National Heart, Lung, and Blood Institute program in
Acute Lung Injury
Specialized Centers of Clinically Oriented Research (ALI SCCOR)

Project 1:  Host determinants of the inflammatory response

Principal Investigators:  Thomas R. Martin, MD and Mark M. Wurfel, MD, PhD

The major scientific goals of this project are to determine the molecular mechanisms controlling inter-individual variability in inflammatory responses to bacterial products, to characterize the role of specific genes in determining this variability, and to determine whether nucleotide variability within these genes explains a portion of the variability seen in the clinical syndromes such as sepsis and ALI/ARDS. The primary hypothesis behind this proposal is that genetic variation that influences in vitro inflammatory responses to bacterial products will influence clinical outcomes in the severe inflammation seen in sepsis and ALI/ARDS. The proposed studies will employ inflammatory responses to bacterial lipopolysaccharide (LPS) ex vivo, an intermediate phenotype likely to contribute a portion of the risk of an individual to the development of sepsis and ALI/ARDS, as a probe to study this variability.

The primary hypothesis of the studies is that factors governing inter-individual variability in host inflammatory responses to bacterial products in vitro also govern a portion of the variability observed in clinical outcomes in sepsis-associated ARDS.

Aim 1:     Identify differences in gene and protein expression in whole blood from individuals who demonstrate hypo or hyper responsiveness to LPS in vitro. 

Aim 2:     Investigate the hereditary component of inter-individual variation in LPS-induced  cytokine responses.

 Aim 3:     Identify associations between LPS responses and SNP allelic haplotypes in genes known to be involved in recognition of LPS and subsequent signaling, and those identified through expression profiling in Aim 1.

 Aim 4:     Identify associations between SNP haplotypes within candidate genes involved in the LPS-recognition and signaling pathway, and clinical outcomes in sepsis-associated ARDS.

 PERFORMANCE SITES 

• University of Washington Medical School, Seattle, WA 
• Veterans Affairs Puget Sound Medical Center, Seattle, WA*
  *One of the research and teaching institutions within the University of Washington system.

KEY PERSONNEL

Name	Organization	Role on Project
MARTIN, Thomas R.	University of Washington	Principal Investigator
WURFEL, Mark	University of Washington	Co-Principal Investigator
RUSSELL, James A.	University of British Columbia	Co-Investigator
JARVIK, Gail P.	University of Washington	Consultant
NICKERSON, Deborah A.	University of Washington	Consultant
BUCHWALD, Dedra S.	University of Washington	Consultant
GOLDBERG, Jack	University of Washington	Co-Investigator

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Project 2:  Improving long-term outcomes in survivors of ALI/ARDS

 Principal Investigator:  Gordon D. Rubenfeld, MD

The overall objective of this project is to create an effective multi-disciplinary intervention that improves health related quality of life, reduces post-traumatic stress, and increases functional status in survivors of acute lung injury.  Current estimates suggest that over 200,000 people in the U.S. each year are afflicted with acute lung injury and the acute respiratory distress syndrome (ALI/ARDS) and that this number will increase as the population ages.  Modern intensive care has reduced hospital mortality in acute lung injury creating more survivors who suffer from depression, post-traumatic stress, weakness, and dyspnea that impair their health related quality of life.  Currently, there is little evidence to guide clinicians on techniques to reduce the morbidity suffered by ALI/ARDS survivors.  The specific aims of this project are (1) To refine and validate a case-management tool to improve the health related quality of life, reduce psychological symptom burden, and increase functional status in survivors of acute lung injury; (2) To evaluate the effectiveness of a case-manager based intervention implementing the tool developed in Aim 1 to improve the health related quality of life, reduce psychological symptom burden, and increase functional status in survivors of acute lung injury; and (3) To identify risk factors for reduced health related quality of life, increased psychological symptom burden, and poor functional status in survivors of acute lung injury.

The experimental approach is a randomized clinical trial of an intervention consisting of a nurse practitioner and social worker supported by a pulmonologist, psychiatrist, and physiatrist who will employ the post-ALI/ARDS case-management tool to identify and treat complications of ALI/ARDS and critical illness.  Treatment will consist of specific therapies provided by the intervention team as well as treatments that are provided within the existing healthcare system by targeted patient referral and follow-up that is managed by the intervention team.

The healthcare needs of survivors of ALI/ARDS have not been addressed.  An effective intervention developed by this project could improve the health related quality of life for thousands of ALI/ARDS survivors and their families each year. 
 

PERFORMANCE SITES University of Washington Medical Center, Seattle, WA Harborview Medical Center, Seattle, WA Virginia Mason Medical Center, Seattle, WA Swedish Medical Center, Seattle, WA Providence Medical Center, Seattle, WA University of Toronto, Toronto, Ontario, Canada
KEY PERSONNEL.
Name	Organization	Role on Project
RUBENFELD, Gordon D	University of Washington	Principal Investigator
CALDWELL, Ellen	University of Washington	Co-investigator
CHAN, Leighton	University of Washington	Co-investigator
DALY, Barbara	Case Western University	Consultant
MITCHELL, Pamela	University of Washington	Consultant
ZATZICK, Douglas	University of Washington	Co-investigator

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Project 3:  Acute lung injury clinical trials incubator unit

Principal Investigator:  Avery B. Nathens, MD, PhD

     Our understanding of the mechanisms underlying the induction, maintenance and resolution of the alveolar inflammatory response in acute lung injury is evolving.  Hypotheses about mechanisms of lung injury have led to a number of clinical trials testing innovative therapies based on experimental models of lung injury, yet none of these have had important biologic or clinical effects in humans.  The failure of many of these clinical trials shows that there is a gap between in vitro or animal studies, and large, expensive phase II/III trials that use clinically important endpoints.  This project is to develop a mechanism to better establish “proof of concept” in humans either at risk for, or with established lung injury.  Proof of concept studies would show that the intervention affects the biologic endpoint(s) of interest in humans and lay the foundation for larger trials of clinical efficacy. 

     The overall objective of this project is to develop a clinical research unit, which we refer to as a Clinical Trials Incubator Unit (CTIU).  The CTIU is a collaborative, multidisciplinary research program with two principal aims: 1) To translate biologically plausible interventions directed at either reducing lung injury or hastening repair into therapeutic strategies of potential clinical benefit using plausible biological endpoints in focused clinical trials to establish proof of concept in humans; and 2)  To use new technological advances in proteomics and genomics to identify patterns of protein and gene expression in the lungs before and after the onset of acute lung injury to better define subpopulations with acute hypoxemic respiratory failure that would benefit from innovative therapies

     The fulfillment of these specific aims would put in place the infrastructure and processes to identify interventions and provide an estimate of the magnitude and direction of any biologic or clinically significant effect, while defining subpopulations with lung injury that are most likely to benefit.  Taken together, this process will serve to “incubate” concepts in preparation for the design and implementation of Phase II/III clinical trials organized through large clinical research networks. 

Project 3 Scientific Advisory Committee

PERFORMANCE SITES  University of Washington School of Medicine, Seattle, WA Harborview Medical Center, Seattle, WA
KEY PERSONNEL.
Name	Organization	Role on Project
NATHENS, Avery B.	University of Washington	Principal Investigator
HUDSON, Leonard D.	University of Washington	Co-Investigator
NEFF, Margaret J.	University of Washington	Co-Investigator
BROUDY, Virginia	University of Washington	Member, CTIU Advisory Committee
CLARK, Joan G.	University of Washington	Member, CTIU Advisory Committee
DELLINGER, E. Patchen	University of Washington	Member, CTIU Advisory Committee
MARSHALL, John C.	University of Toronto	Member, CTIU Advisory Committee
MATTHAY, Michael A.	U of California, San Francisco	Consultant
O’KEEFE, Grant E.	University of Washington	Member, CTIU Advisory Committee
SLUTSKY, Arthur S.	University of Toronto	Member,CTIU Advisory Committee

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Project 4:  Early host: microbial interactions in S. aureus pneumonia

 Principal Investigators:  Craig E. Rubens, MD, PhD and Shawn J. Skerrett, MD

Staphylococcus aureus is the second leading cause of hospital-acquired pneumonia and an important agent of severe community-acquired pneumonia. Pneumonia is both an important preceding event to acute lung injury and the most commonly fatal complication of ARDS. We hypothesize that early host: pathogen interactions in the lung determine whether S. aureus establishes a successful infection or is eliminated by innate defenses.

There is a limited understanding of the pathogenesis of pneumonia, especially the initial microbial: host interaction. Using state of the art in vivo model systems and genomic/proteomic technology we are investigating the early microbial and host responses in the first few hours after bacterial entry into the lungs. The Specific Aims for this project are:  Aim 1: to characterize changes in bacterial gene expression using genomic array analysis in response to the lung environment during the first few hours of infection.  Aim 2: examine the host proteins in airway fluid, which bind to the bacterial surface and correlate these to changes in gene expression examined over the same time frame as observed in Aim 1; characterize the mouse airway fluid proteome, which has yet to be characterized. These studies may identify the host proteins that serve as signals to the organism during the initial phases of infection and further define those which serve as innate defenses against infection for the host.  Aim 3: determine the role of Toll-Like Receptors (TLR) on the interaction of S. aureus with the host, using animals deficient in TLR expression; specifically defective for expressing the downstream signaling molecule MyD88.  These studies will evaluate the importance of the initial inflammatory response to bacterial survival and host immunity.  Aim 4: examine the importance of the bacterial surface associated protein genes with antisense gene inhibition technology in our animal model of pneumonia, to identify those genes and their products which are primarily involved in the initial interactions in the airway.  These studies will yield novel insights into bacterial pathogenesis in the lower respiratory tract by examining both sides of the host: pathogen interface in the early stages of infection. These insights could have important implications for the mechanisms underlying the initiation of pneumonia, acute lung injury, and its sequelae.

PERFORMANCE SITES  University of Washington Medical Center, Seattle, WA Harborview Medical Center, Seattle, WA Children’s Hospital and Regional Medical Center, Seattle, WA
KEY PERSONNEL.
Name	Organization	Role on Project
RUBENS, Craig E.	Children’s Hospital and Regional Medical Center	Principal Investigator
SKERRETT, Shawn J.	University of Washington	Co-Principal Investigator
DUNMAN, Paul M.	Wyeth-Ayerst Research, Pearl River, NY	Consultant
IANDOLO, John J.	University of Oklahoma Health Sciences Center	Consultant
LIGGETT, Denny H.	University of Washington	Consultant
PROJAN, Steven J.	Wyeth-Ayerst Research, Pearl River, NY	Consultant

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Project 6:  Pathogenesis of sepsis-induced lung injury

 Principal Investigator:  W. Conrad Liles, MD, PhD 

The major scientific goal of this project is to determine the pathogenic mechanisms responsible for sepsis-induced acute lung injury (ALI), a leading clinical cause of the acute respiratory distress syndrome (ARDS). Laboratory investigations are being conducted in vivo to elucidate basic immunologic and inflammatory mechanisms of sepsis-related lung injury, including the role of mechanical ventilation in this process. This research project places special emphasis on the roles of apoptosis, especially those mediated by Fas (CD95), and MyD88-dependent in ALI pathogenesis.

The specific aims are: 1) to determine whether mechanical ventilation interacts with sepsis in vivo to synergistically induce ALI; 2) to determine if sepsis and mechanical ventilation promote pro-apoptotic responses in the lung that contribute to ALI; 3) to determine whether mechanical ventilation synergizes with sepsis to induce ALI via potentiation of MyD88-dependent pro-inflammatory mechanisms.

The experimental approach:  Murine models of sepsis are employed to address the specific aims. The relative roles of the Fas apoptotic and the MyD88-dependent pro-inflammatory are assessed in experiments with lpr (Fas-deficient) and MyD88^-/- mice, respectively.

Expected results/significance:  Sepsis alone is not expected to cause significant ALI but is anticipated to prime the lung for tissue injury in response to mechanical ventilation.  Sepsis-induced/ventilator-associated ALI is expected to involve apoptosis of the lung epithelium and MyD88-dependent pro-inflammatory responses.  The experimental approach is unique in that it utilizes a murine model of extra-pulmonary sepsis to study the pathogenesis of ALI and the modulating effects of concomitant mechanical ventilation.  Results are expected to yield fundamental insights into the basic pathogenic mechanisms of sepsis-induced ALI and provide the basis for development of novel anti-apoptotic and anti-inflammatory therapeutic strategies for ALI/ARDS. 

PERFORMANCE SITES  University of Washington School of Medicine, Seattle, WA Veterans Affairs Puget Sound Medical Center, Seattle, WA* *One of the research and teaching institutions within the University of Washington system.
KEY PERSONNEL.
Name	Organization	Role on Project
LILES, W. Conrad	University of Washington	Principal Investigator
ALTEMEIER, William A.	University of Washington	Co-investigator
FREVERT, Charles W.	University of Washington	Co-investigator
MATUTE-BELLO, Gustavo	University of Washington	Co-investigator
SLUTSKY, Arthur	University of Toronto	Consultant

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Biotechnology Core

 Principal Investigator:  Thomas R. Martin, MD 

The major objective of the Biotechnology Core is to provide state-of-the-art methodology in histologic methods, gene expression, and protein analysis to support the clinical and basic projects of the SCCOR.  Dr. Martin will provide overall direction for the Core and will be assisted by Charles Frevert, DVM, ScD, Scientific Director of the Pulmonary Research Laboratories at VA Puget Sound Medical Center.  Core B brings together investigators with state-of-the-art experience in tissue analysis, microarray analysis of gene expression, protein chemistry and proteomics to support the SCCOR clinical and basic investigators.

 The Specific Aims of the Core are:

1.      To provide histologic analysis of tissue specimens provided by the basic science projects.

2.      To provide gene expression analysis for the clinical and basic projects using the expertise and facilities of the University of Washington Microarray Facility, R. Bumgarner, PhD, Director.

To provide:  a) multiplex protein analysis by immunoassay in the Core Laboratory; b) analysis of specific protein modifications in the Protein Analysis Core Facility, University of Washington Department of Medicine; c) complex analysis of proteins in clinical and laboratory samples using proteomics technology available at the Institute for Systems Biology, Seattle, WA. 

PERFORMANCE SITES  University of Washington School of Medicine, Seattle, WA Harborview Medical Center, Seattle, WA VA Puget Sound Medical Center, Seattle, WA Institute for Systems Biology, Seattle, WA
KEY PERSONNEL.
Name	Organization	Role on Project
MARTIN, Thomas R.	University of Washington	Principal Investigator
BASKIN, Denis G.	University of Washington	Consultant
BUMGARNER, Roger E.	University of Washington	Co-Investigator
FREVERT, Charles W.	University of Washington	Co-Investigator
FU, Xiaoyun	University of Washington	Co-Investigator
HEINECKE, Jay	University of Washington	Co-Investigator
LIGGITT, Denny H.	University of Washington	Consultant
SCHNAPP, Lynn	University of Washington	Co-Investigator

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Translational Research Training Program:
Clinical Research Skills Development Core

 Principal Investigators: Lynn M. Schnapp, Gordon Rubenfeld, Richard Goodman, Leonard D. Hudson

Core C, Clinical Research Skills Development Core is a training core with the Specific Aim of developing translational investigators in acute lung injury and related research areas. The philosophy behind our training plan is that a translational investigator will have a primary research discipline, either in basic research or clinical outcomes research, and will enhance this with cross-training (but not equal training) in the other discipline.  It is our hypothesis that this training will markedly improve the translation of basic research advances into clinical benefits for patients with acute lung injury. 

PERFORMANCE SITES  University of Washington School of Medicine, Seattle, WA Harborview Medical Center, Seattle, WA
KEY PERSONNEL.
Name	Organization	Role on Project
HUDSON, Leonard D.	University of Washington	Principal Investigator
GOODMAN, Richard B.	University of Washington	Consultant
RUBENFELD, Gordon D.	University of Washington	Co-Investigator
SCHNAPP, Lynn M.	University of Washington	Co-Investigator

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University of Washington ALI SCCOR Publications in Years 1-2

 PROJECT 1

 Manuscripts

Wurfel MM, Park WY, Radella F, Ruzinski J, Sandstrom A, Strout J, Bumgarner RE, Martin TR.  Identification of high and low responders to lipopolysaccharide in normal subjects: an unbiased approach to identify modulators of innate immunity.  J of Immunol 2005 (in press). 

Abstracts

Wurfel MM, Holden TD, Ruzinski JT, Radella F, Black RA, Martin TR.  Relationship of IRAK4 haplotypes to innate immune inflammatory responses.   Am J Respir Crit Care Med 2005;2:A904.

PROJECT 3

Manuscripts

Swenson ER. Therapeutic hypercapnic acidosis: pushing the envelope.  Am J Respir Crit Care Med 2004;169:8-9.

 Abstracts

Ruzinski JT, Radella F, Wurfel MM, Martin TR.  A new method to measure cytokines in biological fluids: comparison of bead based and conventional assays.   Am J Respir Crit Care Med 2005;2:A74.

Hagimoto N, Matute-Bello G, Kajikawa O, Goodman RB, Park DR, Martin TR.  Modulation of soluble Fas ligand bioactivity in bronchoalveolar lavage fluid from patients with ARDS.  Am J Respir Crit Care Med 2005;2:A579.

PROJECT 4

 Manuscripts

Skerrett SJ, Liggitt HD, Hajjar AM, Wilson CB.  Cutting edge: myeloid differentiation factor 88 is essential for pulmonary host defense against Pseudomonas aeruginosa but not Staphylococcus aureus.  J Immunol 2004;172:3377-3381.

 Skerrett SJ, Liggitt HD, Hajjar AM, Ernst RK, Miller SI, Wilson CB.  Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin.  Am J Physiol: Lung Cell Mol Physiol 2004;287:L143-L152.

 Abstracts

None

PROJECT 6

 Manuscripts

Altemeier WA, Matute-Bello G, Frevert CW, Kawata Y, Kajikawa O, Martin TR, Glenny RW.  Mechanical ventilation with moderate tidal volumes synergistically increases lung cytokine response to systemic endotoxin.  Am J Physiol: Lung Cell Mol Physiol 2004; 287:L533-542.

Matute-Bello G, Liles WC, Frevert CW, Dhanireddy S, Nakamura M, Ballman K, Wong V, Song HY, Witcher DR, Jakubowski JA, Martin TR. Blockade of the Fas/FasL system by improves pneumococcal clearance from lung without preventing bacteremic dissemination to the spleen. J Infect Dis 2005;191:596-606.

Matute-Bello G, Lee JS, Liles WC, Frevert CW, Sutlief S, Martin TR. Fas-mediated acute lung injury requires membrane Fas expression on non-myeloid cells of the lungs. Revision submitted to J Immunol.

Altemeier WA, Matute-Bello G, Gharib SA, Glenny RW, Martin TR, Liles WC.  Mechanical ventilation modulates lung transcriptional response to LPS and promotes lung injury. Manuscript submitted for publication.

Dhanireddy S, Altemeier WA, Matute-Bello G, Glenny RW, Martin TR, Liles WC. ‘Non-injurious’ mechanical ventilation augments intra- and extra-pulmonary inflammation, and promotes acute lung injury, in experimental bacterial pneumonia. Manuscript submitted for publication. 

Three other manuscripts are currently in preparation for publication.

Abstracts

Altemeier WA, Matute-Bello G, Glenny RW, Martin TR, Liles WC.  Mechanical ventilation augments the inflammatory response to lipopolysaccharide in the lung.  Am J Respir Crit Care Med 2004;169:A70.  

Matute-Bello G, Lee JS, Liles WC, Frevert CW, Sutlief S, Martin TR.  Fas-mediated acute lung injury requires membrane Fas expression on non-myeloid cells of the lungs.  Am J Respir Crit Care Med 2004;169:A875. 

Dhanireddy S, Altemeier WA, Matute-Bello G, Glenny RW, Martin TR, Liles WC. Mechanical ventilation augments lung inflammation in bacterial pneumonia.  Am J Respir Crit Care Med 2005;2:A91.

Altemeier WA, Liles WC, An D, Glenny RW. Cytokine expression in lung reperfusion injury is partially medicated by MyD88-dependent signaling.  Am J Respir Crit Care Med 2005;2:A861. 

Gharib SA, Matute-Bello G, Liles WC, Glenny RW, Altemeier WA. Identification of key molecular pathway and transcription factors in a mouse model of acute lung injury.  Am J Respir Crit Care Med 2005;2:A886. 

Tanino Y, Baker C, Mongovin S, Koski A, Wong V, Kajikawa O, Martin TR, Frevert CW.  Modulation proteoglycans in lungs of mice and rabbits with Escherichia coli pneumonia.  Am J Respir Crit Care Med 2005;2:A74.

CORE B

 Manuscripts

Medvedovic M, Yeung KY, Bumgarner RE.  Bayesian mixture model based clustering of replicated microarray data.  Bioinformatics 2004;20:1222-1232.

Yeung KY, Medvedovic M, Bumgarner RE.  From co-expression to co-regulation: how many microarray experiments do we need?  Genome 2004;5:R48.

Wei C, Li J, Bumgarner RE.  Sample size for detecting differentially expressed genes in microarray experiments.  BMC Genomcs 2004;8:87.

Yeung KY, Bumgarner RE, Raftery AE.  Bayesian model averaging: development of an improved multiclass, gene selection and classification tool for microarray data.  Bioinformatics 2005 (Epub ahead of print).

Bergt C, Heinecke JW.  Oxidized amino acids in proteins and tissues.  In: The Encyclopedia of Mass Spectrometry, Vol 2, Biological Applications (Part A), Peptides and Proteins.  Gross ML, Caprioli RM, eds.  Elsevier, 2005, pp 243-249.

Schnapp LM, Donohue S, Chen JZ, Sunde DA, Kelly PM, Ruzinski J, Martin TR, Goodlet DR. Mining the ARDS proteome: identification of IGF/IGFBP-3 pathway in lung injury. Am J Pathol. Vol 169 2006. In Press

Abstracts

Hayashi S, Wurfel MM, Tsuchiya M, Vaisar T, Bumgarner RE, Heinecke JW, Schnapp L, Martin TR.  Protein profiling of lavage fluid from ARDS patients using two-dimensional difference-in-gel electrophoresis (DIGE).  Am J Respir Crit Care Med 2004;169:A350. 

Hayashi S, Matute-Bello G, Wurfel MM, Frevert CW, Martin TR.  Differential effects of TGF- on FasL induced apoptosis in fibroblasts and distal bronchial epithelial cells.  Am J Respir Crit Care Med 2004;169:A795.  

Schnapp LM, Donohue S, Chin J, Goodlett DR, Aebersold R.  Proteomics for acute lung injury.  Am J Respir Crit Care Med 2004;169:A464.  

Schnapp LM, Ekwa-Ekoba C, Carlson C, Nickerson D, Levy B, Diaz G.  Genomic organization and sequence variation of human integrin alpha 8.  Am J Respir Crit Care Med 2004;169:A855. 

Tanino Y, Ballman K, Baker C, Ruzinski J, Selk A, Wong V, Martin TR, Frevert CW.  The glycosaminoglycan-binding domain of interleukin-8 regulates the pulmonary recruitment of neutrophils.  Am J Respir Crit Care Med 2004;169:A806.   

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