Abstracts
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A Rapid Screening Method for Compounds Displaying Synergy for the Treatment of Lung Fibrosis
Polymorphisms in the IL-1 Gene Family and Lung Disease Severity in Patients with Cystic Fibrosis
Theodore J. Kottom1 and Andrew H. Limper1
Hara Levy1,2,3, Amy Murphy2,4, Fei Zou6, Craig Gerard1,2, Barbara J. Klanderman3, Brooke Schuemann3, Ross Lazarus2,3, K. Christopher Garcı´a3, Juan C. Celedo´n2,3, Mitch Drumm7, Mary K. Dahmer8, Michael W. Quasney8, Michael R. Knowles5, Gerald B. Pier2,3, Christoph Lange2,4, and Scott T. Weiss2,3
1 Thoracic Diseases Research Unit, Pulmonary and Critical Care Division, Mayo Clinic College of Medicine, Rochester, Minnesota
Effective treatments for idiopathic pulmonary fibrosis (IPF) are not yet available. Current concepts indicate that multi-agent synergistic drug regimens will be needed to control this lethal condition. The available animal models for lung fibrosis are laborious, variably reproducible, and do not faithfully represent the histopathologic features of IPF. To address this, we developed a new in vitro assay to screen potential drugs for synergistic activity against lung fibrosis. Our group was among the first to implicate transforming growth factor-b1 (TGF-b1) as a principal growth factor in mediating IPF. Additional data indicate roles for platelet-derived growth factor (PDGF) and tumor necrosis factor-a (TNF-a) in the fibrotic process. Accordingly, we cultured IMR90 human lung fibroblasts in the presence of cytokines, such as TGF-b1 (5 ng/ml). The generation of extracellular matrix fibronectin and collagen 1 was quantified by real-time PCR using the iCycler system (Bio-Rad, Hercules, CA), and confirmed with enzyme-linked immunosorbent assay. With this approach we undertook studies of oral agents with activity against cytokinedriven fibrosis. Addition of pentoxifylline (an anti–TNF-a agent) and pirfenidone (an agent with activity against TGF-b1 and PDGF), each individually significantly reduced collagen and fibronectin expression. However, together the combination yielded nearly complete suppression of cytokine-stimulated matrix expression. This synergistic regimen also displayed no abnormal toxicity on the cultured Institute for Medical Research (IMR) lung fibroblasts. Currently, we are testing another drug combination that includes the PPAR-g agonist, Rosiglitazone, along with pentoxifylline, and have shown similar synergistic effects to that of pirfenidone and pentoxifylline. Finally, for both drug combinations Western blotting with a-smooth muscle actin was conducted to determine if these two drug combinations prevented fibroblast to myofibroblast differentiation phenotype. Results from these experiments showed no effect on a-smooth muscle protein levels, suggesting that these drug combinations are acting downstream and independently of SMADs. In conclusion, we believe that this rapid screening approach will be useful to efficiently identify drug combinations with greater activity against fibrosis. Selected combinations can be subsequently tested in available animal models, and ultimately recommended for clinical trials in humans. Conflict of Interest Statement: Neither author has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
(Received in original form August 29, 2007; accepted in final form October 16, 2007) Supported by funds from the Robert N. Brewer Family Foundation. Reprints are not available for this article. Correspondence should be addressed to Andrew Limper, M.D., 8-24 Stabile Building, Mayo Clinic, Rochester, MN 55905. E-mail:
[email protected] Proc Am Thorac Soc Vol 5, 2008 Internet address: www.atsjournals.org
1
Division of Pulmonary Medicine, Children’s Hospital Boston, Massachusetts; 2Harvard Medical School, Boston, Massachusetts; 3 Channing Laboratory, Brigham and Women’s Hospital, Boston, Massachusetts; 4Department of Biostatistics, Harvard School of Public Health, Boston, Massachusetts; 5Division of Pulmonary and Critical Care Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina; 6Department of Biostatistics, University of North Carolina School of Public Health, Chapel Hill, North Carolina; 7 Case Western Reserve University, Cleveland, Ohio; and 8Department of Pediatrics and Critical Care, Children’s Hospital of Wisconsin, Milwaukee, Wisconsin
Variability in pulmonary disease severity is found in patients with cystic fibrosis (CF) who have identical mutations in the CF transmembrane conductance regulator (CFTR) gene. We hypothesized that one factor accounting for heterogeneity in pulmonary disease severity is variation in the family of genes affecting the biology of interleukin-1 (IL-1), which impacts acquisition and maintenance of Pseudomonas aeruginosa infection in animal models of chronic infection. We genotyped 58 single-nucleotide polymorphisms (SNPs) in the IL-1 gene cluster in 808 subjects with CF from the University of North Carolina (UNC) cohort. All were homozygous for DF508, and categories of ‘‘severe’’ (cases) or ‘‘mild’’ (control subjects) lung disease were defined by the lowest or highest quartile of FEV1 for age. After adjustment for age and sex, genotypic data were tested for association with lung disease severity. Odds ratios comparing severe versus mild CF were also calculated for each genotype (with the homozygote major allele as the reference group) for all 58 SNPs. From these analyses, nine SNPs with a moderate effect size, odds ratio (OR) greater than 1, were selected for further testing. To replicate the case–control study results, we genotyped the same nine SNPs in a second population of CF parent–offspring trios in which the offspring had similar pulmonary phenotypes and were recruited from Children’s Hospital, Boston. For the trio analysis, family-based association was performed. SNPs rs1143634 and rs1143639 in the IL1B gene demonstrated a consistent association with lung disease severity categories (P , 0.10) and longitudinal analysis of lung disease severity (P , 0.10) in CF in both the case–control and familybased studies. IL1b is a clinically relevant modulator of CF lung disease. Conflict of Interest Statement: H.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.J.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.C.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.C.C. does not have a financial relationship with a commercial entity that has an interest in the
374 subject of this manuscript. M.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.K.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.W.Q. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.R.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.B.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.T.W. received a grant for $900,065, Asthma Policy Modeling Study, from AstraZeneca from 1997 to 2003. He has been a Co-investigator on a grant from Boehringer Ingelheim to investigate a COPD natural history model which began in 2003. He has received no funds for his involvement in this project. He has been an advisor and chair of the Advisory Board to the TENOR Study for Genentech and has received $10,000 for 2005 to 2006. He received a grant from Glaxo-Wellcome for $500,000 for genomic equipment from 2000 to 2003. He was a consultant for Roche Pharmaceuticals in 2000 and received no financial remuneration for this consultancy. He has also served as a consultant to Pfizer (2000–2003), Schering Plough (1999–2000), Variagenics (2002), Genome Therapeutics (2003), and Merck Frost (2002).
(Received in original form August 31, 2007; accepted in final form October 16, 2007) Correspondence and requests for reprints should be addressed to Hara Levy, M.D., Assistant Professor of Pediatrics, Section of Pulmonary and Sleep Medicine, Children’s Hospital of Wisconsin, Children’s Research Institute, Translational Biomedical Research Center, Medical College of Wisconsin, P.O. Box 26509, Milwaukee, WI 53226. E-mail:
[email protected] Proc Am Thorac Soc Vol 5, 2008 Internet address: www.atsjournals.org
PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL 5
2008
However, VEGF-deficient lungs in vivo are able to maintain surfactant phospholipid levels, but not SP-D expression. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
(Received in original form August 29, 2007; accepted in final form October 16, 2007) Supported by TRDRP 12RT-0062, NIH PO1 HL 17731-28 (Peter D. Wagner), and the Parker B. Francis Foundation. Correspondence and requests for reprints should be addressed to Ellen C. Breen, Ph.D., Division of Physiology, Department of Medicine, University of California, 9500 Gilman Drive, San Diego, La Jolla, CA. E-mail:
[email protected] Proc Am Thorac Soc Vol 5, 2008 Internet address: www.atsjournals.org
Interleukin-1 Receptor Antagonist Intron 2 Polymorphism and Lung Injury in Children with Community-acquired Pneumonia Mary K. Dahmer1, Pallavi P. Patwari2, Peggy A. O’Cain3, Denise M. Goodman2, Julia Krushkal4, Grant W. Somes4, Caiqin Liu4, Melita E. Smith1, and Michael W. Quasney1 1
Vascular Endothelial Growth Factor Is Essential for Maintaining Pulmonary Surfactant Protein D Expression In Vivo Kechun Tang1, Jaret L. Malloy1, and Ellen C. Breen1 1 Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California
The role of vascular endothelial growth factor (VEGF) to regulate surfactant homeostasis was investigated after gene inactivation of murine pulmonary VEGF in isolated type II cells and in vivo. VEGFLoxP type II cells incubated in vitro with cre recombinase adenovirus (Adv/Cre) demonstrated reduced VEGF levels from Days 2 to 5. In vitro VEGF-inactivated type II cells revealed a decrease in total phospholipid, disaturated phosphatidyl choline (DSPC), and DSPC synthesis compared with control uninfected or Adv/LacZ cells. Phorbol myristate acetate (PMA)-induced DSPC secretion was increased in Adv/ Cre-infected cells. The levels of surfactant-associated proteins (SP)-B and -D, and the lipid transporters, ABCA1 and Rab3D, were also decreased in VEGF-deficient type II cells in vitro. These VEGF-dependent changes in surfactant metabolism were accompanied by a 425% increase in apoptotic cells compared with control type II cells. In contrast to these in vitro observations, lung-targeted VEGF gene deletion in vivo did not alter alveolar surfactant or tissue DSPC levels. Further, analysis of alveolar surfactant phospholipid composition revealed a decrease in sphingomyelin. In addition, SP-D expression was decreased in VEGF-deficient lungs. In vitro data suggest a role for VEGF in regulating type II cell surfactant metabolism and apoptosis.
Children’s Research Institute, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin; 2Department of Pediatrics, Northwestern University, Chicago, Illinois; and Departments of 3Pediatrics and 4Preventive Medicine, University of Tennessee Health Science Center, Memphis, Tennessee
An overzealous inflammatory response in some children with pneumonia may lead to the development of more severe lung disease. The inflammatory response is modulated in part by members of the interleukin-1 family. Genetic variations in the interleukin-1 family of genes are associated with variability in inflammatory response and may be involved in the variable degree of lung injury observed in children with pneumonia. Blood samples were obtained from children with community-acquired pneumonia (CAP) who met specific inclusion criteria and had a complete blood count as part of their workup. Genotypes of the variable numbers of tandem repeat (VNTR) polymorphism in intron 2 of the interleukin-1 receptor antagonist (IL-1ra) gene were determined and did not deviate from Hardy-Weinberg equilibrium. Genotypic frequencies of children who required positive pressure ventilation (PPV) were compared with those who did not require PPV. Multivariate logistic regression analysis was performed. Eight hundred fifty children (aged 14 d to 19 yr) with CAP were enrolled; analysis was limited to African American (515) and white (232) patients. Eighty-two subjects required PPV. Patients without a copy of the IL-1ra A1 allele were more likely to need PPV compared with those with 1 or 2 copies of this allele (odds ratio, 3.66; confidence interval, 1.41–9.54). The protective effect of the A1 allele persisted when patients with pulmonary comorbidities (e.g., asthma, chronic lung disease) were excluded from the analysis. Carriage of the A1 allele of the VNTR polymorphic site in intron 2 of IL-1ra is associated with protection from the need for positive pressure ventilation in children with CAP.
