59, or 59%), all within 72 h after endotoxin. The incidence of histamine release (HR) was also 13/22 = 0.59 (59%). The maximum elevation of plasma histamine was measured 30 to 60 s after endotoxin injection. Other criteria for the definition of histamine release in plasma were also fulfilled (Table 11). The conditional probability of fatality with histamine release [P(L/HR)] was 13/13 = 1.0 or 100%; the conditional probability of a lethal outcome without histamine release [P(L/HR)] was 0/9 = 0 (0%). Hence, these data support the view that histamine release may be at least a sufficient determinant for death

+ Fluorometric assay of Anton and Sayre4

? Fluorometric asNot stated say of Hakanson et al.42

3 (40)

4 (16)

+ Immediate chemical assay

? Not stated

Bioassay on ox carotid of Kobold and Thai61

2 (62)

+ Preparation according to Code17

Sampling at the right time?

+ Serum: histamine content comparable to that in plasma9

Whole blood: see comment to Study 1

+ Plasma

? Not stated

+ First sample 4-6 h after onset of shock; further sampling at 1, 2, and 6 h

+ During clinical septic shock pretreatment and 24 h posttreatment with trasylol

+ Whole blood: During fully eshistamine tablished disease content paral- with positive lels number stool culture of basophils

Sample Sampling in preparation relevant body appropriate? fluid?

+ Bioassay on isolated guinea pig ileum of Gaddum, 33 Barsoum and Gaddum6

Histamine assay reliable?

1 (101)

Study no. (Ref.)

+ Test/control (7/8) several prognostic factors stated two control groups

Test/control (20/ 50) not balanced; no prognostic factors stated; no single values stated

Test/control (8/1) not balanced; no absolute histamine values stated

Test/control (17/ 10) not balanced; no information on controls; no prognostic factors stated

Study design appropriate?

+ Positive blood culture confirms systemic sepsis

+ Positive blood culture with definition of organisms

+ Diagnosis gram ( — ) septicemia

+

+

+ Infants 35.8 weeks (x) of age

Man

Man

+

Species clinically relevant?

? Cholera toxin Man shock; no information on patients' anamnestic data

Shock model clinically relevant?

? Control group "respiratory disorders" shows similar findings

? See comment to Study 2

? Data can be caused by other factors such as drugs for supportive treatment

+ Data obtained mostly before starting treatment

HR response caused by the shock itself?

TABLE 12 Analysis of Studies on Histamine Release (HR) Demonstrated in Septic/Endotoxic Shock Conditions In Vivo: Man and Monkey

70 Handbook of Mediators in Septic Shock

+ Fluorometric assay of Lorenz and Neugebauer'42

+ Bioassay on isolated guinea pig ileum of Code17

+ Radioenzymatic assay of Dyer et al.147

5 (136)

6 (112)

7 (137)

Plasma

+

+ Use of EDTA anticoagulated plasma; no details given

Plasma

+

+ Stasis Whole blood: avoided see comment during sam- to Study 1 pie taking!

+ Preparation appropriate; procedure described

? During definite Test/control 41 septic shock or septic shock pt. "clinical septic vs. 76 normal shock"; individuals vs. criteria stated 12 critically ill only one sample/ normotensives pat. vs. 28 pt. with acute localized infections; concomitant sample taking in test and control groups but no control of other confounders

+ Cases with septic shock admitted to a medical ICU

+ Man, no further Plasma histamine details given on values were not patient characincreased due to a teristics histamine methyltransferase-inhibitor in plasma; increase in HR may have been missed also because only one sample taken per patient

+ + + + + Patients had to Test/control (20/ Postoperative pt. Man: 4 females, HR by other factors meet defined 20) prospective with confirmed 16 males in test than sepsis exsepsis criteria;145 cross-sectional sepsis in test group eluded HR is a severity of sepsis study controls: group: 1 1 gram causal factor for trauma pt. with( — ), 8 gram ( + ), lethality from sepwas scored according to Eleout infection; 2 unclear; detailed tic shock control of all clinical data on all bute and Stoner146 10 sam- known sources relevant aspects of pies within 12 h of bias sepsis and one on the following 4 d at 8 a.m. + + + ? Several times in Test/control (?/ Cases with postop- Man Data can be caused agonal states 50) unidentified erative septic by chance or other number of septic shock factors (underlying shock patients disease)

Neugebauer, Rixen, and Lorenz 71

? Paper chromatographic determination of Hinshaw et al.4S ? Whole blood relevance of primates' whole blood is unknown (not investigated)

Study design appropriate?

+ 0-600 min

— No control group; small number of animals (n = 6)

? During single i.v. Only comparison infection of E. to baseline valcoli RE-2 endoues before endotoxin, one samtoxin application pie every 2 h No separate conover an 8 h study trol group period

Sampling at the right time?

? Experiments designed for extreme conditions (see text)

+ Application of endotoxin to man

Shock model clinically relevant?

HR response caused by the shock itself?

? ? Monkey has a No control group; close relationdata can be caused ship to human by chance or other species, but factors (anesHR response thesia, surgery) can differ from man83"

+ 5 Healthy volun- Increase in HR may teers; age: 20have been missed 30 years because of 2 h time difference between sample taking

Species clinically relevant?

Adapted from Neugebauer, E., Lorenz, W., Maroske, D., Barthien, W., and Ennis, M., Theor. Surg., 2, 1-28, 1987. With permission of Springer-Verlag, Heidelberg.

Note: The table contains all questions defined in Figure 1 for investigation of a real existing association between HR and septic/endotoxic shock. Explanation of abbreviations and symbols: the study number corresponds to the number stated in Table 1. Studies 1-8 were in man; study 9 in monkeys; ( + ) indicates a positive answer; ( — ) indicates a negative answer to the question; (?) means either no information given or ambiguous; + and — to the question "histamine assay reliable" is always considered in connection with the body fluids and species; + and - to the question on the "relevant body fluid" is always considered in connection with the species; test/control (n/n) indicates the number of individuals in the test and control group, respectively; DAO = diamine oxidase (EC 1.4.3.6), which catalyzes the oxidative deamination of histamine. For further explanation of the criteria and decisions, see Method, Section III.B.

? Not stated

+

9 (49)

Plasma

+ Radioenzymatic assay of Dyer et al.147

8 (138)

? Not stated

Sample Sampling in Histamine assay preparation relevant body reliable? appropriate? fluid?

Study no. (Ref.)

TABLE 12 (continued) Analysis of Studies on Histamine Release (HR) Demonstrated in Septic/Endotoxic Shock Conditions In Vivo: Man and Monkey

72 Handbook of Mediators in Septic Shock

73

Neugebauer, Rixen, and Lorenz

TABLE 13 Evaluation of Studies on Histamine Release (HR) Demonstrated in Septic/Endotoxic Shock Conditions: Man and Monkey Study HR shown: no. author's Study (Ref.) estimation faultless?

Shortcomings of the study

Evaluation of shortcomings

Study reliable for demonstration of HR?

Differences between — test and control Result explainable by group considerable: changes in baso810 ng/ml vs. 50 ng/ phils (leukocytosis) ml or HR; cholera toxin is not a prototype for septic shock

HR shown: our estimation

1 (101)

+

—

Determination in whole blood; no real cohort study; cholera is not septic shock

2 (62)

+

-

Unreliable assay; No real control group; no really quantiassumed HR varied tative data; samconsiderably beple preparation tween patients; HR not stated; only 1 also shown in the control patient control patient (hemorrhagic shock)

3 (40)

+

-

Determination in Differences between whole-blood; drug test and control group considerable: effects on whole blood histamine 750 vs. 50 ng/ml; (increase of baso- drug effects of this phils) possible; no extent not probable; real cohort (cross- marked differences sectional study) between survivors and nonsurvivors

4 (16)

+

-

Unreliable assay; sample preparation and time of sampling not stated

Assay not specific for plasma/serum histamine by a factor of 10; difference between healthy controls and sepsis patients is small

5 (136)

+

+

A cross-sectional study has a high risk of misinterpretation of findings; HR can be due to a variety of other reasons

All variables with + known influence on Increased plasma hisHR (ICU-treatment, tamine levels due to surgery, primary dis- sepsis and not conease) were carefully comitant conditions; evaluated and exsee detailed causaleluded (Hill ity analysis of Dietz criteria148) et al."">

+

6 (112)

—

—

Determination in Shortcomings not cor— whole blood; no rectable Partially increased real cohort study; and decreased histacases of septic mine values; results shock not sepaby chance rately stated

?

7 (137)

-

-

Only one sample per patient is not sufficient to detect HR; patients

?

Study had a low probability to detect HR

-

?

-

Result explainable by changes in basophils (leukocytosis) or HR

?

-

-

—

74

Handbook of Mediators in Septic Shock

TABLE 13 (continued) Evaluation of Studies on Histamine Release (HR) Demonstrated in Septic/Endotoxic Shock Conditions: Man and Monkey Study HR shown: no. author's Study Shortcomings of (Ref.) estimation faultless? the study

Evaluation of shortcomings

Study reliable for demonstration of HR?

HR shown: our estimation

collected from 1982-1985; degree of removal of inhibitors uncertain 8 (138)

-

-

No separate control Study had a low probgroup; only 5 paability to detect HR tients examined; sampling only once every 2 h

9 (49)

+

—

Fairly insensitive Differences before — and unspecific as- and after endotoxin Increase in blood administration in 3 histamine varies say as shown for dogs;7 determinaof 6 cases considera- considerably tion in whole ble: 0-100 vs. 900 blood; no control ng/ml (at maximum) group; model of doubtful clinical relevance

-

?

?

Note: The evaluation is based on the analysis of several criteria shown in Table 12. The study number corresponds to the number in Table 1. Studies 1-8 were in man; Study 9 was in the monkey; + indicates a positive answer; — indicates a negative answer to the question; ? means an ambiguous answer. Further evaluation of the decisions and comments are given in the text, mainly in the methods section. If the decision is clear, no comment is given. From Neugebauer, E., Lorenz, W., Maroske, D., Barthien, W., and Ennis, M., Theor. Surg., 2, 1-28, With permission of Springer-Verlag, Heidelberg.

1987.

from shock in this experimental model (for calculation see Reference 91). Histamine release might also have been a necessary determinant for death from canine endotoxin shock, but that would be unlikely with a single mediator, and there may be a confounding bias. Unfortunately (Tables 14 and 15), the assay was not reliable (Shore's method is not specific in plasma) nor is the study design appropriate (no control group). A control group showing absence of histamine release under similar experimental, but nonshock conditions would have been of utmost importance. Two further trials (Studies 12 and 13) showed other pitfalls. Study 12 provides an example of a common misleading conclusion, which may also be seen clinically: the effect was mistaken for the cause. Histamine release was apparently a consequence of shock because hypotension (a major feature of shock) occurred immediately after endotoxin administration, but the increase of histamine levels was maximum only after 60 to 180 min. Study 13 did report individual values but could not be evaluated by the multivariate model with conditional probabilities.91 The study was done on a series of ten dogs, which received a second lethal dose of endotoxin after surviving an initial endotoxin injection. Individual values of histamine and the corresponding blood pressures are presented in Table 3 of Spink's study. This special condition, which may be of clinical relevance, revealed that histamine is a sufficient determinant of the hypotensive reaction after the second endotoxin administration. This result

Neueebauer, Rixen, and Lorenz

75

FIGURE 3. Plasma histamine concentrations of patients with and without sepsis and septic shock: results of a prospective clinical cross sectional study.138 (Test group) 20 patients with sepsis and septic shock (inclusion criteria of the VA Coop, study group,145 (Control group) 20 postoperative patients with a peripheral trauma without signs of local or systemic infection. Single values and median values (1 to 3 quartile) from 20 patients per group shown **p 12 h) seen

+ Survival rate in treated group 77 vs. 0% in controls

? Delayed-type hypersensitivity sign improved with H2 blocker; no death in both groups

Development of shock/outcome affected?

TABLE 26 Analysis of Studies with H,- and H2 Antagonists in Septic/Endotoxic Shock Conditions In Vivo Shown to Prevent or Ameliorate Signs and Symptoms of Shock — Authors Estimation of a Positive Effect (Koch-Dale Criterion 4 Fulfilled)

104 Handbook of Mediators in Septic Shock

Diphenhydramine (H,), nonspecific actions

+ + 3 mg/kg bolus + Response tested infusion 1.5 continuously mg/kg/h dose response n.t.

7 + + (204a,b) R (H2); C (H2); Diphenhydramine Response tested diphenhydramine 10 mg/kg, R 25 continuously (H,), all with mg, C 150 mg, for at least 3 h nonspecific acdosage of antions (Table 24) tagonists adequate; no dose response tested

6(203)

+ Chlorphenyramine 10 mg/kg, 30 min after endo(H,) significant antimuscarinic toxin dose reeffects sponse n.t.

5 (202)

+ Response tested before, and 60 and 120 min after treatment

+ + Diphenhydramine 6-8 mg/kg i.v. Response tested (H,), nonspecific 15 min precontinuously actions endotoxin; dose for 4.5-5 h response not until death tested

4 (201)

+ 1.5 mg/kg E. coli endotoxon

+ 4 mg/kg E. coli endotoxin

Dog

Dog

+

+

+ + 6 paired ex0.4-1.0 ptg/kg E. Sheep periments in coli endotoxin 5 animals; every 30 min each sheep is its own control + + 31 Pigs in 5 Continuous infusion Pig groups (n = of pseudomonas 6-8 per aeruginosa 5 x group); 2 10V20 kg/min control groups; antagonists tested in combination with ibuprofen

+ 40 dogs in 4 groups (n = 10 per group); 1 control with endotoxin only

+ 40 Dogs in 4 groups (n = 10 per group); 2 control groups

+ Only lung lymph flow decreased in late phase

+ MAP and SVR sign increased at 60 min other parameters not influenced; at 120 min no significant effect seen

+ Diphenhydramine blocked sustained depression of parameters

+ + BP, PAP, CI, PaO2 Sign temporal imEVLW, pulmonary al- provement of bumin, survival rate shock parameters and survival time in combination with ibuprofen shown

+ PAP, left atrial pressure, lung lymph flow, lymph and plasma protein concentration

+ HR, BP, CVP, CO, SVR, PAP temp, urinary output

+ BP, left ventricular pressure, CO, myocardial contractility

Neugebauer, Rixen, and Lorenz 105

9 (174)

+ 45 mg E. coli endotoxin i.p.

+ 40 mg/kg E. coll endotoxin i.v.

Study design Shock model appropriate? clinically relevant?

+ + + Pretreatment H,: Response tested Saline control 20 mg/kg; H2: continuously and hista80 mg/kg; dosfor 240 min or mine antagoages adequate 24 h (survival) nists tested as tested in alone and in pilot expericombination ments; no dosen = 10 aniresponse tested mals per group too small for survival studies + + + + Test of 3 H, (A, 3 Different dos- 96 h Continuous 3RCT with n D, M) and 3 H2 ages for each observation = 220 ani(C, R, F) antagantagonist mals each 2 onists selection tested control of H] and H2 angroups per tagonists on the study; methbasis of specificylprednisoity lone as positive control, saline as negative control group

Diphenhydramine (H,), C (H2), both with nonspecific actions (Table 24)

Correct temporal relationship?

8(205)

Correct doseresponse relationship?

Antagonist efficient/specific for histamine?

Study no. (Ref.)

Rat

+

+ Rat (conscious model)

Species clinically relevant?

+ Survival rate and time

+ BP, HR, pH, pO2 pCO2, HK, glucose lactate, survival rate (24 h) pathological examinations

Parameters measured adequate?

+ Only R (H2) improved survival rate sign; H, + H2 combinations not effective

+ H, + H2 antagonists prevented: BP, HR, hypoglycemia; survival rate increased with H, + H2 and H2 alone

Development of shock/outcome affected?

TABLE 26 (continued) Analysis of Studies with H,- and H2 Antagonists in Septic/Endotoxic Shock Conditions In Vivo Shown to Prevent or Ameliorate Signs and Symptoms of Shock — Authors Estimation of a Positive Effect (Koch-Dale Criterion 4 Fulfilled)

106 Handbook of Mediators in Septic Shock

0 + A (H,)

+ A 1 mg/kg dose response not tested

+ + + + + + 96 h Continuous Randomized 45 mg E. coli Rat Survival rate and time Beneficial effect observations controlled endotoxin i.p. only for H, anstudy with 2 tagonists shown controls (see Study 9) 20 animals per group 1 1 + + + + ? + + (206) Diphenhydramine 8 Dosages from 6 h Before, 3 h 3 Studies with 36 mg E. coli Mouse Survival rate and time Strong effect on (H,), hydroxy1-5 mg/kg for after endocontrols for endotoxin i.v. survival with zine (H2) (antiki- both drugs 1 h toxin; continudose re(LDg,, dose) hydroxyzine 5 nin) before endoous observasponse and mg/kg toxin tion temporal relationship n = 10 per group, 280 animals 1 2 + + ? + + (207) Diphenhydramine 5 mg/kg pre-, 1 Continuous ob- n = 15 ani- 60 mg/kg Shigella Mouse Survival rate and time Significant in(H,) nonspecific and 2 h postservation mals per sonnei; endotoxin crease in suractions endotoxin group; not typical for vival time groups not sepsis defined, controls included Note: Explanation of abbreviations and symbols: The study number corresponds to the number stated in Table 23. ( + ) indicates a positive answer, (-) indicates a negative answer to the question, (?) means either no information given or ambiguous; (DHT) delayed type hypersensitivity, (BP) arterial blood pressure, (HR) heart rate, (CVP) central venous pressure, (CO) cardiac output, (CI) cardiac index, (PAP) pulmonary artery pressure, (EVLW) extra vascular lung water, (HK) hematocrit, (A) astemizole, (D) dimethindene, (M) mepyramine, (C) cimetidine, (R) ranitidine, (F) famotidine. For further explanation of the criteria and decisions see Methods, Section V.B.

1 (170)

Neugebauer, Rixen, and Lorenz 107

108

Handbook of Mediators in Septic Shock

The most striking shortcoming is the use of nonselective drugs (see Section V.); only two studies had inappropriate study designs.200-203 The evaluation of the shortcomings by our group confirmed the authors estimation of a protective effect of H, blockers in three studies in dogs, mainly because of very strong effects on the parameters measured (Table 27). Lowry et al.200 (Study 3) investigated the pre- and posttreatment of chlorphenyramine after E. coli infusion and found a 75% prevention of the early blood pressure drop compared to controls. The response to intravenous fluid response was also increased. In the pretreatment group, a normal blood pressure was found 6 h after shock induction. The posttreatment group had a typical drop in blood pressure, but when infused with chlorphenyramine a prompt rise was seen. At the end of 6 h, the blood pressure was comparable to pretreatment values and was significantly different from controls. The authors' conclusion is that chlorphenyramine acts twofold — it helps prevent splanchnic venous pooling by prevention of histamine-induced hepatic vein spasm and it also competes against histamine induced arteriolar vasodilatation. Krause and Hess (Study 4) postulated that the initial profound fall in venous return with subsequent fall in cardiac output after E. coli endotoxin administration results from an observed decrease in arterial pressure and coronary perfusion pressures.201 They concluded that this is most probably mediated by the release of histamine, since it is not seen in animals pretreated with diphenhydramine. However, a direct measurement of histamine release was not performed. Furthermore, they presented evidence that the endotoxin induced significant depression of the myocardial contractility could also be blocked by diphenhydramine. The third study with similar early effects on hemodynamics was performed by O'Neil et al.202 (Study 5). chlorphenhydramine pretreatment raised the mean arterial pressure and the systemic vascular resistance up to 60 min after E. coli endotoxin administration. Other parameters however are not affected. The effect is also not significant at 2 h. The study using the sheep model (Study 6)203 was not considered very convincing because the study design was not clearcut and the effect measured was rather small. The authors investigated the effect of diphenhydramine, a nonspecific antagonist on the pulmonary hypertensive response to endotoxin and found a reduction but not prevention of an increased lung vascular permeability. They concluded, that pulmonary hypertension after endotoxemia does not appear to be mediated by histamine. Finally, Studies 2199 and 7204a>b cannot give us an answer on the effect of histamine antagonists on shock development. In both of the studies, combinations of antihistamines with other drugs were used. The study with cats used pyrilamine as an HI antagonist (Table 28). Pyrilamine is a weak and nonspecific antihistamine and it cannot be excluded that the nonsignificant effect on blood pressure and pulmonary-artery pressure change is due to a nonappropriate concentration or administration of the drug (Table 28, 29). 3. Rat and Mouse a. Rat A total of 4 studies were performed in the rat endotoxin model. The only study in which a protective effect of both H,- and H2 blockers (diphenhydramine and cimetidine alone or combined) is postulated in endotoxin shock is that of Brackett et al.205 (Study 8). Following the above treatment modalities, they observed an increased 24 h survival rate and a lessening of intestinal pathology and hypoglycemia in rats. Concerning the survival data, this study has to be criticized because of the low number of animals per group (n = 10). The confidence interval for survival experiments is very high and this result can also be explained by chance. Furthermore, the H,- and H2 antagonists that were used have a high degree of nonspecificity. To establish the pathophysiological significance of histamine in endotoxic shock by studies with H,- and H 2 antagonists, we tried a new approach, selecting three H,- and three

109

Neugebauer, Rixen, and Lorenz

TABLE 27 Evaluation of Studies with H,- and H2 Antagonists in Septic/Endotoxic Shock Conditions Histamine antagonist shown to prevent or Study ameliorate septic/ endotoxic shock: Study Shortcomings of the no. (Ref.) authors estimation faultless? study

Evaluation of shortcomings

Histamine antagonist shown to prevent or ameliorate septic/ endotoxic shock: our estimation

1 (198)

+ Potential beneficial effect of ranitidine in patients with trauma-induced immunosuppression

—

Purpose of study was Study is not in the to evaluate effect of focus of testing the ranitidine in DHT re- Koch-Dale criterion sponse not on the de- 4 veJopment of sepsis; nonspecific effect of ranitidine significant

2 (199)

+ Simultaneous administration of a cocktail of antagonists along with fluid

-

Multiantagonist treat- Study is not dement gives no inforsigned to test the mation on specific H, antagonist sepaantihistamine action; rately nonspecific antagonist

3 (200)

+ H, blocker in doses of 3-10 mg/kg prevented severe early BP drops and improved urinary output

-

Chlorphenyramine has sign antimuscarinic actions; study design inappropriate (too many groups)

Pre- and posttreatment with this drug effectively prevented BP drop (effeet strong); ANOVA statistics for evaluation appropriate

+

4 (201)

+ Prevention of initial hemodynamic depression, and depression of myocardial contractility

-

Diphenhydramine has Protective effect is significant nonspevery strong; effect cific effects at least partly associated with H, blockade

+

5 (202)

+ H, blocker raised MAP and SVR only for a short time

-

Chlorphenyramine has Protective effect is significant nonspevery strong at 60 cific effects min, effects at least partly associated with H, blockade

+

6 (203)

+ Increase in lung vascular permeability partly prevented; histamine not the sole mediator; pulmonary hypertension not histamine mediated

—

Study design not clear Observed effect very cut; diphenhydramine small and can be has significant nondue to nonspecific specific effects actions of H! blocker

—

-

HI and H2 antagonists The positive effects only tested in combi- demonstrated can nation with ibuprobe solely due to fen; nonspecific anibuprofen and/or tagonists tested the nonspecific actions of the antagonists

7 + (204a,b) In combination with ibuprofen

—

-

-

110

Handbook of Mediators in Septic Shock

TABLE 27 (continued) Evaluation of Studies with Hj- and H2 Antagonists in Septic/Endotoxic Shock Conditions Histamine antagonist shown to prevent or Study ameliorate septic/ no. endotoxic shock: Study Shortcomings of the (Ref.) authors estimation faultless? study

Evaluation of shortcomings

8 (205)

+ H, and for H2 improves hemodynamic and metabolic responses pathology and survival

—

Nonspecific antagoResults of survival nists tested; number study not reliable; of animals per group results on hemodytoo small for survival namics and metastudy bolic responses are considered valid

9 (174)

Positive effect of ranitidine on survival not due to H2 antagonistic activity

+

-

10 (170)

+ H! antagonist improves survival time

+

-

11 (206)

+ H2 antagonist improves survival rate

-

Nonspecific antagoThe positive effect nists tested a number of hydroxyzine may of animals per group be due to its antikitoo small for survival nin effect studies

12 (207)

+ Length of survival significant increase

—

Nonspecific antagonist Too many shortcomtested; shortcomings ings and superficial in study design; descriptions endotoxin not typical for sepsis

-

Histamine antagonist shown to prevent or ameliorate septic/ endotoxic shock: our estimation +

+

-

—

Note: The evaluation is based on the analysis of several criteria shown in Table 26. The study number corresponds to the number in Table 23. For further evaluation of the decisions and comments see text of results section. If the decision is clear, no comment is given. DHT = delayed type hypersensitivity; MAP = mean arterial pressure; SVR = systemic vascular resistance.

H2 antagonists on the basis of two criteria: (1) these substances had distinctly different efficacies in binding to H,- or H2 receptors; and (2) the antagonists had distinctly different nonantihistaminic efficacies as inhibitors or stimulators of other shock mediators. Using the same standardized rat model of endotoxic shock we performed three equally structured, randomized, controlled studies of the effects exerted on survival and several observation parameters by H,- and H2 antagonists, first separately (Studies I and II) and then (after the assessment of effective doses) in various combinations (Study III). Each study included nine test groups (three antagonists in three concentrations) and two control groups with 20 animals per group. By selection of the antihistamines on the basis of their graduated efficacies against histamine (H^ astemizole > dimetindene > mepyramine, in doses of 0.1, 1.0, and 5.0 mg/kg b.w. each; H2: famotidine > ranitidine > cimetidine, in doses of 1.0, 10, and 50 mg/kg b.w. each) together with the defined dose ranges, the whole therapeutic spectrum in each class was covered. The survival rate for the saline pretreated endotoxic shock group (control group 1) was 25%. Methylprednisolone pretreatment (second positive control group) completely prevented

0 + Farnotidine (H2)

5 Diphenhydramine H,, burimamide H2 sign; nonspecific effects (antimuscarinic) for H, blocker reported

1 (94)

Note: For explanations of abbreviations and symbols, see note to Table 26.

+ + + + + 1 mg/kg i.v. 30 96 h Continuous Endotoxin 45 mg E. coli min before observation control vs. endotoxin i.p. endotoxin test group (20/20) + + + + ? 15 mg/kg i.p. Response re4 Groups with 0 and 5 mg E. coli (H,) 10 mg/kg corded hourly 48—50 aniendotoxin (63i.p. (H2) 30 for the first 6-8 mals per 90% mortality in min before h; later 18 and group, com48 h) endotoxin 24 h parison to endotoxin control

+

Mouse

Rat

Rat

+ + + 0.05 mg/kg E. coli Cat endotoxin i.v.

+ + + Saline control, 6 mg/kg e. coli n = 5; endotoxin i.v. endotoxin over 1 h control, n = 11; antihistamines, n = 9

1 (170)

+ Response measured every 30 min over 3 h

4 Pyrilamine (H,) metiamide (H2) sign; nonspecific effects of both antagonist

1 (209)

+ Dose response tested 5, 10, and 15 mg/kg i.p. of a combination 30 min before endotoxin

+ ? + Application of 5 Not stated; "ini- Test/control mg/kg 1 h betial acute circu- (5/15) fore endotoxin; latory redose response sponse" not tested

3 Pyrilamine (H,) sign; antiadrenergic effect; weak antagonist

+ No sign; effect on parameters

Development of shock/outcome affected?

Survival

+

+ Survival rate and time

Separate administration of H, and H2 blockers had no effect on mortality

No effect on survival rate and time

+ Cremaster muscle mi- Combined doscrocirculation (Viage of the two deomicroscopy); mean blockers was arterial pressure; surtoxic; vessel vival time (rate) constriction is not prevented

PAP, BP

Species Study design Shock model clinically Parameters measured appropriate? clinically relevant? relevant? adequate?

1 (208)

Correct temporal relationship?

Correct doseresponse relationship?

Study no. Antagonist efficient/ (Ref.) specific for histamine?

TABLE 28 Analysis of Studies with Hj- and H2 Antagonists in Septic/Endotoxic Shock Conditions In Vivo Shown Not to Prevent or Ameliorate Signs and Symptoms of Shock ( — ) or Shown to Have a Negative Effect on These Parameters ( + ) — Authors Estimation of No or a Negative Effect (Koch-Dale Criterion 4 Not Fulfilled)

Neugebauer, Rixen, and Lorenz 111

112

Handbook of Mediators in Septic Shock TABLE 29 Evaluation of Studies with H,- and H2 Antagonists in Septic/Endotoxic Shock Conditions

Histamine antagonist shown to prevent or ameliorate septic/ Study endotoxic shock: no. endotoxic shock: Study (Ref.) authors estimation faultless

Shortcomings of the study

Evaluation of shortcomings

Histamine antagonist shown to prevent or ameliorate septic/ endotoxic shock: our estimation

13 (208)

No significant protection action

-

Nonspecific and weak antagonist used

-

14 (209)

Response not prevented by antihistamines

-

Nonspecific antagonist Carefully designed used, antagonists not study with dose re- A combination is not separately tested sponse relationships effective to prevent endotoxin response and death

10 (170)

No effect on survival rate

+

15 (94)

No effect of H,/H 2 alone and in combination on survival

-

The result supports earlier reports (consistence) from our group Mouse may not be clinically relevant

-

Note: The evaluation is based on the analysis of several criteria shown in Table 28. For further information see legend to Table 26 and 28.

death in the control group. Treatment with the intermediate dose (1 mg/kg) of any of the three H, blockers led to a 30% (nonsignificant) increase in the survival rate. A hypothetical, specific effect of binding to H, receptors, however, should have resulted in a clearly positive dose-response relationship. Despite remarkable differences in the antihistaminic efficacy of the antagonists, no graduated effect related to survival was noted. Neither, however, were differences seen between the nonantihistaminic activities of the drugs, although such differences clearly exist.174 From the H2 antagonists tested, a positive dose-response relationship with a highly significant protective effect was noted with ranitidine pretreatment. With the highest dose (50 mg/kg) only 3 of 20 animals died. However, a negative nonresponse relationship was obtained with famotidine (Table 28). This unexpected result is in contrast to the antihistaminic efficacies of the H2 receptors. Therefore, it is conceivable that the ranitidine effect derives only from a nonspecific activity of this compound. The only reported additional effect of concentrations used in this study is a cholinergic effect. Nine H,IH2 combinations made up of the most effective doses of the separately tested antagonists in each group showed unexpected results. Astemizole and famotidine, the most potent and specific antagonists in each class showed the worst result with only 10% survival rate compared to 25% in the saline-treated endotoxin control. In contrast, ranitidine with astemizole showed a significant protective effect as did dimetindene with famotidine. The results support the concept of nonspecific activities of H/H;, antagonists in this model of endotoxic shock.

Neugebauer, Rixen, and Lorenz

113

The beneficial effect of astemizole (H,) and the detrimental effect of famotidine (H2) has been confirmed in most recent studies with the same model by Rixen et al.170 from our group. Baker and Wilmoth (Study 14) studied the microcirculatory responses of pyrilamine in combination with the H2-antagonist metiamide in three different dosages from 5 to 15 mg/ kg in a standardized endotoxic shock model.209 Pretreatment with the Hj/H 2 blockers did not prevent the vessel constrictions of arterioles and venules subsequent to endotoxin. There were no differences between those rats administered 5 or 10 mg/kg of each blocker. Rats administered 15 mg/kg of each blocker did not survive the 1-h period of endotoxin infusion indicating that this combined dosage of the two blockers was toxic. The study was carefully designed and is considered reliable (Table 29).

b. Mouse

Three studies were performed in mouse endotoxic shock. Study 11 examined diphenhydramine (H,) and hydroxyzine (H2) in an LD80 model.206 The antihistamines were administered 1 h before endotoxin in various doses. Whereas diphenhydramine had only a small effect (20%) on survival, this effect was very strong with the pyrilamine. The survival rate was 100% if pyrilamine was administered simultaneously and less pronounced if given 1 h or more before or after endotoxin. Whether this strong effect is related to its antihistaminic efficacy or a well-known antikinin effect needs to be established.206 A study not considered reliable because of shortcomings in study design and the relevance of the shock model is Study 12.207 Diphenhydramine (H,) significantly increased survival time compared to untreated controls. The final study to be mentioned, is the study of Markley et al.94 (Study 15). They used diphenhydramine (H t ) and burimamide (H2) as antihistamines in endotoxic shock and found that pretreatment with one or both drugs had no influence on mortality. They concluded that histamine is not a lethal factor but may have a compensatory, beneficial effect.Caution, however, must be exercised in the interpretation of this data, since factors such as the animal species studied, the type of shock produced, the lack of specificity of drugs, drug toxicity, and others complicate the validity of the conclusions drawn. D. DESCRIPTIVE STATISTICS ON METHODOLOGICAL SHORTCOMINGS AND THEIR EVALUATION Compared to studies investigating the Koch-Dale criteria 1 and 2 (Tables 18 and 19) it is obvious, that the studies summarized in Table 23 and described above had a higher methodological standard (Table 30). Of the 15 studies, 11 or 73% were estimated to have an appropriate study design; 14 of the studies used appropriate parameters to investigate the effect of antihistamines on shock development. The main problem, except in two studies, was the use of weak and/or nonspecific antagonists. This factor is estimated to be very important, especially after looking at the results of our systematic approach with Hj- and H2 antagonists in the standardized rat endotoxic shock model.174 Although only 3 of the 15 (20%) studies were free of faults by decision tree criteria, 5 of the 15 (33%) were finally considered reliable for demonstration of histamine antagonists preventing of ameliorating signs and/or symptoms of septic/endotoxic shock (Table 31). Looking at the different types of antihistamines, all except one study (Study 8)205 showed a positive effect on hemodynamics only with H! antihistamines. On the contrary, there is a strong trend that the combination of H,/H2 blockers is of no benefit or even detrimental. H2 antagonists alone are considered to influence shock development negatively. For a final decision, however, more studies are needed to verify this postulate.

0 15

13 15

Incidence

2 15

2

0

1

?

R a t a n d mouse (n = 7)

0 7

0

-

Dog, pig, sheep, and cat (n = 7)

+

M a n (n=l)

studies)

Species ("« «f

Antagonist efficient/ specific for histamine?

5

0

14 15

0

6

+

1 15

1

1

-

0 15

7

0

0

?

Correct doseresponse relationship?

0

0

15 15

0

6

+

0 15

0

1

-

0 15

7

1

0

?

Correct temporal relationship?

0

0

11 15

0

5

+

3 15

2

1

-

1 15

5

0

0

?

Study design appropriate?

1

0

14 15

1

7

+

1 15

0

1

-

0 15

6

0

0

?

Shock model clinically relevant?

1

0

12 15

0

7

+

0 15

0

1

-

3 15

4

0

0

?

Species clinically relevant?

0

0

14 15

3

7

+

1 15

0

0

-

0 15

7

0

1

?

Parameters measured adequate?

0

0

12 15

0

+

6

2 15

1

0

-

1 15

6

0

0

?

Development shock/outcome affected?

TABLE 30 Descriptive Statistics on the Methodological Shortcomings of All Studies Listed in Table 23 with Histamine Antagonists in Septic/Endotoxic Shock — Results of the Analysis of Tables 26 and 28

1

1

0

114 Handbook of Mediators in Septic Shock

115

Neugebauer, Rix.cn, and Lorenz

TABLE 31 Descriptive Statistics on the Evaluation of All Studies Listed in Table 23 with Histamine Antagonists in Septic/Endotoxic Shock — Results of the Analysis of Tables 27 and 29

Species inn nf studies)

Histamine antagonists shown to prevent or ameliorate septic/ endotoxic shock: authors estimation + -

M a n ( n= 1 )

Dog, sheep, pig, and cat (n = 7)

1

R a t a n d mouse (n = 7)

Study faultless? + -

0

6

1

1 4

Histamine antagonists shown to prevent or ameliorate septic/ endotoxic shock: our estimation" +

0

0 3

0

7 2

1

3 5

4 2

5

Incidence

11 15

4 15

3 15

12 15

5 15

10 15

%

73

27

20

80

33

67

*

Our estimation includes the evaluation of shortcomings; faults considered not very relevant may lead to a positive estimation of the study.

VI. DISCUSSION For a long time the role of histamine release and blockade in septic and endotoxic shock were assessed only by the global results of the different studies and not by their methodology. Neither the magnitude of the effects found, the quality of the method used, nor the validity of the research designs were evaluated adequately. This unfortunate situation, with many contradictory views, has confused this research field (see Hinshaw's review46 in 1971). Altura and Halevy3 were the first to point to the many deficiencies in most of the previous studies, and a more recent review by Parratt102 ends with the comment: "this is an area that badly needs critical reinvestigation". We have tried to look afresh at this problem with meta-analysis, a new method of analysis and evaluation of data and methods, in combination with a decision tree. Metaanalysis has been considered a significant advance in the methodology of reviewing scientific literature, but is little used both by clinicians and basic scientists.130 Meta-analysis may be a scientific alternative to performing new experiments. There are several fields in this new area of methodology: statistical analysis of a collection of analytic results,24 descriptive and inferential statistical analysis of randomized clinical trials,19-113 and metalevel knowledge, as defined as knowledge about knowledge and used, for instance, in the expert system TEIRESIAS.23 Alas, meta-analysis is inevitably retrospective. Furthermore, the meta-analysis of studies of histamine release and blockade in septic/endotoxic shock has proven to have difficulties in such evaluations. The components of the trials are complex and often nebulous, especially regarding the reliability of the assay used and the relevance of the body fluid assayed in the particular species. This situation must be just as complex for other mediators. In 1987, we recommended that specialists in other mediators of septic/endotoxic shock should use metaanalysis and hold consensus conferences.134 This book is a result of our intention. A round table conference was held in March 1992 in Brussels, organized by the European Society of Intensive Care Medicine. The results are

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Handbook of Mediators in Septic Shock

published in a most recent book by Springer Publishers.223 The aim of this book and especially this chapter was to prove a cause and effect relationship of histamine in septic/endotoxic shock by using the four generally accepted Koch-Dale criteria together with the decision tree approach. This aim has been reached not only for the Koch-Dale criteria 1 and 2 but also for the fourth criterion. The third Koch-Dale criterion could not be analyzed in the same way because the bulk of literature is too extensive. We used the traditional way of evaluation and had to accept the limitation of meta-analysis. Reliable demonstration of histamine release under in vivo septic/endotoxic shock conditions is the first prerequisite which has to be fulfilled before a discussion can start on possible cause-effect relationships. This was sufficiently fulfilled in our opinion only in two studies one in man and one in rats. The main shortcomings were in the assays of histamine and the designs of the studies. These usually neglected points account for most of the controversy on the role of histamine and other mediators in septic shock. Tests under clinical conditions must use reliable assays. The basic rules of randomized controlled trials should be obeyed in both experimental medicine and clinical research;45 otherwise, the studies may be not only scientifically unreliable but also unethical as well as a waste of resources. Our meta-analysis on the first two Koch-Dale criteria recommended only 6 of 27 studies validly demonstrated histamine release in experimental or clinical septic/endotoxic shock conditions. One study, performed in patients with confirmed sepsis (Study 5),136 however, did not differentiate between distinct phases of shock development. From the two studies in dogs:50-129 Study 1250 demonstrated histamine release more as an effect than as the cause of shock, with hypotension as a shock symptom beginning much earlier than histamine release; in Study 10,129 the close relationship of histamine release to hypotension, increase of portal vein pressure, and pooling of blood in the viscera was documented sufficiently in only one case. The two studies in rabbits (Studies 23 and 24) can be criticized because of the dubious clinical relevance of this species.22'106 Finally, the study in rats (Study 26)139 confirmed histamine release but only in the late phase of shock development with the highest histamine levels prior to death. The evaluation of the third Koch-Dale criterion led us to conclude, that histamine, with its different biological effects via Hj/H2- or H3 receptors can exert a positive or a negative effect thus exacerbating or ameliorating the progression of shock. A particular response is always the resultant of multiple components mediated by distinct histamine receptors. Our current hypothesis in septic/endotoxic shock is that histamine contributes to the early cardiovascular change via H^receptor vasoconstriction and to the late hypodynamic phase via H2-receptor vasodilatation and positive inotropic action. Thus a blockade of the early reaction with H, antagonists and a stimulation of the H2 receptors by H2 agonists are worthwhile therapeutic approaches. Our hypothesis was partly confirmed by evaluating the studies performed to prove Koch-Dale criterion 4. A total of 15 studies were performed and revealed a strong support at least for the fact, that H, antagonists exert beneficial effects on shock development. We believe that after more than 60 years of research in this field, with the availability of sufficient, excellent assays for histamine and with sufficiently powerful antagonists, that the role of this biogenic amine should be reexamined in such a common life-threatening disease as septic shock. We expect new insights from further experiments studying H t antagonism, H2 agonisms, cellular models, and experiments investigating the interaction with other mediators.

VII. SUMMARY Histamine was considered to be involved in the pathogenesis of circulatory shock of various etiologies since the beginning of this century. The estimation of its causal role in

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117

septic/endotoxic shock changed over the years, based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. Cause and effect is a relative concept, and a degree of likelihood or probability is sought. However, in biological systems it is not possible to prove causal relationships beyond any doubt. It is only possible to increase one's conviction of a cause and effect relationship by means of empiric evidence, to the extent where, for all intents and purposes, cause is established. In evaluating whether a cause and effect relationship of histamine (and other mediators) and septic/endotoxic shock pathogenesis exists, it is important to rule out bias, chance, and confounding influences. This can be achieved — thus coming to a more valid evaluation of the current status of histamine in septic/endotoxic shock — by using the method of metaanalysis in combination with the model of a decision tree. This methodology was applied to the four classical criteria of Koch-Dale to prove a cause and effect relationship of histamine in septic/endotoxic shock. We started with a comprehensive database of more than 45,000 publications on histamine in health and disease. Only 27 studies in seven different species were found which investigated histamine release in septic/endotoxic shock in vivo (Koch-Dale 1 and 2: presence in disease and absence in health). They were all evaluated by the criteria defined by means of test nodes in the decision tree. Only two of them, one in man and one in rat, were free of faults by decision tree criteria; a total of six were finally considered reliable under conditions of uncertainty for demonstration of histamine release. Unreliable assays, inappropriate sample preparation and serious deficiencies in study design were the main shortcomings. There was a marked disagreement in the conclusion that histamine release had been demonstrated between the authors of the 27 studies and our meta-analysis (74 vs. 19%). It must be concluded, that despite decades of histamine research, we still have a nonconclusive answer to the question of histamine release in septic/endotoxic shock. Over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. The analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via H,-receptor vasoconstriction thus exacerbating (directly and indirectly) the effects of catecholamines. In the later phase of septic shock (low-output, high resistance state), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via H2 receptors. This effect is considered beneficial in counteracting or attenuating the effects of the sympathetic responses of catecholamines and other mediators (Koch-Dale criterion 3). This assessment is supported by the meta-analysis investigating the Koch-Dale criterion 4: blockade of the effects by histamine antagonists. Of a total of 15 studies performed, 5 (33%) were finally considered reliable for demonstration of histamine antagonists preventing or ameliorating signs and/or symptoms of septic/endotoxic shock. With the exception of one study a positive effect on hemodynamics was only demonstrated with Hj antagonists. On the contrary, results strongly support the assumption that the combination of H{/112 blockers does not have a benefit and may even be detrimental. Solely, H2 antagonists are considered to negatively influence shock development. Thus, a blockade of the early reaction with HI antagonists and a stimulation of the H2 receptors by specific H2 agonists are worthwhile therapeutic approaches. This new concept, however, needs further testing in randomized controlled experimental and clinical trials.

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Handbook of Mediators in Septic Shock

REFERENCES 1. Adam, H. M., Hardwick, D. C., and Spencer, K. E. V., Method for determination of histamine in plasma, Br. J. Pharmacol., 12, 397, 1957. 2. Almeida, A. P., Flye, W., Deveraux, D., Horakova, Z., and Beaven, M. A., Distribution of histamine and histaminase (diamine oxidase in blood of various species, Comp. Biochem. Physiol., 670, 187, 1980. 3. Altura, B. M. and Halevy, S., Circulatory shock, histamine and antihistamines: therapeutic aspects, in Handbook of Experimental Pharmacology, Vol. 18, Rocha e Silva, M., Ed., Springer-Verlag, Berlin, 1978, 575. 4. Anton, A. M. and Sayre, D. F., A modified fluorometric procedure for tissue histamine and its distribution in various animals, J. Pharmacol. Exp. Ther., 166, 285, 1969. 5. Arakawa, Y. and Tachibana, S., A direct and sensitive determination of histamine in acid-deproteinized biological samples by high-performance liquid chromatography, Anal. Biochem., 158, 20, 1986. 6. Barsoum, G. S. and Gaddum, J. H., The pharmacological estimation of adenosine and histamine in blood, J. Physiol. (Land.), 85, 1, 1935. 7. Beger, H. G. and Biichler, M., Decision-making in surgical treatment of acute pancreatitis: operative or conservative management of necrotizing pancreatitis?, Theor. Surg., 2, 61, 1986. 8. Beger, H. G. and Stopik, D., Histamine release and hepatic elimination of histamine following abdominal surgery, Klin. Wochenschr., 17, 935, 1982. 9. Beaven, M. A. and Wolde Mussie, E., Histamine in body fluids: its measurement in different clinical states, NER Allerg. Proc., 5, 300, 1984. 10. Beaven, M. A., Jacobsen, S., and Horakova, Z., Modification of the enzymatic isotopic assay of histamine and its application to measurement of histamine in tissues, serum and urine, Clin. Chir. Acta, 37, 91, 1972. 11. Beaven, M. E., Robinson-White, A., Roderick, N. B., and Kauffmann, G. L., The demonstration of histamine release in clinical conditions: a review of past and present assay procedures, Klin. Wochenschr., 17, 873, 1982. 12. Born, G. V. R. and Vane, J. R., The quantitative determination of diffusible histamine in blood, Br. J. Pharmacol., 7, 298, 1952. 13. Brown, M. J., Jud, P. W., Barues, P. J., Jenner, D. A., and Dollery, C. T., A sensitive and specific radiometric method for the measurement of plasma histamine in normal individuals, Anal. Biochem., 109, 142, 1980. 14. Caldwell, R. W., Vick, J. A., and Heiffer, M. H., Modification of experimental endotoxin shock with WR 149,024, Circ. Shock, 2, 265, 1975. 15. Carraway, R., Cochrane, D. E., Lansman, J. B., Leeman, S. E., Paterson, B. M., and Welch, H. J., Neurotensin stimulates exocytotic histamine secretion from rat mast cells and elevates plasma histamine levels, J. Physiol. (Land.), 323, 403, 1982. 16. Cho, C. H., Wu, T. C., Ting, C. W., and Chen, S. W., Gastric and serum histamine in respiratory disorders and in sepsis of newborns, IRCS Med. Sci., 13, 200, 1985. 17. Code, C. F., The quantitative estimation of histamine in blood, J. Physiol. (Land.), 89, 257, 1937. 18. Code, C. F. and Jensen, J. L., A comparison of the histamine content of blood and bone marrow, Am. J. Physiol, 131, 738, 1941. 19. Collins, R., Yusuf, S., and Peto, R., Overview of randomized trials of diuretics in pregnancy, Br. Med. J., 290, 17, 1985. 20. Corrado, A. P., Garcia Lima, E., and Rothschild, A. M., Study of the mechanism of the cardiovascular shock produced by endotoxin of gram-negative bacteria: E. coli and S. typhii, Arch. Intern. Pharmacodyn. Ther., 150, 462, 1964. 21. Dale, H. H., Conditions which are conductive to the production of shock by histamine, Br. J. Exp. Pathol., 1, 103, 1920. 22. Davis, R. B., Bailey, W. L., and Hanson, N. P., Modification of serotonin and histamine release after E. coli endotoxin administration, Am. J. Physiol., 205, 560, 1963. 23. Davis, R. and Buchanan, B. G., Meta-level knowledge, in Rule-Based Expert Systems. The Mycin Experiments of the Stanford Heuristic Programming Project, Buchanan, B. G. and Shortliffe, E. H., Eds., Allison-Wesley, Reading, MA, 1984. 24. De Simonian, R. and Laird, N., Meta-analysis in clinical trials, Contr. Clin. Trials, 1, 177, 1986. 25. Doenicke, A., Ennis, M., and Lorenz, W., Histamine release in anaesthesia and surgery: a systematic approach to risk in the perioperative period, in AnaphylactoidReactions in Anaesthesia, Sage, D. J., Ed., Int. Anaesthesiol. Clin., 23, 41, 1985. 26. Eichler, O. and Barfufi, F., Untersuchungen iiber den Histamingehalt des Bluter bei Infusion von Adrenalin und Histamin, Naunyn Schmiedebergs Arch. Exp. Pathol. Pharmacol., 195, 245, 1940.

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157. Antohe, F., Helitanu, C., and Simionescu, N., Further evidence for the distribution and nature of histamine receptors on microvascular endothelium, Microcirc. Endothelium Lymphatics, 3, 163, 1986. 158. Movat, H. Z., The role of histamine and other mediators in microvascular changes in acute inflammation, Can. J. Physiol. Pharmacol, 65, 451, 1987. 159. Lorenz, W., Ennis, M., Doenicke, A., and Dick, W., Perioperative use of histamine antagonists, J. Clin. Anaesth., 2, 345, 1990. 160. Lorenz, W., Doenicke, A., Meyer, R., Reimann, H. J., Kusche, J., Barth, H., Geesing, H., Hutzel, M., and Weissenbacher, B., Histamine release in man by propanidid and thiopentone: pharmacological effects and clinical consequences, Br. J. Anaesth., 44, 355, 1972a. 161. Lorenz, W., Doenicke, A., Meyer, R., Reimann, H. J., Kusche, J., Barth, H., Geesing, H., Hutzel, M., and Weissenbacher, B., An improved method for the determination of histamine release in man: its application in studies with propanidid and thiopentone, Eur. J. Pharmacol., 19, 180, 1972b. 162. Ind, P. W., Brown, M. J., Lhoste, F. J. M., Macquin, I., and Dollery, C. T., Concentration effect relationship of infused histamine in normal volunteer's, Agents Actions, 12, 12, 1982. 163. Kaliner, M., Shelhamer, J. H., and Ottesen, E. A., Effects of infused histamine: correlation of plasma histamine levels and symptoms, J. Allergy Clin. Immunol, 69, 283, 1982. 164. Hinshaw, L. B., Emerson, T. W., Jr., lampietro, P. F., and Brake, C. M., A comparative study on the hemodynamic actions of histamine and endotoxin, Am. J. Physiol., 203, 600, 1962. 165. Jordan, M. M., Holmes, D. D., and Hinshaw, L. B., Pathophysiological comparisons of histamine and endotoxin shock, J. Trauma, 5, 726, 1965. 166. Sleeman, H. K., Lamborn, P. B., Diggs, J. W., and Emery, L. E., Effects of endotoxin and histamine on serum enzyme activity, Proc. Soc. Exp. Biol. Med., 138, 536, 1971. 167. Schayer, R. W., Evidence that induced histamine is an intrinsic regulator of the microcirculatory system, Am. J. Physiol., 202, 66, 1962. 168. Nagy, S., The role of histamine release in shock, Acta Phys. Hung., 76, 3, 1990. 169. Parratt, J. R. and Sturgess, R. M., The possible roles of histamine, 5-hydroxy tryptamine and prostaglandin F2a]pha as mediators of the acute pulmonary effects of endotoxin, Br. J. Pharmacol., 60, 209, 1977. 170. Rixen, D., Lechleuthner, A., Saad, S., Buschauer, A., Nagelschmidt, M., Thoma, S., Rink, A., and Neugebauer, E., Beneficial effect of H2-agonism and H,-antagonism in endotoxic shock, Circ. Shock, 34, 133, 1991. 171. Hinshaw, L. B., Reins, D. A., and Hill, R. J., Response of isolated liver to endotoxin, Can. J. Physiol. Pharmacol., 44, 529, 1966. 172. Orlidge, A. and Hollis, T. M., Aortic endothelial and smooth muscle histamine metabolism in experimental diabetes, Arteriosclerosis, 2, 142, 1982. 173. Altura, B. M. and Zweifach, B. W., Endogenous histamine formation and vascular reactivity, Am. J. Physiol., 212, 559, 1967. 174. Neugebauer, E., Lorenz, W., Beckurts, T., Maroske, D., and Merte, H., Significance of histamine formation and release in the development of endotoxic shock: proof of current concepts by randomized controlled studies in rats, Rev. Inf. Dis., 9, 585, 1987. 175. Neugebauer, E., and Lorenz, W., A modified Schayer procedure for the estimation of histidine decarboxylase activity: its application on tissue extracts from gastric mucosa of various mammals, Agents Actions, 12, 32, 1982. 176. Neugebauer, E. and Lorenz, W., Identification and measurement of acid (specific) histidine decarboxylase activity in rabbit gastric mucosa: ending an old controversy?, Biol. Chem. Hoppe Seyler, 366, 411, 1985. 177. Neugebauer, E., Beckurts, T., Lorenz, W., Maroske, D., Merte, H., Horeyseck, G., and Dietz, W., Induced histidine decarboxylase in endotoxic shock: identification of the enzyme in rat liver and influence of its inhibitors on survival parameters, Agents Actions, 18, 23, 1980. 178. Altura, B. M., Humoral, hormonal, and myogenic mechanisms in microcirculatory regulatory regulation, in Microcirculation, Vol. 2, Valey, G. and Altura, B. M., Eds., University Park Press, Baltimore, 1978, 411. 179. Ryan, M. and Brody, M. J., Neurogenic and vascular stores of histamine in the dog, J. Pharmacol. Exp. Ther., 181, 83, 1972. 180. McGrath, M. and Shepherd, J. T., Inhibition of adrenergic neurotransmission in canine vascular smooth muscle by histamine, Circ. Res., 39, 566, 1976. 181. Dohlston, M. et al., Scand. J. Immunol., 27, 527, 1988. 182. Vannier, E., Miller, L. C., and Dianarello, C. A., Histamine suppresses gene expression and synthesis of tumor necrosis factor alpha via histamine H2-receptors, J. Exp. Med., 174, 281, 1991. 183. Einstein, R., Mihailidou, A. S., and Richardson, D. P., Positive inotropic effects of histamine in anaesthetized dogs, Br. J. Pharmacol., 92, 445, 1987. 184. Christensen, C. W., Gross, G. J., Havelman, H. F., Brooks, H. L., and Waritier, D. C., Effects of histamine receptor stimulation on regional myocardial blood flow, Am. J. Physiol., 245, H461, 1983.

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185. Miller, W. L. and Bove, A. A., Differential H,- and H2-receptor mediated histamine responses of canine epicardial conductance and distal resistance, coronary vessel, Circ. Res., 62, 226, 1988. 186. Cannion, R. and Parrillo, J. E., Myocardial dysfunction in sepsis — recent insight, Chest, 95, 941, 1989. 187. Rachow, E. C. and Astiz, M. E., Pathophysiology and treatment of septic shock, JAMA, 266, 548, 1991. 188. Felix, S. B., Buschauer, A., and Baumann, G., Structure-activity relationships of histamine H2-agonists, a new class of positive inotropic drugs, in New Perspectives in Histamine Research, Timmermann and van derGoot, Eds., Birkhauser-Verlag, Basel, Agents Actions Suppl. 33, 1991, 257. 189. Buschauer, A. and Baumann, G., Structure-activity relationship of histamine H2-agonists, a new class of positive inotropic drugs, in New Perspectives in Histamine Research, Timmermann and van der Goot, Eds., Birkhauser-Verlag, Basel, Agents Actions Suppl., 1991, 231. 190. McNeil, J. M., Histamine in the heart, Can. J. Physiol. Pharmacol., 62, 720, 1984. 191. Ishikava, S. and Sperelakis, N., A novel class (H3) of histamine receptors on peripheral nerve terminals, Nature, 327, 158, 1987. 192. Bovet, D. and Staub, M. A. M., Action protective des ethers phenoliques au cours de 1'intoxication histaminique, Soc. Biol, 124, 547, 1937. 193. Black, J. W., Duncan, W. A. M., Durant, C. J., Ganellin. C. R., and Parsons, E. M., Definition and antagonism of histamine H2-receptors, Nature, 236, 385, 1972. 194. Ash, A. S. F. and Schild, H. O., Receptors mediating some actions of histamine, Br. J. Pharmacol. Chemother., 27, 427, 1966. 195. Arrang, J. M. et al., Highly potent and selective ligands for histamine H,-receptors, Nature, 327, 117, 1987. 196. Saxena, S. P., Brandes, L. J., Becker, A. B., Simons, K. J., La Bella, F. S., and Gerrard, J. M., Histamine is an intracellular messenger mediating platelet aggregation, Science, 243, 1596, 1989. 197. Cook, D. A., Recent advances in histamine research, Can. J. Physiol. Pharmacol., 62, 738, 1984. 198. Nielsen, H. J., Witt, K., Moesgaard, F., and Kehlet, H., Ranitidine for improvement of delayed hypersensitivity, response in patients with sepsis, Acta Chir. Scand., 155, 445, 1989. 199. Martin, D. S., Kurzeg, F. T., Amor, R. L., Harvey, R., and Daigle, L., Multiantagonist and volume therapy for late endotoxin shock, Surgery, 60, 420, 1966. 200. Lowry, P., Blanco, T., and Santiago-Delpin, E. A., Histamine and sympathetic blockade in septic shock, Am. Surgeon, January 1977, 12. 201. Krause, S. M. and Hess, M. L., Diphenhydramine protection of the failing myocardium during gramnegative endotoxemia, Circ. Shock, 6, 75, 1979. 202. O'Neil, J., Lowry, P. C., and Cruz, N. I., Inhibition of vasoactive substances in experimental endotoxin shock, PRHSJ, 5, 1, 1986. 203. Brigham, K. L., Padove, S. J., Bryant, D., McKeen, C. R., and Bowers, R. E., Diphenhydramine reduces endotoxin effects on lung vascular permeability in sheep, Am. Physiol. Soc., 40, 516, 1980. 204a. Byrne, K., Sielaff, T. D., Carey, D., Tatum, J. L., Blocher, C. R., Vasquez, A., Hirsh, J. I., and Sugerman, H. J., Ranitidine compared to cimetidine in multiagent pharmacological treatment of porcine pseudomonas ARDS, Circ. Shock, 30, 117, 1990. 204b. Byrne, K., Sielaff, T. D., Michna, B., Carey, P. D., Blocher, C. R., Vasquez, A., and Sugerman, H. J., Increased survival time after delayed histamine and prostaglandin blockade in a porcine model of severe sepsis-induced lung injury, Crit. Care. Med., 18, 303, 1990. 205. Brackett, D. J., Schaefer, C. F., and Wilson, M. F., The effects of H, and H, histamine receptor antagonists on the development of endotoxemia in the conscious, unrestrained rat, Circ. Shock, 16, 141, 1985. 206. Wittig, H., Cook, T. J., and Rittmanic, T., Protection against fatal endotoxin shock in mice by antihistamines, Allergol. Immunopathol., VI, 409, 1978. 207. Shenkman, B. Z., Effect of verapamil, cromoglycate, and diphenhydramine on survival of mice after endotoxic shock, Plenum Publishing Corporation, Vol. 104, 10, 1988, 428. 208. Gilbert, R. P., Effect of antihistaminic and antiserotonin drugs on vascular responses to E. coli endotoxin in the cat, P.S.E.B.M., 346, 1959. 209. Baker, C. H. and Wilmoth, F. R., Microvascular responses to E. coli endotoxin with altered adrenergic activity, Circ. Shock, 12, 165, 1984. 210. Altura, B. M. and Altura, B. T., Effects of local anaesthetics, antihistamines and glucocorticoids on peripheral blood flow and vascular smooth muscle, Anaesthesiology, 41, 197, 1974. 211. Bertacchini, G. and Coruzzi, G., H2-receptor antagonists: side effects and adverse effects, Ital, J. Gastroneterol, 16, 119, 1984. 212. Schunack, W., H,- and H2-Rezeptorantagonisten, in Histamin bei Erkrankungen, Bd 1, Speicherungen, Freisetzung und Wirkung an Rezeptoren, Reimann, H. J., Habs, H., Hrg., MD-Verlag, Miinchen, 1986, 39.

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213. Ennis, M. and Lorenz, W., Histamine receptor antagonists, in Discoveries in Pharmacology, Vol. 2, Haemodynamics, hormones and inflammation, Parnham, M. J., and Bruinvels, Eds., Elsevier, Amsterdam, 1984, 623. 214. Kubo, N., Shirakawa, O., Kuno, T., and Tanaka, C., Antimuscarinic effects of antihistamines: quantitative evaluation by receptor binding assays, Jpn. J. Pharmacol., 43, 277, 1989. 215. Coruzzi, G., Poll, E., and Bertacchini, G., Cardiac effects of the new H2-receptor antagonists, Agents Actions, 13, 173, 1983. 216. Neugebauer, E., Dietrich, A., Lechleuthner, A., Bouillon, B., and Eypasch, E., Pharmacotherapy in shock-syndromes: the neglected field of pharmacokinetics and -dynamics, Circ. Shock, 36, 312, 1992. 217. Harmon, J. P., Bossone, C. A., and Wade, C. E., Normal physiological values for conscious pigs used in biomedical research, Lab. Anim. Sci., 40, 293, 1990. 218. Sattler, J., Lorenz, W., Kubo, K., Schmal, A., Sauer, S., and Lueben, L., Food induced histaminosis and diamine oxidase (DAO) blockade in pigs: further evidence of the key role of elevated plasma histamine levels as demonstrated by successful prophylaxis with antihistamines, Agents Actions, 27, 212, 1989. 219. Gahhos, F. N., Chiu, R. C. J., Bethune, D., Dion, Y., Hinchey, E. J., and Richards, G. K., Memodynamic responses to sepsis: hypodynamic versus hyperdynamic states, 1981. 220. Staub, N. C., Blond, R. D., Brigham, K. L., Demling, R., Erdmann, A. J., Ill, and Woolverton, C. W., Preparation of chronic lung lymph fistulas in sheep, J. Surg. Res., 19, 315, 1975. 221. Traber, D. L., Redl, H., Schlag, G., Herndon, D. N., Kimura, R., Prien, T., and Traber, L. D., Cardiopulmonary responses to continuous administration of endotoxin, Am. J. Physiol., 254, H833, 1988. 222. Neugebauer, E., Lechleuthner, A., Rixen, D., and Saad, S., Pharmacotherapy of shock, in The Pharmacological Approach to the Critically III Patient, 3rd ed., Chernow, B., Ed., in press, 1993. 223. Vincent, J. L., Lamy, M., and Thijs, L., Mediator of Sepsis, Springer-Verlag, Berlin, 1993.

Chapter 4

5-HYDROXYTRYPTAMINE AS A CHEMICAL MEDIATOR OF SHOCK James R. Parratt and Brian L. Furman

TABLE OF CONTENTS I.

Introduction A. Distribution of 5-Hydroxytryptamine B. Actions of 5-HT C. Receptors Mediating the Actions of 5-HT D. Assessment of the Role of 5-HT in Shock

128 128 128 128 128

II.

Release of 5-HT during Shock

129

III.

Ability of 5-HT to Mimic Shock A. Production of a Shock-Like State by 5-HT B. Regional Vasoconstriction C. Endothelial Function D. Platelet Function E. Metabolic Changes

131 131 131 131 132 132

IV.

Effects upon Shock of Antagonism or Depletion of 5-HT

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V.

Concluding Remarks

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VI.

Summary

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References

0-8493-3548-5/93/$0.00 + $.50 © 1993 by CRC Press, Inc.

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I. INTRODUCTION A. DISTRIBUTION OF 5-HYDROXYTRYPTAMINE 5-Hydroxytryptamine (5-HT) (serotonin) is distributed widely throughout the mammalian body. It is present in the brain, where its role as a neurotransmitter is well established. 5HT is also present in peripheral structures including the gastrointestinal tract both in enterochromaffin cells1 and enteric neurons,2 platelets,3 the heart,4 the blood vessel wall,4 and in the mast cells of rodents,5 but not of other species.6 The liver clears a substantial proportion of 5-HT released from the gastrointestinal tract,7 the remainder ending up in platelets8 or being taken up and metabolized by endothelial cells, especially of the lung9 and by blood vessel walls, where it is largely disposed of into noradrenergic neurons.10 The bulk of circulating 5-HT is present in the blood platelets and this may be the most important 5-HT in relation to the cardiovascular system. B. ACTIONS OF 5-HT 5-HT has numerous actions. In the vascular system it produces vasoconstriction in large conducting arteries, in veins, and also in resistance vessels.11 Vasoconstriction may be through direct actions on vascular smooth muscle or indirect actions via a depolarizationinduced release of noradrenaline from sympathetic nerve endings.'1 Vasoconstriction induced by 5-HT may also involve amplification of the actions of other vasoconstrictor agents.12 5HT also produces vasodilator responses in many vessels. Again this action is complex involving direct actions on vascular smooth muscle, release of endothelium-derived relaxant factor (EDRF) and inhibition of noradrenaline release.13 The in vivo cardiovascular actions of 5-HT are further complicated by its ability to stimulate sensory nerve endings, thereby activating reflexes which may evoke either hypotension and bradycardia (via epicardial receptors and vagal afferents) or hypertension (via carotid body chemoreceptors).14 Apart from the release of EDRF, 5-HT also modifies endothelial cell function, causing a separation along their junctions,15 an increase in ionic permeability,16 a decrease in albumin permeability,17 a decrease in cell movement in culture,18 and an increased white blood cell adherence.19 5-HT also has widespread actions outside the cardiovascular system. It produces contraction or relaxation of a variety of smooth muscle including that in the gastrointestinal and respiratory tracts where again its actions are a mixture of direct effects and effects involving modulation of neurotransmitter release.20 5-HT also produces an increased intestinal secretion.21 C. RECEPTORS MEDIATING THE ACTIONS OF 5-HT 5-HT research has been greatly facilitated by the recognition of three main receptors, the 5-HTrlike, the 5-HT2, and the 5-HT3 receptors. These have been characterized on the basis of ligand binding and functional studies and as selective agonists and antagonists have become available22 (see Table 1). Analysis of the receptors mediating the actions of 5-HT has revealed a complex situation. For example, vasoconstriction may be mediated by 5-HT, or 5-HT2 subtypes, depending on the vascular bed under consideration and the species studied.11 D. ASSESSMENT OF THE ROLE OF 5-HT IN SHOCK In order to accept a role for 5-HT in the pathophysiology of shock, several criteria must be fulfilled. 1. 2.

5-HT must be shown to be released during shock. 5-HT must be able to mimic one or more of the pathophysiological changes seen in shock.

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TABLE 1 Summary of 5-HT Receptor Classification Nomenclature Previous names Selective agonists Selective antagonists Radioligands

Effector pathways

5-HT1A

5-HT1B

5-HTlc

5-HT1D

—

—

—

—

D

8-OH-DPAT Ipsapirone (partial agonist) —

—

a-methyl-5-HT

—

a-methyl-5-HT 2-Methyl-5HT

—

Ritanserin Pizotifen

—

MDL72222 ICS205930 Ondansetron [3H]GR6563 [3H]quipazinc [3H]zacopride [3H]ICS20592 Cation channel

5-HT,

Ritanserin Ketanserin Pizotifen [3H]8-OH-DPAT [3H]Cyanopindolol [3H)5-HT [3H]5-HT [3H]Ketanserin [3H]5-HT [125I]Lysergide [3H]Spiperone [3H]Ipsapirone

cAMP K + channel (G protein)

cAMP

IP3/DG

cAMP

IP3/DG

5-HT, M

Note: There is also evidence for further subclassification of the 5-HT, receptor, for subtypes of 5-HT2 and 5-HT3 receptors and for the existence of a 5-HT4 receptor. Modified and reproduced from TIPS January 1990, Receptor Nomenclature Supplement. With permission.

3. 4.

Specific pharmacological antagonism of 5-HT should ameliorate shock, especially those aspects that are mimicked by 5-HT. Depletion of 5-HT should ameliorate shock as in (3) above.

These are discussed below. The different forms of shock (cardiogenic, hemorrhagic, traumatic, and septic/endotoxic) are dealt with together, rather than under separate headings.

II. RELEASE OF 5-HT DURING SHOCK Several studies have shown changes in blood and tissue levels of 5-HT during shock, although the results are not consistent. The inconsistencies may relate to the type of shock, the animal species, the method used to determine 5-HT or whether determinations were made using whole blood or plasma. Earlier studies have been reviewed by Kovats,23 Emerson,24 and Urbaschek and Urbaschek.25 Elevation of plasma 5-HT, determined fluorometrically and by bioassay, was found following systemic hemorrhagic hypotension in cats and dogs.26-27 This also occurred following local intestinal ischemia, which together with a relatively greater increase in intestinal venous 5-HT, relative to arterial concentrations, suggested an intestinal origin for the amine.26 The 5-HT could come from the intestinal enterochromaffin cells or from platelets. Similar findings were made during histamine-induced hypotension28 and intestinal vascular occlusion in dogs and rabbits.29'30 Endotoxin shock produced a marked and rapid increase in plasma 5-HT concentrations in rabbits,31 sheep,32 and calves.33 In rabbits the marked (two- to threefold) increase within the first minute of endotoxin administration continued throughout shock, whereas in sheep, the marked initial increase was followed by a significant and sustained decrease (Figure 1), which was attributed to thrombocytopenia.32 There is some evidence that steroids can modify this release.31-34 Endotoxin promotes the rapid release of 5-HT from platelets.35"38 This release requires calcium and a plasma protein factor with properties similar to those of prothrombin;38 the response requires activation of complement and is inhibited by heparin.37 It is unlikely that endotoxin induces the release of 5-HT from intestinal enterochromaffin cells, since

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FIGURE 1. Effects of intravenous infusion of Pasteurella hemolytica endotoxin on plasma levels of 5-HT in sheep. Probability values indicate the levels of significance as compared to zero time (N = 5). (From Emau, P., Giri, S. W., and Brass, M. L., Circ. Shock, 12, 47, 1984. With permission.)

administration of an LD100 dose of endotoxin to piglets did not affect the 5-HT content of their enterochromaffin cells, relative to that measured in sham-shocked controls.39 Endotoxemia resulted in a significant increase in the de novo formation of 5-HT from tryptophan, with a diversion from the other pathways of tryptophan metabolism.40 A positive correlation was found between plasma 5-HT and pulmonary vascular resistance in patients with sepsis-related adult respiratory distress syndrome, suggesting that 5-HT may contribute to pulmonary hypertension in these patients.41 This could, however, reflect a reduction in 5-HT removal by damaged pulmonary endothelial cells. In this context, reduced 5-HT removal by the lung was shown in dogs subjected to hemorrhagic shock 1 and 2 h after resuscitation, although removal was increased during the first hour of shock.42 Increases in lung 5-HT during endotoxin shock in the cat were reported by Naik et al.43 Liver 5-HT was markedly increased in rats injected with endotoxin,44-45 although this was not seen in the cat.43 a-Monofluoromethyldopa, an irreversible inhibitor of aromatic amino-acid decarboxylase, while preventing 5-HTP-induced hepatic 5-HT accumulation, did not modify 5-HT accumulation in response to endotoxin.45 This suggests that endotoxin-induced hepatic 5HT accumulation was from preformed (platelet or gastrointestinal?) 5-HT. Decreases in blood 5-HT concentrations were reported in hemorrhagic shock in dogs46 and endotoxin shock in cats.43 In contrast, Halevy et al.47 found no change in serum 5-HT concentrations in rats after intestinal ischemic shock.

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III. ABILITY OF 5-HT TO MIMIC SHOCK A. PRODUCTION OF A SHOCK-LIKE STATE BY 5-HT The prolonged infusion of 5-HT in dogs48 and rabbits49 results in typical shock-like lesions including: profound systemic hypotension; solidification of the lungs by extravasated plasma and red cells; transalveolar edema with increased pulmonary airways resistance and reduced lung compliance; edema and hemorrhagic ulceration of the gastrointestinal mucosa.49 There is evidence, however, that these lesions were not specific to 5-HT, as they also occurred following prolonged infusion of either histamine or bradykinin. Moreover, the lesions appeared to result from the release into the circulation of endotoxin from the intestine. Endotoxin was detected in high liters in plasma and peritoneal fluid of animals infused with 5-HT. Both the endotoxemia and the severity of the lesions were markedly reduced by pretreatment with an antibiotic.49 Nevertheless, the direct vascular and other direct actions of 5-HT permit its further consideration as a mediator of shock, as discussed below. Oral pretreatment of dogs with tryptophan increased mortality following endotoxin administration but no evidence was presented to support or refute a role for 5-HT in this action.50 One must also consider an isolated report that pretreatment of mice with 5-HT actually protected the animals from burn, tourniquet, or endotoxin shock.51 This protective effect was shared by tryptamine, 5-methyltryptamine, and 5-methoxytryptamine which are now known to have higher affinity for 5-HT; binding sites than for 5-HT2 sites.22 The significance of this protection is unclear, especially as histamine was also protective. B. REGIONAL VASOCONSTRICTION Pulmonary hypertension is a feature of various forms of shock, including septic shock.41 It is, therefore, of interest that 5-HT is a potent pulmonary vasoconstrictor,52"54 an effect probably mediated by 5-HT2 receptors.55 The ability of 5-HT to produce mesenteric vasoconstriction permits consideration of its possible role in the reduction in intestinal blood flow and intestinal mucosal lesions associated with shock. The reported mesenteric vascular effects, however, are variable, species dependent, and sometimes (as in the pig) predominantly vasodilator, especially following local administration.39 5-HT is also a potent renal vasoconstrictor56 and could contribute to the renal lesions seen in shock.57 It must be kept in mind that the responsiveness to various vasoconstrictors and vasodilators may be modified by shock. In porcine splanchnic arterial occlusion shock the pulmonary venous and arterial contractile responsiveness to noradrenaline and, especially, to 5-HT was markedly reduced.58 Both the vasopressor and vasodepressor actions of 5-HT (and other agents) are markedly reduced in endotoxemia in the rat.59 If this altered reactivity occurs differently in different vascular beds it could contribute to the redistribution of blood flow seen in septic/endotoxic shock.60-61 C. ENDOTHELIAL FUNCTION There is considerable evidence for vascular endothelial-cell injury in response to bacterial endotoxin.62 There is also evidence that the endothelium contributes to the loss of vascular reactivity seen in sepsis/endotoxemia.63'64 Recent studies from our own group implicate an endotoxin-induced release of nitric oxide (an EDRF) in this loss of reactivity (see Chapter 18). Moreover, Salvemini et al.65 showed that lipopolysaccharide increased the release of a nitric oxide-like factor from endothelial cells. As indicated above, 5-HT can modify endothelial function, including an enhancement of EDRF (nitric oxide) release. It has also been shown to enhance endothelial-cell damage induced by activated complement/polymorphonuclear leukocytes.66 Stewart67 pointed out the similarity between the effects of

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endotoxin shock on hepatic sinusoids and the actions of 5-HT on endothelial cells in other organs. The 5-HT receptor mediating these various endothelial actions is not known with certainty. However, there is evidence that 5-HTrlike receptors mediate the release of EDRF and 5-HT2 receptors may mediate the changes in endothelial-cell structural characteristics and permeability.20 D. PLATELET FUNCTION Many studies have shown an endotoxin-shock-induced thrombocytopenia that is associated with platelet aggregates localized in pulmonary and hepatic capillaries.62 Endotoxin also induces activation of rat, rabbit, dog, and guinea pig platelets in vitro, although interactions with human platelets are less clear.62 Platelet activation may significantly contribute to the disseminated intravascular coagulation and pulmonary hypertension of endotoxic/ septic shock.68-69 5-HT not only activates platelet aggregation but also augments the effects of adrenaline and adenosine diphosphate.70 The effects of 5-HT on platelets appear to be mediated by 5-HT2 receptors.71 E. METABOLIC CHANGES Marked hypoglycemia is a feature of various types of shock.72-73 Several mechanisms may be involved, but inhibition of hepatic gluconeogenesis may be of major importance.73'74 5-Hydroxytryptophan, the precursor of 5-HT, produces hypoglycemia in mice, an effect probably mediated by 5-HT.45-75'77 As 5-HT also inhibits hepatic gluconeogenesis,78 this could contribute to the hypoglycemia of shock. There was a close relationship between the time course of endotoxin-induced hepatic accumulation of 5-HT and the time course of endotoxin-induced hypoglycemia in the rat.44

IV. EFFECTS UPON SHOCK OF ANTAGONISM OR DEPLETION OF 5-HT 5-HT antagonists have been shown to modify some pathophysiological changes seen in various types of shock. Thus, the 5-HT receptor blocking drug methysergide prevented the increase in small pulmonary venous pressure in dogs subjected to hemorrhagic shock.48 Although the abstract of the paper by Kusajima et al.48 states that methysergide greatly reduced the microscopic and cinemicroscopic lung changes, the main text reports that "addition of methysergide did not change the histologic picture seen in the hemorrhagic shocked lung". Methysergide, but not the antihistamine drug pyrilamine, significantly reduced the hypotensive response to an intravenous injection of killed Escherichia coli organisms, without modifying the thrombocytopenia.79 Endotoxin-induced increases in renal vascular resistance and reductions in renal blood flow in rats were prevented by methysergide but not by aadrenoceptor blockade with phenoxybenzamine.80 However, neither the effects of endotoxin in reducing renal filtration and urine volume nor its effects on cardiovascular function were modified by methysergide. Pulmonary platelet trapping produced by endotoxin-induced shock in dogs was prevented by the potent 5-HT receptor-blocking drug cyproheptadine, although only when the drug was given after induction of shock.81 Cyproheptadine did not modify the endotoxin-induced hypotension. The above studies are all somewhat difficult to interpret because of the relative lack of specificity of the antagonists that were used. Cyproheptadine has potent antihistamine and muscarinic receptor-blocking properties, whereas methysergide produces cardiovascular depression, at least in the cat,52 and also has agonist activity at 5-HT receptors.82 At the beginning of this decade, the introduction of the selective 5-HT2 receptor-blocking drug, ketanserin, facilitated research, although this agent also possesses some aradrenoceptor blocking activity.83

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FIGURE 2. Mean systolic pulmonary artery pressure vs. time postburn in control pigs (Group I) and in pigs receiving ketanserin preburn (Group II, 12 mg i.m. preanesthesia, followed by 2 mg/h postanesthesia and for 24 h postburn) or postburn (Group III, 5 mg i.v. 1 h postburn and then 2 mg/h for 24 h postburn). (From Holliman et al., J. Trauma, 23, 867, 1983. Reproduced with permission.)

Studies with ketanserin, in various models of shock, have yielded mixed results. In a porcine model of burn shock (44% body surface, full-thickness scald burn) ketanserin, administered either before or after the injury, produced marked increases in cardiac index and ameliorated the pulmonary hypertension (Figure 2) in the first 12 h post-burn.84 Body temperature was also lower in the animals receiving ketanserin in the early post-burn period as was the systemic vascular resistance index. Ketanserin also lowered the arteriovenous oxygen difference, indicating an improvement in tissue perfusion. Most of the beneficial effects of ketanserin had disappeared by 12 to 18 h post-burn, despite the continued administration of the drug for 24 h. The mechanisms underlying the beneficial effects of ketanserin were not addressed in this study. Patchy, intermittent renal cortical ischemia, which may be the forerunner of focal or patchy renal necrosis seen after shock, was prevented by ketanserin administered during burn-shock injury in the rat.85 Meuleman et al.7' also showed similar beneficial effects of ketanserin in canine endotoxin shock. Again, ketanserin given before or after endotoxin injection increased cardiac output and reduced pulmonary artery pressure, pulmonary and systemic vascular resistance, and arteriovenous O2 differences. The drug did not modify the severe hypotension, watery diarrhea, or survival. The beneficial effects were attributed to the ability of ketanserin to markedly inhibit in vitro platelet aggregation in response to a mixture of 5-HT and adrenaline. However, no in vivo assessment was made of platelet behavior in response to endotoxemia. Another group86'87 also showed ketanserin to produce a marked suppression of endotoxininduced increases in pulmonary artery pressure and vascular resistance in the dog. Ketanserin, however, did not affect the systemic hemodynamic changes or the fall in cardiac output.86

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FIGURE 3. Pulmonary hypertension (mean pulmonary artery pressure, mmHg) induced by endotoxin (given at time zero) in cats pretreated with either saline (o) or ketanserin (•). The pulmonary hypertension was not reduced by a dose of ketanserin (0.2 mg/kg) which abolished pulmonary responses to exogenous 5-HT. (From Ball, H. A., Parratt, J. R., and Rodger, I. W., Br. J. Pharmacol., 80, 295, 1983. With permission.)

In contrast, a study of ketanserin in feline endotoxin shock showed no effects of ketanserin on the endotoxin-induced systemic hypotension, pulmonary hypertension, reduced lung compliance, systemic hypoxia, or metabolic acidosis (Figure 3).88 Ketanserin did, however, reduce by about 50% the endotoxin-induced increase in pulmonary airways resistance but did not improve pulmonary gas exchange. Although only a single bolus dose of ketanserin was used (in contrast to the infusions used in the other studies), the pulmonary vascular effects of 5-HT and its ability to increase intratracheal pressure were completely prevented by ketanserin. Ketanserin did not modify other cardiovascular effects of 5-HT (bradycardia or hypotension), these being probably reflex in origin and mediated by 5-HT3 receptors.14 Neither the acute pulmonary and systemic vascular changes, nor the leukocytopenia following an infusion of live E. coli organisms in cats were modified by ketanserin.89 Similar negative findings were obtained in a porcine model of septic shock, where ketanserin was not only ineffective, but failed to augment the beneficial effects of a thromboxane synthetase inhibitor.90"92 However, ketanserin did produce some attenuation of the sepsis-induced decreases in antithrombin III and in ot2 macroglobulin indicating a possible protective effect in the coagulation system.91 Ketanserin was also shown to be ineffective in modifying the refractory shock state following intestinal vascular obstruction in the rat.93 Although ketanserin slightly reduced the increased hematocrit accompanying the shock, it failed to influence intestinal fluid leakage, sequestration of platelets in the lungs or intestines, small intestinal mucosal lesions, or mortality. It might have been more valuable, however, to study the

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effect of ketanserin in the cat model of intestinal vascular occlusion, in which the same group had shown an increase in 5-HT release.26 The lack of any marked beneficial effects of ketanserin in the rat and cat models of shock may reflect the different importance of 5HT in shock in different species. One problem inherent in the use of highly selective 5-HT2 receptor-blocking drugs may be the involvement of other receptor types in some effects of 5-HT relevant to shock. Schrauvan et al.39 have made a detailed study of the effects of two newer antagonists, the Janssen compounds R41468 and R50970 on the hemodynamic effects of endotoxin in piglets. These compounds have selective binding affinities for 5-HT! and 5-HT2 receptors, respectively. Both reduced (and in the case of R50970, completely prevented) the endotoxininduced increase in pulmonary vascular resistance. Survival time was also increased with R50970, a finding which may be related to a,-adrenoceptor blocking activity as well as to effects on 5-HT2 receptors. The authors concluded from this study that "5-HT and 5-HT amplifying effects (i.e., interaction of 5-HT with the effects of other vasoactive agents) may play a role in endotoxic shock in the piglet, particularly in the pulmonary vascular bed." The problem of choosing a 5-HT antagonist of the wrong selectivity could be circumvented by the use of selective 5-HT depletion. Apart from the study of Endo44'45 (see Section II) no work appears to have been done in this area. Compound 48/80, which depletes mast cells of histamine and 5-HT, significantly reduced the duration of endotoxin-induced pulmonary hypertension in the cat, without modifying the amplitude of the response.52 It is difficult, however, to relate this observation to the mast cell depleting effects of 48/80 since neither antihistamines (combined H, and H2 blockade, using mepyramine and burimamide) nor 5-HT antagonism (methysergide, see above) modified the response. Studies using tryptophan hydroxylase inhibitors or irreversible aromatic amino-acid decarboxylase inhibitors may be complicated by depletion of 5-HT in the central nervous system and in the case of decarboxylase inhibitors, the concomitant depletion of catecholamines. Moreover, decarboxylase inhibitors may inhibit other pathways of tryptophan metabolism.94

V. CONCLUDING REMARKS 5-HT is clearly released in various forms of experimental shock and its concentrations are elevated in the septic adult respiratory-distress syndrome. Different experimental models have yielded different results in studies using 5-HT receptor-blocking drugs. The question is unresolved concerning which of these models is relevant to human shock states following hemorrhage, burn injury, or septicemia. Perhaps a systematic study is required of a variety of 5-HT receptor-blocking drugs with different selectivities for the main 5-HT receptors. In performing these studies the pharmacokinetics of the antagonists must be carefully considered. It may be necessary to concentrate on porcine or primate models, with the assumption that these "pecies are closer to man than are cats, rats, rabbits, and dogs. The beneficial effects of ketanserin seen in at least two different models of burn shock may justify a clinical trial. In view of the vast number of other active mediators released in shock (thromboxanes, leukotrienes, prostaglandins, histamine, platelet activating factor, and monokines), it is highly unlikely that a blockade of the actions of 5-HT will, by itself, prove to be the solution to this major clinical problem. Nevertheless, 5-HT receptor antagonists may be useful adjuncts to other therapy. Caution, however, would be required in the choice of agent as central 5-HT mechanisms are probably important in regulating glucocorticoid secretion.95 Failure of glucocorticoid secretion in shocked patients is detrimental to survival, as evidenced by the increased mortality among multiple trauma patients treated with the sedative etomidate, an inhibitor of cortisol synthesis.96

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VI. SUMMARY 5-HT is released in experimental hemorrhagic and endotoxin-induced shock and in human septic shock. Moreover, the ability of 5-HT to mimic various aspects of shock, and the beneficial effects of nonselective and 5-HT2-selective antagonists at 5-HT receptors in experimental shock induced by burn injury or by endotoxin, suggests a pathophysiological role for this monoamine.

REFERENCES 1. Polak, J. M., De Mey, J., and Bloom, S. R., 5-Hydroxytryptamine in mucosal endocrine cells of the gut and lung, in 5-Hydroxytryptamine in Peripheral Reactions, De Clerck, F. and Vanhoutte, P. M., Eds., Raven Press, New York, 1982, 23. 2. Gershon, M. D., Dreyfus, C. F., Pickel, V. M., Job, T. H., and Reis, D. J., Serotonergic neurons in the peripheral nervous system: identification in gut by immunohistochemical localisation of tryptophan hydroxylase, Proc. Natl. Acad. Sci., 74, 3086, 1977. 3. Holmsen, H., Platelet activation and serotonin, in Serotonin and the Cardiovascular System, Vanhoutte, P. M., Ed., Raven Press, New York, 1983, 75. 4. Berkovitz, B. A., Lee, C. M., and Spector, S., Disposition of serotoninin the rat blood vessels and heart, Clin. Exp. Pharmacol. Physiol., 1, 397, 1974. 5. Parratt, J. R. and West, G. B., 5-Hydroxytryptamine and tissue mast cells, J. Physiol. (Land.), 137, 169, 1957. 6. Uvnas, B., Chemistry and storage function of mast cell granules, Dermatology, 71, 76, 1978. 7. Tyce, C. M., Flock, E. V., and Owen, C. A., Jr., Uptake and metabolism of 5-hydroxytryptamine by the isolated, perfused rat liver, Am. J. Physiol., 215, 611, 1968. 8. Stolz, J. F., Uptake and storage of serotonin by platelets, Serotonin and the Cardiovascular System, Vanhoutte, P. M., Ed., Raven Press, New York, 1985, 37. 9. Gillis, C. N., Peripheral metabolism of serotonin, in Serotonin and the Cardiovascular System, Vanhoutte, P. M., Ed., Raven Press, New York, 1985, 27. 10. Verbeuren, T. J., Jordaens, F. H., Bull, H., and Herman, A. G., The endothelium inhibits the penetration of serotonin and norepinephrine in the isolated canine saphenous vein, J. Pharmacol. Exp. Ther., 244, 276, 1988. 11. Fenuik, W. and Humphrey, P. P. A., Mechanisms of 5-hydroxytryptamine-induced vasoconstriction, in The Peripheral Actions of 5-Hydroxytryptamine, Fozard, J. R., Ed., Oxford University Press, Oxford, 1989, 100. 12. De La Lande, I. S., Stanton, B. J., and Lungerhausen, Y. K., Receptors mediating sensitising actions of 5-hydroxytryptamine on the rabbit ear artery, Blood Vessels, 20, 189, 1983. 13. Mylecharane, E. J. and Phillips, C. A., Mechanisms of 5-hydroxytryptamine-induced vasodilatation, in The Peripheral Actions of 5-Hydroxytryptamine, Fozard, J. R., Ed., Oxford University Press, Oxford, 1989, 147. 14. McQueen, D. S. and Mir, A. K., 5-Hydroxytryptamine and cardiopulmonary and carotid body reflex mechanisms, in The Peripheral Actions of 5-Hydroxytryptamine, Fozard, J. R., Ed., Oxford University Press, Oxford, 1989, 301. 15. Majino, G. and Polade, G. E., Studies in inflammation. I. The effect of histamine and serotonin on vascular permeability: an electron microscopic study, J. Biophys. Biochem. Cytol., 11, 571, 1961. 16. Olesen, S. P., A calcium-dependent reversible permeability increase in microvessels in frog brain induced by serotonin, J. Physiol., 361, 103, 1985. 17. Bottaro, D., Shepro, D., Petersen, S., and Hechtman, H. B., Serotonin, norepinephrine and histamine mediation of endothelial cells barrier function in vitro, J. Cell Physiol., 128, 1894, 1986. 18. Bottaro, D., Shepro, D., Peterson, S., and Hechtman, H. B., Serotonin, histamine, and norepinephrine mediation of endothelial and vascular smooth muscle cell movement, Am. J. Physiol., 248, C252, 1985. 19. Boogaerts, M. A., Yamada, O., Jacob, H. S., and Moldow, C. F., Enhancement of granulocyteendothelial cell adherence and granulocyte-induced cytotoxicity by platelet release products, in Proc. Natl. Acad. Sci. U.S.A., 79, 7019, 1982.

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20. Cohen, M., 5-Hydroxytryptamine and non-vascular smooth contraction and relaxation, in The Peripheral Actions of 5-Hydroxytryptamine, Fozard, J. R., Ed., Oxford University Press, Oxford, 1989, 201. 21. Donowitz, M., Asakoff, N., and Pike, G., Serotonin-induced changes in rabbit ileal active electrolyte transport are calcium dependent associated with an increased ileal calcium uptake, /. Clin. Invest., 66, 341, 1980. 22. Hoyer, D., 5-Hydroxytryptamine receptors and effector coupling mechanisms in peripheral tissues, in The Peripheral Actions of 5-Hydroxytryptamine, Fozard, J. R., Ed., Oxford University Press, Oxford, 1989, 72. 23. Kovats, T. G., Endotoxin susceptibility and endotoxin hypersensitivity, Monogr. Med. Univ. Szeged, Hungary, 4, 1, 1967. 24. Emerson, T. W., Participation of endogenous vasoactive agents in the pathogenesis of endotoxin shock, Adv. Exp. Med. Biol., 23, 25, 1972. 25. Urbaschek, B. and Urbaschek, R., The inflammatory response to endotoxin, Bibliogr. Anat., 17, 74, 1979. 26. Fara, J. and Haglund, U., Release of serotonin from the small intestine following hypotension in the cat, Eur. Surg. Res., 5, 152, 1973. 27. Luna, A., Villanueva, E., Hernandez-Cueto, C., and Morales, I., Study of 5-hydroxytryptamine (serotonin) in pericardia! fluid in different causes of death. II. Experimental studies of 5-HT levels in two types of shocks (haemorrhagic and septic) in dogs, Z. Rechtsmedizin, 89, 227, 1983. 28. Swank, R. L., Hissen, W., and Fellman, J. H., 5-Hydroxytryptamine (serotonin) in acute hypotensive shock, Bibliogr. Anat., 1, 439, 1964. 29. Kobold, E. E. and Thai, A. P., Quantitation and identification of vasoactive substances liberated during various types of experimental and clinical intestinal ischaemia, Surg. Gynecol. Obstet., 117, 315, 1963. 30. Mustala, O., Alfthan, O., and Peltokallio, P., 5-Hydroxytryptamine concentration of total blood of rabbits following occlusion of superior mesenteric artery, Acta Chir. Scand., 134, 275, 1968. 31. Ebata, T. and Hayasaka, H., Effects of aldosterone and dexamethasone on blood chemical mediators in endotoxin shock, Jpn. J. Surg., 9, 79, 1979. 32. Emau, P., Giri, S. N., and Bruss, M. L., Comparative effects of smooth and rough Pasteurella hemolytica lipopolysaccharides on arachidonic acid, eicosanoids, serotonin and histamine in calves, Circ. Shock, 20, 239, 1986. 33. Emau, P., Giri, S. W., and Bruss, M. L., Role of prostaglandins, histamine and serotonin in the pathophysiology induced by Pasteurella hemolytica endotoxin in the sheep, Circ. Shock, 12, 47, 1984. 34. Erve, P. R., Schuler, J. J., and Schumer, W., Endotoxin-chaJlenged monkeys and rats, glucocorticoid effect on the serotonin level in the blood, Arch. Surg., 113, 561, 1978. 35. Des Prez, R. M., Horowitz, H. I., and Hook, E. W., Effects of bacterial endotoxin on rabbit platelets. I. Platelet aggregation and release of platelet factors in vitro, J. Exp. Med., 114, 857, 1961. 36. Nagayama, M., Zucker, M. B., and Belter, F. K., Effects of a variety of endotoxins on human and rabbit platelet function, Thromb. Diath. Haemorrh., 26, 467, 1971. 37. Nomura, Y. and Takagi, H., Studies on plasma factor involved in 5-hydroxytryptamine release from blood platelets by bacterial endotoxin. I, Jpn. J. Pharmacol., 22, 37, 1972. 38. Nomura, Y., Okada, T., and Takagi, H., Studies on plasma factor involved in 5-hydroxytryptamine release from blood platelets by bacterial endotoxin III, Jpn. J. Pharmacol., 22, 79, 1972. 39. Schrauwen, E., Weyns, A., and Houvenaghel, A., Endotoxic shock in the piglet: beneficial effects of serotonin antagonism, Br. Vet. J., 141, 179, 1985. 40. Morris, K. M. and Moon, R. J., Quantitative analysis of serotonin biosynthesis in endotoxemia, Infect. Immun., 10, 340, 1974. 41. Sibbald, W., Peters, S., and Lindsay, R. M., Serotonin and pulmonary hypertension in human septic ARDS, Crit. Care Med., 8, 490, 1980. 42. Kerstein, M. D., Cronau, L. H., Mandel, S. D., and Gillis, C. N., Effect of hemorrhagic shock on 5hydroxytryptamine removal by the lung, Am. Surgeon, 48, 644, 1982. 43. Naik, P. M., Dadkar, V. N., and Sheth, U. K., Changes in levels of biogenic amines in blood tissues in endotoxin shock in cats, Indian J. Med. Res., 68, 353, 1978. 44. Endo, Y., Induction of hypoglycaemia and accumulation of 5-hydroxytryptamine in the liver after the injection of mitogenic substances into mice, Br. J. Pharmacol., 81, 645, 1984. 45. Endo, Y., Suppression and potentiation of 5-hydroxytryptophan-induced hypoglycaemia by ot-monofluoromethyldopa: correlation with the accumulation of 5-hydroxytryptamine in the liver, Br. J. Pharmacol., 90, 161, 1987. 46. Rosenberg, J. C., Lillehei, R. C., Moran, W. H., and Zimmerman, B., A comparison of the effects of irreversible hemorrhagic and endotoxin shock on serotonin concentration in blood, Surgical Forum, 19, 397, 1959.

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47. Halevy, S., Spector, S., and Altura, B. A., Serum serotonin levels determined by radioimmunoassay in normal male and female rats and after intestinal ischaemia shock, Biochem. Med., 23, 236, 1980. 48. Kusajima, K., Ozdemir, I. A., Webb, W. R., Wax, S. D., and Parker, F. B., Role of serotonin and serotonin antagonist on pulmonary hemodynamics and microcirculation in hemorrhagic shock, J. Thor. Cardiovasc. Surg., 67, 908, 1974. 49. Cuevas, P. and Fine, J., Production of fatal endotoxic shock by vasoactive substances, Gastroenterology, 64, 285, 1973. 50. Boruchow, I. B., Ludwig, G. D., and Wontorsky, D., Potentiation of endotoxin shock by oral Ltryptophan, Am. J. Physiol., 214, 525, 1968. 51. Markley, K., Smallman, E., and Thornton. S. W., Protection against burn, tourniquet and endotoxin shock by histamine, 5-hydroxytryptamine and 5-hydroxytryptamine derivatives, Br. J. Pharmacol., 42, 13, 1971. 52. Parratt, J. R. and Sturgess, R. M., The possible roles of histamine, 5-hydroxytryptamine and prostaglandin F2a as mediators of the acute pulmonary effects of endotoxin, Br. J. Pharmacol., 60, 209, 1977. 53. Chand, N. and Altura, B. M., Serotonin receptors subserve only contraction in canine and rat pulmonary arteries and veins, Artery, 7, 232, 1980. 54. Freeman, W. K., Rorie, D. K., and Tyce, G. M., Effects of 5-hydroxytryptamine on neuroeffector junction in human pulmonary artery, J. Appl. Physiol., 51, 693, 1981. 55. Frenken, M. and Kaumann, A. J., Interaction of ketanserin and its metabolite ketanserinerol with 5-HT2 receptors in pulmonary and coronary arteries of calf, Naunyn-Schmiedeberg'sArch. Pharmacol., 326, 334, 1984. 56. Adler, S., Serotonin and the kidney, in Serotonin in Health and Disease, Vol. 4, Essman, W. B., Ed., Spectrum Publications, New York, 1977, 199. 57. Gumming, A. D., Renal functions in septic shock, in Update in Intensive Care and Emergency Medicine, Vincent, J. L., Ed., Springer-Verlag, Berlin, 1989, 348. 58. Greenberg, S., McGowan, C., and Glenn, T. M., Pulmonary vascular smooth muscle function in porcine splanchnic arterial occlusion shock, Am. J. Physiol., 241, H34, 1981. 59. Guc, M. O., Furman, B. L., and Parratt, J. R., Endotoxin induced impairment of vasopressor and vasodepressor responses in the pithed rat, Br. J. Pharmacol., 101, 913, 1990. 60. Parillo, J. E., Cardiovascular dysfunctions in septic shock: new insights into a deadly disease, Int. J. Cardiol., 1, 314, 1985. 61. Thijs, L. G., Groeneveld, A. B. J., and Schneider, A. J., Changing haemodynamic concepts in septic shock, in Septic Shock and the Adult Respiratory Distress Syndrome, Kox, W. and Bihari, D., Eds., Springer-Verlag, Berlin, 1987, 79. 62. Morrison, D. C. and Ulevitch, R. J., The effects of bacterial endotoxins on host mediator systems, Am. J. Pathol., 93, 527, 1978. 63. McKenna, T. M., Martin, F. M., Chernow, B., and Briglia, F. A., Vascular endothelium contributes to decreased aortic contractility in experimental sepsis, Circ. Shock, 19, 167, 1986. 64. Goligorsky, M. S., Role of endothelium in endotoxin blockade of voltage-sensitive Ca2+ channels in smooth muscle cells, Am. J. Physiol., 257, C875, 1989. 65. Salvemini, D., Korbut, R., Anggard, E., and Vane, J. R., Lipopolysaccharide increases release of a nitric oxide-like factor from endothelial cells, Eur. J. Pharmacol, 171, 135, 1989. 66. Jacob, H. S., Moldow, C. F., Flynn, P. J., Weisdorf, D. J., Vercellotti, G. M., and Hammerschmidt, D. E., Therapeutic ramifications of the interaction of complement, granulocytes and platelets in the production of acute lung injury, N.Y. Acad. Sci., 384, 489, 1982. 67. Stewart, G. J., Effect of endotoxin on the ultrastructure of liver and blood cells of hamsters, Br. J. Exp. Path., 51, 114, 1970. 68. Myrvold, H. E. and Lewis, D. H., Platelets, fibrinogen and pulmonary haemodynamics in early experimental septic shock, Circ. Shock, 4, 210, 1977. 69. Bredenberg, C. E., Taylor, G. A., and Webb, W. R., The effect of thrombocytopenia on the pulmonary and systemic hemodynamics of canine endotoxic shock, Surgery, 87, 59, 1980. 70. Baumgartner, H. R. and Born, G. V. R., Effect of 5-hydroxytryptamine on platelet aggregation, Nature, 218, 137, 1968. 71. Meuleman, T. R., Hill, D. C., Port, J. D., Stanley, T. H., Pace, N. L., and Mohammad, S. F., Ketanserin prevents platelet aggregation and endotoxin-induced pulmonary vasoconstriction, Crit. Care Med., 11, 606, 1983. 72. Hinshaw, L. B., Peyton, M. D., Archer, L. T., Black, M. R., Coalson, J. J., and Greenfield, L. J., Prevention of death in endotoxin shock by glucose administration, Surg. Gynecol. Obstet., 139, 851, 1974. 73. Van de Meer, C., Valkenburg, P. W., and Snyders, P. M., Studies on the site of the block in gluconeogenesis causing severe hypoglycaemia in intestinal ischaemia shock in rats, Circ. Shock, 16, 213, 1985.

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74. Filkins, J. P. and Cornell, R. P., Depression of hepatic gluconeogenesis and the hypoglycaemia of endotoxin shock, Am. J. Physiol., 227, 778, 1974. 75. Lundquist, I., Ekholm, R., and Ericson, L. E., Monoamines in the pancreatic islets of the mouse. 5Hydroxytryptamine as an intracellular modifier of insulin secretion, and the hypoglycaemic action of monoamine oxidase inhibitors, Diabetologia, 1, 414, 1971. 76. Furman, B. L., The hypoglycaemic effect of 5-hydroxytryptophan, Br. J. Pharmacol., 50, 575, 1974. 77. Wilson, G. A. and Furman, B. L., Effects of inhibitors of 5-hydroxytryptamine uptake on plasma glucose and their interaction with 5-hydroxytryptophan in producing hypoglycaemia in mice, Eur. J. Pharmacol., 78, 263, 1982. 78. Smith, S. A., Elliot, K. R. F., and Pogson, C. I., Inhibition of hepatic gluconeogenesis by tryptophan metabolites in rats and guinea pigs, Biochem. Pharmacol., 28, 2145, 1979. 79. Kalter, E. S., Jaspers, F. C., van Dp, W. C., Nijkamp, F. P., de Jong, W., and Verhoef, J., Induction of the early hypotensive phase by Escherichia coli: role of bacterial surface structures and inflammatory mediators, J. Infect. Dis., 152, 493, 1985. 80. Keeler, R., Effects of methysergide or phenoxybenzamine on the renal and cardiovascular responses to endotoxin in rats, Circ. Shock, 8, 361, 1981. 81. Almqvist, P., Skudder, P., Kuenzig, M., and Schwartz, S. I., Effect of cyproheptadine on endotoxininduced pulmonary platelet trapping, Am. Surgeon, 50, 503, 1984. 82. Webb, R. C., Increased vascular sensitivity to serotonin and methysergide in hypertension in rats, Clin. Sci., 63, 73, 1982. 83. Mecca, T. E., Mitchell, J., Bohr, D. F., and Webb, R. C., Effects of serotonin antagonists on blood pressure in mineralocorticoid hypertensive sheep, /. Cardiovasc. Pharmacol., 1, 660, 1985. 84. Holliman, C. J., Meuleman, T. R., Larsen, K. R., Port, J. D., Stanley, T. H., Pace, N. L., and Warden, G. D., The effect of ketanserin, a specific serotonin antagonist, on burn shock hemodynamic parameters in a porcine burn model, J. Trauma, 23, 867, 1983. 85. Haugan, A. and Kirkebo, A., Local renal ischemia during burn shock in rat effected by thromboxane A2 and serotonin, Circ. Shock, 20, 13, 1986. 86. Makabali, G. L., Mandal, A. K., Morris, J. A., Brown, J., Chang, J., and Bankhead, J., An assessment of the participatory role of prostaglandins and serotonin in the pathophysiology of endotoxic shock, Am. J. Obstet., Gynecol, 145, 439, 1983. 87. Makabali, G. L., Mandal, A. K., Morris, J. A., Brown, J., Chang, J., Bankhead, J., and Reeves, B. A., Endotoxemic shock: an implied role for 5-hydroxytrayptamine, in 5-Hydroxytryptamine in Peripheral Reactions, de Clerck, F. and Vanhoutte, P. M., Eds., Raven Press, New York, 1982, 153. 88. Ball, H. A., Parratt, J. R., and Rodger, I. W., The effect of a selective 5-HT2 antagonist, ketanserin, on the pulmonary responses to Escherichia coli endotoxin, Br. J. Pharmacol., 80, 295, 1983. 89. Arvidsson, S., Falk, A., Haglind, E., and Haglund, U., The role of 5-hydroxytryptamine in the feline response to intravenous infusion of live E. coli, Br. J. Pharmacol., 79, 711, 1983. 90. Svartholm, E., Bergqvist, D., Haglund, U., Ljungberg, J., and Hedner, U., Coagulation and fibrinolytic reactions in experimental porcine septic shock: pretreatment with different antiplatelet factors, Circ. Shock, 22, 291, 1987. 91. Svartholm, E., Bergqvist, D., Lindblad, B., Ljungberg, J., and Haglund, U., Pulmonary vascular response to live Escherichia coli: influences of different antiplatelet substances, Circ. Shock, 22, 173, 1987. 92. Svartholm, E., Arvidsson, S., Fait, K., and Haglund, U., Influence of prostanoids on gastrointestinal mucosal injury in experimental septic shock, APMIS, 97, 61, 1989. 93. Haglind, E., Falk, A., and Haglund, U., The effect of 5-HT blockade in graded intestinal vascular obstruction in the rat, Acta Chir. Scand., 149, 273, 1983. 94. Bender, D. A., Inhibition in vitro of the enzymes of the oxidative pathway of tryptophan metabolism and of nicotinamide nucleotide synthesis by benserazide, carbidopa and isoniazid, Biochem. Pharmacol., 29, 707, 1980. 95. Fuller, R. W., Serotonergic stimulation of pituitary-adrenocortical function in rats, Neuroendocrinology, 32, 118, 1981. 96. Kenyon, C. J., McNeil, L. M., and Fraser, R., Comparison of the effects of etomidate, thiopentone and propofol on cortisol synthesis, Br. J. Anaesth., 57, 509, 1985.

Chapter 5

CATECHOLAMINES IN SEPTIC SHOCK: A META-ANALYSIS EVALUATION Reuven Rabinovici and Giora Feuerstein

TABLE OF CONTENTS I.

Introduction

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II.

Methods: Criteria of Test Nodes A. CA Assay B. Sample Preparation C. Source of Sampling D. Timing of Sampling E. Study Design F. Septic/Endotoxic Shock Model G. Species CA Response Caused by Shock Itself? H.

142 142 143 143 146 147 147 148 148

III.

Results A. Analysis of Clinical Studies B. Analysis of Experimental In Vivo Studies 1. Rat Studies Dog and Cat Studies 2.

148 148 154 154 154

IV.

Discussion

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References

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Handbook of Mediators in Septic Shock

I. INTRODUCTION Septic shock is a medical emergency caused by the release into the circulation of bacterial lipopolysaccharide (LPS) endotoxins and associated with profound hemodynamic, hematologic, biochemical, and endocrine derangements ultimately leading to mortality.1"5 Endogenous catecholamines (CA), which are part of the body's homeostatic mechanisms, were suggested for several decades to be involved in the septic-shock syndrome. The major body of evidence to support the role of endogenous CA in this complex disease were obtained by: (1) in vivo experiments demonstrating plasma (Table 1) or tissue levels of epinephrine (E) and norepinephrine (NE) following endotoxin challenge; and (2) clinical studies showing an elevated CA level in septic patients. Nevertheless, a cause-effect relationship between LPS and CA was not yet proven. Reliable demonstration of elevated plasma CA in septic conditions is the first imperative which has to be fulfilled to establish such causal association. In an attempt to determine the current status of E and NE concentrations in septic states, the present chapter reviews the relevant studies using the method of meta-analysis6 in combination with the model of a "decision tree".7 The method provides a quantitative, systematic statistical alternative to the traditional scientific reviews which are largely narrative presentations of studies and their findings ignore the size of the effect found and the strength of research designs. Meta-analysis involves an analysis of previous studies so that the larger numbers obtained by combining studies provide a greater statistical power than any of the individual studies.

II. METHODS: CRITERIA OF TEST NODES The present review utilized the model previously described by Neugebauer et al.7 In brief, the method includes a "decision tree" (Figure 1) with a sequence of hierarchic questions (test nodes) and binary branches (yes-no answers). If the answer to the question at one of the test nodes is no, the study should, strictly speaking, be excluded from further analysis. Therefore, only studies with positive answers to all questions will be classified as suitable for demonstration of a real association between septic shock (disease) and CA (mediator). Since the demand on absolute infallibility became impractical, the shortcomings of the individual studies were estimated in a further step of the analysis. A positive judgment of the shortcomings at a specific question enabled further evaluation of the study at the next test node. A. CA ASSAY Fluorometric, radioenzymatic, or high-performance liquid chromatographic (HPLC) techniques are considered reliable methods for CA assay. CA were first measured in plasma by the fluorometric technique8 which at that time could only determine the sum of total of E and NE. However, the introduction of several modifications resulted in a quantitative and sensitive method which is adaptable to urine, plasma, and tissue9 and which can accurately differentiate between E and NE.10 The radioenzymatic technique11'12 is highly sensitive and permits determination in samples as small as 50 (xl. Disadvantages of the method include the time and expense involved and the lack of specificity in certain circumstances such as low blanks,13'14 probably due to variability in the purity of the isotope. More recently, HPLC with electrochemical detection15 has emerged as the method of choice in cases where somewhat larger CA quantities are being assayed, such as the brain and heart tissue,13-16"18 as well as with large (>2 ml) plasma samples.17'19'23 The larger sample volume has deterred most investigators from applying this technique to routine plasma CA measurement in small laboratory animals. Recent modifications, however, which allow sufficient sensitivity for

Rabinovici and Feuerstein

143

accurate CA determination in plasma samples as small as 100 to 500 jjil,14'24'25 popularized the use of this method in small laboratory animals as well. B. SAMPLE PREPARATION CA are chemically unstable because they are prone to oxidation by dissolved oxygen. To minimize this process and to ensure that any sample actually reflects the true level of CA in the plasma or tissue, several measures should be taken. 1.

2. 3.

Since blood CA were shown to be stable for about 1 h when stored at 4°C,26 blood or tissue samples should be collected with ice-cooled syringes or tubes and the red cells should be separated in a refrigerated centrifuge. In addition, plasma should be stored at — 70°C until assayed. If the plasma proteins are precipitated with 0.3 N perchloric acid prior to freezing, the CA are stable at only — 20°C. Plasma CA stored at these temperatures do not deteriorate significantly for at least 1 year. Keep the sample in a mildly acidic solution to repress the oxidation of CA. Antioxidants such as glutathione or ascorbic acid should be added to the storage tubes to decrease oxidation of plasrha CA.

Also, anticoagulants such as heparin, EDTA, EGTA, and acid-citrate-dextrose can be used in the initial collection tubes to prevent clotting. Only studies that used at least one of these measures were qualified at this test node. All samples should be rapidly centrifuged since the short half-times of adrenalin (1.2 min)27 and noradrenalin (2.5 min)28 lead to a brisk reduction in the plasma concentration of these CA before the beginning of the assay. C. SOURCE OF SAMPLING CA were determined in biological fluids such as plasma, urine, and cerebrospinal fluid, and in tissues, i.e., the brain, adrenal, liver, spleen, heart, kidney, and blood vessels. The rationale for sampling tissues is to measure local CA concentrations. The logic behind sampling plasma and urine is somewhat different; it is assumed that circulating CA levels reflect the sympathetic nervous system activity. Nevertheless, this assumption is difficult to delineate since plasma CA levels can accurately reflect sympathetic nervous system activity only when several criteria are met. First, circulating CA should be proportional to the amount of CA released in the junctional gap or the adrenal medulla. However, since only a small portion of the released CA escapes enzymatic catabolism or re-uptake and diffuses out of the junctional cleft into the blood stream, changes in the rate of release and clearance can obscure the relevance of plasma CA levels to sympathetic activity. This situation is most obvious if both the rate of release and clearance are altered in the same direction.29 Second, CA release, uptake, and diffusion into the circulation, as well as re-uptake, extraction, or clearance should be in "steady state" at the time of blood sampling. Therefore, it is essential to eliminate any stimuli that might activate the sympathetic activity at the time of blood collection. Since practically, these requirements are difficult to control in septic patients and in experimental in vivo models of endotoxic shock, the drawbacks of measuring plasma and urine CA levels should be considered. Nevertheless, although other methods to evaluate sympathetic nervous system function exist, i.e., adrenergic receptor,30'31 exogenous CA absorption,32 and CA apparent release rate and clearance33 studies, currently there are no superior methods to constitute an index of sympathetic nervous system activity. Both arterial and venous sampling are suitable for monitoring CA activity in the plasma. Comparisons between studies, however, should contemplate the source of blood since differences exist between simultaneously collected arterial and venous blood samples. For

Rat (a)

Rat (c)

Rat (c)

Rat (c) Rat (c)

Rat (c)

Rat (c)

Rat (c)

Rat (c)

Rat (c)

Species (anesthesia)

5. enteritidis (bolus iv, 10 mg/kg) S. enteritidis (bolus iv, 100 jig/kg) S. enteritidis (bolus iv, 1 mg/kg) 5. enteritidis (bolus iv, 2 mg/kg) S. enteritidis (bolus iv, 16.7 mg/kg) Cecal ligation and puncture E. coli (iv infusion, 41 mg/kg/min over 4 h) E. coli (iv bolus, 30 mg/kg) S. enteritidis (iv bolus, 3.3 mg/kg) S. enteritidis (iv bolus, 3.3 mg/kg)

LPS (dose, route)

3700 at 90 min

2930 at 90 min

5900 at 45 min

1700 at 41 h 3279 at 1 h

6466 at 60 min

3789 at 30 min

2953 at 30 min

738 at 6 h

292 at 3 h

Max E response (pg/ml)

180 (Control)

40 (Control)

250 (Control)

35 (Control) 329 (Control)

54 (Control)

55 (Control)

66 (Control)

59 (Control)

59 (Control)

Control/basal E (pg/ml)

1890 at 90 min

1580 at 90 min

5000 at 15 min

325 at 20 h 2403 at 4 h

7575 at 5 h

1376 at 5 h

1909 at 6 h

564 at 6 h

286 at 6 h

Max NE response (pg/ml)

TABLE 1 Catecholamine Response to Endotoxin In Vivo

240 (Control)

160 (Control)

80 (Control)

150 (Control) 423 (Control)

701 (Control)

141 (Control)

130 (Control)

130 (Control)

130 (Control)

Control/basal NE (pg/ml)

RE

RE

RE

RE RE

RE

RE

RE

RE

RE

Assay

61

61

54

60 53

52

52

59

59

59

Ref.

144 Handbook of Mediators in Septic Shock

E. coll (iv bolus, 5 mg/kg followed by infusion 2 mg/kg/h) E. coll (iv infusion, 0.5 mg/kg over 5 min) E. coll (iv infusion, 5 mg/kg over 5 min) E. coll (iv, 0.5 mg/kg) E. coll (iv bolus, 0.01 mg/kg) E. coll (iv bolus, 0.05 mg/kg) E. co/i (iv bolus, 0.25 mg/kg) E. co/i (iv bolus, 0.50 mg/kg) E. coll (iv bolus, 2.0 mg/kg) E. coll (iv bolus 7.5 mg/kg) E. coll (iv bolus, 1 .75 mg/kg) E. coll (iv bolus, 7 mg/kg) 295 at 2 h

2291 at 60 min

20,000 at 5 min

5000 at 30 s

3600 at 60 min

3000 at 60 min

3800 at 60 min

11,200 at 5 h 1200 at 60 min

1180 at 60 min

5990 at 60 min

18 nmol/1 at 45 min

42 (Basal)

273 (Basal)

< 1000 (Control)

400 (Control)

500 (Control)

500 (Control)

500 (Control)

400 (Basal) 560 (Control)

610 (Basal)

1675 (Basal)

0 nmol/1 (Basal)

1988 at 2 h

1028 at 2 h

16,000 at 5 min

1 100 at 60 min

600 at 60 min

700 at 60 min

900 at 4 h

3000 at 5 h 700 at 10 min

9830 at 60 min

1070 at 60 min

3 nmol/1 at 45 min

339 (Basal)

213 (Basal)

< 2000 (Control)

300 (Control)

300 (Control)

300 (Control)

300 (Control)

1500 (Basal) 300 (Control)

1090 (Basal)

51 (Basal)

1 nmol/1 (Basal)

RE

RE

Fluorometry

Fluorometry

Fluorometry

Fluorometry

Fluorometry

Fluorometry Fluorometry

Not stated

Fluorometry

HPLC

65

64

51

50

50

50

50

63 50

57

58

62

Note: CA = catecholamines; E = Epinephrine; NE = norepinephrine; c = conscious; a = anesthetized; max = maximal; RE = radioenzymatic technique; HPLC = high-performance liquid chromatography.

Cat (a)

Dog (a)

Dog (a)

Dog (a)

Dog (a)

Dog (a)

Dog (a)

Dog (a) Dog (a)

Dog (a)

Dog (a)

Dog (a)

Rabinovici and Feuerstein 145

146

Handbook of Mediators in Septic Shock

FIGURE 1. "Decision tree" used in combination with meta-analysis for the evaluation of a causal relationship between septic/endotoxic shock and catecholamine response. The structure includes ten questions (test nodes) and nine binary branches. Only a positive answer at each test node qualifies for further evaluation. The "decision tree" was constructed according to Lusted70 and Neugebauer.7

example, in rats kept at room temperature for 5 min after blood sampling, arterial CA concentrations were higher than venous levels.34 Also, a study conducted in subjects undergoing dental procedures reported higher NE concentrations in venous blood and higher E levels in arterial blood.35 D. TIMING OF SAMPLING To enable comparisons between studies and to facilitate the monitoring of temporal changes in the same subject, the basal resting concentration of plasma CA should be defined.

Rabinovici and Feuerstein

147

Since circulating CA levels are influenced by stress (physical and emotional), state of consciousness (sleep vs. awake), anesthetics, and drugs (chlorpromazine, amphetamine, caffeine, nicotine, and others), a true basal value is one obtained after the subject is relaxed, i.e., 15 to 20 min after the needle or catheter insertion, and undisturbed in a quite environment. In addition, since age3637 and posture38-39 affect plasma CA levels, basal values should be posture- and age-matched. The CA response to administration of LPS is immediate with detected increments in plasma levels as early as 2 to 3 min following injection. Therefore, under experimental conditions blood should be sampled before and during the first hour after the administration of LPS. Full time-course study requires sampling for at least 5 to 6 h after endotoxin injection. The optimal timing of blood collection is more problematic in clinical conditions where the exact starting point and quantity of the septic insult is unknown. In intensive-care-unit (ICU) patients, however, hourly or daily samples might be appropriate for the determination of time course or comparison to a control group. E. STUDY DESIGN No study with insufficient numerical details for quantitative statistical analysis could enter the study. Ideally, all studies should be randomized and controlled.40'41 However, to prevent the elimination of almost all studies less rigid criteria were applied to qualify in this test node. First, a "suitable" control group was a prerequisite to classify a study as suitable to proceed along the "decision tree". A "suitable" control group in animal studies was defined as normal saline or sham-operated control group. In clinical studies, there are difficulties in defining or obtaining an optimal control population. In view of the complex release and clearance mechanisms that determine plasma CA levels at steady state, and since CA are subjected to many pathophysiological, physical, and psychological factors which are usually present in septic patients, only a well-matched group of nonseptic patients could be considered as an appropriate control. The second requirement for qualification along the "decision tree" was uniform basal values in the various study groups. F. SEPTIC/ENDOTOXIC SHOCK MODEL Three main approaches are used to produce sepsis in laboratory animals (for review see Reference 42). First, intravenous bolus injection of live bacteria or pure endotoxin is the most commonly used model since it evokes acute hemodynamic, hematologic, and biochemical responses, which are easy to monitor. Its clinical relevance, however, is questionable because most patients are not challenged with such a massive bacterial load at one time, but rather cultivating a septic nidus which is persistently showering the circulation with bacteria. In order to simulate more closely the common clinical situation, and to allow the animals time to respond to the septic insult, an experimental model in which animals were chronically infused with live bacteria or endotoxin was developed. Although this model can provide a state of lethal sepsis, unlike the clinical situation, the host has no chance of isolating the septic focus, since the bacteria are continually being injected through an open vein. This model can, however, provide observations of blood bacterial clearance kinetics, leukocyte, platelet, and febrile responses under conditions of different time-dose relationship. Second, chronic source of infection is an approach which establishes soft tissue or intraperitoneal infection with live bacteria or feces and better mimics the clinical setting of septic shock. However, some models did not cause mortality43"45 and in others no reproducible endotoxin challenge could be obtained. Third, the surgical procedures that partially destroy the normal barriers of the gastrointestinal tract simulate the natural sequence which occurs in the clinical setting (i.e., cecal ligation and puncture or small bowel infarction). The main critics of these clinically relevant models is the inability to control and quantitate the endotoxic challenge.

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Handbook of Mediators in Septic Shock

G. SPECIES Ideally, humans should serve as models for human diseases but such an approach is highly limited at the experiment level. Therefore, investigations of human pathophysiological processes should in principle be conducted in suitable animals which share similarities with the human in respect to: (1) E and NE levels in body fluids; (2) CA synthesis, metabolism, half-life time, and elimination; (3) CA response to LPS/bacteria; and (4) role of sympathoadrenomedullary system in cardiovascular regulation. Alternatively, under specific, limited, and well-controlled circumstances it might be possible to use supersensitive species so the magnitude of the effect could be better monitored. Nonsusceptible strains should be used cautiously as it might lead to underestimating of a relevant phenomenon. On the other hand, the use of unsusceptible strains might contribute to the understanding of biological events. For example, an endotoxin-resistant mouse strain (C3H/HeJ) did not produce tumor necrosis factor-alpha (TNFJ in response to endotoxin challenge,46 and thus provided another line to support the involvement of TNFa in septic shock. The reviewed studies were conducted in the rat (n = 8), dog (n = 7), cat (n = 1), and in the human (n = 5). In general, the more primitive species among land-dwelling vertebrates have the highest CA plasma concentration.47 However, both primitive and highly developed species have similar percent increases in plasma CA levels in response to stress.47 Therefore, most species used in experiments on CA response to endotoxin are suitable to reflect the human CA response in septic states. H. CA RESPONSE CAUSED BY SHOCK ITSELF? The ultimate goal of the present study is to evaluate whether there is solid evidence to conclude that CA are elevated in septic shock and, therefore, might have a pathophysiological role in this syndrome. Before deciding on this matter, however, it is essential to confirm that the elevation of plasma CA was not triggered by factors other than septic shock such as, pain, anesthesia, drugs, and supportive treatment. In addition, it is pertinent to determine whether the CA response resulted from the shock state (i.e., baroreflex elicited by hypotension), a direct action of endotoxin on target cells and organs, or the action of other participating mediators.

III. RESULTS Of the 71 studies dealing with CA in shock, only 22 were judged suitable for analysis according to the "decision tree". The association of endotoxin and CA activity was the direct working hypothesis in 17 studies. The other five studies were designed to test the effects of various drugs on CA activity during endotoxin/septic shock, and therefore, provided only indirect data on the effect of sepsis on plasma or tissue CA. A. ANALYSIS OF CLINICAL STUDIES Five studies on CA response in human septic shock entered the "decision tree" (Table 2). None of the studies completed the '' decision tree'' without major disqualifying drawbacks (Table 2). Three major shortcomings were found. First, there was a lack of nonseptic control group (four studies). The only study with such a control group48 was also inappropriately designed since the control group was not matched for age, sex, site of infection, underlying disease, associated diseases, treatment, and time of entry to the study. It should be noted that three of these studies were not designed to test the causal relationship between septic shock and CA and, therefore, had other control group(s). However, these latter studies require a nonseptic control group so that the actual CA levels could be adequately evaluated. Second, none of the studies could point out septic shock as the sole reason for elevated circulating CA levels since all patients were treated in ICU where they were exposed to a

?

?

?

+

?

Sample preparation appropriate?

+

+ Plasma (v)

+ Plasma (a)

Plasma

+ Plasma (pa)

+ Plasma (v)

Sampling in relevant body fluid?

No consistent timing of sampling

+ 4 Samples within 12 h of drug therapy

+ 2 Samples (before and after naloxone therapy)

+ 6 Samples within 80 min following naloxone therapy

+ One sample per day

Sampling at the right time?

Nonmatched control group

No nonseptic control group (study designed to evaluate effects of naloxone in septic patients) No nonseptic control group (study designed to evaluate effects of naloxone and steroids in septic patients) No nonseptic control group (study designed to evaluate effects of steroids in septic patients)

No nonseptic control group

Study design appropriate?

+

+

+

Shock model clinically relevant?

+

+

+

+

Species clinically relevant?

+ ICU Patients 7 out of 10 patients in septic group had elevated plasma CA without hypotension

ICU Patients (see text)

+ + ICU Patients (see text)

ICU Patients (see text)

ICU Patients (see text)

CA response caused by the shock itself?

Note: CA = catecholamines; RE = radioenzymatic; ( + ) a positive answer; (-) a negative answer; (?) no information or an ambiguous answer; a = arterial; v = venous; pa = pulmonary artery; ICU = intensive care unit.

+ Trihydroxy indole fluorimetry

+

5 (48)

RE

3 (56)

+

+ Trihydroxy indole fluorimetry

RE

2 (55)

+

CA assay reliable?

4 (66)

RE

1 (49)

Study no. (ref.)

TABLE 2 Analysis of Studies on Catecholamine Response in Human Septic Shock

Rabinovici and Feuerstein 149

4

36

(67)

3

34

(52)

32 2

(59)

1

Study no. (ref.)

HPLC

RE

RE

+

+

+

+

CA assay reliable?

?

+

?

+

Sample preparation appropriate?

+

Heart, spleen, liver, muscle, brown adipose tissue, plasma

+

Plasma (a)

+

Plasma (a)

+

Sampling in relevant body fluid?

+

Sampling at only one time point (24 h)

?

Samples at 0, 0.5, 3, 6 h

+

Samples at 0, 0.5, 3, 6 h

+

Sampling at the right time?

u.g/kg)

+

+

Shock model clinically relevant?

+

Weight- and fedmatched NS control group

+

NS control group, also evaluation of plasma CA in chronic vs. acute experiments and in response to low vs. high dose LPS

+

+

+

+

SC, LPS bolus (E. coli 1 mg/ kg)

+

IV, LPS (S. enteritidis 2 or 16.2 mg/kg)

+

Test, n = 8; control, IV, LPS bolus n = 8 (S. enteritidis 10,000 or 1000

+

Study design appropriate?

+

CA response caused by the shock itself?

Rats

+

+

No evidence that LPS had any biological effects

?

Conscious rats Temporal correlation between hypotension and evaluation of plasma CA

+

Conscious rats CA were elevated despite normotension

Species clinically relevant?

TABLE 3 Analysis of Studies on Catecholamine Response in Septic/Endotoxic Shock in the Rat

150 Handbook of Mediators in Septic Shock

8 (61)

44

7 (68)

42

6 (54)

40

5 (53)

38

(60)

+

+

+

+

+

+

?

+

Described only for adrenal samples

+ Sampling at 0, 15, 45 min

+ NS control group

+ + Sampling at 0, 1, NS control group 4, 8 h

Unoperated and sham-operated control groups

+ Plasma (a)

+ Sampling at only one time point (90 min)

+ NS control group

+ ? + Adrenal, heart, Sampling at only NS control group kidney, one time point spleen, liver (5 h)

+ Plasma (v)

+ Plasma

Plasma (a), ad- Sampling at 0, renal 16, 20, 24, 41 h + See Study 2

Rats

+

+

+

+ ? Conscious rats Although there is a marked elevation in plasma CA no direct statistical comparisons between control rats and LPS-treated rats is given

+ Conscious

Conscious rats Within first 24 h CA were elevated despite normotension

+ + IV, LPS (S. enConscious or teritidis 3.3 mg/ anesthesized kg) rats

+ IV, LPS (E. coli 10 mg/kg)

? IV, LPS (E. coli 30 mg/kg = LD100)

+ IV, LPS infusion (£. coli 41 jig/ kg/min for 4 h)

Cecal ligation and puncture

Note: CA = catecholamines; RE = radioenzymatic; ( + ) a positive answer; ( — ) a negative answer; (?) no information or an ambiguous answer; NS = normal saline; a = arterial; v = venous; sc = subcutaneous; LPS = lipopolysaccharide.

RE

HPLC

RE

RE

RE for plasma, trihydroxy indole fluorometrics for adrenal

Rabinovici and Feuerstein 151

4 (63)

3 (57)

2 (58)

1 (62)

Study no. (ref.)

+

+ Hydroxy indole fluorometry

?

+ Hydroxy indole fluorometry

HPLC

CA assay reliable?

?

?

?

+

+ Plasma (a)

+ Plasma (a)

+ Plasma (adrenal v + a)

+ Plasma (a)

Sample Sampling in preparation relevant body appropriate? fluid?

+ Sampling at -20, -5, 2, 30, 60, 120, 180, 240, 300 min

+ Sampling at 0, 1, 6h

+ Sampling at 15, 30, 60, 120, 180 min

? Sampling at only one time point (45 min)

Sampling at the right time?

+ IV, LPS infusion (E. coli 0.5 mg/ kg over 5 min, LAo) + IV, LPS infusion (E. coli 5 mg/kg over 5 min)

+ IV, LPS bolus (E. coli 5 mg/kg) followed by infusion (2 mg/kg/h)

Shock model clinically relevant?

+ No NS control group, IV, LPS (E. coli small groups (n = 0.8 mg/kg, LD50) 3-5)

No NS control group

No NS control group

No NS control group

Study design appropriate?

+ Anesthetized

+ Anesthetized dogs

+ Anesthetized dogs

+ Anesthetized dogs

Species clinically relevant?

See Study 1

See Study 1

See Study 1

Without NS control group there is no evidence on reliability of CA measurement

CA response caused by the shock itself?

TABLE 4 Analysis of Studies on Catecholamine Response in Septic/Endotoxic Shock in the Dog and Cat

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440

Handbook of Mediators in Septic Shock

I. INTRODUCTION Despite considerable advances in the treatment of shock and sepsis in the past decade, high rates of morbidity and mortality still remain associated with these conditions. Obviously, the aim of all researchers and practitioners in this area is to advance the understanding of the condition in order to achieve successful prevention, diagnosis, and therapy of shock states. With regard to this latter point, the relative lack of effective pharmacological interventions highlights the extremely complex biochemical and pathophysiological events which occur in shock and sepsis. Neugebauer and Lorenz1 consider that in the literature about 100 mediators have been described as being "causally related" to shock. The use of the term "causally related" is not particularly helpful in defining the current state of knowledge regarding mediator involvement in shock. It would be appropriate to say that a considerable number of mediators have been implicated in the pathophysiology of shock and sepsis. Neugebauer and Lorenz1 also consider that many investigators "often designate their favorite mediator as being predominant in circulatory shock." The extent of this practice in the literature is debatable, but obviously regarding the pathophysiology of shock from a "personalized" or biased viewpoint contributes little to a better understanding of the pathology. This then may be considered as an "ultra-irrational" approach. At the other extreme, Neugebauer and Lorenz1 have extended the Koch-Dale criteria assessment technique into a method of meta-analysis in combination with a decision tree in an attempt to establish the causal role of a single mediator in shock. This approach could be considered somewhat "ultra-rational". Indeed, after applying meta-analysis to evaluate the role of histamine in shock, Neugebauer et al. 2 conclude that only if release of the mediator (i.e., histamine) can be demonstrated unequivocally (presumably by meta-analysis) in clinical conditions of septic shock, are trials with an antagonist of the mediator (i.e., H ; and H2 antagonists) justified. Obviously, while a critical consideration of the literature is essential, rejection of data from a body of information because it does not absolutely meet specific criteria may not be helpful in advancing the overall understanding of the condition. There are several problems with the "ultra-rational" approach. With regard to the example of mediator release and antagonist trials given above, several points can be raised. First, what is the definition of "unequivocal" identification of a mediator? This depends on the analytical technology and information available at the time. As technology evolves, new and more sensitive methods are constantly being developed, and while some older methods are found to be less valid, others are found to be more so. Thus the situation is in permanent flux. Indeed, in their study on histamine, Neugebauer et al.2 encounter this problem and state "unfortunately, the definition of histamine release was sometimes equivocal". As meta-analysis requires a negative or positive answer at each decision node, Neugebauer et al.2 decide to define histamine release as either an '' increase or decrease of histamine concentration in a body fluid and/or tissue," a criterion which is as arbitrary as any other which could have been chosen. Even if a mediator is unequivocally identified as being present in the clinical condition, it does not necessarily mean that it is relevant or causally related to the condition. The relevance of a mediator in shock with regard to its presence (or absence), concentration, site of release, time of release, movement, interaction with other mediators, and metabolism will only eventually be deduced from the synthesis of a wide range of information. While meta-analysis may be able to clearly demonstrate that no single mediator is causal in shock, it is not a subtle enough method to provide an integrated view of this complex condition. For example, suppose that in the early stages of shock a minute concentration of a mediator is released for a very short time in a location not considered particularly relevant, but by an amplification or priming effect this mediator has a profound influence on the other components of the shock syndrome and thus the outcome of the disease. If examined by

Hosford et al.

FIGURE 1.

441

Single node of a probability matrix. The response to the question can range from 0 to 100% probability.

meta-analysis, this mediator may certainly fail to meet specific criteria such as "unequivocal" demonstration of release or clinical relevance, and thus trials with a suitable antagonist would be rejected. In the clinical situation, however, an antagonist of the mediator provided at the right time and concentration may dramatically improve the outcome of the condition. As previously mentioned, true meta-analysis requires a yes or no response at each test node. Neugebauer et al.2 experience problems with the rigidity of this sytem in their analysis of histamine. In their study it "became obvious that the demand on absolute reliability of the reported data was impracticable" and thus they replace strict meta-analysis with a system which allows some consideration of shortcomings in the reports. This highlights some of the difficulties presented by meta-analysis in the choice of specific criteria with regard to analysis of mediators in shock. If such analytical methods need to be applied to examination of the potential involvement of a mediator in shock, we feel a much more flexible approach is needed. In this paper we will examine the potential involvement of platelet-activating factor (PAF) in shock by a "probability matrix". This matrix can be formulated into any shape, but here for the sake of simplicity we will use a pyramid. Similar to meta-analysis, this model asks questions but the response does not have to be simply negative or positive: it can be anything between 0% probability (i.e., no) and 100% probability (i.e., yes). A single node of the matrix is demonstrated in Figure 1. The pyramid is made up of different levels with a question being asked at each level, and depending on each response, a path descends from the apex of the pyramid to a point on the base somewhere between the left hand and right hand corners. The position on the base is thus the result of "summation" of all the previous probabilities (Figure 2). If the left-hand path of each probability "triangle" is 0% and the right-hand path is 100%, then correspondingly the left-hand corner of the pyramid base will be 0% probability and the right-hand corner 100% probability. Obviously, the choice of questions and the level of their positioning is arbitrary as in any analytical system, but this approach has an advantage over meta-analysis in that it does not exclude any data from the system. In meta-analysis, if the answer at the first decision

442

Handbook of Mediators in Septic Shock

FIGURE 2. Pyramid shaped probability matrix with five levels. In this case the response path after the five decision nodes gives a final response of 50% probability.

node is node is "no" then the report should be excluded, even if it is able to meet all the other following criteria on the decision tree. Using the probability matrix even if the response path from the first test node is 0% (no) the report is still retained within the matrix to be tested at the second level and so no. If such an analysis was applied to a range of mediators it would allow a comparative view of the extent of their involvement in shock. Figure 3 shows the matrix applied in this paper. A quantitative use of the probability matrix has not been attempted herein. This article does not present an exhaustive review of the literature but rather uses a limited amount of information to qualitatively illustrate the principle.

II. ANALYSIS BY THE PROBABILITY MATRIX A. MATRIX LEVEL 1: CAN PAF RELEASE BE INDUCED BY LIPOPOLYSACCHARIDE (LPS) IN VITRO? PAF is a potent autocoid mediator implicated in a diverse range of human pathologies, particularly inflammatory conditions.3"5 Originally isolated from antigen-stimulated rabbit basophils and characterized structurally as l-0-alkyl-2(/?)-acetyl-glycero-3-phosphocholine, the alkyl phospholipid is now known to be produced by, and act on, a variety of cell types including polymorphonuclear (PMN) neutrophils, eosinophils, monocytes, macrophages, platelets, and endothelial cells.3"5 Obviously fundamental to the consideration of PAF in sepsis and endotoxemia is the relationship between PAF and LPS. Monocytes and macrophages provide a major source of tumor necrosis factor (TNF) and other cytokines following stimulation by endotoxin or LPS: thus, numerous in vitro studies have utilized this cell type. Stewart and Phillips6 demonstrated that adherent guineapig macrophages contained cell-associated PAF whose level was increased following stim-

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FIGURE 3. Pyramid shaped probability matrix with six levels used in this analysis to respond to the general question: "Is PAF involved in the pathology of endotoxemia and sepsis?"

ulation by FMLP, endotoxin and ionophore A 23187. However, only endotoxin and A 23187 caused the release of detectable amounts of PAF into the extracellular medium. In contrast, Worthen et al.7 found that while exposure of neutrophils to low concentrations of LPS produced a small but significant increase in intracellular PAF levels, release of the mediator was not detected. Rylander and Beijer8 examined the production of PAF in alveolar macrophages (AM) and neutrophils recovered by bronchial lavage from guinea pigs exposed to aerosolized bacterial LPS. The amount of cell-associated PAF was estimated by measuring serotonin release from rabbit platelets. An increased and dose-related production was found in AM for as long as 2 h after a 40-min exposure. No production was detectable after 4 h and increasing the exposure time did not prolong the response. When a second exposure was given, no PAF could be detected until the time interval between the two exposures was 72 h. Studies on 3H-arachidonic acid turnover in LPS stimulated human monocyte-derived macrophages by Leslie and Detty,9 support the evidence that the bacterial toxin can increase PAF production. The authors found that relative to unstimulated cells, there was a preferential, dose-dependent loss of 3H-arachidonic acid from phosphatidylcholine (PC) and phosphatidylinositol (PI) in cells exposed to LPS, which was maximal between 1 and 3 h after adding the toxin. In addition, LPS induced a 35% decrease in the molar quantity of PI in the macrophages but had no effect on the quantity of PC, phosphatidylethanolamine (PE) or phosphatidylserine (PS). The generation and release of PAF in response to LPS stimulation has also been reported for other cell types, such as human10 and rat glomeruli10-11 and endothelial cells,12 where release of the mediator into the extracellular medium by this latter cell type represented only 4 to 5% of the total produced. Thus, while it seems probable that LPS can increase PAF synthesis in vitro, the amount of mediator released from the cells may be minimal.

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B. MATRIX LEVEL 2: ARE THERE IN VITRO INTERACTIONS BETWEEN PAF AND OTHER SHOCK-ASSOCIATED MEDIATORS? While it is important to consider the direct influence of LPS on PAF production, it is essential to have some understanding of the interactions between PAF and mediators associated with shock. PAF is synthesized and frequently secreted by a variety of cell types in response to mediators and cytokines, particularly TNF. This body of information has been reviewed in depth3'5-13 and does not require further elaboration here, rather it is more pertinent to consider more subtle interactions such as priming and amplification. With regard to cytokines, it has been demonstrated that PAF is able to enhance interleukin 1 (IL-1) production in human monocytes treated with endotoxin.' 4 PAF was shown to increase the release of IL-1 with a multiphasic dose-response curve. When the cells were treated with PAF and endotoxin in combination, these two stimuli interacted synergistically. Immunostaining of proteins isolated from PAF-stimulated cells revealed that PAF and endotoxin increased intracellular IL-1 precursors and thus increased the levels of this cytokine by facilitating its synthesis. The production of TNF by human platelet-free monocytes, isolated by counterflow elutriation, was analyzed following stimulation with endotoxin in the absence or presence of graded concentration ranges of PAF.15 Two concentration ranges showed a significant increase in TNF production. A major enhancement was observed in 10~ 8 to 10~ 6 M which was blocked by BN 52021, while a second enhancement was seen at 10"15 to 10 ~ 1 4 M PAF, which was insensitive to BN 52021. These results suggest that PAF can directly modulate cytokine production by human monocytes possibly by interacting with two types of receptors. The effects of PAF on the induction and priming of TNF secretion in peripheral blood monocytes have recently been studied.16-17 Unlike gamma-interferon (IFN-y) and endotoxin, the addition of PAF to freshly isolated monocytes triggered a rapid, concentration-dependent TNF secretion in the absence of induction of macrophage-mediated cytotoxicity. While biologically active and cytotoxic TNF was detected early after PAF addition, the cytotoxic activity declined thereafter, though the antigenic activity remained constant. Monocytes primed with PAF responded by secreting TNF to both pokeweed mitogen and concanavalin A, representing unspecific stimuli, but responded poorly to specific stimulation by PAF, endotoxin, and IFN-y. These findings suggest that PAF may mediate part of its biological activity via the macrophage, and further, monocyte secretion of PAF can, in turn, regulate monocyte function, thereby contributing to the inflammatory response. Work from our own laboratory has shown that PAF markedly amplifies superoxide production by TNF-stimulated human PMN. PAF antagonists not only inhibited the PAF amplification, but also partially decreased superoxide production elicited solely by TNF, indicating the involvement of endogenous PAF in this process.18 Pretreatment of the PMN with pertussis toxin (PT) or cholera toxin (CT) reduced the PAF amplification of superoxide production in TNF-stimulated PMN, implicating PT and CT sensitive G-proteins in the amplification process.18 Other studies have shown that preincubation of rat spleen macrophages with 10 fM PAF (which had no effect per se) prior to stimulation with 10 fig/ml LPS markedly increased the production of IL-1 by the cells, relative to that induced by LPS alone.19 Association of 1 (Jig/ml PT with PAF suppressed the enhancing effect of PAF on IL-1 release, again implicating PT sensitive G-proteins in the priming process. Furthermore, while, concentrations of PAF lower than 0.1 jjiM were unable to elicit release of leukotriene B4 (LTB4) from human PMN, however, preincubation with granulocyte-monocyte colony stimulating factor (GM-CSF) both increased LTB4 synthesis by tenfold in PMN incubated with 0.1 to 1 |xM PAF and stimulated LTB4 release in cells exposed to 1 nM PAF. Similarly, the authors have examined the effect of PAF on IL-6 release from murine fibroblasts. While a high concentration of PAF (5 jxM) increased IL-6 release from the cells

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by 134%, low concentrations of PAF (1 fM to 1 nM) were ineffective. However, preincubating the fibroblasts for 10 min with low doses of PAF prior to stimulation with 25 [Lgl ml Poly I-C revealed that a concentration of 10-fM PAF was able to prime the cells to produce significantly higher levels of IL-6, relative to that induced by Poly I-C alone.20 With regard to prostanoids, Morris and Moore21 examined whether PAF and endotoxin could stimulate equine macrophage release of thromboxane B2 (TXB2) and prostaglandin I2 (PGI2). Peritoneal macrophages were cultured from clinically normal horses and exposed to various concentrations of PAF, endotoxin, and SRI 63-441. The media concentrations of TXB2 were significantly increased after treatment of macrophages with PAF and endotoxin. In a recent study using isolated, buffer-perfused rabbit lungs, free of plasma and circulating blood cells, Salzer and McCall22 demonstrated that LPS synergized with PAF to injure the lung. In lungs perfused for 2 h with LPS-free buffer (less than 100 pg/ml), stimulation with 1, 10, or 100 nM PAF produced transient pulmonary hypertension and minimal edema. Lungs perfused for 2 h with buffer containing 100 ng/ml of Escherichia coli LPS had slight elevation of pulmonary artery pressure (PAP) but did not develop edema. In contrast, lungs exposed to 100 ng/ml of LPS for 2 h had marked increases in PAP and developed significant edema when further stimulated with PAF. LPS treatment increased capillary filtration coefficient, suggesting that capillary leak contributed to pulmonary edema. LPS-primed, PAF-stimulated lungs had enhanced production of TXB2 and 6-keto-PGFla. These studies indicate that LPS primes the lung for enhanced injury in response to PAF by amplifying the synthesis and release of thromboxane in lung tissue. In general, the above data strongly indicate that important interactions exist between PAF and other mediators that may be operative in the pathology of endotoxemia and sepsis. C. MATRIX LEVEL 3: CAN PAF INDUCE ENDOTHELIAL ALTERATIONS CHARACTERISTIC OF SHOCK? The activity of PAF as an inducer of increased microvascular permeability has been widely documented.3"3'13 A direct agonistic activity of the mediator on endothelial cells has been suggested by experiments showing that PAF stimulates Ca2 + - influx-efflux in cultured human endothelial cells.23 Indeed, recent studies have shown that PAF, but not its deacetylated and biologically inactive metabolite lyso-PAF, dose dependently (0.1 to 10 nM) induces cultured human endothelial cells to retract and lose reciprocal contact and promotes I25 Ialbumin diffusion in endothelial cells grown on fibronectin-coated polycarbonate filters.24 Impairment of endothelial cells by PAF in vivo has also been observed. Following 5 to 10 min superfusion with the autacoid (10 7 Af) in the mesenteric bed of anesthetized guineapigs, retraction of endothelial cells occurs in the area corresponding to the site of application of PAF, resulting in exposure of the subintimal tissue to the blood stream and substantial thrombus formation involving platelets, monocytes, eosinophils, cell infiltration, and diapedesis. Formation of blebs and interstitial edema also accompany these PAF-induced changes.25 In guinea pigs superfused with TNF, a subsequent injection of a low dose of PAF in the mesenteric bed dramatically enhances thrombosis. This enhanced activity of PAF by pretreatment with TNF occurred at doses at which PAF and TNF given alone or PAF prior to TNF did not significantly affect thrombogenesis. BN 52021 or anti-TNF antibodies inhibited this synergism. 13 This result suggests that TNF primes the effect of PAF on the endothelial cell wall, an effect also observed in vitro. In guinea pigs superfused in the mesenteric bed with a nonthrombogenic dose of Salmonella enteritidis LPS, subsequent injection of a low dose of PAF (which does not induce a thrombogenic effect per se) produced an extensive thrombus,25 suggesting that LPS primes the effect of PAF. Vascular endothelial cells synthesize and release PAF in response to various stimuli.26 While in response to stimuli such as thrombin, histamine, ATP, and leukotrienes the release

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of PAF is rapid and transient, a new pathway has recently been elucidated that requires protein synthesis and is primed by IL-1 and TNF.27 In this pathway, PAF is synthesized de novo in endothelial cells, with maximal output occurring a few hours after the addition of the two monokines. Thus, while there is very good evidence showing that at higher concentrations, PAF itself can induce the endothelial alterations apparent in shock, interactions between low concentrations of PAF and TNF (which may be ineffective per se) may be crucial in initiating vascular damage in the very early phases of shock. D. MATRIX LEVEL 4: CAN PAF INFUSION IN VIVO MIMIC SHOCK? Studies on several animal models have shown that an infusion of PAF is able to mimic the shock state. For example, in guinea-pigs PAF administration produces hypotension, bronchoconstriction, thrombocytopenia, and death.28-29 In dogs the mediator induces hypotension, myocardial contractility impairment, decreased coronary artery flow, systemic and pulmonary vascular changes, renal dysfunction, hemoconcentration, and metabolic acidosis.30"32 Leukopenia and thrombocytopenia following PAF infusion have also been observed in this species. The microvascular damage, decrease in blood volume, and increase in plasma extravasation observed in shock are of particular importance to the present discussion. These vascular permeability impairments have also been reported in sepsis in dogs.33"34 PAF induces hemoconcentration, plasma extravasation, and edema formation in rats,35 guinea-pigs,36 and sheep.37 Locally injected, PAF induces plasma extravasation in guinea pigs38 and rabbit skin, and intense plasma leakage and sludge in the rabbit retina.39 A broad range of PAF antagonists protect against shock induced by infusion of the mediator in a variety of animal models,40 for example BN 52021, CV-6209, WEB 2086 and L-652,7314' inhibit PAF-induced hypotension in rats. However, while such in vivo studies confirm the in vitro data on PAFinduction of endothelial damage, they provide little information on complex pathological situation encountered in shock and thus are limited in their value. E. MATRIX LEVEL 5: IS THERE INCREASED PAF PRODUCTION DURING SHOCK? In addition to studies showing that PAF infusion can induce shock, other reports have demonstrated that the mediator is produced in various shock states. Gram-negative sepsis is a major cause of severe circulatory shock, which can be mimicked in animal models by the infusion of either living bacteria or bacterial endotoxin. Inarrea et al.42 showed that shock triggered by intraperitoneal injections in the rat of 2 x 108 colony forming units (CPU) of E. coli produced septicemia with 50% mortality. There was also a dose-dependent increase in vascular permeability accompanied by the appearance of PAF in the peritoneal and spleen cells of the intoxicated animals. Similarly, Doebber et al.43 reported a marked rise in circulating blood levels of PAF 10 min after intravenous injection of endotoxin in rats. The present authors have also recently demonstrated that PAF appears in rat blood samples after intravenous injection of Salmonella enteritidis, while it is not present in control animals. Similar findings have been reported by Buxton et al.44-45 using in situ perfused rat livers challenged with soluble immunoglobulin (Ig)G aggregates. In this experimental system PAF activity was detected in the effluent following IgG challenge. Rylander and Beijer8 showed that inhalation of endotoxin in guinea pigs produced a dose-dependent production of PAF by alveolar macrophages, while Fitzgerald et al.46 noted that sensitized, isolated, and perfused guinea pig lungs released three times more PAF when challenged with antigen relative to control preparations. In addition, both Hsueh et al.47 and Lagente et al.48 have reported markedly elevated levels of PAF production in an experimental animal model where endotoxin has been used to induce intestinal lesions. Finally, several recent reports have demonstrated increased levels of PAF in human shock. Studies by Bussolino et al.49 have shown an elevated intravascular release of the

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mediator in children with sepsis, while Lopez-Diez et al.50 have examined the possible involvement of PAF in human endotoxemia by determining ex vivo PAF binding to platelets of the patients, complemented with extraction and chemical characterization of PAF obtained from these cells. Platelets from 12 healthy human volunteers had 281 ± 6 3 freely accessible PAF high affinity binding sites per platelet, whereas this number was 49 ± 37 PAF-receptors per platelet in a group of 13 patients with septicemia. Patients with sepsis also possessed significant amounts of PAF associated to their platelets, whereas this mediator could not be isolated from platelets of control individuals.50 PAF was also assayed in whole blood samples and found at high concentrations in sepsis patients. In a recent study, Arditi et al.51 detected TNF in the cerebrospinal fluid (CSF) of 33 of 38 children with bacterial meningitis, but not in any of 15 children with viral meningitis. In children with bacterial meningitis, TNF in the CSF correlated with CSF bacterial density, CSF protein, endotoxin (LPS) in gram-negative disease, and consecutive febrile hospital days. TNF levels in the CSF greater than 1000 pg/ml were associated with seizures. Furthermore, a higher proportion of children who died had detectable plasma TNF activity as compared with survivors. PAF in the CSF was also higher in 19 children with Haemophilus influenzae meningitis than in 17 controls and correlated with bacterial density and CSF levels of LPS and TNF. Thus it appears that elevated CSF levels of TNF and PAF are often present in children with bacterial meningitis and are associated with seizures and severity of disease. The above studies demonstrate increased levels of PAF release in human sepsis and that occupancy of PAF receptors and high amounts of platelet-associated PAF are also characteristic of patients with sepsis. The evidence reviewed in this section strongly indicates that increased levels of PAF are a feature of shock and sepsis, and that these elevated levels correlate with the outcome of the condition. F. MATRIX LEVEL 6: CAN PAF ANTAGONISTS PROTECT AGAINST ENDOTOXIC SHOCK IN VIVO? The finding of increased levels of PAF in endotoxemia suggested that PAF antagonists may be effective in counteracting this pathology. Indeed, this hypothesis has been sustained by numerous experimental studies. In shock induced by Salmonella enteriditis endotoxin,52"53 a significant dose-dependent inhibition of the lethality was observed with BN 52021 treatment (5 to 20 mg/kg), total protection being provided by the highest dose of the drug. This effect was associated with a reduced increase in body temperature, suggesting that BN 52021 has an effect on the release of IL-1. In guinea pigs injected with Salmonella typhimurium endotoxin, a similar protection was also afforded by BN 52021 and other PAF antagonists including CV 3988,54 kadsurenone,55 L-652,731,56 SRI 63-072, and SRI 63441,57 WEB 2086,58 and the new hetrapazine-derived antagonists BN 52730 and BN 52739.59 In endotoxemic conscious rats and dogs, Fletcher et al.60 have demonstrated that BN 52021 beneficially attenuates the late systemic hypotension and circulatory dysfunction, respectively, in these species. Similarly in the rat, Rabinovici et al.59 found that pretreatment of the animals with BN 50739 prevented endotoxin-induced hemocentration, reduced 24-h mortality from 100 to 60% and partially protected against the hypotensive response to endotoxin. LPS-induced elevation of thromboxane and TNF was also attenuated, although leukopenia and thrombocytopenia remained unchanged. In addition, the antagonist reduced TNF-induced mortality by 65%, but did not alter hematological responses to the cytokine. BN 50739 was also effective in preventing the early phase of LPS-induced thrombocytopenia and thromboxane elevation in the rabbit.61 The antagonist reduced the 24-h mortality from 75 to 22% and posttreatment with the drug increased the 10-h survival rate from 33 to 87%. However, LPS-induced leukopenia and the increase in plasma TNF levels were not affected in this species by the antagonist.

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In dog endotoxemia, BN 52021 has no beneficial effects on the early hypotension (1 to 2 min) following administration of endotoxin, but beneficially attenuated the continued decrease in blood pressure and cardiac output over time.62 Interestingly, the systemic vascular resistance was slightly decreased by BN 52021 at 1 to 2 min, while the early endotoxininduced increase in pulmonary vascular resistance was inhibited. The finding that the PAF antagonist prevented the early rise in pulmonary vascular resistance relative to that observed in untreated endotoxemic animals suggests that PAF may be directly or indirectly involved as a pulmonary vasoconstrictor in dog endotoxemia. Although the effects of the PAF antagonist on the hemodynamic and metabolic events in dog endotoxemia were variable, pretreatment with BN 52021 dramatically improved the permanent survival rate from 0 to 100% in this model.62 Other studies by this group using endotoxemic, conscious rats have shown that in addition to improving survival, BN 52021 also prevents the release of TXB2 and PGE2 observed in untreated endotoxemic rats.63 These latter data suggest that the actions of PAF in endotoxemia may be partly mediated by the generation of eicosanoids. The protection afforded by PAF antagonists against endotoxin-induced changes in carbohydrate metabolism has been recently investigated by Lang et al.64 In chronically catheterized conscious rats, endotoxin (100 (xg/lOO g b.w.) induced a transient 30 to 35% reduction in mean arterial blood pressure (MAP), while in animals treated with SRI 63-441, a synthetic PAF antagonist containing a tetrahydroduran ring and a quaternary moiety, MAP was only reduced by 14 to 18%. Endotoxin increased plasma glucose and lactate levels, as well as the rate of glucose appearance. The PAF antagonist reduced the hyperglycemia by 60 to 75%, partially inhibited the hyperlactacidemia and significantly reduced the elevation in the rate of glucose appearance. Although a similar degree of hyperglucagonemia and plasma insulin was observed in endotoxemic rats whether treated or not with the PAF antagonist, plasma catecholamine levels were significantly lower (30 to 70%) in endotoxemic rats receiving SRI 63-441. These results indicate that enhanced PAF production following endotoxin administration may be partly responsible for the initial changes in blood pressure, but that the role of PAF as a mediator of endotoxin-induced glucose dyshomeostasis is secondary to its hemodynamic effects.64 Since the demonstration that administration of PAF, either as a bolus65 or continuous infusion,66 induces increases the pulmonary vascular permeability in sheep, several workers have used this model to assess the effects of PAF antagonists against both endotoxin- and PAF-induced shock. An initial study by Toyofuku et al.67 reported that the PAF antagonist, ONO-6240, had little effect against on pulmonary hypertension and lung lymph balance in ovine endotoxemia, but other researchers using structurally different PAF antagonists have reported significant protective effects of the compounds. Sessler et al.68 reported that endotoxin infusion (1.3 (ig/kg over 30 min) caused a rapid, transient rise in PAP and pulmonary vascular resistance (PVR), while arterial pO2 decreased. A 5-h infusion of SRI 63-441 (20 mg/kg/h) blocked the early rise in PAP and PVR and fall in arterial pO2, but had no effect on the late phase pulmonary hypertension or hypoxemia. The drug also abolished the early and attenuated the late increase in lung lymph flow observed in ovine endotoxemia.68 Similar results have been obtained with WEB 2086,69 which attenuated the late increase in permeability-mediated lymph flow, but did not prevent the early pulmonary hypertension after bolus LPS administration. Redl et al70 have reported that BN 52021 is able to attenuate the early rise in PAP, the duration of the pulmonary hypertension and the increased thromboxane levels following LPS administration in the sheep. Increases in lymph flow were also beneficially modulated, although high doses of the drug only achieved a 38% survival rate, whereas dexamethasone treatment resulted in 100% survival. In accordance with the protection afforded by PAF antagonists at the general physiological level, these compounds are able to inhibit the effects of endotoxin on specific organs and tissues. Baum et al.71 have tested the hypothesis that LPS-induced myocardial dysfunction

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is mediated by PAF or cyclooxygenase-derived metabolites of arachidonic acid. These investigators used ether-anesthetized rats which were injected intravenously with normal saline, ibuprofen (cyclooxygenase inhibitor; 15 mg/kg), or the PAF receptor antagonist; SDZ 64688 (5 mg/kg). After 30 min, the rats were injected intravenously with saline or E. coli LPS (20 mg/kg). After 2 h. atria were harvested, connected to an isometric force transduceramplifier-recorder apparatus, and maintained in vitro in oxygenated Krebs-Henseleit buffer. Force of contraction indexed to body weight was significantly lower in the LPS group than in the group which received saline. Pretreatment with ibuprofen did not affect this adverse effect of LPS, however, pretreatment with SDZ 64-688 ameliorated the deleterious effect of LPS on contractility. These results support the notion that LPS-induced myocardial dysfunction in the rat is mediated, at least partly, by PAF. In the guinea pig, endotoxin administered by aerosol induces platelet accumulation and increased vascular permeability in the lung, effects also induced by treatment with PAF. Both these effects of PAF and endotoxin were prevented by WEB 2086, BN 52021, and the ginkgolide mixture, BN 56203.72'74 Furthermore, SRI 63-441, inhibited endotoxininduced pulmonary edema and lung micro vascular damage in the rat75 and sheep.76 Recently, we have shown that lung parenchymal strips from guinea pigs treated with endotoxin exhibit specific desensitization to PAF, an effect not observed in animals also administered BN 52021, which indicates PAF production in endotoxemia.77 In a similar model using E. coli endotoxin, BN 52021 inhibits the endotoxin-induced tracheal hyperreactivity to histamine in the guinea pig, suggesting that PAF may be important in inducing this process.78 PAF antagonists are also effective in other models of endotoxin-induced pulmonary dysfunction. In the pig, intravenous infusion of LPS for 5 h induces a marked increase in PAP. Animals concomitantly treated with WEB 2086 had significantly reduced pulmonary hypertension relative to those not receiving the drug, although the PAF antagonist did not affect the systemic hypotension.79 A sharp decline in peripheral white blood cell counts also occurred following LPS infusion, a process which was retarded by the treatment with WEB 2086. The endotoxin-induced deterioration in both gas exchange and inspiratory pressure were also partially prevented by the drug. PAF antagonists also inhibit the oliguric acute renal failure associated with decreased renal blood flow (RBF) and glomerular filtration rate (GFR) induced by endotoxin in the rat.8o,8i For exampie! both BN 52021 and SRI 63-675 are able to inhibit the endotoxininduced vascular escape and alterations in RBF, thus producing significant improvement in renal function. These findings are in accordance with those of Wang and Dunn,82 who have recently demonstrated that the PAF antagonist L-652,731 has a protective effect during endotoxemia in postpartum rats, further suggesting that PAF mediates endotoxin-induced acute renal insufficiency in this species. Gastrointestinal hemorrhage is another feature associated with septic shock, a condition which can also be produced in several species by injection of PAF or bacterial endotoxin.83'84 Hsueh et al.85 also noted increased production of PAF in the bowel in endotoxemic animals. Accordingly, PAF antagonists, including CV-3988, RO-193704, and BN 52021, are able to significantly reduce the endotoxin-induced gastrointestinal damage in the rat.86'87 Sun and Hsueh88 have demonstrated that rats treated with PAF or endotoxin, develop ischemic bowel necrosis associated with shock. In this model, the morphological changes of TNF-induced bowel lesions were indistinguishable from those caused by PAF. TNF induced PAF production in bowel tissue, and the effects of TNF and endotoxin on PAF production in the intestine were additive. Furthermore, TNF and endotoxin were synergistic in inducing bowel necrosis, and TNF-induced bowel necrosis was partly due to PAF release, since SRI 63-119, had a protective effect.88 In a more recent study Sun et al.89 examined the effect of in vivo priming on TNF and PAF levels in LPS-induced shock. Rats were primed with intraperitoneal injection of zymosan

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24 h before, or Bacillus Calmette-Guerin (BCG) 1 to 15 d before intravenous injection of a low dose of LPS (0.5 mg/g). Results showed that nonprimed animals developed mild hypotension and moderate leukopenia in response to LPS. In contrast, zymosan-primed rats developed shock and marked leukopenia, and more severe bowel injury than nonprimed rats. In addition, following LPS injection, zymosan-primed animals had higher TNF and PAF levels than nonprimed rats. Pretreatment of the animal with the PAF antagonist, SRI 63-441, markedly ameliorated the hypotension and tissue injury. Interestingly, BCG-primed rats did not show aggravation of LPS-induced hypotension, and only levels of TNF (but not PAF) in these animals were increased. Thus, it appears that TNF release alone, without a sufficient increase in PAF, is incapable of causing severe hypotension in this model. The mediatory role of PAF in intestinal inflammation and ulceration has also been investigated. Using the rat model of colitis, the effects of treatment with BN 52021 in comparison with WEB 2086 and WEB 2170 were studied in endotoxin-induced ulceration by measuring PAF biosynthesis and changes in vascular permeability.90 In agreement with previous findings,91-92 PAF antagonists were found to accelerate healing of chronic colitis, thereby suggesting a role for PAF in the mechanism of intestinal ulceration induced by endotoxin. Finally, the effects of endotoxin on the brain include both alterations in cerebral blood flow (CBF) and a fall in respiratory control ratio (RCR; state 3/state 4 of the respiratory chain). In dogs administered intravenously with endotoxin, Ekstrom-Jodal et al.93 observed a decreased CBF and increased cerebral metabolism. In further studies, this group demonstrated increased permeability of the blood-brain barrier following endotoxin treatment.94 Recently, the current authors have also demonstrated that endotoxin decreases RCR values in gerbil brains. Treatment of the animals with BN 52021 improves CBF and normalizes RCR impairments caused by the toxin (B. Spinnewyn, in preparation). The considerable body of evidence reviewed in this section indicates the very high probability that PAF antagonists are capable of protecting against endotoxemia, both with regard to general physiological and hemodynamic alterations, and shock-induced changes experienced by specific organs.

III. CONCLUSIONS In the preceding discussion, the evidence reviewed strongly suggests that PAF is produced during endotoxemia and that PAF antagonists counteract this condition. Indeed, for most of the questions on the decision matrix, with the exception of direct PAF production induced by LPS, the response path would be in the direction of high probability (i.e., 60 to 80%). Thus, in response to the question: "Is PAF involved in pathology of endotoxemia and sepsis?" the final position of the response path at the base of the pyramid (probability matrix) would indicate a high degree of involvement of PAF in the condition. Following from this analysis, we can ask the question: what processes does PAF modulate in shock, and how do PAF antagonists intervene to protect against the alterations? In response to this question, it appears that modulation of two processes are particularly important: PAF/cytokine interactions and PAF stimulation of protease activity. In addition to the numerous vasoactive materials released by endotoxin in shock, a considerable increase in plasma proteolytic activity is observed, for example, during septic shock in man95 and in endotoxin-induced shock in animal models.96 Since PAF injection in the rat induces a rapid significant increase in plasma protease activity,97 part of the beneficial effect of PAF antagonists may result from their indirect inhibition of this process. Endotoxin may stimulate phospholipase A2 restulting in PAF production, which would in-turn lead to cell activation and release of lysomal enzymes. As phospholipase A2 can also be activated by various proteases, a positive feedback cycle is created, increasing the deleterious effects of the toxin.

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PAF antagonists may act by inactivating this feedback loop, indeed, as demonstrated by Etienne et al.,97 BN 52021 inhibits both the PAF- and endotoxin-induced plasma protease activity in the rat. In addition to PAF, endotoxin also induces the release of other mediators, particularly cytokines such as TNF,98 emphasizing the potential importance of PAF/cytokine interactions. The present authors have recently proposed that a complex interaction between PAF, proteases, TNF, and other cytokines is of fundamental importance in initiating the pathological changes not only in shock and sepsis, but also in trauma and diverse ischemic disorders.13-"'100 The fact that PAF and various cytokines can not only induce the release of each other, but also their own generation in vivo indicates that self-generating positive feedback cycles may become established. Indeed, the recently demonstrated priming ability of these mediators underlines the extreme sensitivity of inflammatory processes. The fact that PAF is part of a multicomponent system may explain why PAF antagonists, which inhibit both the direct and amplification and priming effects of the mediator, are effective in diverse pathological models of these conditions. Indeed, PAF antagonists are currently being evaluated clinically and may constitute valuable drugs in conditions such as shock and sepsis, where control of the inflammatory response is a therapeutical requisite.

IV. SUMMARY Despite considerable advances in the treatment of shock and sepsis in the past decade, high rates of morbidity and mortality still remain associated with these conditions. The underlying pathology of these conditions comprises endothelial injury, excessive blood cell infiltration and vascular leakage. Among the various mediators implicated in shock conditions, there is much evidence to suggest that, together with various cytokines, leukotrienes, thromboxane, and proteases, the inflammatory and chemotactic autocoid, platelet-activating factor (PAF), plays an important role. Neugebauer and Lorenz have used meta-analysis in an attempt to establish the causal role of mediators in shock and sepsis. We consider the method of meta-analysis inadequate for the assessment of various mediators in these disorders and propose the use of a probability matrix as more appropriate model. The potential role of PAF in shock is qualitatively analyzed using a probability matrix of six levels and the result indicates a high probability for the involvement of PAF in the pathology. It is emphasized, however, that PAF is only one of a number of mediators active in shock and sepsis, and that effective clinical therapy is likely to comprise antagonists, inhibitors, and antibodies to several of the mediators involved.

REFERENCES 1. Neugebauer, E. and Lorenz, W., Causality in circulatory shock: strategies for integrating mediators, mechanisms, and therapies, in Perspectives in Shock Research, Schlag, G. and Redl, H., Eds., Alan R. Liss, New York, 269. 2. Neugebauer, E., Lorenz, W., Maroske, D., Barthlen, W., and Ennis, M., The role of mediators in septic/endotoxic shock: a meta-analysis evaluating the current status of histamine, Theor, Surg., 2,1, 1987. 3. Braquet, P., Touqui, L., Shen, T. S., and Vargaftig, B. B., Perspectives in platelet-activating factor research, Pharmacol. Rev., 39, 97, 1987. 4. Sturk, A., Ten Gate, J. W., Hosford, D., Mencia-Huerta, J. M., and Braquet, P., Synthesis, catabolism and pathophysiological role of platelet-activating factor, in Advances in Lipid Research, Vol. 23, Paoletti, R. and Kritchevsky, D., Eds., Academic Press, London, 1989, 219. 5. Vargaftig, B. B. and Braquet, P., PAF-acether to day, relevance for acute experimental anaphylaxis, Br. Med. Bull., 43, 312, 1987.

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6. Stewart, A. G. and Phillips, W. A., Intracellular platelet-activating factor regulates eicosanoid generation in guinea-pig resident peritoneal macrophages, Br. J. Pharmacol., 98, 141, 989. 7. Worthen, G. S., Seccombe, J. F., Clay, K. L., Guthrie, L. A., and Johnston, R. B., The priming of neutrophils by lipopolysaccharide for production of intracellular platelet-activating factor, J. Immunol., 140, 3553, 1988. 8. Rylander, R. and Beijer, L., Inhalation of endotoxin stimulates alveolar macrophage production of plateletactivating factor, Am. Rev. Respir. Dis., 135, 83, 1987. 9. Leslie, C. C. and Detty, D. M., Arachidonic acid turnover in response to lipopolysaccharide and opsonized zymosan in human monocyte-derived macrophages, Biochem. J., 236, 251, 1986. 10. Pirotzky, E., Ninio, E., Bidault, J., Pfister, A., and Benveniste, J., Biosynthesis of platelet-activating factor (PAF-acether). Precursors of PAF-acether and acetyl transferase activity in isolated rat kidney cells, Lab. Invest., 51, 567, 1984. 11. Wang, J., Kester, M., and Dunn, M. J., The effects of endotoxin on platelet-activating factor synthesis in cultured rat glomerular mesangial cells, Biochim. Biophys. Acta, 969, 217, 1988. 12. Lynch, J. M. and Benson, P. M., The intracellular retention of newly synthesized platelet-activating factor, J. Immunol., 137, 2653, 1986. 13. Braquet, P., Paubert-Braquet, M., Bourgain, R., Bussolino, F., and Hosford, D., PAF/cytokine autogenerated feedback networks in microvascular immune injury, consequences in shock, ischemia and graft rejection, J. Lipid Mediators, 1, 75, 1989. 14. Barthelson, R., Valone, F. H., and Debs, R., Synergy in interleukin 1 (IL 1) release by human monocytes stimulated with platelet-activating factor (PAF) plus gamma interferon (IFN gamma) or tumor necrosis factor (TNF), FASEB J., 2, 1228, 1988. 15. Poubelle, P. and Rola-Pleszczynski, M., PAF enhances the production of tumor necrosis factor alpha by human monocytes: partial antagonism by BN 52021, in Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives, Vol. 2, Braquet, P., Ed., J.R. Prous, Barcelona, 695, 1990. 16. Bonivada, B., Mencia-Huerta, J. M., and Braquet, P., Effects of platelet-activating factor on peripheral blood monocytes: induction and priming for TNF secretion, J. Lipid Mediators, 2, S65, 1990. 17. Bonavida, B., Jewett, A., and Mencia-Huerta, J. M., Biology of platelet-activating factor (PAF) interaction with human peripheral blood monocytes and induction of tumor necrosis factor (TNF) secretion, in Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives, Vol. 2, Braquet, P., Ed., J.R. Prous, Barcelona, 685, 1990. 18. Paubert-Braquet, M., Hosford, D., Koltz, P., Guilbaud, J., and Braquet, P., Tumor necrosis factor a primes the platelet-activating factor-induced superoxide production by human neutrophils: possible involvement of G proteins, J. Lipid Mediators, 2, SI, 1990. 19. Pignol, B., Henane, S., Chaumeron, S., Mencia-Huerta, J. M., and Braquet, P., Modulation of the priming effects of platelet-activating factor on the release of interleuken-1 from lipopoly saccharide-stimulated rat spleen macrophages, J. Lipid Mediators, 2, S93, 1990. 20. Pignol, B., Lonchampt, M. O., Chabrier, P. E., Mencia-Huerta, J. M., and Braquet, P., Plateletactivating factor potentiates interleukin-1/epidermal cell-derived thymocyte-activating factor release by guineapig keratinocytes stimulated with lipopolysaccharide, /. Lipid Mediators, 2, S83, 1990. 21. Morris, D. D. and Moore, J. N., Equine peritoneal macrophage production of thromboxane and prostacyclin in response to platelet-activating factor and its receptor antagonist SRI 63-441, Circ. Shock, 28, 149, 1989. 22. Salzer, W. L. and McCall, C. E., Primed stimulation of isolated perfused rabbit lung by endotoxin and platelet activating factor induces enhanced production of thromboxane and lung injury, J. Clin. Invest., 85, 1135, 1990. 23. Bussolino, F., Aglietta, M., Sanavio, F., Stacchini, A., Lauri, D., and Camussi, G., Alkyl-ether phosphoglycerides influence calcium fluxes into human endothelial cells, J. Immunol., 135, 2748, 1985. 24. Bussolino, F., Camussi, G., Aglietta, M., Braquet, P., Bosia, A., Pescarmona, G., Sanavio, F., d'Urso, N., and Marchisio, P. C., Human endothelial cells are a target for platelet-activating factor, J. Immunol., 139, 2439, 1987. 25. Bourgain, R. H., Maes, L., Braquet, P., Andries, R., Touqui, L., and Braquet, M., The effect of 1O-alkyl-2-acetyl—sn-glycero-3-phosphocholine (PAF-acether) on the arterial wall, Prostaglandins, 30,185, 1985. 26. Camussi, G., Aglietta, M., Malavasi, F., Tetta, C., Piacibello, F., Sanavio, and Bussolino, F., The release of platelet-activating factor frum human endothelial cells in culture, /. Immunol., 131, 2397, 1983. 27. Bussolino, F., Camussi, G., and Baglioni, C., Synthesis and release of platelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin la, J. Biol. Chem., 263, 11,856, 1988. 28. Vargaftig, B. B., Lefort, J., Chignard, M., and Benveniste, J., Platelet-activating factor induces a platelet-dependent bronchoconstriction unrelated to the formation of prostaglandin derivatives, Eur. J. Pharmacol., 65, 185, 1981.

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29. Kenzora, J. L., Perez, J. E., Bergman, S. R., and Lange, L. G., Effects of acetyl glyceryl ether phosphorylchline (platelet-activating factor) on ventricular preload, afterload and contractility in dogs, /. Clin. Invest., 74, 1193, 1984. 30. Bessin, P., Bonnet, J., Appfel, D., Soulard, C., Desgroux, L., Pelas, I., and Benveniste, J., Acute circulatory collapse caused by platelet-activating factor (PAF-acether) in dogs, Eur. J. Pharmacol., 86, 403, 1983. 31. Handley, D. A., Van Valen, R. G., Melden, M. K., and Saunders, R. N., Evaluation of dose and route effects of platelet activating factor-induced extravasation in the guinea-pig, Thromb. Haemost., 52, 34, 1984. 32. Sybertz, E. J., Watkins, R. W., Baum, T., Pula, K., and Rivelli, M., Cardiac, coronary and peripheral vascular effects of acetyl glyceryl ether phosphoryl-choline in anesthetized dogs, J. Pharmacol. Exp. Ther., 232, 156, 1985. 33. Wedmore, C. V. and Williams, T. J., Platelet-activating factor (PAF), a secretory product of polymorphonuclear leucocytes, increases vascular permeability in rabbit skin, Br. J. Pharmacol., 916, 1981. 34. Van Lambalgen, A. A., Bronsweld, W., Van Den Bos, G. C., and Thus, L. G., Distribution of cardiac output, oxygen consumption and lactate production in canine endotoxic shock, Cardiovasc. Res., 18, 195, 1984. 35. Sanchez-Crespo, M., Alonso, F., Inarrea, P., Alvarez, V., and Egido, J., Vascular actions of synthetic PAF-acether (a synthetic platelet activating factor in the rat, evidence for a platelet independent mechanism, Immunopharmacol., 4, 173, 1982. 36. Hwang, S. B., Lam, M. H., Lee, C. L., and Shen, T. Y., Release of platelet activating factor and its involvement in the first phase of carrageenin-induced rat foot edema, Eur. J. Pharmacol., 120, 33, 1986. 37. Cox, C. P., Mojarad, M., and Attiah, A., Platelet-activating factor (PAF) increases pulmonary vascular permeability in awake sheep, Am. Rev. Resp. Dis., 129, 334, 1984. 38. Morley, J., Page, C. P., and Paul, W., Inflammatory actions of platelet-activating factor (PAF-acether) in guinea-pig skin, Br. J. Pharmacol., 80, 503, 1983. 39. Braquet, P., Vidal, R. F., Braquet, M., Hamard, H., and Vargaftig, B. B., Involvement of leukotrienes and PAF-acether in the increase microvascular permeability of the rabbit retina, Agents Actions, 15, 82, 1984. 40. Braquet, P., Paubert-Braquet, M., Bessin, P., and Vargaftig, B. B., Platelet-activating factor: a potential mediator of shock, in Advances in Prostaglandin, Thromboxane and Leukotriene Research, Vol. 17, Samuelsson, B., Paoletti, R., and Ramwell, P. W., Eds., Raven Press, New York, 1987, 822. 41. Stahl, G. L., Bitterman, H., Terashita, Z., and Lefer, A. M., Beneficial effects of platelet-activating factor receptor antagonists in traumatic shock, Circ. Shock, 24, 241, 1988. 42. Inarrea, P., Gomez-Cambronero, J., Pascual, J., Ponte, M. C., Hernando, L., and Sanchez-Crespo, M., Synthesis of PAF-acether and blood volume changes in gram-negative sepsis, Immunopharmacol., 9, 45, 1985. 43. Doebber, T. W., Wu, M. S., Robbins, J. C., Choy, B. M., Chang, M. N., and Shen, T. Y., Platelet activating factor (PAF) involvement in endotoxin-induced hypotension in rats. Studies with PAF receptor antagonist kadsurenone, Biochem. Biophys. Res. Commun., 127, 799, 1985. 44. Buxton, D., Hanaham, D. J., and Olson, M. S., Stimulation of glycogeno-lysis and platelet-activating factor production by heat aggregated immunoglobulin G in the perfused rat liver, J. Biol. Chem., 259, 13,758, 1984. 45. Buxton, D. B., Fisher, R. A., Hanahan, D. J., and Olsen, M. S., Platelet-activating factor-mediated vasoconstriction and glyco-genolysis in the perfused rat liver, J. Biol. Chem., 261, 644, 1986. 46. Fitzgerald, M. F., Mocada, S., and Parente, L., The anaphylactic release of platelet-activating factor from perfused guinea-pig lungs, Br. J. Pharmacol., 88, 149, 1986. 47. Hsueh, W., Gonzalez-Crussi, F., and Arroyave, J. L., Platelet-activating factor, an endogenous mediator from bowel necrosis in endotoxemia, FASEB J., 1, 403, 1987. 48. Lagente, V., Lidbury, P., Steel, G., Vargaftig, B. B., Wallace, J. L., and Whittle, B. J., Role of PAF as the mediator of endotoxin-induced gastrointestinal damage, Br. J. Pharmacol., 90, 114, 1987. 49. Bussolino, F., Porcellini, M. G., Varese, L., and Bosia, A., Intravascular release of platelet-activating factor in children with sepsis, Thromb. Res., 48, 619, 1987. 50. Lopez-Diez, F., Nieto, M. L., Fernandez-Gallardo, S., Gijon, M. A., and Sanchez-Crespo, M., Occupancy of platelet receptors for platelet-activating factor in patients with septicemia, J. Clin. Invest., 83, 1733, 1989. 51. Arditi, M., Manogue, K. R., Caplan, M., and Yogev, R., Cerebrospinal fluid cachectin/tumor necrosis factor-alpha and platelet-activating factor concentrations and severity of bacterial meningitis in children, J. Infect. Dis., 162, 139, 1990. 52. Etienne, A., Hecquet, F., Soulard, C., Spinnewyn, B., Clostre, F., and Braquet, P., In vivo inhibition of plasma protein leakage and Salmonella enteridis-induced mortality in the rat by a specific PAF acether antagonist, BN 52021, Agents Actions, 17, 368, 1985.

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53. Adnot, S., Lefort, J., Lagente, V., Braquet, P., and Vargaftig, B. B., Interference of BN 52021, a PAF-acether antagonist with endotoxin-induced hypotension in the guinea-pig, Pharmacol. Res. Commun., 18, 197, 1986. 54. Terashita, Z., Imiira, Y., Nishikawa, K., and Sumida, S., Is platelet activating factor (PAF) a mediator of endotoxin shock?, Eur. J. Pharmacol., 109, 257, 1985. 55. Doebber, T. W., Wu, M. S., and Robbins, J. C., Platelet activating factor (PAF) involvement in endotoxininduced hypotension in rats. Studies with PAF receptor antagonist kadsurenone, Biochem. Biophys. Res. Commun., 127, 799, 1985. 56. Inarrea, P., Alonso, F., and Sanchez-Crespo, M., An effector substance of the vasopermeability changes induced by infusion of immune aggregates in the mouse, Immunopharmacol., 6, 7, 1983. 57. Handley, D. A., Van Valen, R. G., and Saunders, R. N., Vascular responses of platelet-activating factor in the Cebus apella primate and inhibitory profile of antagonists SR 63-072 and 63-119, Immunopharmacology, 11, 175, 1986. 58. Casals-Stenzel, J., Muacevic, G., and Heuer, H., Pharmacological actions of WEB 2086, a new specific antagonist of platelet-activating factor, J. Pharmacol. Exp. Ther., 241, 974, 1987. 59. Rabinovici, R., Yue, T. L., Farhat, M., Smith, E. F., Esser, K. M., Slivjak, M., and Feuerstein, G., Platelet-activating factor (PAF) and tumor necrosis factor (TNF) interactions in endotoxemic shock. Studies with BN 50739, a novel antagonist, J. Pharmacol. Exp. Ther., 255, 256, 1990. 60. Fletcher, J. R., Di Simone, A. G., and Earnest, M. A., The effects of BN 52021, a PAF receptor antagonist, on survival and hemodynamic events in endotoxemia, in The Immune Consequences of Shock and Trauma, Faist, E., Ed., Springer-Verlag, Heidelburg, 1989, 322. 61. Yue, T. I., Farhat, M., Rabinovici, R., Perera, P. U., Vogel, S. N., Feuerstein, G., Protective effect of BN 50739, a new PAF antagonist, in endotoxin-treated rabbits, J. Exp. Ther., 254, 976, 1990. 62. Earnest, M. A., DiSimone, A. G., and Fletcher, J. R., The effects of BN 52021, a PAF receptor antagonist, in canine endotoxemia, in Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives, Vol. 2, Braquet, P., Ed., JR Prous, Barcelona, 1990, 463. 63. Fletcher, J. R., DiSimone, B. S., and Earnest, M. A., Platelet-activating factor receptor antagonist improves survival and attentuates eicosanoid release in severe endotoxemia, Ann. Surg., 211, 312, 1990. 64. Lang, C. H., Dobrescu, C., Hargrove, D. M., Bagby, G., and Spitzer, J. J., Attenuation of endotoxininduced increase in glucose metabolism by platelet-activating factor antagonist, Circ. Shock, 23, 179, 1988. 65. Burhop, K. E., Garcia, J. G. N., Selig, W. M., Lo, S. K., van der Zee, H., Kaplan, J. E., and Malik, A. B., Platelet-activating factor increases lung vascular permeability to protein, J. Appl. Physiol., 61, 2210, 1986. 66. Burhop, K. E., van der Zee, H., Bizios, R., Kaplan, J. E., and Malik, A. B., Pulmonary response to platelet-activating factor in awake sheep and the role of cyclooxygenase metabolites, Am. Rev. Resp. Dis., 134, 548, 1986. 67. Toyofuku, T., Kubo, K., Kobayashi, T., and Kusama, S., Effects of ONO-6240, a platelet-activating factor antagonist, on endotoxin shock in unanesthetized sheep, Prostaglandins, 31, 271, 1986. 68. Sessler, C. N., Glauser, F. L., and Signal, B., SRI 63-441, a platelet-activating factor antagonist, prevents endotoxin induced pulmonary and hypoxemia in anesthetized sheep, Am. Rev. Resp. Dis., 135, 187, 1987. 69. Purvis, A. W., Christman, C., and McPherson, C. D., WEB 2086, a platelet activating factor receptor antagonist, attenuates the response to endotoxin in awake sheep, Am. Rev. Resp. Dis., 137, 99, 1988. 70. Redl, H., Gasser, H., Bahrain!, S., Schlag, G., Paul, E., Schiesser, A., Vogl, C., Hopf, R., Khaahpour, S., and Thurner, M., The role of PAF in an ovine model of endotoxin shock, in Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives, Vol. 2, Barquet, P., Ed., JR Prous, Barcelona, 1990, 471. 71. Baum, T. D., Heard, S. O., Feldman, H. S., Latka, C. A., and Fink, M. P., Endotoxin-induced myocardial depression in rats: effect of ibuprofen and SDZ 64-688, a platelet activating factor receptor antagonist, J. Surg. Res., 48, 629, 1990. 72. Barnes, P. J., Chung, K. F., Evans, T. W., and Rogers, D. F., Increased airway vascular permeability induced by platelet-activating factor, effect of specific antagonism and platelet depletion, Br. J. Pharmacol., 89, 764, 1986. 73. Beijer, L., Botting, J., and Crook, P., The involvement of platelet activating factor in endotoxin-induced pulmonary platelet recruitment in the guinea-pig, Br. J. Pharmacol., 92, 803, 1988. 74. Evans, T. W., Rogers, D. F., Dent, G., Aursudki, B., Chung, K. F., and Barnes, P., Effects of plateletactivating factor (PAF) antagonist WEB 2086 on airway microvascular leakage induced by PAF and antigen, Am. Rev. Resp. Dis., 135, 160, 1987. 75. Chang, S. W., Feddersen, C. O., Henson, P. M., and Voelkel, N. F., Platelet-activating factor mediates hemodynamic changes and lung injury in endotoxin-treated rats, J. Clin. Invest., 79, 1498, 1987. 76. Christman, B. W., Lefferts, P., and Snapper, J., Effect of a platelet-activating factor receptor antagonist (SRI 63-441) on the sheep's response to endotoxin, Am. Rev. Resp. Dis., 135, 82, 1987.

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77. Touvay, C., Vilain, B., Carre, C., Mencia-Huerta, J. M., and Braquet, P., Role of platelet-activating factor (PAF) in the bronchopulmonary alterations induced by endotoxin. Effects on beta-adrenoceptors, in Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives, Vol. 1, Braquet, P., Ed., JR Prous, Barcelona, 1989, 477. 78. Nijkamp, F. P., Braquet, P., Kessels, G. C. R., and Heuven-Nolsen, D,, Role of platelet-activating factor in the endotoxin induced tracheal hyperreactivity to histamine in the guinea-pig, Agents Actions, 26, 117, 1989. 79. Siebeck, M., Weipert, J., Jochum, M., and Hoffman, H., A new triazolodiazepine platelet-activating factor receptor antagonist reduces pulmonary dysfunction in endotoxin shock of the pig, Am. Rev. Resp. Dis., 139, 20, 1989. 80. Ha, B., Tolins, J. P., Vercellotti, G., Jacob, H. S., and Raij, L., The role of platelet-activating factor in endotoxemic acute renal failure in the rat, Kidney Int., 33, 358, 1988. 81. Tolins, J. P., Vercellotti, G. M., Wilkowske, M., Ha, B., Jacob, H. S., and Raij, L., The role of platelet-activating factor in endotoxemic acute renal failure in the male rat, J. Lab. Clin. Med., 113, 316, 1989. 82. Wang, J. and Dunn, M. J., Platelet-activating factor mediates endotoxin induced acute renal insufficiency in rats, Am. J. Physiol., 253, 1283, 1987. 83. Gonzalez-Crussi, F. and Hsueh, W., Experimental model of ischemic bowel necrosis. The role of plateletactivating factor and endotoxin, Am, J. PathoL, 112, 127, 1983. 84. Rosam, A. C., Wallace, J. L., and Whittle, B. J., Potent ulcerogenic actions of platelet activating factor on the stomach, Nature, 319, 54, 1986. 85. Hsueh, W., Gonzalez-Crussi, F., and Arroyave, J. L., Platelet-activating factor: an endogenous mediator for bowel necrosis, FASEB J., 1, 403, 1987. 86. Wallace, J. L. and Whittle, B. J. R., Prevention of endotoxin-induced gastro-intestinal damages by CV 3988, an antagonist of platelet-activating factor, Ear. J. Pharmacol., 124, 209, 1986. 87. Wallace, J. L., Steel, G., Whittle, B. J. R., Lagente, V., and Vargaftig, B. B., Evidence for plateletactivating factor as a mediator of endotoxin-induced gastrointestinal damage in the rat. Effects of three platelet-activating factor antagonists, Gastroenterology, 93, 765, 1987. 81. Tolins, J. P., Vercellotti, G. M., Wilkowske, M., Ha, B., Jacob, H. S., and Ray, L., The role of platelet-activating factor in endotoxemic acute renal failure in the male rat, J. Lab. Clin. Med., 113, 316, 1989. 82. Wang, J. and Dunn, M. J., Platelet-activating factor mediates endotoxin induced acute renal insufficiency in rats, Am. J. Physiol., 253, 1283, 1987. 83. Gonzalez-Crussi, F. and Hsueh, W., Experimental model of ischemic bowel necrosis. The role of plateletactivating factor and endotoxin, Am. J. Patho., 112, 127, 1983. 84. Rosam, A. C., Wallace, J. L., and Whittle, B. J., Potent ulcerogenic actions of platelet activating factor on the stomach, Nature, 319, 54, 1986. 85. Hsueh, W., Gonzalez-Crussi, F., and Arroyave, J. L., Platelet-activating factor: an endogenous mediator for bowel necrosis, FASEB J., 1, 403, 1987. 86. Wallace, J. L. and Whittle, B. J. R., Prevention of endotoxin-induced gastro-intestinal damages by CV 3988, an antagonist of platelet-activating factor, Eur. J. Pharmacol., 124, 209, 1986. 87. Wallace, J. L., Steel, G., Whittle, B. J. R., Lagent, V., and Vargaftig, B. B., Evidence for plateletactivating factor as a mediator of endotoxin-induced gastrointestinal damage in the rat. Effects of three platelet-activating factor antagonists, Gastroenterology, 93, 765, 1987. 88. Sun, X. and Hsueh, W., Bowel necrosis induced by tumor necrosis factor in rats is mediated by plateletactivating factor, J. Clin. Invest., 81, 1328, 1988. 89. Sun, X. M., Hsueh, W., and Torre-Amione, G., Effects of in vivo 'priming' on endotoxin-induced hypotension and tissue injury. The role of PAF and tumor necrosis factor, Am. J. PathoL, 136, 949, 1990. 90. Wallace, J. L., Ibbotson, G. C., and Keenan, C. M., Mediatory role of platelet-activating factor in intestinal inflammation and ulceration, in Ginkgolides: Chemistry, Biology, Pharmacology and Clinical Perspectives, Vol. 2, Braquet, P., Ed., JR Prous, Barcelona, 1990, 481. 91. Wallace, J. L., Release of platelet-activating factor (PAF) and accelerated healing induced by a PAF antagonist in an animal model of chronic colitis, Can. J. Physiol. Pharmacol., 66, 422, 1988. 92. Wallace, J. L., Braquet, P., Ibbotson, G. C., Me Naughton, W. K., and Cirino, G., Assessment of the role of platelet-activating factor in an animal model of inflammatory bowel disease, J. Lipid Mediators, 1, 13, 1989. 93. Ekstsrom-Jodal, B., Haggendal, J., Larsson, L. E., and Westerlind, A., Cerebral blood flow and oxygen uptake in endotoxin shock. An experimental study in dogs, Acta Anaesthesiol. Scan., 26, 163, 1982. 94. Ekstsrom-Jodal, B., Haggendal, J., Larsson, L. E., and Westerlind, A., Cerebral hemodynamics, oxygen and cerebral arteriovenous differences of catecholamines following E. coli endotoxin in dogs, Acta Anasthesiol. Scan., 26, 446, 1982.

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95. Caridis, D. T., Reinhold, R. B., Woodruff, P. W., and Fine, J., Endotoxemia in man, Lancet, 1, 1381, 1972. 96. Sardesai, V. M. and Rosenburg, J. C., Proteolysis and bradykinin turnover in endotoxin shock, J. Trauma, 14, 945, 1974. 97. Etienne, A., Hecquet, F., Guilmard, C., Soulard, C., and Braquet, P., A potential link between plateletactivating factor and plasma protease activity inendotoxemia, Int. J. Tiss. Reac., 9, 19, 1987. 98. Beutler, B., Milsark, I. W., and Cerami, A. C., Passive immunisation against cachectin/tumor necrosis factor protects mice from lethal effects of endotoxin, Science, 229, 869, 1985. 99. Hosford, D. and Braquet, P., The potential role of platelet-activating factor in shock and ischemia, J. Crit. Care, 5, 1, 1990. 100. Braquet, P., Paubert-Braquet, M., Koltai, M., Bourgain, R., Bussolino, F., and Hosford, D., Is there a case for PAF antagonists in the treatment of ischemic state?, Trends Pharmacol. Sci., 10, 23, 1989.

Part VI. \7nrin

Chapter 22

POSSIBLE INVOLVEMENT OF OXYGEN FREE RADICALS IN SHOCK AND SHOCK-RELATED STATES Bengt Gerdin and Ulf Haglund

TABLE OF CONTENTS I.

Introduction

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II.

Radicals, Defense Systems, and Damage Due to Radicals A. What is a Radical? 1. Superoxide Anion 2. Hydrogen Peroxide 3. The Hydroxyl Radical 4. Other Radicals B. How Radicals Damage Tissue C. Endogenous Protection Against Radicals

458 458 458 459 461 461 461 462

III.

Pathophysiology A. How to Demonstrate OFR-Mediated Injury B. Reperfusion Damage C. Experimental Shock Studies D. Multiple Organ Failure Systems E. Consideration of Species and Control Conditions

463 463 464 466 467 467

IV.

Summary: Role of OFR in Shock

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References

0-8493-3548-5/93/$0.00 + $.50 © 1993 by CRC Press, Inc.

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I. INTRODUCTION During the past decade, there has been an increased awareness that formation of shortlived highly reactive metabolites, radicals, constitute an important principle of tissue injury which operates in a variety of pathophysiological conditions. There are several sources of information which suggest that an increasing amount of radicals are formed in connection with tissue ischemia and shock, and that these radicals cause damage to cells. In the present review, the authors will first attempt to explain what radicals are, how they are formed, and how they damage tissue. Second, they will discuss the data which suggest that radicals are responsible for some of the pathophysiology of tissue ischemia and shock.

I. RADICALS, DEFENSE SYSTEMS, AND DAMAGE DUE TO RADICALS A. WHAT IS A RADICAL? A radical is defined as a molecule with an unpaired electron in an outer orbit. Such molecules are highly reactive and interact with other molecules with extreme ease. Radicals are usually intermediates in chemical reactions and are normally formed in living cells as part of many chemical reactions. In other words, radicals are normally formed as part of biologic life. Most radicals formed in vivo are derived from oxygen and represent intermediate steps in the reduction of molecular oxygen to water. The three reduction steps and formation of the superoxide anion O2 , hydrogen peroxide (H2O2) and hydroxyl radical (OH') are schematically shown in Figure 1. The oxygen free radicals (OFR) are formed as a result of many different chemical reactions. 1. Superoxide Anion Formation of O2~ will be exemplified in four ways: 1.

2.

3.

Phagocytic cells, e.g., polymorphonuclear (PMN) leukocytes (PMNL) and macrophages, produce Of as part of a tissue destructing biocidal apparatus (for survey see Weiss and de Buglio1)- They all have a membrane bound specific oxidase, NADPHoxidase, which reduces oxygen directly to O2~ under the simultaneous oxidation of glucose-6-phosphate (Figure 2). The NADPH-oxidase is activated when the PMNL are stimulated, e.g., during phagocytosis, and the O2~ that is formed is believed to be crucial for the killing of ingested microbes. Stimulation of phagocytes without simultaneous phagocytosis also results in formation of O 2 ~, and in this case O2~ will appear in the extracellular environment where it will damage neighboring cells and degrade the interstitium. The energy-rich nucleotides are degraded to hypoxanthine and are thereafter further metabolized to uric acid by an enzyme, xanthine dehydrogenase, which uses NAD+ as its electron acceptor. During hypoxia the enzymatic characteristics of xanthine dehydrogenase are altered to that of an oxidase (xanthine oxidase; XO), which oxidizes hypoxanthine to uric acid while the reduction of molecular oxygen is simultaneously reduced to Of (Figure 3). For a survey of the xanthine oxidase-xanthine dehydrogenase conversion see McKelvey et al.2. Superoxide may also be formed by oxidases other than XO.3 6 When oxygen is released from oxyhemoglobin in erythrocytes, a fraction is liberated in the form of Of during the simultaneous formation of methemoglobin (Figure 4). Erythrocytes are thereby continuously exposed to a certain amount of Of ,7-8

Gerduv and Haglund

FIGURE 1.

FIGURE 2.

4.

459

Schematic presentation of the four nonenzymatic reduction steps of oxygen to water.

Schematic presentation of the superoxide producing apparatus of phagocytic cells.

O2~ is also formed in the mitochondria, where a fraction of the available oxygen undergoes univalent reduction.9

2. Hydrogen Peroxide Hydrogen peroxide is by definition not a radical, but takes an intimate part in radical generation and in radical-mediated damage and is therefore discussed here.

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Handbook of Mediators in Septic Shock

FIGURE 3.

Reactions catalyzed by xanthine dehydrogenase and xanthine oxidase, respectively.

FIGURE 4.

Formation of superoxide by hemoglobin and oxygen.

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FIGURE 6.

1. 2. 3.

The Fenton reaction.

Some enzymes simultaneously produce both H2O2 and O^ in proportions which depend on pH, pO2, and substrate concentration. Such is the case for xanthine oxidase.10 O2 dismutates to H2O2 spontaneously or enzymatically. This is the normal way by which O2~ is eliminated (see discussion below). The most important production of H2O2 in vivo occurs in the peroxisomes. There, H2O2 is formed by oxidase-mediated reactions, presumably without previous formation of O2-.n

3. The Hydroxyl Radical 1.

2.

The OH' is so reactive that it reacts within one to five molecular diameters from its formation and has a very short half-life, estimated to be less than 1 nsec in vivo.12 In a water solution, it can be formed by a spontaneous, fairly slow reaction which involves H2O2 and O^. This so-called Haber-Weiss reaction (Figure 5) is presumably of little relevance in vivo, but is the model reaction from which the OH' is formed. In the presence of H2O2 in the solution, OH' is rapidly formed in the presence of transition metals by the so-called Fenton reaction (Figure 6). It has been suggested that the Fenton reaction is the way by which OH' is formed in vivo. The reaction is presumably catalyzed by the trace amount of iron that is always present in the tissues. Iron chelated to ADP (adenosine diphosphate), for instance, is particularly efficient in catalyzing the Fenton reaction.13

4. Other Radicals Superoxide anion functions as a weak base and is in equilibrium with its corresponding acid, HO2 (the perhydroxyl radical). This protonized form is lipid soluble and traverses the cell membrane with ease. Since the pKa value is 4.8,14 only a small fraction of O^ occurs in the form of HO2 in a normally buffered in vivo environment. It has, however, been argued that the local pH in certain microenvironments close to cell membranes can be as low as 4.5 to 5, which would make the endogenous concentration of HO2 higher than originally believed (for a discussion see Freeman and Crapo15). The occurrence of iron-oxygen adducts of various forms, so-called ferryl or perferryl radicals, has been suggested,16 since under experimental conditions iron promotes a radicalmediated damage which is not counteracted by classical scavengers of the hydroxyl radical." B. HOW RADICALS DAMAGE TISSUE Radicals are extremely reactive and interact with other molecules during the formation of new radicals. This leads to a chain reaction (schematically presented in Figure 7) where a continuous formation of new radicals occurs, ultimately resulting in a propagation of

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FIGURE 7.

Principles for radical formation; i.e., for initiation, propagation, and termination.

molecular damage. The chain reaction is stopped when two radicals react with each other in a so-called "termination reaction" (Figure 7). The most studied type of radical-mediated damage to living tissues is the peroxidation of unsaturated fatty acids of the cell membranes (for a survey see Pryor18). This results in an altered structure of the phospholipid bilayer and a decreased "fluidity" of the cell membrane, which in turn has profound effects on cellular functions like cell deformability, locomotion, receptor clustering, etc. Evidence for the occurrence of lipid peroxidation is actually one of the indicators of free radical formation. It is not fully conclusive evidence of radical formation, however, as lipids are also enzymatically peroxidized. In vivo, lipid peroxidation is indicated when elevated levels of malondialdehyde (MDA)19>2° are observed in tissue homogenate and in tissue fluids, or by the appearance of conjugated dienes.21-22 As radicals are normal intermediates in chemical reactions, an accumulation in the vicinity of active sites in enzymes is not uncommon. This imposes a threat to the enzyme molecules themselves, which are destroyed. An example is the autodestruction of cyclooxygenase that occurs during the oxidation of arachidonic acid to prostaglandins under simultaneous formation of O2~.23-24 This type of process is one factor which necessitates a continuous new synthesis of enzymes.25 C. ENDOGENOUS PROTECTION AGAINST RADICALS In environments where there is a continuous high production of radicals, endogenous protecting mechanisms are a prerequisite for continued cellular life. This fact has been repeatedly emphasized and surveyed in other publications26'27 and requires only a brief discussion here. In the intracellular space there is a high concentration of the enzyme, superoxide dismutase (SOD), which reacts with O2~~ during the formation of H2O2 and O2. SOD maintains the intracellular superoxide concentration at a very low level. There is a particularly high concentration of SOD in erythrocytes (cf. discussion above) and in hepatocytes. There is a special type of SOD in mitochondria, where large amounts of O^ are formed in the electron transport chain. In the peroxisomes, where much of the H2O2 production occurs, there is also a high concentration of the enzyme catalase, which converts H2O2 to water (Figure 8).11 H2O2 is also inactivated by glutathione peroxidase (Figure 8). The hydroxyl radical reacts so quickly that no enzymatic elimination is possible. The main biological basis for protection against its action is therefore to avoid its formation or

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FIGURE 8. The function of catalase (top) and of glutathione peroxidase (bottom). The decomposition of hydrogen peroxide occurs in two steps with the formation of an intermediate (Compound I). Glutathione peroxidase reduces both lipid peroxides (LOOM) and hydrogen peroxide under the simultaneous oxidation of reduced glutathione.

to scavenge it once it is produced. A number of low molecular weight compounds, however, act as OH' scavengers. Among these there are amino acids such as cysteine and L-methionine, monosaccharides and uric acid, all in concentrations obtained in vivo. There is a very low SOD-like activity in the extracellular space. The extracellular SOD (EC-SOD) is also different from that found in cytosol28 and has an affinity to endothelium.29 This observation indicates that the endothelial surface is exposed to a certain threat from radicals formed in the circulating blood close to its surface. This could tentatively be O2", or rather HO2, from the erythrocytes, or O2~ produced by PMNLs.

III. PATHOPHYSIOLOGY A. HOW TO DEMONSTRATE OFR-MEDIATED INJURY It has previously been stated in this survey that generation of OFR does occur in normal healthy cells. If effects caused by OFR play a pathophysiological role, the mechanism could be (1) an overproduction of OFR; (2) a decreased local or general endogenous defense to the effects of OFR; or (3) production of OFR in environments where they are not normally formed and where the defense systems do not operate effectively enough. Due to the fact that OFR are very short lived, their existence in vivo is extremely difficult to demonstrate. This also implies that their role in pathophysiological conditions is extremely difficult to prove. The available evidence can be distinguished as based on one or both of the following principles: 1. 2.

The pathophysiological event can be prevented by scavengers or inhibitors of OFR formation. The pathophysiological event is paralleled by evidence of formation of OFR in vitro.

Addressing the first principle, the modulation of pathophysiological events by OFRscavengers or inhibitors of OFR formation is the most commonly used approach to evaluate whether radical-mediated injury is part of the process studied. For instance, the role of free

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radicals in ischemia-reperfusion injury was initially demonstrated by Granger et al.30 who studied intestinal ischemia and showed that animals given SOD intravenously exhibited less intestinal damage than controls. Other scavengers that have been used in this context include catalase to eliminate H2O2, and mannitol and L-methionine to scavenge OH'. Similarly, compounds that eliminate substrates which are required for radical reactions (e.g., desferrioxiamine which chelates the free iron which is required for rapid formation of OH') are used as inhibitors of radical formation. The most commonly used inhibitor of production is probably allopurinol, which inhibits XO, the enzyme considered to be of primary importance for radical formation in ischemia-reperfusion injury (Figure 3).31-32 Addressing the second principle, the formation of free radicals can be more directly indicated by chemiluminescence. Radical formation is followed by emission of ultraweak light chemiluminescence. This light is more than 10,000 times weaker than what can be noticed by the dark adapted human eye. However, using special technology such light can be detected.33'34 This technique is commercially available for in vitro use and has also recently been adopted for in vivo use in the intestine34'35 and in the heart.36 Attenuation of the recorded light by means of SOD or allupurinol, however, is required to positively demonstrate that the recorded light is secondary to radical formation. Another direct way to measure radical formation is by electron spin trapping. The technique is based on the fact that certain substances, "spin traps", are included in the environment where radical reactions occur. The spin traps are attacked by the primary radicals and "radicalized" as part of a propagation reaction (Figure 7). The radical form of the spin traps are detected in a magnetic field. This technique is difficult to use in vivo due to the fact that most spin-trapping substances are extremely noxious. Recently, however, spin trapping has been successfully performed in living gastrointestinal cells in vitro using a tolerable spin trap, OXANOH.37 According to these authors, there was production of superoxide radicals in hypoxic cells following reinstitution of normal extracellular oxygen concentrations. The production occurred extracellularly, as it was abrogated in the presence of SOD added to the solution. After administration of the same spin-trap agent in vivo, it has recently been shown that reperfusion of the previously ischemic small intestine results in the formation of a superoxide dependent signal.38 This information, plus the above-mentioned studies in which chemiluminescence was used34-39 and in which the abolition of injury was proven by means of SOD,30-40 all constitute evidence for the generation of OFR following ischemia and reperfusion in the intestinal wall. B. REPERFUSION DAMAGE It is generally accepted that ischemia causes tissue damage. In addition, it has recently been demonstrated that reperfusion of a previously ischemic organ might in itself be harmful. This was first visualized in the small intestine by Granger et al.30 They showed that there was a significant exacerbation of tissue injury at reperfusion following a certain period of time of ischemia, which in itself had caused only a moderate increase in intestinal permeability. This has also been recently reconfirmed for morphologic injury. Ischemia giving rise to morphologic destruction of the tips of the villi was associated with a dramatic exacerbation of the mucosal damage shortly after perfusion.40'41 This has been termed "reperfusion damage". As will be discussed later, this type of damage has been used synonymously with OFR-mediated tissue damage. It is important to keep in mind, however, that damage occurring in the reperfusion phase does not by definition have to be caused by OFR formation. It has thus been demonstrated that a profound swelling of the microvascular endothelial cells occurs during ischemia.42'43 The endothelial damage caused during the

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ischemic phase might well lead to a longstanding microcirculatory impairment and secondary tissue damage after reperfusion. In general terms, however, "reperfusion damage" can be counteracted by agents such as SOD that scavenge free radicals, and by agents that counteract the formation of OFR, such as allopurinol or the iron chelator desferrioxamine.30'40'41'44 Since SOD is a large protein that is unlikely to get access to the intracellular space rapidly, a therapeutic effect due to its O2~ eliminating ability indicates that O2 generation occurs extracellularly. In support of this, it has recently been demonstrated that endothelial cells do contain XO and may generate OFR on sites at the cellular membrane.45 EC-SOD or intravascularly injected SOD may therefore prevent endothelial cell oxygen radical formation. This may explain why SOD can prevent reperfusion injury even when given just shortly before reperfusion.46 Beneficial effects of compounds preventing radical formation and of scavenging formed radicals have been observed in a number of different model systems and in different tissues including intestine, skin, skeletal muscle, brain, heart, lung, kidney, and liver,2'47 thus suggesting that radical-mediated reperfusion damage is a generalized phenomenon. However, in many reports the effects observed in experimental shock models are minor or moderate (see below). Data have recently been advanced, however, which makes it likely that much of this can be explained if one assumes that tissue damage in ischemia and at reperfusion constitutes a continuum of injury.31 The total injury is thus caused by a number of different pathophysiologic components which, in concert, contribute to the final damage. Oxygen radical-mediated tissue injury presumably predominates among these components only during a certain defined time span following a certain degree of initial (ischemic) tissue injury.31 After a shorter period of ischemia and after more severe ischemic injury, other types of damage and mechanisms are likely to predominate. Authors who have observed a positive effect of scavenging substances have obviously utilized experimental models where injury from oxygen-derived free radicals (the reperfusion component of the total injury) predominates, while those who have obtained negative results apparently have not found that time span. This condition has been termed "the therapeutic window" and is visualized in Figure 9.48 Below are some experimental examples which support the existence of such a therapeutic window in the role of OFR in ischemic damage. Hoshino et al.48 demonstrated that postischemic renal function, measured as creatinine clearance following 1 or 2 h of warm ischemia, was preserved, to a large extent, in animals treated with SOD or allopurinol. The function preservation effect of radical ablation was no longer present following 3 h of warm ischemia. At that time, both treated and untreated kidneys had significantly impaired creatinine clearances. Similarly, following 24 or 48 h of cold ischemia, allopurinol treatment was followed by preserved renal function as compared to untreated controls. However, following 18 h of cold ischemia, both treated and untreated kidneys had normal creatinine clearances, and following 72 h of cold ischemia, both groups showed considerable functional impairment.48 A similar dependence of injury on the effect of SOD and allopurinol has also been demonstrated in cold injury of rabbit ears.49 Following total ischemia and reperfusion in the small intestine, a weak but significant reperfusion injury could be demonstrated in rats where concomitant intestinal venous congestion was prevented, but only if the ischemic period was 50 min. Shorter periods (20 min) or longer periods (90 min) were not followed by a detectable reperfusion component of the injury.50 Reperfusion damage was found following cold ischemia and transplantation of the murine small intestine if the ischemia time was 5 h, but not following 0 or 18 h of ischemia.51 The concept of a "window" of radical mediated reperfusion injury may thus explain much of the confusion regarding the functional importance of OFR formation in ischemia and reperfusion.

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FIGURE 9. Schematic presentation of the relation between ischemic injury, reperfusion injury, and total tissue injury. (Modified from Hoshino, T., Maley, W. R., Bulkely, G. B., and Williams, G. M., Transplantation, 45, 284, 1988. With permission.)

One very important feature of the vascular bed is its ability to actively distribute blood flow in an optimal fashion. It has recently been realized that much of this process is caused by a local shear force-dependent phenomenon, the release of endothelium-derived relaxing factor (EDRF) (for discussion see Vanhoutte52). EDRF is inactivated by O2~53-54 which leads to a considerable loss of autoregulation capacity of various vascular beds. In various experimental ischemia models, SOD has been shown to improve blood flow, which can be related to better protection of the labile and O2~ sensitive EDRF. Furthermore, the role of EDRF as an important distributor of blood flow during hemorrhagic shock has been pointed out in particular.55 C. EXPERIMENTAL SHOCK STUDIES Most experiments with conventional animal shock models have been done with the strategy of investigating whether substances scavenging OFR (such as SOD) or preventing their formation (such as allopurinol) affect the outcome. The beneficial effect of allopurinol in the classical Wiggers hemorrhagic shock model in dogs was reported long ago.56"59 However, it was suggested that the mechanism was a better preservation of the adenylate pool rather than a diminished production of OFR. In recent studies, pretreatment with allopurinol caused a temporary protective effect in the recirculation phase after hemorrhage in dogs;60 this effect was potentiated by the addition of catalase. An interesting aspect observed by Deitch et al.61 is that allopurinol treatment resulted in a decreased bacterial translocation over the rat intestinal mucosa during hemorrhagic shock. Recently, SOD and catalase given prior to bleeding resulted in reduced cardiac functional disturbances and a blunted increase in blood lactate concentration in hemorrhagic shock in dogs.62 SOD also counteracted superior mesenteric artery occlusion (SMAO)-induced shock in rats.63 In baboons, either SOD or allopurinol diminished the gastric mucosal lesions after hemorrhagic shock,54 and in rabbits allopurinol or SOD/catalase was effective.65 Sanan et al. showed that iron contributed to postshock damage, as desferrioxamine decreased liver damage66 and increased survival67 after hemorrhagic shock in dogs.

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The spin trap PEN, which functions as a scavenger, both prevented and was therapeutic for drum traumatic shock in rats.68 Furthermore, it has been shown that lipid peroxides detected by the appearance of fluorescent products in plasma were extinguished by SOD treatment after infusion of live Escherichia coli in dogs.69 As neutrophils are heavy producers of OFR (cf. above), it is tempting to speculate that the recently observed devastating role of intravascular neutrophil accumulation in the irreversibility of hemorrhagic shock in rats70-71 is related to their ability to produce endothelium damaging OFRs.72 D. MULTIPLE ORGAN FAILURE SYSTEMS The late damage to parenchymatous organs after shock is difficult to evaluate in integrated experimental models. Pathophysiology of the lung damage of ARDS has thus conventionally been studied separately from the renal insufficiency and liver failure. Information on the role of OFR in MOF (multiple organ failure) must therefore be evaluated for each organ separately. There are several reports claiming that OFRs contribute to the damage obtained in experimental animal models, mimicking ARDS. The lung damage caused by experimental pancreatitis in rats was thus diminished by neutrophil depletion, and also by SOD and by catalase.73 Catalase and SOD were also protective against the cardiopulmonary alterations following acute hemorrhagic pancreatitis in the dog.74 SOD, catalase, and desferrioxamine were protective against phorbol-induced lung damage.75 SOD and catalase were also effective in preventing a-naphtylthiourea-induced lung damage.76 Furthermore catalase, linked to polyethylene glycol to obtain a longer circulating half-life (but not similarly treated SOD), had a short-term protective effect against lung damage following endotoxemia in pigs.77 SOD, however, protected against thrombin-induced pulmonary damage in sheep.78 The liver damage is difficult to mimic. Here, pretreatment with allopurinol maintained the hepatic function after hemorrhagic shock.58 SOD diminished the peroxidative damage to the liver after endotoxic shock in rats,79 and SOD or allopurinol diminished the hepatic necrosis after endotoxin in rats.80 Similarly there are studies claiming that postischemic renal damage is counteracted by SOD alone81"83 or in combination with other drugs,84 but there are also studies in which no effect was found.85 E. CONSIDERATION OF SPECIES AND CONTROL CONDITIONS There are large species differences in the amount and characteristics of the enzymes which generate radicals and in those which operate in radical elimination. This fact makes extrapolation of experimental results to the human very questionable. To be extreme, it can be argued that much of the experimental work performed so far gives us very little information about the relative importance of OFR in human disease. Roy and McCord86 originally claimed that xanthine dehydrogenase in the rat was converted to O^, producing XO within 1 min of ischemia. This convinced several investigators that only very brief ischemia was sufficient to achieve an environment where radical formation was greatly facilitated. However, later studies have shown that this conversion is in fact considerably slower in the rat intestine,87 in the rat liver,2 and especially in rat skin.88 In isolated rat hepatocytes, cell death actually preceded the conversion,89 which casts doubt on the relevance of the previously described in vivo findings. The question as to whether or not there is a radical producing XO in human organs has been discussed in regard to the heart,90"92 as have consequences for the interpretation of experimental data.93 The recently isolated EC-SOD, which selectively and specifically dismutates O2~ in the extracellular environment, exhibits an affinity to endothelium in most species, although not

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in rats, and the interspecies variation was generally very large.29 The suggested role of this affinity has been to specifically protect endothelium from locally generated radicals from, for instance, erythrocytes or PMNLs. Obviously, there is a considerable difference here between man and rats, one of the animals most commonly used in shock studies. It is also very important to emphasize the role of the animal diet in the endogenous ability to respond to radical generation. Differences between different laboratories, which use models that are similar in other respects, might be explained by differences in the content of selenium, which is part of glutathione peroxidase, or in the content of food preservatives with vitamin E-like inhibitory actions on lipid peroxidation. An important issue in most of the shock and ischemia studies is that improper control conditions have been used. Animals treated with enzymes, like SOD or catalase, have been compared with animals given saline and a beneficial effect has always been attributed to the ability of the enzymes to scavenge radicals. This, however, requires that other possible effects of the enzymes are excluded. Therefore, an inactivated enzyme with unaltered tertiary structure has to be given to the control animals. Heat inactivation is not appropriate here. SOD, for example, can easily be inactivated by exposure to H2O2 in an alkaline environment.94 Commercial preparation of bovine SOD and catalase are often contaminated by endotoxin95 and are often chemically impure. This allows for both false negative as well as false positive results in experimental studies.

IV. SUMMARY: ROLE OF OFR IN SHOCK The available literature is replete with information indicating that OFRs have the ability to influence a number of pathophysiologic steps in the development of shock. First, a reperfusion type of organ damage secondary to peripheral ischemia involves components which are caused by OFR. This has previously been discussed. Second, some parts of the functional collapse of parenchymatous organs, multiple organ failure (MOF) systems, might be due to radical-mediated damage. It has been shown that experimental pulmonary damage mimicking ARDS, which is part of MSOF, can be prevented in various experimental models by treatment with SOD, catalase or with desferrioxamine. Third, ORF inactivates endothelialderived relaxation factor (EDRF), which aggravates vasospasm. What is currently lacking is reproducible evidence that the formation of OFR occurs in integrated shock models, and evidence that shock (or the consequences of shock) is counteracted by treatment with OFR scavengers. This, plus the subsequent delineation of characteristics of radical release and a possible therapeutic window, in stable models, should constitute the main thrust of research today. Are further clinical studies indicated? Critical examination of the experimental data raises many questions which make it difficult to accept the conclusions of investigators and the enthusiastic extrapolations to the clinical arena. Issues such as endotoxin contamination of infused free radical scavenger enzymes, incorrect dosages, the possibility of improper selection of control conditions, species specificity, inadequate consideration of the differences in time spectrums between the experimental situation and human shock, and the possibly transient nature of any benefits of anti-free radical interventions cast doubt on some of the accumulated data and their general applicability to humans. These issues must be satisfactorily addressed before this experimental approach can be fully embraced by the clinician. Causal factors in pathophysiological reactions are often evaluated in terms of the classical criteria of Koch-Dale,96 i.e., the presence of the factor in disease (criterion 1), the absence in health (criterion 2), the ability to elicit disease by exogenous administration (criterion 3), and the ability to block the effect by antagonists and thereby preventing disease (criterion

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FIGURE 10. Decision trees for the investigation of a real existing association between: (A) formation of oxygen free radicals (OFR) and shock/shock-related states; (B) artificial generation of OFR and shock/shock-related states; (C) counter action of shock/shock-related states with antagonists of OFR or OFR release.

4). For one or more of these criteria a decision tree can be constructed96-97 which allows for an evaluation of the results of the available literature. In Figure 10, such decision trees have been constructed, each aiming at evaluating one or more of the Koch-Dale criteria. Information relevant to all questions originated in the decision trees (Figure 10) are presented and commented on in this survery. However, a number of unclear points still exist as the biological effects of OFR, the mode of action of scavenger substances used, as well as bias introduced by the design of many studies. Before experimentors commence further studies on this topic, they should carefully analyze previous studies and put them into the frame of a decision tree given here.

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REFERENCES 1. Weiss, S. J. and deBuglio, A. F., Phagocyte-generated oxygen metabolites and cellular injury, Lab. Invest., 47, 5, 1982. 2. McKelvey, T. G., Hollwarth, M. E., Granger, D. N., Engerson, T. D., Landler, U., and Jones, H. P., Mechanisms of conversion of xanthine dehydrogenase to xanthine oxidase in ischemic rat liver and kidney, Am. J. Physiol., 254, G753, 1988. 3. Aleman, V. and Handler, P., Dihydrorotate dehydrogenase, J. Biol. Chem., 242, 4087, 1967. 4. Massey, V., Strickland, S., Mayhew, S. G., Howett, L. G., Engel, P. C., Matthews, R. G., Schuman, M., and Sullivan, P. A., Direct demonstration of superoxide anion production during the oxidation of reduced flavin and of its catalytic decomposition by erythrocuprein, Biochem. Biophys. Res. Commun., 36, 898, 1969. 5. Hirata, F. and Hayaishi, O., Possible participation of superoxide anion in intestinal tryptophan 2,3dioxygenase reaction, /. Biol. Chem., 246, 7825, 1971. 6. Rajagopolan, K. V., Xanthine oxidase and aldehyde oxidase, in Enzymatic Basis of Detoxication, Vol. 1, Jakoby, W., Ed., Academic Press, New York, 1980, 295. 7. Thomson, A. J., Ligand-binding properties of the haem group, Nature, 265, 15, 1977. 8. Winterbourn, C. C., Free-radical production and oxidative reactions of hemoglobin, Environ. Health Perspect., 64, 321, 1985. 9. Nohl, H. and Jordan, W., The mitochondria! site of superoxide formation, Biochem. Biophys. Res. Commun., 138, 533, 1986. 10. Fridovich, I., Quantitative aspects of production of superoxide anion radical by milk xanthine oxidase, J. Biol. Chem., 245, 4035, 1970. 1 1 . Masters, C. and Holmes, R., Peroxisomes: new aspects of cell physiology and biochemistry, Physiol. Rev., 57, 816, 1977. 12. Pryor, W. A., Oxy-radicals and related species: their formation, life times and reactions, Ann. Rev. Physiol., 48, 657, 1986. 13. Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W., Iron-catalyzed hydroxyl radical formation. Stringent requirement for free iron coordination site, J. Biol. Chem., 259, 3620, 1984. 14. Behar, D., Czapski, G., Rabani, J., Dorfman, L. M., and Schwartz, H. A., The acid dissociation constant and decay kinetics of the perhydroxyl radical, J. Phys. Chem., 74, 3209, 1970. 15. Freeman, B. A. and Crapo, J. D., Free radicals and tissue injury, Lab. Invest., 47, 412, 1982. 16. Ernster, L., Biochemistry of reoxygenation injury, Crit. Care Med., 16, 947, 1988. 17. Youngman, R., Oxygen activation: is the hydroxyl radical always biologically relevant, TIBS, 9, 280, 1984. 18. Pryor, W. A., The formation of free radicals and the consequences of their reactions in vivo, Photochem. Photobiol., 28, 781, 1978. 19. Bird, R. P. and Draper, H. H., Comparative studies on different methods of malonaldehyde determination, Methods Enzymol., 105, 299, 1984. 20. Esterbauer, H., Lang, J., Zadravec, S., and Slater, T. F., Detection of malonaldehyde by high-performance liquid chromatography, Methods Enzymol., 105, 319, 1984. 21. Waller, R. L. and Recknagel, R. O., Determination of lipid conjugated dienes with tetracyanoethylene14C: significance for study of the pathology of lipid peroxidation, Lipids, 12, 914, 1977. 22. Recknagel, R. O. and Glende, E. A., Jr., Spectrophotometric detection of lipid conjugated dienes, Methods Enzymol., 105, 331, 1984. 23. Egan, R. W., Paxton, J., and Kuehl, F. A. Jr., Mechanism for irreversible self-deactivation of prostaglandin synthetase, J. Biol. Chem., 251, 7329, 1976. 24. Gale, P. H. and Eagen, R. W., Prostaglandin endoperoxide synthase-catalyzed oxidation reactions, in Free Radicals Biology, Pryor, W. A. Ed., Academic Press, Orlando, FL, 1984, 1. 25. Wolff, S. P. and Garner, A., Dean RT: Free radicals, lipids and protein degradation, TIBS, 11, 27, 1986. 26. Fridovich, I., Oxygen radicals, hydrogen peroxide and oxygen toxicity in free radicals in biology, in Pryor, W., Ed., Academic Press, New York, 1976, 239. 27. Halliwell, B., Biochemical mechanisms accounting for toxic action of oxygen on living organisms: the key role of superoxide dismutase, Cell. Biol. Intern. Rep., 2, 113, 1978. 28. Marklund, S. L., Human copper-containing superoxide dismutase of high molecular weight, Proc. Natl. Acad. Sci. U.S.A., 79, 7634, 1982. 29. Karlsson, K. and Marklund, S. L., Extracellualr superoxide dismutase in the vascular system of mammals, Biochem. J., 255, 223, 1988. 30. Granger, D. N., Rutili, G., and McCord, J. M., Superoxide radicals in feline intestinal ischemia, Gastroenterology, 22, 1981.

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31. Haglund, U., Bulkley, G. B., and Granger, D. N., On the pathophysiology of intestinal ischemic injury. Clinical review, Acta Chir. Scand., 153, 321, 1987. 32. Granger, D. N., Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury, Am. J. Physiol., 255, H1269, 1988. 33. Boveris, A., Cadenas, E., Reiter, R., Filipkowski, M., Nakase, Y., and Chance, B., Organ chemiluminescence: noninvasive assay for oxidative radical reactions, Proc. Natl. Acad. Sci. U.S.A., 77, 347, 1980. 34. Roldan, E. J. A., Pinus, C. R., Turrens, J. F., and Boveris, A., Chemiluminescence of ischaemic and reperfused intestine in vivo, Gut, 30, 184, 1989. 35. Morris, J. B., Haglund, U. H., Bulkley, G. B., Hall, T. S., Paky, A., Gurtner, G. H., Cadenas, E., and Sies, H., Direct demonstration of oxygen free radical generation from a living, intact organ (the feline intestine), Surg. Forum, 37, 123, 1986. 36. Barsacchi, R., Coassin, M., Maiorino, M., Pelosi, G., Simonelli, C., and Ursini, F., Increased ultra weak Chemiluminescence emission from rat heart at postischemic reoxygenation: protective role of vitamin E, Free Radical Biol. Med., 6, 573, 1989. 37. Nilsson, U. A., Olsson, L. I., Thor, H., Moldeus, P., and Bylund-Fellenius, A. C., Detection of oxygen radicals during reperfusion of intestinal cells in vitro, Free Radical Biol. Med., 6, 251, 1989. 38. Nilsson, U. A., Lundgren, O., Haglind, E., and Bylund-Fellenius, A. C., Radical production during in vivo intestinal ischemia and reperfusion in the cat, Am. J. Physiol., 257, G409, 1989. 39. Morris, J. B., Bulkley, G. B., Haglund, U., Cadenas, E., and Sies, H., The direct, real-time demonstration of oxygen free radical generation at reperfusion following ischemia in the living intact, rat small intestine, Gastroenterology, 92, 1541, 1987. 40. Schoenberg, M. H., Muhl, E., Sellin, D., Younes, M., Schildberg, F. W., and Haglund, U., Postthypotensive generation of superoxide free radicals — possible role in the pathogenesis of the intestinal mucosal damage, Acta Chir. Scand., 150, 301, 1984. 41. Parks, D. A., Bulkley, G. B., Granger, D. N., Hamilton, S. R., and McCord, J. M., Ischemic injury in the cat small intestine: role of superoxide radicals, Gastroenterology, 82, 8, 1982. 42. Gidlof, A., Lewis, D. H., and Hammer sen, F., The effect of prolonged total ischemia on the ultrastructure of human skeletal muscle capillaries. A morphometric analysis. Int. J. Microcirc. Clin. Exp., 1, 67, 1988. 43. Mazzoni, M. C., Borgstrom, P., Intaglietta, M., and Arfors, K. E., Lumenal narrowing and endothelial cell swelling in skeletal muscle capillaries during hemorrhagic shock, Circ. Shock, 29, 27, 1989. 44. Schoenberg, M. H., Fredholm, B. B., Haglund, U., Jung, H., Sellin, D., Younes, M., and Schildberg, F. W., Studies on the oxygen radical mechanism involved in the small intestinal reperfusion damage, Acta Physiol. Scand., 124, 581, 1985. 45. Ratych, R. E., Chuknyiska, R. S., and Bulkley, G. B., The primary localization of free radical generation after anoxia/reoxygenation in isolated endothelial cells, Surgery, 102, 122, 1987. 46. Haglund, U., Morris, J. B., and Bulkley, G. B., Haemodynamic characterization of the isolated (denervated) parabiotically perfused rat jejunum, Acta Physiol. Scand., 132, 151, 1988. 47. Bulkley, G. B., The role of oxygen free radicals in human disease processes, Surgery, 94, 407, 1983. 48. Hoshino, T., Maley, W. R., Bulkley, G. B., and Williams, G. M., Ablation of free radical-mediated reperfusion injury for the salvage of kidneys taken from non-heartbeating donors, Transplantation, 45, 284, 1988. 49. Marzella, L., Jesudass, R. R., Manson, P. N., Myers, R. A. M., and Bulkley, G. B., Morphologic characterization of acute injury to vascular endothelium of skin after frostbite, Plast. Reconstr. Surg., 83, 67, 1989. 50. Park, P. O., Haglund, U., Bulkley, G, B., and Fait, K., The sequence of development of intestinal tissue injury following strangulation ischemia and reperfusion, Surgery, 107, 575, 1990. 51. Park, P. O., Wallander, J,, Tufveson, G., and Haglund, U., Cold ischemic and reperfusion injury in a model of small bowel in the rat Eur. Surg. Res., 23, 1, 1991. 52. Vanhoutte, P. M., Endothelium and control of vascular function. State of the art lecture, Hypotension, 13, 658, 1989. 53. Rubanyi, G. M. and Vanhoutte, P. M., Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor, Am. J. Physiol., 250, H822, 1986. 54. Gryglewski, R. J., Palmer, R. M., and Moncada, S., Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor, Nature, 320, 454, 1986. 55. Miller, V. M. and Vanhoutte, P. M., Endothelium-dependent responses and their potential relevance to the distribution of blood flow during hemorrhagic shock, Resuscitation, 18, 165, 1989. 56. Crowell, J. W., Jones, C. E., and Smith, E. E., Effect of allopurinol on hemorrhagic shock, Am. J. Physiol., 216, 744, 1969. 57. Baker, C. H., Protection against irreversible hemorrhagic shock by allopurinol, Proc. Soc. Exp. Biol. Med., 141, 694, 1972.

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58. Lazarus, H. M., Owens, M. L., and Hopfenbeck, A., Allopurinol protection of hepatic nuclear function during hemorrhagic shock, Surg. Forum., 25, 10, 1974. 59. Cunningham, S. K. and Keaveny, T. V., Effect of a xanthine oxidase inhibitor on adenine nucleotide degradation in hemorrhagic shock, Eur. Surg. Res., 10, 305, 1978. 60. Bond, R. F., Haines, G. A., and Johnson, G., The effect of allopurinol and catalase on cardiovascular hemodynamics during hemorrhagic shock, Circ. Shock, 25, 139, 1988. 61. Deitch, E. A., Bridge, W., Baker, J., Ma, L., Granger, D. N., Grisham, M. B., Specian, R. D., and Berg, R., Hemorrhagic shock, induced bacterial translocation is reduced by xanthine oxidase inhibition or inactivation, Surgery, 104, 191, 1988. 62. Prasad, K., Kalra, J., and Buchko, G., Acute hemorrhage and oxygen free radicals, Angiology, 39, 1005, 1988. 63. Bitterman, H., Aoki, N., and Lefer, A. M., Anti-shock effects of human superoxide dismutase in splanchnic artery occlusion (SAO) shock, Proc. Soc. Exp. Biol. Med., 188, 265, 1988. 64. von Ritter, C., Hinder, R. A., Oosthuizen, M. M., Svensson, L. G., Hunter, S. J., and Lambrecht, H., Gastric mucosal lesions induced by hemorrhagic shock in baboons. Role of oxygen-derived free radicals, Dig. Dis. Sci., 33, 857, 1988. 65. Tsimoyiannis, E. C., Sarros, C. J., Tsimoyiannis, J. C., Moutesidou, K., Akalestos, G., and Kotoulas, O. B., Ranitidine and oxygen derived free radical scavengers in haemorrhagic shock induced gastric lesions, Gut, 29, 826, 1988. 66. Sanan, S., Sharma, G., Malhotra, R., Sanan, D. P., Jain, P., and Vadhera, P., Protection by desferrioxamine against histopathological changes of the liver in the post-oligaemic phase of clinical haemorrhagic shock in dogs: correlation with improved survival rate and recovery, Free Radical Res, Commun., 6, 29, 1989. 67. Sanan, S., Sharma, G., Singh, B., Sanan, D. P., and Vadhera, P., Evaluation of desferrioxamine mesylate on survival, and prevention of histopathological changes of the liver in haemorrhagic shock: an experimental study in dogs, Resuscitation, 17, 63, 1989. 68. Novell!, G. P., Angiolini, P., Tani, R., Consales, G., and Bordi, L., Phenyl-T-butyl-nitrone is active against traumatic shock in rats, Free Radical Res. Commun., 1, 321, 1988. 69. Morgan, R. A., Manning, P. B., Coran, A. G., Drongowski, R. A., Till, G. O., Ward, P. D., and Oldham, K. T., Oxygen free radical activity during live E. coli septic shock in the dog, Circ. Shock, 25, 319, 1988. 70. Barroso-Aranda, J., Schmid-Schonbein, G. W., Zweifach, B. W., and Engler, R. L., Granulocytes and no-reflow phenomenon in irreversible hemorrhagic shock, Circ. Res., 63, 437, 1988. 71. Barroso-Aranda, J. and Schmid-Schonbein, G. W., Transformation of neutrophils as indicator of irreversibility in hemorrhagic shock, Am. J. Physiol, 257, H846, 1989. 72. Hernandez, L. A., Grisham, M. B., Twohig, B., Arfors, K. E., Harlan, J. M., and Granger, N., Role of neutrophils in ischemia-reperfusion-induced microvascular injury, Am. J. Physiol., 253, H699, 1987. 73. Guice, K. S., Oldham, K. T., Caty, M. G., Johnson, K. J., and Ward, P. A., Neutrophil-dependent, oxygen-radical mediated lung injury associated with acute pancreatitis, Ann. Surg., 210, 740, 1989. 74. Chardavoyne, R., Asher, A., Bank, S., Stein, T. A., and Wise, L., Role of reactive oxygen metabolites in early cardiopulmonary changes of acute hemorrhagic pancreatitis, Dig. Dis. Sci., 34, 1581, 1989. 75. Allison, R. C., Hernandez, E. M., Prasad, V. R., Grisham, M. B., and Taylor, A. E., Protective effects of O2 radical scavengers and adenosine in PMA-induced lung injury, J. Appl. Physiol., 64, 2175, 1988. 76. Martin, D., Korthuis, R. J., Perry, M., Townsley, M. L, and Taylor, A. E., Oxygen radical-mediated lung damage associated with alpha-naphthylthiourea, Acta Physiol. Scand., Suppl. 548, 119, 1986. 77. Olson, N. C., Grizzle, M. K., and Anderson, D. L., Effect of polyethylene glycol-superoxide dismutase and catalase on endotoxemia in pigs, J. Appl. Physiol., 63, 1526, 1987. 78. Johnson, A., Perlman, M. B., Blumenstock, F. A., and Malik, A. B., Superoxide dismutase prevents the thrombin-induced increase in lung vascular permeability: role of superoxide in mediating the alterations in lung fluid balance, Circ. Res., 59, 405, 1986. 79. Kunimoto, F., Morita, T., Ogawa, R., and Fujita, T., Inhibition of lipid peroxidation improves survival rate of endotoxemic rats, Circ. Shock, 21, 15, 1987. 80. Arthur, M. J., Bentley, I. S., Tanner, A. R., Saunders, P. K., Millward-Sadler, G. H., and Wright, R., Oxygen-derived free radicals promote hepatic injury in the rat, Gastroenterology, 89, 1114, 1985. 81. Hansson, R., Jonsson, O., Lundstam, S., Pettersson, S., Schersten, T., and Waldenstrom, J., Effects of free radical scavengers on renal circulation after ischaemia in the rabbit, Clin. Sci., 65, 605, 1983. 82. Ouriel, K., Smedira, N. G., and Ricotta, J. J., Protection of the kidney after temporary ischemia: free radical scavengers, J. Vase. Surg., 2, 49, 1985.

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83. Schneider, J., Friderichs, E., and Giertz, H., Equieffective protection by human and bovine SOD against renal reperfusion damage in rats, Basic Life Set., 49, 891, 1988. 84. Bratell, S., Folmerz, P., Hansson, R., Jonsson, O., Lundstam, S., Pettersson, S., Rippe, B., and Schcrslen. T., Effects of oxygen free radical scavengers, xanthine oxidase inhibition and calcium entryblockers on leakage of albumin after ischaemia. An experimental study in rabbit kidneys, Acta Physiol. Scand., 134, 35, 1988. 85. Zager, R. A., Hypoperfusion-induced acute renal failure in the rat: an evaluation of oxidant tissue injury, Circ. Res., 62, 430, 1988. 86. Roy, R. S. and McCord, J. M., Superoxide and ischemia: conversion of xanthine dehydrogenase to xanthine oxidase, in Oxygen Radicals and Their Scavenger Systems: Cellular and Molecular Aspects, Greenwald, R. A. and Cohen, C., Eds., Elsevier, New York, 1983, 145. 87. Parks, D. A., Williams, T. K., and Beckman, J. S., Conversion of xanthine dehydrogenase to oxidase in ischemic rat intestine. A reevaluation, Am. J. Physiol., 254, O768, 1988. 88. Im, M. J., Hoopes, J. E., Yoshimura, Y., Manson, P. N., and Bulkley, G. B., Xanthine: acceptor oxidoreductase activities in ischemic rat skin flaps, J. Surg. Res., 46, 230, 1989. 89. de Groot, H. and Littauer, A., Reoxygenation injury in isolated hepatocytes: cell death precedes conversion of xanthine dehydrogenase to xanthine oxidase, Biochem. Biophys. Res. Commun., 155, 278, 1988. 90. Grum, C. M., Gallagher, K. P., Kirsh, M. M., and Shlafer, M., Absence of detectable xanthine oxidase in human myocardium, J. Mol. Cell. Cardiol, 21, 263, 1989. 91. Downey, J. M., Hearse, D. J., and Yellon, D. M., The role of xanthine oxidase during myocardial ischemia in several species including man, J. Mol. Cell. Cardiol., 20(Suppl. 2), 55, 1988. 92. Wajner, M. and Harkness, R. A., Distribution of xanthine dehydrogenase and oxidase activities in human and rabbit tissues, Biochim. Biophys. Acta, 991, 79, 1989. 93. Cohen, M. V., Free radicals in ischemic and reperfusion myocardial injury: is this the time for clinical trials?, Ann. Intern. Med., I l l , 918, 1989. 94. Blech, D. M. and Borders, C. L., Jr., Hydroperoxide anion, HO"2 is an affinity reagent for the inactivation of yeast Cu,Zn-superoxide dismutase: modification of one histidine per subunit, Arch. Biochem. Biophys., 224, 579, 1983. 95. Nejima, J., Knight, D. R., Fallon, J. T., Uemura, N., Manders, W. T., Canfield, D. N., Cohen, M. V., and Vatner, S. F., Superoxide dismutase reduces reperfusion arrythmias but fails to salvage regional function or myocardium at risk in conscious dogs, Circulation, 79, 143, 1989. 96. Neugebauer, E. and Lorenz, W., Causality in circulatory shock: strategies for integration mediators, mechanisms and therapies, in Perspectives in Shock Research, Adams, H. R. and Chaudry, I. H., Eds., Alan Liss, New York, 1988, 295. 97. Lusted, L. B., Introduction to Medical Decision Making, Charles C. Thomas, Springfield, IL, 1968.

Chapter 23 CALCIUM AS A MEDIATOR IN SEPTIC SHOCK Gary P. Zaloga and Diana Malcolm

TABLE OF CONTENTS I.

Introduction

476

II.

Methods

476

III.

Decision Trees

476

IV.

Data Presentation and Analysis

477

V.

Results Circulating Calcium Levels during Sepsis A. B. Intracellular Calcium Levels during Sepsis C. Calcium Administration during Sepsis D. Calcium Antagonists during Sepsis

477 477 478 480 481

VI.

Discussion

482

VII.

Summary

483

References

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I. INTRODUCTION Calcium is important for normal cellular function.1 Altered calcium levels and fluxes affect protein regulation resulting in abnormal muscle contraction, enzyme secretion, neurotransmitter and hormonal release, blood coagulation, cell division and repair, cardiac action-potential generation, and immune competence. These alterations may contribute to the pathogenesis of sepsis. Calcium has been implicated in the mechanisms of cellular damage during sepsis. Cell dysfunction, injury, and death are postulated to be mediated by a rise in free intracellular calcium content. These postulates are extensions of studies suggesting a role for calcium in ischemic injury.2 Rises in free intracellular calcium are thought to activate enzymatic processes which damage cells (i.e., proteases, nucleases, phospholipases). Increases in free intracellular calcium may also injure mitochondria (diminishing energy production) or cause vasoconstriction and tissue ischemia. Numerous mediators have been implicated to play a role in the physiologic alterations occurring during sepsis. Calcium is one of these mediators. Calcium administration does not itself elicit the sepsis syndrome. Alterations in calcium, however, may be involved in or contribute to the cellular dysfunction occurring during sepsis. For example, exogenous and endogenous factors (i.e., bacteria, endotoxins, tumor necrosis factor) may disrupt cellular activity by increasing cellular calcium permeability leading to an elevation in free intracellular calcium concentration. Understanding the role that calcium plays in the pathogenesis of sepsis is important because calcium administration may be harmful while calcium antagonists may be of therapeutic benefit in treating sepsis. To establish a role for calcium as a mediator of sepsis, calcium levels (extracellular and intracellular) should be altered during sepsis compared with health. In addition, calcium administration should be harmful while calcium antagonism should be beneficial during sepsis. Meeting these criteria would fulfill Koch-Dale criteria 1 through 4 for a mediator being a causal factor in a pathological process.3 Thus, in this paper, we address four questions: 1. 2. 3. 4.

Does sepsis alter circulating calcium levels? Does sepsis alter intracellular calcium levels? Is calcium administration harmful during sepsis? Are calcium antagonists beneficial during sepsis?

II. METHODS A computer search of the literature (MEDLINE® database over the past 15 years) was performed looking for articles listed under the headings "Calcium" and "Calcium antagonists/blockers" versus "Sepsis", "Infection", "Endotoxin", and "Shock". The bibliographies of appropriate articles were also reviewed for additional articles not picked by the computer search. All papers were then evaluated by a decision tree (see below) to determine if they qualified for further analysis.3-4 Sepsis included studies of endogenous infection, administration of exogenous infectious organisms/endotoxin, or cecal ligation.

III. DECISION TREES A. Does sepsis alter circulating calcium levels? 1. Publication of the effect of sepsis on blood calcium? 2. Assay for calcium appropriate (i.e., used ionized calcium)? 3. Appropriate collection of blood sample? 4. Appropriate study design (i.e., control values, not too lethal)? 5. Included in analysis

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B. Does sepsis alter intracellular calcium levels? 1. Publication of the effect of sepsis on intracellular calcium levels? 2. Assay for intracellular calcium appropriate? 3. Appropriate collection and processing of sample? 4. Appropriate study design (i.e., control group, not too lethal of a model)? 5. Included in analysis C. Is calcium administration harmful during sepsis? 1. Publication of calcium administration during sepsis? 2. Study design appropriate (i.e., control group)? 3. Study used whole animal (not isolated organ)? 4. Calcium dose appropriate (blood levels measured)? 5. Outcome measured (organ function or survival)? 6. Included in analysis D. Are calcium antagonists beneficial during sepsis? 1. Publication of a calcium antagonist (i.e., calcium channel blocker, calcium chelator) during sepsis? 2. Study design appropriate (i.e., control group, model not too lethal)? 3. Dose of calcium antagonist adequate to obtain an effect? 4. Outcome measured (i.e., organ function, survival)? 5. Included in analysis

IV. DATA PRESENTATION AND ANALYSIS Circulating ionized calcium levels during sepsis were analyzed by determining the incidence of ionized hypocalcemia during sepsis compared to control values. Hypocalcemia occurs in approximately 2.5% of a normal healthy population, based upon normal values encompassing 95% of the population. Results from measurement of free intracellular calcium levels during sepsis are presented in tabular form. These studies were too variable (i.e., different cells, different models) and too few for performance of meta-analysis. Calcium uptake in organelles from septic cells are also presented in tabular form. These studies were also too variable (i.e., different models, different tissues, different organelles, incomplete data) for performance of metaanalysis. The overall effect of calcium antagonists on survival in prospective randomized casecontrolled animal models of sepsis were evaluated by the technique of meta-analysis. Metaanalysis was performed using the technique of Mantel-Haenszel-Petro. We derived an overall estimate of the relative benefit of calcium antagonists on survival during sepsis. The data are presented as odds ratios (calcium antagonistxontrol) and their associated confidence intervals for survival after sepsis. Significance of the overall combined odds ratio was determined using the Chi-square test.

V. RESULTS A. CIRCULATING CALCIUM LEVELS DURING SEPSIS A total of four animal (Table 1) and six human studies (Table 2) were found which fulfilled the decision criteria.3"14 Low dose endotoxin (5 mg/kg)6 and cecal ligation and puncture (CLP)7 were not associated with ionized hypocalcemia in rats. Doses of endotoxin between 5 and 30 mg/kg produced a dose-dependent decrease in ionized calcium in an additional rat study.5 One other animal study evaluated the effect of Escherichia coli infusion in baboons.8 Bacterial infusion decreased ionized calcium levels by 15%.

478

Handbook of Mediators in Septic Shock TABLE 1 Circulating Calcium during Sepsis — Animal Studies Species/model

Number

Effect on Ca+ +

Ref.

Rat/endotoxin 5-30 mg/kg Rat/endotoxin 5 mg/kg Rat/CLP Baboon/E. coli

24

5

11

Decreased 7-21% (dose dependent) No effect

9 5

No effect Decreased (15%)

7 8

6

TABLE 2 Circulating Calcium during Sepsis — Human Studies Number of patients 62 18 60 18 28 8

Septic Septic Septic Septic Toxic shock Toxic shock

Hypocalcemic (%)

Ref.

76 61 20 100 64 50

9 10 11 12 13 14

TABLE 3 Free Intracellular Calcium Levels during Sepsis Technique

Model

Quin-2

Rat hepatocytes

Quin-2

Rat hepatocytes (saponin permeabilized) Rat hepatocytes Human lymphs Human T cells retrovirus Human B-cells Epstein-Barr virus Rat aorta Fibroblast

Quin-2 Fura-2 Fura-2 Quin-2 Fura-2 Fura-2

Effect on [Ca]

Ref.

1. E bolus decreased [Ca] from 246 to 131 nM 2. E infusion and CLP had no effect on [Ca] 1. E decreased IP3-induced [Ca] increase 2. CLP potentiated IP3-induced [Ca] increase E increased [Ca] from 146 to 525 nM [Ca] elevated in cells from septic patients [Ca] increased in infected cells [Ca] increased in infected cells

15

E decreased K + -induced rise in [Ca] E (in vitro) had no effect on [Ca]

21 22

16 17 18 19 20

Note: E = Endotoxin; IP3 = inositol triphosphate; CLP = cecal ligation and puncture.

Six human studies (four sepsis and two toxic-shock syndrome) evaluated changes in ionized calcium levels during infection (Table 2). All six studies demonstrated the presence of ionized hypocalcemia during infection. The incidence of hypocalcemia ranged from 20 to 100%. One would expect only 2.5% of a healthy population to have an ionized calcium level below the normal range (based upon a normal range of ±2.5 standard deviations). B. INTRACELLULAR CALCIUM LEVELS DURING SEPSIS Free intracellular calcium levels [Ca] were measured using fluorescent dyes (fura-2, quin-2) in eight studies (Table 3). Three studies were performed in rat hepatocytes,1517 three

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TABLE 4 Calcium Uptake during Sepsis — Cardiac Organelle

Increased

In vitro endotoxin (SR) In vitro endotoxin (SL) In vivo endotoxin (SR)

No effect (ref.)

32

In vivo endotoxin (SL) Fecal pellet (SR) S. viridens (SR and mitochondria)

30,31,32 33

Decreased (ref.) 23, 25 25 24, 26, 27, 28 29 34

Note: SR = Sarcoplasmic reticulum, SL = sarcolemma.

TABLE 5 Calcium Uptake during Sepsis — Vascular Organelle In vitro endotoxin (SR) In vitro endotoxin (mitochondria) In vivo endotoxin (SR) In vivo endotoxin (mitochondria) CLP (aorta)

Increased (ref.)

No effect (ref.)

Decreased (ref.) 35 35 36 36 37

Note: SR = Sarcoplasmic reticulum, CLP = cecal ligation and puncture.

in human lymphocytes,18"20 one in rat aorta,21 and one in fibroblasts.22 In vivo endotoxin boluses decreased hepatocyte [Ca] from 246 to 131 nM in one study15 but increased it from 146 to 525 nM in another.17 Resting [Ca] differed substantially between these two studies. In vivo low-dose endotoxin infusion and CLP had no effect on hepatocyte [Ca].15 Endotoxin and CLP decreased the [Ca] response to epinephrine and vasopressin.15-17 In addition, in vivo endotoxin decreased the [Ca] response to inositol triphosphate (IP3) in saponin permeabilized hepatocytes while CLP increased the [Ca] response to IP3.16 All three studies using human lymphocytes demonstrated increases in [Ca] following infection (Table 3). Human lymphocytes from septic patients had higher resting [Ca] levels compared to healthy subjects and other critically ill patients.18 Human lymphocytes infected with retroviruses19 or Epstein-Barr virus20 also demonstrated increases in cytoplasmic [Ca] levels. In addition, endotoxin diminished the rise in [Ca] due to potassium depolarization in rat aorta21 but had no effect on [Ca] in fibroblasts.22 We also evaluated the effect of sepsis on calcium flux (i.e., calcium uptake) (Tables 4 through 8). There were 12 studies identified in which calcium flux was assessed in organelles from cardiac cells (Table 4).23'34 Eight studies reported a decrease in calcium uptake during endotoxin (in vitro and in vivo) exposure and Streptococcus viridens infection in organelles from cardiac tissue.23'29-34 Calcium uptake was decreased in Sarcoplasmic reticulum, sarcolemma, and mitochondria. Four studies found no effect from in vivo endotoxin exposure30 32 or fecal pellet implantation33 on calcium uptake. Calcium uptake in vascular tissue Sarcoplasmic reticulum and mitochondria were reported in three studies (Table 5).35"37 All found a decrease in calcium uptake following endotoxin exposure or CLP.

480

Handbook of Mediators in Septic Shock TABLE 6 Calcium Flux during Sepsis — Liver Increased (ref.)

Organelle In vitro endotoxin (mitochondria) In vivo endotoxin (mitochondria) In vivo endotoxin (ER) CLP (mitochondria) CLP (ER)

No effect (ref.)

Decreased (ref.)

38 (Low endotoxin)

38 (High endotoxin)

39, 40

41

40 40 40

42 41 41

41

Note: ER = Endoplasmic reticulum, CLP = cecal ligation and puncture.

TABLE 7 Calcium Uptake during Sepsis — Skeletal Muscle Model Cecal ligation and puncture (rat) E. coli i.p. (rat)

Increased (ref.)

No effect (ref.)

Decreased (ref.)

43 44

Calcium uptake in liver endoplasmic reticulum and mitochondria were measured in five studies after endotoxin exposure38 42 or CLP40'41 (Table 6). Three studies reported an increase in calcium uptake,38"40 two studies found no effect,41'42 and two studies reported a decrease in calcium uptake.38'41 Skeletal muscle calcium uptake was measured in two studies (Table 7) and found to be increased.43'44 The effect of sepsis on calcium uptake was also reported in four additional tissues (Table 8). In vitro endotoxin exposure increased calcium uptake in fibroblasts at low cell density but decreased calcium uptake at high cell density.45 In vitro endotoxin exposure decreased calcium uptake in neuroblastoma cells46 but had no effect on rat adipocytes.47 On the other hand, in vivo endotoxin exposure increased calcium uptake in rat adipocytes.47 Calcium uptake was increased in chick embryo mitochondria early following infection with Semliki virus but was decreased late after infection.48 C. CALCIUM ADMINISTRATION DURING SEPSIS Calcium administration during sepsis was evaluated in seven studies (Table 9). Hemodynamic responses to calcium were measured in three studies.49~S1 The hemodynamic response to calcium was reduced during sepsis in two studies.50-51 The blood pressure response to the calcium channel agonist, BAYK8644, was increased following endotoxin in one study.49 The greater response to BAYK8644 may have been due to a decreased ability of endotoxin exposed cells to maintain normal [Ca] levels. On the other hand, we have recently found no difference in the blood pressure response to BAYK8644 using intact healthy and CLP animals. Calcium was found to blunt the blood pressure response to epinephrine in a fourth study.6 This blunting effect was greater in endotoxin-exposed animals compared with nonseptic animals. Liver lipid peroxidation was also lower in endotoxin treated mice receiving a low calcium diet compared to a diet higher in calcium,52 suggesting that calcium intake

481

Zaloga and Malcolm TABLE 8 Calcium Uptake during Sepsis — Miscellaneous Model

Increased (ref.)

Chick embryo semliki virus (mitochondria) Rat adipocytes in vivo endotoxin; in vitro endotoxin Neuroblastoma cells, in vitro endotoxin Fibroblasts in vitro endotoxin

No effect (ref.)

48 (Early infection) 47 (In vivo endotoxin)

Decreased (ref.) 48 (Late infection)

47 (in vitro endotoxin) 46

45 (Low cell density)

45 (High cell density)

TABLE 9 Calcium Administration during Sepsis (Whole Animal) Model

Variable

Rat, endotoxin (i.v.)

Blood pressure

Cat, endotoxin

Rat, endotoxin Mice, endotoxin Rat, endotoxin

LV dP/dT, blood pressure, cardiac output Blood pressure, cardiac output Blood pressure Liver lipid peroxidation Survival

Rat, cecal ligation

Survival

Dog, E. coli (i.v.)

Result

Ref.

Response to BAYK8644 increased during sepsis Response to Ca reduced during sepsis (5 & 10 mg/kg/min) Response to Ca reduced during sepsis

49

Response to epinephrine inhibited by Ca Peroxidation reduced by low Ca/vit D diet Ca reduced survival from 80 to 20%; EGTA improved survival Ca reduced survival from 100 to 44%

6 52 53

50 51

7

modulated lipid peroxidation in response to endotoxin. Two animal studies evaluated the effect of calcium administration on survival following endotoxin exposure53 and CLP.7 Both studies demonstrated a decrease in survival following calcium. No human studies fulfilling the entrance criteria (i.e., controlled study of calcium administration during sepsis) have been reported. D. CALCIUM ANTAGONISTS DURING SEPSIS Pretreatment with diltiazem, a calcium channel blocker, attenuates the rise in [Ca] due to endotoxin in skeletal muscle54 and liver.17 Diltiazem also attenuates E. coli induced increases in skeletal muscle calcium uptake.44 Diltiazem reverses the effect of endotoxin on the rise in [Ca] stimulated by IP3 in hepatocytes16 and verapamil attenuates endotoxin induced lipid peroxidation in mouse liver.52 On the other hand, verapamil and nifedipine are ineffective in blocking increases in [Ca] due to interleukin-1. Verapamil reverses depressed cardiovascular function in dogs55 and rats56 following endotoxin. In addition, nilvadipine and nitrendipine inhibit endotoxin-induced disseminated intravascular coagulation in rats.57 We are aware of six studies evaluating the effects of calcium channel antagonists on survival in sepsis (Table 10). One study involved administration of live E. coli5* and five employed the administration of endotoxin.53"56'58-59 The studies used dogs, rats, and mice. No human studies of calcium antagonists during sepsis have been reported. Calcium channel antagonists improved survival in all six animal studies (Table 10). Overall, survival increased from 52% (193 of 371) in control septic animals to 75% (321 of 427) in septic animals treated with calcium antagonists (p P(A,|B,,) •

The event B,. is a genuine cause of the event A,, if B,, is a prima facie cause of A,, but B,. is not a spurious cause of At.

In all definitions it is assumed that the conditional probabilities are well defined. This approach is a generalization of Hume's concept of probabilistic relationships, and therefore allows a frequency interpretation because part of the concept itself is to claim relative frequency of co-occurrence of cause and effect.'' Wulff et al.12 do not incorporate the time aspect in their chapter on causality in medicine. Time per se is not essential for their concept of causality, but it is important to study the temporal relationship between the occurrence of different events as a means to discover the underlying mechanism, i.e., to confirm a causal assessment. Necessary and sufficient causes are introduced with nonprobabilistic methods. An event X is a necessary cause of the event Y, if Y is always preceded by X, and we say that X is a sufficient cause of Y, if Y always succeeds X. In other words: "X is a necessary cause of Y" means "If X had not occurred, Y would not have occurred". "X is a sufficient cause of Y" means "If Y had not been going to occur, X would not have occurred".12 In multicausal processes a discrimination into redundant and nonredundant factors is mentioned. A factor is nonredundant, if it is an indispensable part of the effective causal complex, i.e., the sum of factors which generated the desired response, and it is redundant, if that is not the case. Mackie13 deals in a very similar way with a complex of factors and an effect which occurs whenever some conjunction of certain factors occur. If the conjunction of factors ABC is sufficient but not necessary for the effect P, and furthermore, ABC is a minimally sufficient condition, i.e., none of its conjuncts (e.g., AB) is redundant; then each single factor, such as A, is an insufficient but nonredundant part of an unnecessary but sufficient condition. This is abbreviated as an INUS condition (using the first letters of the italicized words).

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III. FROM EVIDENCE TO CAUSE A. ASSOCIATION VERSUS CAUSATION Spitzer and Horwitz14 reiterate that association does not mean causation. Confirming a convincing evidence for causation, ordinarily, is only possible through experimental approaches. In a probabilistic model, dependent events are called associated. This, immediately, implies that every causation is also an association, but the reverse statement does not hold true. An interesting example is the well-known strong association (with a correlation coefficient of nearly 1) between the fall of the birth rate and the decrease of stork populations in the last 20 years in the Federal Republic of Germany, but nobody would seriously claim a causal relationship. Armitage and Berry15 report the following example: "There is a strong association between the number of divorces made absolute in the United Kingdom in each of the last 100 years or so and the amount of tobacco imported. It does not follow either that tobacco is a serious cause of marital discontent, or that those whose marriages have broken down turn to tobacco for solace. Association does not imply causation." An important point which helps distinguish between association and causation is to realize if biases and confounding3 are present. With the design of an experiment it should be assured that all forms of biases, especially bias in selection or measurement, are excluded. If the aim is to establish a cause-effect relationship for a specific population, the participants of a trial have to be representative for the target population. When the purported cause and the effect are due to an event different from both, we are confronted with confounding. A further explanation for an association which is noncausal is random variation. The possibility of accepting an apparent association erroneously as causal, can be reduced if there is a clear research question, definitions of procedures and equipment used, standardization and quality control. "Blind" or "double-blind" studies are very useful to guarantee standardization and to reduce observers' variation. Control of confounding can be achieved by matching individuals across different groups, restricting the study population by more selective inclusion criteria, and assessment of subjects to different groups by randomization. When different models are presented in the following sections, there will also be discussion of experimental methods for the confirmation of a causal association. The difference between association and causation of two events is also evident in the classification of the two relations according to the basic properties of "reflexivity, symmetry, and transitivity". Causation is always transitive, but not symmetric and not reflexive. Event A being a cause of B and B being a cause of C implies that A is a cause of C (transitivity). From the fact that A causes B, we cannot conclude that B also causes A (symmetry). According to reflexivity, it is impossible to consider an event A as the cause of itself. On the other hand, if A is associated with B, of course, B is also associated with A, and each event can be considered associated to itself. B. CAUSALITY IN MEDICINE Important goals of medicine are the cure of patients and the improvement of diagnosis, treatment, and prognosis. Therefore the medical community has an utmost interest in establishing physiological and pathological mechanisms, characterizing adverse reactions, identifying risk factors, etc. Etiology, as a whole, is devoted to the assessment of causal factors. A physician equipped with information on causal interrelationships is able to make predictions; Carr16 mentions that one of the most fundamental properties of thought is its power of predicting events. Prophylactic treatment is only feasible with the knowledge of existing causal interrelationships. For instance, physicians and patients will only agree to use a prophylactic drug to block the release of a mediator during a surgical procedure if the drug suppresses the effect of the mediator release and if the mediator itself causes unwanted

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reactions which lead to a bad outcome of surgery. Health service management needs such predictions to organize a health care system. Usually biological systems relevant for disease concepts are complex, e.g., there is a variety of causal and noncausal interrelationships and associations, and they include many sources of variation. Therefore, uncertainty is, in some situations, inherent to medical knowledge. It seems appropriate to create sophisticated models like causal probabilistic networks and expert systems for causality assessment. These tools are not only employed to aid diagnosis and prognosis, but they can be also used for simulation, which can save time, money, and prevent subjects from dangers.17 C. EXPERIMENTAL DESIGNS The test for a causal relationship is preferably performed by an experiment. Individuals are assigned to different groups according to a defined protocol. It is desirable that the different groups are identical or comparable with respect to all relevant influence factors except the hypothetical causal factor. After observation of the outcome inference about causality from the comparison of the different groups is warranted. The main feature for the strength of a causal association is the quality of the underlying trial. But what is a "good" trial? In a clinical environment, groups of members of trials cannot be formed in such a way that all but one relevant characteristics are identical. Therefore, the investigator tries to minimize unwanted differences and to avoid bias. This can be done by randomization and blinding techniques. But there are situations where these techniques cannot be employed, because of ethical reasons, legal regulations, or financial constraints. If one is investigating surgical techniques, it is obvious that the surgeon cannot be "blind" about the technique in use. In prospective studies, the only recorded events are those which happen after inclusion into the trial, a retrospective study uses information from the time before data collection. The most important designs for trials in medical research are cohort, case-control, and crosssectional studies. In a cohort study, the groups are exposed to different levels of the purported causal factor (where level 0 indicates absence of this factor), and then the target variable is determined. In case-control studies, the direction of this comparison is reversed, this is the reason why such studies are sometimes called trohoc studies (that is just the word cohort written from right to left). In a cross-sectional study, a population is selected and the exposure as well as the outcome is determined at a fixed point in time. In the following, measurements of association are illustrated for these different types of studies. Sitter et al.18 report about a prospective randomized trial, where intravenously injected histamine causes increased plasma histamine levels was investigated. The control group in this trial received a saline injection (placebo). Other causes for elevated plasma histamine levels could have been stress or artefacts due to the injection. The results of the study are shown in Table 1. Measures of association for Table 1 are the relative risk = 93.5/3.4 = 27.5, and the odds ratio = (29/2)7(1/28) = 406. Arranging the results from a case-control study in a matrix like Table 2, according to El wood2 it is not meaningful to look at the totals of the rows (a + b respective of c + d), because the inclusion into the trial of the individuals who show the target variable is completed before the presence or absence of the causative factor is recorded. Therefore, the odds ratio ( = ad/bc) is recommended as a measure of association (cf. Table 2). Dietz19 presents the results of a cross-sectional study dedicated to the question: Is histamine a mediator in human septic shock? Here one can calculate the relative risk as well as the odds ratio, where the relative risk is preferable to characterize association, because the odds ratio depends on the prevalence of septic shock. Usually one cannot expect to have such a balanced design as in Table 3. Interpretation of a cross-sectional study for a causal

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Sitter et al. TABLE 1 Contingency Table from a Randomized Controlled Trial i.v. Histamine "disease"

i.v. Saline "absence of disease"

Total

Prevalence of disease (%)

29 1 30

2 28 30

31 29 60

93.5 3.4 50

Increased PHL Not increased PHL Total

RR = 93.5/3.4 = 27.5 OR = (29/2)7(81/28) = 406 Note: Determination of strength of association by risk calculations.18 PHL = plasma histamine level; RR = relative risk; OR = odds ratio.

TABLE 2 Contingency Table from a CaseControl Study Group 1

Group 2

a c a + c

b d b + d

Relevant factor Present Absent Total

OR = (a/b)/(c/d) = ad/bc Note: Assessment of strength of association. Calculation of totals of rows inappropriate (for reasons see text). OR = odds ratio.

TABLE 3 Contingency Table from a Cross-Sectional Study

PHL elevated Yes No Total

Septic shock

Controls

Total

Prevalence septic shock (%)

10 10 20

2 18 20

12 28 40

83.3 35.7 50

RR = 83.3/35.7 = 2.3 OR = (10/2)/(10/18) = 9 Note: Calculation of risk for investigation of association between elevated PHL and septic shock in humans.19 PHL = plasma histamine level; RR = relative risk; OR = odds ratio.

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assessment raises a lot of troubles, there being no evidence if the supposed factor causes the outcome or if the so-called outcome causes the event which is considered as the causative factor.20 In the example from Table 3, additional information is needed to decide whether histamine release causes septic shock, or whether septic shock causes histamine release? A prospective randomized controlled trial, which is a special form of a cohort study, is the gold standard for the experimental design in causality analysis. Other types of trials are listed in the order of decreasing strength of association as follows: (1) cohort studies, (2) case-control studies, (3) cross-sectional studies, (4) case series, and (5) case report. Besides taking care of an optimal design, the investigator has to ensure other items for increasing the strength of an association. The comparison of two trials on the question if blood histamine concentrations are elevated in humans with septic shock,21-22 resulted in the following list of important items for an appropriate design: (1) completion of study protocol before start of trial; (2) elimination of bias; (3) well-defined inclusion criteria; (4) statement about the assignment of individuals to the groups; (5) prognostic factors; (6) report on differences between groups in diagnostic information; (7) random assignment of blood samples in the laboratory; (8) quality control of the histamine assay; and (9) role of interfering drugs. D. INTERNAL AND EXTERNAL VALIDITY To guarantee the internal validity which characterizes how evident a causal explanation is for the study members, we have to avoid the manipulation of the results by observation bias, confounding, and chance variation.2 Furthermore, for internal validity, we will gather affirmative items for causation depending on the model. In the subsequent chapters, different models for this assessment are introduced; an example for increasing the probability of a causal interrelationship is the confirmation of the Hill criteria. For internally valid studies which confirm causality, generalizations of the results are desirable. For the check of the external validity, information about the selection of the study members is required. Only if the study population is representative for the source population, or if there are other populations for which the results are applicable, the findings can be generalized.

IV. DETERMINISTIC MODELS A. KOCH-DALE CRITERIA In 1882 Koch described four postulates for identifying an infectious agent as the cause of a disease: (1) the organism must be present in every case of the disease; (2) the organism must be isolated and grown in pure culture; (3) the organism must, when inoculated into a susceptible animal, cause the specific disease; and (4) the organism must then be recovered from the animal and identified.(This formulation is cited according to Fletcher et al.)3 Evans23 mentions that the original idea for this concept goes back to Henle who published the basic ideas about 40 years earlier.24 Henle's pupil Koch expressed the criteria more rigorously. The following adaption of these postulates to more general situations is called Koch-Dale criteria,25 Dale26 transferred them to chemical factors, especially transmitters. These modified Koch-Dale criteria, for the determination of the causal role of a mediator for a disease, include the presence of a chemical factor in disease, its absence in health, elicitation of the disease in experiments by this mediator, and preventing its effect by a specific antagonist (cf., Table 4). The examination of these criteria in a real situation depends heavily on the technology which is used by the investigator; e.g., if an antagonist of the mediator is not known at the time of investigation, criterion 4 cannot be checked. Koch noted that he himself did not consider his postulates as rigid; he would have had accepted an association as a causal relationship even if not all of his postulates have been fulfilled. Neugebauer et al.27 show

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Sitter et al. TABLE 4 Koch-Dale Criteria 1. 2. 3. 4.

Presence in disease Absence in health Eliciting disease by exogenous administration Blocking disease presentation by antagonists

Note: Koch-Dale criteria for confirmation of causal enrollment of a mediator in a disease concept.

the importance of technological and methodological equipment for the investigation of the problem: Is histamine a causal factor for septic shock? For checking the Koch-Dale criteria, they designed a decision tree with the following nodes: (1) reliability of the assay for histamine determination; (2) correct preparation of samples; (3) choice of the relevant body fluid for mediator release; (4) appropriate timing for sampling; (5) suitable study designs (prospective randomized controlled trials as gold standard); (6) clinical relevance of shock model; and (7) clinical relevance of species in animal trials. The acceptance and the use of the Koch-Dale criteria by the scientific community was a real breakthrough in causality analysis, because thereby the determination of different kinds of bacteria as responsible for certain diseases and the definition of pathological mechanisms was possible. Nevertheless, this concept is not applicable in all situations because it is based on the assumption that a particular disease is caused by a single factor and that the presence of this factor results in only one disease. A limitation of these postulates of causation is the fact that in medicine, the manifestation of many diseases can be described only by multicausal processes. For instance, a mediator in septic shock which is a causal factor can release other mediators which themselves influence the disease presentation and probably also have influence via feedback loops on the first mediator. The Koch-Dale criteria are in some cases not applicable to viruses and parasites. The most striking example for such a situation is the acquired immunodeficiency syndrome, where Duesberg28 questions the causal role of human immunodeficiency virus, and shows that it does not meet the KochDale postulates. B. HILL CRITERIA In 1965, Sir Bradford Hill introduced a set of criteria which facilitates the decision whether an association is a causation or not.29 This concept applies also if experimental designs for analyzing causality are not available, because in a certain sense they formalize the judgement based on evidence.3 Sir Bradford Hill proposed nine different aspects which guide an investigator to the conclusion that causation is the most likely interpretation of an established association. These nine different features are (1) strength of association, (2) consistency, (3) specificity, (4) temporality, (5) biological gradient, (6) plausibility, (7) coherence, (8) reversibility, and (9) analogy (cf. Table 5). Establishing a causal relationship depends on the strength of the association, which can be expressed by a large relative risk or odds ratio. It is much easier to believe a causal relationship, if there is a tenfold increase in the frequency of an effect compared to only a threefold increase. But even if the strength of an association is only slight, a causal relationship might be present, and to confirm this we have to check the other items on this list. An observed association is more likely considered as causation, if the phenomenon has been observed repeatedly by different persons, in different places, circumstances, and times, i.e., if it is consistent. If different trials have produced the same result, it is easy to accept causality; but it is well known that often different studies about the same subject lead to

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Handbook of Mediators in Septic Shock TABLE 5 Hill Criteria Strength of association Consistency Specificity Temporal relationship Biological gradient Biological plausibility Coherence of evidence Reversibility Analogies Note: Hill criteria for causality analysis.

different and sometimes contradictory results and conclusions. Pooling results from different studies should be done by meta-analysis,30 where different studies are weighted according to the confidence of their results. The specificity of an association is a characteristic in favor of causation. If there is one cause and one effect, then specificity is an appropriate criterion. In other cases one should not pay too much attention to this criterion, as Hill29 states "we must not, however, overemphasize the importance of the characteristic.'' Because many processes should be described by multicausation more appropriately, we cannot rule out causation if there is no high specificity. But conversely, if high specificity exists, we are prepared to believe in a causal relationship. The temporal relationship between a cause and an effect is the fourth criterion and expresses the fundamental principle that a cause has to precede the effect in time. At first glance, this seems to be a very simple postulate, but it is not always easily proven. For instance, it is often not clear, if a certain adverse reaction releases a mediator, or if the release of the mediator causes the adverse reaction. Certain types of studies, like crosssectional studies, (where exposure and effect are measured at the same point in time) or case-control studies, are difficult to analyze with respect to temporality. Two events which coincide in time may be caused by a third, not known or not observed factor, this influence is known as confounding. This kind of bias is also called the cart vs. horse bias and is dominant in diseases with slow development. For example, a particular diet may lead to a disease or the disease may lead to particular dietetic habits where the beginning of one process initiates the beginning of the other and vice versa. Many authors3'29 emphasize that temporality is absolutely necessary for causality assessment, but that the fact of temporal sequence alone is not very convincing. Nevertheless, there are authors who do not believe that a temporal direction is absolutely necessary for establishing a causal relationship. Scriven31 recognizes also that causes are not separable from effects, neither with respect to time nor to space. Furthermore, causes need not be logically separable from their effects, and then the relation between cause and effect need not be empirical. An example31 is the act of closing a door causes, and its description entails, the door's closing. So, this notion of cause depends on the context, in which an association is considered. If it is possible to determine a biological gradient, or a dose-response curve, there is good evidence for a causal relationship. Dose-response curves give a quantitative relation between a factor and an effect and have a simple interpretation. Trials about cigarette smoking show that incidence of lung cancer death is higher in male patients with a large amount of cigarettes per day in contrast to patients with a low dose of cigarettes or to nonsmokers. Even if this is strong evidence for a causation, this fact alone does not prove the causal

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relationship, because there might be another factor which causes lung cancer and at the same time leads people to habits of heavy smoking. This would be an example for a confounding bias. Biological plausibility is very helpful for the confirmation of a causal association, but it cannot be demanded as a necessary prerequisite, because it depends on the biological knowledge of the present time. There might well be a causal relationship which is biologically not plausible if the biological knowledge and the biological models are not optimal. In such a case, one should look for other characteristics which enable us to decide if a causative factor is present. Biological implausibility means either that there is no causal relationship or that there is a lack of biological and medical knowledge. If results and data from a causality analysis are coherent with generally known facts from natural history and biology, the presence of a causal factor is confirmed. Both, the increase in lung cancer and the increases in cigarette smoking are coherent and support the hypothesis that smoking causes lung cancer. Comparisons with results from animal trials are also important for checking coherence. Reversibility is present, if the removal of a suspected casual factor results in a lower frequency of the effect. However, a reversible association can also be due to confounding. Analogies with well-known causal relationships facilitate our judgment. If people know examples of mechanisms or disease concepts which are analogous to the problem under investigation, it is much easier for them to believe in the suggested causation. But analogy in general is only weak evidence for cause.3 The more of the nine different Hill criteria that are fulfilled in a particular situation, the more convincing is the causal relationship. But it is obvious that one cannot claim that all points have an affirmative answer in a cause-effect relation. They can give more or less strength to the role of purported causes in a model and they can help to refute other events as causal factors. Similar to the Koch-Dale criteria, the Hill criteria are designed for assessment of unicausal factors. Saltier et al.32 mention that deterministic quantitative mathematical relations in most cases are only used in a unicausal setting.

V. STOCHASTIC MODELS A. NECESSARY, SUFFICIENT, AND CONTRIBUTORY CONDITIONS In the introduction we have already pointed out that probabilistic reasoning is a tool for causality assessment. These models are more appropriate for multicausal relationships than the concepts from the previous chapter. We are considering an example where infection, the release of the mediator histamine, and septic shock are investigated. Figure 1 gives a schematic representation of six different situations for probabilistic causality analysis.33-34 Probabilities are assigned to all events, and the role of histamine release as a causal factor for septic shock is investigated. In the first chart of Figure 1 histamine release is a necessary determinant for septic shock. With the probability of one, septic shock is not seen if no histamine release has taken place. The conditional probability of septic shock under the condition that no histamine release takes place equals zero. Of course, it is possible that histamine release also causes other reactions, this is illustrated by the two arrows pointing into other directions than to septic shock. As a second possibility, histamine release, after an infection, can be a sufficient determinant for septic shock. Histamine release alone is able to produce the septic shock but other mediators beside histamine are also potent enough to elicitate septic shock; this is indicated in Figure 1 by the two arrows pointing from other directions than histamine release to septic shock. Histamine release as a sufficient determinant for septic shock means that the probability of the septic shock under the condition of histamine release equals one. In the third example, histamine is released after infection as a contributory

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FIGURE 1. Probabilistic model for causal associations of histamine in the development of septic shock. The arrows symbolize elapse of time. IN = infection (etiological factor); HR = histamine release; SE = septic shock. The complement of event X is denoted by X.

condition for septic shock. Other mediators as well as histamine are able to produce septic shock and histamine release can also be responsible for other reactions. We call histamine release a contributory condition for septic shock, if the probability of septic shock under the condition of a histamine release is greater than the probability of septic shock under the event of no histamine release. After estimation of the probabilities which are used in these definitions, the classification of histamine release as a necessary or sufficient determinant or a contributory condition is possible. The sequence of the events can also differ from our previous assumption that infection is followed by histamine release and histamine release is followed by septic shock. The fourth graph in Figure 1 has the infection as a common cause for histamine release and septic shock. Histamine release and septic shock are independent events without a causal relation. In diagram 5 (of Figure 1), histamine release is not the cause for septic shock but it is a concomitant factor of the infection. The events of infection and histamine release are independent. In the last diagram of Figure 1, the time course of our three events is first the infection, then septic shock, and then histamine release. Here histamine release is a consequence of septic shock, and septic shock is a necessary and a sufficient determinant for

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FIGURE 2. Calculation of conditional probabilities for assessing causality between histamine release and cardiac instability (adverse reaction). IS = cardiac instability, HR = histamine release. The complement of event X is denoted by X. (a) Histamine release is a causal factor for cardiac instability after nalbuphine. (b) The model shows no causal relationship between histamine release and adverse reaction after fentanyl. (c) After alloferine, flunitrazepam, and thiopentone, histamine has a protective effect.

histamine release. The probability of histamine release under the condition that there is no septic shock equals zero (necessary determinant), and the probability of histamine release under the condition of septic shock equals one (sufficient determinant). Obviously, in diagram 6 we can incorporate the situations from the diagrams 1 to 3 in Figure 1, accordingly, and can identify septic shock as a necessary, sufficient, or contributory condition. Figure 2 shows estimates of the probabilities from trials which dealt with the question: do anesthetic drugs release histamine?35 Three different anesthetics which are used for the preparation of a surgical patient were investigated and cardiac instabilities (tachycardia as well as bradycardia) were recorded. The three drugs were (a) nalbuphine, (b) fentanyl, and (c) the combination of alloferin, flunitrazepam, and thiopentone. The comparison of the estimated probabilities from Figure 2 shows that histamine release is not a contributory condition after fentanyl, or alloferin, flunitrazepam, and thiopentone. In the case (c) with the drug combination, histamine release acts as a protective factor against cardiac instability.

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Handbook of Mediators in Septic Shock TABLE 6 Relationship Between Histamine and Death in Septic Shock Lethal outcome

H R HR Total

Incidence LE Incidence HR Conditional probability Conditional probability P (LE/HR) > P (LE/HR);

+

-

8 1 9

1 10 11

P P P P

(LE) (HR) (LE/HR) (LE/HR)

Total 9 11 20

= 9/20 = 9/20 = 8/9 =1/11

= = = =

0.45 0.45 0.89 0.09

HR is a contributory determinant.

Note: Estimation of conditional probabilities for establishing a causal relationship between elevated plasma histamine levels and lethal outcome in septic shock. HR = histamine release; HR = complement of HR = no histamine release; LE = lethal outcome.

Several authors consider the problem of histamine in human patients with septic shock still an open question.36 Dietz19 reports on a cross-sectional trial where 20 patients with septic shock and 20 patients with peripheral trauma staying in an open ward were compared, and plasma histamine levels were determined in all patients before the surgical procedure. The septic shock group shows elevated plasma histamine levels which are (statistically) significantly different from the control group (p E) + P(B|A -/> E) P(A -+> E)

where A —» E signifies that A causes E, and A -/» E denies A —* E. The prior probability P(A —» E) is updated to the posterior probability P(A —> E|B) because of the additional knowledge of B. The situation where not just one factor is supposed to be causal for the effect E, can also be handled with a natural generalization of the above formula. With this method one can derive a quantitative value for the desired probability in multicausal processes. For estimating the probabilities on the righthand side of the formula, data have to be acquired in a trial or gathered from the literature. Of course, the quality of the determination of the strengh of association depends on the quality of the data used as input for the estimations. Lane et al.38 describe this concept, give examples for causality assessment of adverse drug reactions, and claim that this approach is, through the ability of providing quantitative probability statements, superior to many standardized assessment models in adverse drug reaction research. Most important features of background information B for adverse drug reactions are the patient's condition, the time horizon, and characteristics of the adverse event. D. REGRESSION MODELS A regression model specifies a functional relationship f between an outcome variable y and supposed relevant factors x,, x2, ..., xn:

y = f(x,, x2, . . ., xj

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The special case of a linear function f yields the equation: y = ao + HjX, + a2x2 + . . . + anxn The parameters a,,, a,, ..., a,, are estimated from a data set usually with the least squares technique (the sum of the squared deviations of the function f from the collected data at the points of measurement is minimized). There are no unambiguous recommendations for the choice of the function f, but this choice is crucial for an optimal fit of the model to reality. This article does not present further details of regression methods and estimation of regression parameters, because these subjects can be found in every standard text book on medical statistics.15 Regression models do not only determine the strength of an association, but they give a quantitative relation between the outcome and the causative factors in a multivariate setting. This property predesignates these models for prediction. In applications it is often unclear which factors, x,, ..., x n , should be chosen from all possible factors for analysis. This problem is tackled by selection procedures based on statistical tests. Regression analysis is applied mainly in establishing dose-response curves and scoring systems.39

VI. CAUSAL NETWORKS A. GRAPH REPRESENTATIONS Now a basic issue of further methodology for causality assessment will be introduced. Causal networks are also called causal probabilistic networks, Bayes networks, believe networks, relevance diagrams, and knowledge nets. A causal network is a directed acyclic graph which represents medical knowledge in a specific domain. Such a network is a basic framework for investigating complex relationships under a variety of conditions.40 Uncertainty is handled by probabilistic methods and implemented into expert systems. Expert systems based on causal networks combine statistical reasoning with experience rating. As a result of this procedure probabilities are obtained that, out of a set of putative causal factors, one factor alone or every possible combination of factors causes the effect. For example, we want to clarify the role of two putative causes A and B for a specified effect E. An arrow symbolizes a causal relationship, e.g., A —> E denotes that A causes E. The medical setting is characterized by information on the environment in which A, B, and E occur; this background information is denoted by S. According to Spiegelhalter et al.41 and Cowell et al.,42 we choose a Bayesian probabilistic approach, which gives an update of the prior odds. The prior odds for A are P(A -> E|S) P(B -> E|S)

After the acquisition of knowledge of additional findings D (e.g., blocking of mediator release by antagonists, or stimulation of a mediator by injection of a releaser) the posterior odds are expressed P(A -» E|D,S) _ P(D|A -> E,S) P(B -> E|D,S) ~~ P(D|B -» E,S)

X

P(A -> E|S) P(B -» E|S)

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FIGURE 4. Chart of the diagnostic process being modeled. After knowledge of the outcome, P(C ! D, S) can be calculated with Bayes' theorem and the arrow between C and D is reverted. C depends on S, and D depends on S and C. By observing S and D we want to infer on C.

The posterior odds on the lefthand side are expressed as the product of the likelihood ratio and the prior odds. Figure 4 is a schematic representation of this model. A simplified graph for association between the anesthetic premedication of a surgical patient and adverse reactions via the intermediate state of elevated plasma histamine levels is depicted in Figure 5. A surgical patient is exposed to perioperative stress, which is supposed to be responsible for histamine release, as well as to various other mediators with interactions to histamine. Histamine release is followed by the binding of free histamine to receptors and the induction of adverse reactions (e.g., cardiovascular instability). If one can give estimates of probabilities for the links, we can build a network and calculate the probability of an adverse reaction for a specific patient. This model can be implemented on a computer as an expert system. It is also possible that the data which are used for deciding a causal relationship for one patient can be stored and used for updating of the probabilities. This is a feature which accounts for experience. These models are very suitable for analyzing the effects of different drugs and plasma substitutes. Such a situation is routinely encountered during anesthesia of a surgical patient. Lauritzen and Speigelhalter43 demonstrated that it is possible to use only local computations for the estimation of the conditional probabilities. In a directed acyclic graph consisting of nodes and arrows, the node A is called the parent node for another node B, if A is connected with B by an arrow (A —» B). The Lauritzen-Spiegelhalter algorithm transforms the initial graph in the following way: first, all parent nodes belonging the same "child" node are connected by a link — this step is called marrying parents; second, all directions of the arrows in the network are dropped; and third, if a path connects node A via several intermediate nodes with itself, then new connections are introduced which provide a shortcut of maximum length three for a cycle — this step is called triangulation. The transformed graph is labeled as a full moral graph. The graph of the network from Figure 5 is transformed to a full moral graph in Figure 6. After this procedure it is possible to update the conditional probabilities only through local computations, and it is not necessary to calculate all conditional probabilities, which saves a lot of computational effort in large networks. B. EXPERT SYSTEMS The estimation of the probabilities in a causal network is usually performed by expert systems. HUGIN (handling uncertainty in general influence networks) is an expert system

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FIGURE 5. Network for histamine release during the perioperative period. This is a simplified schematic representation of influence factors on histamine release during the perioperative period. The arrows indicate time course of the events.

shell which is based on the above described algorithm and can be applied to deal with causality analysis in a specific medical problem.44 MUNIN45 (muscle and nerve inference network) is an expert system which was built with the shell HUGIN and consists of approximately 1000 nodes, each having several, up to 21, states. The MUNIN system aids the diagnosis of neurological diseases through analysis of bioelectrical signals from muscle and nerve tissue. The artificial words HUGIN and MUNIN are abbreviations.46 Besides this explanation, there is another etymological reason for the names HUGIN and MUNIN. In the Norse mythology, Hugin and Munin are two ravens embodying the soul of the god Odin living in Valhalla. Hugin represents his capacity to reason and understand, and Munin his capacity to acquire and memorize knowledge of the world.47

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FIGURE 6. Histamine release during perioperative period. This is a full moral scheme for the network from Figure 5, obtained by marrying parents, dropping directions, and triangulation.

The problem with the routine use of expert systems incorporating causal networks is that there are only very few systems dealing with a realistic medical problem and using sound and evaluated databases for probability estimation. In addition to MUNIN, another system of this kind, called Pathfinder,48'49 is currently in use by practicing pathologists and for teaching purposes. The fact that more than 60 lymphnode diseases are modeled in Pathfinder gives an idea of the complexity of the networks underlying such expert systems. Lauritzen50 shows that local computations with probabilities, means, and variances are possible for mixed qualitative and quantitative variables, with the restriction that it is not allowed for a quantitative node to have qualitative children. The great advantage of causal networks is their suitability for modeling multicausal processes. There is no restriction on the number of putative causal factors and conditions in the network. Nevertheless, the computational effort can rise considerably if there are many elements in the decomposition by the triangulation.

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VII. FUTURE DEVELOPMENTS During the last two decades, a lot of research has been carried out in dynamic systems and chaos theory. Biology and medicine is full of examples where deterministically started processes end up in, what colloquially would be called, chaotic behavior. Chaos theory, which can be considered as a new branch in the philosophy of science, really does not accept a difference between deterministic and stochastic (predictable as well as unpredictable) events.51 The word 'chaos' is of Greek origin and has the meaning of openness and gaping.52 Mandelbrot,53 one of the pioneers in this field, investigated the behavior of the iteration of the following mathematical transformation: g(z) = z2 + c. Where z and c are complex numbers; c is usually called the control parameter and is an arbitrary but fixed complex number. We start the iteration process with the complex number z = z0 and calculate z, as z, = g(z0) = z2, 4- c. If we have calculated /;_,, step i in the iteration gives z( = g(Zj__[), and we have the sequence z0, z 1( z2, .... If we are now changing the start value z0 and investigate the limit behavior of the respective sequences under certain assumptions, we notice an interesting phenomenon: there is one set of starting values where the associated sequences converge to a fixed point, and there is another set of starting values for which the sequences tend to infinity, and in between these two sets exists a border of infinitely small width.54 The border is called a Julia-set. For different choices of the control parameter c, we have two possibilities for the corresponding Julia-set: (1) the Julia-set is (topologically) connected in the space of complex numbers; (2) the Julia-set is a "cloud" of separate points (a so-called Cantor-set).54 The famous Mandelbrot set is the set of all control parameters c with a connected Julia-set. A generalization of the current concept of dimensionality (a straight line is one-dimensional, a plane is two-dimensional, etc.) allows the definition of a dimension for Julia-sets and the Mandelbrot set. These dimensions are no longer positive integers, but fractals. Computer simulations produce visualizations of the Mandelbrot set and of Julia-sets (see Plate 1*). If we magnify a part of the Mandelbrot set by advanced computer technology, we recognize after a sufficient number of magnification steps a tiny copy of the original picture, and once more this process can be iterated, there exists reflexive similarity. Structures with such reflexive similarity properties are also encountered in biological and medical systems, i.e., as models of the lung, the nervous system, or the heart. Winfree55 describes a chaos model for ventricular fibrillations, which the author uses for computer simulation. The sequence of heart strokes follows a fractal rhythm. Each stroke is similar but not identical to the previous one, which might be the cause for two different pathological findings: first, if the heart beats are too regular, there is a danger of heart failure by stagnation; and second, a too aperiodic rhythm causes ventricular fibrillation. Thus, a physiological heart rhythm oscillates between order and chaos. "Since phenomena are so complex there is no a priori rational way to determine their laws" (Zajicek56), this new chaos approach looks very promising for the future, but one has clearly to admit that at the present stage of development chaos theory is no serious competitor for the causality assessment models presented in the previous chapters. Applications of chaos theory to nonmedical domains are also under investigation, and Briggs et al.51 conjecture that some day chaos theory will be able to identify the wing stroke of a butterfly in China as the cause of a hurricane in the U.S. a few weeks later. That is the theory, but in practice nobody would be able to name the corresponding butterfly.

*

Plate 1 follows page 522.

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Sitter et al.

VIII. SUMMARY The investigation of causal interrelationships in medicine is important for prevention, diagnosis, and treatment. Large parts of etiology are dedicated to the identification of causal factors and to the analysis of mechanisms governed by them. The confirmation of causality in the research of adverse drug reactions incorporates legal aspects. If an association is identified as causation, predictions can be based on this finding. Causality assessment is performed in two steps: in the first step, a model is built which represents a slice of the real world suitable for handling the problem under consideration, and in the second step, the relationships of the elements of the established model are analyzed. The same methodology and tools for analysis can be applied to causal processes in individuals and in whole populations, which is a common ground for medical decision making and epidemiology. The study of causation is a prominent topic in philosophy which includes the discussion of a definition of "causality". A short survey on selected philosophical views relevant in medicine was presented. Experiments are used to prove causal relations. The willingness to believe the strength of an association depends on the quality of the experimental investigations. But legal, ethical, or financial aspects may enforce the use of nonexperimental methods. Methods for causality analysis are divided into deterministic and stochastic models. Stochastic approaches facilitate the model building process and have the advantage of producing quantitative results in the form of probabilities. A further development of traditional stochastic models are computer implementations of causal probabilistic networks which combine learning by experience with statistical techniques. Expert systems using this technology also yield quantitative results. Chaos theory seems to be suitable for the explanation of medical phenomena which looked, until now, too ill-structured for analysis with established methods of causality assessment.

ACKNOWLEDGMENTS This research was supported by a grant from Deutsche Forschungsgemeinschaft (Lo 199/ 16-2). Plate 1 was prepared by J. Lorenz (Munich) to whom we are most grateful for placing it at our disposal.

REFERENCES 1. Casti, J. L., Alternate Realities. Mathematical Models of Nature and Man, John Wiley & Sons, New York, 1989. 2. Elwood, J. M., Causal Relationships in Medicine. A Practical System for Critical Appraisal, Oxford University Press, Oxford, 1988. 3. Fletcher, R. H., Fletcher, S. W., and Wagner, E. H., Clinical Epidemiology — The Essentials, Williams & Wilkins, Baltimore, 1988, chap. 11. 4. Velanovich, V., Causality concepts in surgery, Theor. Surg., 1, 197, 1992. 5. Holland, P. W., Statistics and causal inference, J. Am. Stat. Assoc., 81, 949, 1986. 6. Taylor, R., Causation and determinism, in The Encyclopedia of Philosophy, Vol. 2, Edwards, P., Ed., MacMillian Publishing, New York, 1967. 7. Locke, J., An Essay Concerning Human Understanding, Book II, 1690, chap. 26. 8. Hume, D., A Treatise on Human Nature, Selby-Bigge, L. A., Ed., Clarendon Press, Oxford, 1888. 9. Hume, D., An Inquiry Concerning Human Understanding, 1748. 10. Mill, J. S., A System of Logic, 1843.

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11. Suppes, P., A Probabilistic Theory of Causality, North-Holland, Amsterdam, 1970, 1. 12. Wulff, H. R., Andur Pedersen, S., and Rosenberg, R., Philosophy of Medicine — An Introduction, Blackwell Scientific Publications, Oxford, 1986, 61. 13. Mackie, J. L., The Cement of the Universe. A Study of Causation, Clarendon Press, Oxford, 1980, 1. 14. Spitzer, W. O. and Horwitz, S. M., Selected nonexperimental methods: an orientation, in Principles and Practice of Research. Strategies for Surgical Investigators, 2nd ed., Troidl, H., Spitzer, W. O., McPeek, B., Mulder, D. S., McKneally, M. F., Wechsler, A. S., Balch, C. M., Eds., Springer-Verlag, New York, 1991, 104. 15. Armitage, P. and Berry, G., Statistical Methods in Medical Research, 2nd ed., Blackwell Scientific, Oxford, 1987. 16. Carr, D. B., Causality, predictability and explanation, Theor. Surg., 4, 88, 1989. 17. Oppel, U. G. and Moser, W., Kausal-probabilistische Netze zur Konstruktion medizinischer Expertensystems fur Diagnose, Simulation und Prognose, Biomet. Inf. Med. Biol., 2, 84, 1992. 18. Sitter, H., Lorenz, W., and Doenicke, A., The clinical and biological signs of histamine release during induction of anaesthesia and preparation of the sugical patient: a farewell party for the classical manifestations of anaphylaxis, Agents Actions (Special Conference Issue), C219, 1992. 19. Dietz, W., Neue Wege in der Entscheidungsfindung fur eine Histaminprophylaxe beim chirurgischen Eingriff: Identifizierung von Risikogruppen mil hohen Plasmahistaminspiegeln und Nachweis der Wirksamkeit gegen verschiedene Mediatoren auch bei schwersten lebensbedrohlichen Reaktionen, Habilitationsschrift, University of Marburg, 1989. 20. Flanders, W. D., Lin, L., Pirkle, J. L., and Caudill, S. P., Assessing the direction of causality in crosssectional studies, Am. J. Epidemiol., 135, 926, 1992. 21. Lorenz, W., Thon, K., Stoltzing, H., Neugebauer, E., Lindlar, R., Sattler, J., and Weber, D., Histamine and the stomach: chemical histamine assays, Scand. J. Gastroenterol., 26 (Suppl. 180), 9, 1991. 22. Jacobs, R., Kaliner, M., Shelhamer, J. H., and Parrillo, J. E., Blood histamine concentrations are not elevated in humans with septic shock, Crit. Care Med., 17, 30, 1989. 23. Evans, A. S., Causation and disease: a chronological journey, Am. J. Epidemiol., 108, 249, 1978. 24. Henle, J., On Miasmata and Contagie (transl.), Johns Hopkins Press, Baltimore, 1938. 25. Lorenz, W., Rdher, H. D., Doenicke, A., and Ohmann, C., Histamine release in anaesthesia and surgery: a new method to evaluate its clinical significance with several types of causal relationship, Clinics Anaesthesia!., 2, 403, 1984. 26. Dale, H. H., Croonian lectures of some chemical factors in the control of circulation, Lancet, i, 1285, 1929. 27. Neugebauer, E., Lorenz, W., Maroske, D., and Barthlen, W., Mediatorenvielfalt beim septischen Schock, in Sepsis — Experimented Befunde, klinische Erfahrungen, Schulte am Esch, J., Ed., W. Zuckschwerdt Verlag, Munchen, 1987. 28. Duesberg, P. H., Human immunodeficiency virus and acquired immunodeficiency syndrome: correlation but not causation, Proc. Natl. Acad. Sci. U.S.A., 86, 755, 1989. 29. Hill, A. B., The environment and disease, Proc. R. Soc. Med., 58, 295, 1965. 30. Neugebauer, E., Lorenz, W., Maroske, D., Barthlen, W., and Ennis, M., The role of mediators in septic/endotoxic shock. A meta-analysis evaluating the current status of histamine, Theor. Surg., 2, 1, 1987. 31. Scriven, M., The logic of cause, Theor. Decision, 2, 49, 1971. 32. Sattler, J., Lorenz, W., Lindlar, R,. and Schafer, U., Histamine in duodenal ulcer, stress-induced lesions, and upper gastrointestinal bleeding: causality analysis, in Handbook of Experimental Pharmacology, Vol. 97, Uvnas, B., Ed., Springer-Verlag, Berlin, 1991, chap. 13. 33. Lorenz, W., Dietz, W., Ennis, M., Stinner, B., and Doenicke, A., Histamine in anaesthesia and surgery: causality analysis, in Handbook of Experimental Pharmacology, Vol. 97, Uvnas, B., Ed., Springer-Verlag, Berlin, 1991, chap. 12. 34. Wulff, R. W., Rational diagnosis and treatment, Blackwell Scientific, Oxford, 1976. 35. Dick, W., Lorenz, W., Heintz, D., Sitter, H., and Doenicke, A., Histaminfreisetzung bei der Einleitung von Kombinationsnarkosen mit Nalbuphin oder Fentanyl, Anaesthesist, 41, 239, 1992. 36. Hinshaw, L. B., Histamine in septic/endotoxic shock: pathophysiological considerations, Theor. Surg., 4, 91, 1989. 37. Hilden, J., Causal networks, not clinical trials, as a model for experimental shock research, Theor. Surg., 4, 100, 1989. 38. Lane, D. A., Kramer, M. S., Hutchinson, T. A., Jones, J. K., and Naranjo, C., The causality assessment of adverse drug reactions using a Bayesian approach, Pharm. Med., 2, 265, 1987. 39. Spiegelhalter, D. J., Statistical methodology for evaluating gastrointestinal symptoms, Clin. Gastroenterol., 14, 489, 1985.

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40. Spiegelhalter, D. J. and Lauritzen, S. L., Techniques for Bayesian Analysis in expert systems, Ann. Math. Artif. Intell, 2, 353, 1990. 41. Spiegelhalter, D. J., Daw id, A. P., Hutchinson, T. A., and Cowell, R. G., Probabilistic causality assessment after a suspected adverse drug reaction: a case study in Bayesian network modelling, BAIES Report BR-ll, 1990. 42. Cowell, R. G., Dawid, A. P., Hutchinson, T., and Spiegelhalter, D. J., A Bayesian expert system for the analysis of an adverse drug reaction, Artif. Intell. Med., 3, 257, 1991. 43. Lauritzen, S. L. and Spiegelhalter, D. J., Local computations with probabilities on graphical structures and their application to expert systems, J. R. Stat. Soc., 50, 157, 1988. 44. Andersen, S. K., Olesen, K. G., Jensen, F. V., and Jensen, F., HUGIN — a shell for building belief universes for expert systems, Proc. 11th Int. Joint Conf. Artificial Intelligence, 1989. 45. Andreassen, S., Woldbye, M., Falck, B., and Andersen, S. K., MUNIN — a causal probabilistic network for interpretation of electromyographic findings, Proc. 10th Int. Joint Conf. Artificial Intelligence, 1987, 366. 46. Jensen, F. V., Lauritzen, S. L., and Olesen, K. G., Bayesian updating in causal probabilistic networks by local computations, Comput. Stat. Q., 4, 269, 1990. 47. Andreassen, S., Jensen, F. V., and Olesen, K. G., Medical expert system based on causal probabilistic networks, Res Rep R-91-6, Inst. Elect. Systems, Aalborg University, Denmark, 1991. 48. Heckerman, D. E., Horvitz, E. J., and Nathwani, B. N., Toward normative expert systems. I. The pathfinder project, Meth. Inform. Med., 31, 90, 1992. 49. Heckerman, D. E. and Nathwani, B. N., Toward normative expert systems II. Probability-based representations for efficient knowledge acquisition and inference, Meth. Inform. Med., 31, 106, 1992. 50. Lauritzen, S. L., Propagation of probabilities, means and variances in mixed graphical association models, Res Rep R-90-18, Inst. Elect. Systems, Aalborg University, Denmark, 1990. 51. Briggs, J. and Peat, F. D., Die Entdeckung des Chaos, Carl Manser Verlag, 1990 (German translation of Turbulent Mirror. An Illustrated Guide to Chaos Theory and the Science of Wholeness, Harper & Row Publishers, New York, 1989.). 52. Gross, R., Systemwissenschaft in der Medizin, Deutsches Arzteblatt, 88, B-1720, 1991. 53. Mandelbrot, B. B., The Fractal Geometry of Nature, Freiman, New York, 1982. 54. Jiirgens, H., Peitgen, H. O., and Saupe, D., Fraktale — eine neue Sprache Fur komplexe Strukturen, in Spektrum der Wissenschaft (German edition of Scientific American), Chaos und Fraktale, Spektrum der Wissenschaft Verlagsgesellschaft, Heidelberg, 1989, 106. 55. Winfree, A. T., Sekundenherztod: Hilfe von der Topologie?, in Spektrum der Wissenschaft (German edition of Scientific American), Chaos und Fraktale, Spektrum der Wissenschaft Verlagsgesellschaft, Heidelberg, 1989, 92. 56. Zajicek, G., Chaos and biology, Meth. Inform. Med., 30, 1, 1991.

PLATE 1. In this three-dimensional computergraphical representation, points of the Mandelbrot set are associated with the spatial dimension depth. In this dimension, for each point of the line between the coordinates (-0.83, 0.25) and (-0.83, -0.25), the corresponding Julia-set is drawn in the two dimensions of height and width. The different colors indicate different numbers of iteration steps of the computations. (Courtesy of J. Lorenz, Munich).

Chapter 26

META2 — AN ANALYSIS OF META-ANALYSES OF MEDIATORS IN SEPTIC SHOCK John W. Holaday, Edmund Neugebauer, and Daniel B. Carr

TABLE OF CONTENTS I.

Introduction

524

Summary and Analysis of Individual Mediators

524

III.

Methods of Determining Causality

525

IV.

The Meta-Analysis Approach: Strength and Limitations in Defining Causal Relationships of Mediators in Septic Shock

530

Alternative Methods to Evaluate a Causal Role of a Single Mediator in Septic Shock

531

Where to Go in the Future

533

II.

V. VI.

Acknowledgment

534

References

534

0-8493-3548-5/93/SO.OO + $.50 O 1993 by CRC Press, Inc.

523

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I. INTRODUCTION Over the past century of research directed at uncovering the causes and treatments of septic shock, many important advances have been made. Pathophysiological events that were once medical mysteries now can be attributed to mechanisms that involve the specific effects of biological mediators. Despite this wealth of accumulating knowledge, application of this emerging technology in the clinic has yet to alter significantly the morbidity and mortality of septic shock. Perhaps the insights gathered by you, the reader of this handbook, will serve to break this impasse. It is clear that septic shock, now recognized as one form of a "systemic inflammatory response syndrome" (SIRS),10 is a multifactorial process that involves a cascade of mediators whose actions over time lead to a sequence of pathological events from which the patient either recovers or dies. In sepsis or SIRS, the usual orchestration of biological processes that characterizes homeostasis is disrupted, resulting in the biological cacophony, or dyshomeostasis, that characterize this syndrome. Although there are hundreds of books and symposia proceedings that deal with the intricacies of individual "sepsis" mediators, to the best of our knowledge, none of these published works provides an integrated perspective that describes how these many mediators interact with each other over time to produce the signs and symptoms that are pathognomic of this disease. Understandably, most of the authors who have contributed to earlier publications have chosen to emphasize their own favorite mediator, and in so doing, they have largely presented supportive evidence obtained from their own collaborative studies. An evaluation of these myriad books and symposia leaves the reader with a sense that, although many pieces of a complex shock "puzzle" may have been presented, no clue was provided to indicate how the pieces of the puzzle fit together. To add to the confusion, in these publications, often different scientific strategies have been used by authors in their attempts to prove a causal role for their favorite mediator, making it difficult to assess objectively the strengths or weaknesses of supportive evidence.

II. SUMMARY AND ANALYSIS OF INDIVIDUAL MEDIATORS In this handbook, we asked the contributing authors to conduct a very difficult, thorough, and time-consuming meta-analysis of many of the individual mediators of septic shock in an attempt to provide an objective assessment of causality. This initially required an exhaustive evaluation of the sufficiency of the published data from the available literature bases. Then, only after individual publications were judged to have complied with rigorous scientific criteria, could they be used to support or refute the branches of the Koch-Dale decision tree. It was hoped that the meta-analysis process presented in this handbook would provide an objective, state-of-the-art picture of present day knowledge about the mediators of septic shock. Despite this noble intention, it became apparent that some of the "mediators" chosen by the editors for evaluation were not amenable to this rigorous meta-analysis technique. For example, as one progresses down the nodes of the Koch-Dale decision tree,12'13 the failure to meet an individual criterion at an early node may have precluded further evaluation, despite the fact that other subsequent criteria may have been satisfied by existing data. Table 1 provides an analysis of each chapter (mediator) according to the type of analysis conducted by the authors and the sufficiency of data for satisfying the Koch-Dale criteria. It is also important to note that the perspective that one chooses in evaluating a mediator strongly affects the outcome of even an objective meta-analysis. For example, most if not all of the mediators represented in this handbook have important beneficial roles in maintaining homeostasis in healthy organisms. It is the disruption of the normal patterns of mediator release over time, as well as alterations of the amounts of mediators that are released, that may dictate

Holaday, Neugebauer, and Carr

525

a pathological or beneficial outcome during septic shock. As a specific example, adrenal glucocorticoids (e.g., cortisol) have profound effects on glucose homeostasis, immunity, and various other critical aspects of cellular function. Although presently disputed, it is this panoply of potentially therapeutic effects that has resulted in the use of glucocorticoids for the treatment of shock. Could it also be that high concentrations of glucocorticoids have immunosuppressive actions that contribute to the anergy that characterizes septic shock, possibly even resulting in an increased likelihood of subsequent infection of patients by a nosocomial organism? If so, how does one evaluate this "good" and "bad" mediator using a classic Koch-Dale meta-analysis for causality? For these reasons and others, a few of the chapter authors proposed creative modifications of the Koch-Dale-decision tree approach or alternative techniques for objective causality analysis (Table 1). On a chapter-by-chapter basis (with apologies to the authors), we have attempted to provide a subjective analysis of the available evidence to support a cause-and-effect relationship for each mediator, with our assessment of the relative strength of evidence (Table 2). In summary, among all the 24 chapters representing single mediators (e.g., histamine) or categories of mediators (e.g., interleukins), only nine were judged by us as supported by strong evidence for causal involvement in endotoxic/septic shock (Table 2). These included endotoxin, endorphins, complement, interleukins, tumor necrosis factor, cyclooxygenase metabolites, leukotrienes, platelet activating factor, and calcium. It is important to note that although some of the other mediators will ultimately be proven to play a causal role (e.g., nitric oxide), such judgment cannot be made at present (based upon the evidence provided in this handbook) due to insufficiency of data, limited species used, inability to measure the mediator in question, or due to other reasons that preclude a full meta-analysis (Table 1). Even if this handbook successfully represented all of the putative mediators of septic shock and clearly established or refuted their causal roles, our task would only have been partially successful. The relative "importance" ascribed to each of these mediators must be considered relative to the other mediators with which they interact over time. Ultimately, a therapeutic strategy for septic shock or SIRS based upon enhancing or diminishing mediator functions must involve a number of drugs as part of a temporal cocktail whose ingredients are modified over time to treat the changing medical circumstances.

IE. METHODS OF DETERMINING CAUSALITY In assembling this handbook, it is apparent that physiology may be on the verge of becoming the victim of its own success, in that the analytical techniques used to disclose the many putative mediators of septic shock have outpaced the integrative approaches that can make sense of this complexity for the practical physiologist or the clinician. The collective energies directed towards the meta-analyses in this handbook are therefore fueled in part by a restlessness, a dissatisfaction with outdated modes of explanation that are no longer adequate in our attempts to understand a complex web of causality in septic shock. Perhaps guidance can be found in the responses of physical scientists during the past century as they have been forced to adapt, clarify, and invent new concepts of explanation and causality to overcome theoretical or experimental dilemmas.2'6-14 Further guidance can be sought in the achievements of econometricians in modeling complex interactive phenomena even though in the real world it is impossible to observe the isolated effects of changes in single variables.7 James Clerk Maxwell pointed out that "To bring a quality within the grasp of exact science, we must conceive it as depending on the values of one or more variable quantities, and the first step in our scientific progress is to determine the number of these variables which are necessary and sufficient to determine the quality."20 Here, the clear disadvantages of septic shock studies compared with classical electrodynamics are apparent, for "new" mediators are constantly being

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TABLE 1 Evaluation of All Articles According to the Type of Analysis and the KOCH-DALE Criteria Considered

No.

Mediator

Metaanalysis with decision tree(s)

KOCH-DALE criteria considered

Classical review

Comments

1

Endotoxin

+

\-4

-

Meta-analysis for anti-LPS antibody (polyclonal and monoclonal) studies in animals and in man and meta-analysis on the characteristics of antiLPS antibodies that contribute to protective activity

2

Exotoxin

-

-

+

Meta-analysis not possible because (a) measurement in body fluids not possible and meaningless (b) scarcity of data

3

Histamine

+

1-4

-

Meta-analysis considered impossible for KOCH-DALE criterion 3 because of too many investigations: literature goes back to 1920

4

Serotonin

-

1-4

+

Meta-analysis should be possible

5

Catecholamines

+

1+2

-

Evidence proven for the rat only

6

Vasoactive intestinal peptide (VIP)

+

1+2

-

Only 7 original investigations published till now

7

Kallikrein-kinin system

-

l^t

+

Plasma not glandular kallikreinkinin systems are activated Kinin inhibition can have negative effects on the course of shock

8

Vasopressin

+

1-4

9

Endorphins

+

l^t

-

Evaluation of studies performed by one observer Publication bias cannot be excluded One decision tree for all four KOCH-DALE criteria constructed — 64 studies analyzed

10

Complement (C3a,C5a)

(+)

1-4

-

The article is structured according to decision tree criteria

527

Holaday, Neugebauer, and Can

TABLE 1 (continued) Evaluation of All Articles According to the Type of Analysis and the KOCH-DALE Criteria Considered

No.

Mediator

Melaanalysis with decision tree(s)

KOCH-DALE criteria considered

Classical review

Comments

11

Renin-angiotensin

+

1-4

12

Neuropeptide Y (NPY)

-

1-4

+

Literature is extremely scarce, Article is structured according KOCH-DALE criteria, NPY may have a beneficial effect

13

Interleukins 11, I12 I16

+ + +

1^ 1-4 1-2

-

11r I12, 116 also assessed for a causal role as a contributory or necessary determinant for multiple organ failure (MOF) Difficulties to assess them separately

14

Tumor necrosis factor (TNF)

-

1-4

+

Meta-analysis should be possible

15

Fibronectin

+

1—4

—

New faults (bias) may be produced by the metaanalysis approach Fibronectin is considered a protective non-specific mediator KOCH-DALE criteria had to be reversed

16

PMN-elastase Cathepsin B

—

1-4

+

All important decision tree questions fulfilled KOCH-DALE criteria 1-4 considered

17

Phospholipase A2

-

1-4

+

Meta-analysis difficult

18

EDRF/Nitric oxide

-

-

+

Summary of in vitro and in vivo data of current knowledge

Endothelin

-

-

+

19

Cylco-oxygenase metabolites (TXA2, PGF2a, PGE2, PGI2)

-

1-4

+

Systematical evaluation of the 4 KOCH-DALE criteria not performed

20

Peptidoleukotrienes (LTC4-E4)

-

1-4

+

Article analysed carefully decision tree and KOCHDALE criteria

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Handbook of Mediators in Septic Shock

TABLE 1 (continued) Evaluation of All Articles According to the Type of Analysis and the KOCH-DALE Criteria Considered

No.

Mediator

Metaanalysis with decision tree(s)

KOCH-DALE criteria considered

Classical review

Comments

21

Platelet-activating factor (PAF)

(+)

1-4

-

Method of meta-analysis in combination with decision tree to establish the causal role of single mediators is "ultra-rational" and not flexible enough. Authors suggest a probability matrix with the advantage of that it does not exclude any data from the system.

22

Oxygen free radicals (OFR)

-

1,2,4

+

In vivo existence difficult to demonstrate, use of OFRscavengers is the most commonly used approach Careful analysis of previous studies with decision trees presented is recommended

23

Calcium

+

1-4

24

Cortisol

-

\-4

Meta-analysis for all 4 KOCH-DALE criteria with decision trees performed +

Review on glucocorticoid therapy and cytokine interactions

identified and thus the number of variables operant in septic shock is always expanding. One possible multivariate approach to this problem might be to perform a cumulative meta-analysis of the relative contributions of incrementally considered mediators of a particular response within septic shock. In theory, the unexplained residual gap of each response can thereby be reduced to a negligible amount as more mediators are successively considered. As cumulative metaanalyses converge in their predictive power, the point is reached at which it is fruitless to expand further the universe of mediators. Assuming that the number of mediators may one day reach a plateau, i.e., become "close enough" to the number at which introducing individual variables offers negligible incremental predictive power, the challenge to the intuition to grasp this interaction will be formidable. Feynman, writing of physical science,3 has stated that

"the next great era of awakening of human intellect may well produce a method of understanding the qualitative content of equations. Today, we cannot. Today we cannot see that the water flow equations contain such things as the barber pole structure of turbulence that one sees between rotating cylinders. Today we cannot see whether Schrodinger's equation contains frogs, musical composers, or morality — or whether it does not."

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TABLE 2 Types of Evidence for a Cause and Effect Relationship of Single Mediators in Septic/ Endotoxic Shock and Their Relative Strength Type and quality of evidence according to KOCH-DALE criteria No. 1 2 3 4 5 6 7 8 9 10 11 12 13

14 15 16 17 18 19 20 21 22 23 24

Mediator Endotoxin Exotoxins Histamine Serotonin Catecholamines Vasoactive intestinal peptide (VIP) Kallikrein Vasopressin Endorphins Complement (C3a,C5a) Renin-angiotensin Neuropeptide Y Interleukin 11, I12 I16 Tumor necrosis factor (TNF) Fibronectin PMN-elastase, Cathepsin B Phospholipase A2 ERDF/Nitricoxide Endothelin Cyclo-oxygenase metabolites (TXA2, PGF2a,PGE2,PGI2) Peptidoleukotrienes (LTL4-E4) Platelet-activating factor (PAF) Oxygen free radicals (OFR) Calcium Cortisol

Strength of evidence (our estimation)

1/2

3

4

Strong

III 0 II II I I I I

III II II II ? ? ? ?

III 0 II I

+

I I II II III

? ? 0 II II

I II II

I II

I 0

II I

II II II III

II II II III

II I I II

II III

I I

II II

II I I III

I 0 I

II II I II

+

II

II

II

(+)

II

II

II

(+)

I

0

II

II II

II III

II I

Medium

Weak +

+ + + + + + (+) + + + (+) (+) (+) +

+ + + + +

+ (+) +

( ) Means absence of category III evidence. Our estimation on the strength of evidence in 1992 is based upon the critical evaluation of the different chapters presented by the authors of this book. Analysis according to the KOCH-DALE criteria 1-4. The quality of evidence is indicated by three different categories: III Evidence from at least one properly designed controlled clinical trial. II Evidence from at least one well designed clinical or experimental trial with controls (from cohort or case controlled analytic studies). I Evidence from descriptive studies without adequate controls, mainly animal studies. 0 Analogy, biological plausibility, intuition, etc. ? No information.

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Thus, assuming that a formal multivariate expression of interactions among mediators becomes feasible, there is little likelihood that those who read it could intuit the dynamic causal flow embodied within it. Among Feynman's many contributions was the integration of simple yet rigorous graphical methods ("Feynman diagrams") within theoretical particle physics; similarly, we may look forward to the extension of such methods into physiological depictions of causality. In this handbook, Braquet and colleagues5 have sketched out visual analogies in an effort to render explicit a common perception among contributors to this volume of the complex nature of causal flow between mediator release and in vivo outcome during septic shock. Because graphical analogies such as these are often helpful adjuncts to the psychological process of accepting an explanation of complex phenomena, it might be helpful to adapt approaches outlined in recent monographs by Tufte17-18 on the visual display of quantitative information, or Klir's text8 on systems problem solving, in an effort to ease perception of a model of causality and to catalyze future discussion on this issue (Figures 1).

IV. THE META-ANALYSIS APPROACH: STRENGTH AND LIMITATIONS IN DEFINING CAUSAL RELATIONSHIPS OF MEDIATORS IN SEPTIC SHOCK How do the analyses presented in this volume fit into the broader, more dynamic picture of causality that emerges? At the outset, several disclaimers are in order. First, Professor Chalmers (one of the fathers of meta-analysis) has already pointed out that the "decision tree" algorithm presented in the meta-analysis of the role of histamine as the mediator of septic/endotoxic shock by Neugebauer, Lorenz and colleagues12 "is not what is usually considered a meta-analysis, in that it does not combine data from the selected primary papers to arrive at conclusions that could not be arrived at without the combination".1 Nonetheless, this approach (again, to quote from Chalmers) "accomplishes what most meta-analyses recognize if they are any good, namely, that there are graven defects in the majority of original research projects that come to light only if an attempt is made to combine the data with those of other research." Still, an important distinction remains: the conclusion of the decision tree approach is simply to identify whether it is plausible that a specific mediator contributes to the pathogenesis of septic shock, but does not quantitate the strength of its contribution, much less the time course. Neugebauer, Lorenz, and colleagues clearly recognized this restriction, and have expressed the need for "a multivariate model recognizing the importance of a single mediator, such as histamine, in the presence of some or many other determining factors in septic shock".12'13 As pointed out earlier, a second limitation of meta-analysis as advanced in this volume is some degree of imprecision in determining which features and outcomes of septic shock are assessed according to the different experimental strategies used in each study (Table 1). Thus, possible outcomes range from survival versus mortality, to hypotension, to hyperthermia, etc. Hence, there will always be some degree of "fuzziness" to the inferred interactions among independent (i.e., putative predictive) variables when the dependent variable is not held uniform. Third, on the narrower matter of meta-analysis per se, Chalmers1 has also accurately pointed out that blinding of the meta-analysts as to the source and results of each article being evaluated can add materially to the quality of the process, a precaution that has not proven feasible for the contributors to this handbook. Finally, the accessibility of certain mediators to assay in plasma is questionable as an essential criterion for meta-analysis when their local or paracrine actions (e.g., nitric oxide) may be of primary importance, and their systemic levels are low and may not reflect accurately their primary biological target.

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FIGURE 1. An ST-function characterizing the metabolism of aclass of bacteria. This is but one example of a generative system used by Klir in his text of system problem solving; details may be found in Reference 8. (From Klir, G. J., in Architecture of Systems Problem Solving, Plenum Press, New York, 1985. With permission.)

V. ALTERNATIVE METHODS TO EVALUATE A CAUSAL ROLE OF A SINGLE MEDIATOR IN SEPTIC SHOCK A concise survey of models applicable to causality assessment has been provided above by Sitter and colleagues in masterful fashion in the preceding chapter.16 This section will expand upon their closing comments concerning the relevance of newer, nonlinear dynamic methods to causality assessment, for these appear to have importance for the future of this area (see below). One can find examples within other, nonbiological branches of the sciences wherein attempts to

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Handbook of Mediators in Septic Shock

describe and predict reality have led scientists to re-examine their views of causal mechanisms, often leading them to counter-intuitive and/or unforeseen models and conclusions. Turning again to physics, it has been recognized for over a century that while the two-body problem (calculating position and velocity at later times of two mutually orbiting point masses in space based upon their initial values) is capable of exact solution, the three-body problem is not. To quote from a recent essay on chaos theory in pharmacology, this failure of determinism is "because such systems are very sensitive to small changes in the initial conditions and to small perturbations".19 In a mathematical sense, the key feature of the three-body problem that renders it insoluble, and that is shared by other likewise deceptively simple but nonetheless insoluble problems, is its expression as a group of coupled nonlinear equations. To cite another well-known example that arose from the applied science of weather forecasting, the three simple coupled equations: dx/dt = -lOx + lOy dy/dt = 28x - y - z dz/dt = xy - 8/3 z were recognized by Lorenz to generate chaotic patterns when he applied repeated iterations as a means to refine initial estimates of their solutions.16 Tiny, apparently insignificant changes in the precise initial values for x, y, or z generated wild disparities in the resultant solutions, although these solutions, however unstable, tended over time to trace out a stable surface in space. This stable surface in space, or the corresponding surface towards which the system evolves in "phase space" (that is the derivative of state space), are termed "attractors". Mathematical techniques that consider the coefficients and exponents of the specific equations describing the system allow one to decide if an attractor exists for a particular system, and if so to define it a priori without the need for cumbersome numerical calculations. The paradigm of a linked set of variables (e.g., mediators) that evolves over time to define an attractor is more feasible as an explanatory model than is a conventional multivariate analysis, in which time need not be a parameter, and in which the variables are typically viewed as having a linear relationship. In biological systems, concentrations of mediators do not have a linear relationship to biological effect; in fact, as with most biological processes and pharmacological responses, the secretion of mediators, the administration of mediators, or the administration of drugs that affect the release of mediators results in a logarithmic (i.e., non-linear) response. As an added complication, the biological release of mediators is largely phasic, not tonic, and the nature of the relationship between mediator concentrations and biological response obviously changes over time, often irreversibly, as homeostasis breaks down. To capture catastrophic outcomes in a mathematical sense, we propose a novel formulation of the process. In such a formulation, as the system loses its homeostatic behavior, this loss occurs when the dimension of the system's attractor increases from zero or one to greater than two. Equivalently, the entropy of the system (roughly speaking, the rate at which information is lost from the system) increases as septic shock evolves. What sorts of steps might be taken to accomplish such a quantitative formulation of the linkages between mediators, other mediators, and outcome variables in septic shock? First, as emphasized earlier, there must be agreement upon and specific definition of the dependent outcome variables, whether these be cardiac output, regional blood flow, blood pressure, urine output, or probability of succumbing or surviving during an incremental time period. The process of deciding upon such dependent variables may well entail empirical construction of a surface in a multidimensional state space, such that within this surface (defined by cardiac output, regional blood flow, and so on) the probability of survival in the absence of treatment is less than 5 or 10%. Next, a series of dose:response and dose:variability-in-response studies must be accomplished

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to define the coefficients of the linear terms of the differential equations that link dependent to independent variables. Then, just as one might proceed empirically to investigate many-body problems in physics, one might seek to define the coefficients of nonlinear, double-product terms by co-administering mediators, blocking one mediator but not a second, or blocking pairs of mediators to examine the effect upon dependent variables, as well as holding one or another independent variable (cf. the Lorenz equations above). Beyond this, selected triple-product terms could be explored for those variables that demonstrate the greatest influence in single- or doublevariable studies. While admittedly this task appears daunting, advances in the techniques of cumulative meta-analysis and statistical forecasting, and falling costs of hardware and software with which to accomplish modeling, may well render it feasible in the near future. Such tasks have already been successfully undertaken in disciplines as disparate as ecology, weather forecasting, and econometrics.

VI. WHERE TO GO IN THE FUTURE Nelkin11 has called attention to the dilemma that "in most problems in physics, we have a subtle mixture of uncertainty about the validity of the equations we study, and uncertainty about how to solve them". This dilemma is magnified for those physiologists and clinicians who seek to define and manipulate the causal flow between sepsis (or endotoxemia) and shock. Also at issue is which formalism to use to generate which equations to be solved by which method. Equally unsettling is the lack of a clear explanatory structure to account for the reversibility of phenomena involving one mediator studied in vitro or on a small scale in vivo, and the irreversibility, even to mortality, of individual instances of the cascade of events that occur in septic shock in the whole animal or patient. The transition from microscopic reversibility to macroscopic irreversibility is at the heart of modern thermodynamics, and has been the subject of numerous and ongoing reviews from the viewpoint of dynamics9 and physical chemistry15 as well. At present, the language and mathematical formalism of nonlinear dynamics is very much in vogue, for several reasons. Chaotic attractor theories: provide a rigorous model for the transition between regular and irregular behavior in a physiological system, introduce time within mathematical descriptions of the behavior of the system, have flexibility by virtue of the distinct characteristics of distinct attractors, and allow defined pathways by which a system may approach chaos.3 It is particularly relevant to septic shock that such formalisms lend themselves to descriptions of bifurcations between system states in which continuous oscillations of one or more variables occur (such as one may see in health as regards hormone secretion, cardiac muscle depolarization, or baroceptor function) and states in which oscillations either build up uncontrollably or end. The evolution of mediator studies in septic shock, from the initial identification of circulating factors to a questioning of the philosophical underpinnings of causality in the biological sciences, has been rapid and impressive. This movement will predictably take on unpredictable forms as advances in the mathematical modeling of complex realities are applied to a field where once they would have been viewed as unrelated, if not completely alien. As Heisenberg pointed out,4 "It is probably true quite generally that in the history of human thinking the most fruitful developments frequently take place at those points where two different lines of thought meet. These lines may have their roots in quite different times...or religious traditions; hence if they actually meet, that is, if they are at least so much related to each other that a real interaction can take place, then one may hope that new and interesting developments may follow."

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ACKNOWLEDGMENT I thank Maria M. Ruiz and Wendy A. Mills for their assistance in preparing this manuscript.

REFERENCES 1. Chalmers, T. C., The quality of primary and secondary research and meta-analysis, Theor. Surg., 2, 40, 1987. 2. Gushing, J. T. and McMullin, E., Philosophical Consequences of Quantum Theory: Reflections on Bell's Theorem, University of Notre Dame Press, Notre Dame, Indiana. 1989. 3. Garfinkel, A., A mathematics for physiology, Am. /. Physiol., 245, (Regulatory Integrative Cornp. Physiol, 14), R455, 1983. 4. Heisenberg, W., Physics and Philosophy, Harper, New York, 1958. 5. Hosford, D., Koltai, M., Paubert-Braquet, M., and Braquet, P., Analysis of platelet-activating factor in endotoxic shock and sepsis using probability matrix: a critique of meta-analysis, in Handbook of Mediators in Septic Shock, Neugebauer, E. and Holaday, J. W., Eds., CRC Press, Boca Raton, FL, 1993. 6. Jammer M., The Conceptual Development of Quantum Mechanics, McGraw-Hill, New York, 1966. 7. Kalman, H. E., Identifiability and problems of model selection in econometrics, in Advances in Econometrics, Hildenbrand, W., Cambridge University Press, London, 1982. 8. Klir, G. J., Generative systems, Architecture of Systems Problem Solving, Plenum Press, New York, 1985. 9. Lebowitz, J. L., Microscopic dynamics and macroscopic laws, Horton, C.W., Reichl, L.E., Szebehely, V.G., Longtime Prediction in Dynamics, John Wiley, New York, 1983. 10. Members of the American College of Chest Physicians/Society of Critical Care Medicine consensus conference committee, Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis, Crit. Care Med., 20, 864, 1992. 11. Nelkin, M., In what sense is turbulence an unsolved problem?, Science, 255, 566, 1992. 12. Neugebauer. E., Lorenz, W., Maroske, D., Barthlen, W., and Ennis, M., The role of mediators in septic/ endotoxic shock: a meta-analysis evaluating the current status of histamine, Theor. Surg., 2, 1, 1987. 13. Neugebauer, E., Rixen D., and Lorenz, W., Histamine in septic/endotoxic shock, in Handbook of Mediators in Septic Shock, Neugebauer, E. and Holaday, J.W., CRC Press, Boca Raton, FL, 1993. 14. Pagels, H. R., Perfect Symmetry, New York, Simon & Schuster, 1985. 15. Prigogine, I., From Being to Becoming: Time and Complexity in the Physical Sciences, Freeman, San Francisco, 1980. 16. Sitter, H., Lorenz, W., Klotter, H. J., and Lill, H., Models for causality assessment, in Handbook of Mediators in Septic Shock, Neugebauer, E., and Holaday, J. W., Eds., CRC Press, Boca Raton, FL.1993. 17. Tufte, E. R., The Visual Display of Quantitative Information, Graphics Press, Cheshire, Connecticut, 1983. 18. Tufte, E. R., Envisioning Information, Graphics Press, Cheshire, Connecticut, 1990. 19. Van Rossum, J. M., de Bie JEGM, Chaos and illusion, Trends Pharmacol. Sci., 12, 379, 1991. 20. Winfree, A. T., The Geometry of Biological Time, New York, Springer, 1980.

Appendix

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Appendix

Discussion forum Meta-analysis: from classical review to a new refined methodology Introduction to the discussion forum about an example of meta-analysis in basic surgical research: The role of mediators in septic/endotoxic shock [Theor Surg (1987) 2:1-28] E.Neugebauer1 andW.Lorenz2 1 Biochemical and Experimental Division, 2nd Department of Surgery, University of Cologne, Ostmerheimer Strasse 200, D-5000 Cologne 91, Federal Republic of Germany Institute of Theoretical Surgery, Centre of Operative Medicine I, Philipps University, Baldinger Strasse, D-3550 Marburg/Lahn, Federal Republic of Germany

Key words: Meta-analysis - Mediators - Histamine

Introduction Meta-analysis is a term used for a quantitative method of combining and integrating findings from different independent research studies. It takes a more structured approach to literature review than does the traditional narrative review and in this way can be more helpful in evaluating scientifically the accumulated evidence. However, the methodolgy for applying this research strategy to clinical questions, particularly to biomedical research, is still evolving. Our example of a meta-analysis evaluating the current status of the mediator histamine as a causal chemical factor in the pathogenesis of septic/endotoxic shock is, to our knowledge, the first one in basic shock research. Besides histamine, there are many other mediators said to be causally associated with the pathogenesis of the septic shock syndrome. The results of studies on other individual mediators are as contradictory as those of studies on the mediator histamine [10]. However, little effort has been made to address causality in septic, or more generally in circulatory shock using a disciplined and structured analysis of mediator mechanisms and therapies. Our suggestion, therefore, was to reanalyse the current literature on each of the mediators by asking experts in the field to apply the , , . , . , , . . . methods of meta-analysis and the decision-tree approach used in our paper [10].

After publication of our article [10] and presentation of the methods and results to different groups and audiences in basic and clinical research (initially at the First International Conference on Shock (Montreal, June, 1987), [9] a controversial discussion started. This circumstance, as well as different opinions on the usefulness and limitations of meta-analysis, together with some recent methodological developments in this field, encouraged us to invite widely accepted scientists from different basic and clinical research areas to discuss this new approach to the mediator field. A list of contributors with their main specialities is given in Table 1. Table 1. Contributors to the discussion forum, and their main working disciplines Contributor

Main working disciplines

Oettinger, W. General surgery, clinical shock research Carr, D.B. Anaesthesiology, causality analysis Hinshaw.L.B. Physiology, clinical and experimental shock research, histamine Green, J.P. Pharmacology, receptor research, neurotransmitters, histamine Chalmers,!.C. Statistics, controlled clinical trials, metaanalysis Hilden J -Biostatistics, medical decision analysis, causality analysis Wulff.H.R. Philosophy (epistemology) of medicine, clinical methodology in allfields,internal 'medicine

Reprinted from Theoretical Surgery, 4, 79-85, 1989. With the kind permission of Springer-Verlag © 1989.

536

Decision tree and main results of the meta-analysis on histamine release in septic/endotoxic shock To enable the readers of the subsequent discussion forum to understand and to evaluate the comments properly, a short description of the scope, the methods and the main results of our meta-analysis [10] is given here. To establish a causal relationship between mediator release and shock symptoms several noncausal associations must be excluded, such as bias in selection of patients and animals, bias in measurements, chance and confounding bias. Our model was a decision tree (Fig. 1) with a sequence of hierarchical questions (test nodes) and binary branches (yes and no answers). Published studies investigating the presence of histamine release in septic/endotoxic shock in vivo and its absence in a state of health were evaluated by the criteria defined by the test nodes in the tree. All criteria and methodological standards at the test nodes in the decision tree (Fig. 1) were tabulated and set up in detail before the analysis was started. The primary data base for retrieval of studies published on histamine release in septic/endotoxic shock was the manual research library system of the Institute of Theoretical Surgery in Marburg; it consisted of 45 352 publications on histamine in health and disease at the time of analysis. To minimize publication bias

Handbook of Mediators in Septic Shock

we tried to include all available publications using different research library systems and secondary literature cited in original papers and review articles. A first retrieval on the more general topic of "histamine in shock conditions" produced 1222 publications; a second retrieval on the specific topic of "histamine release in septic endotoxic shock conditions in vivo" revealed only 25 publications, of which 23 on seven different species reported sufficient numerical details for the meta-analysis with the decision tree. All these papers were listed and then analysed systematically according to the criteria set up in the decision tree, This analysis was performed by a consensus conference of the whole research group. The overall results of this analysis are summarized in Table 2. Because no study reached the end of the decision tree process without failures, their shortcomings were further evaluated in a second step. After the main shortcomings of each study had been listed, their relevance for a 'yes' or 'no' decision was carefully evaluated. As a result, only four of the studies (two in dogs and two in rabbits) were judged reliable under conditions of uncertainty for demonstration of histamine release in septic/ endotoxic shock. However, in one of the two studies in dogs, histamine release is considered an effect rather than a cause of shock [6] and the second study demonstrated a close relationship of histamine release and shock symptoms in only one dog [14]. In the eval-

537

Appendix Table 2. Main results of the meta-analysis of studies on histamine release in septic/endotoxic shock [10]; results from 23 studies of 7 different species

the therapeutic effectiveness or to plan is little known . , , . . . , . . ,. • , , in the field of basic clinical research. To overcome the weakness of previous methods No study reached the end of the decision tree without failures - Only 26% (6/23) of the studies used a reliable assay (test for research integration, clear, generally accepted node !) guidelines are mandatory. Each meta-analysis should - Only half of the studies used a correct sample preparation be conducted like a scientific experiment, beginning procedure (test node 2) or the relevant body fluid (test node 3) with a dear plan of ,he question to be answered and - Only 35% (8/23) of the studies fulfilled a minimum standard tne methods to be employed [8]. At present, however, of study design (test node 5) there {& nQ single «correct» method for performing a - Only 35% (8/23) of the studies demonstrated reliably a hismeta-analysis [8]. Several attempts have been made to tamine release response caused by shock itself (test node 8) j^ ^ ^ methodologica, issues rf each meta.

uation of the two studies in rabbits [3, 12] all the shortcomings except the clinical relevance of the species itself were considered capable of being compensated for. Unlike human beings, rabbits are relalively insensitive to released histamine and have atypically high levels of histamine in their platelets. This meta-analysis suggested that, despite decades of histamine research, we still have no acceptable answer to whether histamine plays a pathogenetic role in septic/ endotoxic shock. We therefore doubt whether there is any more certainty for any other "modern" mediators. Meta-analysis: methodological elements and critical issues In 1976, Glass [5] was the first to refer to this type of research as meta-analysis. In recent years an increasing number of publications have appeared in the medical literature that attempt to evaluate and to combine results of previous studies [15]. Presently, the methodology ist most often used in the field of therapy evaluation from clinical trials, either to draw conclu-

sjons about

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analysis. Two recent examples were the Workshop on "Methodological issues in overviews of randomized chnicaltrias , sponsored by the Nat onal Heart, Lung and Blo od , IT'?'6 f t ?f',T,0ofm! T,?' 1 ethes f7ersalll ,f' ,Maryland'f ^F'16' ^ [^' ^ the V es Meeting of the Meta-analysis Cooperative Gro u , P< °cter 1985< m Fr,ance M' Recently> Sacksf et a '' ^ evaluated the quahty of 86 meta-analyses of ™domized controlled trials m the English-language llterature Usm a sconn me thod that 8 S f c°nsldered the most im ortant elements f m ta anal sis makm P ° . r!, - y o ( g *erePort a meta-meta-analysis [2]) .Figure 2 presents the generally accepted issues, which are summarized in the following paragraphs, accompanied by a list of selected references (see Appendix).

1. Aim Each meta-analysis should be conducted like a scientific experiment, beginning with a clear plan of the question to be answered and the methods to be employed. Almost any clinical question or controversy can be subjected to meta-analysis. In current practice, meta-analyses proceed from one of two different approaches: (a) to increase the precision of al-

Fig. 2. Methodological elements of a meta-analysis (modified from [8])

538 ready formulated findings from a series of trials, and (b) to test a preformulated hypothesis relative to the efficiency of a treatment or an intervention strategy for the sake of making decisions [1]. 2. Literature search A valid meta-analysis includes as many relevant studies as possible. The authors should provide details of their search procedures. At present it is insufficient to rely solely on computer searches of the literature, since they may yield less than two thirds of the relevant studies [4]. Efforts to minimize this "publication bias" include working from references of published studies, searching data bases of unpublished material and questioning experts in the particular field. The meta-analysis will be clear only if the studies are chosen according to predefined inclusion and exclusion criteria, the rationale of which must be stated. Each meta-analysis should list the studies analysed, as well as those excluded and the reasons for exclusion [13]. 3. Study characteristics ., , j. i_ i_ ., j j , • After the studies have been collected and selected, it is helpful to record key descriptive characteristics of eocAtAid? onfeta abstract fora;. The reader needs to be convinced that the results of separate tnals have been meaningfully combined. Common methodological characteristics included in meta-analysis are type and year of publication; study design; type, dosage, route, frequency and duration of treatment; and control, sample size, subject loss and randomization. In general, the meta-analyst should note any deferences between the primary studies and discuss how these differences affect the conclusions. 4 Study outcomes Similar data abstract forms are useful for recording study outcome characteristics. The outcome of interest , ,. . . ,, , may be measured by a continuous variable such as blood pressure, by categorical variables such as mortality or complications, by an ordinal variable such as tumour stage or by time-related variables expressed in life tables. In performing data abstraction it is advisable to record raw numbers instead of proportions [8]. The data abstraction process is a subject of potential bias. The ideal way to control this type of bias is to have the data extracted by more than one observer, with each observer blinded to the various treatment groups through a coded photocopying process, and then to measure the interobserver agreement [13]. 5. Statistical analysis Meta-analysis includes transformation of multiple study findings into a common matrix. The several

Handbook of Mediators in Septic Shock methods used are controversial. Whatever statistic is used, a systematic quantitative procedure to accumulate results across studies should include: (a) a summary of descriptive statistics across studies and the averaging of those statistics; (b) calculation of the variance of a statistic across studies (i.e. tests of heterogeneity); (c) correction of the variance by substracting sampling error; (d) correction in the mean and variance for stud y artefacts other than sampling; and (e) comparison of the corrected standard deviation with the mean to assess the size of the potential variation across studies 7 t ]- However, statistical analysis is not nl or the ° yf statistician - the experienced physician should als be asked or a dinical ° f interpretation and about the significance of the results obtained! 6. Sensitivity analysis Sensitivity analysis is a subsequent important element meta-analysis methodology. Here the meta-analyst asks how sensitively the meta-analysis results react to changes in the way that the meta-analysis was done. The results on the same set of data may vary depend^ and Qn ; on ^ Qverall number of rf whether certain ^^ sub of tients or other ; rtam variables have been excluded or ch d [1 3 ] . ThuSjSensitivityanalysiscanhe ip t oresolveclin• , t . in

7

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y quality assessment ^^ of ^ tQ b£ combined shou,d bg assessed and introduced into ,he sensitivity

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anal js The resuki condusion win be less re|iable B jf th *originaltrials are poor Severa,methods exis, for

examining quantitatively the studies published (see reference list). . a. Conclusions and recommendations The extraction and pooling of data from published remethodology of meta-analysis is time consuming and tedious. At the end of the process the meta-analyst must put the results in perspective and, if the data are not decisive, should recommend appropriate future studies,

ports w j tn t h e

Conclusion xhe many poorly understood problems make metaanalysis difficult to apply properly. Misuse of this powerful tool could give misleading conclusions. However, it offers opportunities for analyses that are not possible from single studies through examination

539

Appendix of covariables and aspects of study design and analysis. Meta-analysis is a methodolgy that requires testing and empirical evaluation in different fields of clinical and basic research. Our meta-analysis, as the first in the field of basic shock research, differs in some aspects from the methodolgy outlined above. The element of adecisiontreeisanewoneand,ofcourse,needstobe examined. For this purpose other mediators will be , , .' . - u r - i j T T . analysed in the same way by experts in the field. The results will be published in a Handbook of mediators in septic shock (E. Neugebauer, J. Holaday, eds.) which will be available within the next 2 years.

References 1. Boissel JP, Perrieux JC, Panak E, Blanchard J, Leizorovicz A, Sacks H and the Meta-analysis Cooperative Group (1989) Guidelines for meta-analysis of clinical trials. (Submilled for publication) 2. Brennan S (1987) Meta-analysis: letter to the editor. N Engl J Med 317:575 3. Davis RB, Bailey WC, Hanson P (1963) Modification of serotonin and histamme release after E. coh endotoxm admmstration. Am J Physiol 205:560-566 4. Dickersin K, Hewitt P, Mutch I, Chalmers I, Chalmers TC (1985) Perusing the literature: comparison of MEDLINE searching with a perinatal trials data base. Controlled Clin Trials 6:306-317 5. Glass GV (1976) Primary, secondary and meta-analysis of research. Educ Res 5:3-8 6. Hinshaw LB, Jordan MM, Vick JA (1961) Mechanism of histamine release in endotoxic shock. Am J Physiol 200:987-989 7. Hunter JE, Schmidt FG, Jackson GB (1982) Meta-analysis: cumulating research findings across studies. California Sage Publications, Beverly Hills 8. L'Abbe KA, Detsky AS, O'Rourke K (1987) Meta-analysis in clinical research. Ann Intern Med 107:224-233 9. Neugebauer E, Lorenz W (1988) Causality in circulatory shock: strategies for integrating mediators, mechanisms undtherapies.ProgClinBiolRes264:295-305 10. Neugebauer E, Lorenz W, Maroske D, Barthlen W, Ennis M (1987) The role of mediators in septic/endotoxic shock. A meta-analysis evaluating the current status of histamine. TheorS e2'l-28 11. Proceedings of~"Methodological Issues in Overviews of Randomized Clinical Trials" (1987) Stat Med 6:217-409 12. Rampart M, Bulf H, Herman AG (1982) Contribution of complement activation to the rise in blood levels of 6-oxoprostaglandin Fl during endotoxin-induced hypotension in rabbits. Eur J Pharmacol 79:91-99 13. Sacks HS, Berrier J, Reitman D, Ancona-Berk VA, Chalmers TC (1987) Meta-analyses of randomized controlled trials. N Engl J Med 316:450-455 14. Weil MH, Spitz WW (1957) A comparison of shock due to endotoxin with anaphylactic shock. J Lab Clin Med 50:501515 15. Wolf FM (1987) Meta-analysis: letter to the editor. N Engl J Med 317:576 Received 7 April 1989

Appendix suggested references for further information on methodologiCal elements of meta-analysis: L Am

°f ^'"-"""'y^ 1- Cooper HM (1982) Scientific guidelines for conducting te grativ e " ^ "™I*SJ Rfev Educ Res 52:291~302D 2. ™ t - Jackson GB (1980) Methods for mtegrative reviews. Rev Ec)uc Res 50.433.4^0 3. Louis TA, Fineberg HV, Mosteller F (1985) Findings for public health from meta-analysis. Ann Rev Public Health *>: 1-20

2. Literature search l Cooper HM (1982) Scientific guidelines for conducting integrative research reviews. Rev Educ Res 52:291-302 2 Dickersin K , Hewitt R, Mutch L, Chalmers I, Chalmers TC (1985) Perusing the literature: comparison of MEDLINE searching with a perinatal trials data base. Controlled Clin Trials 6.306-317 3 Haynes DB? McKibbon KA, Walker CJ, et al. (1985) Computer searching of the medical literature: an evaluation of MEDLINE search systems. Ann Intern Med 103:812-816 4 Hewitt P, Chalmers TC (1985) Using MEDLINE to peruse the literature. Controlled Clin Trials 6:75-83 5 Hewit, P chaimers TC (1985) Perusing the literature: methods of accessing MEDLINE and related data bases, Controlled Clin Trials 6:168-177 6 Horowitz GL, Bleich HL (1981) Paperchase: a computer program to search the medical literature. N Engl J Med 305: 924-930 7 Poynard T _ C onn HO (1985) The retrieval of randomized clinical trials in liver disease from the medical literature: a comparison of MEDLARS and manual methods. Controlled Clin Trials 6 • 217-279 8 Stock WA ORun MA Hanng MJ Miller w K inney C, Ceurvorst RW (1982) Rigor in data synthesis: a case of reliability jn meta-analysis. Educ Res 11:10-14 3. Study characteristics „ _ „ ,.„„ L Glass GV McGraw B Smith MC 1981 ' < > Meta-analysis in sodal research Sa e BCT Hllls - 8' »ly 2 L Abbe KA Detsk AS ' ' ' y - O'Rourke K (1987) Meta-analysis in clinical research. Ann Intern Med 107:224-233 3 ' Sacks MS, Berrier J, Reitman D, Berk AA, Chalmers TC Meta-analyses of randomized controlled trials. N tnglJ Med 316:450-455 4 S m l t h MC Glass GV ' ' - ™M" TI Ben f fits of psychotherapy. Johns Hopkins University Press, Baltimore 4 S f

,

oulcomes

1. Cochran WG (1954) The combination of estimates from different experiments. Biometrics 10:101-129 2. Furberg CD, Morgan TM (1987) Lessons from overviews of cardiovascular trials. Stat Med 6:295-303 3. Goldsmith JR, Beeser S (1984) Strategies for pooling data in occupational epidemiological studies. Ann Acad Med Singapore 13 [Suppl 2]: 297-307 4. Sacks HS, Chalmers TC, Berck AA, Reitman D (1985) Should mild hypertension be treated? An attempted meta-

540 analysis of the clinical trials. Mt Sinai J Med (NY) 52:265270 5. Shapiro DA, Shapiro D (1983) Comparative therapy outcome research: methological implications of meta-analysis. J Consult ClinPsychol 51-.42-53 6. Solomkin JS, Meakins JL, Allo MD, Dellinger EP, Simmons RL (1984) Antibiotic trials in intra-abdominal infections. Ann Surg200-.29-39 7. Wagner G (1975) Datenkontrolle. In: Roller S, Wagner G (eds) Handbuch der medizinischen Dokumentation und Datenverarbeitung. Schattauer, Stuttgart 8. Yusuf S, Peto R, Lewis J, Collins R, Sleight P (1985) Beta blockade during and after myocardial infarction: an overview of the randomized trials. Prog Cardiovasc Dis 27,5: 335-371 5. Statistical analysis

Handbook of Mediators in Septic Shock entific discipline. II. Replicate variability and comparison of studies that agree and disagree. Stat Med 6:733-744 5. Demets DL (1987) Methods for combining randomized clinical trials: strengths and limitations. Stat Med 6:341-348 6. Dickersin K, Chan S, Chalmers TC, Sacks HS, Smith H (1987) Publication bias and clinical trials. Controlled Clin Trials 8:343-353 7. L'Abbe KA.Detsby AS, O'RourkeK (1987) Meta-analysis in clinical research. Ann Intern Med 107:224-233 8. Rosenthal R (1978) Combining results of independent studies. Psychol Bull 85:185-193 9. Rosenthal R (1979) The "file drawer problem" and tolerance for null results. Psychol Bull 86:638-641 10. Simes RJ (1987) Confronting publication bias: a cohort design for meta-analysis. Stat Med 6:11-29 7

- Study quality assessment

1. Aalen OO (1988) Heterogeneity in survival analysis. Stat 1- Blackburn BA, Smith H, Chalmers TC (1982) The inadequate evidence for short hospital stay after hernia or Med7:1121-1137 2. Abraham IL, Schultz IIS (1983) Univariate statistical modvaricose vein stripping surgery. Mt Sinai J Med (NY) 49: els for meta-analysis. Nurs Res 32:312-315 383-390 2 3. Abt K (1987) Descriptive data analysis: a concept between - Chalmers TC, Smith H, Blackburn B, Silverman B, confirmatory and exploratory data analysis. Methods Inf Schroeder B, Reitman D, Ambroz A (1981) A method for Med 26:77-88 assessing the quality of a randomized control trial. Con4. Bulpitt CJ (1987) Confidence intervals. Lancet 1:494-497 trolled Clin Trials 2:31-49 3 - DerSimonian R, Charette LJ, McPeek B, Mosteller F 5. Cox DR (1970) The analysis of binary data. Chapman and Hall, London, pp 1-142 (1982) Reporting on methods in clinical trials. N Engl J Med 306: 6. Gelber RD, Goldhirsch A (1987) Interpretation of results 1332-1337 4 from subset analyses within overviews of randomized clini- Emerson JD, McPeek B, Mosteller F( 1984) Reporting clinical trials in cal trials. Stat Med 6:371-378 general surgical journals. Surgery 95:572-579 5 7. Hedges LV (1982) Estimation of effect size from a series of - Goodman CS (1985) Guide to comparative clinical trials, Assessing medical technology. In: Oilman AG, Goodman independent experiments. Psychol Bull 92:490-499 Ls Ral1 TW 8. Hedges LV, Olkin I (1985) Statistical methods for meta' Murad F (eds) The pharmacological basis of analysis. Academic Press, New York, pp 1-369 therapeutics. Macmillan, New York, pp 490-501 6 9. Hedges LV, Olkin I (1984) Nonparametric estimators of - Pocock SJ, Hughes MD, Lee RJ (1987) Statistical problems in the reporting of clinical trials. N Engl J Med 317:426-432 effect size in meta-analysis. Psychol Bull 96:573-580 7 10. Kraemer HC, Andrews G (1982) A nonparametric tech- Spilker B (1984) Systems of evaluating published data. In: Guide to nique for meta-analysis effect size calculation. Psychol Bull clinical interpretation of data. Raven, New York 8 91:404-412 - Thacker SB (1985) Quality of controlled clinical trials. The case of imaging ultrasound in obstetrics: a review. Br J 11. Littell RC, Louv WC (1981) Confidence regions based on methods of combining test statistics. J Am Stat Assoc 76: Obstet Gynaecol 92:437-444 125-130 12. MantelN,HaenszelW(1959)StatisticalaSpectsoftheanalg Conclusions and recommendations ysis of data from retrospective studies of disease. JNCI 22:719-748 L Louis TA Fineberg Hv Mosteller F (1985) Findings for 13. Rosenthal R, Rubin DB (1979) Comparing significance blic health from meta-analyses. Annu Rev Public Health levels of independent studies. Psychol Bull 86:1165-1168 g. j_2Q 14. Tarone RE (1981) On summary estimators of relative risk. 2 Parloff M (1979) Can psychotherapy research guide the polJ Chronic Dis 34:463-468 icy maker? Am Psychol 4:296-306 3. Sacks HS, Berrier J, Reitman D, Berk AA, Chalmers TC (1987) Mela-analyses of randomized controlled trials. N 6. Sensitivity analysis EnglJ Med 316:450-485 4. Wittes RE (1987) Problems in the medical interpretation of 1. Begg CB (1985) A measure to aid in the interpretation of overviews. Stat Med 6:269-276 published clinical trials. Stat Med 4:1-9 2. Chalmers TC, Celano P, Sacks HS, Smith H (1983) Bias in treatment assignment in controlled clinical trials. N Engl J 9. Examples of meta-analyses Med 309:1358-1361 3. Chalmers TC, Levin H, Sacks HS, Reitman D, Berrier J, 1. Bassan MM, Shalev O, Eliakim A (1984) Improved progNagalingam R (1987) Meta-analysis of clinical trials as a scinosis during long-term treatment with beta-blockers after entific discipline. I. Control of bias and comparison with myocardial infarction: analysis of randomized trials and large co-operative trials. Stat Med 6:315-325 pooling of results. Heart Lung 13:164-168 4. Chalmers TC, Berrier J, Sacks HS, Levin H, Reitman D, 2. Baum ML, Anish DS, Chalmers TC, Sacks HS, Smith H, Nagalingam R (1987) Meta-analysis of clinical trials as a sciFagerstrom RM (1981) A survey of clinical trials of antibiot-

541

Appendix ic prophylaxis in colon surgery: evidence against further use of no-treatment controls. N Engl J Med 305:795-799 3. Canner PL (1983) Aspirin in coronary heart disease. Isr J Med Sci 19:413-423 4. Chalmers TC, Malta RJ, Smith H, Kunzler AM (1977) Evidence favoring the use of anticoagulants in the hospital phase of acute myocardial infarction. N Engl J Med 297:1091-1096 5. Colditz GA, Tuden RL, Oster G (1986) Rates of venous thrombosis after general surgery: combined results of randomised clinical trials. Lancet 1:143-146 6. Collins R, Langman M (1985) Treatment with histamine H2-antagonists in acute upper gastrointestinal hemorrhage. N Engl J Med 313:660-666 7. Collins R, Yusuf S, Peto R (1985) Overview of randomised trials of diuretics in pregnancy. Br Med J 290:17-23 8. Collins R, Scrimgeour A, Yusuf S, Peto R (1988) Reduction in fatal pulmonary embolism and venous thrombosis by perioperative administration of subcutaneous heparin. N EnglJMed318:1162-1173 9. Conn HO, Blitzer BL (1976) Nonassociation of adrenocoricosteroid therapy and peptic ulcer. N Engl J Med 294: 473-479 10. Conn HO, Poynard T (1985) Adrenocorticosteroid administration and peptic ulcer: a critical analysis. J Chronic Dis 38:457-468 11. Cuzick J, Stewart H, Feto R, Baum M, Fisher B, Host H, Lythgoe JP, Ribeiro G, Scheurlen H, Wallgren A (1987) Overview of randomized trials of postoperative adjuvant radiotherapy in breast cancer. Cancer Treat Rep 71:15-29 12. Detsky AS, Baker JP, O'Rourke K, Goel V (1987) Perioperative parenteral nutrition: a meta-analysis. Ann Intern Med 107: 195-203 13. Felson DT, Anderson J (1984) Evidence for the superiority of immunosuppressive drugs and prednisone over prednisone alone in lupus nephritis. N Engl J Med 311:1528-1533 14. Freiman JA, Chalmers TC, Smith H, Kuebler RR (1978) The importance of beta, the type-II error and sample size in the design and interpretation of the randomized control trial. N Engl J Med 299:690-694 15. Himel HN, Liberal! A, Gelber RD, Chalmers TC (1986) Adjuvant chemolherapy for breast cancer. A pooled estimate based on published randomized control trials. JAMA 256:1148-1159 16. Lam W, Sacks HS, Sze PC, Chalmers TC (1987) Meta-analysis of randomised controlled trials of nicotine chewinggum. Lancet II: 27-29 17. Leiboff AR, Soroff HS (1987) The treatment of generalized peritonitis by closed postoperative peritoneal lavage. Arch Surg 122:1005-1010 18. Leizorovicz A, Boissel JP (1983) Oral anticoagulant in patients surviving myocardial infarction. A new approach to old data. Eur J Clin Pharmacol 24:333-336 19. Messer J, Reitman D, Sacks HS, Smith H, Chalmers TC (1983) Association of adrenocorticosteroid therapy and peptic-ulcer disease. N Engl J Med 309:21-24 20. Patel B, Kloner RA (1987) Analysis of reported randomized trials of streptokinase therapy for acute myocardial infarction in the 1980s. Am J Cardiol 59:501-504 21. Sacks HS, Chalmers TC, Berk AA, Reitman D (1985) Should mild hypertension be treated? An attempted metaanalysis of the clinical trials. Mt Sinai J Med (NY) 52:265270

22. Tran ZV, Weltman A (1985) Differential effects of exercise on serum lipid and lipoprotein levels seen with changes in body weight. JAMA 254:919-929 23. Tran ZV, Weltman A, Glass GV, Mood DP (1983) The effects of exercise on blood lipids and lipoproteins: a metaanalysis of studies. Med Sci Sports Exerc 15:393-402 24. Yusuf S, Peto R, Lewis J, Collins R Sleight P (1985) Beta blockade during and after myocardial infarction: an overview of the randomized trials. Prog Cardiovasc Dis 27:335371 10. Review articles and books on meta-analysis (selection) 1. Cooper HM (1987) The integrative research review. A systematic approach. Appl Soc Res Methods Ser 2:1-44 2. Collins R, Gray R, Godwin J, Peto R (1987) Avoidance of large biases and large random errors in the assessment of moderate treatment effects: the need for systematic overviews. Stat Med 6:245-250 3. Curlette WL, Cannella KS (1985) Going beyond the narrative summarization of research findings: the meta-analysis approach. Res Nurs Health 8:293-301 4. Einerson TR, McGhan WF, Bootman JL, Sabers DC (1985) Meta-analysis: quantitative integration of independent research results. Annu J Hospital Pharmacy 42:19571964 5. L'Abbe KA, Detsky AS, O'Rourke K (1987) Meta-analysis in clinical research. Ann Intern Med 107:223-224 6. Light RJ, Pillemer DB (1984) Summing up. The science of reviewing research. Harvard University Press, London, pp 1-191 7. Peto R (1987) Why do we need systematic overviews of randomized trials? Stat Med 6:233-240 8. Proceedings of the Workshop on methodology issues in overviews of randomized clinical trials (1987) Stat Med 6: 217-409 9. Rosenthal R (1978) Combining results of independent studies. Psychol Bull 85:185-193 10. Rosenthal R (1984) Meta-analytic procedures for social research. Appl Soc Res Methods Ser 6:1-149 11. Smith MC, Naftel DC (1984) Meta-analysis: a perspective for research synthesis. J News Scholarship 16:9-13 12. Strube MJ, Hartmann DP (1982) A critical appraisal of meta-analysis. Br J Clin Psychol 21:129-139 13. Taylor Halvorsen K (1986) Combining results from independent investigations. Meta-analysis in medical research. In: Bailar JC III, Mosteller F (eds) Medical uses of statistics. NEJM Books 14. Thacker SB (1988) Meta-analysis: a quantitative approach to research integrations. JAMA 259:1685-1689 15. Wilson GT, Rachman SJ (1983) Meta-analysis and the evaluation of psychotherapy outcome: limitations and liabilities. J Consult Clin Psychol 51:54-64 16. Wolf MF (1986) Meta-analysis. Quantitative methods for research synthesis. Series Quantitative Applications in the Social Sciences: Sage University Paper. Sage, Beverly Hills, pp 1-65 17. Wood-Dauphinee S, McPeek B (1986) Systematically reviewing previous work. In: Troidl H, Spitzer WO, McPeek B, Mulder DS, McKneally MF (eds) Principles and practice of research. Strategies for surgical investigators. Springer, Berlin Heidelberg New York, pp 45-52

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Meta-analysis: only a part of the story on mediators in septic shock Discussion about an example of meta-analysis in basic surgical research: The role of mediators in septic/endotoxic shock [Theor Surg (1987) 2:1-28] W.Oettinger Department of Surgery, University of Ulm, Steinhovelstrasse 9, D-7900 Ulm, Federal Republic of Germany

Key words: Decision tree - Shock mediator - Shock models

Introduction Dr. Neugebauer et al. made an enormous effort to scrutinise knowledge about the issue "histamine and septic/endotoxic shock" by means of meta-analysis. Derived from the greek word meta-ana-lyein which means "to resolve by looking backwards and taking apart," the method refers to a decision tree on the basis of a questionnaire allowing the answers "yes" or "no" at each step of decision-making. The final goal of that decision-making is to obtain real data to allow conclusions on whether or not a presumed shock mediator (in this case histamine) is associated with septic/ endotoxic shock. The final question of the decision tree, interestingly, asks for "association," not for "causative relation." The decision-tree approach: is there a real benefit? The advantage of meta-analysis as compared with the narrative approach is the use of numerical details in sufficient quantity to eliminate as much bias as possible. The method is retrospective in nature, but in my opinion, its merit is the provision of prospective clues as to how to improve and how to design experimental or clinical studies today. However, I am not sure whether the elaborate work of screening more than 40000 pap-

ers is the appropriate and necessary means to achieve little more than a commonplace imperative: to avoid the shortcomings of a possible defective assay and sample preparation, wrong body compartment, wrong species and irrelevant shock model. In other words, all questions of the decision tree (except no. 1: Is it an issue-related paper?) can and indeed should be raised by every responsible investigator during the design of his particular study, and to do so he needs not only meta-analytic experience but also state-of-the-art knowledge. There is not a single shortcoming in the "Results" section of the paper which can be regarded as an exclusive result, obtainable only by meta-analysis. This method, of course, helps to identify objective knowledge and to separate it from beliefs on the basis of biological plausibility. However, meta-analysis disregards the historical environment of the various investigations and investigators. Most of the few clinical papers cited, all of which were lacking the quality standards of meta-analysis, were published more than 15 years ago. Most of what we could only "narrate" about then we can measure today. For instance, the definition of sepsis and improvement of patient selection today are highly dependent on advanced methods made available only in recent years. At least as important as taking the meta-analytic approach is our application of these methods in a prospective way to eliminate bias, again on the basis of numerical data. I personally prefer to compare objective data (including baseline levels and controls) rather than to find consensus by discussion of historical entities. The latter approach was alledgedly necessary, for example, to decide about "yes" or "no" at some tree nodes.

Reprinted from Theoretical Surgery, 4, 86-87, 1989. With the kind permission of Springer-Verlag © 1989.

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Appendix The question of relevant shock models, for exampie, is by no means answerable only by meta-analytic approach; we rather must make the most careful use possible of the methods available to monitor clinical conditions and also make the effort to imitate them experimentally as closely as possible. The sensitivity to histamine and its content in basophils of various animals is just one relevant component of the model: the clinical/haemodynamic exposition of shock and its time-mediator presence-effect interrelation is another. This is indeed an aspect of major concern in the literature [3]. Only a tiny minority of septic/endotoxic shock models provide a minimum of haemodynamic qualifications; some resort to mean arterial blood pressure only, few present clinically less relevant hypodynamic shock characteristics, and only a handful of papers in the past few years have tried to cope with the clinically more relevant condition of hyperdynamic response in early septic shock. Another problem in the shock literature is to agree on what we call a "shock mediator". Although there are some objective approaches (biochemical identifiability, reproducibility of concentrations to be associated quantitatively and timewise with certain shock patterns, absence of the mediator in healthy conditions, etc.), papers on unidentifiable mediators or those identifiable only for privileged laboratories (depressant, activating, dilating, etc.) still appear and probably will continue to appear because of uncontrollable creativty. I wonder, however, how this problem can be demystified by meta-analysis alone [1,2]. Again, learning to cope experimentally with clinical problems is the result of applying currently available methods (whenever possible quantitative) on qualified comparison between clinical findings and , . . , , .,.,.. r™. .their experimental reproducibility. This, of course, seems to be done best in combination with looking at what has been done wrong or what could not have been done better in the past, perhaps because of lack of appropriate methods and knowledge. This learning process and its successful application in the search for the truth also requires intellectual accuracy, nurtured by constant education to scientific

honesty and modesty in drawing conclusions: "Being associated with" rather than "caused by." "Meta-analysis" can only be a part of this story. It may cover the theoretical essentials of experience and historical judgement, but it cannot replace prospeclive synthesis on the basis of present-day ideas and methods. There is one other beneficial "side effect" of meta-analysis: It provides insight into everything that has been done - well or faulty - on a certain issue and automatically puts it into a historical framework. This may help to answer the crucial question: Is the problem worth further study? Three of the mere five clinical papers on histamine and septic/endotoxic shock are more than 25 years old. Conclusion The educational momentum and resulting contribution of historical experience is the major advantage of meta-analysis. The decision tree provides a helpful checklist to every researcher, ranking from the fellow to the world-travelling senior scientist, for evaluating his individual study design and the resulting data, The decision tree should be on the boards of every medical journal when submitted papers and their particular conclusions are screened. It is doubtful, however, whether meta-analysis can solve the problem of the cause-effect relationship on its own, i.e. without prospective methodological elements, References l Ha lund u

8 (1983) T°*ic factors in shock- In: L^'5 DH, Hagmnd U (eds) Shock research. Elsevier, Amsterdam, pp 191-202 2. Lefer AM (1973) Blood-borne humeral factors in the pathophysiology of circulatory shock. Circ Res 32:129-135 3- Wichterman K, Baue A, Chaudry I (1980) Sepsis and septic Res29~ll8-W "* 'ab°rat0ry m°dels a"d proposals'J Surg -

Received 29 November 1988

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Handbook of Mediators in Septic Shock

Causality, predictability and explanation Discussion about an example of meta-analysis in basic surgical research: The role of mediators in septic/endotoxic shock [Theor Surg (1987) 2:1-28] D.B.Carr Department of Anesthesia, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

Key words: Concepts of causality - Computer modeling approach

Introduction Neugebauer and his colleagues in Marburg deserve congratulations for their uniquely comprehensive and rigorous meta-analysis [13] of the current status of histamine in septic shock. This type of analysis addresses one question concerning causality within complex physiological responses: whether published studies of a single mediator fulfill objective criteria for methodological validity. Yet by tabulating multiple classes of candidate mediators within their report, they implicitly acknowledge that causation is a web, not a thread. They have elsewhere noted [12] that if reality indeed consists of "a mixture of causal factors... acting in concert," then the traditional "one cause-one disease" concept must be extended to determinants that are neither necessary nor sufficient, but rather contribu.r Causality and prediction of external processes Before we can reach a consensus on causality within some process or response, we must first agree upon what form a satisfying explanation of that process ,.. _, . r . ... , would have. That is, the terms in which we evaluate causality are inseparable from what we consider to be a psychologically or philosophically acceptable expla-

nation of the meaning of causality in that situation [14], Craik has reminded us that one of the most fundamental properties of thought is its power of predicting events, although the process of prediction is not unique to minds [2]. Following Craik's lead, we might as c not ' What kind of thing is causality?" but instead "What kinds of structures and processes are required in [^ system to enable il to imitate corre«'y and to Predlct external processes?" Building on earlier work by Wiener, the econometnc >an Granger [5] has formalized the concept of causality as predictive value. If, knowing all earlier values of x our estimate of x at time lsim roved b ' ' P y knowing all earlier values of y, then a relationship of "Granger causality" exists between y and x. One's Psychological discomfort at equating mere predictabllit with true y causality echoes Aristotle's distmctlon berween the realm of forms and therealm of thm &' whlch have both matter and form 3 Certalnl I !' y> at flrst blush ll a ears PP contnved to declare a mediator to be a contributing cause, say, of septic shock, simply if '"eluding it in a multivanate regression equation reduces the standard error of estimate. Yet precisely this formulation has been embraced in recent years in disciplines whose existence centers upon modelling and prediction [4, 6, 8]. Kalman [10], for example, has remarked that Economics deals with complex interactive phenomena. It is impossible to study quantitative relationships between important var ables without l ^rence to the context. Nor is it possible to perform experiments or make direct observations that would at £ast dimin|;h the noise ,evel under which these effccts can be observed. We possess, however, innumerable time series engendered by the very economic forces we wish to uncover. By

Reprinted from Theoretical Surgery, 4, 88-90, 1989. With the kind permission of Springer-Verlag © 1989.

545

Appendix constructing models for time series we may hope to gain indirect access to the desired quantitative relationships because these are intrinsic in the models and therefore should be recoverable from the structure and parameters of the models. Does this not sound like the situation physiologists face when confronted with a proliferation of mediators between which there exist many potentially meaningful interactions?

Causality and system science Other disciplines such as ecology [15] have also evolved techniques for casting complex interrelationships into mathematical forms that express the variable tightness of linkages between elements of a system. Common to such disciplines is their convergence, at some level of abstraction, to the mathematical descriptions of relationships between elements, i.e., to systems science. At present, under the headings of systems science or operations research, methodological tools for analyzing complex networks are readily available [11], some as microcomputer software. Indeed, low-cost software programs for statistical analysis including time series forecasting are currently advertised in popular magazines for microcomptuer users.

geometrical [20]. Lest this be perceived as an alarming f rom reality, the reader might be , . „', . .. , . ... reassured to recall that the change in conceptual^tion of motion from linguistic (even sacred) terms to mathematical terms during early medieval times paved the way for Gallileo's and Newton's later advances [3]. More recently, philosophical antideterminism and preoccupation with causality in the late nineteenth and early twentieth centuries were keys to the development of quantum mechanics and relativity [9]. Second, as computers become more powerful and their predictive algorithms more "expert," we may see increasing numbers of accurate, predictive, yet unconscious machines enter our daily lives [17,18]. Whether "causality engines" may unsettle us after the fashion of computer chess players remains to be seen. Third, even in the face of limitless amounts of computational power, intrinsic limits probably exist as to the precision with which one can predict the behaviour of increasingly complex, self-interactive systems (Zadeh, cited in [11]). Defining these limits will be a challenge for theoretical biologists, for beyond them, even the best dynamic methods break down into unpredictable chaos [16,19].

turn of events away

References Causality and communication Within bioscience certain disciplines such as endocrinology have, in their focus upon patterns of interor6 ... , . ... i (u gan communication, emphasised the role of hormones as signals and at times described hormone secretion in purely communicational terms [7, 21]. Application of a slightly refined version of lagged simultaneous equation modelling of beta-endorphin levels in closely timed . . . i ^ j j • •^ serial plasma samples after endotoxm administration in sheep has, for us, demonstrated the feasibility of this approach to relate mediator responses to vital sign changes [1]. One wonders whether the present gap between the sophistication of modelling in other disci,. j ., , .... , , « i_ • i • phnes and that within whole-animal physiology is not more the reflection of unfamihanty with methods, and unavailability of computing power, than intrinsic unsuitability. If so, then the situation should change in the near future, as techniques diffuse outwards and hard- and software decline in COS,. Conclusion • , . ., . , ... 0 T- . .. . TT How might these changes be manifest? First, the description of mediator secretion during shock could change from concrete and tabular to more abstract or

Carr DB, Jones KJ, Bergland RM, Hamilton A, Kasting NW, Fisher JE, Martin JB (1985) Causal links between plasma and CSF endorphin levels in stress: vector-ARMA analysis. Peptides 6 (Suppl 1): 5-10 2. Craik KJW (1967) The nature of explanation. Cambridge University Press London 3. Gaukroger S (1978) Explanatory structures: a study of concepts of explanation in early physics and philosophy. Humanities Press, Atlantic Highlands, NJ 4 Gew ' ecke J (1982) Causality, exogeneity and inference. In: Hildenbrand w (ed) Advances in econometncs. Cambridge University Press London 5. Granger J (1982) Generating mechanisms, models, and causality. In: Hildenbrand W (ed) Advances in econometrics, Cambridge University Press, London 6 Horton CW Reichl LE ' ' ' Szebehely VG (1983) Long-time prediction in dynamics. Wiley, New York 7 f berall AS Soodak H Hajer p (19?g) A field and drcuit thermodynamics for integrative physiology. II. Power and communicational spectroscopy in biology. Am J Physiol 3: R3-R19 8 ' '^"S"ss Computer modeling of complex biologi,_ ^M^nT^^ development of quantum mechanics. McGraw-Hill, New York 10. Kalman HE (1982) Identifiability and problems of model selection in econometrics. In: Hildenbrand W (ed) Advances in econometrics. Cambridge University Press, London 11. Klir G (1985) Architecture of systems problem solving. plenum New York 12. Neugebauer E, Lorenz W (1988) Causality in circulatory shock: strategies for integrating mediators, mechanisms and L

546 therapies. In: Bond RF, Adams HR, Chaudry IH (eds) Perspectives in shock research. Liss, New York (Progress in clinical and biological research, vol 264, pp 295-303) 13. Neugebauer E, Lorenz W, Maroske D, Barthlen W, Ennis M (1987) The role of mediators in septic/endotoxic shock. A meta-analysis evaluating the current status of histamine. Theor Surg 2:1-28 14. Nozick R (1981) Philosophical explanations. Belknap, Cambridge, Mass 15. Odum HT (1988) Self-organization, transformity, and information. Science 242:1132-1139 16. Prigogine I (1980) From being to becoming: time and complexity in the physical sciences. Freeman, San Francisco 17. Rosen R (1985) Anticipatory systems: philosophical, mathematical and methodological foundations. Pergamon, Oxford

Handbook of Mediators in Septic Shock 18. SegelL (1984) Modeling dynamic phenomena in molecular and cellular biology. Cambridge University Press, Cambridge 19. Thompson JMT, Stewart HB (1986) Nonlinear dynamics and chaos. Wiley, New York 20. WinfreeAT(1980)Thegeometryofbiologicaltirne.Springer, Berlin Heidelberg New York 21. Yates FE (1981) Analysis of endocrine signals: the engineering and physics of biochemical communication systerns. Biol Reprod 24:73-94

Received 5 December 1988

547

Appendix

Histamine in septic/endotoxic shock: pathophysiological considerations Discussion about an example of meta-analysis in basic surgical research: The role of mediators in septic/endotoxic shock [Theor Surg (1987) 2:1-28] L.B.Hinshaw Oklahoma Medical Research Foundation and University of Oklahoma, Oklahoma City, Oklahoma 73104, USA

Key words: Histamine - Local histamine release Histamine release response - Decision-tree approach j

.

.

__ . , „, . . „, . . The constituents of a decision tree (meta-analysis , . . > , . , j , v , , \, j. logistics), described by Neugebauer and arranged in sequence through sampling procedures, study design, type/kind of shock model, and species, concluding with the responses themselves, are very useful in determining whether a direct association exists between a shock mediator (e.g., histamine) and septic/ endotoxic shock in vivo. The foregoing statements elaborate on the paper of Neugebauer and hopefully contribute to the value of meta-analysis according to the "decision-tree" approach in evaluating the role of a shock mediator, specifically histamine. Dr. Neugebauer and colleagues recently estimated that about 100 different mediators, including histamine, have been described in association with the septic shock syndrome [8]. Without a doubt, a multitude of endogenously released vasoactive agents are activated in response to injections of endotoxin in experimental animal models. These agents may be released into the circulating blood or may be activated locally at sites of vascular smooth muscle [3]. In the latter instance they may not circulate in the blood and therefore will miss detection by the experimental means described in Neugebauer's analysis [8]. Furthermore, the kinds and concentrations of involved agents may vary be-

tween the species of animals, may be modified as a function of the dose of endotoxin, and probably vary in accordance with time of release during the postendotoxin period [3]. Without question, the roles of agents released into the blood, or released at the site of action (e. g., hepatic venules in dogs) without reaching s the circulation, are of enormous importance in the , . , ' . . , . , , pathoeenesis o f septic/endotoxic shock, F 6 F

Histamine and endotoxin: similarities and dissimilarities in pathophysiological actions I would like to discuss ramifications of the last step in the decision tree, "response caused by the shock itself," as described in Dr. Neugebauer's paper [8]. The significance of comparative studies is involved, which we have carried out contrasting the pathophysiological effects of endotoxin with those of histamine, each administered separately in dogs [4,6]. The results of these studies are quite illuminating, particularly in highlighting the differences between the effects of endotoxin and histamine. In separate experiments dogs were intravenously infused with either endotoxin, histamine, or the histamine releaser, 48/80 [4, 6]. Similarities of actions of each agent were as follows: (a) an early increase in portal venous pressure coincident with a decrease in venous return, vascular pooling, and a rapid decrease in systemic arterial pressure and (b) eventual increases in foreleg vascular resistance, foreleg small vein pressure, leg weight, and

Reprinted from Theoretical Surgery, 4, 91-93, 1989. With the kind permission of Springer-Verlag © 1989.

548 hematocrit. The early responses to endotoxin were greatly altered when 48/80 was administered prior to endotoxin; this suggested a common underlying mechanism for the action of each agent [4]. Histamine appears to serve as a triggering device for the sustained release of adrenergic-like agents which superimpose their effects on those of histamine [4], However, differences in the responses of dogs given endotoxin vs. histamine are apparent [6]. The histopathological effects of even massive doses of histamine are much less than those of endotoxin alone. However, the large amounts of histamine required to mimic the endotoxin response [6] do not necessarily diminish its possible importance in endotoxin shock, but are compatible with the view of Schayer [9] that histamine, as a microcirculatory regulator, is released in close proximity to its sites of action, including the hepatic veins. Therefore, a highly concentrated amount of histamine released in a confined region (e.g., hepatic venules) could have major impacts on the circulatory responses to endotoxin administration. The use of an alpha-adrenergic- and histamineblocking agent [7], phenoxybenzamine (Dibenzyline), in canine endotoxin shock studies [1] demonstrated a basic similarity between the early vascular responses to histamine and endotoxin in the hepatoportal system. A major dissimilarity with the vascular effects of histamine was the absence of an early drop in total peripheral resistance after endotoxin. Again, it may be that histamine released by endotoxin shows its maximal action at the site of release (i.e., hepatic veins), and its peripheral dilator effect is offset by the presence of vasoconstrictor agents also known to be released by endotoxin. These several studies [1, 4, 6, 7, 9] point out the problem which may exist in placing undue emphasis on the concentration of histamine in circulating blood by whatever methodology, and total lack of emphasis on the probable local release (noncirculating) of histamine at the sites of vascular action, in which condition circulating levels of histamine are not applicable and may be irrelevant to the intimate vascular response to endotoxin. Evidence for and against the role of histamine in endotoxin shock has been discussed [2]. There is strong research support for the early release of histamine after endotoxin and its important role in the development of vascular pooling and systemic hypotension. Responses of the dog and nonhuman primate to injected histamine are markedly different; within each species, however, responses to histamine and endotoxin are remarkably similar in certain respects [2]. The vascular responses to endotoxin are exceedingly complex, and unexplainable differences between the actions of histamine and endotoxin clearly exist. It is possible that the elaboration of multiple agents, each

Handbook of Mediators in Septic Shock exerting its own effect, together with inter-actions between the agents, complicates the interpretations of an individual agent's effects in the in vivo model. Histamine release in septic shock: still an open question A recent study provides inadequate support for the role of histamine in human patients with septic shock [5]. In this report, measurement of plasma histamine from human subjects in septic shock failed to confirm an increase in circulating histamine. On the contrary, nearly all of the subjects had unmeasurable values due to the presence of an "inhibitor" in the plasma which interfered with the assay employed. After removal of the inhibitor from plasma, septic shock plasma histamine levels were normal. The authors considered the possibility that histaminemia in sepsis could be transient and thereby missed by sampling error; however, their samples were drawn during the persistent hypotension of human septic shock. If histamine were involved in the pathogenesis of this hypotension, some of the histamine levels should have been elevated, as they surmised, but they were not. The authors concluded that it is unlikely that histamine release from mast cell activation is a major factor in the pathogenesis of septic shock. This finding would appear to close the issue of the role of histamine in human septic shock; however, questions still remain. The degree of the removal of the "inhibitory" factor in their studies is uncertain; The precise identity of the factor remains unknown, and the mechanisms for its release are not identified. Another problem with the authors' conclusion that histamine performs an irrelevant role in septic shock is concerned with the timing of the sampling. Many hours may have elapsed following an early release of histamine , since the patients were in an advanced stage of shock. The endogenous stores of histamine could have been depleted hours earlier and the pathophysiological status of mast cell metabolism and function might have been depressed to the point of nonfunction for replenishment of histamine stores. Endogenous actions destroying or degrading circulating histamine may have removed it from the circulation at an earlier time, yet concentrations at local sites in the vascular system could have remained high but unmeasurable because of the lack of histamine circulating where it could be sampled, if present. The authors have contributed significantly to the concept that elevated levels of circulating histamine, sustained during the period of septic shock, cannot be documented; however, the problems of timing of sampling, local noncirculating release of histamine, and a possible remain-

549

Appendix ing factor of inhibition of histamine analysis suggest caution in accepting their conclusion that histamine release is not an important factor in the pathogenesis of human septic shock. Conclusion The thesis of Neugebaueretal. regarding meta-analysis is a sound approach to identifying the role of a "shock mediator" in endotoxic/septic shock. The various branches of the decision tree appear logical sequen.. n T . ., . ., , ^ t £., ., , tially. I suggest that the last step of the authors process concerning "responses" could be extended to utilize the tools and procedures of experimental pharmacology and physiology and possibly placed at earlier points in the analysis

References 1. Brake CM, Emerson TE, Wittmers LE, Hinshaw LB (1964) Alteration of vascular responses of endotoxin by adrenergic blockade. Am J Physiol 207:149-151

2. Hinshaw LB(1964)Thereleaseofvasoactiveagents by endotoxin In: Bacterial endotoxins (symposium). Institute of "f^*1"8"5 ^ University' New Brunswick' 3 Hinshaw LB (1971) Release of vasoactive agents and the vascular effects of endotoxin. In: Kadis S (ed) Microbial toxins, vol 5. Academic Press, New York, pp 209-260 "*• Hinshaw LB, Emerson TE Jr, lampietro PF, Brake CM (1962) A comparative study of the hemodynamic actions of histamine and endotoxin. Am J Physiol 203:600-606 5 Jacobs R Kaliner M _ shelhamer JH, Parrillo JE (1989) Blood histamine concentrations are not elevated in humans with septic shock. Crit Care Med 17:30-35 6 Jordan MM Holmes DD Hinshaw ' LB (1965) Pathophysiological comparisons of histamine and endotoxin shock 3 Trauma 5 -726-737 7 KindLS(1954)Inhibitionofhistaminedeathinpertusssis-inoculated mice by Dibenzyline, an adrenergic blocking agent, J Allergy 25:33-35 "• NeugebauerE,LorenzW,MaroskeD,BarthlenW,EnnisM (1987) The role of mediators in septic/endotoxic shock. A meta-analysis evaluating the current status of histamine. TheorSurg2:l-28 9. Schayer RW (1963) Induced synthesis of histamine, microcirculatory regulation and the mechanism of action of the adrenal glucocorticoid hormones. Prog Allergy 7:187-212 Received 1 February 1989

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Association and cause: problems with the Koch-Dale criteria Discussion about an example of meta-analysis in basic surgical research: The role of mediators in septic/endotoxic shock [Theor Surg (1987) 2:1-28] J. P. Green Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York, NY 10029, USA

Key words: Decision-tree approach - Biological plausibility - Causality - Plasma histamine

Introduction It is a pleasure to be invited to offer comments regarding the paper on the role of histamine in shock, by Neugebauer and his associates [35], This work exemplifies the value and cautions of meta-analysis [6,43,45]. Implicit is the appraisal of all papers published on the subject. From the meta-analysis could have emerged a positive or negative statement of the validity of the hypothesis that histamine is a mediator in shock. As from the conventional anecdotal literature review, but with more persuasiveness, a firm conclusion could discourage, even obviate, redundant experiments. An advantage of this meta-analysis is that the authors first provide a decision tree, explicitly stating their criteria for accepting a study. The decision tree evolved from a consensus conference composed of people [34] other than themselves and knowledgeable about the research problem. The components of the decision tree are reasonable, for they are based on documented facts. The components therefore cannot be said to be unbiased in the sense that being unbiased is to be unknowing. Rather, the components derive from awareness of the critical issues in probing the hypothesis (e.g., problems with the specificity of the assays for histamine) which, in this context, is a most welcome "bias." It should be noted that being excluded from the analysis [35] on the basis of this decision tree is no

shame, for some of the facts that formed the criteria embodied in this decision tree were learned fairly recently. This meta-analysis, like others, was retrospective. Therefore, it almost certainly excludes many negative experiments which many authors and most journals are not enthusiastic to publish. The authors' lament — "Alas, meta-analysis is inevitably retrospeclive." - may not be deserved; meta-analysis can be and is being done prospectively [6]. The pervasive value of the decision tree and other aspects of this work [35] is that it imposes stringent standards a priori that are sustained simply by respecting the strictures of its formality, thus compelling analysis in a systematic and rigorous way. At the same time, it identifies problems, and it demands quantitalive data. All biomedical research can profit from this example, Biological plausibility: a weak type of evidence in assessing causation This approach can help to avoid the quick and sometimes false attributions that have been made in both basic and clinical research, which, as implied by Neugebauer etal. [35], rest on examinations of plausibilities. It was plausible to infer that the most conspicuous sign of a malignant carcinoid tumor [47], the flush, was attributable to 5-hydroxytryptamine (i.e., serotonin) since in many patients the flush was accompanied by increased plasma levels of 5-hydroxytryptamine, sometimes by increased urinary levels of its metabolite 5hydroxyindoleacetic acid, and 5-hydroxytryptamine

Reprinted from Theoretical Surgery, 4, 94-97, 1989. With the kind permission of Springer-Verlag © 1989.

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Appendix was reported in carcinoid tumors, even nonmalignant ones found by chance at necropsy. It was a most plausible conjecture that the flush may be attributable to the release of 5-hydroxytryptamine into the systemic circulation (not into the portal circulation, where the liver would destroy it). But intravenous infusions of 5-hydroxytryptamine failed to produce the flush, and carcinoid patients were "emphatic" that the symptoms produced by intravenous 5-hydroxytryptamine "were distinct from any exerienced during spontaneous flushes..." [38]. Despite this and other evidence [47], the spurious causal connection persisted. (Failure to show that the flush and high plasma levels of 5-hydroxytryptamine were conjoined was sometimes ascribed to the "exception that proves the rule," an oxymoron, since an exception probes a rule, announcing that the validity of the rule needs to be tested.) Work showed that other substances may account for or contribute to the flush [25, 36, 47]. The plausible hypothesis, when hastily elevated to a fact without supporting evidence, has important consequences when applied to patient management. The plausibility that strict bed rest for 6 weeks was desirable after a myocardial infarction was laid to rest after the regimen was examined [30,31], as was the prolonged rest prescribed for acute infectious hepatitis, after that tenet was examined [7]. A baseless method for treating frostbite took a long time to be refuted [17]. Only 11 years ago it was said, "Very few of the hypotheses involving the management of the bulk of disorders now affecting the adult population... have been adequately tested for or, if so tested, supported by unequivocal evidence." [17]. But the probing is going on. The adage that impotence is psychogenic "in considerably more than 90 percent" of the patients was offered without evidence [41] and widely embraced, until a study showed that 33% of patients with impotence (including one who developed impotence after his pituitary adenoma had been treated with radiotherapy) had disorders of the hypothalamic-pituitarygonadal axis, and that 89% of these had restoration of function after hormonal therapy [40]. Measurements of TSH may offer entry to therapy [9] for some patients whose chronic fatigue has been attributed to psychological reasons (in most cases without a psychiatric consultation). The illness characterized by fatigue, weakness, myalgia, memory loss, and depression is now being considered a syndrome of viral origin [26, 42] rather than - too often earlier - of hysteria; the virus remains to be identified. Problems in establishing causation The difficulty in establishing causal connections and the elusiveness of certainties in clinical medicine have

been lucidly presented [48, 49]. These humblings apply not only to research on living laboratory animals but also to research on much simpler systems. For example , work on hippocampal membranes in vitro showed that the 5-hydroxytryptamineiA receptor is linked to stimulation of adenylate cyclase activity [39]. In the same laboratory, work showed that in the same tissue membranes, the same receptor is linked to inhibition of adenylate cyclase activity [18]. The two different results found in homogenates may be more paradoxical than contradictory, not an example of nonmonotonic reasoning [46] or of an antinomy, for the agonist-activated receptor may, in a disassembled tissue, conceivably interact with either the guanine nucleotide binding protein, Gs, that stimulates adenylate cyclase or with GI, which inhibits adenylate cyclase, the experimental design determining which effect is elicited. The question, then, was: Which effect occurs in a more organized system, the hippocampal slice? Electrophysiological experiments on the slice suggested that the effect of 5-hydroxytryptamine was due to inhibition of adenylate cyclase, supported by a correlation of the two effects [8]. But other experiments then showed that the electrophysiological effect was insensitive to intracellular injections of cyclic AMP and that instead the G-protein activated by the stimulated receptor is directly linked to a K+ channel [1]. Thus, whatever linkage the receptor has to adenylate cyclase, neither the activation nor the inhibition of adenylate cyclase appears to be related to the electrophysiological event that was measured. Are plasma histamine measurements reliable to demonstrate histamine release? The question asked by Neugebauer et al. [35] has far more complexities, burdened with many issues. The unacceptability of some of the methods used to measure histamine was one of the frequent reasons for their rejection of a study. The problem is not only the selection of a method but also correct performance. Analysis of the same plasma samples by the same method in different laboratories showed large variations among laboratories [22], although the method used (radioenzymatic assay) is proved to be specific. A consideration for future work is the value of measuring histamine correctly, and at the same time, measuring its metabolites. As pointed out by the authors [35], plasma histamine in man usually has a half-life of about 2min, but high levels have been noted as long as 8h after it is released from cells. The short half-life introduces the risk of missing a rise. The variability in the half-life, probably due to variability in the metabolism, introduces a confounding factor in comparison

552 of results between different animals or people. These sources of obfuscation could be avoided by measurements of both histamine and its main metabolites [24], which are te/e-methylhistamine, tefe-methylimidazoleacetic acid, and imidazoleacetic acid. Even if measurements of histamine and histamine metabolites were unchanged, histamine could play a role in septic/endotoxic shock. Endotoxins of Brucella and Escherichia coli increase the sensitivity of tissues of some animals to histamine [44]; the hypersensitivity produced by Bordetella pertussis is counteracted by treatment with epinephrine and norepinephrine [4], which may be released in shock. Airway responses to histamine are increased by a host of other substances, e.g., some prostaglandins and leukotrienes [28] and even by increased salt intake [27]. Obviously, if factors increasing histamine sensitivity are increased in shock, then histamine could play a role in shock that its plasma levels would not reflect. Among the 100 other substances that Neugebauer et al. [35] stated (some of which they tabulated) were proposed as mediators of shock are some that have been shown to alter the response of tissues to histamine.

. . ,. --.... . Multiplication and Plurality: necessary to solve the problem It is reasonable, even plausible, that more than one mediator contributes to the syndrome. The likelihood of more than one substance being liberated in injury was suggested by Dale [13] and amplified by Lorenz et al. [33] and Neugebauer et al. [35]. With regard to the carcinoid syndrome, Page [37] said "...it is always dangerous to attribute an entire syndrome to one substance or mechanism. While serotonin is doubtless intimately involved in the carcinoid system, there is good evidence that bradykinin is also concerned, probably both to varying degrees. But there may be a third and fourth substance as well." Since a carcinoid tumor releases more than one substance, as mentioned above, , . . , . , . , . ,. , . . it is even more likely that in shock, which has immense dimensionality, multiple substances are released. This suggestion does not spurn the razor of Occam that "entities are not to be multiplied without necessity" [21]. The multiplication and the plurality may be essential , , ,. .,.. _i • »i_ » . to solve the problem, even if it were certain that plasma histamine levels rise in shock, because histamine influences the release of other mediators. Histamine releases catecholamines from the adrenal medulla [5], Histamine stimulation of the H2-receptor in the myenteric , , .. .. i «V. j . » • plexus of the ileum releases 5-hvdroxvtrvptamine, acetylcholine, a peptide(s), and a prostaglandm(s) [3]. Histamine stimulates prostacyclin synthesis in endo-

Handbook of Mediators in Septic Shock thelial cells [2]. a-Adrenergic stimulation, like leukotrienes, can enhance histamine release [19, 29].

K*««e"»Mit «f «*• nucleotides in shock Substances that especially need to be reassessed for a adenine nucleotides, prominent in this context 40 years ago. They were shown to produce hypotension, and it was proposed that ATP is released b Y hypoxic cells during shock [23]. ATP and other adenosine derivatives act on defined purinergic (or adenosine) receptors [10, 15]. Among their actions which ma be V relevant to shock are an enhancement of histamine release from mast cells [32], an increase in histamine sensitivity [11], stimulation of prostacyclin formation in endothelial cells [20], and an interaction with the Hrreceptor in stimulating cyclic AMP formatlon 14 16 t - 1-

role in shock are

Conclusion These complexities, which merely suggest the scope of the problem, are beyond those that Neugebauer and his assodates thdr attention to [35] Appropriately, necessarily, they asked a precise, seemingly tractable question that, all would hope, would yield an unequivoca, answer It is sad to have to agree with their con. elusion that the suggestion made nearly 70 years ago that histamine ;s a mediator in shock [12] remains inchoate „ L Andrade R Malenka RC NicoU RA (19g6) A Q protdn

couples serotonin and GABA-B receptors to the same channels in hippocampus. Science 234:1261-1265 2. Baenziger NL, Fogerty FJ, Mertz LF, Chernuta LF (1981) Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells. Cell 24:915-923 3 Barker LA Eberso]e BJ (19g2) Histamine HZ receptors on guinea-pig ileum myenteric plexus neurons mediate the release of contractile agents. J Pharmacol Exp Ther 221:6975 4 ' Bergman RK, Munoz J (1966) Protection against histamine shock by catecholamines in Bordetella pertussis-tteated, adrenalectomized, or adrenergic blocked mice. Proc Soc £xp Biol Med 122:428-433 5. Burn JH, Dale HH (1926) The vasodilator action of histamine and its physiological significance. J Physiol (Lond) 61:185-214 6. Chalmers TC, Buyse ME (1988) Meta-analysis. In: Chalmers TC (ed) Data analysis for cl'inicai medicine: the quantitative approach to patient care in gastroenterology. International University Press, Rome, pp 75-84

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Appendix 7. Chalmers TC, Eckhardt RD, Reynolds WE, Feifenstein RW, Deane N, Smith CW, Cigarroa JG, Davidson CS (1955) The treatment of acute infectious hepatitis. Controlled studies of the effects of diet, rest, and physical reconditioning on the acute course of disease and on the incidence of relapses and residual abnormalities. J Clin Invest 34:11631234 8. Clarke WP, DeVivo M, Beck SG, Maayani S, Goldfarb J (1987) Serotonin decreases population spike amplitude in hippocampal cells through a pertussis toxin substrate. Brain Res 410:357-361 9. Cooper DS (1987) Subclinical hypothyroidism. JAMA 258: 246-247 10. Cooper I, Dermot MF (1988) Adenosine receptors. Liss, New York 11. CronholmLS, FishelCW(1968)Histaminehypersensitivity of mice induced by 5'-AMP. Proc Soc Exp Biol Med 127: 1178-1180 12. Dale HH (1920) Conditions which are conducive to the production of shock by histamine. Br J Exp Pathol 1:103-114 13. Dale HH (1929) Croonian lectures on some chemical factors in the control of the circulation. Lancet 1:1285-1290 14. Daly JW (1977) Cyclic nucleotides in the nervous system. Plenum, New York 15. Daly JW (1982) Adenosine receptors: targets for future drugs. J Med Chem 25:197-207 16. Daum PR, Hill SJ, Young JM (1982) Histamine Hragonist potentiation of adenosine-stimulated cyclic AMP accumulation in slices of guinea-pig cerebral cortex: comparison of response and binding parameters. Br J Pharmacol 77:347357 17. Dawber TR (1978) Annual discourse-unproved hypotheses. NEnglJ Med 299:452-458 18. DeVivo M, Maayani S (1986) Characterization of the 5hydroxytryptamineiA receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes. J Pharmacol Exp Ther 238: 248-253 19. Fantozzi R, Moroni F, Masini E, Blandina P, Mannaioni PF (1978) Modulation of the spontaneous histamine release by adrenergic and cholinerigc drugs. Agents Actions 8:347358 20. Forsberg EJ, Feuerstein G, Shohami E, Pollard HB (1987) Adenosine triphosphate stimulates inositol phospholipid metabolism and prostacyclin formation in adrenal medullary endothelial cells by means of P2-purinergic receptors. Proc Natl Acad Sci USA 84:5630-5634 21. Fremantle A (1957) The age of belief. Houghton Mifflin, Boston, p 202 22. Gleich GJ, Hull WM (1980) Measurement of histamine: a quality control study. J Allergy Clin Immunol 66:295-298 23. Green HN, Stoner HB (1950) Biological actions of adenine nucleotides. Lewis, London 24. Green JP, Prell GD, Khandelwal JK, Blandina P (1987) Aspects of histamine metabolism. Agents Actions 22:1-15 25. Gustafson J, Boesby S, Man WK (1988) Histamine in carcinoid syndrome. Agents Actions 25:1-3 26. Holmes GP, Kaplan JE, Gantz NM, Komaroff AL, Schonberger LB, Straus SE, Jones JF, DuBois RE, CunninghamRundles C, Pahwa S, Tosato G, Zegans LS, Purtilo DT, Brown N, Schooley RT, Bros I (1988) Chronic fatigue syndrome: a working case definition. Ann Intern Med 108: 387-389 27. Javaid A, Cushley MJ, Bone MF (1988) Effect of dietary salt on bronchial reactivity to histamine in asthma. Br Med J297:454

28. Joad J, Casale TB (1988) Histamine and airway caliber. Ann Allergy 61:1-7 29. Kaliner MK, Orange RP, Austen KF (1972) Immunological release of histamine and slow-reacting substance of anaphylaxis from human lung. IV. Enhancement by cholinergic and alpha-adrenergic stimulation. J Exp Med 136:556-567 30. Levine SA (1952) Myth of strict bed rest in treatment of heart disease. Acta Med Scand 142 (Suppl 266): 671-679 31. Levine SA, Lown B (1952) "Armchair" treatment of acute coronary thrombosis. JAMA 148:1365-1369 32. Lohse MJ, LotzKN, SalzerMJ.SchwabeU (1988) Adenosine regulates the calcium sensitivity of mast cell mediator release. Proc Natl Acad Sci USA 85:8875-8879 33. Lorenz W, Rbher HD, Doenicke A, Ohmann CH (1984) Histamine release in anaesthesia and surgery: a new method to evaluate its clinical significance with several types of causal relationship. Clin Anaethesiol 2:403-426 34. McKneally MF, McPeek B, Mulder DS, Spitzer WO, Troidl H (1986) Chairing panels, seminars and consensus conferences. In: Troidl H et al (eds) Principles and practice of research - strategies for surgical investigators. Springer, Berlin Heidelberg New York, pp 249-253 35. Neugebauer E, Lorenz W, Maroske D, Barthlen W, Ennis M (1987) The role of mediators in septic/endotoxic shock. A meta-analysis evaluating the current status of histamine. TheorSurg2:l-28 36. Dates JA, Pettinger WA, Doctor RB (1966) Evidence for the release of bradykinin in carcinoid syndrome. J Clin Invest 45:173 37. Page IH (1968) Serotonin. Year Book Medical Publishers, Chicago, 111, pp 104-109 38. Robertson JIS, Peart WS, Andrews TM (1962) The mechanism of facial flushes in the carcinoid syndrome. Q J Med 31:103-127 39. Shenker A, Maayani S, Weinstein H, Green JP (1987) Pharmacological characterization of two 5-hydroxytryptamine receptors coupled to adenylate cyclase in guinea pig hippocampal membranes. Mol Pharmacol 31:357-367 40. Spark RF, White RA, Connolly PB (1980) Impotence is not always psychogenic. Newer insights into hypothalamicpituitary-gonadal dysfunction. JAMA 243:750-755 41. Strauss EB (1950) Impotence from a psychiatric standpoint. Br Med J 1:697-699 42. Swartz MN (1988) The chronic fatigue syndrome - one entity or many? N EngI J Med 319:1726-1728 43. Thacker SB (1988) Meta-analysis. A quantitative approach to research integration. JAMA 259:1685-1689 44. Urbaschek B, Versteyl R (1965) Increase of the effect of histamine by E. coli endotoxin on the smooth muscle. Nature 207:763-764 45. Wachter LW (1988) Disturbed by meta-analysis? Science 241:1407-1408 46. Waldrop MM (1987) Causality, structure, and common sense. Science 237:1297-1299 47. Warner RRP (1985) Carcinoid tumor. In: Berk JE (ed) Bockus gastroenterology, vol 3,4th edn. Saunders, Philadelphia, pp 1874-1886 48. Wulff HR (1981) Rational diagnosis and treatment. An introduction to clinical decision-making, 2nd edn. Blackwell, Oxford 49. Wulff HR, Pedersen SA, Rosenberg R (1986) Philosophy of medicine. An introduction. Blackwell, Oxford

Received 16 February 1989

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The quality of primary and secondary research and meta-analysis Discussion about an example of meta-analysis in basic surgical research: The role of mediators in septic/endotoxic shock [Theor Surg (1987) 2:1-28] T.C.Chalmers Technology Assessment Group, Department of Health Policy and Management, Harvard School of Public Health, 677 Huntington Avenue, Boston, Massachusetts 02115, USA, and Boston Veterans Administration Medical Center, Boston, Massachusetts 02115, USA

Key words: Meta-analysis - Secondary analysis Decision tree approach - Observer variability - Bias

Introduction The following commentary is a response to the request by the authors of the article published in Theoretical Surgery [6]: "The role of mediators in septic/endotoxic shock. A meta-analysis evaluating the current status of histamine." The request, with its implication that meta-analysis may not be scientific, gives this author an opportunity to comment on the validity of all science related to human diseases. Meta-analysis does have its unreliable aspects, largely stemming from the fact that it is retrospective research, but its performance spotlights the defects of the original research it seeks to combine. Meta-analysis, secondary analysis and decision-^ approach From the outset it should be recognized that the article by Neugebauer et al. is not what is usually considered a meta-analysis, in that it does not combine data from the selected primary papers to arrive at conclusions that could not be arrived at without the combination. Instead, it is more like what Glass has classified as a secondary analysis [5], in that the validity of the conelusions of the original articles is examined by critically appraising the data. In doing so the experimental design and analysis are examined in a most thorough way. The article accomplishes what most meta-analyses recognize if they are any good, namely that there are grave defects in the majority of original research projects that come to light only if an attempt is made to combine the data obtained with those of other research.

A most notable aspect of the scholarly examination of the evidence that histamine might cause irreversible shock is the distressingly poor quality of almost all of the relevant research. The decision-tree approach to secondary analysis that the authors use is, to my mind, original. Although it may be difficult for a paper to pass all nine tests of scientific quality, it is remarkable that none pass them all One ordinari i y thinks of re_ search with animals as relatively easy to design as foo,_ proof as necessaryj because the subjects do not have to be treated for their diseases at the same time, From the evidence in Ms paper it appears that the basic-science investigators carrying out these experiments have a ,ot ,o lgam from dinical investigators carrying out randomized control trials. The scien tjfic method is the same whether applied to human or to animal research. The iate David Rut. stein first chairman of the Department of Preventive Medicine at Harvard Medical School, used to love to quote his favorite teacher, L.J.Henderson, to wit: "Sci*nce is *e collection of reproducible facts." The truth 1S . ^producible. If investigators use assays of questionable validity, do not prepare their samples a PProPriate1)', do not sample relevant body tissues at the "«*" tlme' f°' ow an /™PP«>P™te studV deslf' or sesh ck mode s rammal s eclesthat " ° ' ° P f no< cllnlcall re evant and lf ihe cannot y ' ' ? Pr°ve