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Final Report: A Model to Study the Development of Persistent Environmental Airway Disease
EPA Grant Number: R826711C004Subproject: this is subproject number 004 , established and managed by the Center Director under grant R826711
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: University of Iowa Children's Environmental Airway Disease Center
Center Director: Hunninghake, Gary W.
Title: A Model to Study the Development of Persistent Environmental Airway Disease
Investigators: Schwartz, David , Hunninghake, Gary W.
Institution: University of Iowa
EPA Project Officer: Fields, Nigel
Project Period: January 1, 1998 through January 1, 2002
Project Amount: Refer to main center abstract for funding details.
RFA: Centers for Children's Environmental Health and Disease Prevention Research (1998)
Research Category: Health Effects , Children's Health
Description:
Objective:This project addressed a fundamental issue in childhood asthma: why only a minority of children who wheeze at an early age develop persistent airway disease that continues throughout their life. Epidemiological studies in children suggest that recurrent episodes of airflow obstruction and airway inflammation result in persistent airway disease. Although thickening and fibrosis of the subepithelial region beneath the basement membrane is a consistent histologic feature of asthma that is related directly to the clinical severity of this disease, the biological factors that lead to a localized fibrotic response following reversible airway inflammation have not been well defined. Understanding the biological relationship between reversible airway inflammation and structural changes of the airway will enhance our understanding of childhood asthma. The overall hypothesis of this investigation is that many of the biologic features of acute and reversible airway inflammation are fundamental to the development of chronic grain dust -induced airway disease. The objective of this research project was to determine which specific elements of the acute inflammatory response to inhaled grain dust are essential to the development of chronic grain dust- induced airway disease, defined as persistent airway hyperreactivity and airway remodeling.
Summary/Accomplishments (Outputs/Outcomes):The following aims were designed to address our overall hypothesis.
Specific Aim 1:
Determine whether the initial inflammatory response to endotoxin, a key biological component of inhaled grain dust, affects the development of chronic grain dust -induced airway disease.
Specific Aim 2:
Determine whether amplification and localization of an inflammatory response in the airway lumen is essential to the development of chronic grain dust- induced airway disease.
Specific Aim 3:
Determine whether anti- inflammatory cytokines (IL-1ra or IL-10) modulate the severity of chronic grain dust -induced airway disease.
We have made substantial progress in our project in the areas described below.
Endotoxin is One of the Principal Components of Grain Dust That Causes Acute Reversible Airflow Obstruction and Neutrophilic Airway Inflammation
To determine if endotoxin responsiveness influences the development of chronic grain dust-induced airway disease, physiologic and airway inflammation/remodeling parameters were evaluated following an 8 -week exposure to corn dust extract (CDE) and again following a 4 week recovery period in a strain of mice sensitive to (C3H/HeBFeJ) and one resistant to endotoxin (C3H/HeJ). Following the CDE exposure, both strains of mice had equal airway hyperreactivity to a methacholine (MCh) challenge, however, airway hyperreactivity persisted only in the C3H/HeBFeJ mice after the recovery period. Only the C3H/HeBFeJ mice showed significant neutrophilic inflammation of the lower airway following the 8 week exposure to CDE. Following the recovery period, this inflammatory response completely resolved. Lung stereologic measurements indicate that an 8 -week exposure to CDE results in persistent expansion of the airway submucosal cross-sectional area only in the C3H/HeBFeJ mice ( Figure 1). Collagen type III and an influx of cells into the subepithelial area participate in the expansion of the submucosa. Our findings demonstrate that subchronic inhalation of grain dust extract results in the development of chronic airway disease only in mice sensitive to endotoxin but not in mice that are genetically hyporesponsive to endotoxin, suggesting that endotoxin is important in the development of chronic airway disease.
Figure 1. Results of 8 Week Exposure to CDE
Subchronic Inhalation of Lipopolysaccharide (LPS) Causes Persistent Airway Disease, Reversible Airway Inflammation and Airflow Obstruction, and Persistent Airway Hyperreactivity and Airway Remodeling
To test the hypothesis that endotoxin alone causes airway remodeling, we have compared the response of two inbred mouse strains to subchronic endotoxin exposure. Physiological and biological parameters were evaluated after 1 day, 5 days, or 8 weeks of exposure to endotoxin (LPS) in endotoxin-sensitive (C3HeB/FeJ) and endotoxin-resistant (C3H/HeJ) mice. After 5 days or 8 weeks of LPS exposure, only C3HeB/FeJ had elevated airway hyperreactivity to inhaled MCh. Only the C3HeB/FeJ mice had significant inflammation of the lower respiratory tract after 1 day, 5 days, or 8 weeks of LPS exposure. Stereological measurements of small, medium, and large airways indicate that an 8 -week exposure to LPS resulted in expansion of the submucosal area only in the C3HeB/FeJ mice. Cell proliferation as measured by BrdU incorporation contributed to the expansion of the submucosa and only was elevated significantly in C3HeB/FeJ mice actively exposed to LPS. C3HeB/FeJ mice had significantly elevated levels of total and active transforming growth factor β1 in whole lung lavage fluid after 5 days of LPS exposure. Our findings demonstrate that subchronic inhalation of LPS results in the development of persistent airway disease in endotoxin responsive mice.
Neutrophils are Essential to Development of LPS -Induced Persistent Airway Hyperreactivity
The role of neutrophils in endotoxin-induced airway disease was evaluated by systemic neutrophil depletion of C3H/HeBFeJ mice and exposing them to 4 week LPS inhalation challenge. Mice were made neutropenic with intraperitoneal injections of anti neutrophil serum (rabbit antimouse neutrophil antiserum; Accurate Chemical & Scientific Corporation) started prior to and continued throughout the whole exposure. Age-matched controls were injected with control serum and exposed to filtered air, respectively. Physiologic, biologic, and morphologic assessments were performed following a 4-week exposure and again following a 4 week recovery. After the 4 week exposure, LPS-induced inflammation of the lower airways was attenuated significantly in the neutropenic mice ( Figure 2; A = nonneutropenic, B = neutropenic), although airway responsiveness (AR) to MCh remained unchanged. Following the recovery period, LPS-exposed neutrophil-replete mice had increased AR to MCh when compared with the LPS-exposed neutropenic animals. Figure 3 illustrates the sensitivity to MCh expressed as the dose required to provoke a 200 percent increase in baseline Penh (ED200 of MCh; * p < 0.005).
Figure 2. LPS-Induced Inflammation of the Lower Airways
Figure 3. Sensitivity to Methacholine
Neutrophils are Critical for LPS -Induced Airway Remodeling
Airway morphometry demonstrated that the subepithelial area (calculated per length of basal membrane) of the medium size airways in the LPS-exposed, control serum-treated group was increased significantly (p < 0.05) compared with corresponding air-exposed control animals. At the same time point, mice that were LPS-exposed but neutropenic had significantly reduced (p < 0.005) expansion of the subepithelial area for the medium airways in comparison to the control serum-treated, LPS-exposed mice ( Figure 4). Following the 4 week recovery period, LPS-exposed, neutrophil-sufficient animals showed moderate thickening of the lung interstitial areas and much less cellular infiltration, but collagen deposition in the airway wall compared with the samples prepared from neutropenic mice. These microscopic and morphometric findings were confirmed by Masson Trichrome staining, which demonstrated an increase in the concentration of collagen under the epithelial basement membrane, seen as blue-green staining in specimen A (non neutropenic) versus specimen B (neutropenic) in the histologic specimen shown in Figure 5.
Figure 4. Airway Size
Figure 5. Masson Trichrome Staining Demonstrates an Increase in Collagen
TGF-β1 Activity Is Induced by LPS Exposure and Mediated by Recruited Neutrophils
TGF-β1 secreted by airway epithelia and inflammatory cells may play a role in the development of subepithelial fibrosis with collagen deposition. Both TGF-β1 expression in the mouse conducting airways, as well as levels of total and active forms of TGF-β1 in the lung lavage are increased after subchronic LPS inhalation challenge. We found that tissue expression is dependent on neutrophilic infiltration. Bronchial epithelial and subepithelial TGF-β1 expression were diminished in the LPS challenged neutropenic mice as compared to the neutrophil-sufficient mice ( Figure 6; A = neutropenic; B = nonneutropenic; brown staining is TGF-β1). TGF-β1 activation is induced by LPS exposure and mediated by neutrophils recruited into the tissue ( Figure 7 ).
Figure 6. TGF-β1 Expression
Figure 7. Semiquantitative Analysis of Tissue TGF-β1 After 4 Week Challenge With
Inhaled LPS
Oxidative Burst and LPS Induced Airway Disease
Following 4 hour inhalation challenge with LPS, gp91phox (NADPH oxidase subcomponent) deficient mice, we found that gp91phox deficient mice are producing significantly less TGF-β1 (see Figure 8) and TNF-α. We have experience measuring the oxidative burst in whole lung lavage cells and preliminary data suggest that this is involved in LPS -induced airway disease. It previously has been demonstrated that oxygen radicals can both up regulate the expression of and the activation of TGF-β1. Primed and activated neutrophils responding to stimulation with LPS or fMLP produce a profound oxidative burst. It previously has been demonstrated that priming with IL-6 predisposes polymorphonuclear neutrophils (PMNs) to produce an oxidative burst in response to further stimulation by fMLP in an in vitro assay. In Figure 9 , we show that PMN lavaged from mice deficient in IL-6 exposed to LPS for 4 hours produce a significantly reduced oxidative burst when compared to PMN from similarly exposed wild type mice.
Figure 8. TGF-β1 Production Following LPS Challenge
Figure 9. Oxidative Burst Assay. 2.5 x 105 PMN from C57BL/6 and C57BL/6IL-6-/- mice were incubated for 5 minutes at 37°C in 250 μl DMEM/10% FBS containing 50 μM lucigenin. RLU/s were determined in a Zylux Femtomaster FB15 luminometer for 10 seconds per sample.
LPS -Induced Airway Lesion Is Associated With Fibrin Deposition
In a chronic LPS experiment, we compared the development and evolution of the airway inflammatory lesion in plasminogen activator inhibitor deficient (C57BL/6JPAI-1-/-) mice and wild-type (WT) C57BL/6J mice. Following LPS exposure, WT animals show neutrophilic infiltrates together with fibrin-rich material under the airway epithelium, whereas C57BL/6JPAI-1-/- animals develop PMNs-rich but fibrin-poor infiltrates in the airway wall (see Figure 10).
Figure 10. Histologic Sample of Airway Epithelium
Active Fibrinolysis Accelerates the Resolution of the Pathological Matrix in the Airway Subepithelium, Observed Following Chronic Inhalation of LPS
To examine the role of the fibrinolytic system in LPS -induced airway disease, we compared the effect of a chronic LPS challenge in C57BL/6JPAI-1-/- mice and WT C57BL/6J mice. Immediately following the 8 week LPS exposure, WT mice had increased estimates of airway reactivity to MC h when compared to C57BL/6JPAI-1-/- mice (see Figure 11) ; however, airway inflammation was similar in both C57BL/6JPAI-1-/- and WT LPS -exposed groups. Significant increases in both active TGF-β1 and active matrix metalloproteinase 9 (MMP-9) was detected following LPS exposure in WT mice but not in C57BL/6JPAI-1-/- mice. C57BL/6JPAI-1-/- animals showed significantly less TGF-β1 (total and active) in the lavage than WT mice at end of the exposure as well as 4 weeks later (see Figure 12). Following LPS exposure, only WT (not C57BL/6JPAI-1-/- mice) had substantial expansion of the subepithelial area of the medium (d = 90-129 μm) and large (d > 129 μm) size airways when compared to filtered air (FA) exposed mice (see Figure 13). Subepithelial fibrin deposition was prevalent in WT mice but greatly diminished in C57BL/6JPAI-1-/- mice (see Figure 13). PAI-1 expression by non ciliated bronchial epithelial cells was enhanced in the airways of LPS- exposed WT mice when compared to the FA -exposed group. Four weeks following inhalation of LPS the increased airway reactivity and the expansion of the subepithelial area in the medium and large size airways persisted in WT mice but not in the C57BL/6JPAI-1-/- mice. We conclude that an active fibrinolytic system can alter substantially the development and resolution of the postinflammatory airway remodeling that is observed following chronic inhalation of LPS.
Figure 11. Airway Reactivity to Methacholine
Figure 12. TFG-β1 Expression
Figure 13. Expansion of Subepithelial Area
Importance of Matrix Metalloproteinase 9 (MMP-9)
MMPs are part of the extracellular proteolytic/fibrinotic system and their activation may lead to accelerated degradation of ECM proteins and, therefore, increased turnover of ECM. Among C57BL/6J (WT) mice, 8 week LPS exposure resulted in elevation of the amount of active MMP-9 in the lung tissue homogenates when compared with FA-exposed mice (see Figure 14). PAI-1 deficient mice have marked up regulation of MMP-9 in the lung compared to WT animals. High levels of MMP-9 activity were observed in the lung tissue after either an 8 week exposure to FA or LPS, but no difference was observed in MMP-9 activity between these two exposures. Even among LPS- exposed WT mice, however, the level of MMP-9 was significantly less than observed in LPS -exposed C57BL/6JPAI-1-/- mice.
Figure 14. Amount of Active MMP-9 in Lung Tissue Homogenates
Importance of ECM Deposition in Chronic LPS-Induced Airway Disease
TGF-β1 is associated with chronic LPS -induced airway disease. TGF-β1 increases the expression of ECM genes and proteins in vitro and has been associated in vivo with fibrotic lung disease in the asbestos, bleomycin, silica, and LPS -induced airway disease models (see above). We have found that after inhalation of LPS for 8 weeks, the airways from C57BL/6 IL-1 R1 -/- mice were not different from baseline unexposed mice, wh e reas C57BL/6 mice developed substantial expansion of the submucosal (subbasement membrane) tissue (see Figure 15). We also have found that the submucosal expansion in the C57BL/6 mice is associated with increased type III collagen gene expression (see Figure 16).
Figure 15. Airway Size
Figure 16. Collagen III Gene Expression
The Importance of Anti protease Activity in Chronic LPS-Induced Airway Disease
In addition to increasing the expression of ECM genes and proteins, TGF- β1 also contributes to fibrogenesis by increasing the expression of protease inhibitors such as TIMP1 and PAI-1. We have experience in measuring expression of mRNA of protease inhibitors as shown in Figure 17. Here we demonstrate that in C57BL/6 mice TIMP1 mRNA is increased significantly over that of C57BL/6 IL-1 R1 -/- and that this is associated with submucosal expansion in these LPS -exposed mice, again suggesting that protease inhibitors play a role in the development of airway remodeling following chronic inhalation of LPS.
Figure 17. TIMP1 Expression
Significant Achievement
We have found that subacute exposure to grain dust and LPS cause an asthma phenotype, reversible airflow obstruction and airway inflammation, and persistent airway hyperreactivity and airway remodeling. Moreover, these lesions are dependent on the presence of specific adhesion molecules, PMNs, and TGF-β1; TNF-α and IL-1β do not appear to have an essential role in mediating the physiologic or inflammatory response to inhaled LPS. Our findings also suggest that LPS induced airway hyperreactivity can occur in the absence of airway inflammation.
Journal Articles on this Report: 19 Displayed | Download in RIS Format
| Other subproject views: | All 19 publications | 19 publications in selected types | All 19 journal articles |
| Other center views: | All 33 publications | 32 publications in selected types | All 32 journal articles |
| Type | Citation | ||
|---|---|---|---|
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Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M, Frees K, Watt JL, Schwartz DA. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nature Genetics 2000;25(2):187-191. |
R826711 (Final) R826711C001 (2000) R826711C002 (2000) R826711C004 (Final) |
not available |
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Brass DM, Savov JD, Gavett SH, Haykal-Coates N, Schwartz DA. Subchronic endotoxin inhalation causes chronic airway disease. American Journal of Physiology-Lung Cellular and Molecular Physiology 2003;285(6):L755-L761. |
R826711 (Final) R826711C004 (Final) |
not available |
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George CL, Jin H, Wohlford-Lenane CL, O’Neill ME, Phipps JC, O’Shaughnessy P, Kline JN, Thorne PS, Schwartz DA. Endotoxin responsiveness and subchronic grain dust-induced airway disease. American Journal of Physiology-Lung Cellular and Molecular Biology 2001;280(2):L203-L213. |
R826711 (Final) R826711C001 (2000) R826711C002 (2000) R826711C004 (Final) |
not available |
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George CL, White M, O’Neill ME, Thorne PS, Schwartz DA, Snyder JM. Altered surfactant protein A gene expression and protein metabolism associated with repeat exposure to inhaled endotoxin. American Journal of Physiology-Lung Cellular and Molecular Physiology 2003;285(6):L1337-L1344. |
R826711 (Final) R826711C004 (Final) |
not available |
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Hollingsworth JW, Cook DN, Brass DM, Walker JK, Morgan DL, Foster WM, Schwartz DA. The role of toll-like receptor 4 in environmental airway injury in mice. American Journal of Respiratory and Critical Care Medicine 2004;170(2):106-107. |
R826711 (Final) R826711C004 (Final) |
not available |
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Kline JN, Jagielo PJ, Watt JL, Schwartz DA. Bronchial hyperreactivity is associated with enhanced grain dust-induced airflow obstruction. Journal of Applied Physiology 2000;89(3):1172-1178. |
R826711 (Final) R826711C001 (2000) R826711C002 (2000) R826711C004 (Final) |
not available |
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Lorenz E, Jones M, Wohlford-Lenane C, Meyer N, Frees KL, Arbour NC, Schwartz DA. Genes other than TLR4 are involved in the response to inhaled LPS. American Journal of Physiology-Lung Cellular and Molecular Physiology 2001;281(5):L1106-L1114. |
R826711 (Final) R826711C004 (Final) |
not available |
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Moreland JG, Fuhrman RM, Wohlford-Lenane CL, Quinn TJ, Benda E, Pruessner JA, Schwartz DA. TNF-α and IL-1β are not essential to the inflammatory response in LPS -induced airway disease. American Journal of Physiology-Lung Cellular and Molecular Biology 2001;280(1):L173-L180. |
R826711 (Final) R826711C001 (2000) R826711C002 (2000) R826711C004 (Final) |
not available |
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Moreland JG, Fuhrman RM, Pruessner JA, Schwartz DA. CD11b and intercellular adhesion molecule-1 are involved in pulmonary neutrophil recruitment in lipopolysaccharide-induced airway disease. American Journal of Respiratory Cell and Molecular Biology 2002;27(4):474-480. |
R826711 (Final) R826711C004 (Final) |
not available |
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Quinn TJ, Taylor S, Wohlford-Lenane CL, Schwartz DA. IL-10 reduces grain dust-induced airway inflammation and airway hyperreactivity. Journal of Applied Physiology 2000;88(1):173-179. |
R826711 (Final) R826711C001 (2000) R826711C002 (2000) R826711C004 (Final) |
not available |
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Savov JD, Gavett SH, Brass DM, Costa DL, Schwartz DA. Neutrophils play a critical role in the development of LPS-induced airway disease. American Journal of Physiology-Lung Cellular and Molecular Physiology 2002;283(5):L952-L962. |
R826711 (Final) R826711C004 (Final) |
not available |
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Savov JD, Brass DM, Berman KG, McElvania E, Schwartz DA. Fibrinolysis in LPS -induced chronic airway disease. American Journal of Physiology-Lung Cellular and Molecular Physiology 2003;285(4):L940-L948. |
R826711 (Final) R826711C004 (Final) |
not available |
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Savov JD, Whitehead GS, Wang J, Liao G, Usuka J, Peltz G, Foster WM, Schwartz DA. Ozone-induced acute pulmonary injury in inbred mouse strains. American Journal of Respiratory Cell and Molecular Biology 2004;31(1):69-77. |
R826711 (Final) R826711C004 (Final) |
not available |
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Schwartz DA. Does inhalation of endotoxin cause asthma? American Journal of Respiratory and Critical Care Medicine 2001;163(2):305-306. |
R826711 (Final) R826711C004 (Final) |
not available |
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Schwartz DA, Christ WJ, Kleeberger SR, Wohlford-Lenane CL. Inhibition of LPS -induced airway hyperresponsiveness and airway inflammation by LPS antagonists. American Journal of Physiology-Lung Cellular and Molecular Physiology 2001;280(4):L771-L778. |
R826711 (Final) R826711C004 (Final) |
not available |
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Walker JK , Fong AM, Lawson BL, Savov JD, Patel DD, Schwartz DA, Lefkowitz RJ. Beta-arrestin-2 regulates the development of allergic asthma. The Journal of Clinical Investigation 2003;112(4):566-574. |
R826711 (Final) R826711C004 (Final) |
not available |
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Warshamana GS, Pociask DA, Sime P, Schwartz DA, Brody AR. Susceptibility to asbestos-induced and transforming growth factor-beta 1-induced fibroproliferative lung disease in two strains of mice. American Journal of Respiratory Cell and Molecular Biology 2002;27(6):705-713. |
R826711 (Final) R826711C004 (Final) |
not available |
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Whitehead GS, Walker JK , Berman KG, Foster WM, Schwartz DA. Allergen -induced airway disease is mouse strain dependent. American Journal of Physiology-Lung Cellular and Molecular Physiology 2003;285(1):L32-L42. |
R826711 (Final) R826711C004 (Final) |
not available |
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Zeldin DC, Wohlford-Lenane C, Chulada P, Bradbury JA, Scarborough PE, Roggli V, Langenbach R, Schwartz DA. Airway inflammation and responsiveness in prostaglandin H synthase-deficient mice exposed to bacterial lipopolysaccharide. American Journal of Respiratory Cell and Molecular Biology 2001;25(4):457-465. |
R826711 (Final) R826711C004 (Final) |
not available |
pollutants, airway disease, airway inflammation, allergen, assessment of exposure, asthma, biological markers, childhood respiratory disease, children, children’s health, disease, endotoxin, exposure, grain dust, human exposure, inhalation, model, rural communities, sensitive populations,
,
Scientific Discipline, Health, RFA, Susceptibility/Sensitive Population/Genetic Susceptibility, Biology, Risk Assessments, genetic susceptability, Health Risk Assessment, Epidemiology, Children's Health, Environmental Chemistry, Allergens/Asthma, allergen, inhalation, air contaminant exposure, rural communities, assessment of exposure, childhood respiratory disease, toxics, endotoxin, agricultural community, sensitive populations, grain dust, air pollution, airborne pollutants, airway disease, biological markers, children, disease, exposure, children's vulnerablity, asthma, human exposure, model
Progress and Final Reports:
Original Abstract
Main Center Abstract and Reports:
R826711 University of Iowa Children's Environmental Airway Disease Center
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R826711C001 Mechanisms that Initiate, Promote, and Resolve Grain Dust/LPS Induced Inflammation
R826711C002 Multi-component Intervention Study of Asthma in Children from Rural Communities
R826711C003 Role of RSV Infection and Endotoxin in Airway Inflammation
R826711C004 A Model to Study the Development of Persistent Environmental Airway Disease