Disease Markers in Exhaled Breath: Basic Mechanisms and Clinical Applications (Nato: Life and Behavioural Sciences, 346)

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DISEASE MARKERS IN EXHALED BREATH

NATO Science Series A series presenting the results of scientific meetings supported under the NATO Science Programme.

The series is published by 10s Press and Kluwer Academic Publishers in conjunction with the NATO Scientific Affairs Division. Sub-series

I.

Life and Behavioural Sciences Mathematics, Physics and Chemistry 111. Computer and Systems Sciences IV. Earth and Environmental Sciences V. Science and Technology Policy

II.

10s Press Kluwer Academic Publishers 10s Press Kluwer Academic Publishers 10s Press

The NATO Science Series continues the series of books published formerly as the NATO AS1 Series. The NATO Science Programme offers support for collaboration in civil science between scientists of countries of the Euro-Atlantic Partnership Council. The types of scientific meeting generally supported are “Advanced Study Institutes” and “Advanced Research Workshops”, although other types of meeting are supported from time to time. The NATO Science Series collects together the results of these meetings. The meetings are co-organized by scientists from NATO countries and scientists from NATO’s Partner countries - countries of the CIS and Central and Eastern Europe.

Advanced Study Institutes are high-level tutorial courses offering in-depth study of latest advances in a field. Advanced Research Workshops are expert meetings aimed at critical assessment of a field, and identification of directions for future action. As a consequence of the restructuring of the NATO Science Programme in 1999, the NATO Science Series has been re-organized and there are currently five sub-series as noted above. Please consult the following web sites for information on previous volumes published in the series, as well as detail; of earlier sub-series: http://www.nato.int/science http://www.wkap.nl http://www.iospress.nl http://www.wtv-books.de/nato-pco.htm

-*-

Series I: Life and Behavioural Sciences - Vol. 346

ISSN: 1566-7693

Disease Markers in Exhaled Breath Basic Mechanisms and Clinical Applications

Edited by

Nandor Marczin National Heart and Lung Institute, Imperial College, London, United Kingdom

and

Magdi H. Yacoub National Heart and Lung Institute, Imperial College, London, United Kingdom

/OS

Press Ohmsha

Amsterdam • Berlin • Oxford • Tokyo • Washington, DC Published in cooperation with NATO Scientific Affairs Division

Proceedings of the NATO Advanced Study Institute on Disease Markers in Exhaled Breath 22 June- 1 July 2001 Limin Hersonissou, Crete, Greece © 2002, IOS Press All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior written permission from the publisher. ISBN 1 58603 273 9 (IOS Press) ISBN 4 274 90532 2 C3045 (Ohmsha) Library of Congress Control Number: 2002109991

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LEGAL NOTICE The publisher is not responsible for the use which might be made of the following information. PRINTED IN THE NETHERLANDS

Preface This monograph contains the contributions of invited speakers and participants at the NATO Advanced Study Institute on Disease Markers in Exhaled Breath: Basic Mechanisms and Clinical Applications, held at the Knossos Royal Village in Crete, Greece, June 22-July 1, 2001. This ASI was designed to summarise and disseminate expert knowledge regarding this rapidly evolving field of lung biology. Breath testing dates from the earliest history of medicine and puzzled brilliant scientific minds including Linus Pauling. It has provided us with powerful techniques of immense medical and commercial applicability such as capnography and ethanol breath testing. Despite this, a wider concept of breath analysis only recently moved from scientific curiosity to clinical reality. The major events responsible for promoting the current knowledge of exhaled breath testing relate to evolution of the idea that molecules such as nitric oxide (NO) and carbon monoxide (CO), previously viewed only as environmental toxicants, are naturally occurring compounds and play a principal role in the normal and pathological regulation of lung function. Furthermore, we have made tremendous progress in our understanding of the biochemistry and molecular biology of disease processes and bioproducts, which are either released from or consumed by the tissues resulting in changes in exhaled breath. Finally, advances in biomedical engineering now allow us to reliably and sensitively identify and quantify these molecules in the parts per billion concentration range and more than 3000 volatile organic compounds (VOCs) at picomolar concentrations. These developments hold enormous promise that analysis of exhaled breath could open a valuable new window onto human metabolism and illuminate its functions in health and disease. Accordingly, analysis of NO, CO and VOCs in exhaled breath has become a primary focus of respiratory research and essential aspect of investigations into many systemic diseases both in the laboratory and in the clinics. These activities are reflected in the rapid expansion of presentations at international meetings, scientific and clinical publications, editorials and recommendations by major respiratory societies. With the rapid expansion of this field, we felt it was timely to organise this institute and to purposefully bring together technical and basic mechanistic aspects of breath analysis through interaction of industry representatives and scientific investigators in order to explore further its potential from basic science to clinical practice. Accordingly, plenary lectures, short oral communications, small group tutorials, poster sessions, lively pro and contra debates and technical discussions were devoted to discuss basic mechanisms underlying production and release to the gas phase of the proposed major disease markers such as NO, CO and VOCs. The remaining daily lectures examined the mechanisms of altered regulation of these molecules and pathways and evidence for the recommendation to use these changes as biomarkers of the broad spectrum of clinical problems. These included acute and chronic lung inflammation with major emphasis on asthma and chronic obstructive pulmonary disease, acute lung injury such as occurring during thoracic organ transplantation and related end stage lung disease, mechanisms of acute and chronic rejection, ischaemia-reperfusion injury and systemic inflammation. It is concluded that NO, CO and VOCs are important molecular players in many of these conditions and analysis of these molecules in the exhaled breath might provide

important insights into pathogenesis, progression and therapeutic responses of patients suffering from these conditions. The tremendous success of the meeting reflects the tireless work of many people. We are especially thankful for the prominent Organising Committee, which consisted of internationally renowned leaders covering expertise on NO, CO and VOCs. The members of the Organising Committee, which included Peter J. Barnes (London), Augustine M.K. Choi (Pittsburgh), Serpil Erzurum (Cleveland), Sergei A. Kharitonov (London) and Charis Roussos (Athens), assisted by the selected Scientific Advisory Panel have been essential in setting the main objectives of the ASI and putting together a stellar cast of lecturers and eminent scientific program. We would like to take this opportunity to thank the members of the Local Organising Committe: Christina Gratsiou, Antonia Koutsoukou, Stylianos Orfanos and Andreas Papapetropoulos for all their hard work. We are also grateful for the help of our close colleagues, Tamas Kovesi and Marianna Imre throughout the meeting and for their editorial assistance. A very special thanks to Ruth Bundy-Marczin for her efficient and meticulous help and passionate support not only during but much before and after the conference. We are especially indebted to Lydia Argyropoulou for working day and night for making sure that every aspect of the organisation was executed smoothly. Finally the organisers would like to salute all speakers and participants for making this event so special and a landmark in the history of dialogues on exhaled breath markers. Nandor Marczin Magdi H. Yacoub

We wish to extend our sincere thanks and gratitude to the following companies for their generous support of this NATO ASI.

SPONSORS

THE SCIENTIFIC AFFAIRS DIVISION OF NATO, Brussels, Belgium ABBOTT LABORATORIES, Greece AIR LIQUIDS HELLAS, Greece AEROCRINE AB, Smidesvagen, Sweden ASTRAZENECA, Prague, Czech Republic BOEHRINGER INGELHEIM, Greece ERICH JAEGER GMBH, Hochberg, Germany GLAXO WELCOME (Allen Pharmaceuticals), Greece LOGAN RESEARCH LTD., Rochester, UK NOVARTIS (HELLAS) A.E.B.E., Athens, Greece PAPAPOSTOLOU, Greece RESPIRATORY RESEARCH INC, Charlottesville, VA, USA START PROMOTION, Milan, Italy SERVIER POLSKA, Warsaw, Poland THE THORAX FOUNDATION, Athens, Greece

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Participants Participantsofofthe theNATO NATOASI ASIononDisease DiseaseMarkers MarkersininExhaled ExhaledBreath, Breath,Greece, Greece,2001 2001


List of Participants Ian Adcock Thoracic Medicine National Heart and Lung Institute, Imperial College. Dovehouse St London SW3 6LY UNITED KINGDOM [email protected] Christofer L. Adding, MD, PhD Division of Physiology Departement of Physiology and Pharmacology Von Eulers vag 6 171 77 Karolinska institute! Solna Stockholm SWEDEN T: 08 728 7226 F: 08 33 20 47 [email protected] Veronica Agrenius, MD PhD, Acting chief, Thoracic Medicine, Karolinska Hospital, 17176 Stockholm SWEDEN Ernst Altman Senior Researcher St Petersburg State University St Petersburg RUSSIAN FEDERATION altniaii@ea4603. spb.edu Adam Antczak MD Med. Univ. Lodz, POLAND T: +48 42 6787505 F:+48 42 6782129 [email protected] Zoe Athanasa, MD Resident and Clinical Fellow Asthma and Allergy Center, Pulmonary and Critical Care Department, Medical School, Athens University, Evgenidio Hospital, 20 Papadiarnantopoulou,! 1528, Athens, GREECE T: (301)7236743 F: (301)7242785

Fritz H. Bach, MD Lewis Thomas Professor Harvard Medical School 99 Brookline Ave. Ste. 370D Boston MA. 02215 USA T: (617) 632-1199 F:(617)632-1198 Fritzhbach @ aol .com Beatrix Balint MD Dept. of Thoracic Medicine University of Szeged Deszk, Alkotmany u 36. HUNGARY T:+36-62-271-411 F:+36-62-271-344 [email protected] George Baltopoulos Professor of Critical Care & Pulmonary Diseases Athens University School of Nursing Director, ICU at "KAT" Hospital of Kifissia 2 Nikis Street Kifissia 14561 Athens GREECE T: 00301-6218381 F: 00301-6218381 [email protected] Adam Barczyk Department of Pneumology Silesian Medical Academy ul. Medykow 14 40-752 Katowice POLAND F:+48-32-2523831 [email protected] Peter J Barnes Professor of Thoracic Medicine National Heart and Lung Institute, Imperial College, Dovehouse St London SW3 6LY UK T:0207 351 8174 F: 0207 351 5675 [email protected]

Gunther Becher FILT Lung and Chest Diagnostics Ltd. Robert-Rossle-Str. 10 Haus 79 (Erwin-Negelein-Haus) 13125 Berlin GERMANY T:+49-30-94892114 [email protected] Ion Belenis Head of Dept of Thoracic and Vascular Surgery, Evangelismos Hospital, Athens, GREECE T:+30-17224449 F:+30-17201506 [email protected] Michael J. Berry Ph.D Chief Scientist, Quadrivium LLC, P.O.Box 1421, Pebble Beach, CA 93953. USA T: (831)625-1177 F: (831) 647-1777. MBcrry717 @cs.com Jorge Boczkowski Unite INSERM 408 Faculte X. Bichat BP416 75870 Paris CEDEX 18 FRANCE T:33 1 44856251 F: 33 1 42263330 [email protected] Nigel Bough ton-Smith AstraZeneca R&D Charnwood Discovery BioSceince, Bakewell Road, Loughborough, Leics. L E i l 5RH, UK T:+44 (0)1509 644332 F:+44 (0)1509 645574 [email protected] Ruth Bundy Heart Science Centre Imperial College Harefield Hospital UB9 6JH, Middlesex UNITED KINGDOM T: 44-(0)l 895-823-737 ext 5044 F:44-(0) 1895-828-900 ruth, bundv@ harcfield.nthames.nhs.uk

John D. Catravas, Ph.D. Regents Professor and Director Vascular Biology Center Medical College of Georgia Augusta, GA 30912-2500 USA T: 706-721-6338

F: 706-721-9799 [email protected] Vladimir Cerny, MD, PhD, FCCM Charles University, Faculty of Medicine University Hospital Dept. of Anesthesiology and Intensive Care 500 05 Hradec Kralove CZECH REPUBLIC T: 42 49 583 ext. 2147 or 3218 F: 42 49 583 2022 [email protected] Augustine M.K. Choi, M.D. Professor of Medicine, Chief, Pulmonary, Allery and Critical Care Medicine University of Pittsburgh School of Medicine MUH 628 NW, 3459 Fifth Ave Pittsburgh, PA 15213 USA T: (412) 692-2117 F: (412) 692-2260 pager (412) 958-6054 choiam @ msx. upmc.cdu Alexander G. Chuchalin Academician, Director Institute of Pulmonology, 32/61, 11 -th Parkovaya Street Moscow 105077, RUSSIAN FEDERATION T: +7 095 465 5208 F: +7 095 465 5364 aisanov @ telemed.ru Eric Demoncheaux, PhD Division of Clinical Sciences (CSUH) Section of Medicine and Pharmacology Respiratory Medicine Unit University of Sheffield Medical School Floor F, Beech Hill Road Sheffield, S10 2RX, UK T: 44(0)114 271 2451 F: 44(0)114 271 1711 [email protected] Christiana Dimitropoulou, Ph.D. Department of Pharmacology and Toxicology Medical College of Georgia Augusta, GA 30912-2300 USA T: 706-721-7361 F: 706-721-2347 cd i [email protected]

Raed A. Dweik, M.D. Staff Physician Pulmonary and Critical Care Medicine Cleveland Clinic Foundation Cleveland, OH, 44195 USA T: 216-445-5763 P: 216-445-8160 [email protected] Serpil C. Erzurum, MD, FCCP Director, Lung Biology Program, Pulmonary and Critical Care Medicine, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue A90, Cleveland, Ohio 44195 USA T: (216)445-5764, F: (216)445-6624

Dr.Cristian Falup-Pecurariu Department of Neurology "Transilvania" University 2200 Brasov ROMANIA cristp@di'urocon.sult.n) Dr. Oana Falup-Pecurariu Assistant, Depart, of Pediatrics "Transylvania" University, Brasov, ROMANIA. [email protected] Gerasimos S. Filippatos MD Attending Cardiologist Evangelismos Hospital, 28 Doukissis Plakentias 1 1523 Athens GREECE Tel: 30-1-6745222 Fax: 30-1-6745222 geros@compulink,gr Bryan Flaherty, Ph.D. Vice President, Research and Development Natus Medical Inc. USA T: 650-801-7262 [email protected] Ben Gaston, MD. Division oi'Pediatric Respiratory Medicine Box 800386 University of Virginia Charlottesville, VA 22908 USA [email protected]

Nakos George MD, Director, ICU of University Hospital of loannina loannina GREECE T: + 30651 99353, F:+30 651 99279 [email protected] Gianfranco Giubileo ENEA, Laboratory of Molecular Spectroscopy Via E.Fermi 45, 00044-Frascati(RM), ITALY T: +39-06-94005768 F: +39-06-94005400 [email protected] Liza Goderdzishvili Department of Intensive Care Central Military Hospital Navtlugi St. No 6, 380013, Tbilisi, GEORGIA T: 995 32 962356 F:995 32958016 eli/.aveua [email protected] Christina Gratziou Head of Asthma and Allergy Center, Pulmonary and Critical Care Department, Medical School, Athens University Evgenidio Hospital, 20 Papadiamantopoulou,! 1528, Athens, GREECE T: (301)7236743 F: (301)7242785 [email protected] Lars E Gustafsson Professor Department of Physiology and Pharmacology Karolinska Institutet 171-77 Stockholm SWEDEN T: 46 8 728 7226 F: 46-8 332 047 [email protected] Nihal El Habashi. MD. Assistant Lecturer, Department of Physiology, Faculty of Medicine, Alexandria University EGYPT [email protected]

Tryggve Hemmingsson, Project leader Aerocrine AB Smidesvagen 12 S-171 41 SOLNA SWEDEN T: +46 8 629 07 80

F: +46 8 629 07 81 [email protected]

Timothy W Higenbottam, MD MA DSc FRCP Professor Global Clinical Expert, AstraZeneca R&D Charnwood, Clinical Sciences, Bakewell Road, Loughborough, Leics LEI 1 5RH, UK T:+44 (0)1509 644846 F:+44 (0)1509 645563 tim.higenbottam @ astrazeneca.com Markus Hofer MD Fellow, Lung Transplant Program CHOER 19 University Hospital CH-809! Zurich SWITZERLAND T:+41-1-255-1 1 1 1 , markus.holcM @DlM.us/..ch) Marieann Hogman, PhD, Dept of Medical Sciences, Uppsala University, SWEDEN maricann.hognian(°'mcdsci.uu.se

Jiri Homolka,M.D.,Ph.D. Assoc. Professor First Lung Department Foundation Katerinska 19 120 00 Prague 2 CZECH REPUBLIC T/F:+4 20-2-24941500 [email protected] Dr Adriana Hristea National Institute of Infectious Diseases "Prof dr. Matei Bals" 1 Dr. Grozovici Sect 2, 72204 Bucharest ROMANIA [email protected] Dr Razvan Hristea Institute of Aeronautical Medicine Str M.Vulcanescu nr 88 Bucharest. ROMANIA [email protected]

John Hunt, MD Division of Pediatric Respiratory Medicine Box 800386 University of Virginia Charlottesville. VA 22908 USA JFH2M@hscmail. nice, virginia.edu Marianna Imre, MD Assistant Professor Dept. Radiology Medical University of Pecs 7643 Pecs, Ifjusag u. 13 HUNGARY [email protected] Tamas Jilting, MD Assistant Professor Department of Pediatrics The Evanston Hospital Northwestern University Medical School 2650 Ridge Ave. Evanston, IL 60201 USA T: 847-570-1643 F: 847-570-0231 [email protected] Lena Kajland Wilen Marketing Director Aerocrine AB Smidesvagen 12 S-171 41 SOLNA SWEDEN T: +46 8 629 07 80 F:+46 8 629 07 81 [email protected] Miklos KeUermayer Professor, Director Dept.Clinical Chemistry Medical University of Pecs 7643 Pecs, Ifjusag u. 13 HUNGARY T: 36-72-536-122 F: 36-72-536-121 [email protected] Irina A. Kharitonova MD Department of Sleep Disorders, Centre of Medical Rehabilitation of the President of Russian Federation, Poselok Getsena, 143088 Moscow RUSSIAN FEDERATION.

Sergei A. Kharitonov, MD PhD Lecturer, Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, Royal Brompton & Harefield NHS Trust, Dovehouse Street, London SW3 6LY, UK T: +44 020 7352 8 1 2 1 pager 0025 F: +440207351 8126 [email protected] Anastasia Kotanidou Critical Care Department, Evangelismos General Hospital, Medical School, University of Athens, GREECE. akotanicKg'mcd.noa.gr Nick Koulouris MD. PhD. Senior Lecturer in Respiratory Medicine Respiratory Medicine Dept National University of Athens Medical School "Sotiria" Hospital for Diseases of the Chest 152 Mesogion Ave. Athens GR-11527

GREECE"

T: 0030 1 777 8827 F: 0030 1 777 0423 koulniCfl-'.vtre mc.gr Antonia Koutsoukou Critical Care Department, Evangelismos General Hospital, Medical School, University of Athens, GREECE. T: +30-1-7201913 F: +30-1-7244941 [email protected]

Andrew B. Lindstrom, Ph.D. Exposure Methods and Monitoring Branch (MD44) National Exposure Research Laboratory U.S. Environmental Protection Agency Research Triangle Park, NC 2771 1 USA T: 919-541-0551 F: 919-541-3527 [email protected] Ron Logan-Sinclair Director Logan Research Ltd., Unit B2, Spectrum Business Centre, Anthony's Way, Rochester Kent, ME2 4NP. UK. T:+44(0) 1634294900 F: 444(0) 1634294906 [email protected] Stelios Loukidis MD Chest Physician Head of Pneumonology Dept Athens Army General Hospital GREECE T:003018954603 F: 003017494095 ssat@hol.«r Katerina Marathias, M.D. Associate Director Intensive Care Unit Onassis Cardiac Surgery Center 356 Sygrou Ave, Athens GREECE T: (01) 9493000 Beeper #142 F: (01)9493331

v [email protected] Tamas Kovesi, MD Head of Anaesthetics Dept.Paediatrics Medical University of Pecs 7643 Pecs, Jo/.sel'A. u7 HUNGARY Tamas [email protected] Dan Laskovvski Cleveland Clinic Foundation, 9500 Euclid Avenue A90 Cleveland, Ohio 44195 USA T: (216)445-5764, F: (216)445-6624 [email protected] Marilcna Lekka Dep. Of Biochemistry, University of loannina, loannina, GREECE

Sadis Matalon, Ph.D. Associate Dean for Post Doctoral Education Alice McNeal Professor of Anesthesiology Professor of Physiology and Biophysics, Pediatrics, Comparative Medicine and Environmental Health, University of Alabama at Birmingham, 940 THT, 619 19th St. S, Birmingham, AL 35249-6810 USA T:(205)-934-4231 F: (205)-934-7437 [email protected]

Karen McRae, MDCM, FRCP Head of Thoracic Anaesthesia, Department of Anaesthesia, The Toronto General Hospital 585 University Ave Toronto, Ontario, M5G 2C4 CANADA T: I 4163405164 F: 1 4163403698 Karen. McraeCq:uhn.on.ca Wolfram Miekisch, PhD Dept. of" Anaesthesia and Intensive Care Med. University Hospital of Rostock, Schillingallee 35 18057 Rostock GERMANY T:+49-381-494 6114, F:+49-381-4946434 [email protected] Manfred Muertz, Ph.D. Institute of Laser Medicine Diisseldorf University, Medical Department GERMANY F:+49-211-811-1374 rnuerlz @ uni-duesscldorl.de

Andreas Papapetropoulos, PhD GP LIVANOS Research Laboratory Ploutarchou 3 10 675 Athens, GREECE T: (301) 721 7467 F: (301)721 94 17 [email protected] Lara Pizurki, PhD GP LIVANOS Research Laboratory Ploutarchou 3 10 675 Athens, GREECE T: (301) 721 7467 F: (301)721 94 17 lara [email protected] Mieczysiaw Pokorski Professor Dept. of Neurophysiology Medical Research Center ' Polish Academy of Sciences 5, Pawinskiego St. 02-106 Warsaw POLAND mpokorski@medres,cmdik. pan.pl

Pavlos Myrianthefs, MD Attending Physician Athens University School of Nursing, ICU at "KAT" Hospital, Nikis 2, Kifissia, 14561, Athens GREECE T: 00301/6280685 F: 00301/6280702 pavlos myrianlhcfs(fthotmail.com

Victoria PolyakovaPhD, Senior Researcher Institute of Human Ecological Pathology Vasylkivska Street, 45 Kiev, 03022 UKRAINE T: +380 44 266 98 03, 266 98 48 F: +380 44 227 66 13 victoria [email protected]

Lothar Neumann Produktmanagment Pneumologie ERICH JAEGER GmbH Leibnizstr. 7 D-97204 Hochberg GERMANY T:+49 931/4972-189 [email protected]

Cristina Popescu, MD, Specialist National Institute of Infectious Diseases "Prof dr. Matei Bals" 1 Dr.Grozovici Sect 2, 72204 Bucharest ROMANIA [email protected]

Stylianos Orfanos, MD., PhD Dept. of Critical Care University of Athens Medical School, Athens, GREECE T: 30-1-7201919 [email protected]

Gabriel A. Popescu, MD, PhD Senior Physician National Institute of Infectious Diseases "Prof dr. Matei Bals" I Dr.Grozovici Sect 2, 72204 Bucharest ROMANIA [email protected]

Alfred Priftanji Professor UHC "Mother Teresa"; 372, Dibra Sir. Tirana. ALBANIA. T/F: +355 4 224590 pri ftan ji («\san.corn.al Jacques Rami PhD Service d'exploration fonctionnelle respiratoire CHU de Rangueil-Larrey 1 Avenue Jean Poulhes, 31062 Toulouse Cedex, FRANCE T: 33.05.61.32.28.42 (Hospital) F: 33.05.62.88.90.75 (University) [email protected] Fabio Ricciardolo, MD, PhD Department of Respiratory Disease Ospedali Riuniti di Bergamo Largo Barozzi, 1 24100 Bergamo ITALY T: +39-035-269989 F: +39-035-266825 Trice iardoloCq'ospcdaliriuniti.bergarnu.it Terence H. Risby, Ph.D. Professor of Toxicological Sciences Department of Environmental Health Sciences Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe Street Baltimore MD 21205 USA T: (410)955-0024 F: (410) 955-0027. Pager: (410) 283-7078 [email protected] Giovanni Rolla, MD Clinical Investigator Dipartimento di Scienze Biomedtche e Oncologia Uinana, Universita di Torino Divisione di Immunologia Clinica e Allergologia Ospedale Mauriziano Umberto I largo Turati 62, 10128 Torino ITALY F: +39011 5682588 i>rolht@mauri/iano.ii

Charis Roussos MD, PhD Professor Critical Care Department. Evagelismos General Hospital 45-47 Ipsilandou Str. Athens 10675 GREECE T: 30-1-72-43-320 F: 30-1-72-16-503 croussos holinail .com

Werner Steinhauser Marketing Manager Produktmanagment Pneumologie ERICH JAEGER GmbH Leibnizstr. 7 D-97204 Hochberg GERMANY T: +49 93174972-319 DS @jaeger-toennies.com Gregory Stratakos Dept. of Critical Care, University of Athens Medical School, Athens, GREECE [email protected] Steven Sunshine, Ph.D. CEO Cyrano Sciences, Inc. Pasadena, CA USA T: (626) 744-1700x226 http. //www.cvranosciences.com Camille Taille INSERM U408 Faculte de Medecine Xavier Bichat 16, rue Henri Huchard 75018-Pans FRANCE T: (33) 1 44.85.62.48 F: (33) I 42.26.33.30 [email protected]

Miroslava Tilkian Department of Anaesthesia and Critical Care Plovdiv 4000 "Leonardo da Vinchi" str.66 BULGARIA F:+35932238107 miratil(°) vahoo.co.uk Tatiana Tkacheva, PhD Senior Researcher, Toxicology department Institute of Occupational Health of Russian Academy of Medical Sciences Prosp.Budennogo, 31 Moscow 105275, RUSSIAN FEDERATION T: +7 095 365 1000 F: +7 095 366 0583 latkachcva Helv. Chim. Ada 7, 1201-1206. [31]Koppenol, W. (1999) in Metal tons In Biological Systems, eds. Sigel, A. & Sigel, H. (Marcel Dekker. New York), pp. 597-619. [32]Rochelle, L., Fischer, B., Adler, K. (1998) Free Radio. Biol. Med. 24, 863-868. [33]Pryor, W.A., Lemercier, J.N., Zhang, H., Uppu, R.M., Squadrito, G.L. (1997) Free Radic. Biol. Med. 23,331-338. [34] Fang, K., Johns, R., Macdonald, T., Kinter, M., Gaston, B. (2000> Am J Physiol. Lung Cell Mol. Physiol.23,Lll6-L72\. [35] Schmidt, H.H.H.W., Hofrnan, H., Schindler, U., Shutenko, Z.S., Cunningham, D.D., Feeelisch, M. Proc. Natl. Acad. Sci. USA. 93, 14492-14497. [36]Guo, F.H., Uetani, K., Haque, J., Williams, B.R.G., Dweik, R.A., Thunnissen, F.B.J.M., Calhoun, W.. Erzurum, S.C. (1997) J. Clin. Inv. 100, 829-838. [37] Uetani, K., Der, S.D., Zamanian-Daryoush, M., de La Motte, C., Lieberman, B.Y., Williams, B.R.. Erzurum, S.C. (2000) J. Immunol. 165, 988-996. [38] De Sanctis, G.T., MacLean, J.A., Hamada, K., Mehta, S., Scott, J.A., Jiao, A., Yandava, C.N., Kobzik, L., Wolyniec, W.W., Fabian, A.J., Venugopal, C.S., Grasemann, H., Huang, P.L., Drazen, J.M. (1999) J. Exp. Med. 189, 1621-1630. [39] Schuiling, M., Meurs, H., Zuidhof, A.B., Venema, N., Zaagsma, J. (1998) Am. J. Respir.Crit Care. Med.158, 1442-1449. [40] Ricciardolo, F.L., Geppetti, P., Mistretta, A., Nadel, J.A., Sapienza, M.A., Belloflore, S., Di Maria, G.U. (1996) Lancet. 348, 374-377.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

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Regulation of pH in the Human Airway: Mechanisms and Monitoring John F. HUNT Division ofPediatric Respiratory Medicine, Box 800386 The University of Virginia Health System, Charlottesville, VA 22908, USA Phone: 804-924-1820; Fax: 804-243-5392. Email: jfh2m @ Virginia, edu Abstract. The pH of exhaled breath condensate (EBC) is measurable and stable after deaeration with Argon and is found to be moderately alkaline in healthy subjects. In contrast, in patients with asthma, EBC is prominently acidic and normalizes during corticosteroid therapy. Depletion of ammonia (NH3) in exhaled air is necessary but not sufficient for EBC acidification. Human airway epithelial cells respond to extracellular acidic challenge with upregulation of glutaminase activity and resultant release of ammonia. This process serves a pH homeostatic role that may be dysregulated by pro-inflammatory cytokines, allowing airway pH disturbance in inflammatory lung diseases. The measurement of EBC pH and NH3 allows simple non-invasive assays of biochemical processes relevant to lung disease in humans.

1. Introduction Nebulized acidic challenge to the airway is used in the research setting to induce bronchoconstriction, cough and pulmonary function changes[l-7] . Gastro-esophageal reflux disease is causally associated with chronic asthma symptoms by a mechanism attributable to microaspiration of acidic gastric fluid[8]. Inhalation of acid fog has been reported to be a risk factor for asthma hospitalization, bronchial hyperreactivity and decreased pulmonary function[9-15]. In the process of clarifying potential adverse effects of environmental acidic insult, investigators have identified that acidic airway pH (below 6.8) diminishes ciliary function[16], causes epithelial damage and sloughing[17], and alters mucous viscosity[18]. Eosinophils subjected to acidic stress in vitro undergo necrosis and release of inflammatory granule products [19]. Importantly, the chemistry of diverse chemical reactions—including those involving nitrogen oxides[20, 21]—is altered by protonation of the reactants. These observations are consistent with recent findings concerning airway pathophysiology in several lung diseases—most specifically asthma—and led to the hypothesis that airway acidification might also occur endogenously within the lung. In this regard, multiple biochemical processes release acids in the airway, especially so jn the setting of inflammation [22-25]. The condensable portion of exhaled breath (EBC) can be assayed for pH, and in addition, specific acids and bases can be measured that together contribute to final fluid pH.

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9 i 8 -

7 '

Exhaled Breath Condensate pH

fr I. — m

-

•

6 -

,

j-

5 • A

-

***

f

r B

C

Figure 1. Exhaled breath condensate pH is low in acute asthma. After EBC collection, samples were deaerated with Argon until pH stabilized. Control EBC pH (A) was 2-3 orders of magnitude higher than the EBC pH of acutely ill asthma patients (B) but similar to asthma patients treated for > 48 hours with systemic corticosteroids. Amer. J. Respir. Crit Care Med. 2000: 161(3): 694-9.

These measurements have identified prominent changes in condensate chemistry in various disease states. In particular, we have reported that the pH of EBC is prominently acidic during acute exacerbations of asthma—with a mean pH of 5.2—and rises to normal (mean pH of 7.7) during resolution of symptoms with systemic corticosteroid treatment [Figure 1][26]. Subjects with stable asthma have normal to subtly low EBC pH. We fortuitously identified two asthmatic patients whose EBC pH was prominently low while they were asymptomatic, but each of these patients developed symptomatic airway obstruction 48-72 hours later, while maintaining the low EBC pH. The large effect size of these findings (change in hydrogen ion concentration of up to 3 log orders), the possibility that a simple pH assay might be suitable to predict asthma exacerbations, and the potential that airway acidbase disturbance might contribute to asthma pathophysiology has prompted continued investigations into the factors underlying EBC acidity. 2. Methods of Collecting and Processing Exhaled Breath Condensate for pH Assay Our initial method of collecting EBC—using home built devices consisting of Tygon® (Norton, USA) tubing surrounded by a frozen high specific heat gel—relied on gravity and exhaled airflow to propel condensed fluid down the collecting surface wall to an attached test tube[26, 27]. This system was functional for small studies but was encumbered by size, some difficulty with portability, and the necessity for 10-minute collections to be able to obtain sample. In order to ease collections from hospitalized patients, enable use in the clinic, home and work environment, and allow for more automated assays of pH, we developed systems (RTube™, Respiratory Research, Inc, USA, along with a version modified for rapid pH measurement: the pHTube™) that are readily portable and incorporate a one-way exhalation valve that serves also as a syringe-style plunger used to collect fluid off the condenser walls for quick aggregation of sample. These devices have allowed simple collections of sufficient EBC volumes in as little as one minute. Using this system, we have collected samples from

J. F. Hunt / Regulation ofpH in the Human Airway

1 69

spontaneously breathing patients through the mouth and through tracheostomies. Additionally, we have collected multiple samples from intubated patients with the RTube connected directly to endotracheal tubes. Samples have also been collected unsupervised in subjects' homes and at school-based clinic. The pH of an EEC sample is affected by carbon dioxide (CO2) diffusing in and out of solution. Thus, pH measured during or immediately after collection in healthy individuals has a pH of between 6.3 and 7.2, and tends to rise slowly as dissolved CC»2 is spontaneously evolved from the fluid. To obtain assay stability, we employ a system that evolves COi rapidly out of EBC samples by deaeration. This is accomplished by bubbling Argon gas at 700 ml/min (in the pHTube system) or 350 ml/min in 300 uL of EBC in a 2 ml microcentrifuge tube. As CO2 is evolved from the EBC, protons are consumed and pH rises until no further CC»2 evolves and pH becomes stable. We have found in multiple comparisons that this final, deaerated pH is essentially identical to the pH of secretions suctioned from the airway in matched samples (although very limited data exist from matched tracheobronchial secretions from patients with low pH). Beyond the simplicity of the assay, pH measurements in EBC are also robust. The assay is highly reproducible with a coefficient of variation of 4% among healthy subjects. Duration of sample collection does not significantly affect EBC pH. Samples can be left capped at room temperature for months with no change in pH (healthy subjects), and minimal rise in pH (acute asthmatic subjects). The minimal rise that occurs with asthmatic samples at room temperature amounts to less than 0.3 log order, and may relate to loss of protons to nitrite, forming nitrous acid, and subsequent inorganic generation of nitric oxide. We find that samples frozen at -80° C maintain reproducible pH even after years of storage. Transportation or storage of unfrozen samples is not a barrier to reproducible and useful pH assays. Some small effect of salivary pH on EBC pH is to be expected since there is airflow through the mouth during collection. However, salivary pH in matched samples does not significantly correlate with EBC pH[26], and pronounced experimental acidification of saliva with citric acid only minimally effects EBC pH (0.2 log order) [28]. As others have shown, we have been unable to detect salivary amylase in our samples, suggesting that gross contamination is not a concern. Nonetheless it is conceivable that certain foods/beverages ingested just prior to EBC collection, if containing a highly volatile acid, could affect EBC pH, and therefore caution needs to be exercised when performing collections until such issues are more clearly defined. 3. A Review of pH as it Relates to Airway and Exhaled Breath Condensate Chemistry The concentration of hydrogen ions (protons) can be measured by proton-selective electrodes, colorimetric indicator dyes, and solid-state devices. It has become accepted to present these concentrations as the negative log of the hydrogen concentration, or 'pH.' A pH of 7 therefore represents a hydrogen ion concentration of 100 nanomolar (1 x 10 ~ ), a pH of 6 being 1 micromolar (1 x 10 ~6), etc. Although it is functional and simple to consider protons in solution, the reality is that hydrogen ions are mixed with hydronium ions and other higher order complexes of H2O. In this chapter, the terms "hydrogen ions" or "protons" are used, and will be assumed to include these higher order complexes with KÔ. A solution with a pH of 5 by definition has a 10 micromolar concentration of protons, but that provides little indication of the availability of protons for reactions. For depending on the amount and type of buffers in the solution, as protons are used up in reactions, additional protons can be released from buffering compounds in large numbers. At pH 5 the available protons in a solution of a strong acid, such as hydrochloric acid, is smaller than the

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available protons of a weak acid, such as acetic acid, at the same pH. This is because the concentration of acetic acid needed to achieve a pH of 5 is much greater than that of hydrochloric acid. This represents the concept of titratable acidity: it's the available inventory of protons that is important, not just the number that are in solution at a given moment. A discussion of the carbonic acid (^COs) buffer system is warranted because of its potential role in condensate and airway lining fluid chemistry. Buffers maintain pH stability best within 0.8 log order of the pKa of the relevant proton. The FhCOa buffer system (including its components HCOa" and COj2") is the most important buffer in the human blood system, helping to maintain the pH at 7.4. However, the two protons of HzCOs have pKa's of 6.1 and 10.3, seemingly making this system poorly suitable to buffer at pH 7.4. However, this buffer system is not a classic buffer. First, when equilibrium is perturbed by addition of a proton to a solution containing HCOs", H2CO3 is formed that is then directly dehydrated to CO2 [Figure 2]. If the CO2 molecule formed is removed from solution—as would occur in the blood as the respiratory system responds with increased ventilation—the proton is successfully neutralized. This process can continue to neutralize protons as long as there is HCOs" present and this compound therefore serves as a neutralizing base, not a buffer system. Likewise, if the deviation in equilibrium is caused by continuous removal of CC>2 from solution, the reaction continues to the right until 1) CC>2 is at sufficiently low concentrations no longer to be volatile from the solution (and therefore equilibrium is achieved), or 2) the HCOs" is completely consumed. The consumption of HCOs" requires the de novo formation of protons from water, with concomitant release of hydroxide anion (OH") in a process that is alkalinizing. The reaction to form H2CO3 of course slows as the concentrations of reactants (HCO3~ and H+) falls. The key element of the bicarbonate buffer system in blood is that the human senses the concentrations of CC>2 and protons, and responds with a physiologic change (by altering respiratory rate or renal ammonia, phosphate or bicarbonate excretion) to maintain blood pH at 7.4. The utility of H2CC)3 as a pH buffering system in human blood depends on these abilities to maintain homeostasis of the individual components of the system. In a compartment that does not sense and respond to alterations in the system's components, the H2CC>3 system has limited utility as a buffer. For the bicarbonate system to be pH regulatory in the airway lining fluid requires not only an excretion mechanism, but one that is responsive to pH aberration. In the human airway, much controversy exists as to the mechanisms of HCCV excretion, and its concentration and availability to neutralize acid loads in airway lining fluid are uncertain.

pKa=10.3

pKa = 6.1

HCO3 +rf10 ppb at baseline has a positive predictive value of 83% for an improvement in FEVi of >15%, and therefore may be useful in predicting the response to a trial of oral steroid in asthma [24]. A key question is why has it been so difficult to show a dose-dependent effect of inhaled corticosteroids in the treatment of asthma? First, it is possible that the small change in doses makes it difficult to detect changes in asthma symptoms and lung function (FEVi). Secondly, the currently recommended doses may be at the upper end of the dose-response curve, making it difficult to detect a relatively small change in dose. In view of concerns about systemic effects and the better effects of adding an inhaled longacting P2~agonist compared to doubling the dose of inhaled steroid, there is now a trend towards use of lower doses of inhaled corticosteroids. Exhaled NO as an inflammatory marker sensitive to corticosteroids may be the ideal tool to demonstrate a dose-response effect and to adjust the dose in clinical practice. It may also be useful in patients using a fixed combination inhalers (corticosteroids and long acting Pi-agonist) to ensure that inflammation is controlled, as this may be difficult to assess from symptoms when a longacting bronchodilator is taken.

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In fact, inhaled corticosteroids reduce exhaled NO in asthmatic patients [5] and this effect is dose-related [11]. However, a plateau effect on exhaled NO measured after 6 to 12 h since the last treatment may be seen at a dose of 400 u,g budesonide and higher [11,25] in contrast to dose-related improvements in adenosine monophosphate and metacholine reactivity up to 1600 [ig in patients with mild-to-moderate asthma [14,26]. The effect of inhaled steroids on exhaled NO is very rapid and may occur within 6 hours after a single high-dose (8 mg) budesonide (Pulmicort Respules®) in symptomatic moderate asthma [9]. Therefore, chronic and acute reduction in exhaled NO may be of a different magnitude. Recently, it has been shown that the onset of action of inhaled BUD on exhaled NO and the time to reach the maximal reduction were also dose-dependent [14]. A gradual reduction in exhaled NO is seen during the first week of regular treatment [5,13,14] with maximal effect between 3 [5,12] or 4 [10,11] weeks. It is still uncertain whether exhaled NO is useful to direct changes in asthma therapy. Recently, it has been shown that exhaled NO values above 13 ppb had a sensitivity of 0.67 and a specificity of 0.65 to predict a step up in therapy [27], but clearly more studies needed using exhaled NO to direct therapy. Corticosteroids may reduce exhaled NO by directly inhibiting the induction of NOS2 [28] or by suppressing the proinflammatory cytokines that induce NOS2. There is inhibition of NOS2 immunoreactivity with inhaled corticosteroid treatment in asthmatic patients and a parallel reduction in immunoreactivity for nitrotyrosine, which may reflect local production of peroxynitrite from an interaction of NO and superoxide anions [29].

2.2. p2-agonists. Neither short-acting [5,22,30-33] nor long-acting [22,25,30,32,34] Pi-agonists reduce exhaled NO. This is consistent with the fact that they do not have any anti-inflammatory effects in asthma, although it has been shown that regular treatment with inhaled formoterol reduces inflammatory cells in the mucosa of asthmatic patients [35]. There may even be a short-term increase in exhaled NO after Pi-agonists, which may be due to opening up of airways with higher local NO concentrations [36]. 2.3. Anti-Ieukotrienes. The leukotriene receptor antagonist pranlukast blocks the increase in exhaled NO when inhaled corticosteroids are withdrawn [37], and montelukast rapidly reduce exhaled NO by 15-30% in children with asthma [38]. Anti-leukotrienes have a moderate effect in patients with asthma and seasonal allergic rhinitis [39,40], Both formoterol and zafirlukast were equally effective in maintaing asthma control, and zafirlukast caused a significant reduction in exhaled NO [31]. 2.4 NOS inhibitors. Nebulized L-NMMA and L-NAME, which are non-selective inhibitors of NOS, both reduce exhaled NO in asthmatic patients, although this is not accompanied by any changes in lung function [19,41]. Aminoguanidine, a more selective inhibitor of NOS2, reduces exhaled NO in asthmatic patients, but has little effect in normal subjects, indicating that NOS2 is an important source of the increased exhaled NO in asthma [42].

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2.5. Prostaglandins. Prostaglandin (PG)E2 down-regulates NOS2 expression [43] and inhaled PGE2 and PGF2a decrease exhaled NO in normal and asthmatic subjects [44].

2.6. Other drugs. The immunosuppressive drugs, cyclosporin and rapamycin, inhibit NOS2 expression [45], suggesting that exhaled NO can be used to monitor their effect. Ibuprofen, a cyclooxygenase inhibitor, reduces the elevated levels of exhaled NO in normal subjects after i.v. administration of endotoxin [46], and indomethacin partially prevents an increase in exhaled NO and asthma symptoms in patients whose dose of steroids was reduced [47] A low dose of theophylline has no effect on exhaled NO levels in asthmatic patients [48]. Nebulized IL-4 receptor (altrakincept) reduces exhaled NO in patients with moderate asthma [49]. 3. Breath condensate Several non-volatile chemicals, including proteins, have now been detected in breath condensates (Figure 1).

Figure 1. Exhaled breath condensate: Panel A: diagram of the apparatus (EcoScreen, Jaeger, Germany);

3.1. Hydrogen peroxide H202 has been detected in exhaled condensate in healthy adults and children with increased concentrations in asthma [50-53]. There is no correlation between the levels of exhaled H2O2 and age, gender, or lung function in healthy children [53]. However, exhaled H2O2 concentration is related to the number of sputum eosinophils and airway hyperresponsiveness in asthma of different severity, and is elevated in severe unstable asthmatics, although exhaled NO is significantly reduced by the treatment with corticosteroids [51]. This may be related to the fact that neutrophils, prevalent in severe asthma [17], generate higher amounts of superoxide radicals and therefore H2O2 [54].

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Asthmatic patients also exhale significantly higher levels of thiobarbituric acid-reactive products (TBARs), which indirectly reflect increased oxidative stress [50].

3.2. Leukotriens Leukotrienes (LTs), a family of lipid mediators derived from arachidonic acid via the 5lipoxygenase pathways, are potent constrictor and pro-inflammatory mediators that contribute to pathophysiology of asthma. Detectable levels of LTB4, C4, D4, E4 and F4 have been reported in exhaled condensate of asthmatic and normal subjects [55,56]. Elevated exhaled condensate levels of LTB4 have been found in healthy calves during an experimental chest infection [57]. There have been attempts to measure leukotrienes in urine and increased levels of LTE4 have been reported in some asthmatic patients, but they are not consistently increased after allergen challenge [58]. Allergen provocation increases LTC4 and LTE4 concentrations in BAL and in urine during early and late asthmatic responses [59]. However, measurement of airway mediators in urine is problematic because of dilution of the lung-derived signal and delay in excretion. Increased levels of LTE4 have also been found in induced sputum during the late response to allergen in patients with mild asthma [60]. In mild asthmatic patients levels of LTE4, LTC4, LTD4 levels in exhaled condensate are increased during the late asthmatic response to allergen challenge [61]. The levels of leukotrienes LTE4,C4,D4 in breath condensate are elevated significantly in patients with moderate and severe asthma [56], and steroid withdrawal in moderate asthma leads to worsening of asthma and further increase in exhaled NO and the concentration of LTB4, LTE4, LTC4, LTD4 in exhaled condensate [61] (Figure 2)-

80-,

r800

Stable asthma Unstable asthma padrenoceptor genotype. Am J Med 109 (2000) 114-121. [32] D.H.Yates et al.. Effect of short- and long-acting inhaled beta2-agonists on exhaled nitric oxide in asthmatic patients. Eur. Respir. J. 10(1997) 1483-1488. [33] P.Garnier et al.. Exhaled nitric oxide during acute changes of airways calibre in asthma. Eur. Respir J. 9(1996) 1134-1138. [34] G.Fuglsang et at.. Effect of salmeterol treatment on nitric oxide level in exhaled air and dose-response to terbutaline in children with mild asthma. Pediatr. Pulmonol. 25(1998) 314-321. [35] A.Wallin et al.. The effects of regular inhaled formoterol, budesonide, and placebo on mucosal inflammation and clinical indices in mild asthma. Am J Respir Crit Care Med \ 59 (1999) 79-86. [36] L.P.Ho et al.. The current single exhalation method of measuring exhales nitric oxide is affected by airway calibre. Eur. Respir. J. 15 (2000) 1009-1013. [37] H.Kobayashi et al.. Decreased exhaled nitric oxide in mild persistent asthma patients treated with a leukotriene receptor antagonist, pranlukast. Jpn. J. Physiol. 49 (1999) 541-544. [38] H.Bisgaard et al.. NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast. Am. J. Respir. Crit. Care. Med. 160(1999) 1227-1231. [39] A.M.Wilson et al.. Antiasthmatic effects of mediator blockade versus topical corticosteroids in allergic rhinitis and asthma. Am J Respir Crit Care Med 162 (2000) 1297-1301. [40] D.L.Bratton et al.. Exhaled nitric oxide before and after montelukast sodium therapy in school-age children with chronic asthma: A preliminary study. Pediatr. Pulmonol. 28 (1999) 402-407. [41] F.P.Gomez et al.. Effect of nitric oxide synthesis inhibition with nebulized L-NAME on ventilationperfusion distributions in bronchial asthma. Eur Respir J 12 (1998) 865-871. [42] D.H.Yates et al.. Endogenous nitric oxide is decreased in asthmatic patients by an inhibitor of inducible nitric oxide synthase. Am. J. Respir. Crit. Care. Med. 154 (1996) 247-250. [43]

F.D'Acquisto et al.. Prostaglandins prevent inducible nitric oxide synthase protein expression by inhibiting nuclear factor-kappaB activation in J774 macrophages. FEBS. Lett. 440 (1998) 76-80.

[44]

S.A.Kharitonov et al.. Prostaglandins E 2 and F2a reduce exhaled nitric oxide in normal and asthmatic subjects irrespective of airway calibre changes. Am. J. Respir. Crit. Care. Med. 158 (1998) 1374-1378.

[45] M.G.Attur et al.. Differential anti-inflammatory effects of immunosuppressive drugs: cyclosporin, rapamycin and FK-506 on inducible nitric oxide synthase, nitric oxide, cyclooxygenase-2 and PGE2 production. Inflamm. Res. 49 (2000) 20-26. [46]

R. W. Vandivier et al.. Down-regulation of nitric oxide production by ibuprofen in human volunteers. J. Pharmacol. Exp. Ther. 289(1999) 1398-1403.

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[47] J.Tamaoki et al.. Effect of inhaled indomethacin in asthmatic patients taking high doses of inhaled corticosteroids. J Allergy Clin Immunol 105 (2000) 1134-1139. [48] B.Oliver et al.. The effect of low dose theophylline on cytokine production in alveolar macrophages in patients with mild asthma. Am. J. Respir. Crit. Care. Med. 161 (2000) A614. [49] L.C.Borish et al.. Interleukin-4 Receptor in Moderate Atopic Asthma. A phase i/ii randomized, placebocontrolled trial. Am. J. Respir. Crit. Care. Med. 160 (1999) 1816-1823. [50] A.Antczak et al.. Increased hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients. Eur. Respir. J. 10 (1997) 1235-1241. [51] I.Horvath et al.. Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma. Am. J. Respir. Crit. Care. Med. 158(1998) 1042-1046. [52] A.W.Dohlman et al.. Expired breath hydrogen peroxide is a marker of acute airway inflammation in pediatric patients with asthma. Am. Rev. Respir. Dis. 148 (1993) 955-960. [53] Q.Jobsis et al.. Hydrogen peroxide in exhaled air of healthy children: reference values. Eur. Respir. J. 12(1998) 483-485. [54] A.Antczak et al.. Hydrogen peroxide in expired air condensate correlates positively with early steps of peripheral neutrophil activation in asthmatic patients. Arch. Immunol. Ther. Exp. (Warsz, ). 47 (1999) 119-126. [55] G.Becher et al.. Breath condensate as a method of noninvasive assessment of inflammation mediators from the lower airways. Pneumologie. 51 Suppl 2:456-9 (1997) 456-459. [56] T.Hanazawa et al.. Increased Nitrotyrosine in Exhaled Breath Condensate of Patients with Asthma. Am J Respir Crit Care Med 162 (2000) 1273-1276. [57] P.Reinhold et al.. Breath condensate--a medium obtained by a noninvasive method for the detection of inflammation mediators of the lung. Berl. Munch. Tierarztl. Wochenschr. 112(1999) 254-259. [58] R.Dworski, Sheller JR. Urinary mediators and asthma. Clin. Exp. Allergy. 28 (1998) 1309-1312. [59] S.O'Sullivan et al.. Urinary excretion of inflammatory mediators during allergen-induced early and late phase asthmatic reactions. Clin. Exp. Allergy. 28 (1998) 1332-1339. [60] A.J.Macfarlane et al.. Sputum cysteinyl leukotrienes increase 24 hours after allergen inhalation in atopic asthmatics. Am. J. Respir. Crit. Care. Med. 161 (2000) 1553-1558. [61] T.Hanazawa et al.. Nitrotyrosine and cystenyl leukotrienes in breath condensates are increased after withdrawal of steroid treatment in patients with asthma. Am. J. Respir. Crit. Care. Med. 161 (2000) A919. [62] J.D.Morrow, Roberts LJ. The isoprostanes: unique bioactive products of lipid peroxidation. Prog. Lipid Res. 36(1997) 1-21. [63] P.Montuschi et al.. Increased 8-Isoprostane, a Marker of Oxidative Stress, in Exhaled Condensate of Asthma Patients. Am. J. Respir. Crit. Care. Med 160 (1999) 216-220. [64] P.Montuschi et al.. 8-Isoprostane as a biomarker of oxidative stress in interstitial lung diseases. Am. J. Respir. Crit. Care. Med 158 (1998) 1524-1527. [65] L.J.Roberts, Morrow JD. Measurement of F(2)-isoprostanes as an index of oxidative stress in vivo. Free. Radio. Biol. Med. 28 (2000) 505-513. [66] T.A.Mori et al.. Effect of dietary fish and exercise training on urinary F2-isoprostane excretion in noninsulin-dependent diabetic patients. Metabolism. 48 (1999) 1402-1408.

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[67] C.T.Carpenter et al.. Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS. Chest. 114 (1998) 1653-1659. [68]

K.Marangon et al.. Comparison of the effect of alpha-lipoic acid and alpha-tocopherol supplementation on measures of oxidative stress. Free. Radic. Biol. Med. 27 (1999) 1114-1121.

[69] L.M.Landino et al.. Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 15069-15074. [70]

L.G.Wood et al.. Lipid peroxidation as determined by plasma isoprostanes is related to disease severity in mild asthma. Lipids 35 (2000) 967-974.

[71] R.Dworski et al.. Allergen-induced synthesis of F(2)-isoprostanes in atopic asthmatics. Evidence for oxidant stress. Am J Respir Crit Care Med 160 (1999) 1947-1951.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) /OS Press, 2002

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Extended NO analysis applied to patients with known altered values of exhaled NO Marieann HOGMAN Department of Medical Cell Biology; Section of Integrative Physiology, Uppsala University, Box 571,SE 75123 Uppsala, SWEDEN Abstract. If exhaled NO is measured at multiple flow rates it is possible to partition NO into airway and alveolar NO-levels. A further step can be taken by dividing the airway NO into wall concentration and diffusion rate of NO when an iterative NO analysis is applied. With this analysis it is shown that allergic asthmatics as well as patients with allergic rhinitis have increased diffusion rates of NO. Patients with COPD have increased alveolar levels of NO and these patients can be divided into two groups with regards to NO diffusion rate. A quality control feature is also introduced in order to gain acceptance for these measurements in clinical practice.

1. Introduction A new research field opened up when nitric oxide (NO) was discovered in exhaled breath [ 1 ]. It became even more interesting when NO was found in increased levels in inflammatory diseases such as asthma [2], allergic rhinitis [3], and chronic obstructive pulmonary disease (COPD) [4]. Scientists stared to argue about the source of the NO production. The NO measurements only reflected the whole respiratory system like a black box. One step forward was taken with the discovery of the flow dependence of the NO signal [5,6]. This led to the standardisation of the NO measurements, first in Europe [7] then in North America [8]. However, these recommendations did not spread any light on the source of NO in the respiratory system.

2. Extended NO analysis - theory Several research groups started to apply different models to the exhaled NO measurements [9,10]. An attempt to distinguish between the NO generated in the alveolar region and in the airways was made by Tsoukias & George [11]. A plot of the exhaled volume of NO versus the expiratory flow rate was used, where the slope of the curve represents the alveolar concentration and the intercept the airway NO-flux. The mechanism behind the flow dependency of the NO output from the lower airways has been suggested to be diffusion-related [11]. Applying a simplified model (Figure 1) and mathematics based on the classical Pick's 1st law of diffusion [12] to the concept of Tsoukias and George, we derived an equation for the NO concentration as a function of exhaled flow The model has three unknown variables FAA NO, FawNO and Daw NO. Here FAA NO L[13]. J represents the alveolar NO concentration, FawNO the NO concentration in the airway wall

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tissue and DawNO the airway transfer rate of NO. If enough data points of NO concentration at various flows are measured, it is possible to find the value of these three variables by a recursive least square fitting (LSF) algorithm [13-15]. However, LSF method can give erroneous values quite frequently. We therefore wanted to develop a simple and robust solution algorithm as an alternative for the LSF. Since the NO-measurements are meant to be a clinical tool we also wanted to introduce a data quality control feature. The model consists of an alveolar compartment, and a conductive airway compartment modelled as a cylindrical tube with a diffusion barrier layer between the tissue and airway (Figure 1). For the algorithms and the validation of the iteration NO analysis the reader is referred to reference [19]. The iteration algorithms can be run from a simple spreadsheet.

r

FANO - * • - > - > 4 4 4

-+•

-+

FENO

4

Figure 1. The model consists of an alveolar compartment with the NO fraction FANO. The airways are brought together into one big tube with an airway wall fraction, F,WNO with a diffusion rate D1WNO.

3. Extended NO analysis - practice When the slope-intercept method [11] was applied to exhaled NO values in patients with allergic asthma it was found that the increase in exhaled NO was generated in the airways and that the alveolar region had normal NO levels [16]. This is not surprising since asthma is an airway disease. In smokers, airway NO is decreased [17] and in alveolitis the alveolar NO is increased [18]. For the iteration NO analysis the guidelines for NO measurements [7] were followed, except for using three flows (0.005, 0.1 and 0.5 L-s"1) and no vital capacity manoeuvre since a deep breath with slow inhalation was found sufficient [13]. The analysis was applied to patients with known alterations in exhaled NO, e.g. asthma, allergic rhinitis, COPD, smokers and healthy subjects [19]. Patients with Sjogren's syndrome were also investigated. From Table 1 it can be seen that the reason for an increase in exhaled NO at an expiratory flow of 0.1 L-s"1 (FeNOo i) in asthma is due to an increased fraction in the airway wall together with an increased diffusion rate. Interestingly, the diffusion rate in allergic rhinitis was also increased but the airway wall showed normal values. Hence the increase in exhaled NO that Henriksen et al. found in patients with allergic rhinitis [3] might be explained by an altered diffusion rate.

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Table 1. Extended NO analysis applied to patients with known altered exhaled NO values. Data are from reference [19], except for the SjOgren's patients, and given in meaniSEM. Controls n=40 FENO0.i ppb FANO ppb DawNO nL'S" 1 FawNO ppb Quality value

Allergic rhinitis n=15

9±1

1312

21 1

211

8±0

12± 1* 98+ 10 8± 1

98 ±7 12± 1

allergic asthma n=15 18±3*

2± 1 12± 1* 144121*

5± 1

Sjdgren's syndrome n=5

12±3 4±1* 71 1 121 ±34 13± 1

ANOVA, different from control * pCl">r>F", (b) a single channel conductance of 5-10 pS, (c) linear current/voltage relationship under symmetrical ion concentrations, (d) regulation by the phosphorylation of the R domain by protein kinase A, (e) requirement for the presence of ATP at the cytoplasmic side for channel activity, (f) unlike several other Cl" channels, CFTR conductance is not blocked by disulfonic stilbenes. 5. CFTR functions other than apical Cl" channel in epithelial cells CFTR protein is expressed predominantly by epithelial cells lining the airways, gastrointestinal tract and the urogenital tract. The best characterized, and therefore the one fully accepted cellular function of CFTR is a cAMP-regulated Cl" channel function in the apical plasma membranes of epithelial cells. Additional proposed functions of CFTR can be divided into three groups: (a) Conductance of other molecules in addition to chloride, (b) regulation of transepithelial ion transport via interaction with other ion channels, (c) Cl" channel function in intracellular membranes, and (d) apparently ion transport-independent functions.

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Conductance of bicarbonate, ATP and glutathione: CFTR has been shown to conduct HCO3~, in addition to Cl" [22]. The HCCV conductance has been shown to manifest in transepithelial HC(V secretion in airway epithelial cells [23]. This finding suggests a potential physiological role for CFTR in the regulation of pH on epithelial surfaces, such as the airway lining fluid. Given that pH affects many physiological functions, including the regulation of mucus viscosity, ciliary beat frequency and the regulation of phagocytes to internalize and destroy pathogens, this observation might have further implications regarding the pathology of CF. Bicarbonate conductance by CFTR also has an effect on the regulation of intracellular pH, and this might have implications in the regulation of apoptosis in CFTR-expressing cells [24]. CFTR expression has been linked to the ability of cells to secrete ATP, perhaps via an interaction with another transporter [25]. CFTR also conducts glutathione [26], CF cells appear to be defective of glutathione secretion [27], and the use of a synthetic Cl" channel peptide can overcome this defect at the cellular level [28]. The absence of this function of CFTR might have implications in the inflammatory state of the CF mucosa. Regulation of transepithelial ion transport via interactions with other channels: CFTR has been shown to regulate amiloride-sensitive Na* channels [29], and the outwardly rectifying chloride channel (ORCC) [30]. The regulation of Na+ channels by CFTR appears to involve an interaction with the actin cytoskeleton [29]. The inhibitory regulation of Na" channels by CFTR is thought to have pathological significance, however despite apparent success in an early pilot study [31], clinical trials aimed at reducing the increased activity of Na+ channels in the CF airways using amiloride failed to demonstrate any clinically significant improvement in lung function [32-34]. The physiological, or pathological significance of ORCC regulation by CFTR is yet to be determined. CFTR Cl~ channel function in intracellular membranes: Several studies indicated that a substantial pool [35-37], in some cells as much as 50% [36,37] of all fully matured (i.e., fully glycosylated; band C) CFTR is localized in intracellular membrane compartments, and that CFTR is rapidly recycles through the endocytic recycling compartment [17,35,38,39]. There are at least two frequently voiced hypotheses regarding a potential role of intracellular CFTR: 1) The intracellular pool of CFTR could provides a reserve, from which CFTR can be recruited to the cell surface upon stimulation by cAMP [37,40] and 2) CFTR plays a functional role in intracellular membranes [37,40-45]. Such proposed intracellular functions for CFTR include a role in endosomal and TON acidification, sialylation of glycoproteins, regulation of endocytosis and membrane recycling. The hypothesis that CFTR has intracellular functions is surrounded by controversy, since many of the observations that were made in one model system could not be reproduced using other model systems. A major part of this controversy might be due to the diversity of cellular systems that were used for studies addressing the role of intracellular CFTR. Studies that investigated the intracellular functions of CFTR in epithelial cells usually found a correlation between CFTR expression, TON acidification or the regulation of endocytosis and membrane recycling by cAMP [37,40-42,46], while studies addressing the same issues using heterologous expression of CFTR in fibroblasts, or other unpolarized cells found no role for CFTR in either endosomal acidification or in the regulation of membrane traffic [47-50]. Therefore, it is possible that CFTR-dependent regulation of organelle acidification exists only in the context of a polarized epithelial cell. Further complication is that the bicarbonate status of cells, and the expression of other Cl" channels in certain cell types might have an effect on endosome, or TON pH. In order to resolve the controversy over the role of CFTR in pH regulation of intracellular organelles, more carefully designed expression systems in polarized epithelial cells, and a better accounting for the role of CFTR bicarbonate conductance will be required. A CFTRdependent regulation of endosome fusion was also reported [51]. The regulation of

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227

endosome fusion was shown to be dependent on CFTR Cl" channel function, however, without any dependence on endosomal acidification. Ion transport-independent functions of CFTR: CFTR has been proposed to function as a receptor for certain bacteria, including Pseudomonas aeruginosa [21]. This function has been proposed to directly underlie the susceptibility of CF airways to Pseudomonas colonization. 6. CFTR Mutations Disease-causing mutations in CFTR that have been reported enumerate almost 1,000, although only a small fraction of these have been functionally characterized. Up-to-date information on all mutations can be found at the Cystic Fibrosis Mutation Database (http://www.genet.sickkids.on.ca/cftr/)) which is maintained by the Cystic Fibrosis Genetic Analysis Consortium. CFTR mutations can be categorized into four groups based on their functional consequences. Class I mutants are nonsense, splice, and frameshift mutants that encode truncated or aberrant forms of CFTR (e.g., G542X). These mutants in general associate with greatly reduced mRNA expression and protein levels. Many of these mutants cause severe pathology including pancreatic insufficiency. Interestingly, some premature stop mutations in the CFTR coding region (G542X and R553X) can be overcome by low doses of the aminoglycoside antibiotics neomycin and gentamicin [52]. Treatment of cells harboring these alieles with aminoglycosides results in read-through of the inefficient stop codons generated by these mutations. For the limited number of CF patients affected by these mutations, chronic low dose aminoglycoside therapy might provide an efficient therapy [53]. Class II mutants, which are defective in their posttranslational folding, and/or processing constitute the most prevalent disease causing alieles including the DF508 mutation, accounting for approximately two-thirds of all CF alieles. This class of mutations was discovered, similar to many other discoveries, as a consequence of a controversy. While initial attempts failed to detect functional DF508 CFTR in mammalian expression systems [10], there were several reports indicating that DF508 CFTR is functional based on experiments conducted in insect cells [54] and xenopus oocytes [55]. This controversy was resolved by the realization that insect cell cultures and oocytes are maintained at, or near ambient temperature, and mammalian cells had to be cooled to similar temperatures to observe functional DF508 CFTR [56]. Based on these results, and on in vitro findings indicating a folding defect in DF508 CFTR NBD synthetic peptide, the notion emerged that the temperature sensitive CFTR mutants represent a folding defect in the endoplasmic reticulum [57]. DF508, and other similar mutants obtain their final and stable conformation slower, and therefore, are recognized by the ER quality control machinery, become polyubiquitinated, and targeted for degradation in the proteasome [58]. Class II mutations mostly, but not exclusively represent single amino acid substitutions or deletions in one of the two NBDs. CFTR is an inefficiently processed protein. Only approximately 25% of wild type CFTR nascent chain is folded and processed completely, the rest is degraded during the process. Various class two mutants decrease this low efficiency even further, but to varying degrees. Consequently, disease severity within this class correlates with the amount of mutant protein that can be released from the ER. Almost none of DF508 mutant CFTR processes completely, thus, this mutation is associated with severe disease. Some other calls II mutants, such as A455E and P574H process more efficiently, and are associated with milder disease.

228


Class III and class IV mutants are fully glycosylated and are targeted to the plasma membrane but either exhibit defective channel regulation (class HI) or ion conduction (class IV). Class III mutants generally exhibit mutations in the NBDs that lead to compromised channel activation. Class IV mutants represent a small group of amino acid substitutions in the membrane-spanning domains (e.g., R117H and R347P). The consequence of these mutations is reduced single channel conductance. 7. CFTR mutations as therapeutic targets: The discovery of CFTR gene in 1989 generated great anticipation of the forthcoming gene therapy for CF. Although it is still likely that such therapy eventually will be available in the future, research targeting this problem has shown us that accomplishing clinically useful CF gene therapy faces extraordinary challenges. However based on the emerging details of the cell biology and physiology of CFTR's cellular functions, and based on the understanding how the various CFTR mutations cause disease several alternative therapeutic approaches have been proposed. Developing some of these novel therapeutic concepts into practical therapies will be useful even if gene therapy becomes available, as they might allow the alleviation of airway obstruction that might be a prerequisite for the access of gene therapy vehicle to the lower airways. The discovery of the folding defect of Class II mutations, and the realization that these mutants once folded are functional, fueled the development of several therapeutic concepts. Since a miniscule proportion of DF508 CFTR is processed to functional form, boosting its expression level using butyrate [59] promises to have at least partial correction of the defect. Moreover, several observations indicated that the use of chaotropic agents such as DMSO [60] can increase the efficiency of DF508 CFTR processing in the ER, perhaps by not allowing the misfolded conformation to stabilize. Based on these in vitro data, finding a biologically safe and clinically usable combination of chemicals that boost expression levels and facilitate processing could provide a novel therapy for CF. As mentioned previously, certain class I mutants represent inefficient stop codons generated by single base substitutions in the CFTR gene. Aminoglycoside antibiotics can facilitate read through these stop codons, allowing the translation of full length, functional CFTR in cells where at least one of the mutant alleles falls into this category [61]. Furthermore, there is clinical evidence that such treatment strategy can alleviate the Cl" transport abnormality in the airways of CF patients as evidenced by a partial correction of the abnormal nasal transepithelial potential difference following parenteral gentamicin treatment for 7 days [53]. In addition to therapies aimed at increasing the amount of functional CFTR, various additional therapeutic approaches have been considered. Since the majority of CF scientists agree that a correction of the ion transport defect in the CF lung would alleviate the CF lung pathology, pharmacological manipulation of airway epithelial cell ion transport pathways is considered as therapy. These approaches include (a) CFTR openers that might increase the amount of Cl" transported by the available small amount of CFTR, (b) openers of alternative Cl" conductance pathways, such as synthetic channels and openers of Ca+ activated Cl" channels, (c) activators of K+ channels that increase the driving force for Cl- across the airway epithelial monolayer [62] and (d) inhibitors of Na+ channels [31].


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8. Inflammation in CF It has been known that the CF lung is continuously in an exaggerated state of inflammation [63]. There are two aspects of this inflammatory state that will be discussed here: (a) Inflammation might be a direct consequence of the CF defect, and not only a reaction to the infections that are present in the lung, and (b) inflammation reduces CFTR expression and function via multiple mechanisms. (a) Inflammation might be a direct consequence of the CF defect: Inflammation is present in airways of CF infants even under conditions when infectious agents could not be detected [64,65]. Additionally, an increased accumulation of mast cells and macrophages can be observed during fetal development in CF [66]. Furthermore, it has been shown that sustained dosage of Ibuprofen lowered the rate of lung function deterioration of CF patients over the period of 4 years [67]. These findings suggest that an exaggerated inflammatory response to pathogens is directly related to the CF defect, and might underlie the deterioration of lung function during the progress of the disease. There have been several proposed mechanisms that might explain the connection between mutant CFTR and inflammation at the cellular and molecular level. The expression of trafficking mutants of CFTR in cells has been correlated with an activation of the nuclear factor DB (NFDB) [68], NFDB activation has been generally considered to be an underlying mechanism of inflammatory processes at a cellular level. Cultured CF airway epithelial cells exhibit an exaggerated production of the inflammatory cytokines IL-6 and IL-8 upon stimulation with TNFD, when compared to normal, or corrected CF cells [69]. The mechanism of this abnormal response is not known. As discussed earlier, CFTR can also conduct glutathione. In CF airways the relative absence of glutathione might contribute to the lack of protection from inflammatory insults [70]. The exact contribution of epithelial secretory products to the inflammatory balance of the airway mucosa is not completely understood, but it is known that epithelial cells have the capacity to secrete a wide variety of molecules with inflammatory or antiinflammatory activity such as cytokines, surfactants, antiproteases. We have shown that in a CFTR-expressing epithelial HT29-CL19A cell line the regulation of the apical constitutive secretory pathway by cAMP is Cl"-dependent, and it likely involves the regulation of TGN acidification [45]. One of the main secretory products of HT29CL19A cells is D1 antitrypsin, an important antiinflammatory protein on mucosal surfaces. In the same cell line the inhibition of CFTR expression by the inducible expression of antisense CFTR mRNA [71], or by the cytokines IL-4 and IFND dramatically changes the secretion profile of apically secreted proteins, and eliminates the regulation of this apical secretion by cAMP [72]. These findings suggest that the regulation of the epithelial apical macromolecule secretion machinery might be affected in CF, and that this effect might have an impact on the inflammatory balance on mucosal surfaces. (b) Inflammation reduces CFTR expression and function via multiple mechanisms: Epithelial CFTR expression has been shown to be inhibited by various inflammatory mediators, including IFND, IL-4, and TNFD. [73-76]. Interestingly, IL-1D increases CFTR expression via a mechanism that is dependent on NFkB activation [77]. Furthermore, exposure of stably transduced cells to exogenous nitric oxide, dramatically reduces heterologously expressed CFTR [78]. On the other hand, treatment with Snitrosoglutathione, which promotes S-nitrosylation of proteins, as opposed to tyrosine nitration by nitric oxide in the presence of superoxide, increases DF508 CFTR expression and improves processing [79]. Understanding the exact relationship between CF mutations and the inflammatory response in the lung might aid the design of better therapies for CF. Additionally, in vitro data regarding inflammatory regulation of CFTR expression suggest that temporary downregulation of CFTR expression might have pathological implications in lung diseases other than CF when inflammation is present.

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9. Summary Since the first description of cystic fibrosis in 1936 an enormous amount of knowledge accumulated regarding the pathology of cystic fibrosis. The discovery of CFTR in 1989 has accelerated the studies aimed at the understanding of cellular level CF defects. However, the direct relationship between the various cellular level defects and disease pathology needs clarification. Most agree that ultimately CF will be cured by gene replacement therapy, but its practical application appears to be far in the future. A better understanding between the cellular and molecular CF defects, and the resulting clinical disease should aid the design of better pharmacotherapy for CF.

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[68JA.J. Weber, G. Soong, R. Bryan, S. Saba and A. Prince Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function, Am J Physiol Lung Cell Mol Physio! 281 (2001 )L71-78. [69JA.A. Stecenko, G. King, K, Torii, R.M. Breyer, R. Dworski, T.S. Blackwell, J.W. Christman and K.L. Brigham Dysregulated cytokine production in human cystic fibrosis bronchial epithelial cells, Inflammation 25 (2001)145-155. [70] V.M. Hudson Rethinking cystic fibrosis pathology: the critical role of abnormal reduced glutathione (GSH) transport caused by CFTR mutation, Free Radic Biol Med 30 (2001) 1440-1461. [71]T. Jilling, E. Weber, E.J. Sorscher and K.L. Kirk Modulation of CFTR expression in HT29-CL19A colonic cells via the regulated expression of antisense mRNA, Fed. Pulm. Supplement 13, A#71 (1996) 229. [72] T. Jilling, C.J. Venglarik, E.J. Sorscher and K.L. Kirk CFTR expression correlates with the regulation of apical constitutive protein secretion in polarized epithelial cells, Fed. Pulm. Supplement 13, A#68 (1996) 228. [73] T. Jilling, C.J. Venglarik, E.J. Sorscher and K.L. Kirk IFN gamma and IL-4 regulate CFTR expression in HT29-CL19A colonic epithelial cells, Fed. Pulm. Supplement 13, A#70 (1996) 228. [74] H. Nakamura, K. Yoshimura, G. Bajocchi, B.C. Trapnell, A. Pavirani and R.G. Crystal Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene, FEBS Lett 314 (1992) 366-370. [75] F. Besancon, G. Przewlocki, I. Baro, A.S. Hongre, D. Escande and A. Edelman Interferon-gamma downregulates CFTR gene expression in epithelial cells, Am J Physiol 267 (1994) C1398-1404. [76JS.M. Fish, R. Proujansky and W.W. Reenstra Synergistic effects of interferon gamma and tumour necrosis factor alpha on T84 cell function, Gut 45 (1999) 191-198. [77] E.G. Cafferata, A.M. Guerrico, O.H. Pivetta and T.A. Santa-Coloma NF-kappaB activation is involved in regulation of cystic fibrosis transmembrane conductance regulator (CFTR) by interleukin-lbeta, J Biol Chem 276 (2001) 15441-15444. [78] T. Jilling, I.Y. Haddad, S.H. Cheng and S. Matalon Nitric oxide inhibits heterologous CFTR expression in polarized epithelial cells, Am J Physiol 111 (1999) L89-96. [79] K. Zaman, M. McPherson, J. Vaughan, J. Hunt, F. Mendes, B. Gaston and L.A. Palmer Snitrosoglutathione increases cystic fibrosis transmembrane regulator maturation, Biochem Biophys Res Commun 284 (2001)65-70.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub{Eds.) IOS Press, 2002

Exhaled markers in cystic fibrosis Beatrix BALINT1, Sergei A. KHARITONOV3, Ildiko HORVATH2, Peter J. BARNES3 Dept. of Thoracic Medicine, Medical Faculty of University of Szeged, Hungary, 2Dept. of Pathophysiology at the National Kordnyi Institute, Budapest, Hungary, 3Dept. of Thoracic Medicine, Imperial College School of Medicine at National Heart and Lung Institute, London, United Kingdom 1. Introduction Cystic fibrosis (CF) is a genetic disorder caused by mutation of CFTR gene leading a life-long chronic airway inflammation. Lung disease in CF is the primary cause of morbidity and mortality due to recurrent airway infection. The diagnosis of acute respiratory infection in individuals with CF is difficult because conventional measures of acute infection such as fever, raised leukocyte count, deterioration in lung function, and positive sputum culture are not always helpful [1]. The chronicity of lung disease in CF and the tendency for acute respiratory infections to present in an atypical fashion, poses problems for research design. Sputum is readily available in CF and does not require the patient to undergo a moderately invasive procedure, although measurement of cytokines and inflammatory mediators in CF sputum did not prove to be helpful for identifying acute exacerbations [2]. Invasive diagnostic method, such as bronchoscopic examinations and sampling via bronchoalveolar lavage (BAL) require a high deal of expenditure, cause strain to the patients and modify the samples to be taken as they irritate the airways [3,4]. It would be useful to have some other indicators of infection and inflammation as a diagnostic tool and as a way to monitor disease and guide therapy. Analysis of exhaled breath constituents may be a non-invasive method of monitoring inflammation and oxidative stress in the lungs. Measurement of exhaled nitric oxide (FENO) is the most widely investigated method for monitoring airway inflammation in several inflammatory lung diseases, such as asthma [5, 6], bronchiectasis [7], unstable chronic obstructive lung diseases (COPD) [8], viral and bacterial airway infection [9]. Surprisingly, in CF patients FENO and nasal nitric oxide (NO) are significantly lower than in normal subjects, despite the intense neutrophilic inflammation in the airways [10-14]. Carbon monoxide (CO), another exhaled marker may be more useful in CF, because it is elevated significantly in CF patients and increases further during acute exacerbation [15, 16]. Analysis of exhaled breath condensate is a new non-invasive method for detection of changes in lung metabolism and the inflammatory status of the lung. Several compounds of exhaled breath condensate have been recently investigated in CF, such as hydrogen peroxide (fyOi) [17, 18], NO-related products [11, 19-21], eicosanoids [22] and cytokines [23]. The aim of this publication to overview data regarding exhaled breath and exhaled breath condensate analysis in CF. We will discuss our experience regarding NO metabolites (nitrotyrosine) and cytokine (IL-8) in exhaled breath condensate from CF patients with and without acute exacerbation.

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2. Measurement of exhaled breath in CF Exhaled Nitric Oxide NO is produced in the respiratory system and is detectable by chemiluminescence analyser in airway gas. Concentrations are higher in the upper than the lower airways. Several different tissues in the lung have been identified as capable of NOS expression and NO formation. Generally FENO is increased in inflammatory condition such as asthma [5, 6], bronchiectasis [7], unstable COPD [8], upper airway infection [9], probably due to iNOS induction. In asthma, measurement of FENO may serve as a marker of airway inflammation [8, 24]. Surprisingly, despite chronic inflammation in CF lungs, exhaled and nasal NO levels are significantly lower in patients with CF either being stable or having acute exacerbation [1014]. The level of FENO in CF is not influenced by the age of patients [13, 14, 17], use of antibiotics [17], use of corticosteroids [11] as well as CF genotype [14]. Besides some technical issues, the possible explanations for the paradoxical reduction in FENO may be complex. On one hand, a reduced expression of iNOS may occur in CF lung [25], which can contribute to the decreased concentration of FENO. In inflammatory lung diseases expression of iNOS is induced by inflammatory signals such as cytokines and lipid polysacharide (LPS) [26, 27]. In contrast, CF epithelial cells when stimulated by cytokine mix and co-cultured with activated neutrophils, have reduced iNOS expression compared to normal epithelial cells [27]. On the other hand, an increased metabolism of NO to reactive nitrogen intermediates would account for the low levels of nasal and exhaled NO. NO can be oxidized rapidly to form reactive nitrogen intermediates such as nitrite (NOi"), nitrate (NOs") and peroxynitrite (ONOO") [25]. The microenvironment of CF lung - viscous mucus secretion and increased reactive oxygen species release from inflammatory cells - may facilitate the reaction of NO with inflammatory oxidants causing an increased formation of reactive NO metabolites. In CF airways NO in its different oxidative states can be trapped in the mucus. Measurement of free NO could therefore be misleading and may not reflect to the total NO production in CF airways. Exhaled Carbon Monoxide In contrast to FENO exhaled CO levels are markedly increased in patients with CF [15]. CO is a product of heme degradation by heme-oxygenase (HO). Two isoforms of HO have been described: the constitutive HO-2 and the inducible HO-1, which is ubiquitously distributed. HO-2 can be upregulated by oxidative stress and proinflammatory cytokines [27, 28] and is part of the protective response to oxidative stress [29], HO is present in the pulmonary vascular endothelium and alveolar macrophages [30, 31]. CF is characterized by increased oxidative stress in the airways, so probably elevated level of CO in exhaled breath reflects the increased HO-1 expression by the pulmonary vascular endothelium and alveolar macrophages. Furthermore high level of CO may inhibit iNOS activity [32] and therefore reduce the level of FENO. Exhaled CO elevates further during acute exacerbation suggesting that exhaled CO not only a marker of oxidative stress but is also a marker of disease activity [16]. It has been observed that exhaled CO can be decreased by corticosteroid therapy [15] and patients homozygous for delta508 mutation have higher exhaled CO concentration, than heterozygous patients [15].

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Taken together, measurement of exhaled CO seems to be a useful tool of detecting and monitoring cytokine mediated inflammation and oxidant stress in the lower respiratory tract and of assessing the efficacy of therapy. CO measurement is simple and non-invasive, so it can be repeated as needed, and is suitable for using at patients with advanced diseases as well as at children. Exhaled Hydrocarbons Hydrocarbons are non-specific markers of lipid peroxidation, which is one of the consequences of the constant and inevitable formation of oxygen radicals in the body. During the process of peroxidation of polyunsaturated fatty acids hydrocarbons are distributed in the body, partly metabolized and excreted in the breath. Ethane is produced from lipid peroxidation and can be measured in the exhaled breath. Exhaled ethane can be collected into a reservoir and can be analyzed by chromatograpy. It is elevated in CF compared to normal subjects and reduced by steroid-treated patients. The level of exhaled ethane correlates with exhaled CO and lung function parameter (RVYTLC) [33]. It may be a useful noninvasive marker of oxidative stress. 3. Measurement of exhaled breath condensate in CF NO-related products NO reacts with superoxide to yield peroxynitrite and it can be trapped by thiolcontaining molecules such as cysteine and glutathione, to form S-nitrosothiols or can be oxidized to nitrite (NO2~) and nitrate (NOs") [34]. NO metabolites, such as NOi", and NOs", can be detected in airway aspirates, BAL fluid and exhaled breath condensate from normal subjects [12]. Inflammatory conditions are associated with enhanced NO formation reflecting to the increased concentrations of its metabolites. NO metabolites have been measured is sputum from CF patients [35] as well as in exhaled breath condensate [11] and probably reflect better to NO metabolism than FENO in CF [11 ]. Nitrite, S-nitrosothiols and Nitrotyrosine Nitrite levels in exhaled breath condensate are significantly higher in patients with stable CF compared to normal subjects [11, 20, 23]. NO2~ concentration in breath condensate correlates positively with circulating plasma leucocytes and neutrophils, and does not correlate with FENO and lung function parameters. It supports the hypothesis, that in inflamed airways, a significant proportion of NO from the lower airways may have been degraded by oxidation to NO2" and/or NO3". S-nitrosothiols are formed by interaction of NO with glutathione and may limit the detrimental effect of NO. It can be detected in exhaled breath condensate in several inflammatory diseases such as asthma, COPD and cystic fibrosis [20]. It has been published that S-nitrosothiols values are elevated in adult patients with more severe CF during both stable period and acute exacerbations [20]. Nitrotyrosine is a marker of protein nitration and can be detected by using a specific nitrotyrosine antibody. Tyrosine nitration can be mediated by multiple pathways under different conditions, suggesting that nitrotyrosine may be considered as a collective indicator for the involvement of reactive nitrogen species [36]. Nitration of tyrosine could impact deleteriously on cellular function and viability because this specific modification is known to alter protein function in vitro [36].

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Peroxynitrite is a potent oxidant, formed by the rapid reaction of the free radicals NO and Oi" and causes tyrosine nitration in lung tissue [37]. The toxicity of peroxynitrite is due to the direct reactions of the anion (ONOO"), as well as the reactivity of the acid (ONOOH). Activation of inflammatory cells, such as neutrophils, eosinophils and macrophages induces a marked production of superoxide facilitating the formation of peroxynitrite [38]. In chronic inflammation, or other inflammatory cell-mediated process, the myeloperoxidase (MPO)dependent pathways must be considered. Large numbers of polymorphonuclear neutrophils (PMN) accumulate in airways of CF patients, and lead to increased MPO activity [39]. Activated human PMNs can convert NOi" into inflammatory oxidants through MPO pathway [36]. It has been suggested that MPO-catalysed nitration in the presence of IHhOi to form nitrating intermediates from NO2~, a main end-product of NO, is an alternative mechanism of protein nitration, which is independent of peroxynitrite [36]. The other pathways of the formation of nitrotyrosine which are detected in vitro, including direct oxidation of NO2~ by HiOi or hypochlorous acid or reaction of NO or nitrogen dioxide with tyrosyl radicals in vivo have not been completely elucidated [37]. We found increased levels of nitrotyrosine in exhaled breath condensate from stable CF patients compared to normal subjects [21]. It has overlapped with the finding of elevated level of nitrotyrosine in CF sputa [23]. In our study there was no correlation between FENO and nitrotyrosine concentration, although there was an inverse correlation between nitrotyrosine levels and disease severity measured by lung function. There was no significant difference in nitrotyrosine levels in breath condensate between patients treated or not treated with steroids. In this study we have provided evidence that oxidative stress induced by inflammation produces nitrotyrosine, which presumably reflects increased direct nitration by granulocyte peroxidases. Nitration of proteins by MPO is, perhaps a major source of nitrotyrosine in patients with CF who have, a low NO production. p < 0.001

p < 0.05

50 ! IS) 40 c

g 30-

|,j |j 10 0J

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steroid Treated

Normal Subjects

CF steroid Untreated Treated

Figure 1. FENO in stable CF patient and normal subjects (right panel). Nitrotyrosine in exhaled breath condensate from stable CF patient and normal subjects (left panel)

Hydrogen peroxide Activation of inflammatory cells, including neutrophils, macrophages and eosinophils, results in an increased production of superoxid, which by undergoing spontaneous or enzymcatalyzed dismutation leads to formation of H2O2. As HzOz is less reactive than other reactive oxygen species, furthermore it is soluble, increased H2O2 in the airway equilibrates with air [40]. In CF breath condensate H2O2 level proved to be not elevated in patients with stable CF [18], although it has been reported being increased in CF with acute exacerbation, and

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decreased during intravenous antibiotic treatment [17]. Measurement of HaC^ in exhaled breath condensate may serve as a useful noninvasive parameter of airway inflammation. Eicosanoids Eicosanoids are inflammatory mediators, can cause vasodilatation or vasoconstriction, plasma exudation, mucus secretion, bronchoconstriction or bronchodilatation and inflammatory recruitment. Several types of biomolecules with either opposite effects belong to this group. All of them derive from the arachidonic acid cascade and include prostaglandins, thromboxane, isoprostanes and leukotrienes. Several of them can be detected in exhaled breath condensate providing an opportunity to assess the eicosanoid profile in lung diseases. Isoprostanes are a novel class of prostanoids formed by free radical-catalyzed lipid peroxidation of arachidonic acid [41]. 8-Isoprostane is elevated in plasma as well as in exhaled breath condensate in stable CF. Its level negatively correlates with FEV] and positively correlates with exhaled CO [22]. Leukotrienes, prostaglandines in exhaled breath condensate have been investigated in asthma and COPD, but there are no published data available regarding their levels in CF. Cytokines Chronic and acute inflammation is associated with activation of pro-inflammatory cytokine network. Several pro- and anti-inflammatory cytokines can be detected in sputum [42] as well as in BAL fluid in different inflammatory diseases including CF [43]. Interleukin-8 (IL-8) is a neutrophil-activating peptide associated with acute and chronic inflammation is produced by a wide variety of cells, including bronchial epithelial cells, monocytes, alveolar macophages, endothelial cells, fibroblasts and PMNs. IL-8 levels are elevated in sputum and BAL fluid in CF [42, 44], promoting the destructive inflammatory process in the lung. It has been reported to play a major role in the early inflammatory pathogenesis in the airways of CF patients before the manifestation of bacterial infection [45]. Recent publication has shown that IL-8 could be detected in exhaled breath condensate of children with CF [23]. We assessed the level of IL-8 in exhaled breath condensate from clinically stable and unstable adult CF patients. Furthermore, we investigated the alteration of IL-8 level in exhaled breath condensate in unstable CF after recovery from acute respiratory tract infection. We compared the levels of IL-8 in breath condensate from CF patients to other non-invasive inflammatory marker like FENO, as well as lung function and blood test. Subjects and Methods: 18 clinically stable and 12 unstable CF patients were recruited into the study along with 11 healthy normal subjects. 7 CF patients with acute exacerbation were followed after 10-14 days of complex treatment and IL-8 was detected again after the recovery from the acute symptoms. For all subjects FENO, lung function measurement and collection of exhaled breath condensate as well as blood test were performed. IL-8 levels in exhaled breath condensate were measured by sandwich ELISA (R & D Systems Europe, Abingdon, UK). Initially, all samples from the breath condensate were concentrated fourfold, using a freeze dryer (Modulyo, Edwards, Crawley, UK), and then analyzed according to the manufacturer's instructions. Detection was performed with tetramethylbenzidine (R & D Systems Europe. Abingdon, UK) following R & D instructions. The lower limit of detection for this assay was 16pg/ml.

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Results: FENO was significantly lower in stable CF patients than in normal subjects (3.9 ±0.5 vs 6.1 ± 1.0 ppb, p < 0.05) and did not alter significantly during the acute exacerbation. There was no significant difference in the levels of IL-8 in exhaled breath condensate between normal subjects and stable CF patients, although it was significantly elevated in unstable CF compared with normal subjects (36.1 ± 5.1 vs 17.4 ± 2.8 pg/mL, p < 0.01). 7 patients were followed after 10-14 days of antibiotic treatment and IL-8 decreased significantly to the baseline level after recovery (34.3 ± 2.0 vs 21.8 ± 3.9 pg/ml, p < 0.05). Discussion: Our study demonstrated that IL-8 can be detected in exhaled breath condensate of normal subjects as well as in adult CF patients. IL-8 levels were not different in exhaled breath of stable CF patients from normal subjects, but it was significantly higher in CF with acute exacerbation than in non-smoking healthy controls. IL-8 is an important proinflammatory cytokine, which has an outstanding role in CF pathogenesis. In CF patients endogenous signal may be generated, leading to an intense inflammatory response with the production of factors, which could damage the airway surface and favor infection and bacterial colonization. This signal may be directly linked to the abnormal CFTR and may be associated with a dysregulated inflammatory response. Conclusion: Our data indicate that IL-8 in exhaled breath condensate may be useful noninvasive marker of airway inflammation in CF exacerbation. p < 0.01 ns

p < 0.05

ns

50-

50-

25-

25-

0-

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Stable Unstable CF

Admission

Day 10

Figure 2. IL-8 in exhaled breath condensate from CF patient with and without acute exacerbation (right panel). IL-8 in exhaled breath condensate from CF patient with acute exacerbation at the onset of exacerbation and at the time of recovery (left panel)

4. Summary This chapter has overviewed the recent developments of assessment of exhaled breath and exhaled breath condensate in CF patient with and without acute exacerbation. NO concentrations in the expired air of patients with stable CFare significantly decreased compared to normal subjects. Furthermore there is no significant alteration in FENO during acute exacerbation. This is likely to be on one hand due to the decreased iNOS expression by the epithelial cells, and on the other hand due to the increased consumption of NO by superoxide in CF airways. Measurement of exhaled CO may be promising non-invasive tool in CF, because its level is significantly increased in stable CF and elevates further during exacerbation. Analysis of exhaled breath condensate is a new, currently studied, non-invasive research procedure, which may have an important place in the diagnosis and management of inflammatory lung diseases such as CF. NO-related products in exhaled breath increased

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significantly in CF patients reflecting the increased oxidative metabolism of NO in CF airways. Eicosanoids, proteins and cytokines, electrolytes and pH of breath condensate in CF have been investigated, or the topic of the present research, which may offer a possibility to us to monitor the airway inflammation and to assess the efficacy of therapy. Acknowledgements This study was supported by the Hungarian Respiratory Society, Foundation for Patients with Lung & Heart Diseases (Hungary) and the British Lung Foundation (NHLI, UK). References 1. A. L. Smith, G. Redding, C. Doershuk, D. Goldmann, E. Gore, B. Hilman, M. Marks, R. Moss, B. Ramsey, T. Rubio, et al. Sputum changes associated with therapy for endobronchial exacerbations in cystic fibrosis, JPediatr 112 (1988) 547-554. 2. J. M. Wolter, R.L. Rodwell, S. D. Bowler, J. G. McCormack. Cytokines and inflammatory mediators do not indicate acute infection in cystic fibrosis, Clin and Diagnostic Lab Immunol 6 (1999) 260-265. 3. M. W. Konstan, K. A. Milliard, T. M. Norvell, M. Berger. Broncoalveolar lavage findings in cystic fibrosis patients with stable, clinically mild lung disease suggest ongoing infection and inflammation, Am J Respir Crit Care Med. 150(1994) 448-54 4. K. Balough, M. McCubbin, M. Weinnnberger, W. Smits, R. Ahrens, R Pick, The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis, Pediatr Pulmonol 20 (1995)63-70. 5. K. Alving, E. Weitzberg and J. M. Lundberg, Increased amount of nitric oxide in exhaled air of asthmatics. Ear Respir J. 6(1993) 1368-1370. 6. S. A. Kharitonov, D. Yates, R. A. Robbins, R. LoganSinclair, E. A. Shineboume and P. J. Barnes, Increased nitric oxide in exhaled air of asthmatic patients, Lancet343 (1994) 133-135. 7. S. A. Kharitonov, D. A.U Wells, B. J. Oconnor P. J. Cole, D. M. Hansell, LoganSinclair, P. J. Barnes. Elevated levels of exhaled nitric oxide in bronchiectasis, Am J Respir Crit Care Med 151 (1995) 18891893. 8. W. Mazaik, S. Loukides, S. V. Culpitt, P. Sullivan, S. A. Kharitonov and P. J. Barnes, Exhaled nitric oxide in chronic obstructive pulmonary disease, Am J Respir Crit Care Med 157 (1998) 998-1002. 9. S. A. Kharitonov, D. H. Yates, P. J. Barnes, Increased nitric oxide in exhaled air of normal human subjects with upper respiratory infections, Eur Respir J. 12 (1995) 295-297. 10. H. Grasemann, E. Michler, M. Wallot, F. Ratjen. Decreased concentrations of exhaled nitric oxide (NO) in patients with cystic fibrosis. Pediatr Pulmonol. 24 (1997) 173-177. 11. Ho LP, Innes JA, Greening AP. Nitrite levels in breath condensate of patients with cystic fibrosis is elevated in contrast to exhaled nitric oxide. Thorax 53 (1998) 680-684. 12. H. Grasemann, F. Ratjen. Cystic fibrosis lung disease: The role of nitric oxide. Pediatr Pulmonol 28 (1999)442-448. 13. H. E. Elpick, E. A. Demoncheaux, S. Ritson, T. W. Higenbottam, M. L. Everard, Exhaled njtric oxide is reduced in infants with cystic fibrosis, Thorax 56 (2001) 151-152. 14. S. R. Thomas, S. A. Kharitonov, S. F. Scott, M. E. Hodson, P. J. Barnes, Nasal and exhaled nitric oxide is reduced in adult patients with cystic fibrosis and does not correlate with cystic fibrosis genotype, Chest 117(2000)1085-1089. 15. P. Paredi, P. L. Shah, P. Montuschi, P. Sullivan, M. E. Hodson, S. A. Kharitonov, P. J. Barnes, Increased carbon monoxide in exhaled air of patients with cystic fibrosis, Thorax 54 (1999) 917-920. 16. J. D. Antuni, S. A. Kharitonov, D. Hughes, M. E. Hodson, P. J. Barnes, Increase in exhaled carbon monoxide during exacerbations of cystic fibrosis, Thorax 55 (2000) 138-142. 17. Q. Jobsis, H. C. Raatgeep, S. L. Schhellekens, A. Kroesbergen, W. C. Hop, J. C. de Jongste, Hydrogen peroxide and nitric oxide in exhaled air of children with cystic fibrosis during antibiotic treatment, Eur /?esp .716 (2000) 95-100. 18. L P Ho, J. Faccenda, J. A. Innes, A. P. Greening, Expired hydrogen peroxide in breath condensate of cystic fibrosis, Eur Respir J 13 (1999) 103-106. 19. Grasemann H, loannidis I, Tomkiewicz RP, de Groot H, Rubin BK, Ratjen F. Nitric oxide metabolites in cystic fibrosis lung disease. Arch Dis Child 78 (1998) 49-53.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) 1OS Press, 2002

Lung Cancer Screening by Breath Analysis Michael J. Berry Quadrivium, L.L.C., P. O. Box 1421, Pebble Beach, CA 93953 USA Abstract. Laser photoacoustic detection of biomarkers in exhaled breath samples may be suitable for lung cancer screening in the general population.

1. Background Lung cancer (LC) is a major cause of death worldwide, accounting for more than 1.1 million deaths (ca. 2.1% of all causes) annuallyf'!. In the United States (US), the five-year survival rate following LC diagnosis is only 14% for all patients!2), but improves to 60% or better for patients diagnosed with early-stage (Stage 0 - carcinoma in situ or Stage IA small localized tumor) disease!3). A simple, inexpensive LC screening test that can identify early-stage LC patients with high sensitivity (i.e., a low percentage of false negatives) and high specificity (i.e., a low percentage of false positives) is needed to provide the basis for a significant reduction in LC deaths. 2. Previous Studies Over 1700 endogenous volatile organic compounds (VOCs) have been identified in human exhaled breatW4!; these VOCs are primarily excreted metabolic products. Malignant cells exhibit differences in metabolism compared to normal cellsl5'; these differences may produce VOC biomarkers. Candidate VOC biomarkers have been identified by several groups using analyses of LC patient and control breath samples by gas chromatography/ mass spectrometry (GC/MS^12!. Results of these studies are summarized as follows. Gordon, et al. (1985)l6); In a retrospective study involving w=12 LC patients and m=9 control subjects, statistical analysis of GC/MS data by a discriminant model yielded 93% classification (LC vs. control) accuracy using relative concentrations of only three VOCs (acetone, 2-butanone, and 1-propanol). Preti, et aL (1988)171: In a retrospective study involving w=10 LC patients and m=16 controls (in two subgroups: age-matched and younger subjects), statistically significant (p Thl

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Fig.l Role of NO in autoimmune diseases. Intracellular antigens may be released as a result of direct cytotoxicity and increased apoptosis in target organs. A decrease in autoreactive lymphocytes apoptosis, combined with an expansion of CD4+ Th2 lymphocytes lead to B cells activation and autoantibodies production. The immunocomplexes may eventually upregulate NO synthase in macrophages and in endothelial cells with resulting increased NO production.

2. Systemic Lupus Erythematosus (SLE). SLE is a disease of unknown etiology in which tissues and cells are damaged by pathogenic autoantibodies and immune complexes. The disease is generally multisystemic, even if at onset SLE may involve only one organ and additional manifestations occur over time. Most patients experience exacerbations interspersed with periods of relative quiescence. A murine model of lupus-like disease is accompanied by increased urinary excretion of nitrite, enhanced macrophage NO production, an increased immuno reactive NO synthase in spleen and renal tissue. In the same murine model, the oral administration of the NOS inhibitor L-NG monomethyl arginine (LNMMA) after the onset of disease, reduced proteinuria and the addition of dietary arginine restriction to L-NMMA also significantly improved renal pathology scores (5). An increased production of NO has been observed in patients with SLE in comparison to normals by Wanchu et al. (6), who also observed highest values of nitrites in patients with more active disease. The activity of disease is calculated on the basis of a score which takes into account clinical, laboratory, instrumental activity indexes referred to the various organs and systems (SLE Disease Activity Index or European Consensus on Lupus Activity Measurement). The clinical manifestations of activity in Wanchu series were mainly articular and dermatologic. Similar results have been obtained by Gilkesson et al , who measured also serum levels of nitrotyrosine (a metabolite not affected by diet) (7). Belmont et al. (8) have demonstrated that SLE is characterized by an increased production of NO, as reflected by elevation of serum nitrites (37 ± 6 uM/1 in 46 patients with SLE compared to 15 ± 7 uM/1 in controls, p< 0.01). The same authors showed that patients with active disease had more elevated serum nitrites compared with those with inactive disease. Serum nitrite levels correlated with disease activity (r = 0.47, p= 0.04), evaluated by SLE disease activity index (SLEDAI), and with level of

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antibodies to dsDNA (r = 0.35, p= 0.02). Endothelial and keratinocyte expression of iNOS were significantly elevated in SLE patients compared with controls, and were higher in patients with active disease as compared to those with inactive disease. Exhaled NO. Higher than normal expired NO concentrations have been reported in SLE patients (9). Mean values of peak concentration of NO in exhaled air were 64.8 ± 27.5 ppb in 27 SLE patients compared to 31.6 ± 7.7 ppb in controls, p< 0.001. (see Fig.2)

Fig.2. Mean +/- SE values of exhaled NO in patients with SLE and in normal controls.

In the same patients NO concentration was directly related to ECLAM activity score (r = 0.42, p< 0.05) . No patient had a major respiratory problem (pleurisy, pneumonia, radiologic evidence of interstitial lung disease), but 15/27 and 17/27 respectively had a significant decrease in lung diffusing capacity and hi MEF25, a test that reflects small airway function, and a significant inverse correlation was observed between eNO and MEF25 (r = -0.44, p< 0.05). Even in the absence of overt respiratory disease, the studied patients had a high prevalence of functional lung impairment, in agreement with literature data. A mixed pattern of functional lung damage, characterized by mild restrictive and diffusion abnormality, along with a decrease of MEF25, is commonly reported in SLE (10). Respiratory function abnormalities in patients with SLE have been related to inflammatory reactions. Alveolar lymphocytosis has been correlated with lung diffusion decrease (11), while peribronchial and bronchial wall inflammation, together with interstitial mononuclear cell infiltrate, have been suggested as the pathologic basis for small airway obstruction (12). The inverse correlation between eNO and MEF25 may support the hypothesis that respiratory tract inflammation causes increased eNO in patients with SLE, but direct investigation (i.e. BAL, lung biopsies) is needed to clarify this point.

Origin and Significance of NO in SLE. The identity of cells producing NO in SLE and the role of NO in the pathogenesis of the disease are important questions. An emerging concept is that in active SLE there is a


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widespread activation of the vascular endothelium. Activated endothelial cells may release excessive amounts of NO, correlated with evidence of increased disease activity, based on SLEDAI, anti-DNA levels and C3a levels (13). Activated endothelial cells have also been shown to increase the expression of adhesion molecules like E-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM1), the scenario for tissue injury in the disease (13). Immune stimuli which may account for endothelial cell activation in SLE include immune complexes, complement components, antiphospholipid antibodies (13). The correlation between exhaled NO and ECLAM score of disease activity (9) may thus depend either upon respiratory tract inflammation or upon inflammatory cytokines and other circulating factors released in sites other than the respiratory system. In conclusion, NO production is increased in patients with SLE proportionally to the activity of the disease. The ultimate clinical significance of this observation is presently undefined. The measurement of NO in the exhaled air is the simplest and more rapid test which may document the increased NO production in SLE. Whether eNO measurement is merely one of the many available nonspecific markers of inflammation or whether it may help in the follow up of patients, particularly those with lung involvement, remains to be assessed. 3. Rheumatoid arthritis (RA). Rheumatoid arthritis is a chronic disease characterized by joint swelling, synovial inflammation and cartilage destruction. The importance of NO in the development of RA is increasingly recognized. Nitric oxide is thought to be a critical mediator of the inflammatory cascade which leads to synovial proliferation and mononuclear cell infiltration, which are the characteristics of involved joints in RA. Adjuvant-induced arthritis is a murine model of human RA, whose symptoms are preceded by elevated production of nitrates and nitrites A significant increase of urinary nitrate excretion was found in rats 20 days after induction of adjuvant arthritis compared with non-arthritic rats and L-NAME decreased NO biosynthesis, paw swelling and histopathologic changes in ankle joints (14). These data suggest that in adjuvant arthritis endogenous NO formation is clearly enhanced and related to joint inflammation. Parrel et al. (15) first reported an increased concentration of nitrite in synovial fluid and in serum of patients with RA, compared to serum nitrite concentration of healthy controls. Nitrite concentration in synovial fluid were higher than in serum samples. This finding is consistent with the presence, within inflamed synoviurn, of NO generating cells, such as fibroblasts, chondrocytes, macrophages, endothelial cells and granulocytes. Widespread synovial inflammation might increase serum nitrite by equilibration with the vascular compartment, but this may not entirely account for the whole serum nitrite concentration. A possible source of increased nitrite is the systemic vasculature, where the induction of NO syntehsis may occur, as an effect of circulating cytokines (16). Stichtenoth et al. (17) found the urinary nitrate excretion of 10 patients with RA to be 2.7-fold greater (p< 0.001) than that of healthy controls. After 2-week therapy with prednisolone (0.5 mg/Kg) , when inflammatory activity (as indicated by C reactive protein, ESR, swelled joint count, early morning stiffness) was significantly reduced, urinary nitrate excretion decreased significantly by 28%. By chemiluminescence after nitrite reduction, Ueki et al. (18) found that the serum concentration of NO was significantly higher in patients with RA as compared with patients with osteoarthritis and healthy subjects (291.4± 108.5 33.4 + 4 and 35.9 + 4.5 nM/1 respectively, p< 0.01).

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Furthermore, the serum concentration of NO was significantly higher in patients with active RA than in patients with inactive disease (670.9 ± 44.7 vs 97.7 ± 11 nM/1, p< 0.001) and serum NO levels were significantly correlated with the duration of morning stiffness, the number of tender or swollen joints, CRP, serum TNF-a and IL-6 levels. Peripheral mononuclear blood cells (PMBC) from RA patients had increased NOS activity and increased iNOS antigen content compared to those from normal subjects, and responded to interferon-y with increased NOS expression and nitrite/nitrate production in vitro (19). NOS activity of freshly isolated blood mononuclear cells correlated significantly with disease activity, as assessed by tender and swelled joint counts. These findings demonstrate that patients with RA have systemic activation of iNOS expression, and that the degree of activation correlates with disease activity. Clinical trials of TNF-a neutralizing antibodies or a soluble recombinant TNF-a receptor fusion protein (p75) in RA patients demonstrate that TNF-a blockade leads to reduction in disease activity (20, 21). Since TNF-a can promote iNOS expression and increased NO production, Perkins et al. (22) investigated if a chimeric monoclonal antibody against TNF-a might decrease iNOS protein expression and NOS enzyme activity in PBMC from RA patients. They found that the elevated levels of baseline iNOS protein and NOS enzyme activity in PBMC from RA patients were significantly reduced by the anti- TNF-a treatment. Changes in NOS activity following treatment correlated significantly with changes in the number of tender joints. These findings clearly indicate that the anti-inflammatory affects of anti-TNF-a treatment may be mediated by a reduction of NO overproduction. Role of NO in Rheumatoid Arthritis. The evidence that NO play a proinflammatory role in RA may be summarized as it follows. 1- Elevated NO levels in the serum and synovial fluid of patients with RA have been found. 2- Increased production in synovial tissue and overexpression of iNOS antigen and NOS activity of PBMC have been shown in RA patients. 3- The production of NO and the NOS activity have been shown to be related to clinical activity of the disease. 4- Therapeutic interventions (corticosteroids, anti- TNF-a) which decrease clinical activity of the disease, also decrease NO production and NOS activity. NO from synovial fibroblasts has been suggested to enhance proinflammatory cytokine production by macrophages which in turn may upregulate iNOS expression, thereby generating a positive feedback loop (23). Additionally, NO may upregulate matrix metalloprotease production (24) and is implicated in IL-1B mediated inhibition of proteoglycan synthesis (25).

4. Sjogren's Syndrome (SS). Sjogren's syndrome is a chronic, slowly progressive, polysystemic disease, characterized by lymphocyte infiltration of the exocrine glands, which results in diminished or absent glandular secretion and mucosal dry ness, particularly of mouth and eyes (sicca

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syndrome). SS can occurr alone (primary SS) or in association with other rheumatic diseases such as RA, SLE and SSc . NO and Sicca Syndrome. NO has been measured in human saliva (26) demonstrating that it is released from salivary glands rather than derived from the activity of oral bacteria. NO may influence parasympathetic vasodilation and salivary flow by regulating peptide release and second messenger system for both VIP and acetylcholine, as reported in pigs and rats (27). Physiologically NO is produced in normal salivary glands mainly by constitutively expressed neural and endothelial NOS isoforms, and may contribute to the many events that regulate vascular reactivity, salivary flow, and neuropeptide release. Increased concentration of nitrites (mean ± SEM 307+51 /xM vs 97±16 /ttM, p< 0.05) and output (166±46 nmoles/min vs 37 ±7 nmoles/min) have been reported in SS patients compared with healthy control subjects (28). The increased salivary NO production in SS patients has been found to be mediated by iNOS upregulation, induced by cytokines, such as TNF-a, IL-1 and IFN-y , which are produced by the salivary gland epithelial cells and/or activated T lymphocytes (29, 30). In conclusion, salivary NO production is greatly increased in SS patients and may contribute to the diminished salivary flow, characteristic of sicca syndrome, either through cytotoxic mechanism and increased rate of apoptosis (31, 32) or through functional impairment of secretion. Exhaled NO. Pulmonary involvement has been reported both in primary and secondary SS, the most common respiratory symptoms being chronic dry cough and dyspnea (33). Evidence of airway obstruction has been shown in patients with SS by several authors (34, 35) and bronchial hyperresponsiveness to methacholine has been observed in 60 % of the patients. An increased production of exhaled NO has been reported in patients with SS compared to healthy controls (147 ± 82 vs 88 ± 52 nL/min, p = 0.04) by the Swedish BHR study group (36). Exhaled NO was correlated to age and serum human neutrophil lipocalin, a newly recognized secretory protein of the secondary granules of neutrophil granulocytes (37). Exhaled NO was not found to be correlated with bronchial responsiveness, respiratory symptoms or lung function. The mechanisms underlying the increased exhaled NO in SS patients were not investigated. The elevated NO levels may derive from inflammatory cells in the bronchial mucosa and/or from epithelial cells or macrophages activated by cytokines released from lymphocytes. An increased number of CD4+ T lymphocytes has been shown in the bronchial mucosa of patients with SS (38). The possibility of a contribution to exhaled NO from nonenzymatically formed NO derived from nitrites in saliva cannot be excluded in SS patients (39).

5. Systemic Sclerosis (SSc). Systemic sclerosis is a multisystemic disease characterized by Raynaud's phenomenon,

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vascular lesions and widespread fibrosis of the skin and visceral organs. The accumulation of inflammatory mononuclear cells is frequently detected in early cutaneous and visceral SSc lesions (40), followed by excessive collagen deposition. Yamamoto et al. (41) found that serum of patients with SSc contains increased amount of nitrites compared with controls (47.8 ± 17 uM vs 25.6 ± 10.3 fiM respectively, p< 0.01). The same authors using immunohistochemical techniques found positive staining for iNOS in infiltrating mononuclear cells and in fibroblasts of patients with SSc, while fibroblasts in normal skin showed only faint staining. Inducible NOS messenger RNA expression was detected in scleroderma derived culture fibroblasts, while negative expression was detected on cultured normal fibroblasts. These results suggest that SSc fibroblasts are already activated by exposure to inflammatory cytokines. Actually patients with SSc are exposed to the stimulation of various cytokines, such as IL-1, TNF-a, platelet-derived growth factor (PDGF) and TGF-B. While IL-1 and TNF-a increase NO production in a wide variety of cells (42), both PDGF and TGF-B decrease NO production, by inhibiting iNOS gene expression and by suppressing IL-1 induced NO production in macrophages by post-transcriptional mechanism (43). Peripheral blood mononuclear cells from patients with SSc have been shown to produce higher levels of NO spontaneously and in response to IL-1 stimulation (44). In conclusion, the majority of studies show that scleroderma mononuclear cells and fibroblast may escape the downregulation of NO production, and the resulting excessive NO or its reaction products may be cytotoxic to host cells. Exhaled NO. Pulmonary involvement is common in SSc and pulmonary hypertension is the most frequent cause of death (45). More than 30% of SSc patients may develop pulmonary hypertension (46), either in association with extensive interstitial lung disease or as a consequence of prominent vascular lesions of the pulmonary arterioles. In 23 patients with SSc Kharitonov et al. (47) found peak eNO levels significantly higher than in normal controls. They also found that the six patients with pulmonary hypertension had eNO values significantly lower than SSc patients without pulmonary hypertension (20 ± 6 ppb vs 149 ± 19 ppb respectively, p< 0.001) and normal controls (80 ± 7 ppb, p< 0.002). There was no relationship between eNO and lung function tests, while a negative correlation was observed between PaO2 and peak eNO in patients with SSc without pulmonary hypertension. In 47 patients with SSc, 19 with limited and 28 with diffuse form of disease, higher levels of eNO (plateau value) than in controls (16.6 ± 9 . 1 ppb vs 9.9 ± 2.9 ppb respectively, p< 0.0001) were observed (48). Patients with lung involvement (defined on the basis of a CT score for interstitial lung disease > 5, associated with a TLC or VC < 80% of predicted) had the highest value of eNO, while patients with isolated decreased DLco and patients without lung involvement had mean eNO not significantly different from controls. In agreement with previous observation by Kharitonov et al (47), eNO was significantly lower in the 16 patients with pulmonary hypertension (mean PAP 40.7 ± 15.4 mmHg, range 30-93 mmHg) than in patients without pulmonary hypertension (10.7 ± 5 ppb vs 19.6 ± 9 ppb, p< 0.001) and there was a significant inverse correlation between PAP and eNO in all the patients (r = -0.53, p = 0.004, Fig.3). In 17 patients with fibrosing alveolitis associated with SSc (FASSc), Paredi et al.

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(49) found eNO levels higher than in controls (9.8 ± 1 ppb vs 6.9 ± 0.5 ppb, p< 0.05) and the eight patients who had active BAL (defined as either lymphocyte > 14%, neutrophils > 4% or eosinophils > 3%) had significantly higher NO levels than patients who had inactive BAL ( 13.2 ± 1.8 ppb and 6.7 ± 1.2 ppb respectively, p< 0.05). There was a significant correlation between eNO and lymphocyte cell count in patients with FASSc (r = 0.58, p< 0.05). Recently, Moodley and coll. (50) found that patients with SSc and associated ILD had normal value of exhaled NO, but all their patients had pulmonary hypertension (PAP > 30 rnmHg). Origin and Significance of eNO in SSc. All studies concerning eNO measurement in SSc patients report values higher than in controls. The inflammatory process underlying interstitial lung disease in SSc is the most likely explanation for the increased NO production by respiratory tract. On the other hand patients with pulmonary hypertension associated with SSc have been shown to have normal or low values of exhaled NO (47, 48, 51). In SSc pulmonary hypertension may develop either in association with extensive pulmonary fibrosis or as a consequence of prominent vascular lesions of the pulmonary arterioles with minor parenchymal fibrosis.. Damage of endothelial cells either primary or linked to substances circulating in the serum of patients with SSc (52) may explain the low levels of exhaled NO in patients with pulmonary hypertension not associated with interstitial lung disease. These patients have been shown to have eNO values even lower than patients with pulmonary hypertension associated with parenchymal lung involvement (48). The diminished production of NO could favour the unopposed action of vasoconstrictor substances such as endothelin-1 and lead to obstruction and proliferation changes in the pulmonary arteries (53). The capability to increase NO production by respiratory system after Larginine administration has been proposed as a test of endothelial health, which could anticipate the response of pulmonary artery pressure to vasodilating drugs in patients with SSc (51, 54) (Fig.3). 2005/3 JD

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In conclusion, the measurement of exhaled NO may be a useful non-invasive marker of activity in patients with SSc and interstitial lung disease as well as an useful test to identify SSc patients with pulmonary hypertension. References I- Taylor Robinson AW, Liew FY, Severn A et al. Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells. Eur J Immunol 1994; 24: 980-984 2-Bauer H, Jung T, Tsikas D, Stichtenoth DO, Frolich JC, Neumann C. Nitric oxide inhibits the secretion of T-helper 1- and T-helper 2-associated cytokines in activated human T cells. Immunology 1997; 90: 205211 3- Mannick JB, Asano K, Izumi K, Kieff E, Stamler JS. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 1994; 79: 1137-1146 4- Geller DA, Billiar TR. Molecular biology of nitric oxide synthases. Cancer Metastasis Rev 1998; 17: 723 5- Gates JC, Ruiz P, Alexander A, Pippen AM, Gilkeson GS. Effect of late modulation of nitric oxide production on murine lupus. Clin Immunol Immunopathol 1997; 83: 86-92 6- Wanchu A, Khullar M, Deodhar SD, Bambery P, Sud A. Nitric oxide synthesis is increased in patients with systemic lupus erythematosus. Rheumatol Int 1998; 18: 41-3 7- Gilkeson G, Cannon C, Goldman D, Petri M. Correlation of a serum measure of nitric oxide production with lupus disease activity measures (abstract). Arthritis Rheum 1996; 39 Suppl 9: S251 8- Belmont HM, Levartovsky D, Goel A, Amin A, Giorno R, Rediske J, Skovron ML, Abramson SB. Increased nitric oxide production accompanied by the up-regulation of inducible nitric oxide synthase in vascular endothelium from patients with systemic lupus erythematosus. Arthritis Rheum 1997; 40: 1810-16 9- Rolla G, Brussino L, Bertero MT, Colagrande P, Converse M, Bucca C, Polizzi S, Caligaris-Cappio F. Increased nitric oxide in exhaled air of patients with systemic lupus erythematosus. J Rheumatol 1997; 24: 1066-71 10- Rolla G, Brussino L, Bertero MT, Bucca C, Converse M, Caligaris-Cappio F. Respiratory function in systemic lupus erythematosus: relation with activity and severity. Lupus 19%; 5: 38-43 II- Groen H, Aslander M, Bootsma H, van der Mark ThW, Kallenberg CGM, Postma DS. Bronchoalveolar lavage cell analysis and lung function impairment in patients with ststemic lupus erythematosus. Clin Exp Immunol 1993; 94: 127-33 12- Grennan DM, Howie AD, Moran F, Buchanan WW. Pulmonary involvement in systemic lupus erythematosus. Ann Rheum Dis 1987; 37: 536-9 1997; 403: 273-8 13- Belmont HM, Abramson SB, Lie JT. Pathology and pathogenesis of vascular injury in systemic lupus erythematosus: interactions of inflammatory cells and activated endothelium. Arthritis Rheum 19%; 39: 922 14- Stefanovic-Racic M, Meyers K, Meschter C, Coffey JW, Hoffman RA, Evans CH. N-monomethylarginine, an inhibitor of nitric oxide synthase, suppresses the development of adjuvant arthritis in rats. Arthritis Rheum 1994; 37: 1062-69 15- Farrell AJ, Blake DR, Palmer RMJ, Moncada S. Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases. Ann Rheum Dis 1992; 51: 1219-22 16- Kilbourn RG, Belloni P. Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumour necrosis factor, interleukin-1 or endotoxin. J Natl Cancer Inst 1990; 82: 772-6 17- Stichtenoth DO, Fauler J, Zeidler H, Frolich JC. Urinary nitrate excretion is increased in patients with rheumatoid arthritis and reduced by prednisolone. Ann Rheum Dis 1995; 54: 820-4 18- Ueki Y, Miyake S, Tominaga Y, Eguchi K. Increased nitric oxide levels in patients with rheumatoid arthritis. J Rheumatol 1996; 23: 230-6


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42- Kovacs EJ, Di Pietro LA. Fibrogenic cytokines and connective tissue production. FASEB J 1994; 8: 854-861 43- Vodovotz Y, Bogdan C, Paik J, Xie Q, Nathan C. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor ###. J Exper Med 1993; 178: 605-13 44- Yamamoto T, Sawada Y, Katajama I, Nishioka K. Increased production of nitric oxide stimulated by interleukin-l### in peripheral blood mononuclear cells in patients with systemic sclerosis. Br J Rheumatol 1998; 37: 1123-25 45- Lee P, Langevitz P, Alderdice CA, Aubrey M, Baer PA, Baron M, Buskila D, Dutz JP, Khostant I, Piper S. Mortality in systemic sclerosis (scleroderma). Quart J Med 1992; 82: 139-48 46- Ungerer RG, Tashkin DP, Furst D, Clements PhJ, Gong H, Bein M, Smith JW, Roberts N, Cabeen W. Prevalence and clinical correlates of pulmonary artery hypertension in progressive systemic sclerosis. Am J Med 1983; 75: 65-74 47- Kharitonov SA, Cailes JB, Black CM, duBois RM, Barnes PJ. Decreased nitric oxide in the exhaled air of patients with systemic sclerosis with pulmonary hypertension. Thorax 1997; 52: 1051-55 48- Rolla G, Colagrande P, Scappaticci E, Chiavassa G, Dutto L, Cannizzo S, Bucca C, Morello M, Bergerone S, Bardini D, Zaccagna A, Puiatti P, Fava C, Cortese G. Exhaled nitric oxide in systemic sclerosis: relationship with lung involvement and pulmonary hypertension. J Rheumatol 2000 (in press) 59- Paredi P, Kharitonov SA, Loukides S, Pantelidis P, du Bois RM, Barnes PJ. Exhaled nitric oxide is increased in active fibrosing alveolitis. Chest 1999; 115: 1352-56 50- Moodley YP, Lalloo UG. Exhaled nitric oxide is elevated in patients with progressive systemic sclerosis without interstitial lung disease. Chest 2001; 119: 1449-54 51- Rolla G, Colagrande P, Brussino L, Bucca C, Bertero MT, Caligaris-Cappio F. Exhaled nitric oxide and pulmonary response to iloprost in systemic sclerosis with pulmonary hypertension. Lancet 1998; 351: 1491-92 52- Etoh T, Igararhi A, lozunii K, Ishibashi Y, Takehara K. The effects of scleroderma sera on endothelial cell survival in vitro. Arch Dermatol Res 1990; 282: 516-19 53- Rolla G, Caligaris-Cappio F. Nitric oxide in systemic sclerosis lung: controversies and expectations. Clin Exper Rheumatol 1998; 16: 522-24 54- Rolla G, Colagrande P, Bucca C, Dutto L, Audano G, Caligaris-Cappio F. Exhaled nitric oxide (NO) after L-arginine may predict the effect of aerosolized iloprost in pulmonary hypertension associated with systemic sclerosis (SSc). Eur J Clin Invest 1999; 29 (Suppl 1): 82 (abstract)

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) [OS Press, 2002

Nitric Oxide in Hepatopulmonary Syndrome Giovanni ROLLA Universita di Torino (Italy) - Immunologia Clinica e AllergologiaOspedale Mauriziano Umberto I di Torino, Largo Turati 62, 10128 Torino (Italy) Abstract. Hepatopulmonary syndrome is characterised by severe hypoxemia due to intrapulmonary vascular dilatations in patients with liver disease. An imbalance between vasodilating and vasocostricting substances is supposed to lead to oxygenation abnormalities in patients with liver disease. Experimental and clinical data support the hypothesis that increased production of nitric oxide plays a key role in determining hypoxemia in hepatopulmonary syndrome.

Impaired arterial oxygenation, particularly increased alveolar-arterial oxygen gradient (AaO2), is frequent in patients with liver cirrhosis, even if the true frequency has not been established. However, studies of patients referred for liver transplantation indicate that as many as 20 % are hypoxaemic at rest, with 3-7 % having severe hypoxaemia (PaO2 < 60 mrnHg) (1), while a widened AaO2 gradient is reported in up to 60% (2). In 1977 Kennedy et al. (3) first used the term hepatopulmonary syndrome (HPS) to characterise the association of severe hypoxaemia with intrapulmonary vascular dilatations in hepatic failure. 1. Definition The syndrome can be defined as a clinical triad of: a) liver disease b) increased AaO2 gradient (> 15 mmHg) while breathing room air c) evidence of intrapulmonary vascular dilatations (commonly by contrast-enhance echocardiography, see below). 2. Clinical findings Liver diseases that have been associated with HPS include most commonly cirrhosis (cryptogenertic, alcoholic, post-viral hepatitis and biliary), but also noncirrhotic portal hypertension (4). No relationship has been found between HPS and biochemical indexes of hepatic function, ascites or gastrointestinal bleeding. On the other hand, the number of cutaneous spider naevi have a strong association with HPS (5). Dyspnea is a common symptom, and it may be the presenting symptom in 18 % of patients (4). Platypnea, defined as dyspnea induced by the upright position and releived by recumbency and orthodeoxia, defined as a decrease (> 10 %) of PaO2 when changing from the supine to the standing position (6), are characteristic, but not unique, of the HPS and may be seen in 5% of patients with cirrhosis. Platypnea and orthodeoxia may also be found in other clinical contexts such as intracardiac shunts, post-pneumonectomy, recurrent pulmonary emboli, chronic lung disease (7). A hyperdynamic circulation characterised by systemic

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vasodilatation and elevated cardiac output (often > 7 1/min), associated with low pulmonary vascular resistance is the haemodynamic pattern more commonly observed in patients with HPS (8). 3. Oxygenation impairment Impaired oxygenation is a hallmark of the HPS, ranging from a widened AaO2 gradient to severe hypoxaemia. The most important mechanism underlying impaired gas exchange in HPS include ventilation/perfusion inequality (9), alveolar-capillary diffusion limitation and intrapulmonary shunts (10). Precapillary pulmonary vascular dilatations and direct arterio-venous communications (11) are the pathological bases for oxygenation impairment. These intrapulmonary vascular dilatations range from 15 to 500 fim in diameter. As supplemental oxygen enhances oxygenation more than it would be expected with "true anatomic" shunts, a new mechanism, called diffusion-perfusion impairment, has been hypothesised to explain the hypoxaemia associated with HPS. As the capillary is dilated, O, molecules from adjacent alveoli cannot diffuse to the centre of the dilated vessel to oxygenate the erythrocytes at the central stream of venous blood (12). Supplemental oxygen provides enough driving pressure to overcome the relative diffusion defect. Intrapulmonary vascular dilatations include two types of vascular abnormalities: vascular dilatations at the pre-capillary level which cause oxygenation impairment responsive to supplemental oxygen (PaO2 > 500 mmHg when breathing 100% oxygen for 15 min) and larger arterio-venous communications, which cause hypoxaemia, poorly responsive to 100 % oxygen breathing (4). Contrast-enhanced (CE) echocardiography is the most frequently used technique to detect intrapulmonary vascular dilatations. It is based on the observation that microbubbles (agitated saline solution injected through a peripheral vein) can pass through dilated pulmonary vessels (> 15 (im) and can be detected as echogenicity in the left heart chambers (11). 4. NO theory NO is a powerful local vasodilator, which contributes to the normally low pulmonary vascular tone. Vallance et al (12) postulated that an increased NO production may account for the hyperdynamic circulation of liver cirrhosis. An increased production of pulmonary NO may contribute to oxygenation abnormalities through abnormal pulmonary vasodilatation as well as through the inhibition of hypoxic vasocostriction. It has been suggested that patients with cirrhosis may have a continuous stimulation of endothelial NO-synthase by circulating endotoxins and/or circulating cytokines,such as TNF-a (13 ). In a rat model of HPS, Fallen and coll. (14) showed that the endothelial NO synthase content of lung homogenates progressively increased and the increase was closely correlated with the development of hypoxemia. Increased NO output in exhaled air has been reported in patients with advanced cirrhosis, in whom exhaled NO was associated with systemic circulatory disturbances (15). Exhaled NO was reported to be raised almost threefold in three patients with HPS, compared with normal volunteers and with normoxemic cirrhotic patients (16). In one case of severe HPS we reported that i.v. methylene blue (a dye that inhibits the effect of NO on soluble guanylate cyclase and thereby


prevents the cascade of events leading to vasodilation) acutely improved oxygenation, through a marked decrease in pulmonary shunting (17). After the patient had been breathing 100 percent oxygen, PaO2 was 325 mmHg (normal value > 500 mmHg) while she was in the supine position, and it decreased to 115 mmHg while she was standing. Twenty minutes after the i.v. administration of methylene blue (3 nig/Kg) PaO2 during oxygen breathing was 480 mmHg in the supine position and 390 mmHg in the standing position. Very recently (18) Schenk and coll showed that i.v. methylene blue improved hypoxemia and hyperdynamic circulation in 7 patients with liver cirrhosis and severe HPS. We hypothesised that NO locally produced in the lung may play an important role in determining oxygenation abnormalities in patients with cirrhosis. To this aim, we investigated the relationship between NO production in the lung, assessed by exhaled NO measurement, and oxygenation abnormalities in patients with advanced cirrhosis (19), In 45 cirrhotic patients we showed mean values of exhaled NO output and serum NO2V NO3 significantly higher than in normal controls (252 + /117 vs 75.5 +/- 19 nL/min/m2, p< 0.0001 and 47.5 +/- 29.4 vs 32.9 +/- 10.1 îmol/L, p< 0.02, respectively). In all patients there was a significant correlation between exhaled NO and arterial-alveolar oxygen gradient (r= .78, p< 0.001). The nine patients who met the criteria for the diagnosis of HPS had also the highest values of exhaled NO (331 +/- 73.2 vs 223 +/- 118.4 nL/min/m2, p< 0.05). To further investigate the association between NO produced in the lung and oxygenation abnormalities in patients with cirrhosis, we measured exhaled NO and oxygenation measures before and after liver transplantation in a selected group of 18 patients with cirrhosis who did not have obvious cardiorespiratory diseases (20). Before transplantation, the mean exhaled NO was higher in patients than in normal controls (13 +/- 4.9 ppb compare with 5.75 +/- 1.9 ppb, p< 0.001). After transplantation, the AaO2 gradient significantly decreased (from 17.3 +/- 7.1 mmHg to 9 +/- 5.2 mmHg, p< 0.001), as did the exhaled NO concentration (from 13 +/- 4.9 ppb to 6.2 +/- 2.8 ppb, p< 0.001). The decrease in exhaled NO was significantly correlated with the decrease in AaO2 gradient (r = 0.56, p = 0.014). Five patients met the criteria for the diagnosis of HPS before transplantation and the syndrome was cured by transplantation. The correlation between the decrease in exhaled NO after liver transplantation and the improvement in oxygenation reinforces the hypothesis that NO is an important mediator of impaired oxygenation in patients with cirrhosis. In a murine model, chronic inhibition of lung NO production , by L-NAME administration, could prevent the development of HPS and associated hemodynamic alterations (21). Very recently, we reported that smoking, by decreasing respiratory NO, apparently contributed to improve oxygenation in a 44-year-old man with cirrhosis, complicated by severe HPS, who underwent liver transplantation (22). In conclusion, experimental and clinical data support the theory that NO plays a major role in oxygenation abnormalities of patients with liver cirrhosis, complicated by hepatopulmonary syndrome. References [1] Krowka MJ, Cortese DA. Pulmonary aspects of chronic liver disease and liver transplantation. Mayo Clin Proc 1985; 60: 407-18

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[2] Hourani JM, Bellamy PE, Tashkin DP, Batra P, Simmons MS. Pulmonary dysfunction in advanced liver disease: frequent occurrence of an abnormal diffusing capacity. Am J Med 1991; 90: 693-700 [3] Kennedy TC et al. Exercise-aggravated hypoxemia and orthodeoxia in cirrhosis. Chest 1977; 72: 305-9 [4] Krowka MJ, Cortese DA. Hepatopulmonary syndrome. Current concepts in diagnostic and therapeutic considerations. Chest 1994; 105: 1528-37 [5] Agusti AGN et al. The lung in patients with cirrhosis. J Hepatol 1990; 12: 262-3 [6] Robin ED et al. Platypnea related to orthodeoxia caused by true vascular lung shunts. N Engl J Med 1976; 294:941-3 [7] Seward JB. Platypnea-Orthodeoxia: clinical profile, diagnostic workup, management, and report of seven cases. Mayo Clin Proc 1984; 59: 221-3 [8] Naeije R et al. Pulmonary hemodynamics in liver cirrhosis. Semin Respir Med 1985; 7: 164-170 [9] Hedenstiema G, SOderman C, Eriksson LS, Wahren J. Ventilation-perfusion inequality in patients with non-alcoholic liver cirrhosis. Eur Respir J 1991; 4: 711-7 [10] Edell ES, Cortese DA, Krowka MJ, Rehder K. Severe hypoxemia and liver disease. Am Rev Respir Dis 1989; 140: 1631-35 [11] Krowka MJ, Tajik J, Dickson ER, Wiesner RH, Cortese DA. Intrapulmonary vascular dilatations (IPVD) in liver transplant candidates. Screening by two-dimensional contrast-enhanced echocardiography. Chest 1990; 97: 1165-70 [12] Vallance P, Moncada S. Hyperdynamic circulation in cirrhosis: a role for nitric oxide ? Lancet 1991;337:776-778 [13] Khoruts A, Stahnke L, McClain CJ, Logan G, Allen JI. Circulating tumor necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients. Hepatology 1991; 13: 267-276 [14] Fallen MB, Abrams GA, Luo B, Hou Z, Dai J, Ku DD. The role of endothelial nitric oxide synthase in the pathogenesis of a rat model of hepatopulmonary syndrome. Gasstroenterology 1997; 113:606-614 [15] Matsumoto A, Ogura , Hirata Y, Kakoki M, Watanabe F, Takenaka K, Shiratory Y, et al. Increased nitric oxide in the exhaled air of patients with decompensated liver cirrhosis. Ann Intern Med 1995; 123: 110-113 [16] Cremona G, Higenbottam TW, Mayoral V, Alexander G, Demoncheaux E, Borland C, et al. Elevated exhaled nitric oxide in patients with hepatopulmonary syndrome. Eur Respir J 1995; 8: 1883-1885 [17] Rolla G, Bucca C, Brussino L. Methylene blue in the hepatopulmonary sindrome. N Engl J Med 1994; 331: 1098 [18] Schenk P, Madl C, Rezaie-Majd S, Lehr S, MUlier C. Methylene blue improves the hepatopulmonary syndrome. Ann Intern Med 2000; 133: 701-706 [19] Rolla G, Brussino L, Colagrande P, Dutto L, Polizzi S, Scappaticci E, Bergerone S, et al.Exhaled nitric oxide and oxygenation abnormalities in hepatic cirrhosis. Hepatology 1997; 26: 842-847 [20] Rolla G, Brussino L, Colagrande P, Scappaticci E, Morello M, Bergerone S, Ottobrelli A,-et al. Exhaled nitric oxide and impaired oxygenation in cirrhotic patients bifore and after liver transplantation. Ann Intern Med 1998; 129: 375-378 [21] Nunes H, Lebrec D, Heller Y, Mazmanian M, Zerbib E, Herve P. Prevention of hepatopulmonary syndrome by inhibition of nitric oxide synthase. Am J Respir Crit Care Med 1999; 159: A523 (abstract) [22] Rolla G, Brussino L, Dutto L, Ottobrelli A, Bucca C. Smoking and hypoxemia caused by hepatopulmonary sindrome before and after liver transplantation. Hepatology 2001; 34: 430-431

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Pathological Changes in the Airways Epithelium of Liquidators of the Chernobyl Catastrophe

Victoria POLYAKOVA Institute of Human Ecological Pathology, Vasylkivska Street 45, Kyiv, 03022, Ukraine Abstract. By method of transmission electron microscopy (TEM) bronchial biopsies of liquidators of the Chernobyl catastrophe and persons of control nosological group were studied. Statistically significant distinctions in the manifestation degree of pathological changes of the bronchial epithelium were found in the main groups in comparison to control parameters. Obtained results testify stereotype reactions in the epithelium of airways on the effect of the "Chernobyl factor".

1. Introduction In spite of recently closing of the Chernobyl Nuclear Power Station a lot of medical and biological problems caused by the accident of 1986 still remained. Among them the prevalence of chronic obstructive pulmonary diseases in persons which had taken part in the liquidation of the Chernobyl disaster consequences is of a great importance. The main subject of our research was superficial bronchial epithelium as it plays the leading role in the pathogenesis of lung diseases. 2. Material and methods Bronchial biopsies obtained in 1989-1999 from persons with chronic bronchitis: 110 liquidators of the Chernobyl accident and from 23 patients of control nosological group without radiation factor in an anamnesis were studied. All patients were not older than 40 years old. A chronic bronchitis was diagnosed to all of them after 1986. Liquidators were in region with high radiation in 1986-87 (irradiation doses mainly under 10 cSv). Samples were proceed with routine technique for TEM and observed in JEM 100CX electron microscope. All investigated cases of liquidators were divided into two periods of observations: 1989-1992- I group (48 persons) and 1994-1999 - II group (62 persons).

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3. Results and Discussion In the control group sites of normal unchanged bronchial epithelium were more often determined (52%) whereas in the main groups a percentage of such places was extremely decreased (27% - I group, 20% - II group). Similarly in relation to control observations (48%) the number of epithelial loci with normal ratio of main cell types was reduced (32%-I group, 15% - II group). The decrease of bronchial endocrine cell number was also observed. If in the control material presence of APUD-cells were found out in 68% of observation in the main groups they were seen quite seldom (11% - I group and 14% - II group). Besides most of endocrine cells of liquidators had dystrophic changes: dilatation of perinuclear space, vacuolation of cytoplasm,

essential decrease of specific granules and intracellular

organoides quantity. High frequency of bronchial basal cell hyperplasia was a typical feature of a researched material but it was more significantly marked in the main groups (61% - control group, 77% - I group, 92% - II group). Alongside in liquidators reliable increase of basal cells rows number was revealed, whereas thickness of an epithelial layer remained practically constant. It allows to assume the activization of proliferative processes and acceleration of cellular population turnover in bronchial epithelium, probably, as a result of contraction of a cell cycle growth phase. In the II group squamous metaplasia of bronchial epithelium was found more often (43%) than in the control group (30%). Besides in the control group squamous metaplasia was detected only in elderly and senile persons. In the main groups such changes were revealed even in young people that can indicate the acceleration of involution. Dystrophic changes of bronchial ciliated cells such as: a vacuolation of cytoplasm, accumulation of a large number of myelin-like structures, appearance of swollen mitochondria with the clarified matrix and paniculate or complete cristal degeneration and often loss of cilia were observed more often in the I group (69%) in spite of control parameters (43%). Numerous ciliary damages like disorientation of cilia, winding ciliary membrane, their swelling, pathology of microvilis were found. The percentage of ciliary defects significantly grew up in liquidators of II group (66%) as against nosological indices (30%). In addition, the increased number of microtubular ciliary disturbances was observed. Besides in the second group different pathological changes of basal bodies such as disturbances of their location and structure were observed. Probably, the increased cellular

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turnover leads to the acceleration of the ciliogenesis processes and causes the formation of pathological basal bodies that give rise to abnormal cilia. The changes found in bronchial basal cells deserve our special interest. These cells in early terms were characterized by a hypertophy of intracellular organoids with the subsequent progressing development of dystrophic changes. The features of basal cells population, probably, made for fenotype changes of mature epithelial ceils such as disturbances of polarity in goblet cells with accumulation of mucous granules in both apical and basal loci of cytoplasm (fig.l) and appearance of epithelial cells that had morphological signs of ciliated cells and II type pneurnocytes (fig.2), Such transformation must testify the realization of basal cells genome injuries and its turn are important in carcinogenesis respect. Our subsequent investigations of liquidators upper airways indicate similar pathological changes in the nasal epithelium with the only difference in their manifestation degree.

Figure 1. Disturbance of polarity in goblet cells of bronchial epithelium of II group liquidators. There are mucous granules both in apical and basal loci of cytoplasm. X 3,600.

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Figure 2. The lamellar bodies of II type pneumocytes in cytoplasm of bronchial ciliated cell of II group liquidator. X 7,200.

Thus, obtained results testify there is certain dynamics in the development of pathological reactions in the epithelium of airways. More expressed in comparison with the control data epithelial pathology in liquidators was caused by the effect of stronger than in usual inflammation pathogenic inductor. The permanent impact of the "chernobyl factor" promote the intensification of structural and functional systems of the airways epithelium that leads to exhaustion of compensatory and adaptive mechanisms and to deterioration of the protective properties of the epithelium. These changes are stereotype for both upper and lower airways with the different expression and have an important diagnostic and prognostic significance.

Part IV. Transplantation


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Heme Oxygenase-1 and/or Carbon Monoxide can Promote Organ Graft Survival Miguel P. SCARES12, Lukas GUENTHER, Pascal BERBERAT and Fritz H.BACH1 ' Immunobiology Research Center,Beth Israel Deaconess Medical Center, Department of Surgery, Harvard Medical School, Boston, MA 02215 Înstituto Gulbenkian de Ciencia, Apartado 14, 2781-901 Oeiras, Portugal Abstract. Endothelial cells (EC) play a pivotal role in regulating inflammatory reactions such as those involved in the rejection of transplanted organs. This occurs through the expression of a series of pro- and anti-inflammatory genes that are associated with the activation of these cells. Expression of pro-inflammatory genes promotes events that lead to graft rejection while expression of anti-inflammatory (protective) genes suppresses those events and thus contribute to sustain graft survival. Understanding how the expression of these genes is regulated and their mechanism of action are important issues for the development of new therapeutic strategies to suppress graft rejection. The main thesis of our work is to exploit the mechanisms that are physiologically used by an organ to contribute to its own survival. This concept expands our previous vision that the main, if not only, way of sustaining graft survival is to suppress the anti-graft immune response of the recipient. We have studied protective genes and molecules using experimental models of transplantation in rats. We discuss here data that supports the concept that grafts can express " protective genes" and their products that are both anti-apoptotic and antiinflammatory (protective). The anti-inflammatory response mitigates inflammatory reactions leading to graft rejection. The data reviewed focus on the role of one of such genes, i.e. heme oxygenase-1 (HO-1), a stress responsive gene. One product of HO-1 action on heme is the production of carbon monoxide (CO), which can suppress graft rejection and lead to long-term graft survival.

1. Introduction The success of organ transplantation is largely due to the development of potent but largely non-specific immunosuppressive drugs that block T cell mediated events involved in graft rejection. However, we have found that the fate of a transplanted organ depends not only on the immune response against the graft but also on the ability of the graft to protect itself from immune mediated injury. We have suggested that the survival of such grafts depends in a critical manner on the ability of the graft vascular endothelium to protect itself against injury. The data reviewed here supports the concept that the vascular endothelium of a graft can express a series of anti-apoptotic, anti-inflammatory (protective) genes that act to help overcome graft rejection.

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2. Grafts can protect themselves from rejection The combination of anti-organ graft antibodies that deposit on the graft endothelium plus activated complement normally leads to rapid rejection of the organ. In some situations, however, the graft can survive despite the presence of the antibodies plus the complement. We refer to such situations as "graft accommodation", i.e. the survival despite the presence of the anti-graft antibodies and complement. We have tested specifically whether immediately-vascularized transplants could develop "resistance" to rejection caused by anti-graft antibodies and complement. To do so, we have used as an experimental model the transplantation of mouse or hamster hearts into rats, a model of xenotransplantation (transplantation between different species). Naive grafts undergo acute vascular rejection, which occurs around day 3 to 4 after transplantation, when elicited anti-graft antibodies are synthesized in the presence of complement [1-3]. This type of rejection is characterized by a series of inflammatory lesions involving EC activation [4], graft infiltration by activated host monocyte/macrophages and NK cells [5], coagulation and platelet aggregation associated with vascular occlusion and tissue necrosis [4]. We have shown that accommodated grafts express one or more protective genes in their vascular endothelium and smooth muscle cells [6]. That accommodated grafts are protected from rejection was formally demonstrated by the observation that a naive graft transplanted into a recipient carrying a first "accommodating" graft for 10 days undergoes rejection in few minutes while the first graft survives long-term [7. 8]. In addition, we found that the expression of a single protective gene on the vascular endothelium of these grafts, i.e. heme oxygenase-1 (HO-1), acted directly to suppress rejection [9]. Normal mouse hearts transplanted into immunosuppressed rats up-regulate the expression of HO-1 within hours after transplantation and survive indefinitely while HO-1"" deficient mouse hearts transplanted under the same immunosuppressive regimen are rejected in 3-7 days [9]. These results provide direct evidence that a single protective gene, i.e. HO-1, expressed in the vasculature of a graft can function to prevent graft rejection and to allow the development of accommodation. While the mechanism(s) underlying this effect of HO-1 is still not entirely clear, we believe that it is related to the ability of HO-1 to protect EC from apoptosis and from (immune-mediated) injury leading to graft rejection.

3. Protective Genes in Prevennting Rejection There are several genes that are expressed in endothelial cells and smooth muscle cells of grafts that accommodate. These include A20, a gene actively studies by our colleague, Dr. Christiane Ferran, and other genes that are both anti-apoptotic and anti-inflammatory. It is our purpose here to discuss heme oxygenase-1 (HO-1) and especially one of the products generated when HO-1 acts on heme: carbon monoxide as protective gene/molecules.

4. Heme oxygenases The heme oxygenase system is composed of three proteins referred to as heme oxygenases-1 (HO-1), -2 (HO-2) and -3 (HO-3) (reviewed in [10-12]). Among these only HO-1 and -2 are thought to act as enzymes that catabolize heme into biliverdin, free iron and the gas carbon monoxide (CO) [13]. Biliverdin is subsequently

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catabolyzed into bilirubin by biliverdin reductase (reviewed in [14]) and free iron induces the expression of ferritin [15, 16]. HO-2 is expressed constitutively in most cell types, including EC, while HO-1 is expressed at low or undetectable levels but is rapidly up-regulated under stress conditions such as upon UV radiation [17] or exposure to hydrogen peroxide, heavy metals, cytokines (IL-1, IL-6 or TNF-a), bacterial lipopolysaccharide, shear stress or heat shock (reviewed in [I I, 14]). Expression of HO-1 is rapidly up-regulated in the vascular endothelium of transplanted organs [9, 18]. In this situation the primary stimuli leading to HO-1 expression is not clear but this is likely to occur through the exposure of EC to circulating heme, a potent activator of HO-1 in most cell types [16]. The reason for this is that endothelial cells in the vasculature of a graft are probably exposed to high levels of free heme following transplantation. There are at least two potential "reservoirs" of pre-synthesized heme that can contribute to this effect. These are heme derived from hemoglobin and myoglobin when these proteins are released from red blood cells or myocytes through hemolysis or necrosis, respectively [19]. Once released, free heme can intercalate into EC cytoplasmic membranes and by this route become incorporated into the intracellular compartments of EC where it acts as a potent pro-oxidant [14, 16, 19]. A number of studies have suggested that acute exposure to heme is a highly cytotoxic [16, 20], probably related to the fact that Fe2" in the core of the heme becomes available to participate in the generation of free radicals through the Fenton reaction [14]. Generation of free radicals in this manner initiates a variety of signal transduction pathways that can induce both the expression of pro-inflammatory genes associated with EC activation as well as EC apoptosis. These events are highly deleterious and presumably can contribute to initiate the rejection of transplanted organs. Under these circumstances the only known mechanism by which EC can clear pro-oxidant free heme is to up-regulate the expression of HO-1. Once expressed at sufficiently high levels, HO-1 acts as the ratelimiting enzyme in the clearance of pro-oxidant heme and generates bilirubin as well as CO that suppress the pro-oxidant effects of heme [14]. In addition, HO-1 action on heme releases free iron, which up-regulates the expression of the iron sequestering protein ferritin (reviewed in [21]). The combined action of bilirubin, CO and ferritin are thought to contribute in a crucial manner to the cytoprotective response of EC exposed to extracellular heme.

5. HO-1 derived CO can prevent graft rejection We have recently shown that CO can account in large or full measure for the protective effects of HO-1 in terms of preventing graft rejection [18]. As described above, mouse to rat cardiac transplants survive long-term under transient complement depletion in combination with T cell immunosuppression [2]. HO-1 expression by the graft vasculature is critical to achieve long-term graft survival in this experimental model since HO-1 deficient mouse hearts transplanted under the same conditions are rapidly rejected [9]. We showed that the HO-1 protective effect was attributable to CO [18]. Under the same immunosuppressive regimen that allows mouse to rat cardiac transplants to survive long-term, inhibition of HO-1 activity by the specific HO inhibitor tin protoporphyrin (SnPPIX), precipitates graft rejection in 3-7 days [18], a model similar to using the HO-1 deficient mouse heart. Graft rejection under inhibition of HO activity by SnPPIX is associated, with microvessel platelet

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sequestration, thrombosis of coronary arterioles, myocardial infarction and apoptosis of EC as well as cardiac myocytes. Under inhibition of HO-1, exogenous CO suppresses graft rejection and restores long-term graft survival [18]. This effect of CO is associated with inhibition of platelet aggregation, thrombosis, myocardial infarction and apoptosis [18]. CO appears to bring the situation back to the normal. The exact mechanism by which HO-1 derived CO prevents graft rejection is not clear. CO is a signaling molecule that exerts a large spectrum of biological functions in several cell types, including in EC. The biological functions attributed to CO are thought to result directly or indirectly from binding of CO to iron in various proteins. Presumably this accounts for the ability of CO to modulate the activation of several signal transduction pathways, including guanylyl cyclase/cGMP [22], p38 mitogen activated protein kinase (MAPK) [23, 24], p21cipl [25] and thus regulate the expression of vasoconstrictor [25, 26], pro-inflammatory [23] as well as procoagulant molecules [27]. This broad action of CO is thought to account for its ability to promote vasodilation [25, 28] as well as to inhibit inflammation [23], apoptosis [24, 29], cell cycle progression [25, 30] and thrombosis [31, 32]. As for NO, another gaseous signaling molecule, CO also inhibits platelet activation/aggregation through activation of guanylyl cyclase and subsequent generation of cGMP [31, 33]. This is also thought to contribute in a critical manner to the ability of CO to suppress graft rejection. There are additional features of CO that may contribute to its protective function in terms of suppressing graft rejection. CO inhibits the pro-inflammatory phenotype associated with the activation of monocyte/macrophages (M0) [23, 34]: CO inhibits the generation of M0 derived proinflammatory molecules such as TNF-a while increasing that of anti-inflammatory molecules such as IL-10 [23]. The mechanism by which CO acts to modulate M0 activation in this manner is not dependent on activation of the guanylyl cyclase signal transduction pathway. Rather CO acts through a signaling pathway that involves activation of the p38 MAPK [23]. CO can also act through the p38 MAPK signal transduction pathway. The ability of CO to suppress EC apoptosis likely also contributes to suppressing graft rejection [24].

6. EC Apoptosis in graft rejection Under acute inflammatory conditions such as those associated with the rejection of a transplanted organ, widespread EC apoptosis can occur and thus enhance the pro-inflammatory environment leading to graft rejection [35]. EC apoptosis can lead to disruption of the integrity of the vascular endothelium with exposure of the pro-coagulant sub-endothelial matrix and subsequent induction of thrombosis, hypoxia, and tissue necrosis. Apoptosis of EC can also promote thrombosis directly through the exposure of pro-coagulant apoptotic bodies [36] as well as through the activation of the classical pathway of complement [37] and the activation of circulating platelets [38].

7. HO-1 derived CO suppresses EC apoptosis When cultured in vitro EC can be induced to undergo apoptosis when exposed to TNF-a in the presence of the transcription inhibitor Actinomycin D (Act.D). We have used this well-established experimental system to analyze whether expression of

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HO-1 in EC could suppress TNF-ct mediated apoptosis. When exposed to extra-cellular heme, EC up-regulate the expression HO-1 and as a result of that become resistant to TNF-ct mediated apoptosis [24]. Similar results are observed when HO-1 is overexpressed in EC [9, 24]. The protective effect of HO-1 requires its enzymatic activity, indicating that this effect involves the generation of one or several end-products of heme catabolism by HO-1, i.e. Fe2\ bilirubin and/or CO [24]. Since heme derived Fe2+ up-regulates the expression of the iron sequestering protein ferritin [15, 16], we tested if Fe2+ sequestration/elimination could account for the antiapoptotic effect of HO-1 in EC. Our data [24] as well as that of others [39] indicates that this is the case: the anti-apoptotic effect of HO-1 can be mimicked by the exogenously-administered iron chelator Desferoxarnine [24] and/or by overexpressing the heavy chain of ferritin (P. Berberat et al., manuscript submitted). Our data, however, suggests that in addition to the induction of systems that lead to iron sequestration [39], HO-1 generates physiological levels of CO that act to suppress EC apoptosis [24]. This notion is supported by the observation that exposure of EC to exogenous CO suppresses EC apoptosis [24]. At least in EC, the anti-apoptotic action of CO is strictly dependent on the activation of the p38 MAPK signal transduction pathway: HO-1 derived CO enhances p38 MAPK activation in EC and inhibition of p38 MAPK activation abrogates the anti-apoptotic effect of HO-1 and/or CO in these cells [24]. How HO-1 derived CO acts to modulate the activation of this specific signal transduction pathway and how this acts to suppress EC apoptosis remains to be fully elucidated.

8. Other actions of CO in transplant related models We have evaluated the role of CO in ischemia-reperfusion injury by transplanting a rat heart to a syngeneic recipient after the heart was held under ischemic conditions for 24 hours. Control hearts all failed within 24 hours. Hearts treated with CO had various rates of survival after 1, 7 or 14 days. Treatment of the donor alone led to 50% 1 day survival and 33% at 7 and 14 days; treatment of the heart while ex vivo alone had a similar effect as treating the donor; the combination of these two treatments led to 100% 1 day, 83% 7 day 66% 14 day; and the combination of these two treatments plus treatment of the recipient led to 100% survival on day 1 and 83% on days 7 and 14. Clearly the CO treatment improves the function of a heart not susceptible to immune rejection but subject to ischemia-reperfusion injury without CO treatment.

9. Concluding remarks The findings reviewed hereby show that transplanted organs can express a series of protective genes in their vasculature that may contribute to promote the survival of such organ. We found that one of these protective genes, HO-1, acts in such a manner. The mechanism by which HO-1 suppresses graft rejection is still not clear. Our data suggests that this protective effect relies in large measure on the ability of HO-1 to catabolize pro-oxidant heme, as it accumulates following transplantation, into the gas CO. HO-1 derived CO has a series of biological effects that can contribute to suppress graft rejection. These include its anti-apoptotic function that has yet to be fully elucidated in terms of its molecular basis. Our data suggests that

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HO-1 derived CO suppresses EC apoptosis through a mechanism that is dependent on the activation of p38 MAPK and the transcription factor NF-icB. These findings raise important questions related not only the mechanism of action of CO in terms of preventing graft rejection but also to the development of new strategies aimed to promote graft survival. Based on the observation that inhibition of NF-icB impairs the anti-apoptotic effect of CO, it is possible that the protective effect of CO in terms of suppressing graft rejection may also be impaired upon inhibition of NF-icB activation. Given that inhibition of NF-KB activation is perceived as a potential therapeutic tool to suppress the expression of pro-inflammatory genes that promote graft rejection; our present data raises significant questions about the relative therapeutic values of the two approaches. Footnotes This paper was taken in large part from a recent review written from our laboratories. The work from our laboratories was supported by a grant from the Roche Organ Transplantation Research Foundation (ROTRF; 998521355) awarded to MPS, NIH grants (HL67040) awarded to MPS and (HL58688) awarded to FHB. Fritz H. Bach is the Lewis Thomas Professor at Harvard Medical School and is a paid consultant to Novartis Pharma, Basel, Switzerland. Abbreviations ActD: Actinomycin-D; CO: Carbon monoxide; EC: Endothelial cell; HO-1: Heme oxygenase-l; IicBa: Inhibitor nuclear factor-tcBa; MAPK: Mitogen-activated Protein Kinases; Ma; Monocyte macrophage. MnSOD: Manganese Superoxide Dismutase; NF-KB: Nuclear Factor-icB; TNF: Tumor Necrosis Factor.

References 1. Bach, F. H., Winkler, H., Ferran, C., Hancock, W. W. & Robson, S. C. (1996) Immunology Today 17,379-84. 2. Koyamada, N., Miyatake, T., Candinas, D., Mark, W., Hechenleitner, P., Hancock, W. W., Scares, M. P. & Bach, F. H. (1998) Transplantation 65,1210-5. 3. Scares, M. P., Lin, Y., Sato, K., Takigami, K., Anrather, J., Ferran, C., Robson, S. C. & F.H.Bach (1999) Current opinion in organ transplantation 4, 80-89. 4. Bach, F. H., Robson, S. C., Ferran, C., Winkler, H., Millan, M. T., Stuhlmeier, K. M., Vanhove, B., Blakely, M. L., van, der, Werf, Wj, Hofer, E. & et, a. 1. (1994) Immunological Reviews 141, 5-30. 5. Fryer, J. P., Leventhal, J. R., Dalmasso, A. P., Chen, S., Simone, P. A., Jessurun, J., Sun, L. H., Reinsmoen, N. L. & Matas, A. J. (1994) Transplant Immunology 2, 87-93. 6. Bach, F. H., Ferran, C., Hechenleitner, P., Mark, W., Koyamada, N., Miyatake, T., Winkler, H., Badrichani, A., Candinas, D. & Hancock, W. W. (1997) Nature Medicine 3, 196-204. 7. Lin, Y., Soares, M. P., Sato, K., Takigami, K., Csizmadia, E., Smith, N. & Bach, F. H. (1999) Journal of Immunology 163,2850-2857. 8. Soares, M. P., Lin, Y., Sato, K., Stuhlmeier, K. M. & Bach, F. H. (1999) Immunology Today 20,434-437. 9. Soares, M. P., Lin, Y., Anrather, J., Csizmadia, E., Takigami, K., Sato, K., Grey, S. T., Colvin, R. B., Choi, A. M., Poss, K. D. & Bach, F. H. (1998) Nature Medicine 4, 1073-1077. 10. Willis, D. (1999) in Inducible enzymes in the inflammatory response, eds. Willoughby, D. A. & Tomlinson, A. (Birkhauser, Basel), pp. 55-96. 11. Choi, A. M. & Alam, J. (1996) American Journal of Respiratory Cell & Molecular Biology 15,9-19. 12. Maines, M. D. (1997) Annual Review of Pharmacology & Toxicology 37,517-54. 13. Kutty, R. K., Daniel, R. F., Ryan, D. E., Levin, W. & Maines, M. D. (1988) Archives of Biochemistry & Biophysics 260,638-44. 14. Ryter, S. W. & Tyrrell, R. M. (2000) Free Radical Biology & Medicine 28, 289-309.

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Mechanisms of and Clinical Efforts to Minimise Perioperative Lung Injury I. Gavin WRIGHT and Nandor MARCZIN Department of Anaesthesia and Critical Care Royal Brompton and Harefield NHS Trust Harefield Hospital Harefield, Middlesex. UB9 6JH Abstract The success of clinical lung transplantation is constrained by a shortage of suitable donor organs. In addition, even suitable donor lung grafts exhibit significant perioperative dysfunction and are at risk to develop lung injury throughout the procedure. Significant recent progress into the cellular and molecular mechanisms of pulmonary ischemia-reperfusion injury provides a good opportunity for implementation of this understanding into clinical practice. On the basis of recent evidence that the endothelium plays an essential role in regulating the dynamic interaction between pulmonary vasodNatation and vasoconstriction and is a major target during lung injury due to enhanced leukocyte-endothelial interactions, we at Harefield Hospital intend to implement protective strategies to limit perioperative lung injury during lung transplantation. This review summarises the major mechanisms potentially contributing to lung injury and the currently available surgical and anaesthetic protective strategies with a focus on clinical applicability.

1. Introduction Lung transplantation has become an established therapeutic modality for end-stage lung diseases worldwide due to improvement in surgical technique, advances in the anaesthetic management and pharmacological repertoire including immunosuppression. Although lung transplantation now offers a realistic opportunity for mid term survival in selected patients with end stage pulmonary disease, the practice is constrained by alarming shortage of suitable donor organs, significant primary graft failure and chronic rejection in the form of obliterative bronchiolitis. This is illustrated in the recent survival figures reported by the International Society for Heart and Lung Transplantation (ISHLT) [1] and can be summarised as follows 1) Heart transplantation has achieved good long term survival with 5 year survival rates in the 70% range, whereas combined heart lung transplantation is 40%. Isolated Ltx is also in the 40-50% range, suggesting that lung transplantation at the moment can only offer mid term survival. The most important factor adversely affecting long-term survival after lung transplantation is bronchiolitis obliterans syndrome, which is generally considered to be a complex end result of a chronic rejection process. 2) At least 50 % of the 5 year mortality occurs during the 1st year post transplant in both heart and lung transplantation with a significant number of death taking place perioperatively.

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3) Although there is a small but significant improvement in late mortality presumably due to better immunosuppression, no improvement in perioperative mortality can be seen when compared to data during the early 80's. 4) Analysis of the causes of perioperative death reveal that the majority of death is due to primary organ failure. The high incidence of loss or compromise of transplanted organs in the immediate posttransplantation period due to primary graft dysfunction prompted many research groups to study mechanisms of lung injury and transplant centres to re-evaluate donor management, and preservation/reperfusion protocols. Lung injury can occur at any stage of this complex procedure with distinct mechanisms that might provide a number of opportunities for surgical and anaesthetic efforts to minimise lung damage. 2. Organ damage during transplantation Much recent evidence indicates that components of the alveolocapillary unit are the major targets during acute lung injury with the micro vascular endothelium being the most susceptible element. During the injury process, endothelial cells become activated and more permeable with characteristic loss of function it plays as an essential regulator of pulmonary vasoreactivity, intravascular coagulation, inflammatory response and gas exchange [2]. In addition, the composition, function, and metabolism of pulmonary surfactant produced by alveolar type II epithelial cells are increasingly being recognised as important factors in acute lung injury. Although Type I epithelial cells appear to be more resistant to inflammatory response and injury and their barrier and metabolic function might remain intact even in high permeability pulmonary oedema, long-term outcome may depend upon Type I epithelial cell survival. Thus donor evaluation should include cellular and molecular assessment of endothelial and epithelial cell integrity and organ management, rescue and preservation should provide strategies to maintain structural and functional integrity of these cells in the face of multiple insults to the alveolocapillary unit. Donor lung injury Many donor lungs exhibit severe hypoxemia and diffuse infiltrates on chest roentgenograms owing to oedema, infection, aspiration, contusion, or ventilator-induced injury. The highpermeability pulmonary oedema that is prevalent in many lung donors often results from neurogenic causes, in addition to iatrogenic overhydration during the resuscitation attempts. Neurogenic pulmonary oedema occurs after a sudden, massive sympathetic neural discharge, which engenders a "blast injury" to the pulmonary circulation and may disrupt the anatomic integrity of the pulmonary capillaries. Recent studies have confirmed that in the hours after brain death there is significant increase in right ventricular hydraulic power and pulmonary artery blood flow These changes, along with increases in systemic and pulmonary vascular pressures, may lead to pulmonary endothelial cell injury, impaired lymphatic drainage, and an increase in pulmonary extravascular lung water [2]. Even in donors who do not have overt lung injury by standard criteria, measurements of intrapulmonary shunt may be highly abnormal and fiberoptic bronchoscopy may reveal aspiration of a significant volume of gastric contents or blood.

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In addition to injury during the hyperdynamic phase of brain death, the following period of haemodynamic instability and restoration manoeuvres might also contribute to lung damage. Hypovolemic shock might result in microvascular pulmonary occlusion by platelet and leukocyte aggregates, pulmonary endothelial injury and pulmonary parenchymal neutrophil sequestration. These changes are further aggravated by ventilator induced injury. It is now widely accepted that improper ventilator management has greatly compromising effects on the lung in the setting of an impending injury, through a mechanism which is related to biochemical upregulation of an inflammatory response mediated by a pro-and anti-inflammatory cytokine imbalance within the lung. This mechanism in concert with a systemic inflammatory response resulting from polytrauma with increased levels of cytokines might underlie the upregulation of vascular adhesion molecules even before harvesting, which might have implications for post ischaemic lung injury. Thus it can be argued that a significant number of cases of severe ischaemia-reperfusion injury probably result from evolving or subclinical donor lung dysfunction that was not readily apparent during the preharvest assessment [2]. Ischaemia-reperfusion Survival as the balance of microvascular vasoactive and cytotoxic/protective bioactivities Under basal conditions as well as during evolving lung injury, the function of the pulmonary endothelium depends on a dynamic balance between endothelial protective and injurious substances. Three important protective agents produced by endothelial cells are prostacyclin, nitric oxide, and adenosine all primarily acting by modulating cellular cyclic nucleotide (cAMP and cGMP) levels. Prostacyclin is a potent eicosanoid that causes vasodilation, prevents neutrophil adherence, inhibits platelet aggregation, and stabilises lysosomal membranes. Nitric oxide is produced in endothelial cells by nitric oxide synthase, a calcium- and calmodulin-dependent enzyme. The effects of nitric oxide are mediated by activation of guanylate cyclase, resulting in the formation of cyclic guanosine monophosphate (GMP). Nitric oxide has a shorter biological half-life than prostacyclin (10 to 20 seconds), is able to quench superoxide radicals produced by endothelial cells, and causes vasodilatation, decreased neutrophil adherence, and inhibition of platelet aggregation. A third key protective agent formed by the endothelium is adenosine, which produces its effects by activation of purinergic receptors coupled to adenylate cyclase by a guanosine triphosphate-dependent mechanism. Adenosine is rapidly metabolized by the enzymes adenosine deaminase and adenosine kinase. Like prostacyclin and nitric oxide, adenosine is a potent vasodilator, decreases neutrophil adherence to endothelial cells, and diminishes neutrophil cytotoxicity . Adenosine also inhibits superoxide radical production by neutrophils, and like prostacyclin and nitric oxide has produced beneficial effects in a variety of models of ischaemia-reperfusion injury. In addition to cytoprotective agents, the endothelium generates substances that have a marked vasoconstrictor and thrombogenic effect, thus promoting cell injury. These include endothelin-1, the phospholipid platelet activating factor, which causes the release of various leukotrienes and thromboxanes, all of which are vasoconstrictors. Oxidativc stress During the past few years many of the cellular and molecular events modulating the inflammatory response to ischaemia-reperfusion injury have been elucidated. Although

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changes in the energy homeostasis within the lung has received much initial attention, recent studies have confirmed that lung dysfunction after storage and reperfusion is associated with significant direct oxidative injury to endothelial cell constituents including lipids, protein, and DNA [3-4]. While many components of the oxidative stress are initiated during the ischaemic and hypoxic phase, the critical step mediating this injury appears to be the burst in the production of oxygen and nitrogen derived oxidative species at the onset of uncontrolled reperfusion [5-7]. Role of neutrophils Although in some models lung injury occurred even after neutrophil depletion, most studies suggest that endothelial and lung injury depends upon enhanced interaction between white cells particularly neutrophils and the endothelium [8,9]. During reperfusion after pulmonary ischaemia, neutrophils may contribute to lung injury in several ways. 1) Neutrophils are the major source of reactive oxygen metabolites such as the superoxide anion and hydrogen peroxide, which can damage pulmonary endothelial cells. 2) Due to cytoskeletal reorganisation, activated neutrophils are stiffer than quiescent cells, making them less able to undergo deformation as they circulate through the pulmonary capillaries and thus more likely to induce adherence to endothelial cells and capillary plugging. 3) Activated neutrophils can produce TNF, IL-1, IL-6, and IL-8 contributing to cytokine imbalance. 4) In addition, neutrophils can generate leukotriene 84, a potent chemotactic agent that activates neutrophils and promotes their adherence to the endothelium. 5) Finally, upon degranulation neutrophils release elastase and other proteases, which directly injure pulmonary endothelial and parenchymal cells. A recent study has identified heparin binding protein as one of the major neutrophil product causing changes in endothelial permeability [10]. Neutrophil protease inhibitors have been shown to attenuate experimental lung injury. The potential role of neutrophil-endothelial interactions in the clinical setting is important because many pharmacological and mechanical strategies can target these interactions that have been shown to be beneficial in many animal models with clinical applicability. However, in order to devise optimal strategies to reduce the injurious effects of neutrophils, one has to consider the origin of the harmful neutrophils and the consequence of total body elimination of neutrophil activity in the globally immunosuppressed patient. a) The number of neutrophils in the normal lung is approximately three times the circulating pool of neutrophils and due to their marginalisation, a significant number of neutrophils may reside in close proximity to endothelial cells even after flushing [2,11]. b) Postoperative infections are important factors in early mortality following transplantation, therefore systemic attenuation of neutrophil function is undesirable. This makes it difficult to adopt techniques aimed at depleting the circulating neutrophil pool or preventing neutrophil activation and migration through the endothelium in the long run. However recent data suggest that temporary attenuation of neutrophil function for as short as 15-30 minutes during reperfusion offers considerable benefit. This might be achievable with pharmacological therapy such as antioxidants, and agents modulating neutrophil

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function such as cAMP elevating agents and NO. In addition, an interesting model has been recently proposed to selectively perfuse the pulmonary circulation with a separate circuit, which includes a neutrophil depleting filter. This strategy can be easily considered on lung transplants performed utilising cardiopulmonary bypass. Cytokine network It is now evident that many of the cellular and molecular processes in the lung are controlled by a vast network of cytokines, which function as extracellular signalling proteins. Cytokines such as IL-1 and TNF play critical roles in the inflammatory response to ischaemia-reperfusion injury. Both of these cytokines cause endothelial cell activation, a phenotype which is markedly adhesive for neutrophils and other white cells. These cytokines induce cytoskeletal reorganisation, expression of leukocyte adhesion molecules and vasoactive and prothrombotic factors. They also induce neutrophil activation characterised by increased neutrophil phagocytosis, respiratory burst activity, and degranulation. All these events can cause pulmonary vascular endothelial cell injury both in vitro and in vivo. Studies utilising bronchoalveolar lavages in animal lung transplant models have demonstrated early release of pro-inflammatory cytokines. Similarly, IL-6 has been detected in the clinical situation, and peak serum IL-6 level has been correlated with the severity of preservation injury [16]. However the levels of other cytokines in bronchoalveolar lavage and serum during the first few days after clinical lung transplantation remains unknown. However, despite the postulation of their role in lung injury, no single cytokine has been identified to explain the onset, extent and outcome of acute lung injury. It is becoming evident that in addition to the pro-inflammatory, and pro-apoptotic cytokines there is a phased anti-inflammatory and cytoprotective response. Current efforts are increasingly focused on the characterisation of the actual net imbalance in these activities by utilising a number of bioassay s . It is thus evident that pulmonary ischaemia-reperfusion injury has multifaceted effects on the complex cytokine network and balance within the lungs which remains to be established. Although administration of anti-inflammatory cytokines such as IL-10 or monoclonal antibodies directed against selected cytokines involved in the early stages of this injury may ultimately prove useful clinically, enthusiasm is diminished in light of the complexity of the system and recent clinical trials in other forms of sepsis and lung injury. It is more convincing that a global strategy to interfere with cytokine response would be more attractive and simpler for example in the form of administration of high dose methylprednisolone to the donors prior to organ harvest. Epithelial injury and protection, surfactant The pulmonary alveolar epithelium is composed of two cell types, elongated type I cells, which cover most of the alveolar surface, and cuboidal type II cells, which predominate in the alveolar corners. Both type I and II cells exist in close proximity to pulmonary capillary endothelial cells and play important roles in pulmonary surfactant production and in modulating host defense in the alveolar space [2]. It is evident that the endogenous surfactant system undergoes profound alterations after pulmonary ischaemia and transplantation that are qualitatively similar to the changes in surfactant that occur in nontransplant lung injury models. Also there are many studies showing that exogenous surfactant therapy can mitigate pulmonary dysfunction in a wide array of experimental models. Although case reports show beneficial effects of surfactant

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after reperfusion, most studies suggest that for surfactant to have a maximum impact on lung injury after prolonged storage, donor surfactant therapy is probably essential [2]. The role of Type I function has received considerably less attention. However, recently it is suggested that these cells have a paramount role not only in modulating permeability but also in resolution of pulmonary oedema after lung injury in humdns. By monitoring alveolar fluid clearance in transplant patients through determination of alveolar lining fluid and plasma protein concentrations, a recent study established the capacity of the alveolar epithelium to reabsorb fluid from the alveolar space. This appeared to be a marker of less severe reperfusion injury on the basis of histological and clinical criteria and outcome [15]. 3. Strategies to limit lung injury Hypothermic preservation Flush perfusion of pulmonary grafts with cold modified EuroCollins solution supplemented by prostaglandin treatment was introduced clinically 10 years ago. During the last decade, much experimental work has led to reports of alternative storage solutions, differing storage conditions, and pharmacologic interventions that improve pulmonary graft performance. A world-wide survey of the 125 centres performing lung transplantation revealed that most centres continue to use EuroCollins solution of whom 69% include prostaglandin therapy and 32% donor steroid treatment [16]. University of Wisconsin solution (UW) is used by 15 centres (13.5%), of which 10 (67%) use prostaglandin and seven (47%) use donor steroids. Nine centres use Papworth solution and one uses donor core cooling. This report suggests that there has been a trend toward the use of UW solution and a slightly warmer storage temperature. However, for most centres, graft storage techniques have changed little over the last decade. Laboratory work showing that high-potassium storage solutions impair vascular endothelial cell function prompted studies to evaluate low-potassium-dextran pulmonary flush solution (LPD). It has been shown that LPD provides excellent 12-hour lung preservation in different animal models of experimental lung transplantation and exhibits favourable profile on alveolar cells injury and survival. The Toronto Program has now adopted LPD preservation solution into clinical practice after approval was obtained for the use of LPD in clinical lung transplantation in 1998. In a recent article they have reported their initial experience with the use of LPD in comparison with EC in 94 lung clinical lung transplant procedures [17]. Vasodilator and protective strategies These agents are used in selected centres as part of donor management and in the preservation flush solution Prostagiandins Similarly to many studies showing endothelial cytoprotection by prostaglandins, recent animal work has demonstrated that a continuous prostaglandin Ej infusion in the recipient, starting before reperfusion, was associated with improved oxygenation and less lung oedema after transplantation. Although clinical studies have not addressed this organ protection, there is evidence for efficient haemodynarnic effects of aerosolized prostacyclin

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in human lung transplantation. Also there is increasing numbers of centres using prostacyclin as part of their preservation protocol. Nitric oxide donors, nitroglycerin, inhaled NO As discussed above, NO is one of the major endogenous protective modalities of the lung. It exerts the majority of its effects via activation of guanylate cyclase and the resultant formation of cyclic GMP. A number of studies have documented characteristic changes in NO and NO bioactivity in the setting of ischaemia reperfusion. Most cellular studies including our own suggests that NO is consumed during oxidative stress. This has been demonstrated in animal models showing that both nitric oxide and cyclic GMP levels decreased markedly during ischaemia and at the onset of reperfusion . Finally this has been observed in ARDS and by us in lung transplantation [18]. Thus, decrease in NO levels might be a sensitive index of lung dysfunction and prevention of the decrease in endogenous NO or supplementation with exogenous NO should be a major aspect of lung protection strategies. Stimulation of guanylate cyclase activity can be achieved by exogenous nitric oxide, nitroglycerin, or nitroprusside. The ability of these agents to increase cyclic GMP levels was first noted almost 20 years ago. Recently, inhaled nitric oxide has been used as a potent and selective pulmonary vasodilator in many pathological situations including ARDS. In the setting of lung transplantation, nitric oxide is theoretically attractive because of its ability to quench superoxide radicals and to protect pulmonary endothelial cell function. Nonetheless, the use of nitric oxide can actually contribute to lung injury as a result of its combination with superoxide to form peroxynitrite. Peroxynitrite can generate the highly toxic hydroxyl anion and is a strong oxidant in its own right; it readily catalyses membrane lipid peroxidation, reacts with metals to form toxic nitrosylating species, and oxidises sulfhydryl groups on cellular proteins. Thus inhaled NO can only be recommended in the presence of strong antioxidant supplementation where the potential for peroxynitrite toxicity is reduced. Phosphodiesterase inhibitors Levels of cAMP and cGMP not only depend on stimulated production but also by the rate of their degradation. Inhibition of the degradation pathways through inhibition of phosphodiesterase enzymes is one of the major developments in haemodynamic management and organ preservation. These agents exhibit beneficial haemodynamic profile in heart failure, can reduce cathecolamine requirement and offer cytoprotection in many systems. Pentoxifylline is a methylxanthine derivative that has been used as a hemorrheologic agent for the treatment of peripheral vascular disease. In vitro studies have demonstrated that pentoxifylline has a marked inhibitory effect on neutrophils, particularly neutrophils activated by inflammatory cytokines. In view of its myriad antineutrophil effects and beneficial hemorrheologic properties, as well as its long track record of use in humans, pentoxifylline has clearly been recommended in the preservation of clinical lung grafts for transplantation [2]. Antioxidants In addition to a vast literature in animal models, the compromised antioxidant status of lung transplant recipients before and after transplantation is being increasingly recognised. Before surgery, the antioxidant status of patients was poor as serum ascorbate and total thiol concentrations were significantly lower man control subjects [19]. Two weeks post-surgery.

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ascorbate and total thiol concentrations were still low and urate concentrations had fallen compared to control subjects. These data provide solid support for perioperative antioxidant administration to these patients. One problem, however, is that there are distinct reactive species and it is likely that an antioxidant mixture including different components would be more effective than single antioxidants. Ascorbic acid Numerous studies have confirmed the beneficial effect of ascorbic acid in multiple oxidative stress conditions both in animal models and humans. Regarding lung transplantation ascorbic acid was able to weaken reperfusion injury in an in situ autotransplantation model in sheep [20]. N-acety 1-1 -cysteine (NAC), an oxidant scavenger, promotes glutathione in its reduced form (GSH) that is depleted during ischaemia. Numerous studies have recently demonstrated its efficacy in protecting lungs in animal reperfusion injury. In addition NAC has been shown to modulate inflammatory gene transcription and cytokine secretion in many cell types. Allopurinol: It is the only inhibitor of the enzyme xanthine oxidase, whose activation is involved in free radical generation. In addition to intracellular mechanisms, circulating xanthine oxidase activity has been linked to lung reperfusion injury. This circulating enzyme could be an important target of allopurinol. Its efficacy is well established in reducing the inflammatory components in gout. Superoxide dismutase: Recombinant human superoxide dismutase (rh-SOD) has been shown the potential to mitigate free radical-mediated reperfusion injury-induced acute endothelial cell damage that potentially may contribute to the process of chronic obliterative rejection arteriosclerosis. In a prospective randomized double-blind placebo-controlled trial, the effect of rhSOD, given in a dose of 200 mg intravenously during surgery to cyclosporine-treated recipients of cadaveric renal allografts, on both acute and chronic rejection events as well as patient and graft survival was investigated. The results obtained show that rh-SOD exerts a beneficial effect on acute rejection events and significant improvement of the actual 4-year graft survival rate in rh-SOD-treated patients [21]. Anticytokine therapy Corticosteroids are theoretically attractive drugs to control many inflammatory events in a wide number of pathological situations. This is likely related to inhibition of proinflammatory cytokines and pro-apoptotic activities. In addition to this theoretical advantage, the clinical benefit of steroid administration has recently been confirmed in donor lung viability. A retrospective study of all thoracic organ donors procured by the California Transplant Donor Network investigated which donor management factors were associated with an increased likelihood of successful lung procurement. Corticosteroid usage and initially clear breath sounds were independent predictors of successful procurement by multivariate analysis [22]. In addition to contribution of donor lung viability, corticosteroids might modulate the inflammatory response during ischaemia/reprefusion. A number of clinical studies have demonstrated that administration of methylprednisolone attenuated the inflammatory response to cardiopulmonary bypass, although the clinical benefit remains to be established [23]

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Complement attenuation Activation of the contact as well as the complement system is regulated by a common inhibitor, Cl-esterase inhibitor (Cl-INH). In addition to case reports where severe uncontrollable capillary leak syndrome after lung transplantation was successfully managed by Cl inhibition, there are data from a randomised, double-blinded, placebocontrolled multicenter trial at five North American centres regarding the influence of TP10 (soluble C receptor 1 inhibitor). TP10 led to a significant increase in early extubation of patients. Although there was no difference in PaO2/fraction of inspired oxygen between groups, total time receiving mechanical ventilation both tended to be shorter in the TP10 group but did not achieve statistical significance [24]. Serine protease inhibition: aprotinin The addition of aprotinin to EC and UW solutions increases endothelial cell viability in hypoxic cold storage conditions. In terms of whole organ function, aprotinin has been shown to improve lung preservation as demonstrated by increased oxygenation and compliance, and decreased capillary permeability. These observations are clinically applicable as there is already extensive experience with the use of aprotinin in heart and lung transplant recipients, in addition to its routine use in conventional cardiac operations in reducing perioperative blood loss [25]. The risk to develop lung injury continues in the postoperative period and thus lung protective strategies also have to be extended to this period. It is now well accepted that subclinical injury can be augmented with improper ventilation strategy. Thus currently proven protective ventilation strategies including low tidal volume and PEEP above lower inflection point are desired if tolerated in this condition. However during this period donor/recipient size mismatch, differential lung compliance especially during single lung transplantion remains a clinical challenge which might recuire sophisticated strategies including differential lung ventilation. References. [ 1 ] http://www.ishlt.org/regist_heart-lung_main.htm [2]

Novick RJ, Gehman KE, AH IS, Lee J. Lung preservation: the importance of endothelial and alveolar type II cell integrity. Ann Thorac Surg 1996; 62:302-314.

[3]

Granger DN. Ischemia-reperfusion: mechanisms of microvascular dysfunction and the influence of risk factors for cardiovascular disease. Microcirculation 1999; 6:167-178.

[4]

Garden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol 2000;

[5]

Verrier ED, Morgan EN. Endothelial response to cardiopulmonary bypass surgery. Ann Thorac Surg 1998;66:S17-S19.

[6]

Boyle EM, Jr., Pohlman TH, Cornejo CJ, Verrier ED. Endothelial cell injury in cardiovascular surgery: ischemia-reperfusion. Ann Thorac Surg 1996; 62(6): 1868-1875.

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Boyle EM, Jr., Canty TG, Jr., Morgan EN, Yun W, Pohlman TH, Verrier ED. Treating myocardial ischemia-reperfusion injury by targeting endothelial cell transcription. Ann Thorac Surg 1999; 68(5): 1949-1953.

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[8]

Bando K, Pillai R, Cameron DE, Brawn JD, Winkelstein JA, Hutchins GM et al. Leukocyte depletion ameliorates free radical-mediated lung injury after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99(5):873-877.

[9]

Eppinger MJ, Deeb GM, Boiling SF, Ward PA. Mediators of ischemia-reperfusion injury of rat lung. Am J Pathol 1997; 150:1773-1784.

[10]

Gautam N, Olofsson AM, Herwald H, Iversen LF, Lundgren-Akerlund E, Hedqvist P, Arfors KE, Flodgaard H, Lindbom L. Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability. Nat Med. 2001 Oct;7(10):l 123-7.

[11]

Gee MH, Albertine KH. Neutrophil-endothelial cell interactions in the lung. Annu Rev Physiol 1993;55:227-48

[12]

Halldorsson AO, Kronen M, Allen BS, Rahman S, Wang T, Layland M, Sidle D. Controlled reperfusion prevents pulmonary injury after 24 hours of lung preservation. Ann Thorac Surg. 1998 Sep;66(3):877-84

[13]

Pham SM, Yoshida Y, Aeba R, et al. Interleukin-6, a marker of preservation injury in clinical lung transplantation. J Heart Lung Transplant 1992;! 1:1017-24.

[14]

Martin TR. Cytokines and the acute respiratory distress syndrome (ARDS): a question of balance. Nat Med. 1997Mar;3(3):272-3.

[15]

Ware, L. B., Golden, J. A., Finkbeiner, W. E., Matthay, M. A. Alveolar Epithelial Fluid Transport Capacity in Reperfusion Lung Injury after Lung Transplantation. Am J Respir Crit Care Med . 1999; 159:980-988

[16]

Hopkinson DN, Bhabra MS, Hooper TL. Pulmonary graft preservation: a worldwide survey of current clinical practice. J Heart Lung Transplant. 1998 May;17(5):525-31.

[17]

Fischer S, Matte-Martyn A, De Perrot M, Waddell TK, Sekine Y, Hutcheon M, Keshavjee S. Lowpotassium dextran preservation solution improves lung function after human lung transplantation. J Thorac Cardiovasc Surg. 2001 Mar;121(3):594-6.

[18]

Marczin N, Riedel B, Gal J, Polak J, Yacoub M. Exhaled nitric oxide during lung transplantation [letter]. Lancet 1997; 350(9092):1681-1682

[19]

Williams A, Riise GC, Anderson BA, Kjellstrom C, Schersten H, Kelly FJ. Compromised antioxidant status and persistent oxidative stress in lung transplant recipients. Free Radic Res. 1999;30(5):383-93

[20]

Demertzis S, Scherer M, Langer F, Dwenger A, Hausen B, Schafers HJ. Ascorbic acid for amelioration of reperfusion injury in a lung autotransplantation model in sheep. Ann Thorac Surg. 2000 Nov;70(5): 1684-9.

[21]

Land W, Schneeberger H, Schleibner S, Illner WD, Abendroth D, Rutili G, Arfors KE, Messmer K. The beneficial effect of human recombinant superoxide dismutase on acute and chronic rejection events in recipients of cadaveric renal transplants. Transplantation. 1994 Jan;57(2):211-7.

[22]

McElhinney DB, Khan JH, Babcock WD, Hall TS. Thoracic organ donor characteristics associated with successful lung procurement. Clin Transplant. 2001 Feb;15(l):68-71.

[23]

Chaney MA. Corticosteroids and cardiopulmonary bypass : a review of clinical investigations. Chest. 2002Mar;121(3):921-31.

[24]

Martin R. Zamora et al. Complement inhibition attenuates human lung transplant reperfusion injury Chest 1999;116:46S

[25]

Royston D. Preventing the inflammatory response to open-heart surgery: the role of aprotinin and other protease inhibitors. Int J Cardiol. 1996 Apr 26;53 Suppl:Sl 1-37

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.). IOS Press, 2002

Condensate Inflammatory Markers in Lung Transplantation Karen McRAE Director of Anesthesia for Thoracic Surgery and Lung Transplantation 585 University Avenue Toronto, Ontario Canada M5G 2C4 Abstract. In human lung transplantation, ischemia-reperfusion (IR) injury occurs in 15-30% of recipient patients after lung engraftment. IR injury is the primary cause of death in the early postoperative period. Additionally, there is increasing evidence indicating a relationship between early IR injury and chronic graft dysfunction. Traditional respiratory monitoring techniques have failed to predict the occurrence of IR injury in the transplanted lung. The collection of breath condensate has been suggested as a technique to acquire samples representative of the interstitial fluid compartment of the lung parenchyma, and has been shown to contain protein macromolecules including cytokines. Cytokines play a critical role in modulating inflammatory processes and in enhancing cellular infiltration in injured tissue, including ischemia-reperfusion.

1. Introduction In clinical lung transplantation, the kinetics of cytokine release at the time of lung reperfusion, and the resulting clinical implications are the focus of ongoing investigation. In animal models of a variety lung injuries, plasma cytokine levels have failed to predict severity of injury. Tissue cytokines, specifically increased levels of the pro-inflammatory cytokine IL-8, have been demonstrated to correlate to worse clinical outcome in human lung transplantation [1], but tissue cannot be serially sampled. In this pilot study, three cytokines (TNF-a, IL-8 both pro-inflammatory and the anti-inflammatory IL-10) were measured in lung tissue, plasma and breath condensate in a porcine single lung transplant model throughout four hours of reperfusion, unmodified by immunosuppression. The goals of the study were: 1. To determine the relative amounts of cytokines measurable in samples taken from the three compartments of interest: lung parenchymal tissue, arterial plasma, and breath condensate. 2. To determine the kinetics of cytokines release throughout the transplant procedure and reperfusion, in each compartment. 3. To compare the amounts of cytokines measured in each compartment of the transplanted lung (allograft) and the native lung.

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2. Methods Left single lung transplantation was performed in five 60-70kg Yucatan swine. Allografts were harvested from donors after perfusion with a low potassium dextran (LPD) preservation solution and maintenance at 4°C for 24 hours. Anesthesia was induced and maintained with intravenous agents. Recipient swine were intubated with a single lumen endotracheal tube and ventilated with 50% oxygen, PEEP of 5cmH2O, to maintain peak airway pressure of less than 30cmH2O. Respiratory rate was adjusted to maintain PaCO2 of 37±5mmHg. Norepinephrine infusion was used as needed to maintain mean blood pressure of 60 mmHg throughout the reperfusion period of four hours. Lung parenchymal tissue was sampled from the allograft after cold ischemia, prior to and hourly after reperfusion, and from the native lung hourly after reperfusion. Plasma was sampled prior to and after thoracotomy, 10 and 30 minutes after reperfusion and hourly thereafter from arterial blood. Breath condensate was collected from exhaled gases of both lungs and each lung separately hourly. Lung separation was achieved by positioning of a bronchial blocker in the contralateral bronchus. Breath condensate was collected by the diversion of exhaled gas through a 20cm length of 1 cm diameter Tygon tubing encased in ice. Analysis for cytokines was performed in tissue, plasma and breath condensate using ELISA kits (Biosource, Camarillo California). For tissue samples, a supranatant was created and protein content was determined by the Bradford method [2]. Results of cytokine analysis in tissue are expressed in picograms per milligram of protein. For all data mean ± standard deviation are presented in graphical form.

3. Results Transplanted lungs appeared progressively hyperemic throughout reperfusion and a moderate amount of pulmonary edema was observed in several but not all recipient animals. Arterial oxygenation was relatively stable after the first hour of reperfusion, as seen in Figure 1. Fluid resuscitation and norepinephrine infusion was required in all animals, reflecting a systemic inflammatory response.

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3.1. TNF-a Tissue TNF-a in the left lung (allograft) was highest at the end of cold ischemic time (CIT), prior to engraftment, and steadily decreased throughout the four hour reperfusion period (Figure 2). Tissue TNF-a in the right (native) lung was in a similar range to the allograft (not shown). Plasma TNF-a exhibited two peaks (Figure 3). The first peak occurred after thoracotomy incision. Plasma TNF-a then significantly increased over the four hour reperfusion period long after tissue levels had reached a plateau, suggesting extrapulmonary production. TNF-a measured in the breath condensate collected from both lungs simultaneously increased after thoracotomy, then remained essentially stable during reperfusion Figure 4). TNF-a measured in breath condensate from the left lung showed a trend to being higher that levels obtained from the right lung (not shown). It must be noted that in breath condensate the range of TNF-a observed was close to the reported sensitivity of the ELISA assay.

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Tissue IL-10 was highest at the end of CIT in the left lung, and steadily decreased (Figure 7). This may represent a washout of the cytokine from tissue. Tissue IL-10 in the native right lung was in a similar range to the allograft and appeared to decrease initially after reperfusion, but the trends are not significant. Thoracotomy alone produced a striking increase in plasma IL-10 measured in arterial blood, a decrease during lung engraftment with no significant alteration during reperfusion (Figure 8). In contrast, IL-10 measured in the exhaled breath condensate collected from both lungs simultaneously increased after thoracotomy, and appeared to peak one hour after reperfusion (Figure 9). Analysis of the condensate collected separately from each lung revealed the same trend in the breath condensate from the left lung (allograft). IL-10 in the breath condensate from the right (native) lung remained in the range of baseline measurements.

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4. Discussion 1) This pilot study presents the time course of the cytokines TNF-a, IL-8 and IL-10 detected in various sample compartments of a porcine model of single lung transplantation; some but not all cytokines are detected in breath condensate. 2) The time courses of cytokines measured in tissue, plasma and breath condensate vary considerably. Cytokine levels detected in breath condensate were unlike those in plasma or tissue samples in magnitude and trend, Thoracotomy alone produced a significant increase in the TNF-a and IL-10 detected in plasma, the degree to which plasma levels of cytokines influenced measurements in breath condensate is unclear. 3) Animal studies may be limited by a lack of species specific ultrasensitive ELISA kits, particularly for measurement of low concentrations of cytokines in breath condensate. 4) Separate measurement of IL-10 in breath condensate from each lung in a single lung transplant model suggests that the IL-10 from the transplanted lung most influences the measurement for both lungs together. This may reflect increased droplet formation from injured small airways of the allograft, as compared to the native lung. 5) The clinical usefulness of the analysis of breath condensate for inflammatory mediators in lung transplantation requires further study.

Acknowledgement. Many thanks to collaborators Marc De Perrot, Stefan Fischer and Shaf Keshavjee of the Thoracic Surgery Research Laboratory, Toronto General Hospital and the University of Toronto

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References 1. De Perrot M, Sekine Y, Fischer S, Waddell T, McRae K, Wigle D, Keshavjee S. IL-8 release during early reperfusion predicts outcoe in human lung trapslantation. In press Am J Resp Crit Care Med 2002. 2. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-54, 1976.


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Exhaled Nitric Oxide (NO) in Human Lung Ischeniia-Reperfusion (I/R) Nandor MARCZIN Department of Anaesthetics, Royal Brompton and Harefield Hospital and Department of Cardiothoracic Surgery, Faculty of Medicine, National Heart and Lung Institute, Imperial College, Heart Science Centre, Harefield Hospital, Harefield, United Kingdom, Abstract. This review emphasizes recent progress in the direct evaluation of endogenous NO through bedside monitoring of NO concentrations in the expired air of patients subjected to ischaemia and reperfusion during cardiothoracic surgery. There has been recent progress in our understanding of the determinants of exhaled NO, the anatomical, cellular and molecular origin of NO in the expired air. The scientific community has widely accepted that NO levels in the gas phase reflect in an accurate and qualitative manner the dynamics of NO production and consumption in the airways, especially in the microenvironment of epithelial cells. The contribution of vascular compartments to exhaled NO has been debated and it appears that changes negatively affecting NO metabolism in the microvasculature remain largely undetectable with exhaled NO. We have provided evidence that augmented vascular NO from endogenous metabolism of GTN can be detected in the expired air in humans and have postulated that this phenomenon could be used to assess vascular NO consumption in ALL In the setting of ischaemia and reperfusion related ALI, we have obtained intriguing data, which might have implications to mechanisms, extent and management of ALI associated with cardiothoracic surgery.

1. Introduction: lung ischaemia-reperfusion in cardiothoracic surgery Complete and prolonged lung ischaemia up to several hours is unavoidable during lung transplantation with dire consequences. Transbronchial biopsies performed after lung transplantation showed characteristic histologic features of diffuse alveolar damage in 35 % of patients, even when implantation was performed without CPB [1], This is associated with severe graft dysfunction in about 20% of lung transplant recipients with the clinical manifestation of progressive hypoxemia, decreased pulmonary compliance, high permeability pulmonary edema, and widespread alveolar densities on chest radiographs [24], The early lung allograft dysfunction remains the primary cause of early mortality in lung transplantation. Although severe graft dysfunction can be reversible, it is often associated with the need for prolonged mechanical ventilation, intensive care and hospital stay and compromised recovery among survivals [5]. In addition to this morbidity, ischaemiareperfusion injury may also predispose grafts to acute and chronic rejection via upregulation of class II major histocompatibility complex antigens, release of endothelial cell antigens potentially triggering antiendothelial antibody production and via generation of proinflammatory mediators including cytokines and growth factors [6],

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2. Mechanisms of ischemia-reperfusion injury Role ofmicrovascular endothelial and epithelial cells and neutrophils in I/R Significant progress has been made in understanding the complex cellular and molecular events that mediate and modulate ischaemia-reperfusion lung injury [2;7;8]. Much recent evidence indicates that in addition to arteriolar and postcapillary venular alterations, components of the alveolo-capillary unit are the major targets during acute lung injury with the microvascular endothelium being the most susceptible element. During the injury process, endothelial cells become activated and more permeable with characteristic loss of their normal function as essential regulators of pulmonary vasoreactivity, intravascular coagulation, and inflammatory response and gas exchange. In addition, the composition, function, and metabolism of pulmonary surfactant produced by alveolar type II epithelial cells are increasingly being recognized as important factors in lung injury [2;9]. Although Type I epithelial cells appear to be more resistant to inflammatory response and injury, and their barrier and metabolic function might remain intact even in high permeability pulmonary edema, long-term outcome might depend upon Type I epithelial cell survival. It is now established that human endothelial cells are substantially altered either during hypoxia associated with ischaemia or during reestablishment of blood flow and oxygen (reperfusion and reoxygenation) or in response to inflammatory mediators resulting in an activated phenotype [10]. This includes changes in the profile of vasoregulatory endothelium dependent factors, and the expression of activities that initiate and amplify inflammation and coagulation. Prolonged hypoxia may lead to severe depletion of energy stores causing cellular energetic failure. Immediately after reperfusion, there appears to be a burst of oxidant production within the hypoxic endothelial cells. This together with complement fragment activation on the surface of these cells cause transient expression of preformed proteins and release of mediators that promote leukocyte-endothelial cell interactions, coagulation and cytoskeletal rearrangement which might underlie transient increase in permeability [11]. Alternatively, this oxidative stress can initiate signal transduction events to activate a delayed transcriptional program of several genes resulting in the translation and prolonged surface expression of leukocyte adhesion molecules and cytokines that mediate further recruitment of neutrophils to sites of inflammation [12]. There has been considerable circumstantial evidence both from animal and clinical studies implicating the neutrophil as a potentially important mediator of the early changes in lung endothelial and epithelial permeability following ischaemia reperfusion. The enhanced neutrophil-endothelial interactions might promote microvascular injury by multiple mechanisms. First, activation of neutrophils in the close proximity of endothelial cells might accentuate and prolong oxidative stress resulting in oxidative stress signaling in endothelial cells to further enhance pro-inflammatory phenotype and sustain cytoskeletal reorganization [13]. Neutrophils can produce significant quantities of a number of pro-inflammatory cytokines contributing to cytokine imbalance, potent chemotactic agents that further promote their adherence to the endothelium. Finally, they can release elastase and other proteases, which might contribute to direct pulmonary cell injury [14]. Role of NO in I/R Beyond oxygen centered free radicals and oxidants, nitric oxide appears to play a multifaceted role in ischaemia-reperfusion and to modulate the biological effects of reactive oxygen species. Under normal conditions, there is a considerable release of NO both in the

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microvasculature and airways to elicit a number of bioactivities through either direct signaling or via a guanylate cyclase and cGMP dependent process. In the normal lung these include 1) regulation of pulmonary arteriolar and bronchial tone by relaxing smooth muscle; 2) prevention of platelet aggregation and thrombus formation; 3) modulation of multiple aspects of lung inflammation through attenuation of the adhesive interactions between leukocytes and the endothelial or epithelial cell surface, leukocyte trafficking and reduction of oxidative stress by effectively scavenging the low intracellular levels of superoxide anion (8). NO production and bioactivity is subjected to great alterations during hypoxia, ischaemia and reperfusion. Enzymatic NO production exhibits a characteristic O2 dependence and thus hypoxia reduces enzyme activity to synthesize NO. This phenomenon has been demonstrated in both cells in culture and animal and human lungs [15; 16] . Hypoxia however might increase NO generation from non-enzymatic sources. This involves non-enzymatic reduction of inorganic nitrite to NO, a reaction that takes place predominantly during acidic/reducing conditions [17]. Thus hypoxia and ischaemia might alter NO concentrations and bioactivity by multiple and sometimes opposing mechanisms. Nevertheless, animal studies suggest that under conditions of hypoxia and ischaemia of the lung the predominant effect appears to be reduction in NO concentrations. In an orthotopic rat model of lung transplantation, NO release at the surface of the lung was diminished during hypothermia storage [18]. In addition to changes in NO availability during ischaemia, reperfusion can cause further consumption of NO through interactions with superoxide. In this situation NO undergoes radical-radical reactions with superoxide at near diffusion-limited rates to yield peroxynitrite, a potent oxidizing agent to lipids, aromatic amino acid residues, protein sulfhydryls and DNA [19]. Peroxynitrite has been shown to initiate lipid peroxidation in biological membranes at rates that are a thousand-fold higher than for hydrogen peroxide. However, NO displays a dual action with lipids: in addition to pro-oxidant characteristics through peroxynitrite mediated oxidation reactions it has potent capability to inhibit lipid radical chain propagation [20]. Thus although NO can serve both as antioxidant (by inhibiting lipid free radicals) or an oxidant (by contributing to peroxinitrite formation), both of these reactions will lead to consumption of NO and reduced levels of bioactivity to elicit normal signaling and biological functions in the lung. Following the acute phase of NO-superoxide interactions, the redox milieu is further complicated by transcriptional induction of iNOS and various antioxidant enzymes [21 j. The resulting reactions will again depend on relative quantities of NO and superoxide and the local redox microenvironment. It is conceivable to believe that in case of continuous ongoing superoxide production, increased NO synthesis may contribute to further peroxynitrite formation, however increased NO may attenuate the extent of cellular injury through inhibition of apoptosis or may restore endothelial function if concomitant superoxide generation had subsided. All these considerations predict that I/R will be associated with a complicated picture of NOS expression, NO generation and consumption. Actual NO concentrations will be different accordingly to the dynamically changing cytokine environment, nature of microvascular and airway inflammation, neutrophil activation, concomitant production of reactive oxygen species and acidity in the immediate environment of endothelial and airway epithelial cells. Understanding of these reactions and their consequences in lung microvascular or airway damage or protection may provide a more rational basis for new therapeutic strategies towards better preservation of organ viability and function during and following I/R.

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Given the pivotal importance of all of these fluid phase reactions in acute lung injury, monitoring changes in the production and bioavailability of NO in the lung would be extremely desirable. However due to the short half life of NO and peroxynitrite and the nature of the fluid phase reactions analytical repertoire has been limited to detect stable end products of NO metabolism such as nitrite and nitrate and footprints of peroxynitrite such as nitrotyrosine. The mindful discovery of Gustafsson and colleagues, followed by recent technological developments allowing direct measurements of NO in the expired air, however, have provided an exciting opportunity to evaluate changes in NO production and consumption in the clinical setting [22]. 3. Physiology of exhaled NO Although measurements of exhaled NO provide important direct information regarding NO concentrations in the gas phase, interpolation of these data to in vivo NO metabolism is far from straightforward. The biological reactions of NO are likely related to local NO concentrations in the fluid phase which is influenced by many processes including generation rate, fluid phase reactions such as autooxidation, and consumption by a variety of mechanisms including interaction with hem-iron groups, proteins and scavenging by hemoglobin and interactions with superoxide. Since many of these processes appear to be anatomical site-dependent within the lung and they are likely to be differentially altered by dynamically changing pathological processes, it is of crucial importance to consider the implications of the anatomical origin of NO in exhaled air to molecular pathology of acute lung injury (ALI). As discussed in details elsewhere in the book, the anatomic site and the type of cells responsible for the release of NO into the gas phase remains a matter of debate. On one hand, there is evidence that under certain conditions vascular mechanisms could contribute to exhaled NO. In particular, infusion of endothelium-dependent vasodilators increased exhaled NO in isolated perfused lung models suggesting that a fraction of microvascular NO may diffuse into the alveolar compartment contributing to exhaled NO. However, elegant studies by Sartori et al utilizing an inhaled or infused NO synthase inhibitor suggest that exhaled NO is mostly of airway epithelial rather than of vascular endothelial origin [23]. On the basis of these considerations they have concluded that exhaled NO may not be used as a marker for vascular NO production and/or endothelial function in healthy humans. These observations and conclusions provide a solid basis for current promotion to use exhaled NO as a diagnostic tool to monitor inflammatory responses affecting primarily the conducting airways in asthma [24]. In contrast, the same considerations indicate major limitations of exhaled NO as a marker of ALI, which is primarily characterised by microvascular and alveolar dysfunction. The major implications are that changes in vascular NO metabolism in ALI likely remain undetectable by exhaled NO measurements and detected changes in exhaled NO would probably reflect altered epithelial NO generation and consumption. We have recently suggested a potential solution to this problem by utilizing exhaled NO responses following intravenous administration of nitroglycerin (GTN), which elicits its biological effect by NO release mediated by thiol-dependent enzymatic biotransformation. Shortly after the original observations of Persson et al in animal models regarding increased exhaled NO levels following vascular metabolism of intravenous nitric oxide donors [25], we have established and characterized this phenomenon in humans [26,27]. We concluded that a fraction of nitroglycerin is metabolized in the pulmonary microvasculature to NO,

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which then diffuses into the alveolar space, giving rise to exhaled NO. On the basis of these considerations, we have suggested that GTN-induced exhaled NO might be a useful tool to monitor metabolic function of the pulmonary microvasculature. 4. Exhaled NO following CPB and lung transplantation At Harefield Hospital, we have studied these issues in the setting of clinical ischaemiareperfusion [28]. We have measured endogenous NO in the expired air as a means to assess bronchial epithelial function and we have examined GTN-induced exhaled NO indicating vascular and alveolar metabolic function. We have performed these studies in the setting of complete and prolonged lung ischaemia and reperfusion during lung transplantation and compared them to those occurring with transient and incomplete lung ischaemia during routine open-heart surgery for coronary artery bypass grafting (CABG) utilizing CPB. Breath to breath measurements of NO concentrations in the lower airways were performed using a real-time, computer-controlled and integrated system (Logan Research Ltd. 2000 and 3000 series). Inspired and expired samples for analysis of NO and COi were continuously withdrawn directly from the main lower airways through a thin Teflon sampling tube at a flow rate of 150 ml/minute. Since detected concentration of exhaled gases depends on both the production rate and ventilation parameters, ventilation was standardized for inspired gas (100% 02), tidal volume (5 ml/kg), respiratory rate (10/min) and inspiratory and expiratory ratio (1:2). To eliminate the influence of positive end expiratory pressure on gas phase NO, PEEP was set to zero. Baseline measurements were performed prior to CPB to evaluate endogenous levels of exhaled NO. After the baseline measurements, three increasing boluses of 1, 2 and 3 fig/kg GTN were administered to the patient via the central venous catheter with exhaled NO and haemodynamic response recorded. Between each boluses of GTN a short period of time was allowed for both the haemodynamic and exhaled gas parameters to return to the baseline values. The similar protocol was repeated 1, 3 and 6 hours after CPB. Arterial blood was simultaneously collected for haemoglobin, blood gas, electrolytes and full blood count analysis. In all 12 patients undergoing myocardial revascularisation involving cardiopulmonary bypass, NO was detectable in the exhaled air before CPB as a characteristic oscillating signal which appeared to increase with expiration as judged by the COi. Intravenous bolus administration of 1, 2 and 3 fig/kg GTN resulted in a rapid, transient and dose-dependent increase in exhaled levels of NO. Associated with the transient increase in exhaled NO following administration of GTN boluses, systolic arterial blood pressure transiently and concomitantly decreased. Endogenous exhaled NO levels remained unchanged 1 and 3 hours after CPB in these patients. Although measurements were performed in the intubated patients 6 hours after the operation, at this time point the majority of patients already made some spontaneous breathing efforts. There were characteristic changes in GTN-induced response in exhaled NO after CPB. The dose-dependent increases in exhaled NO by GTN were significantly smaller at 1 hour and 3 hours after CPB when compared to levels measured before CPB. There was no characteristic exhaled NO signal, such as seen with CABG patients in the majority of the lung transplant recipients during the post CPB period. This was either due to reperfusion induced loss of a detectable signal during the ischaemia period in two patients, who exhibited characteristic NO signals before reperfusion of their lungs (measured during CPB after completion of airway anastomoses. In most of the patients, however this was unrelated to reperfusion, since no detectable signals were obtained during ischaemia. A

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comparable NO signal to CABG patients was only seen in two out of the 10 lung transplant recipients during the post reperfusion period. When considered as a group and compared to exhaled NO levels in CABG patients, both peak expired NO and NO output were lower in lung transplant recipients after reperfusion. GTN-induced increases in exhaled NO were generally absent or appeared very small in lung transplant recipients after reperfusion. Furthermore, total NO output over 30 seconds was also profoundly reduced. Interestingly, GTN-induced exhaled NO was attenuated even in those patients whose endogenous exhaled NO was preserved. In addition GTN-induced exhaled NO recovered slowly in the postoperative period (> 24 hours) despite earlier normalisation of endogenous exhaled NO.

Figure 1. Representative traces of GTN-induced exhaled NO in routine heart surgery patient (top panel) and in lung transplant recipient following reperfusion (lower panel). Please note the absence of detectable basal and GTN-induced exhaled NO in the transplant recipient.

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This series of investigations aimed to clarify the influence of open-heart surgery and/or CPB on NO concentration in the expired air. Currently there are contradicting published results showing no change, increase or decrease in NO concentrations by different groups of investigators using a variety of methodological approaches [29-32]. The two principal observations of this part of the study are the unchanged basal concentrations of expired NO during the immediate postoperative period and decreased GTN-induced exhaled NO after surgery. Our interpretation of these data is that basal and GTN-induced exhaled NO represents distinct anatomical compartments and physiological mechanisms contributing to exhaled NO and that these mechanisms are differently affected by CPB and heart surgery. Our conclusion is that in the clinical setting of routine open-heart surgery, CPB-induced inflammatory response and ischaemia-reperfusion injury do not reach sufficient levels to compromise endogenous NO mechanisms to produce exhaled NO, (events that likely reflect airway epithelial processes). Similarly, the pulmonary and systemic haemodynamic response to a challenge with a bolus of GTN is also preserved, yet the characteristic increase in evolution of NO into expired air from GTN (which likely reflects lung microvascular events) is impaired in the early post reperfusion period. This might have clinical implications to heart surgery and CPB induced pulmonary microvascular injury and support our original idea to further evaluate this response as a bedside test of the metabolic function of the lung. Although routine CPB and open heart surgery is associated with a degree of clinically significant pulmonary dysfunction this rarely fulfills the criteria of ALL This might be related to the transient and incomplete nature of ischaemia and reperfusion. In contrast, lung transplantation is frequently associated with perioperative ALI, which might be related to prolonged and complete lung ischaemia. In accordance with the greater potential to ischaemia-reperfusion injury, lung transplantation was associated with a profound loss of GTN metabolism to produce exhaled NO. In addition and in contrast to open-heart surgery we found a variable decrease in endogenous exhaled NO levels. The ability to measure exhaled NO levels during ischaemia, reperfusion and after operation allows the elucidation of distinct mechanisms contributing to loss of exhaled NO. In conclusion, our findings provide additional evidence that during even in clinically successful lung transplantation ischaemia-reperfusion injury may reach sufficient levels to routinely compromise vascular mechanisms at least those responsible for pulmonary metabolism of organic nitrates, transport and release of NO to the air space. In addition, there is evidence of epithelial dysfunction in releasing NO into the gas phase. In light of recent observations suggesting the critical role of epithelial cells in the resolution of acute lung injury [33], exhaled NO might be a useful bedside tool to monitor the onset, extent and resolution of vascular and epithelial injury and the involvement of the NO pathways.

Acknowledgements. This work has been supported by a MRC Clinician Scientist Fellowship to Dr. Nandor Marczin. Magdi Yacoub is a British Heart Foundation Professor of Cardiothoracic Surgery. The contribution of the Julia Polak Transplant Fund is greatly appreciated.

References

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Khan SU, Salloum J, O'Donovan PB, Mascha EJ, Mehta AC, Matthay MA et al. Acute pulmonary edema after lung transplantation: the pulmonary reimplantation response. Chest 1999; 116:187-194.

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Kundu S, Herman SJ, Winton TL. Reperfusion edema after lung transplantation: radiographic manifestations. Radiology 1998; 206:75-80.

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Christie JD, Bavaria JE, Palevsky HI, Litzky L, Blumenthal NP, Kaiser LR et al. Primary graft failure following lung transplantation. Chest 1998; 114(1):51-60.

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Serrick C, Giaid A, Reis A, Shennib H. Prolonged ischemia is associated with more pronounced rejection in the lung allograft. Ann Thorac Surg 1997; 63:202-208.

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Granger DN. Ischemia-reperfusion: mechanisms of micro vascular dysfunction and the influence of risk factors for cardiovascular disease. Microcirculation 1999; 6:167-178.

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Boyle EM, Jr., Pohlman TH, Comejo CJ, Verrier ED. Endothelial cell injury in cardiovascular surgery: ischemia-reperfusion. Ann Thorac Surg 1996; 62(6): 1868-1875.

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Boyle EM, Jr., Canty TG, Jr., Morgan EN, Yun W, Pohlman TH, Verrier ED. Treating myocardial ischemia-reperfusion injury by targeting endothelial cell transcription. Ann Thorac Surg 1999; 68(5): 1949-1953.

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Bando K, Pillai R, Cameron DE, Brawn JD, Winkelstein JA, Hutchins GM et al. Leukocyte depletion ameliorates free radical-mediated lung injury after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990; 99(5):873-877.

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Dweik RA, Laskowski D, Abu-Soud HM, Kaneko F, Hutte R, Stuehr DJ et al. Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism. J Clin Invest 1998; 101(3):660-666.

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Phelan MW, Faller DV. Hypoxia decreases constitutive nitric oxide synthase transcript and protein in cultured endothelial cells. J Cell Physiol 1996; 167(3):469-476.

(17) Zweier JL, Samouilov A, Kuppusamy P. Non-enzymatic nitric oxide synthesis in biological systems. Biochim Biophys Acta 1999; 1411(2-3):250-262. (18) Pinsky DJ, Naka Y, Chowdhury NC, Liao H, Oz MC, Michler RE et al. The nitric oxide/cyclic GMP pathway in organ transplantation: critical role in successful lung preservation. Proc Natl Acad Sci U S A 1994; 91(25): 12086-12090. (19) Freeman BA, White CR, Gutierrez H, Paler-Martinez A, Tarpey MM, Rubbo H et al. Oxygen radicalnitric oxide reactions in vascular diseases. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Adv Pharmacol 1995; 34:45-69.

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(20) O'Donnell VB, Freeman BA. Interactions between nitric oxide and lipid oxidation pathways: implications for vascular disease. Circ Res 2001; 88:12-21. (21) Liu M, Tremblay L, Cassivi SD, Bai XH, Mourgeon E, Pierre AF et al. Alterations of nitric oxide synthase expression and activity during rat lung transplantation. Am J Physiol Lung Cell Mol Physiol 2000;278(5):L1071-L1081. (22) Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991; 181(2):852857. (23) Sartori C, Lepori M, Busch T, Duplain H, Hildebrandt W, Bartsch P et al. Exhaled nitric oxide does not provide a marker of vascular endothelial function in healthy humans [see comments]. Am J Respir Crit Care Med 1999; 160:879-882. (24)

Kharitonov SA, Barnes PJ. Nitric oxide in exhaled air is a new marker of airway inflammation. Monaldi Arch Chest Dis 1996; 51(6):533-537.

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Marczin N, Riedel B, Royston D, Yacoub M. Intravenous nitrate vasodilators and exhaled nitric oxide Lancet 1997; 349:1742-1742.

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Marczin N, Riedel B, Royston D, Yacoub M. Exhaled nitric oxide and pulmonary response to iloprost in systemic sclerosis [letter; comment]. Lancet 1998; 352(9125):405-406.

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Marczin N, Riedel B, Gal J, Polak J, Yacoub M. Exhaled nitric oxide during lung transplantation [letter]. Lancet 1997; 350(9092): 1681-1682.

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Beghetti M, Silkoff PE, Caramori M, Holtby HM, Slutsky AS, Adatia I. Decreased exhaled nitric oxide may be a marker of cardiopulmonary bypass-induced injury. Ann Thorac Surg 1998; 66:532-534.

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Hill GE, Snider S, Galbraith TA, Forst S, Robbins RA. Glucocorticoid reduction of bronchial epithelial inflammation during cardiopulmonary bypass. Am J Respir Crit Care Med 1995; 152:1791-1795.

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Brett SJ, Quinlan GJ, Mitchell J, Pepper JR, Evans TW. Production of nitric oxide during surgery involving cardiopulmonary bypass [see comments]. Crit Care Med 1998; 26(2):272-278.

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Ishibe Y, Liu R, Hirosawa J, Kawamura K, Yamasaki K, Saito N. Exhaled nitric oxide level decreases after cardiopulmonary bypass in adult patients. Crit Care Med 2000; 28:3823-3827.

(33) Ware LB, Golden JA, Finkbeiner WE, Matthay MA. Alveolar epithelial fluid transport capacity in reperfusion lung injury after lung transplantation. Am J Respir Crit Care Med 1999; 159(3):980-988.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub(Eds.) IOS Press, 2002

In Situ Lung Autotransplantation Model in Pigs E. N. KOLETSISl, A. CHATZIMICHALIS1, K. KOKINIS^, V. FOTOPOULOS 2,1. BELLENIS1, D. DOUGENIS2. 1

Dept. of Thoracic Vascular Surgery, Evagelismos Medical Center, 2 Dept. of CardioThoracic Surgery & 3 Dept of Anesthesiology and Intensive Care Unit University ofPatras. Abstract. Lung transplantation is a well accepted treatment for patients with end stage pulmonary disease [1]. Early graft dysfunction remains one of the major causes of early morbidity and mortality, with reperfusion injury (RI) being the most responsible mechanism [2, 8}. The exact pathophysiology of RI in lung transplantation has not been fully evaluated and understood [2]. Experimental transplantation after cold storage has been so far unable to duplicate the complete clinical picture of RI, such as, hypoxia, severe impairment of endothelial permeability, and frank alveolar oedema. On the basis of our previous experimental works with pigs, this paper describes a single lung transplantation model [3,4,5], which might be useful in assessing ischaemia and reperfusion injury without the potential interference of acute rejection.

1. Introduction. Our aim was to create a steady and reproducible experimental protocol that could demonstrate several parameters associated with the mechanisms of reperfusion injury, including impaired gas exchange, elevated pulmonary vascular resistant, local [6,7] and systemic aspects of the reperfusion syndrome, but without the interference of the pathology concerning acute graft rejection. [2] 2. Methods Female Pigs with a body weight between 25 and 30 kg were used for the experiments. All animals received humane care in compliance with the word wide accepted quidlines for care and use of laboratory animals. [3] The experimental protocol was approved by the Patras University Ethical Committee. Anaesthesia The animals were premedicated with midazolam 5mg and atropine sulfate 0.5 mg both given by intramuscular (IM) injection. A venous line was established by puncturing an auricular vein. Induction of anesthesia was performed with sodium thiopental 250 - 400 mg intravenously. The animals were initially intubated with an 6.5 mm internal diameter

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orotracheal tube followed by a proper tracheostomy to gain better access to the tracheobronchial tree and facilitate bronchoscopy. Volume-controlled mechanical ventilation was instituted (Siemens Servo 900 C respirator). Initially tidal volume was set to 10 mL/kg body weight, respiratory rate to 14 breaths per minute with a FiO2of 0.5. The respirator settings were subsequently adjusted to achieve a Pco2 of 40 to 45 mm Hg and arterial oxygen saturation of more than 90%. Anaesthesia was maintained with 300 mg sodium thiopental and 0.2 mg fentanyl [9] both given as IV bolus injections every 15 to 30 minutes. For muscular relaxation 4 mg pancouronium bromide was added intravenously as appropriate. Perioperative antibiotic medication consisted of cefuroxime IV 750 mg bolus injection. Central and core temperatures were recorded. [3,4,5,9]. Surgical Technique Following dissection of the right femoral artery and vein, a central venous catheter was inserted in the right femoral vein using the Seldinger technique and a Swan-Ganz catheter was placed subsequently. An arterial catheter for invasive blood pressure monitoring was placed in the in right femoral artery in the same fashion. A Foley catheter was introduced by cystostomy. [3]. The animal was placed in the right lateral position. A posterolateral thoracotomy incision was performed in the left fifth intercostal space. The incision was extended anteriorly toward the sternum. Meticulous control of bleeding was used all through the surgical procedure. A thoracoplasty type procedure was used by removing 2 or 3 ribs, so as to achieve a wide opening exposure for careful and safe manipulation of the left lung. With the lung retracted inferiorly, the pleura overlying the left pulmonary artery is opened with careful dissection of the left pulmonary artery. The hemiazygos was liquated and all lymphatic nodes of the hilum, paratracheal and aortopulmonary window space were removed. The left main pulmonary artery and bronchus were isolated in the pulmonary hilum. The lung was elevated superiorly, exposing and dividing the inferior pulmonary ligament. The inferior pulmonary vein was isolated. The pericardium was opened and the origin of the left pulmonary vein was dissected and isolated. A tape was passed around the left main bronchus, which was stripped by all bronchial arteries in order to ablate the bronchial circulation.The pulmonary veins were further dissected at their entrance into the left atrium and the hemiazygos was dissected intrapericardialy. During these steps of the surgical procedure particular care was taken to minimize manipulation of the prone to damage and particular fragile ventilated left lung. A Swan-Ganz catheter was inserted through the inferior vena cava and placed in the right pulmonary artery, the proper position of the Swan-Ganz catheter in to the right pulmonary artery was verified by palpating the left pulmonary artery, and it is notable that the pulmonary catheter had been positioned in the right place all through the experiments. Heparin was given intravenously (300 lU/kg). A purse-string stitch 6-0 Prolene suture (Ethicon, Hamburg, Germany) was placed in the pulmonary artery; taking care not to penetrate into the lumen. An artery cannula 18 G was carefully inserted in the pulmonary artery. The artery was proximally occluded by a tourniquet. A side-biting clamp was placed on the left atrium central to the left pulmonary veins and a minor incision was made for fluid drainage (vent). The left lung was then flushed with cold modified Euro-Collins solution (60 mL/kg). Pulmonary artery pressure during flushing was kept at 15 mm Hg approximately. (Fig 1). Ventilation was continued during flush perfusion. After completion of perfusion, the main left bronchus was crossclamped with bronchial clamp and the lung kept semi-inflated. The lung was left in situ and covered with cold swabs inside an isotherm and waterproof bug. The temperature was measured continually in the left interlobar space and when exceeded 8°C, additional cold normal saline and ice was applied to the towels over the isotherm bag, which isolated the lung from the pleural space. Warm normal saline at 38,5°C was added into the pleural

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space with a control way, when ever the central temperature dropped to less than 36.5°C so that the central temperature was kept between 37 and 38.5°C. Additional measurements were used to maintain animal's normal temperature according to the core temperature. Total ischemic time of the left lung was set at 3 hours. Reperfusion was initiated by removing the tourniquet from the pulmonary artery. Vent from the pulmonary veins was kept until fresh red blood was coming out ensuring that all Euro-Collins lung preservation fluids had been flushed out by the pulmonary circulation. Following that, the incision of the left atrium vent was repaired with a 5-0 Prolene suture (Ethicon, Hamburg, Germany) and subsequently the left atrium and the bronchial clamp were removed and ventilation to the left lung restored.

Fig 1. Surgical field after complete preparation. LIPV: Left Inferior Pulmonary Vein, LSPV Left Superior Pulmonary Vein, LA: Left Atrium as well as LPA Left Pulmonary Artery is isolated. * Vent, ** artery cannula

Cardiopulmonary assessment. Cardiopulmonary function assessment consisted of the following measurements: heart rate, cardiac output by thermodilution, pulmonary artery pressure, wedge capillary pressure, central venous pressure, arterial pressure, continues SVO2 measurement, arterial blood gases, urine output [3,7,11].

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Subsequently the arterio-alveolar oxygen difference (AaDO2 ) and the pulmonary vascular resistance (PVR) were calculated according to the following formulas: Effective AaD(>2 : Fio2 x (Pbar - P H2o ) - Paco2 - Pao2 where Pbar is barometric pressure, P H2o is partial pressure of water vapour, Fio2 is inspiratory oxygen fraction, and Paoi and Paco2 are arterial partial pressures for O2 and CO2 ). PVR: (MPAP - LAP)/CO x 80 where MPAP is pulmonary artery mean pressure, LAP is left atrial pres-sure, and CO is cardiac output. [9] We also assessed static compliance (Cst) using the following formula: Cst =Vt / (PinsPexp) where Vt is tidal volume, Pins is pressure at inspiratory hold and Pexp is pressure at expiratory hold. The evaluation time points were at the start of the experiment, after completion of instrumentation and hilar preparation, 60, 120, and 180 minutes after ischemia and 60, 120. and 180 minutes after reperfusion. 3. Conclusion This experimental model can offer a steady and reproducible environment that could be used to assess the ischaemia and reperfusion injury without the potential interference of acute rejection mechanism of graft failure. In this model, various pharmaceuticals manipulations could be used to evaluate reperfusion injury in lung transplantation with the view to reducing local and systemic complication[l,12]. References: 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12.

Meyers B, Patterson A. Lung transplantation: Current status and future prospects. World J. Surg. 23,1156-1162,1999 Palazzo R, Hamvas A, Shuman T, Kaiser L, Cooper J, Schuster DP. Injury in nonischemic lung after unilateral pulmonary ischemia with reperfusion. J Appl Physiol 1992; 72:612-20 Dougenis D, Tzorakoelefterakis E., KS Filos, et al. Experimental single lung transplantation in pigs. Intern J Artif Organ 1992; 15(9): 533 Koletsis E, Melachrinou M, Filos K, Dougenis D, Androulakis J. Experimental Lung Transplantation. Initial results. 3rd Pan-Hellenic Scientific Medical Students Congress; Thessaloniki March 1993 Dougenis D, Tzorakoelefterakis E, Filos K, Goudas L, Melachrinou M, Moschos S, Kyriakopoulou T, Poulopoulou M, Koletsis E, Androulakis J. Single lung transplantation: An experimental study in pigs. Acta Chirurgica Hellenica 1993; 65(1): 50-6 Novick RJ, Gehman KE, Ali IS, Lee J. Lung preservation: the importance of endothelial and alveolar type II cell integrity. Ann Thorac Surg 1996; 62:302-14. Novick RJ, Menkis AH, McKenzie FN. New trends in lung preservation: a collective review. J Heart Lung Transplant 1992; 11:377 King CR, Binns O, Rodriguez F et al. Reperfusion injury significantly impacts clinical outcome after pulmonary transplantation. Ann. Thorac Surg 2000; 69:1681-5 Swindell MM, Anesthetic and perioperative techniques in swine. Wilmington, MA: Charles River Laboratories, CRL Tech. Bull, (winter) 1991 Merin RG, Verdouw PD, Jong JW. Myocardial functional and metabolic responses to ischemia in swine during halothane and fentanyl anesthesia. Anesthesiology 56:84-92,1982 Filos K, Dougenis D, et al. The prognostic value of continuous SvO2 monitoring by fiberoptic oxymetry in single lung transplantation. A pilot study. Intens Care Med 1992; 18 (suppl): 117. Cooper JV, Vreim CE. Biology of lung preservation for transplantation. Am Rev Resp Dis 1992; 146:803

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub {Eds.) IOS Press, 2002

Pathogenetic Mechanisms Leading to Obliterative Bronchiolitis Erkki A. KALLIO*, Jussi M. TIKKANEN*, Petri K. KOSKINEN*, Karl B. LEMSTR6M*, and Magdi YACOUBf ^Transplantation Laboratory, University of Helsinki and Helsinki University Central Hospital. Hartmaninkatu 3, FIN-00014 University of Helsinki and f Department ofCardiothoracic Surgery, Imperial College, Faculty of Medicine, Heart Science Centre, Harefield, United Kingdom

Abstract Obliterative bronchiolitis causes significant mortality and morbidity among lung transplant recipients. Obliterative bronchiolitis presents as gradual deterioration of graft function in the absence of any other cause such as infection. The clinical diagnosis of Obliterative bronchiolitis syndrome is based on decline in forced expiratory volume in 1 second. Obliterative bronchiolitis syndrome manifests as obstructive ventilatory defect, leading to shortness of breath, wheezing, and gradually to hypoxia, hypercapnia, and ultimately, to death. Pathologically, Obliterative bronchiolitis is characterized by dense eosinophilic hyaline fibrous plaques in the submucosa of small airways resulting in partial or complete luminal compromise of membranous and respiratory bronchioles. This bronchiolar scar tissue may be concentric or eccentric, and may extend through smooth muscle wall to peribronchial interstitium. Currently, Obliterative bronchiolitis is considered as a form of chronic rejection of lung allografts. Although clinical and experimental studies have provided significant insight into risk factors and pathophysiology of Obliterative bronchiolitis, the underlying pathogenetic mechanisms are largely unknown. In this communication, we review our experience of the pathogenesis of experimental Obliterative bronchiolitis in rat heterotopic tracheal allografts.

1. Rat heterotopic tracheal transplantation as a model for Obliterative bronchiolitis Rat heterotopic tracheal allografts develop similar histological changes as seen in human obliterative bronchiolitis (OB) [1]. These changes are not observed in syngeneic grafts, indicating that they are not due to ischemia or interruption of blood vessels and lymphatics. Tracheal epithelium strongly expresses major histocompatibility complex (MHC) class II enabling direct antigen presentation, also observed in human lung allografts developing OB [2, 3]. Submucosal and peritracheal inflammation resembles that seen in lymphocytic bronchitis/bronchiolitis (LBB) and acute cellular rejection [4], with submucosal inflammatory cell infiltrates, focal epithelial necrosis and neutrophil infiltration. There is progressive loss of normal respiratory epithelium, first replaced by cuboidal and squamous epithelium, progressing to complete loss of epithelium. Similar sequence of events has been observed also by others using heterotopic tracheal transplantation model [5, 6]. The inflammatory cell subsets in tracheal allografts are similar to those in human lung allografts

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consisting first of CD4+ T cells and later, in increasing numbers, of CD8+ T cells and macrophages, which have been suggested to be required for the development of OB [7]. Epithelial injury and airway inflammation lead to ingrowth of granulation tissue consisting of mononuclear inflammatory cells and intense proliferation of a-smooth muscle actin positive myofibroblast-like cells causing gradual occlusion of airway lumen. Airway inflammation and epithelial injury have been suggested to initiate the development of OB in human lung allografts [8, 9] and prevention of epithelial injury has been shown to ameliorate OB in experimental models [10]. Heteropic tracheal transplantation may be considered as a model for human OB which has its benefits, but also limitations. Tracheal transplantation is technically simple and reproducible. In addition, no baseline immunosuppression is required, making it possible to investigate different drugs without drug interactions. On the other hand, the anatomy of trachea and bronchioli differ considerably, and pathological changes of OB seen in bronchioli are not observed in large airways in human. There is no airflow, which may affect epithelial function and pathology. The blood supply comes from systemic circulation, in contrast to lung allografts which receive their blood mainly from pulmonary circulation. Further, tracheal allograft is not a vascularized organ, but is revascularized as capillary network infiltrates the graft, making it difficult to investigate the very early events associated with ischemia and reperfusion, and possibly affecting the infiltration of inflammatory cells. The immunogenecity of tracheal allografts may be lower than in orthotopic lung transplants as tracheal allografts contain less lymphoid tissue. On the other hand, tracheal allografts develop obliterative changes in accelerated fashion. 2. Alloimmune response causes graft injury and the development of OB Cyclosporine A (CsA) inhibits the development of experimental OB in dose-dependent fashion, and inhibition of OB correlates with CsA trough levels [11, 12]. Similar results have been obtained in other experimental studies using mouse and rat tracheal allografts [13, 14]. Also other immunosuppressive drugs which block the proximal events leading to acute rejection, i.e., IL-2R activation and cytokine transcription, such as tacrolimus, leflunomide, and rapamycin, have been effective in inhibition of experimental OB [13-15], demonstrating that inhibition of alloimmune induced graft injury prevents the development of experimental OB. However, the level of immunosuppresssion needed to prevent experimental OB has been relatively high, and drugs affecting later steps of alloimmune response, such as mycophenolate mofetil and 15-deoxyspergualin, have been relatively ineffective in preventing OB. Role of the early events of alloimmune activation in the development of OB, is further emphasized by the effect of blockade of T cell costimulation. Blockade of CD28/B7-1 and CD28/B7-2 T cell costimulatory pathways with CTLA4Ig fusion protein reduces intragraft expression of tumor necrosis factor (TNF)-ct, interleukin (IL)-2, and interferon (IFN)-y as well as epithelial injury and graft occlusion [16]. Interestingly, selective blockade of CD28/B7-1 pathway does not affect graft cytokine profiles or the development of experimental OB [16]. Clinical studies demonstrate that rejection, especially multiple acute rejection episodes, late acute rejection episodes, and LBB are most important risk factors for OB and bronchiolitis obliterans syndrome (BOS) [17-19]. In addition, low level of immunosuppression has been associated with the development of BOS [19]. Improved immunosuppression with new immunosuppressive drugs has delayed the onset of BOS and

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augmentation of immunosuppression stops or delays the progression of BOS [20, 21]. Taken together, both clinical and experimental studies demonstrate the important role of alloimmune induced graft injury in the pathogenesis of OB, and that sufficient immunosuppression may partially prevent or delay the development of OB.

3. Cytomegalovirus infection enhances the development of OB Cytomegalovirus (CMV) infection has been identified as a risk factor for OB in man [18, 22, 23], In our studies, both chronic (latent) and acute recipient rat Cytomegalovirus (RCMV) infection significantly enhanced the development of experimental OB. Enhancing effect of RCMV infection has been demonstrated also in aortic, heart, and kidney allograft models of chronic rejection [24-26] and OB [27]. CMV may enhance OB by several mechanisms. CMV pneumonitis may contribute to epithelial injury and thereby enhance the development of OB. However, CMV seropositivity, without clinical infection, has also been associated with enhanced OB [22]. It is likely that CMV infection modulates the alloimmune response to facilitate chronic rejection and OB. In vitro studies have demonstrated that CMV infection directly induces MHC class I expression [28, 29]. CMV encodes a protein with sequence homology and cross-reactivity with MHC class II. Expression of MHC class II may also be indirectly induced by CMV activated CD4+ T cells [30, 31]. CMV infection induces endothelial and epithelial ICAM-1 expression in vitro and in vivo [32, 33] and causes endothelialitis in allograft vascular wall [34]. In addition, CMV infection may upregulate IL-2 and IL-2 receptor gene expression thus inhibiting the effect of CsA [35, 36]. CMV may also induce TNF-ct [37] as well as IFN-y [38] expression by monocytes and macrophages. These events may enhance acute rejection, and thus facilitate OB development. On the other hand, cytokines, especially TNF-a, produced during acute rejection and released during antibody treatments, may activate latent CMV infection [39], promoting alloreactivity that leads to a vicious circle. In addition, CMV may directly induce mesenchymal cell proliferation by inhibiting cell growth suppressor genes, leading to manifestations of chronic rejection by inducing transcription of different growth factors and inactivating growth suppressors [4042]. In rat tracheal allografts, both acute and latent rat CMV infection enhances the development of experimental OB. RCMV infection induces epithelial MHC class II expression, the number of graft infiltrating CD4+ T cells, and macrophages, as well as alloimmune activation of graft infiltrating inflammatory cells. RCMV infection upregulates epithelial platelet-derived growth factor (PDGF)-AA and alfa-receptor expression [43]. Increased inflammation leads to epithelial injury, inducesing the reparative prosesses culminating to myofibroproliferation and enhanced graft occlusion. In RCMV-infected grafts, treated prophylactically with ganciclovir or anti-CMV hyperimmune serum, the degree of obliteration is similar to that of non-infected controls [44]. On the other hand, anti-RCMV treatment, initiated a few days after infection, is not effective in inhibiting the RCMV-enhanced experimental OB. Interestingly, increased immunosuppression is similarly effective in inhibiting RCMV-enhanced OB in rat tracheal allografts. Our observations indicate, that in order to prevent the enhancing effects of CMV infection on the development of OB, CMV infection should be treated prophylactically, or at least preemptively. In addition, CMV infection is associated with induction of various proinflammatory cytokines, which may be prevented by increasing the level of immunosuppression during CMV infection [43, 44].


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The effect of CMV on the development of OB may be summarized as bidirectional and biphasic. Bidirectional because CMV infection augments alloimmune responses to the graft leading to OB, and, on the other hand, alloimmune activation may activate latent CMV infection. Biphasic because CMV infection affects alloimmune responses in both acute and chronic rejection [45]. 4. Protective role of nitric oxide in the pathogenesis of OB Nitric oxide (NO) may have different roles in various stages of transplantation in regard of pathophysiological effects and therapeutic implications. During reperfusion NO functions as a scavenger of free radicals [46], and NO supplementation improves graft preservation [47, 48], In addition, inhaled NO has been effective in treatment of early graft dysfunction after lung transplantation [49]. We investigated the role of NO in the pathogenesis of OB, by treating tracheal allograft recipients with either a inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine, or by supplementing NO production with L-arginine, the substrate for iNOS [50]. We observed, that epithelial iNOS expression and NO production were decreased during the development of OB. Inhibition of NO production enhanced graft inflammation and occlusion, whereas suplementation of NO induced a switch towards Th2 type immune response and reduced graft obliteration [50]. During acute rejection, macrophage- and T cell-derived cytokines, such as TNF-a, IL-1, IL-2, and IFN-y may induce iNOS expression in macrophages producing of large quantities of NO. Macrophage-derived NO and superoxide anion (O2-) may form peroxynitrite anion (ONOO-), a free radical, which may cause tissue damage [46]. Myocyte death by apoptosis in acute cardiac allograft rejection has been associated with induction of iNOS expression and NO production [51 ] and inhibition of NO improves rat cardiac allograft survival by downregulation of apoptosis [52]. Inhibition of iNOS with aminoguanidine ameliorates acute lung allograft rejection in the rat [53]. Functional studies using iNOS knockout mice heart transplantation model show that reduced NO production inhibits inflammation and cell injury during acute rejection [54] suggesting a damaging role for NO in acute rejection. In chronic rejection, iNOS is upregulated in rat [55] and human [56] cardiac allografts, and rat aortic allografts [57]. In human lung allografts, induction of iNOS and peroxynitrite are associated with epithelial damage and the development of OB [58]. Similarly in our study, iNOS expression was induced, but NO production was reduced by the loss of continuously expressed epithelial iNOS as epithelium was damaged during the development of experimental OB. Our results demonstrate, that inhibition of iNOS accelerates and supplementation of iNOS inhibits the development of OB [50]. In rat aortic allografts, inhibition of NO production accelerated allograft arteriosclerosis [59], whereas, transduction of iNOS expression with adenoviral vector suppressed the development of allograft arteriosclerosis [59]. In iNOS knockout mice, lack of iNOS enhanced the development of chronic cardiac allograft rejection [54]. In addition, supplementation of iNOS pathway with L-arginine prevented allograft arteriosclerosis in rabbit heart allografts [60]. NO may modulate immune responses and graft homeostasis in several ways that may downregulate OB. NO inhibits leukocyte adhesion [61] and platelet aggregation [62]. NO prevents proliferation Thl cells, but not Th2 cells [63]. We observed a switch from Thl towards Th2 type alloimmune response with inhibition of IL-2 expression and induction of IL-10 expression in rat tracheal allografts [50]. Th2 type responses have been associated

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with enhanced graft survival and inhibition of experimental OB [64, 65]. L-arginine treatment inhibited myofibroproliferation, which may be mediated by NO directly by increasing intracellular cyclic-GMP levels [66, 67], and indirectly by inhibiting growth factor release [68, 69]. Further, NO induces apoptosis of smooth muscle cells, which may reduce proliferative responses in chronic rejection [70], In conclusion, NO may have different roles in acute and chronic rejection. While NO behaves as a cytotoxic effector molecule contributing to graft injury during acute rejection, it seems to be protective in chronic rejection and OB.

5. Inhibition of complement activation prevents epithelial injury and graft occlusion Our studies demonstrate that complement activation takes place during the development of experimental OB, and that inhibition of complement activation with human recombinant soluble complement receptor type 1 (sCRl) reduces epithelial injury and myofibroproliferation, leading to ameloriation of OB. In the transplant, complement activation may occur via both classical and alternative pathways [71]. Complement mediates much of the tissue damage occuring in ischemia-reperfusion injury [72, 73]. In addition to early complement induced graft injury leading to enhancement of reparative processes and myofibroproliferation, complement components may facilitate chronic rejection and OB by several other mechanisms. Complement may enhance antigen presentation and T cell proliferation promoting cell-mediated rejection [74, 75]. Complement activation components increase leukocyte chemotaxis by upregulating expression of endothelial adhesion molecules [76, 77] and production of chemoattractants monocyte chemoattractant protein, macrophage inflammatory protein-la and IL-8 by monocytes and macrophages [78, 79]. In addition, complement may induce release of IL-1 and TNF-a from monocytes and macrophages [80, 81], and induce release of bFGF and PDGF from endothelial cells [82]. All these factors have been implemented to contribute to alloimmune induced graft injury ultimately leading to OB. Complement receptor type 1 (CR1) is an endogenous regulatory protein of complement, which inhibits C3 and C5 convertases, thus inhibiting activation of both classical and alternative pathways [83]. Recombinant human soluble CR1 (sCRl) has the same capacity to inhibit complement activation as CR1 [84]. sCRl effectively protects against complement mediated tissue damage in ischemia/reperfusion injury as well as in immune complex, thermally and cobra venom factor induced injury models [84, 85]. In addition, sCRl effectively inhibits hyperacute rejection in xenograft models [86] and in allografts [87], as well as vascular injury and inflammation in acute rat renal allograft rejection [88]. Although complement is an important mediator of acute lung injury [73], little data exists on its role in acute lung allograft rejection or OB. In our study, sCRl-treatment reduced epithelial injury, inflammatory cell proliferation, and graft occlusion, that are the hallmarks of OB. sCRl inhibited ICAM-1 and IL-8 expression and neutrophil infiltration, previously associated with the development of OB [89]. In addition, sCRl upregulated IL10 expression, which was recently shown to prevent experimental OB [65]. 6. Regulatory role of platelet-derived growth factor in the pathogenesis of OB In our study, platelet-derived growth factor (PDGF)-AA and platelet-derived growth factor receptor-a (PDGF-Ra) were identified as key regulatory molecules in the

E.A. Kallio et al. / Ohliterative Bronchiolitis

pathogenesis of experimental OB. Upregulation of PDGF-AA, PDGF-Ra, and PDGF-Rp protein expression was demonstrated during the early phases of the development of OB, while PDGF-BB expression was downregulated in allografts compared to syngeneic grafts. Inhibition of signaling downstream of PDGF receptors with CGP 53716, protein-tyrosine kinase inhibitor selective for PDGF receptors [90], markedly inhibited the development of experimental OB. In human lung allografts with OB, increased concentrations of PDGF in BAL fluid, upregulation of PDGF-AB protein expression in epithelial cells and mononuclear cells, and PDGF-B mRNA expression in macrophages have been observed [91]. Locally applied PDGF induces OB-like changes in heterotopic tracheal syngeneic grafts [92]. In experimental models of lung fibrosis and inflammation, overexpression of PDGF-B gene increases fibroproliferation and collagen deposition [93] and exogenous PDGF-BB induces mesenchymal cell and epithelial cell proliferation [94]. In vitro, PDGF-BB and -AB are more potent chemoattractants and mitogens for non-stimulated lung fibroblasts than PDGFAA [95, 96]. However, fibroblasts stimulated with cytokines such as IL-1|3 and TNF-a, express more PDGF-AA and PDGF-Ra [97, 98]. Expression of PDGF-Ra, to which PDGF-AA binds, appears to be required for maximal chemotaxis and proliferation of lung fibroblasts [99]. In this study, upregulation of PDGF-AA and -Ra occured concomitantly with the peak of inflammation and myofibroproliferation. A specific role for cytokine activated expression of long chain PDGF-AA, which is required for full activation of PDGF-Ra protein-tyrosine kinase, has been suggested in the pathogenesis of chronic heart allograft rejection in human [100]. These findings suggest that although PDGF-BB may have a significant role in normal physiology and in the pathogenesis of several fibroproliferative disorders, PDGF-AA and PDGF-Ra may be more important in mediating myofibroproliferation in chronic rejection and OB. Inhibition of signal transmission of PDGF-R by CGP 53716 effectively attenuated neointimal formation in carotid denudation model of atherosclerosis [101] and chronic rejection in rat heart allografts [102]. In our study, the compound inhibited myofibroproliferation and airway occlusion but had no effect on airway wall inflammation indicating that it is not immunosuppressive, but rather that its effects are mediated by inhibition of myofibroproliferation. However, the inhibition of OB by CGP 53716 was not complete, indicating that also other growth factors in addition to PDGF, operate in the disease process. 7. "Response-to-injury" hypothesis of the pathogenesis of obliterative bronchiolitis Accumulating body of evidence derived from both clinical and experimental studies support the the hypothesis that epithelial injury and airway wall inflammatory responses activate reparative processes leading to OB. Graft injury is initiated already at donor brain death, and enhanced by ischemia and reperfusion, where neutrophils, free radicals, and complement cause the tissue damage. Acute rejection, mediated by CTLs, T helper cells and macrophages, augmented by antibody induced responses and NK cells, further damages epithelium. CMV infection may contribute to the development of OB by inducing epithelial injury and promoting acute rejection. As acute rejection subsides, alloimmune activation may be maintained by activated T cells and macrophages. Macrophages release cytokines and growth factors which induce myofibroproliferation. Graft injury and alloimmune activation induce growth factor release in several cell types in the graft. Growth factors released by epithelial cells, endothelial cells, smooth muscle cells, and fibroblasts may act

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in autocrine and paracrine fashion and induce phenotypic changes in fibroblasts favoring proliferation. Growth factors, especially PDGF, enhance migration and proliferation of myofibroblasts leading to gradual occlusion of the airway lumen. Our studies suggests several sites for intervention in order to prevent the development of OB, starting with reducing early graft injury caused by reperfusion and acute rejection, further with diminishing the constant alloimmune activation and inflammation by effective immunosuppression and CMV prophylaxis, and finally by inhibiting mesenchymal cell proliferation.

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[81] R. L. Kaspar, and L. Gehrke. 1994. Peripheral blood mononuclear cells stimulated with C5a or lipopolysaccharide to synthesize equivalent levels of IL-lp mRNA show unequal IL-1P protein accumulation but similar polyribosome profiles. J. Immunol. 153:277-286. [82] L. R. Benzaquen, A. Nicholson-Weller, and J. A. Halperin. 1994. Terminal complement proteins C5b-9 release basic fibroblast growth factor and platelet-derived growth factor from endothelial cells. J. Exp. Med. 179:985-992. [83] B. P. Morgan. 1995. Complement regulatory molecules: application to therapy and transplantation. Immunol. Today. 16:257-259. [84] H. F. Weisman, T. Bartow, M. K. Leppo, et al. 1990. Soluble human complement receptor type 1: In vivo inhibitor of complement suppressing post ischemic myocardial inflammation and necrosis. Science. 249:146-151. [85] M. S. Mulligan, C. G. Yeh, A. R. Rudolph, et al. 1992. Protective effects of soluble CR1 in complementand neutrophil-mediated tissue injury. J. Immunol. 148:1479-1485. [86] S. K. Pruirt, W. M. Baldwin III, H. C. Marsh Jr., et al. 1991. The effect of soluble complement receptor type 1 on hyperacute xenograft rejection. Transplantation. 52:868-873. [87] S. K. Pruitt, and R. R. Bellinger. 1991. The effect of soluble complement receptor type 1 on hyperacute allograft rejection. J. Surg. Res. 50:350-355. [88] J. R. Pratt, M. J. Hibbs, A. J. Laver, et al. 1996. Effects of complement inhibition with soluble complement receptor-1 on vascular injury and inflammation during acute renal allogarft rejection in the rat. Am. J. Pathol. 149:2055-2066. [89] B. DiGiovine, J. P. Lynch III, F. J. Martinez, et al. 1996. Bronchoalveolar lavage neutrophilia is associated with obliterative bronchiolitis after lung transplantation. Role of IL-8. J. Immunol. 157:4194-4202. [90] E. Buchdunger, J. Zimmermann, H. Mett, et al. 1995. Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class. Proc. Natl. Acad. Sci. USA. 92:2558-2562. [91] M. I. Hertz, C. A. Henke, R. E. Nakhleh, et al. 1992. Obliterative bronchiolitis after lung transplantation: a fibroproliferative disorder associated with platelet derived growth factor. Proc. Natl. Acad. Sci USA. 89:10385-10389. [92] G. A. Al-Dossari, J. Jessurun, R. M. Bolman III, et al. 1995. Pathogenesis of obliterative bronchiolitis: Possible role of platelet derived growth factor and basic fibroblast growth factor. Transplantation. 59:143145. [93] M. Yoshida, J. Sakuma, S. Hayashi, et al. 1995. A histologically distinctive interstitial pneumonia induced by overexpression of the interleukin 6, transforming growth factor pi, or platelet-derived growth factor B gene. Proc. Natl. Acad. Sci. USA. 92:9570-9574. [94] E. S. Yi, H. Lee, S. Yin, et al. 1996. Platelet-derived growth factor causes pulmonary cell proliferation and collagen deposition in vivo. Am. J. Pathol. 149:539-548. [95] J. C. Bonner, A. R. Osornio-Vargas, A. Badgett, et al. 1991. Differential proliferation of rat lung fibroblasts induced by the platelet-derived growth factor-AA, -AB, and -BB isoforms secreted by rat alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 5:539-547. [96] E. R. Osornio-Vargas, A. L. Goodell, N. A. Hernandez-Rodriguez, et al. 1995. Platelet-derived growth factor (PDGF)-AA, -AB, and -BB induce differential chemotaxis of early passage rat lung fibroblasts in vitro. Am. J. Respir. Cell Mol. Biol. 12:33-40. [97] E. J. Battegay, E. W. Raines, T. Colbert, et al. 1995. TNF-a stimulation of fibroblast proliferation. Dependence on platelet-derived growth factor (PDGF) secretion and alteration of PDGF receptor expression. J. Immunol. 154:6040-6047. [98] P. M. Lindroos, P. G. Coin, E. R. Osornio-Vargas, et al. 1995. Interleukin-lp (IL-lp) and the IL-lp-a2macroglobulin complex upregulate the platelet-derived growth factor a-receptor on rat pulmonary fibroblasts. Am. J. Respir. Cell Mol. Biol. 13:455-465.


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Part V. ARDS and Oxidative Stress



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Clinical Pathology of ARDS Stylianos E. ORFANOS, Antonia KOUTSOUKOU, Irene MAVROMMATI, Ekaterini PSEVDI, loanna KOROVESI, Anastasia KOTANIDOU, and Chans ROUSSOS Department of Critical Care and Pulmonary Medicine, University of Athens Medical School, Evangelismos Hospital, Athens, Greece Abstract. Acute lung injury (ALI) is an acute, diffuse and severe alteration of lung structure and function that occurs after exposure to noxious endogenous or exogenous agents, ALI represents a pathologic continuum characterized by impairment of arterial oxygenation and diffuse chest x-ray abnormalities. The most severe extreme of this continuum is the acute respiratory distress syndrome (ARDS) an overt non-cardiogenic pulmonary edema that carries high morbidity and mortality. ALI/ARDS is associated with a variety of etiologies and, although its pathogenesis is still partly understood, pulmonary endothelium appears to play a major role in the syndrome development. Numerous biological markers of ARDS have been investigated in an effort to identify pathogenic and prognostic significance. Among them, pulmonary endothelium-bound angiotensin converting enzyme activity, assessed at the bedside by means of indicatordilution techniques: i. offers a direct and quantifiable index of pulmonary endothelial dysfunction, ii. is altered early in ALI, and iii. correlates with the severity of lung injury. ARDS treatment remains mainly supportive, with positive-pressure mechanical ventilation always playing a major role in the treating process. Studies on lung mechanics have offered new insights into the clinical pathology of the syndrome, allowing the clinician to apply appropriate ventilatory management in an effort to increase survival.

1, Definitions Acute lung injury (ALI) is a pathologic continuum characterized by acute respiratory distress, severe impairment of oxygenation, and noncardiogenic pulmonary edema. ALI varies in severity and acute respiratory distress syndrome (ARDS) is a term applied to patients with more severe manifestations of ALI. Both terms are used to reflect a relatively specific form of lung injury to the lung occurring from a wide variety of causes or associated conditions. This acute injury involves the alveolar epithelium, the alveolar capillary endothelium and the pulmonary interstitium, and it is caused by an acute inflammatory response, usually with a tremendous influx of neutrophils, leading to a breakdown of the lung barrier and gas exchange functions. Initially this results in flooding of the alveolar spaces with protein-rich edema fluid, leading to severe gas exchange and lung compliance abnormalities. If the process is sustained, fibroproliferation occurs with collagen deposition and lung remodeling. ALI and ARDS are defined in terms of the associated clinical, physiologic, and radiologic manifestations. The current definitions are those introduced by the AmericanEuropean Consensus Conference (AECC) on ARDS published in 1994 [1]. This international group limited the criteria for ALI to: i. oxygenation abnormality with an

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arterial partial pressure of oxygen to inspired oxygen fraction ratio (PaO2/FiO2) < 300 mm Hg, ii. bilateral opacities on chest radiograph, and iii. pulmonary arterial occlusion pressure (PAOP) < 18 mm Hg, or no clinical evidence of left heart failure. An additional criterion is the acute onset of the syndrome. ARDS is the most severe part of ALI, with PaO2/FiO2 being < 200 mm Hg. 2. Epidemiology The incidence of ALI/ARDS is not clear. A National Institutes of Health (NIH) panel in 1972 estimated the incidence of ARDS to be approximately 75cases/100000 population/year [2]. This number has been widely used since that time, without confirmation from epidemiological studies. Data on the incidence of either ARDS or ALI using AECC criteria have only recently been published. One such study screened patients for an 8-week period in most intensive care units in Sweden, Denmark and Iceland, and identified incidences of 17.9 cases/100000 population/year for ALI and 13.Scases/100000 population/year for ARDS [3]. In contrast, a 3-year study in one center in Finland, using the AECC definition for ARDS plus the requirement of a known triggering cause, found an incidence of 4.9 cases ofARDS per 100000 population per year [4]. Clinical risk factors can be categorized mechanistically as causing either direct (primary) or indirect (secondary) injury. The latter refers to an extrapulmonary injury that affects the lungs through activation of systemic inflammatory pathways, mainly cytokines and other biochemical and cellular mediators [1,5]. Direct risk factors include pneumonia, aspiration of gastric contents, pulmonary contusion, smoke inhalation, and near drowning. Indirect risk factors include sepsis, trauma, pancreatitis, multiple transfusions, and shock. Factors shown to increase ARDS risk, following predisposing conditions include: age, severity of illness, cigarette smoking, chronic alcohol abuse, and combination of risk factors [6]. Attempts to predict which patients at risk will develop ARDS are mostly based on biochemical measurements in blood or bronchoalveolar lavage (BAL) fluid. Obtaining BAL fluid in all patients at risk for ARDS, in order to identify those with early or mild lung injury, is not a practical approach and is limited essentially to research studies. Measurement of blood biochemical markers, including cytokines reputedly associated with lung injury, has not been very successful. The prevalence of any given risk condition varies considerably by geography, and by the clinical population seen at a given institution. In general however sepsis is the commonest, with aspiration of gastric contents being relatively common, and trauma less common but still important. Diffuse pneumonia also appears to be a relatively common risk condition, regardless of being classified as pneumonia or as sepsis originating from the lungs [6]. Sepsis as a risk for ARDS is generally associated with a considerably higher mortality rate than most other common risks, including trauma and aspiration of gastric contents [5]. Older patients (> 65 years of age), have an increased mortality rate when compared to younger patients [7]. ARDS severity at the time of first diagnosis, given by the degree of hypoxemia, has not been constantly associated with poor outcome, except perhaps at extreme abnormalities [8]. Montgomery et al [9] found that patients dying with ARDS appeared to die primarily of multiple organ failure and sepsis, rather than respiratory causes such as hypoxia or uncontrollable respiratory acidosis. Recent studies have confirmed the above observations [4,10]. Through most of the 1980s, ARDS-related mortality was in the range of 60% or higher. Since that time, mortality appears to be decreased. Abel et al [11] found that mortality in the years 1993-96 was decreased as compared to 1990-93 (30 to 60%. respectively). More recent studies have reported ARDS mortalities of 37% and 41.2% [12]. The exact reasons for this improvement in survival remain unclear, but they may relate to


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improved treatment of the underlying cause(s) and improved supportive care of patients with ALI/ARDS; the latter includes changes in ventilatory management. 3. Pathogenesis ofARDS The pathophysiologic hallmark of ALI/ARDS is a progressive injury of the alveolar-capillary membrane that leads to increased vascular permeability and pulmonary edema. Although the pathogenesis of ALI/ARDS is still partly understood, extensive basic and clinical research performed during the last 25 years revealed the existence of an inflammatory syndrome, where numerous mediators and cell types participate in multiple cascades that interact with each other, either promoting or suppressing inflammation. The way this "inflammation-balance" will finally move, will determine patients' outcome. A detailed analysis of all the known pathogenic mechanisms is not within the scope of this chapter. Briefly, pro-inflammatory cytokines are released following pulmonary and/or extra-pulmonary stimuli, such as endotoxin [13]. They amplify the inflammatory process by releasing additional mediators from macrophages and other cell-types, and by activating the complement system, neutrophils and other blood components that aggregate in the pulmonary circulation. Activated neutrophils adhere to the pulmonary endothelium and release proteases and oxidative products that injure the vascular wall and increase permeability. The release of several vasoactive substances, lipids and peptides is either induced or altered, while the endothelial vascular layer becomes thrombogenic. Ongoing endothelial injury appears to be a key component of the pathogenic process. Studying ALI/ARDS pathogenesis has an additional important role: identify potential biological markers that could predict either ARDS development in high-risk patients, or outcome from the already established syndrome. Such markers would allow better triage and better patient care.

4. Inflammatory mediators Among the earliest events of the inflammatory process is the release of tumor necrosis factor-alpha (TNF-a), interleukin-1 (IL-1), as well as interleukins-6 and -8 (IL-6, IL-8), following pulmonary or extra-pulmonary insults. All these mediators are involved in inflammation, their production and release are stimulated by multiple relevant mediators, including endotoxin [14], and they are regulated by nuclear factor kappa B (NFicB) [15]. They have all been shown to be implicated in the development of ALI in animal models, and they are all present in patients at risk for and with established ARDS. TNF-a was the first cytokine to be extensively studied in patients at risk for and with ARDS. TNF-a is increased in BAL and pulmonary edema fluid from patients with ARDS, but it is not specific for ARDS and does not correlate with morbidity and mortality [16]. The likely source of TNF-a in the lung is the alveolar macrophage, although there could be leak from the circulation as well. IL-1, IL-6, and IL-8 have been measured in both the circulation and lung fluid from patients at risk for and with ARDS. IL-1 levels are increased in the BAL fluid obtained from patients with ARDS [17]. Alveolar macrophages from these patients release higher IL-1 levels, both at the baseline and following lipopolysaccharide (LPS) stimulation, than alveolar macrophages obtained from either normal subjects or patients with other lung diseases [18]. There is evidence that IL-1 likely accounts for the majority of lung proinflammatory cytokine activity in ARDS [19]. Both TNF-a and IL-1 stimulate the production of IL-8, which is a potent neutrophil chemoattractant. In light of its chemoattractant activity, it is likely that IL-8 contributes more to the acute inflammatory

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process in the lung than in the circulation. IL-8 levels are increased in both BAL and pulmonary edema fluid in ARDS [19] and correlate with neutrophil concentrations within the lung. This supports further the hypothesis that IL-8 contributes to ALI, at least in part, through its neutrophil chemoattractant capacity. IL-8 levels are higher in the lungs of patients at risk who ultimately develop ARDS [20], while higher IL-8 levels may be associated with higher mortality [21]. IL-6 has both pro-inflammatory and anti-inflammatory properties [22]. Plasma IL-6 appears to be the best predictor of ARDS morbidity and mortality, from all the proinflammatory cytokines measured to date. Several studies have found that IL-6 levels are significantly increased in patients at risk for ARDS, while they are higher and persist longer in patients who die [23]. However, there is no specific IL-6 level that predicts which patient will die. Consequently, measuring IL-6 is more likely to be useful in identifying groups of patients at high risk for mortality. Recent investigations have focused on the role of a more proximal common regulatory factor, namely NFicB. NFicB is the regulatory transcription factor for TNF-a, IL1, IL-6, and IL-8, as well as for many other protein compounds that could be involved in ARDS development [15]. In humans, NFicB activation is significantly increased in alveolar macrophages obtained from patients with ARDS, as compared to macrophages of patients without lung injury [24]. Further studies should determine the role of NF-icB in ALI/ARDS development, and its utility as a target for novel therapeutic options.

5. Neutrophils The focus on the role of neutrophils in ALI arose from early observations that in ARDS, neutrophils accumulate in the airspaces and the lung interstitium [25]. In addition, BAL fluid from these patients is characterized by neutrophilia [26]. Moreover, several studies suggest that circulating neutrophils are activated in ARDS [27], while after ARDS has been developed the severity of lung injury, estimated by gas exchange abnormalities, correlates with the extent of neutrophil influx into the airspaces [28]. In some patients, the persistence of the initial neutrophilic inflammatory response is associated with higher mortality [29]. Neutrophils injure the lung by at least two potential mechanisms: the release of proteases (elastase, collagenase, gelatinase), and the production of reactive oxygen species (hydrogen peroxide, hydroxyl radicals and superoxide anions) [30]. Neutrophil adherence to endothelium is thought to be an early event that leads to subsequent infiltration of lung parenchyma. Several cell surface adhesion molecules are involved in neutrophil adhesion and migration into the extravascular spaces [31]. Such molecules include the selectin family, which mediates the rolling of neutrophils on endothelial cells, and the beta 2 integrins (GDI 1/CD18) that mediate the firm adherence to the endothelium via interaction with endothelial ligands such as intercellular adhesion molecule-1 (ICAM-1) [31]. The mechanisms for regulating neutrophil sequestration and adherence to the lung are somewhat different from those reported in the systemic circulation, and include physical factors such as neutrophil deformability, as well as several specific adhesion molecules [32]. 6. Pulmonary Endothelium Pulmonary endothelium possesses numerous physiologic and pharmacokinetic functions. These functions may be altered early in ALI and further contribute to ARDS development, mainly through the promotion of cell-cell adhesion and endothelial

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permeability, and through endothelial metabolic alterations [33]. The pulmonary endothelium is a major metabolic organ that synthesizes, releases and degrades vasoactive peptides, and possesses antithrombogenic-thrombolytic activities [34]. Ectoenzymes responsible for many of these functions are located on the luminal endothelial cell surface, with their catalytic sites exposed to the blood stream; they are directly accessible to bloodborne substrates, and their activities may be measured in vivo by means of indicatordilution techniques [35]. One such ectoenzyme is angiotensin converting enzyme (ACE) [36]. Several investigations on endothelium-related mediators of ALI/ARDS have provided insights into the pathogenesis of the syndrome, and have examined potential predictors of either ARDS development or outcome [33]. In this respect, Morel et al, [37] have shown that the pulmonary endothelium-mediated extraction of serotonin in humans with ARDS correlates with the severity of the syndrome, while pulmonary propranolol extraction is decreased in patients at risk. More recently, the net balance between pulmonary clearance and release of endothelin-1 was found to be decreased early in ALL while it reversed in patients who recovered [38]. Other endothelium-related markers altered in patients with ALI/ARDS include plasma levels of von Willebrand factor antigen [39], soluble endothelial-derived adhesion molecules, such as E- and P-selectins and ICAM-1 [40,41], as well as plasma soluble ACE (sACE) activity [42]. Endothelial markers in plasma, however, are surrogate indices of endothelial function and may not be directly linked to pulmonary endothelial dysfunction. In addition, they have not been useful to predict ARDS development in multiple at-risk patients [33]. Consequently, the need for methods that will directly assess pulmonary endothelial function in humans is still present. Pulmonary capillary endothelium-bound (PCEB) ACE activity Estimating pulmonary endothelial ectoenzyme activity in vivo or in situ, by means of indicator-dilution techniques, offers direct assessment and quantification of endothelial function [34,35]. Applying indicator-dilution techniques we and others have studied pulmonary endothelium-bound ACE activity in different animal models, by measuring, under first order reaction conditions, the single pass transpulmonary hydrolysis of synthetic substrates highly specific for ACE [35,36]. The most widely used synthetic ACE substrate is benzoyl-Phe-Ala-Pro (BPAP), radiolabeled with 3H [36]. This technique allows estimations of very rapid interactions between substrate and the endothelium-bound enzyme, thus minimizing the contribution of sACE. Furthermore, ACE located on alveolar capillaries with a diameter < 20 um appears to be responsible for the great majority of the product formed, due to the very high local enzyme concentration. Consequently in this type of studies, monitoring pulmonary endothelial ACE activity is in practical terms equal to monitoring PCEB ACE activity [36]. Substrate hydrolysis is expressed as either percent metabolism (%M) or v (=enzyme concentration x capillary transit time x k^/K^), where kcat is the catalytic rate constant and K,n is the Michaelis-Menten constant [43]. The modified kinetic parameter A^/K™ (=enzyme mass x kcat/K,,,) may be additionally calculated [44]. Under normal conditions Amax/Km is an index of dynamically perfused capillary surface area (DPCSA) in animals [34,35,45] and humans [46,47], while under pathological conditions Amax/Km is an index of functional capillary surface area (PCS A) related to both enzyme mass (available for reaction) and functional integrity [46]. Both v and %M are reflections of ACE activity per capillary, while A^/K™ reflects ACE activity per vascular bed. An additional advantage of this method is that it may help distinguish between abnormalities secondary to endothelial dysfunction per se and decreased pulmonary vascular surface area [46,48].

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Figure 1. Top: PCEB-ACE activity expressed as BPAP hydrolysis (v) and A „„/!(,,, in 33 mechanicallyventilated patients belonging to high risk groups for ARDS development, divided in those who have no acute lung injury (NoALI) and those who have ALI/ARDS according to the American-European Consensus Criteria [1]; "p2 in EBC has been described in asthma, COPD, ARDS, CF and lung cancer and in healthy smokers. Levels of ^2®"! m ^BC were further increased in exacerbation of COPD and asthma and decreased after steroid treatment and long-term treatment with N-acetylcysteine in COPD patients. Products of lipid peroxidation (thiobarbituric acid -reactive substances and 8-isoprostane) have been increased in asthma, COPD and smokers. Nitrosothiols are elevated in asthma, CF and COPD and nitrotyrosine in asthma, CF and smokers. Increased total nitrite/nitrate levels have been described in asthma. As concentrations of markers of oxidative stress in EBC vary in the different airway diseases and increase with the severity of some and decrease during the treatment, their measurement may have some clinical relevance as a non-invasive biomarker of inflammation.

1. Oxidative stress Reactive oxygen species (ROS) are produced by several inflammatory and structural cells within the airways. An imbalance of oxidant/antioxidant in favour of oxidants, oxidative stress, has been implicated in the pathophysiology of a number of respiratory disorders such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) [1]. Activation of inflammatory cells such as eosinophils, neutrophils, and macrophages induces a respiratory burst resulting in the production of ROS. When liberated ROS cause tissue damage and cell death or apoptosis. Lung cells, in particular alveolar epithelial type II cells, are particularly susceptible to the injurious effects of oxidants. Therefore oxidative stress and overall inflammatory response are fundamental processes involved in the pathogenesis of many lung diseases [2]. New techniques such as exhaled breath condensate (EBC) might be utilized in detecting and assessing oxidative stress non-invasively and might offer an opportunity to gain insights into the local processes in the airways. EBC provides an easy to perform method to study the airways, without the need to undertake invasive procedures, such as bronchoscopy. It is very well tolerated by patients and no adverse events have been reported. Growing body of evidence suggests that it is a useful way to monitor markers of inflammation and oxidative stress in various respiratory tract diseases, such as asthma, COPD and cryptogenic fibrosing alveolitis.

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2. Markers of oxidative stress 2.1. H2O2 An important source of H2®2 1S me phagocytes. Activated neutrophils, eosinophils and macrophages generate Oi.", which is converted to H2Oz by superoxide dismutase. and hydroxyl radical, fonned non-ezymatically in the presence of Fe2*. ROS are highly reactive and, when close to cell membranes, oxidize membrane lipids (lipid peroxidation), which continue as a chain reaction [3]. This potentially leads to membrane damage by disruption of its function or cell death. An intense airway inflammation can be caused either by H2O2 alone or newly generated hydroxyl radical [3]. H2O2 is released in extracellular fluid and in the airways a part of H2C>2, which has not been decomposed by antioxidant enzymes, can be exhaled with exhaled breath. H2O2 is elevated in EBC in various inflammatory lung disorders such as asthma [4,5], cystic fibrosis (CF) [6], bronchiectasis [7], ARDS and acute hypoxemic respiratory failure [8], cigarette smoking [9], and COPD [10]. Cigarette smoking is a strong pro-inflammatory and prooxidant factor and therefore high levels of FhOi have been found in EBC from smokers compared to non-smoking subject [11]. Dohlman described an increased H2O2 level in EBC in a relatively small group of asthmatic children, mainly in those with acute disease, however elevated H2O2 in stable asthmatics has been also observed [12]. As postulated by Horvath and co-workers, measurement of H2O2 in EBC and exhaled NO in asthmatic patients provides complementary data for monitoring the disease activity [13]. Not only as a diagnostic test, but exhaled H2O2 may be used to guide anti-inflammatory treatment. Inhaled beclomethasone dipropionate in low dose has been shown to decrease H2O2 in EBC following a 2-week treatment [14]. This has also been observed in children with stable asthma, some of them receiving inhaled cortiosteroids in a daily regimen [5]. There was significant difference in median H2C»2 concentration between asthmatics without antiinflammatory treatment and healthy controls. A study in ARDS patients treated with corticosteroids showed a tendency towards lower levels of H2O2 in the expired air condensate as compared to steroid-naive ARDS patients [15]. In a recent study it has been observed that long-term treatment of N-acetylcysteine (600 mg daily) decreases FbO2 exhalation in subject with COPD [16]. Both, asthma and CF patients with an acute pulmonary exacerbation have abnormally high levels of exhaled FhOi, which decrease during antibiotic treatment [17]. 2.2. Products of lipid peroxidation 2. 2. 7. Thiobarbituric acid - reactive substances Measurement of thiobarbituric acid - reactive substances (TEARS) seems to be the most simple, but non-specific method to assess lipid peroxidation damage in tissue, cells and body fluids. Levels of TBARS are increased in exhaled breath condensate in asthma [4] and COPD [18] and they increase during exacerbations [17]. These results are consistent with those of Ignatova who found increased levels of conjugated dienes in exhaled breath condensate and bronchial biopsies from patients with COPD and simple chronic bronchitis [19].

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2.2.2. 8-isoprostane 8-isoprostane, a stable prostaglandin-like arachidonate product formed on membrane phospholipids by the action of reactive oxygen species is postulated to be a reliable biomarker of lipid peroxidation caused by oxygen reactive species and represents a quantitative measure of oxidant stress in vivo [20]. 8-isoprostane appears to reflect oxidative stress in EBC and is progressively increased with the severity of asthma and appears to be very high in aspirininduced asthma [21,22]. It is also reported to be increased in EBC in COPD patients [23] and further increases are observed with exacerbation of COPD [24]. There was also a positive correlation between exhaled H2O2 and 8-isoprostane levels, which might reflect the causeand-effect relationship (A. Antczak - not published data). 8-isoprostane has also been detected in EBC from patients with ARDS [25]. 3. Nitrogen-reactive species 3.1. Nitrotyrosine Nitrotyrosine formation in EBC may be a marker of oxidative stress in airways. The reaction of nitric oxide (NO) and superoxide anions in the airways leads to the formation of peroxynitrite, a highly reactive oxidant species. Peroxynitrite in reaction with tyrosine residues in proteins forms the stable product nitrotyrosine. Nitrotyrosine concentrations have been detected in EBC of normal subjects, and were increased significantly in patients with mild asthma [26]. However, the levels of nitrotyrosine in EBC have been lower in patients with moderate and severe asthma. Moreover, there was a significant correlation between nitrotyrosine in EBC and exhaled NO in patients with mild asthma [26]. Exhaled NO is decreased in cystic fibrosis (CF) patients, perhaps because it is metabolised to oxidative end products. 3-nitrotyrosine may indicate local formation of reactive nitrogen species. As shown by Balint and co-workers nitrotyrosine levels in EBC were increased significantly in stable CF patients, compared with normal subjects. There was an inverse correlation between the levels of nitrotyrosine and the severity of lung disease [27]. Production of nitrotyrosine may reflect increased formation of reactive nitrogen species such as peroxynitrite or direct nitration by granulocyte peroxidases, indicating increased oxidative stress in airways of cystic fibrosis patients [27]. 3.2. Nitrosothiols Nitrosothiols (RS-NOs) are formed by interaction of nitric oxide (NO) with glutathione and may limit the detrimental effect of NO. RS-NOs are detectable in EBC of healthy subjects and are increased in patients with inflammatory airway diseases. RS-NOs in EBC were higher in subjects with severe asthma compared with normal control subjects and with subjects with mild asthma [28]. Elevated RS-NOs were also found in CF patients and in smokers. In current smokers a correlation between RS-NOs values and smoking history (pack/year) was observed. As RS-NOs concentrations in EBC vary in the different airway diseases and increase with the severity of asthma, it is postulated that their measurement may have clinical relevance as a non-invasive biomarker of oxidative-nitrosative stress. 3.3. Nitrites/nitrates NO may be oxidized to nitrite (NO2) and nitrate (NOs), both of which are end products of NO metabolism. Total EBC NO2/NO3 concentrations were significantly higher in CF patients and

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smokers compared to healthy controls [29, 30]. They were also higher in patients with asthma than in normal subjects [31]. Moreover, patients who were on inhaled steroid therapy had significantly lower values compared to steroid-naive subjects. There was a significant positive correlation between NO2/NO3 levels and H2C>2 concentration in EBC [31]. Measurement of expired NC^/NOa levels and FÔz may be clinically useful in the management of oxidation and inflammation mediated lung injury. 4. Conclusions There is accumulating evidence that abnormalities in exhaled breath condensate composition may reflect biochemical changes of airway lining fluid. Detecting and monitoring of biomarkers in exhaled breath condensate may be helpful in diagnosis and follow-up of patients with various pulmonary diseases. References 1) 2) 3) 4)

5) 6)

7)

8) 9) 10)

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12) 13) 14)

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D. Kietzman, R. Kah, M. Muller, H. Burchardi, D. Kettler, Hydrogen peroxide in expired breath condensate of patients with acute respiratory failure and with ARDS, Intensive Care Medicine 19 (1993)78-81. M. Kasielski, D. Nowak, Long-term administration of N-acetylcysteine decreases hydrogen peroxide exhalation in subjects with chronic obstructive pulmonary disease, Respiratory Medicine 95(2001)448-456. A. Antczak, P. Gorski, Antibiotic treatment decreases hydrogen peroxide and thiobarbituric acid reactive substances in expired air condensate in infectious exacerbation of bronchial asthma, International Review of Allergology and Clinical Immunology 6 (2000) 52-55. D. Nowak, M. Kasielski, T. Pietras, P. Bialasiewicz, Cigarette smoking does not increase hydrogen peroxide levels in expired breath condensate of patients with stable COPD, Monaldi Archives of Chest Diseases 53(1998) 268-273. G.L. Ignatova, LA. Volchegorskii, E.G. Volkova, E.L. Kazachkov, O.L. Kolesnikov, [Lipid peroxidation processes in chronic bronchitis], Terapii Arkhiv 70 (1998) 36-37. Russian. P. Montuschi, S.A. Kharitonov, G. Ciabattoni, M. Corradi, L. van Rensen, D. M. Geddes, ME. Hodson, PJ. Barnes, Exhaled 8-isoprostane as a new non-invasive biomarker of oxidative stress in cystic fibrosis, Thorax 55 (2000) 205-209. P. Montuschi, M. Corradi, G. Ciabattoni, J. Nightingale, S.A. Kharitonov, P.J. Barnes, Increased 8isoprostane, a marker of oxidative stress, in exhaled condensate of asthma patients, American Journal of Critical Care and Respiratory Medicine 160 (1999) 216-220. A. Antczak, S.A. Kharitonov, P. Montuschi, P. Gorski, P.J. Barnes, Increased 8-isoprostane in aspirin-induced asthma, European Respiratory Journal 18 (2001) 247s. P. Montuschi, J.V. Collins, G. Ciabattoni, N. Lazzeri, M. Corradi, S.A. Kharitonov, P.J. Barnes, Exhaled 8-soprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers, American Journal of Critical Care and Respiratory Medicine 162 (2000)11751177. A. Antczak, P.Gorski, Increased cysteinyl-leukotrienes, leukotriene B4 and 8-isoprostane in exacerbation of COPD, European Respiratory Journal 18 (2001) 248s. C. Carpenter, P. Price, and B. Christmas, Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury and ARDS, Chest 114 (1998) 1653-1659. T. Hanazawa, S.A. Kharitonov, P.J. Barnes, Increased nitrotyrosine in exhaled breath condensate of patients with asthma, American Journal of Critical Care and Respiratory Medicine 162 (2000) 1273-1276. B. Balint, S.A. Kharitonov, T. Hanazawa, L.E. Donnelly, P.L.Shah, M.E. Hodson, P.J. Barnes, Increased nitrotyrosine in exhaled breath condensate in cystic fibrosis, European RespiratoryJournal 17 (2001) 1201 -1207. M. Corradi, P. Montuschi, L.E. Donnelly, A. Pesci, S.A. Kharitonov, P.J. Barnes, Increased nitrosothiols in exhaled breath condensate in inflammatory airway diseases, American Journal of Critical Care and Respiratory Medicine 163 (2001)854-858. L.P. Ho, J.A. Innes, A.P. Greening, Nitrite levels in breath condensate of patients with cystic fibrosis is elevated in contrast to exhaled nitric oxide, Thorax 53 (1998)680-684. B. Balint, T. Hanazawa, S.A. Kharitonov, L.E. Donnelly, P.J. Barnes, Increased nitric oxide metabolites in exhaled breath condensate after exposure to tobacco smoke, Thorax 56 (2001)456461. K. Ganas, S. Loukides, G. Papatheodorou, P. Panagou, N. Kalogeropoulos, Total nitrite/nitrate in expired breath condensate of patients with asthma, Respiratory Medicine 95 (2001)649-654.

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Volatile Organic Compounds as Prognostic Markers in ARDS JK Schubert, W Miekisch, GFE Noldge-Schomburg Department of Anaesthesia and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock Germany Abstract. Exhaled volatile substances in ARDS can be linked to different pathways of inflammation and to membrane metabolism. As concentrations of such markers show a specific pattern along the course of ARDS, diagnosis of different stages of the disease should be possible by means of breath analysis. Future progress of analytical techniques and a better understanding of the relationship between expired substance concentrations and the clinical status will certainly help to improve diagnosis, to stimulate basic research, and to guide and evaluate the therapy of ARDS.

1. ARDS: Definition, underlying diseases, lung pathology, clinical aspects First described by Ashbough et al [1] in 1967, the acute respiratory distress syndrome (ARDS) represents a uniform response of the lung to direct or indirect injury. According to the AmericanEuropean consensus conference it is characterised by an acute onset of respiratory failure, a PaO2/FiO2 < 200 mmHg regardless of positive end expiratory pressure (PEEP), bilateral infiltrates on frontal chest radiograph, and pulmonary artery wedge pressure (PC WP) < 18 mmHg or lack of clinical evidence of left atrial hypertension [2]. The incidence is about 75 cases/100,000 population, meaning 150,000 cases annually in the United States [3]. Various clinical conditions may precipitate the onset of ARDS. ARDS may arise from mechanical injury to lung parenchyma, from bacterial, viral or mycotic pulmonary infection or may result from inhalation of toxic gases such as nitrous oxide (NC>2), hydrochloric acid (HC1) or sulphuric oxide (SO2). Indirect lung injury may be elicited by multiple trauma without lung injury, by sepsis, by ischemia/reperfusion or by mass transfusion. Pathophysiology of ARDS is closely related to inflammatory responses including cellular and humoral components. The early stage of ARDS is characterised by neutrophil sequestration into the lung. Activation of these neutrophils causes liberation of oxygen derived free radicals and numerous mediators. Increased concentrations of superoxide, hydrogen peroxide or hypochlorous acid [4] have been found in the blood of ARDS patients. Inflammatory mediators such as complement fragments, thromboxane, leukotrienes, proteases, cytokines or platelet activating factor (PAF) are variably present in both patients at risk for, as well as those with ARDS. Recent work has shown that at the same time hydrogen peroxide, serum catalase. manganese-superoxide dismutase and ceruloplasmine are increased, glutathione is decreased. Findings such as these suggest that the balance between oxidants and anti-oxidant capacity must be important [5]. Reactive oxygen species and inflammatory mediators released from neutrophils have a direct effect on endothelial and alveolar cells and may simultaneously precipitate a general inflammatory response (SIRS = systemic inflammatory response syndrome) in the body. These remote effects contribute significantly to the mortality of ARDS. In fact, many patients die because of multiple organ failure (MOF) rather than of respiratory failure. Direct injury to endothelial and alveolar cells results in increased alveolar-capillary permeability, lung oedema.

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pulmonary hypertension and respiratory failure. Hypoxic pulmonary vasoconstriction is deranged and shunt fraction is consecutively elevated contributing to refractory hypoxemia. During later stages of ARDS fibrosis of the lung tissue determines the course and outcome of the disease. Despite new methods in the treatment such as inhalation of NO, instillation of surfactant, ventilation in prone position or extracorporeal membrane oxygenation (ECMO) mortality of ARDS remains high. Many of the mechanisms in the pathogenesis of ARDS have not yet been elucidated. As a result, a specific therapy of ARDS is lacking. Actual therapy consists of controlling the underlying disease, avoiding hypoxemia by means of lung protective ventilatory support and supportive care. Diagnostic parameters indicating risk or onset of the disease and characterising the course of ARDS are lacking. The association between lung injury and the systemic inflammatory response (SIRS) very often triggered by an ARDS has not been completely understood. Specific prognostic criteria are still to be defined. In ARDS inflammatory processes take place near or in the alveoli. Elevated concentrations of volatile substances generated through the effect of radicals or cytokines on cellular structures should, therefore, be found in the alveolar gas. If these substances can be identified and linked to a specific event in the course of ARDS, analysis of exhaled air will provide valuable information for diagnosis, treatment control and prognosis in ARDS. 2. Analytical procedures in mechanically ventilated patients Many ARDS patients require mechanical ventilation because of respiratory insufficiency and refractory hypoxia. Breath sampling in mechanically ventilated patients requires techniques which are different from those that can be used in spontaneously breathing individuals. Gas sampling in those patients is affected by the high water content of the samples, the respiratory circuit and possible contaminants coming out from tubings or the gas supply. Therefore, substance concentrations have to be corrected for inspiratory values. Special attention has to be paid to the mode of gas sampling. Only alveolar concentrations reflect concentrations in blood, whereas mixed expired concentrations may be diluted and contaminated by dead space gas [6], Due to the very low substance concentrations in the exhaled air preconcentration is mandatory. This can be done by adsorption onto activated charcoal [7], organic polymers, e.g. Tenax [8,9] or via solid phase micro extraction (SPME) [10]. Desorption from the adsorbents takes place through microwave energy or some sort of thermal treatment. Substances are separated by gas chromatography and detected by flame ionisation and identified by mass spectrometry. 3. Marker substances in ARDS A considerable number of substances have been described that could be related to some aspect of acute lung injury (ALI)/ARDS. Hydrogen peroxide levels in expired air condensate were increased in patients with respiratory failure and were highest in ARDS patients [11]. Concentrations decreased after clinical improvement. NO from alveolar macrophages and superoxide from membrane bound NADPH oxidase can combine to form peroxynitrite a potent oxidant capable of damaging cell structures by peroxidation. Peroxynitrite has to be determined via nitrotyrosine in lung sections or breath condensate [12, 13]. Toxic levels of peroxynitrite have been found in ALI/ARDS.

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Isoprostanes, prostanoid compounds formed nonenzymatically via lipid peroxidation are markers of in vivo oxidant stress, and can be determined in breath condensate. Expired isoprostane levels were elevated in patients with ALI or ARDS when compared to healthy controls [14]. Elevated isoprostane concentrations in plasma were predictive of increased mortality in ARDS [15]. A correlation between isoprostane concentrations in breath condensate and the severity of lung injury may, therefore, be assumed. Since many ARDS patients require mechanical ventilation sampling of breath condensate is affected by the respiratory circuit and the mode of gas humidification. Hence quantitative analysis in breath condensate of mechanically ventilated patients is difficult and prone to error, standards of sampling are still to be defined. Exhaled NO concentrations are elevated in patients with inflammation of the airways [16,17]. Inflammatory insults induce an increase in the expression of the inducible isoform of nitric oxide synthase (iNOS) in pulmonary tissue [18]. Since inflammatory processes play an important role in ARDS, elevated exhaled NO concentrations are expected in those patients. Surprisingly, Brett et al [19] found lower exhaled NO concentrations in ARDS patients when compared to mechanically ventilated patients without lung injury. Changes in diffusion capacity of NO or formation of peroxynitrite from NO and superoxide are considered the cause of this finding. Alkanes such as ethane and n-pentane are believed to be markers of lipid peroxidation [20 - 23] and have been demonstrated in a variety of pathological conditions. Pentane arises from peroxidation of co-6 polyunsaturated fatty acids [24], ethane from peroxidation of co-3 fatty acids (e.g., linolenic acid) [25]. Elevated ethane concentrations that could be reduced by radical scavengers were found in ARDS patients. [26]. Since pentane is believed to be a marker of lipid peroxidation elevated exhaled pentane concentrations are expected in ARDS. However, no difference in the pentane production could be observed between ARDS and non-ARDS patients in our first studies (Table 1) [27, 28]. Table 1: Substance exhalation (nmol/m2/min) in patients with and without ARDS Substance Acetone Pentane Isoprene

ARDS Median (95% CI) 119(52-270) 5.1 (1.4- 18.6) 21.8(13.9-41.4)

Non-ARDS Median (95% CI) 149(113-485) 4.15(3.7-9.3) 9.8(8.2-21.6)

P 0.25 0.37 0.04

CI confidence interval, ARDS: Acute Respiratory Distress Syndrome

This finding may be due to the fact that the analysis of air was not performed at the very beginning of ALI/ARDS where inflammatory activity is highest. Furthermore, patients in the non-ARDS group covering a wide range of diagnoses were not completely free of inflammation. In a group of patients who developed pneumonia during their ICU stay, it could be shown that pentane concentrations did increase at the very beginning of an inflammatory process [27]. In order to get a better understanding of the role of pentane in ARDS exhaled breath markers were determined in healthy volunteers, in patients with and being at risk for ARDS and in patients with head injury (Table 2). Pentane concentrations were significantly increased in ARDS patients and in those being at risk to develop ARDS when compared with healthy volunteers or with patients with cranio-facial trauma without lung injury. Isoprene, (2-methylbutadiene-l,3) is always present in human breath, and is thought to be formed along the mevalonic pathway of cholesterol synthesis [28]. This reaction involves mevalonate, isopentenyl pyrophospate and dimethylallyl pyrophosphate (DMPP).

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Table 2: Exhaled breath markers in 65 critically ill patients and 10 healthy volunteers Control group (1)

Substance Acetone Median 25-75% P [nmol/1] Isoprene Mean 95%CI [nmol/1] n-Pentane Median 25-75% P [nmol/1]

Head Injury

(ID

ARDS (III)

At risk (IV)

33.2 20.8 - 38.6

51.4 14.5-76.6

50.0 19.6-72.3

26.0 14.1-54.4

5.99f 3.53 - 8.45

7.53* 5.02- 10.05

2.18*st 1.1 -3.89

6.44§ 5.04-7.85

0.12*s 0.10-0.16

0.33 0.11-0.83

01.00s 0.26- 1.72

0.49* 0.30 - 0.99

Values are given as medians and 25% - 75% percentiles or as mean and 95% confidence interval, respectively. *, §, t indicate results that are significantly different ( p< 0.05).

The rate limiting step of sterol synthesis, i.e. the formation of mevalonate is catalysed by hydroxymethylglutaryl(HMG)-CoA. Although some details of isoprene formation in humans are still unknown there is experimental evidence that isoprene exhalation may be related to oxidative damage to fluid lining of the lung [30] and the body [31]. Surprisingly, isoprene concentrations in the breath of laboratory animals are considerably lower than in human breath [32]. In our studies [27] ARDS patients produced over 50 % less isoprene than those without ARDS (Table 2). In 151 mechanically ventilated patients with various diagnoses a negative correlation between exhaled isoprene concentrations and the severity of lung injury (Murray score) [33] was found (Fig. 1). Decreasing isoprene concentrations in ALI/ARDS may be due to a reduction of cholesterol synthesis in the lung, to reactions of isoprene with other reactive species like peroxynitrite or to a decreased exhalation because of ventilation/perfusion mismatch. Fig. 1 Exhaled isoprene concentration vs. Murray score

§ 15,00 I

10,00

TT t-i 0,00 i 0,00

i « 1,50

2,00

2,50

Murray Score

Correlation coefficient = - 0.300, p < 0.01, N = 151

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There were no correlations between exhaled concentrations of acetone, sulphur containing compounds like dimethylsulfide or €82 [34] or aldehydes like hexanal or nonenal and ALI/ARDS. As for breath condensate, standards of quantitative analysis in exhaled air are lacking and very often results show a wide variation. Some of the very volatile substances, such as ethane require sophisticated analytical techniques [9]. Other substances, such as pentane may be present in ambient air. When standardised alveolar sampling techniques are used as described above (including correction for inspiratory concentrations) analysis of exhaled volatile organic compounds (VOCs) seems to be more reliable than other methods. 4. Requirements to define prognostic markers Due to the complex pathophysiology of ARDS and a lack of knowledge on origin and physiological meaning of exhaled substances for the time being it is not possible to define a single prognostic breath marker in ALI/ARDS. But different markers are known that are specifically linked to single aspects of airway inflammation and lung injury. Isoprene is linked to cholesterol metabolism, ethane and pentane are linked to lipid peroxidation, NO is linked to airway inflammation in a very complex way and isoprostanes are linked to the arachidonic acid metabolism. In order to obtain information on prognosis of ALI/ARDS a set of breath markers has to be used. These parameters have to be chosen in a way that the different aspects of inflammation are taken into account. The upcoming progress in analytical procedures will enlarge our knowledge on exhaled breath markers concerning physiology, mechanisms and kinetics of exhalation. Thus, in the near future it should be possible to define a set of parameters characterising onset, course and prognosis of ALI/ARDS. References 1. Ashbaugh DG, Biglow DB, Petty TL et al. Acute respiratory distress syndrome in adults. Lancet 1967;ii:319323 2. Bernard GR, Artigas A, Brigham K.L, Carlet J, Falke K, Hudson L et al. The American-European consensus conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 1994; 149:818-824 3. American Lung Program. Respiratory Diseases. Task force report on problems, research approaches, needs. The Lung Program. National Heart and Lung Institute. Washington DC, US Government Printing Office, 1972. DHEW Publication No. (NIH) 73-432, pp 165-180 4. Tate RM, Regine JE. Neutrophils and the adult respiratory distress syndrome. Am Rev Respir Dis 1983; 125:552-559 5. Brigham KL. Role of free radicals in lung injury. Chest 1986; 89:859-863 6. Schubert JK, Spittler K-H, Braun G, Geiger K, Guttmann J. COyControlled Sampling of Alveolar Gas in Mechanically Ventilated Patients. J Appl Physiol 2001; 90:486-492 7. Muller WPE,. Schubert JK, Benzing A, Geiger K. Method for the analysis of exhaled air by microwave energy desorption coupled with gas chromatography -. flame ionisation detection - mass spectrometry. J. Chromatogr. B 1998; 716:27-38 8. Miekisch W, Schubert JK, Mailer WPE, Geiger K: Analysis of Exhaled Air As a New Means of Critical Care Testing. In "Advances in Critical Care Testing", W.F. List, M.M. MQller, M.J. McQueen (Eds.), Springer Verlag 1999, p 202 9. Risby TH, Sehnert SS. Clinical application of breath biomarkers of oxidative stress status. Free Rad Biol Med 1999; 27:1182-1192 10. Grote C, Pawliszyn J: Solid-phase micro extraction for the analysis of human breath. Anal Chemistry 1997; 69:587-596 11. Kietzmann D, Kahl R, MQller M, Burchardi H, Kettler D. Hydrogen peroxide in expired breath condensate of patients with acute respiratory failure and with ARDS. Intensive Care Med 1993; 19:78-81. 12. Haddad IY, Pataki G, Hu P, Galliani C, Beckman JS, Matalon S. Quantitation of nitrotyrosine levels in lung section of patients and animals with acute lung injury. J Clin Invest 1994; 94:2407-13

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13. Haddad IY, Ischiropoulos H, Holm BA, Beckman JS, Baker JR, Matalon S. Mechanisms of peroxynitriteinduced injury to pulmonary surfactants. Am J Physiol 1993; 265:L555-L564. 14. Carpenter CT, Price PV, Christman BW. Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS. Chest 1998; 114:1653-1659 15. Delanty N, Reilly M. 8-epi-PGF2 a: specific analysis of an isoeicosanoid as an index of oxidant stress in vivo. Br J Clin Pharmacol 1996; 42:15-19 16. Olopade CO, Christon JA, Zakkar M, Hua C, Swedler I, Scheff PA, Rubinstein I: Exhaled pentane and nitric oxide levels in patients with obstructive sleep apnea. Chest 1997; 111:1500-1504 17. Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatics. Lancet 1994; 343:13-135 18. Liu SF, Barnes PJ, Evans TW. Time course of lipopolysaccharide-induced inducible nitric oxide synthase mRNA expression in the rat in vivo. Am J Respir Crit Care Med 1996; 153:A186 19. Brett SJ, Evans TW. Measurement of endogenous nitric oxide in the lungs of patients with the acute respiratory distress syndrome. Am J Respir Crit Care Med 1997; 156:993-7 20. Phillips M, Greenberg J: Ion-trap detection of volatile organic compounds in alveolar breath. Clin Chem 1992; 38:60-5 21. Van Gossum A, Decuyper J: Breath alkanes as an index of lipid peroxidation. Eur Respir J 1989; 2:787-91 22. Van-Rij AM, Wade CR: In vivo lipid peroxidation in man as measured by the respiratory excretion of ethane, pentane, and other low-molecular-weight hydrocarbons. Anal Biochem 1985; 150:1-7 23. Morita S, Snider MT, Inada Y: Increased n-pentane excretion in humans: A consequence of pulmonary oxygen exposure. Anesthesiology 1986; 64:730-33 24. Frankel EN. Volatile lipid oxidation products. Prog Lipid Res 1982; 22:1-33 25. Do BQ, Harinder BS, Garewal S, Clements Jr NC, Peng Y, Habib MP. Exhaled Ethane and antioxidant vitamin supplements in active smokers. Chest 1996; 110:159-64 26. Ortolani O, Conti A. Protective effects of N-acetylcysteine and rutin on the lipid peroxidation of the lung epithelium during the adult respiratory distress syndrome. Shock 2000; 13:14-18 27. Schubert JK, Muller WPE, Benzing A, Geiger K. Application of a new method for analysis of exhaled gas in critically ill patients. Intensive Care Med 1998; 24: 415-421 28. Miekisch W, Schubert JK, Miiller WPE, Geiger K. Analysis of Exhaled Air As a New Means of Critical Care Testing. Clin Chem Lab Med 1999; 37:S347 29. Stone BG, Besse TJ, Duane WC, Evans CD, DeMaster EG: Effect of regulating cholesterol biosynthesis on breath isoprene excretion in men. Lipids 1993;28:705-8 30. Foster MW, Jiang L, Stetkiewicz PT, Risby TH: Breath isoprene: Temporal changes in respiratory output after exposure to ozone. J Appl Physiol 1996; 80:706-10 31. Mendis S, Sobotka PA, Euler DE: Expired hydrocarbons in patients with acute myocardial infarction. Free Radical Res 1995; 23:117-22 32. Cailleux A, Cogny M, Allain P: Blood isoprene concentrations in humans and some animal species. Biochem Med Metabol Biol 1992; 47:157-160 33. Murray JF, Matthay MA, Luce JM, Flick MR. An expanded definition of the adult respiratory distress syndrome. Am Rev Respir Dis 1988; 138:720-723 34. Phillips M, Saba M, Greenberg J: Increased pentane and carbon disulfide in the breath of patients with schizophrenia. Clin Pathol 1994; 46:861-864

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Do Reactive Oxygen-Nitrogen Intermediates Contribute to the Pathogenesis of ARDS? Judy M. HICKMAN-DAVIS1, Ian C. DAVIS2, Phillip O'REILLY3, Philip IVÂRDLE1 and Sadis MATALON1*2'4 'Departments of Anesthesiology, 2Genomics and Pathobiology, 3Pulmonary and Critical Care Medicine and 4Physiology and Biophysics, University of Alabama at Birmingham, 619 South 19th Street, Birmingham, AL 35233-6810 Abstract: Inhaled nitric oxide (NO) has been proven effective in lowering pulmonary arterial pressure and improving gas exchange in patients with pulmonary hypertension. Furthermore, because of its potential anti-inflammatory properties, inhaled NO* therapy can be a potentially useful adjunct in the treatment of a number of inflammatory conditions, including acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). However, NO is also a free radical and can therefore combine with reactive oxygen species to form highly reactive oxygen-nitrogen intermediates, which may overwhelm antioxidant defenses and cause significant damage to alveolar epithelial cells. Nitrotyrosine, a stable end product of reactive oxygen-nitrogen species interactions, is commonly detected in infectious and inflammatory diseases. Nitration and oxidation of a variety of crucial proteins present in the alveolar space has been shown to be associated with diminished function in vitro and has also been identified ex vivo in proteins sampled from patients with ALI/ARDS. Oxidant-mediated tissue injury is therefore likely to be important to the pathogenesis of ARDS. The purpose of this chapter is to review the results from various studies that demonstrate increased levels of NO* and its reactive intermediates in the alveolar spaces of patients with ALI/ARDS, or which show that various proteins are nitrated and or oxidized, and discuss the physiological consequences of protein nitration. 1. Introduction Inhaled nitric oxide (NO») has been proven effective in lowering pulmonary arterial pressure and improving gas exchange in patients with pulmonary hypertension [1]. Furthermore, because of its potential anti-inflammatory properties, inhaled NO* therapy can be a potentially useful adjunct in the treatment of a number of inflammatory conditions, including acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). However, NO* is also a free radical and can therefore combine with reactive oxygen species (such as superoxide and peroxyl radicals) to form highly

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reactive oxygen-nitrogen intermediates which may overwhelm antioxidant defenses and cause significant damage to alveolar epithelial cells, resulting in functional and structural abnormalities. In addition, S-nitrosothiols, formed by the interaction of NO intermediates with thiols present in hemoglobin, albumin or other proteins, may be capable of producing systemic vasodilation [2]. Potential sources of NO in the lungs include activated alveolar macrophages (AMs) [3;4], neutrophils [5], alveolar type II cells [6;7], endothelial cells, and airway cells [8]. Both neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS) have been detected in human lungs [8]. nNOS is localized to nerve terminals that likely contribute to nonadrenergic/noncholinergic airway innervation, and is also present in human and rat airway epithelial cells [8]. eNOS is localized to pulmonary endothelium and bronchial epithelium [9]. Studies have suggested that inducible NOS (iNOS) is constitutively expressed in human upper airway epithelium [10] and occasional AMs [8], but this may be a result of chronic exposure of these cells to inhaled pollutants and microbes [11], Expression of iNOS in other regions of the normal lung is believed to be minimal. However, iNOS has been immunolocalized to airway cells and human lung tissue obtained from patients with ARDS [12;13], bacterial pneumonia [14], lung cancer [15], pulmonary sarcoidosis [16], idiopathic pulmonary fibrosis [17], asthma [18] and tuberculosis [19]. These findings raise the possibility that during lung inflammation, an increased amount of NO' may be released into the epithelial lining fluid, where it may have both beneficial (antimicrobial) and detrimental (tissue-damaging) effects. 2.

Reactive oxygen-nitrogen intermediates contribute to lung injury in experimental models of ARDS

There is now substantial experimental evidence that reactive oxygen-nitrogen species (RNS) may be involved in pulmonary epithelial injury in a variety of pathological situations. Induction of immune complex alveolitis in rat lungs results in increased alveolar epithelial permeability, which is associated with the presence of elevated concentrations of NO* decomposition products in bronchoalveolar lavage (BAL) fluid [20]. Alveolar instillation of the NOS inhibitor L-NMMA ameliorates NO* production and alveolar epithelial injury. Similarly, both paraquat-induced [21] and ischemia-reperfusion-induced [22] lung injury are associated with stimulation of NO* synthesis, and are abrogated by NOS inhibitors. Tracheal epithelial cytopathology induced by Bordetella pertussis is associated with the induction of NO* synthesis, and is remarkably attenuated by inhibition of NOS [23]. Likewise, influenza virus-induced lung pathology in mice results from increased expression of iNOS and increased generation of NO* [24]. Administration of NOS inhibitors significantly improves survival of influenza-infected mice. Additional evidence that RNS play a role in pulmonary inflammation is derived from studies utilizing transgenic Nos2~'~ mice. Lung damage induced by either injection of lipopolysaccharide (LPS) [25], influenza virus infection [26], or hemorrhage and resuscitation [27], is markedly reduced in these mutant mice. Likewise, in an experimental murine model of allergic airway disease, deletion of the Nos2 gene results in a significant decrease in eosinophil infiltration into the lungs [28]. However, levels of NO* produced by inflammatory cells vary widely among species; thus extrapolation of animal data to human disease states is not warranted. Herein we will: (1) review the results from various studies demonstrating increased levels of NO* and its reactive intermediates in the alveolar spaces of patients with ALI/ARDS; (2) show that various proteins are nitrated and or oxidized and (3) discuss the physiological consequences of protein nitration.

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3. Increased levels of NO* in the BAL and edema fluid of patients with ARDS ARDS is a disease process characterized by diffuse inflammation in the lung parenchyma. Nitrate and nitrite (NOX), the stable breakdown by-products of NO, can be measured in biological fluids using the Greiss reaction. While this assay is relatively simple to perform, it is important that any nitrate present is first chemically or enzymatically reduced to nitrite (in most biological fluids, nitrite is usually converted to nitrate because of slow oxidation by hemoglobin). If this reduction step is not performed, levels of NOX production are likely to be grossly underestimated. For example, NOX concentrations were significantly higher than normal in the BAL fluid from patients at risk for developing ARDS as well as those with ARDS [12] and remained elevated throughout the course of ARDS. In all cases, the majority of the products detected were in the form of nitrate (>90% nitrate, and < 0.005 vs. normal subjects. Reprinted with permission from reference [12].

In comparing patients at-risk for ARDS and patients with established ARDS, there was no statistically significant difference in BAL NOX concentration, either at the onset of ARDS or at any subsequent time. However, the patients studied on day 21 after the onset of ARDS, a time when the course of ARDS was waning, had the lowest concentrations of NOX in BAL fluid. While there were no statistically significant differences in the BAL NOX concentrations from patients at-risk for the development of ARDS, the NOX concentrations in the BAL of patients with ARDS who subsequently died were significantly higher on days 3 and 7.

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Levels of NOX in the epithelial lining fluid of these patients cannot be easily estimated since they are diluted considerably (more than 50 fold) by the BAL fluid. To address this issue, we measured NOX levels in pulmonary edema fluid (EF) and plasma samples from patients with ALI/ARDS and for comparison, in samples from patients with hydrostatic pulmonary edema. All of these patients were admitted to the intensive care units at the University of California at San Francisco (UCSF) or San Francisco General Hospital between 1985 and 1998. Pulmonary EF was collected from each patient within 30 min after endotracheal intubation by passing a standard 14 Fr tracheal suction catheter through the endotracheal tube into a wedged position in a distal airway as described previously [29]. Pulmonary EF from patients with ALI had significantly higher levels of NOX compared to pulmonary EF from patients with hydrostatic pulmonary edema (108 ± 13 uM vs. 66 + 9 uJVI; Means ± SEM; p < 0.05). In addition, patients with shock had higher plasma NOX levels than those without shock (79 ± 11 uM vs. 53 ± 12 uM, p < 0.05). The ratios of nitrite to nitrate in 11 edema and 9 plasma samples were 0.01 ± 0.005 vs. 0.008 ± 0.004, indicating that more than 90% of NOX were present as nitrate, in agreement with our BAL data (see above). Acidemia and increased anion gap, markers of systemic hypoperfusion, were also associated with two fold higher plasma NOX levels. TABLE 1. Patient population, (reprinted from reference [12] with permission) Characteristic

N Age, yr.

Gender (% male)

- —At Risk—

—

-

--ARDS

Day 21

Day 1

Day 3

Day 1

Day 3

Day 7

Day 14

19

14

36

41

30

16

11

48.0

48.2

42.5

45.1

46.3

44.6

42.6

58

64

67

59

63

50

46 3 (27.3)

Primary Risk Sepsis, n (%)

8(42.1)

4 (28.6)

10(27.8)

15(36.6)

10(33.3)

4 (25.0)

Trauma, n (%)

1 1 (57.9)

10(71.4)

15(41.7)

15(36.6)

14(46.7)

8 (50.0)

5 (45.5)

0(0)

0

1 1 (30.6)

1 1 (26.8)

6 (20.0)

4 (25.0)

3 (27.3)

Other, n (%) Apache II Score

21.8+1.4

14.6 + 2.1

21.1 + 1.1

21.0+1.1

18.8+ 1.4

18.9+1.3

15.5+ 1.8

PO,/Fi02 ratio

209.6 + 23.8*

243.8 + 30.0

152.5 + 8.3*

162.7 + 8.8

203.1 +14.0

197.2+13.1

207.7 + 18.7

2(10)

1(7)

8(22)

8(20)

3(10)

1(7)

0

Mortality, n (%)

Data are the mean + SE P02/Fi02 ratio. Ratio of arterial oxygen tension to inspired fraction of oxygen. * p = 0.03 for the comparison of Day 1 At Risk vs. Day 1 ARDS.

An additional benefit of sampling undiluted pulmonary EF rather than diluted BAL fluid is the opportunity to measure alveolar fluid clearance by following serial changes in the protein concentration in the pulmonary EF. Patients with ALI who were able to concentrate alveolar protein (as a result of active sodium reabsorption) had a better prognosis than those that did not [29;30]. Our results indicate that increased levels of NOX in EF samples were associated with slower rates of alveolar fluid clearance (Figure 2). One possible explanation for this finding is that the generation of reactive oxygen and nitrogen species in the alveolar compartment leads to nitration, oxidation and inactivation of proteins important in alveolar epithelial sodium transport, such as the epithelial sodium channel. Indeed, intra-tracheal instillation of DETANONOate, an NO- donor, decreased amiloride-sensitive fluid clearance in rabbit lungs (31).

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J.M. Hickman-Davis el al. /Pathogenesis ofARDS 250

200 -

150 -

i 100 -\ c 0}

5

50 H

Submaximal/lmpaired Maximal Alveolar Fluid Clearance (%/hr)

Figure 2. Box-plot summary of mean interval EF nitrate and nitrite concentration versus two categories of alveolar fluid clearance. Maximal alveolar fluid clearance is > 14%/h. Impaired/Sub-maximal alveolar fluid clearance is < 14%/h. The horizontal line represents the median, the box encompasses the 25* to 75* percentile and the error bars encompass the 10* to 90* percentile. *p < 0.05 by Mann Whitney U-test. Reprinted with permission from reference [30].

4. Evidence for the existence of nitrated proteins in vivo Several studies have provided evidence that nitration reactions occur in vivo during inflammatory processes. 3-nitrotyrosine residues, products of the addition of a nitro-group (NOi) to the ortho position of the hydroxyl group of tyrosine, are stable end products of RNS mediated reactions. They therefore serve as footprints of RNS action, which are readily detectable by immunohistochemistry, ELISA or high-pressure liquid chromatography (HPLC) [32]. Nitrotyrosine is commonly detected in tissues infiltrated by neutrophils and monocytes during infectious and inflammatory processes [12;33]. In vitro, proteins can be nitrated either by peroxynitrite or by reactive intermediates generated by the myeloperoxidase-catalyzed reaction of reactive species released from activated neutrophils [34;35]. Irnmunohistochemical studies showing evidence of nitrotyrosine residue formation on proteins in cells taken from lung tissues of pediatric patients with ALI, were first reported by Haddad et al. [33]. Oxidant mediated tissue injury is likely to be important in the pathogenesis ofARDS [36;37]. Protein nitration and oxidation by reactive oxygen nitrogen species in vitro has been associated with diminished function of a variety of crucial proteins present in the alveolar space, including ai-proteinase inhibitor and surfactant protein A (SP-A) [38;39]. Gole el al. [40] reported the presence of nitrated ceruloplasmin, transferrin, a i-protease inhibitor, ai-anti-chymotrypsin and p-chain fibrinogen in the plasma of patients with ALI/ARDS. Using quantitative ELISA and HPLC, we detected significant levels of protein-associated nitrotyrosine (~400 - 500 pmol/mg protein) in EF samples from both ALI/ARDS and hydrostatic edema patients [41] and in the BAL from patients with ARDS [12]. These levels of nitrotyrosine are at least one order of magnitude higher than those found in proteins in normal human BAL fluid (28 pmol/mg protein) [42], or normal rat lung tissue (-30 pmol/mg protein) [43], or than those found in normal human serum albumin (-30 pmol/mg protein) [44], and normal human plasma low-density lipoprotein (-85 pmol/mg protein) [44]. Lamb et al. [45] also measured nitrotyrosine content in the BAL fluid of

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349

patients with severe ARDS and healthy volunteers using HPLC, although their values were considerably higher than those reported by Sittipunt et al. [12] and Zhu et al. [41]. Nitrated pulmonary SP-A was also detected in the EF but not in the plasma of patients with ALI after immunoprecipitation with a specific antibody against this protein (Figure 3). Although we previously demonstrated that SP-A is nitrated and oxidized in vitro, using LPS-stimulated rat AMs as the source of reactive species [46] (see below), this is the first in vivo evidence for nitration of a specific protein in the alveolar spaces of human lung. Results of previous in vitro studies indicated that nitrated SP-A loses its ability to enhance the adherence of Pneumocystis carinii to rat AMs [39] and inhibits killing of Mycoplasma pulmonis by mouse AMs (Hickman-Davis et al.; unpublished observations). Also, nitration of human SP-A by peroxynitrite or tetranitromethane inhibited its lipid aggregation and mannose binding activities [47]. Finally, SP-A isolated from the lungs of lambs exposed to high concentrations of inhaled NO* had decreased ability to aggregate lipids. Thus, nitration of SP-A may be one of the factors responsible for increased susceptibility of patients with ARDS to nosocomial infections [48]. Interestingly, despite being present at high concentrations in the epithelial lining fluid of patients with ARDS, albumin was nitrated to a much lesser degree than SP-A [41].

Figure 3. Nitration of SP-A in pulmonary EF samples from ALI/ ARDS patients. SP-A was immunoprecipitated and Western blotting used to identify SP-A (A) and nitrotyrosine (B). SP-A was detected in the pulmonary EF but not in the plasma of all patients. E!-E5: pulmonary EF samples from 5 different ALI/ARDS patients; Pi-Pa: plasma samples from 3 different ALI/ARDS patients; C: purified human SP-A from a patient with alveolar proteinosis. Notice the lack of nitration in the control sample. (Reprinted with permission from reference [41].

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5. Nitration of SP-A is sufficient to inhibit function In addition to being a nitrating agent, peroxynitrite is a strong oxidant. The inactivation of al-antitrypsin inhibitor by peroxynitrite has been attributed to the oxidation of a methionine in its active site (38). Likewise, SP-A isolated from the epithelial lining fluid of patients with ARDS was shown to be oxidized as well as nitrated [41]. How then can one be sure that tyrosine nitration, instead of oxidation, was responsible for the observed loss of SP-A function? Several pieces of evidence support the hypothesis of nitration-mediated protein inactivation: first, exposure of SP-A to tetranitromethane at pH 6.5, at which pH it acts as an oxidizing agent, did not decrease the ability of SP-A to aggregate lipids or bind to mannose [49], while exposure of SP-A to TNM at pH 7.5, when it functions as a nitrating agent, decreased its function. Second, carbon dioxide (CC^) (0 - 1.2 mM) augmented peroxynitrite (0.5 mM)-induced SP-A nitration and decreased oxidation in a dose-dependent fashion, as assessed by western blotting. Peroxynitrite also decreased the ability of SP-A to aggregate lipids and this inhibition was augmented by 1.2 mM CO2, in spite of the CO2-mediated decrease in oxidation [46]. Finally, exposure of SP-A to generators of reactive oxygen intermediates (such as xanthine and xanthine oxidase), did not decrease SP-A function [50]. 6. Conclusions Data presented herein indicate that stable decomposition products of both NO* and intermediated generated by its reaction with reactive oxygen species, are detected in high concentrations in both the BAL and EF of patients who are at risk of developing ARDS or who have established ARDS. Levels of reactive species correlate both with the outcome of the disease and the severity of injury to the alveolar epithelium. Finally, significant levels of nitrated SP-A and fibrinogen are detected in the EF and plasma of patients with ARDS. Although in vitro studies indicate that nitration of both proteins leads to diminished function it still needs to be established whether there are sufficient levels of nitration in vivo to contribute to the pathogenesis ofARDS. Acknowledgements This work was supported by grants RR00149 (J.M.H.-D.), HL31197 and HL51173 (S.M.) from the National Institutes of Health. Dr. Ian Davis is a Parker B. Francis Foundation Fellow.

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42. de AndradeJ.A., Crow,J.P., Viera,L., Bruce,A.C., Randall,Y.K., McGiffin,D.C., Zorn,G.L., Zhu,S., Matalon,S.( and Jackson,R,M. 2000. Protein nitration, metabolites of reactive nitrogen species, and inflammation in lung allografts. Am.J.Respir.Crit Care Med. 161:2035-2042. 43. Tanaka S, Choe,N., Hemenway D.R, Zhu,S., Matalon,S., and Kagan,E. 1998. Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura of the rat. J.Clin.lnvest. 102:445-454. 44. Khan,J., Brennand,D.M, Bradley,N., Gao,B., Bruckdorfer,R., Jacobs,M., and Brennan,D.M. 1998. 3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method. BiochemJ. 330:795-801. 45. Lamb,N.J., Gutteridge,J.M., Baker,C., Evans,T.W., and Quinlan,G.J. 1999. Oxidative damage to proteins of bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome: evidence for neutrophil-mediated hydroxylation, nitration, and chlorination. Crit Care Med 27:1738-1744. 46. Zhu,S., Basiouny,K.F., Crow.J.P., and Matalon,S. 2000. Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages. Am.J.Physiol Lung Cell Mol.Physiol 278-.L1025-L1031. 47. Zhu,S., HaddadJ.Y., and Matalon,S. 1996. Nitration of surfactant protein A (SP-A) tyrosine residues results in decreased mannose binding ability. Arch.Biochem.Biophys. 333:282-290. 48. Fagon.J.Y., Chastre,J., Domart,Y., TrouilletJ.L., Pierre,J., Dame,C., and Gibert,C. 1989. Nosocomial pneumonia in patients receiving continuous mechanical ventilation. Prospective analysis of 52 episodes with use of a protected specimen brush and quantitative culture techniques. Am.Rev.Respir.Dis. 139:877-884. 49. HaddadJ.Y., Zhu,S., lschiropoulos,H., and Matalon,S. 1996. Nitration of surfactant protein A results in decreased ability to aggregate lipids. Am.J.Physiol. 270:L281-8. 50. Haddad,I.Y., Crow,J.P., Hu,P., Ye,Y., Beckman.J., and Matalon,S. 1994. Concurrent generation of nitric oxide and superoxide damages surfactant protein A. Am.J.Physiol. 267:L242-9.

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Disease Markers in Exhaled Breath N. Marczin andM.H. Yacoub (Eds.) IOS Press. 2002

Modulation of Active Alveolar Na+ Transport by Reactive Oxygen-Nitrogen Intermediates Karin M. HARDIMAN1, Ahmed LAZRAK2, Vance NIELSEN2 and Sadis MATALON1*2 1 Departments of Physiology and Biophysics and 2Anesthesiology, University of Alabama at Birmingham, 619 South 19th Street, Birmingham, AL 35233-6810 Abstract. Active sodium transport across the alveolar epithelium creates an osmotic force, which contributes to the reabsorption of edema fluid across the alveolar epithelium. Various studies in animals and humans have demonstrated the importance of active Na* transport in limiting alveolar flooding in acute lung injury and the reabsorption of fetal lung fluid shortly after birth. Macroscopic measurements of Na* transport across alveolar have shown that Na* ions enter the cytoplasm of alveolar cells mainly through amiloride-inhibitable Na* channels. Molecular biology studies have shown the existence of three Na* channel subunit mRNAs and proteins (a, ji and y-rENaC) in adult alveolar type II (ATII) cells. Patch clamp studies have demonstrated the existence of various types of amilorideinhibitable Na+ channels, located in the apical membranes of ATII cells. The activities of these channels are modulated by a variety of agents including cAMP, glucocorticoids, mineralocorticoids and oxygen. Increased levels of reactive oxygen-nitrogen intermediates, secreted in the alveolar space by activated inflammatory cells, down regulate the activity of these channels and decrease Na transport in vivo, by increasing cGMP, and/or interacting with key residues of channel proteins.

1. Introduction In order for gas exchange to occur optimally, the alveoli must remain open and free from fluid. In utero, the fetal lung is filled with fluid which is removed shortly after birth, mainly because active transport of sodium ions (Na+) across the alveolar epithelium, starting shortly before birth, creates an osmotic force favoring reabsorption of alveolar fluid [1,2]. Studies showing reabsorption of intratracheally instilled isotonic fluid or plasma across the alveolar spaces of adult anesthetized animals and resected human lungs, and the partial inhibition of this process by amiloride and ouabain, indicate that adult alveolar epithelial cells are also capable of actively transporting sodium (Na*) ions [reviewed in [3]]. Whether or not active Na+ transport plays an important role in maintaining the normal alveoli free of fluid, remains to be established. On the other hand, a variety of studies have clearly established that active Na+ transport limits the degree of alveolar edema in pathological conditions in which the alveolar epithelium has been damaged. For example, intratracheal instillation of a Na* channel blocker in rats exposed to hyperoxia, increased the amount of extravascular lung water [4]. Conversely, intratracheal instillation of adenoviral vectors containing copies of the Na+,K+-ATPase genes increased survival of rats exposed to hyperoxia [5]. Patients with Acute Lung Injury (ALI) who were able to concentrate alveolar protein (as a result of fluid efflux secondary to active Na+ reabsorption) had a better prognosis than those that did not [6,7]. In addition, p-agonists, which upregulate Na* transport across the alveolar epithelium

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of animal and resected human lungs [8] by increasing cAMP levels, reversed the decrease in amiloride-sensitive alveolar fluid clearance in rats exposed to hypoxia [9]. Thus, p-agonists may prove useful in the amelioration of high altitude pulmonary edema. 2. ATII cells contain functional Na+ transporters. Additional insight into the nature and regulation of transport pathways has been derived from electrophysiological studies in freshly isolated and cultured alveolar type II (ATII) ceils. These cells, which make up 67% of the alveolar epithelial cells but constitute only 3% of the alveolar surface area in the adult lung, can be isolated at high purity from rats and grown on permeable supports to form confluent monolayers [10,11]. Based on the results of a variety of biophysical studies we know that Na+ ions diffuse passively into ATII cells mainly through apically located amiloride-sensitive, amiloride-insensitive and cGMP-gated cation channels with conductances of 4-25 pS [12],[13,14] and are extruded across the basolateral membranes by the ouabain-sensitive Na+,K+-ATPase [15]. To preserve neutrality, chloride (Cl") ions move from the apical to the basolateral compartments either through the paracellular junctions and/or through chloride channels located in alveolar epithelial cells [16,17]. The three subunits of the epithelial Na+ channel (aENaC, fiENaC, and yENaC) have been found in freshly isolated and cultured ATII cells using western blotting and RT-PRC, and anti-sense oligonucleotides directed against aENaC mRNA caused a decrease in ATII cell channel activity [13]. In situ hybridization studies identified the presence aENaC and yENaC but not pENaC in the alveolar region of both fetal and adult lungs [18]. Recently alveolar type I (ATI) cells have been isolated from adult rat lungs, and like ATII and FDLE cells, have been shown to contain aquaporins and possess very high permeability to water [19-21]. Preliminary (currently unpublished) data from a number of laboratories indicate that ATI cells contain proteins antigenically related to ENaC and Na,K-ATPase subunits. Thus, it seems likely that ATI cells are also capable of vectorial Na+ transport, although no direct evidence exists at present. 3. Sources of Reactive Oxygen and Nitrogen Species in the Lung. Because of their location, alveolar epithelial cells are often exposed to increased intracellular and extracellular concentrations of reactive oxygen and nitrogen species (RONS) present in cigarette smoke, environmental pollutants, oxidant gases or generated by activated inflammatory cells. Reactive oxygen species are formed as intermediates of the incomplete reduction of oxygen in mitochondrial electron-transport systems, by microsomal metabolism of endogenous compounds and xenobiotics, or by various enzymatic generators, such as xanthine oxidase. Neutrophils and other inflammatory cells generate and release reactive oxygen species via an NADPH oxidase-dependent mechanism which is mediated by membrane receptor activation of protein kinase C and phospholipase C[22]. Nitric oxide («NO) synthesis involves the five-electron oxidation of the guanidino nitrogen of L-arginine by nitric oxide synthases (NOS) [23]. The three enzymes that make •NO are endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS). Potential sources of -NO include both rat and human activated alveolar macrophages [24,25], neutrophils [26], alveolar type II ceils [27,28], and airway cells [29], Increase iNOS levels have been found in airway cells and human lung tissue obtained from patients with ARDS [30-32] and other inflammatory lung diseases. The biological effects of *NO depend on its concentration, the biochemical composition of the target, and the presence of other radicals. Nitric oxide may bind to the heme group of soluble guanylate cyclase resulting in increased cellular cGMP levels [33]; it

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may react with superoxide ((V) at diffusion limited rates (6.7 x 109 M"1 x s"1) to produce peroxynitrite (ONOO") [34] and higher oxides of nitrogen; or, in the presence of an electron acceptor, it may react with thiols to form nitrosothiols (RS-NO) [35]. RONS may cause extensive cellular injury by initiating iron-independent lipid peroxidatation, sulfhydryl oxidation, DNA strand scission, tyrosine nitration, apoptosis and cellular necrosis [36]. Herein we will review the results of various studies indicating that over-production of reactive oxygen-nitrogen intermediates damage ATII cell ion channel function, and decrease vectorial Na+ transport across alveolar epithelial cells both in vitro and in vivo. 4. Assessment of Na+ transport across ATII cells in vitro. There are various methods of showing the presence of active vectorial Na+ transport in freshly isolated and cultured ATII cells, including: (1) detection of whole-cell and single channel currents across single ATII cells by patch clamp; (2) measurements of 22Na+ and 86 Rb* fluxes across ATII cells, and the extent to which these fluxes are inhibited by amiloride and ouabain respectively; and (3) quantitation of short circuit current (Isc) across confluent monolayers of ATII cells mounted in Ussing chambers. The effects of reactive oxygennitrogen intermediates are then assessed by measuring changes in these variables following exposure of ATII cells to -NO, ONOO'. Results of studies from various laboratories are discussed below. a. Patch clamp studies: This technique relies on the formation of an electrically tight seal between a glass micropipette and the membrane, with a resistance around 5-50 Gft (1 gigaohm = 109 Q). Jain et al [37] isolated rat ATII cells to high purity, grew them on transparent filters and patched them in the cell attached mode. The authors found that most ATII cells expressed cation channels, equally selective to Na* and K* ions with a conductance of 20 pS. Addition of S-nitrosoglutathione (GSNO) and S-nitroso-Nacetylpenicillamine (SNAP), agents that generate oxides of nitrogen, into the bath, increased ATII cell cGMP content and significantly reduced the open probability (Po) this channel; pretreatment of ATII cells with a PKG inhibitor prevented the inhibitory effects of GSNO on this channel; incubation of ATII cells with a cell-permeable analogue of cGMP (8-BrcCMP) also decreased the Po. They concluded that »NO decreased the activity of this channel by activating a cGMP-dependent protein kinase. It should be noted however that GSNO has been shown to alter ion channel function by nitrosating channel proteins[38]. Only a small fraction of ATII cells patched in the cell-attached mode have single channel activity. Furthermore, the contribution of various types of channels with different conductances and densities to overall Na+ transport is difficult to ascertain. Additional information can be obtained from measurements of current-voltage relationships across the entire ATII cell membranes. In this case, after the cell is patched in the cell attached mode, an electrical pulse and or suction is applied rupturing the cell membrane and allowing equilibration between the contents of the pipette and cytoplasm. Movements of ions into or out of the cytoplasm, in response to altering the membrane potential, create inward and outward currents respectively. Lazrak et al. [39] reported that A459 cells, an adenocarcinoma cell line which has some properties similar to those of ATII cells, when patched in the whole cell mode, exhibited significant levels of amiloride-sensitive Na* currents. Perfusion of A549 cells with PAPANONOate (a «NO donor) decreased the amiloride-sensitive component of the Na+ whole cell current in a rapid and reversible fashion (Figure 1).

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Figure 1. (A) Time course recording of whole-cell inward (Na+) current, evoked by -100 mV voltage pulses every 10 s across an A549 cell. Initially, the cell was perfused with a solution containing (in mM): NaCl 145, KC1 2.7, CaCl2 1.8, MgCl2 2, Glucose 5.5, N- [2-HydroxyEthyl] Piperazine-N'- [2-EthaneSulfonic acid] (HEPES) 10, pH 7.4, 323 milliosmoles). At the interval indicated by the horizontal bar, the solution was changed to one containing 100 uM PAPANONOate. The pipette was filled with a solution containing: (mM): K-methylsulphonic acid 135, KC1 10, NaCl 6, Mg2ATP 1, Na3ATP 2, glucose 5.5, HEPES 10, EGTA 0.5. Whole-cell I-V relations before (B) and five min post PAPANONOate perfusion, when the steady state currents were seen (C) are shown. The whole-cell current inhibited by NO (NO sensitive) was calculated by digitally subtracting the currents at the steady state effect of NO (as shown in C) from the current before the perfusion with NO containing SES (as shown in B). Mean I-V relationships for the total and NO-sensitive currents are shown in panel D. For panel D, values are means ± 1 SEM (n=6). (from reference [39] by permission).

Significant inhibition was seen with as low as 300 nM »NO, well below the levels expected to be present in the alveolar epithelial space during lung inflammation. Pre-incubation of A549 cells with 3 uM ODQ (a potent inhibitor of soluble guanyl cyclase) for 30 min prior to perfusion with NO donors, totally prevented the reduction of the Na+ currents. As in the studies of Jain et al. [37], these authors also found that perfusion of A549 cells with 100 uM 8-Br-cGMP markedly inhibited the inward (Na+) but not the outward (K+) currents. In additional studies, they also demonstrated that -NO release by PAPANONOate markedly decreased single channel activity in the cell-attached patches of A549 cells [39]. These results are in agreement with in vivo data showing that intratracheal instillation of «NO donors in rabbits decreases levels of amiloride-sensitive transport in vivo [40] (see below). However, intratracheal instillation of dibutyryl-cGMP (1 mM) in anesthetized rats increased the amiloride-insensitive fraction of Na+ transport across the alveolar epithelium [41]. Thus •NO may be inhibiting Na+ transport via cGMP-independent pathways. Duvall et al. [42] reported that peroxynitrite, but not »NO, decreased amiloride sensitive currents across Xenopits oocytes injected with cDNA's of a, p and y ENaC, the three subunits of the amiloride sensitive sodium channel. Thus, RONS may damage either Na+ channel proteins directly or cytoskeletal proteins (such as actin and fondrin) which are required for proper Na+ channel function [43,44]. b. Short-circuit current measurements: Guo et al. [45] examined the mechanisms by which »NO decreased vectorial Na+ transport across confluent monolayers of rat ATII cells grown on permeable supports. Two different sets of experiments were performed: In the first, spontaneous potential difference (PD) and transepithelial resistance

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KM. Hardiman etal. /Modulation of Active Alveolar Na* Transport

(Rt) across ATII cell monolayers were measured daily. Forty-five minutes following addition of either of two »NO donors (spermine NONOate (200 uM) or PAPANONOate (100 uM) in the apical and basolateral media bathing the monolayers, the equivalent current (leq; calculated from the Ohm's law under open-circuit conditions) was decreased by -80% whereas the Rt increased by -30%, consistent with the inhibition of a conductive pathway across the epithelium. Under these conditions, the effect of -NO on the Ieq was concentration dependent (ICso = 0.4 uM), well within the concentrations of »NO likely to be present in the alveolar epithelial lining fluid during inflammation. In the presence of the »NO scavenger oxy-hemoglobin (50 uM), -NO donors did not increase »NO levels above baseline and Ieq remained at baseline. Moreover, the Ieq recovered when monolayers were treated with oxyhemoglobin 45 min after administration of the »NO donors (Figure 2). These findings demonstrated that »NO specifically inhibited transepithelial Na+ transport across ATII monolayers.

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