Inhalation Of Endotoxin Research Articles

Inhaled endotoxin induces a systemic neutrophil response without affecting cardiovascular measures in a randomized cross-over exposure study

Objective The gram-negative bacterial cell wall component endotoxin (lipopolysaccharide, LPS) is a key component of particulate matter (PM). PM exposure is associated with cardiovascular morbidity and mortality. However, the contribution of individual components of PM to acute and chronic cardiovascular measures is not clear. This study examines whether systemic inflammation induced by LPS inhalation causes acute changes in cardiovascular physiology measures. Materials and Methods In this double blinded, placebo-controlled crossover study, fifteen adult volunteers underwent inhalation exposure to 20,000 EU Clinical Center Reference Endotoxin (CCRE). Peripheral blood and induced sputum neutrophils were obtained at baseline and six hours post-exposure. Blood pressure, measures of left ventricular function (ejection fraction (LVEF) and global longitudinal strain (LVGLS)), and indices of endothelial function (flow mediated dilation (FMD) and velocity time integral during hyperemia (VTIhyp)) were measured before and after treatment. Wilcoxon sign-rank tests and linear mixed models were used for statistical analysis. Results In comparison with normal saline, LPS inhalation resulted in significant increases in peripheral blood and sputum neutrophils but was not associated with significant alterations in blood pressure, LVGLS, LVEF, FMD, or VTIhyp. Discussion and Conclusions In healthy adults, systemic inflammation after LPS inhalation was not associated with acute changes in cardiovascular physiology. Larger studies are needed to investigate the effects of other PM components on inflammation induced cardiovascular dysfunction.

Indoor air filtration could lead to increased airborne endotoxin levels

Stand-alone portable air purifiers (APs) have become an increasingly popular method of controlling individual inhalation exposure. Exposure to bacterial endotoxins has a causative role in respiratory inhalation health. Here, we studied the changes in endotoxin levels in indoor air before and after purification by a portable AP equipped with HEPA (high-efficiency particulate air) filters. An increase in endotoxins was observed when a previously used AP was turned on to clean the air. Replacing the HEPA filters in the AP helped to mitigate the increase in endotoxins of larger sizes but not endotoxins of smaller sizes. Consequently, the use of APs could lead to increased endotoxin deposition in airways, especially in the alveolar region. The endotoxin concentrations on the HEPA filters were well correlated with the free DNA concentrations on the HEPA filters. This correlation indicates that the disrupted bacteria, which released free DNA, could also release endotoxins, thus making HEPA filters a source of indoor airborne endotoxins. Our results illustrate a potential endotoxin inhalation risk associated with HEPA-APs as an air cleaning strategy and highlight the importance of composition-specific air cleaning while reducing the particle number/mass.

Mesenchymal stem cells’ therapeutic potential for endotoxin-induced brain and spleen injuries in rats

Background and objective Inhalation of bacterial endotoxin induces an acute inflammation in various organs, especially the brain and spleen. This study examined the therapeutic effects of bone marrow mesenchymal stem cells (BM-MSCs) in lipopolysaccharide (LPS)-induced brain-spleen injuries in rats as compared with dexamethasone. Materials and methods A total of 32 male Wistar albino rats, weighing 180–200 g, were used in the study and were divided into four groups. Group 1 (normal) rats received 20 μl of saline in each nostril for two consecutive days. Group 2 animals received LPS (20 μl of LPS of Escherichia coli in each nostril for 2 consecutive days) that induced brain-spleen injuries and served as a positive control group. Group 3 animals were injected with dexamethasone (2 mg/kg, once, intraperitoneal). Group 4 animals received (1×106) BM-MSCs in 500 μl PBS/rat via intraperitoneal injection once before acute injury induction with LPS. At the end of the experiment, the authors studied the sickness behavior by assessing open field behavior and measured oxidative and inflammatory parameters. Results and conclusion LPS-induced open field behavior impairments (decreased locomotion and rearing and increased immobility), with elevation of a number of inflammatory cells, especially neutrophils. Moreover, LPS-induced elevation of lipid peroxidation along with reduction of both reduced glutathione and superoxide dismutase in brain and spleen tissues and increased interleukin-1β and myeloperoxidase contents in rats, compared with normal group. These harmful effects were hindered after treatment with MSCs. In conclusion, MSCs prevented both sickness and depressive-like behavior via neuroinflammatory pathway and could be a novel approach to therapy for LPS-induced serious injuries in rats but needs further clinical studies.

Effects of chlorination and combined UV/Cl2 treatment on endotoxin activity and inhalation toxicity of lipopolysaccharide, gram-negative bacteria and reclaimed water

Disinfection processes were applied in reclaimed water plant to eliminate pathogens and control the related health risk during water reuse. However, extra problems might emerge such as the released free endotoxins from the ruptured cell wall of gram-negative bacteria. Endotoxins can induce lung inflammatory responses after inhalation, which has been neglected in the water quality regulation, and the removal of endotoxin was not under consideration in the process of reclamation. In the present study, two well-known disinfection processes, chlorination and combined UV/chlorine (UV/Cl2), were performed to test the removal efficiency of endotoxin activity, as well as the inflammation inducing ability. In the pure LPS solution, UV/Cl2 treatment significantly reduced both endotoxin activity and lung inflammation responses with better oxidizability of the generated hydroxyl radical. However, its performance on bacteria liquid and real secondary effluent was more complicated. The cell wall-bound LPS have lower endotoxin activities and inflammation inducing ability. Immediately after the cell wall was destroyed, the bound LPS were released to the solution to be free LPS, which dramatically increased both the endotoxin activity and inflammation inducing ability of the water. When these free endotoxins were continuously oxidized, the endotoxin activity and inflammatory response decreased again but not to the background level. Therefore, the inflammation inducing ability of reclaimed water could not be removed efficiently. These results suggest that in spite of its high oxidability, UV/Cl2 treatment is not capable of removing the endotoxin-based toxicity, and other technologies are necessary to control endotoxin levels in reclaimed water.

Adaptation to Occupational Exposure to Moderate Endotoxin Concentrations: A Study in Sewage Treatment Plants in Germany.

Acute or chronic inhalation of endotoxin may lead to changes of lung function and inflammatory markers in the airways. Adaptation to workplace exposure may be possible. In this study, we investigated the possible difference in inflammatory markers assessed in nasal lavage fluid (NALF) in chronical exposure compared to voluntary subjects exposed acutely to endotoxin. We sought to define the variability of inflammatory markers in NALF and the dose-related changes after moderate exposure in naïve subjects. Endotoxin exposure (4-1039 EU/m3) resulted from routine work during one shift in sewage treatment plants. Subjects were matched to pairs (8 workers escorted by 10 students). Inflammatory markers were investigated before, directly after, and 16h after the shift end. Additional NALF samples were collected in students without any specific exposure after 3days. In NALF, total cell count, and interleukin (IL)-8 and IL-1β concentrations were significantly higher in workers than in students at all times pointing to workplace-related long-lasting exposure resulting in adaptation. However, concentration of inflammatory markers without specific exposure in students showed a great variability, covering the whole range of values recorded in the workers. The findings of this study make us to recommend a repeated assessment of inflammatory markers in healthy volunteers before the investigation of exposure-related changes and a sample size adequate for statistical analysis.

Attenuation of pulmonary ACE2 activity impairs inactivation of des-Arg9 bradykinin/BKB1R axis and facilitates LPS-induced neutrophil infiltration.

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase with important functions in the renin-angiotensin system and plays a critical role in inflammatory lung diseases. ACE2 cleaves single-terminal residues from several bioactive peptides such as angiotensin II. However, few of its substrates in the respiratory tract have been identified, and the mechanism underlying the role of ACE2 in inflammatory lung disease has not been fully characterized. In an effort to identify biological targets of ACE2 in the lung, we tested its effects on des-Arg9 bradykinin (DABK) in airway epithelial cells on the basis of the hypothesis that DABK is a biological substrate of ACE2 in the lung and ACE2 plays an important role in the pathogenesis of acute lung inflammation partly through modulating DABK/bradykinin receptor B1 (BKB1R) axis signaling. We found that loss of ACE2 function in mouse lung in the setting of endotoxin inhalation led to activation of the DABK/BKB1R axis, release of proinflammatory chemokines such as C-X-C motif chemokine 5 (CXCL5), macrophage inflammatory protein-2 (MIP2), C-X-C motif chemokine 1 (KC), and TNF-α from airway epithelia, increased neutrophil infiltration, and exaggerated lung inflammation and injury. These results indicate that a reduction in pulmonary ACE2 activity contributes to the pathogenesis of lung inflammation, in part because of an impaired ability to inhibit DABK/BKB1R axis-mediated signaling, resulting in more prompt onset of neutrophil infiltration and more severe inflammation in the lung. Our study identifies a biological substrate of ACE2 within the airways, as well as a potential new therapeutic target for inflammatory diseases.

Endotoxin exposure and changes in short-term pulmonary function among sewage workers.

The inhaled endotoxin is considered as a causative factor in the process of acute bronchial obstruction, which can be measured by a decrease in forced expiratory volume in 1 s (FEV1). The aim of this study was to assess endotoxin exposure among sewage treatment plant workers (STPW) and its effect on across-shift changes in respiratory airflow. A group of 78 STPW from a large sewage treatment plant was studied. Inhalable dust for endotoxin assessment was collected using personal aerosol samplers. Endotoxin was assayed with the kinetic, chromogenic Limulus amebocyte lysate test. Across-shift spirometric measurements were performed on Mondays, after 2-days absence from work, with the use of portable spirometer. The forced vital capacity (FVC), and FEV1 parameters were analyzed. Multifactor regression modeling was performed to determine parameters significantly associated with endotoxin exposure. The concentration of inhalable dust and endotoxin ranged from 0.01-1.38 mg/m3 and 0.68-214 endotoxin units per cubic meter of air (EU/m3), respectively. Endotoxins were characterized with the skewed distribution (arithmetic mean (AM) = 38.8 EU/m3, geometric mean (GM) = 15.4 EU/m3, geometric standard deviation (GSD) = 4.21). Through the use of multifactor analysis, which excluded the main confounders (inhalable dust and smoking habit) it was found that, despite low levels of endotoxin, it had significant impact on the observed across-shift decline in FEV1 (p = 0.044). For this parameter, the regression slope was additionally calculated (r = -0.017, p = 0.071). Relatively low levels of endotoxin among sewage treatment plant workers may cause small, but significant across-shift declines in FEV1. The observed relationship was independent of organic dust concentrations and smoking habit. The respiratory protection should be provided for STPW.

Anti-TNF inhibits the airways neutrophilic inflammation induced by inhaled endotoxin in human.

BackgroundInhaled endotoxin induces airways’neutrophilia, in human. TNF-a being a key cytokine in the response to endotoxin, the effect of anti-TNF on the endotoxin-induced neutrophilic response was evaluated among healthy volunteers.MethodsAmong a population of 30 healthy subjects, an induced-sputum was collected 2 weeks before, and 24 hours after an inhalation of 20 mcg endotoxin (E coli 026:B6). Then, the subjects were randomized into 3 parallel groups treated with control, oral methylprednisolone 20 mg/day during 7 days or anti-TNF (adalimumab, Humira®, Abbott) 40 mg SC. One week later, an induced-sputum was sampled, 24 hours after an inhalation of endotoxin.ResultsAfter endotoxin inhalation, the number of total cells, neutrophils and macrophages was significantly increased (p <0.001). Compared to the response to endotoxin among the control group, anti-TNF inhibited the endotoxin-induced neutrophil influx, both in relative (51.3 (±6.4)% versus 26.2 (±5.3)%, p <0.002) and in absolute values (1321 (443–3935) cells/mcL versus 247 (68–906) cells/mcL, p <0.02). The endotoxin-induced neutrophilic response was not significantly modified among the control group and oral corticosteroid group.ConclusionsWhile oral corticosteroid had no effect, anti-TNF inhibited the neutrophil influx in sputum, induced by inhalation of endotoxin, in human subject. The endotoxin model could be an early predictor of clinical efficacy of novel therapeutics.Trial registrationClinicalTrials.gov NCT02252809 (EudraCT2008-005526-37)

Expression of calgranulin A/B heterodimer after acute inhalation of endotoxin: proteomic approach and validation

BackgroundThe acute inhalation of endotoxin mimicks several aspects of the inflammation related to chronic obstructive pulmonary disease (COPD). The aim of the current study was to identify and to validate biomarkers of endotoxin-induced airways’ inflammation.MethodsThe cellular count in the induced-sputum, was measured before and after an inhalation of 20 mcg endotoxin, in 8 healthy volunteers. A proteomic analysis was applied to identify the more relevant proteins expression, before measurement by ELISA. The amplitude and the repeatability of the markers were evaluated among another population of 12 healthy subjects.ResultsThere was a significant rise of viable cells (p <0.01), macrophages (p <0.05), and neutrophils (p <0.02) 24 hours after endotoxin inhalation, and of neutrophils (p <0.02) and lymphocytes (p <0.05) at 6 hours. Among the highest amplitude responses, the two dimensional electrophoretic separation shown proteolytic activity and overexpression of protein spots. By MALDI-TOF mass spectrometry, the last were identified as calgranulin A and B. The expression of the bioactive A/B heterodimeric complex was confirmed by ELISA both in the sputum (p <0.01) and at the blood level (p <0.01). The intra-subject repeatability of the sputum calgranulin A/B was highly significant (p <0.0001).ConclusionIn healthy subjects, the inhalation of endotoxin induced expression of sputum calgranulin A/B that could be a biomarker of the endotoxin response/exposure.

Low-dose endotoxin inhalation in healthy volunteers - a challenge model for early clinical drug development

BackgroundInhalation of endotoxin (LPS) induces a predominantly neutrophilic airway inflammation and has been used as model to test the anti-inflammatory activity of novel drugs. In the past, a dose exceeding 15–50 μg was generally needed to induce a sufficient inflammatory response. For human studies, regulatory authorities in some countries now request the use of GMP-grade LPS, which is of limited availability. It was therefore the aim of this study to test the effect and reproducibility of a low-dose LPS challenge (20,000 E.U.; 2 μg) using a flow- and volume-controlled inhalation technique to increase LPS deposition.MethodsTwo to four weeks after a baseline sputum induction, 12 non-smoking healthy volunteers inhaled LPS on three occasions, separated by at least 4 weeks. To modulate the inflammatory effect of LPS, a 5-day PDE4 inhibitor (Roflumilast) treatment preceded the last challenge. Six hours after each LPS inhalation, sputum induction was performed.ResultsThe low-dose LPS inhalation was well tolerated and increased the mean percentage of sputum neutrophils from 25% to 72%. After the second LPS challenge, 62% neutrophils and an increased percentage of monocytes were observed. The LPS induced influx of neutrophils and the cumulative inflammatory response compared with baseline were reproducible. Treatment with Roflumilast for 5 days did not have a significant effect on sputum composition.ConclusionThe controlled inhalation of 2 μg GMP-grade LPS is sufficient to induce a significant neutrophilic airway inflammation in healthy volunteers. Repeated low-dose LPS challenges potentially result in a small shift of the neutrophil/monocyte ratio; however, the cumulative response is reproducible, enabling the use of this model for “proof-of-concept” studies for anti-inflammatory compounds during early drug development.Trial registrationClinicaltrials.gov: NCT01400568

Endotoxin inhalation alters lung development in neonatal mice.

Childhood asthma is a significant public health problem. Epidemiologic evidence suggests an association between childhood asthma exacerbations and early life exposure to environmental endotoxin. Although the pathogenesis of endotoxin-induced adult asthma is well studied, questions remain about the impact of environmental endotoxin on pulmonary responsiveness in early life. We developed a murine model of neonatal/juvenile endotoxin exposures approximating those in young children and evaluated the lungs inflammatory and remodeling responses. Persistent lung inflammation induced by the inhalation of endotoxin in early life was demonstrated by the influx of inflammatory cells and pro-inflammatory mediators to the airways and resulted in abnormal alveolarization. Results of this study advance the understanding of the impact early life endotoxin inhalation has on the lower airways, and demonstrates the importance of an experimental design that approximates environmental exposures as they occur in young children.

Unrecognized Bomb Hidden in the Babies' Room: Fatal Pulmonary Damage Related with Use of Biocide in Humidifiers

Unrecognized Bomb Hidden in the Babies' Room: Fatal Pulmonary Damage Related with Use of Biocide in Humidifiers

Pulmonary Endotoxin Tolerance Protects against Cockroach Allergen-Induced Asthma-Like Inflammation in a Mouse Model

Background: Compounds which activate the innate immune system, such as lipopolysaccharide, are significant components of ambient air, and extremely difficult to remove from the environment. It is currently unclear how prior inhalation of endotoxin affects allergen sensitization. We examined whether lung-specific endotoxin tolerance induction prior to sensitization can modulate the response to allergen. Methods: Endotoxin tolerance was induced by repeated intratracheal exposure to endotoxin. All mice were then sensitized and challenged by direct intratracheal instillation of cockroach allergen. Results: After allergen sensitization and challenge, endotoxin tolerant mice had significantly decreased airways hyperresponsiveness to methacholine challenge, which was confirmed by invasive lung function tests. Decreased goblet cell hyperplasia and mucus production were also found by histological assessment. Tolerant mice were protected from airway eosinophilia through the mechanism of reduced CCL11 and CCL24. Interestingly, endotoxin tolerant mice had only a modest reduction in cockroach-specific IgE; however, total IgE was significantly reduced. Conclusions: These data show that induction of endotoxin tolerance prior to sensitization protects against the hallmark features of asthma-like inflammation, and that transient modulation of innate immunity can have long-lasting effects on adaptive responses.

TLR4 signalling in pulmonary stromal cells is critical for inflammation and immunity in the airways

Inflammation of the airways, which is often associated with life-threatening infection by Gram-negative bacteria or presence of endotoxin in the bioaerosol, is still a major cause of severe airway diseases. Moreover, inhaled endotoxin may play an important role in the development and progression of airway inflammation in asthma. Pathologic changes induced by endotoxin inhalation include bronchospasm, airflow obstruction, recruitment of inflammatory cells, injury of the alveolar epithelium, and disruption of pulmonary capillary integrity leading to protein rich fluid leak in the alveolar space. Mammalian Toll-like receptors (TLRs) are important signalling receptors in innate host defense. Among these receptors, TLR4 plays a critical role in the response to endotoxin.Lungs are a complex compartmentalized organ with separate barriers, namely the alveolar-capillary barrier, the microvascular endothelium, and the alveolar epithelium. An emerging theme in the field of lung immunology is that structural cells (SCs) of the airways such as epithelial cells (ECs), endothelial cells, fibroblasts and other stromal cells produce activating cytokines that determine the quantity and quality of the lung immune response. This review focuses on the role of TLR4 in the innate and adaptive immune functions of the pulmonary SCs.

Time Course of Endotoxin-Induced Airways’ Inflammation in Healthy Subjects

Few data are available on the kinetic of the airways' inflammation induced by inhaled endotoxin in a given subject. The purpose of this study was to evaluate in healthy subjects the time-related endotoxin-induced airways' inflammation. The cells counts from the induced-sputum were evaluated before, 6 and 24 h, and 7 days after an exposure to 20 mcg inhaled endotoxin, in eight pre-selected volunteers. To avoid interference of the induced-sputum procedure on the response to endotoxin, each time-point was evaluated in randomized order at 2-weeks interval after three separate inhalations of endotoxin. A significant rise of the relative number of lymphocytes (p<0.05) and polymorphonuclear neutrophils (PMN; p<0.02) and of the absolute number of PMN (p<0.05) occurring at 6 h, followed by an increase of the absolute number of the total viable cells (p<0.01), macrophages (p<0.001), neutrophils (p<0.01), and lymphocytes (p<0.05) at 24 h after endotoxin inhalation. The inflammatory response recovered totally after 7 days. In human beings, the inhalation of endotoxin induced a transient airway inflammation after 6 h, peaked at 24 h and recovered after 7 days. When repeated endotoxin inhalations are used as a model of inflammation, a wash-out period of at least 7 days should be applied between each exposure in each subject.

Airborne endotoxin associated with particles of different sizes and affected by water content in handled straw

High exposures to endotoxin are observed in environments where organic materials are handled and lower exposures are found in e.g. indoor air. Inhaled endotoxin contributes significantly to the induction of airway inflammation and dysfunction. The size of an inhaled particle influences the deposition in the airways and the following health symptoms. The objective is to characterise the distribution of endotoxin on airborne particles of different sizes in straw storage halls with high exposure and in other environments with lower exposure levels to endotoxin. Furthermore we have studied the influence of water content of handled straw on the size distribution of endotoxin containing particles. Total, inhalable, thoracic and respirable endotoxin and particles have each been quantified in aerosols from boiler rooms and straw storage halls at 24 power plants, including 21 biofuel plants. Inhalable, thoracic and respirable endotoxin have been quantified in aerosols from offices and outdoor air. The endotoxin concentration was higher in airborne thoracic dust than in airborne 'total dust'. The median respirable fraction in the straw storage halls, boiler rooms at biofuel plants, boiler rooms at conventional plants, offices and outdoors was respectively 42%, 9%, 19%, 24% and 34%. Thoracic endotoxin per number of thoracic particles was higher than respirable endotoxin per number of respirable particles at the biofuel plants. In straw storage halls the fraction of endotoxin of respirable size was highest on the days with lowest water content in the received straw. Furthermore the exposures to all endotoxin fractions were highest on days with the lowest water content in the received straw. In conclusion the highest exposures and concentrations of endotoxin occur or tend to occur from thoracic dust. A high variation in endotoxin concentrations and in fractions of respirable or thoracic size is found in the different working areas. This is important in the risk assessment and makes attempts to influence the endotoxin exposure a possibility. Water content in straw affected the concentration, exposure level and size distribution of airborne endotoxin.

Effect of endotoxin inhalation on airway responsiveness in guinea pigs

Effect of endotoxin inhalation on airway responsiveness in guinea pigs

Reproducibility of Sputum Endpoints after Repeated Inhaled Endotoxin Challenges

RATIONALE: We have developed a low level (20,000 EU) endotoxin inhalation challenge to model the effect of environmental bioaerosols on airway inflammation and as a tool to assess anti-inflammatory effects of pharmaceutical agents. In this study, we assess the intrasubject reproducibility of sputum endpoints in subjects that have undergone repeated endotoxin challenge.

Increase in proinflammatory cytokines in peripheral blood without haemostatic changes after LPS inhalation

Introduction Bronchoalveolar fibrin deposition is a characteristic of various lung disorders including acute lung injury, acute respiratory distress syndrome and sepsis. It is secondary to the activation of coagulation and inhibition of fibrinolysis in the alveolar space, and can be stimulated by lipopolysaccharide (LPS) inhalation. The aim of this study was to determine the relation between compartmental stress in the lung and systemic response after LPS inhalation by measuring haemostatic parameters. Patients and methods 12 healthy subjects underwent a bronchial challenge test with LPS; sequential dosages were performed for 5 biological markers (Interleukin-6 (IL-6), C-Reactive Protein (CRP), Prothrombin Fragments 1 and 2 (F 1+2), cortisol and Plasminogen Activator Inhibitor 1 (PAI-1) before endotoxin inhalation and 2, 4, 6, 8 and 24 hours afterwards. Results IL-6 and CRP levels in the peripheral blood were higher after LPS inhalation; there was no activation of coagulation and no increase in PAI-1 level. Conclusion This study confirms that despite systemic release of proinflammatory cytokines, LPS inhalation does not induce systemic haemostatic response to LPS challenge.

Early‐life co‐administration of cockroach allergen and endotoxin augments pulmonary and systemic responses

Environmental exposures to cockroach allergen and endotoxin are recognized epidemiological risk factors for the early development of allergies and asthma in children. Because of this, it is important to examine the role of early-life concurrent inhalation exposures to cockroach allergen and endotoxin in the pathogenesis of allergic airways disease. We examined the effects of repeated concomitant endotoxin and cockroach allergen inhalation on the pulmonary and systemic immune responses of newborn and juvenile mice. C3H/HeBFeJ mice were exposed to inhaled endotoxin and cockroach allergen via intranasal instillation from day 2 to 21 after birth, and systemic and pulmonary responses were examined in serum, bronchoalveolar lavage fluid, and lung tissue. Cockroach allergen exposures induced pulmonary eosinophilic inflammation, total and allergen-specific IgE, IgG(1), and IgG(2a) production, and alveolar remodelling. Co-exposures with endotoxin and cockroach allergen significantly increased serum IgE and IgG(1), lung inflammation, and alveolar wall thickness, and decreased airspace volume density. Importantly, compared with exposures with individual substances, the responses to co-exposures were more than additive. Repeated inhalation exposures of neonatal and juvenile mice to endotoxin and cockroach allergen increased the pulmonary inflammatory and systemic immune responses in a synergistic manner and enhanced alveolar remodelling in the developing lung. These data underscore the importance of evaluating the effect of multiple, concurrent environmental exposures, and of using an experimental model that incorporates clinically relevant timing and route of exposures.