Exhaled breath condensate profiles of U.S. Navy divers following prolonged hyperbaric oxygen (HBO) and nitrogen-oxygen (Nitrox) chamber exposures.

Prolonged exposure to hyperbaric hyperoxia can lead to pulmonary oxygen toxicity (PO2tox). PO2tox is a mission limiting factor for special operations forces divers using closed-circuit rebreathing apparatus and a potential side effect for patients undergoing hyperbaric oxygen (HBO) treatment. In this study, we aim to determine if there is a specific breath profile of compounds in exhaled breath condensate (EBC) that is indicative of the early stages of pulmonary hyperoxic stress/PO2tox. Using a double-blind, randomized 'sham' controlled, cross-over design 14 U.S. Navy trained diver volunteers breathed two different gas mixtures at an ambient pressure of 2 ATA (33 fsw, 10 msw) for 6.5 h. One test gas consisted of 100% O2(HBO) and the other was a gas mixture containing 30.6% O2with the balance N2(Nitrox). The high O2stress dive (HBO) and low O2stress dive (Nitrox) were separated by at least seven days and were conducted dry and at rest inside a hyperbaric chamber. EBC samples were taken immediately before and after each dive and subsequently underwent a targeted and untargeted metabolomics analysis using liquid chromatography coupled to mass spectrometry (LC-MS). Following the HBO dive, 10 out of 14 subjects reported symptoms of the early stages of PO2tox and one subject terminated the dive early due to severe symptoms of PO2tox. No symptoms of PO2tox were reported following the nitrox dive. A partial least-squares discriminant analysis of the normalized (relative to pre-dive) untargeted data gave good classification abilities between the HBO and nitrox EBC with an AUC of 0.99 (±2%) and sensitivity and specificity of 0.93 (±10%) and 0.94 (±10%), respectively. The resulting classifications identified specific biomarkers that included human metabolites and lipids and their derivatives from different metabolic pathways that may explain metabolomic changes resulting from prolonged HBO exposure.

Novel LC-MS-TOF method to detect and quantify ascorbic and uric acid simultaneously in different biological matrices.

Ascorbic acid (AA) and uric acid (UA) are known as two of the major antioxidants in biological fluids. We report a novel liquid chromatography-mass spectrometry with time-of-flight (LC-MS-TOF) method for the simultaneous quantification of ascorbic and uric acids using MPA, antioxidant solution and acetonitrile as a protein precipitating agent. Both compounds were separated from interferences using a reverse phase C18 column with water and acetonitrile gradient elution (both with formic acid) and identified and quantified by MS in the negative ESI mode. Isotope labeled internal standards were also added to ensure the accuracy of the measures. The method was validated for exhaled breath condensate (EBC), nasal lavage (NL) and plasma samples by assessing selectivity, linearity, accuracy and precision, recovery and matrix effect and stability. Sample volumes below 250 µL were used and linear ranges were determined between 1 - 25 and 1 - 40 µg/mL for ascorbic and uric acid, respectively, for plasma samples, and between 0.05 - 5 (AA) and 0.05 - 7.5 (UA) µg/mL for EBC and NL samples. The new method was successfully applied to real samples from subjects that provided each of the studied matrices. Results showed higher amounts determined in plasma samples, with similar profiles for AA and UA in EBC and NL but at much lower concentrations.

Comparison of nonparametric and parametric methods for time-frequency heart rate variability analysis in a rodent model of cardiovascular disease.

The aim of time-varying heart rate variability spectral analysis is to detect and quantify changes in the heart rate variability spectrum components during nonstationary events. Of the methods available, the nonparametric short-time Fourier Transform and parametric time-varying autoregressive modeling are the most commonly employed. The current study (1) compares short-time Fourier Transform and autoregressive modeling methods influence on heart rate variability spectral characteristics over time and during an experimental ozone exposure in mature adult spontaneously hypertensive rats, (2) evaluates the agreement between short-time Fourier Transform and autoregressive modeling method results, and (3) describes the advantages and disadvantages of each method. Although similar trends were detected during ozone exposure, statistical comparisons identified significant differences between short-time Fourier Transform and autoregressive modeling analysis results. Significant differences were observed between methods for LF power (p ≤ 0.014); HF power (p ≤ 0.011); total power (p ≤ 0.027); and normalized HF power (p = 0.05). Furthermore, inconsistencies between exposure-related observations accentuated the lack of agreement between short-time Fourier Transform and autoregressive modeling overall. Thus, the short-time Fourier Transform and autoregressive modeling methods for time-varying heart rate variability analysis could not be considered interchangeable for evaluations with or without interventions that are known to affect cardio-autonomic activity.

The Organophosphorus Pesticide Chlorpyrifos Induces Sex-Specific Airway Hyperreactivity in Adult Rats.

Occupational and environmental exposures to organophosphorus pesticides (OPs) are associated with increased incidence of asthma and other pulmonary diseases. Although the canonical mechanism of OP neurotoxicity is inhibition of acetylcholinesterase (AChE), it was previously reported that the OP chlorpyrifos (CPF) causes airway hyperreactivity (AHR) in guinea pigs at levels that do not inhibit lung or brain AChE. The guinea pig is considered to have inherently hyperresponsive airways, thus, cross-species validation is needed to confirm relevance to humans. Additionally, sex differences in asthma incidence have been demonstrated in the human population, but whether OP-induced AHR is sex-dependent has not been systematically studied in a preclinical model. In this study, 8-week old male and female Sprague Dawley rats were administered CPF at doses causing comparable AChE inhibition in whole lung homogenate (30 mg/kg in males, 7 mg/kg in females, sc) prior to assessing pulmonary mechanics in response to electrical stimulation of the vagus nerves at 24 h, 48 h, 72 h, 7 d or 14 d post-exposure in males, and 24 h or 7 d post-exposure in females. CPF significantly potentiated vagally induced airway resistance and tissue elastance at 7 d post-exposure in males, and at 24 h and 7 d post-exposure in females. These effects occurred independent of significant AChE inhibition in cerebellum, blood, trachealis, or isolated airway, suggesting that AChE independent OP-induced airway hyperreactivity is a cross-species phenomenon. These findings have significant implications for assessing the risk posed by CPF, and potentially other OPs, to human health and safety.

Ultrafine Particulate Matter Combined With Ozone Exacerbates Lung Injury in Mature Adult Rats With Cardiovascular Disease.

Particulate matter (PM) and ozone (O3) are dominant air pollutants that contribute to development and exacerbation of multiple cardiopulmonary diseases. Mature adults with cardiovascular disease (CVD) are particularly susceptible to air pollution-related cardiopulmonary morbidities and mortalities. The aim was to investigate the biologic potency of ultrafine particulate matter (UFPM) combined with O3 in the lungs of mature adult normotensive and spontaneously hypertensive (SH) Wistar-Kyoto rats. Conscious, mature adult male normal Wistar-Kyoto (NW) and SH rats were exposed to one of the following atmospheres: filtered air (FA); UFPM (∼ 250 µg/m3); O3 (1.0 ppm); or UFPM + O3 (∼ 250 µg/m3 + 1.0 ppm) combined for 6 h, followed by an 8 h FA recovery period. Lung sections were evaluated for lesions in the large airways, terminal bronchiolar/alveolar duct regions, alveolar parenchyma, and vasculature. NW and SH rats were similarly affected by the combined-pollutant exposure, displaying severe injury in both large and small airways. SH rats were particularly susceptible to O3 exposure, exhibiting increased injury scores in terminal bronchioles and epithelial degeneration in large airways. UFPM-exposure groups had minimal histologic changes. The chemical composition of UFPM was altered by the addition of O3, indicating that ozonolysis promoted compound degradation. O3 increased the biologic potency of UFPM, resulting in greater lung injury following exposure. Pathologic manifestations of CVD may confer susceptibility to air pollution by impairing normal lung defenses and responses to exposure.

Nonhuman Primate Models of Respiratory Disease: Past, Present, and Future.

The respiratory system consists of an integrated network of organs and structures that primarily function for gas exchange. In mammals, oxygen and carbon dioxide are transmitted through a complex respiratory tract, consisting of the nasal passages, pharynx, larynx, and lung. Exposure to ambient air throughout the lifespan imposes vulnerability of the respiratory system to environmental challenges that can contribute toward development of disease. The importance of the respiratory system to human health is supported by statistics from the Centers for Disease Control and Prevention; in 2015, chronic lower respiratory diseases were the third leading cause of death in the United States. In light of the significant mortality associated with respiratory conditions that afflict all ages of the human population, this review will focus on basic and preclinical research conducted in nonhuman primate models of respiratory disease. In comparison with other laboratory animals, the nonhuman primate lung most closely resembles the human lung in structure, physiology, and mucosal immune mechanisms. Studies defining the influence of inhaled microbes, pollutants, or allergens on the nonhuman primate lung have provided insight on disease pathogenesis, with the potential for elucidation of molecular targets leading to new treatment modalities. Vaccine trials in nonhuman primates have been crucial for confirmation of safety and protective efficacy against infectious diseases of the lung in a laboratory animal model that recapitulates pathology observed in humans. In looking to the future, nonhuman primate models of respiratory diseases will continue to be instrumental for translating biomedical research for improvement of human health.

Early life allergen and air pollutant exposures alter longitudinal blood immune profiles in infant rhesus monkeys.

Early life is a critical period for the progressive establishment of immunity in response to environmental stimuli; the impact of airborne challenges on this process is not well defined. In a longitudinal fashion, we determined the effect of episodic house dust mite (HDM) aerosol and ozone inhalation, both separately and combined, on peripheral blood immune cell phenotypes and cytokine expression from 4 to 25weeks of age in an infant rhesus monkey model of childhood development. Immune profiles in peripheral blood were compared with lung lavage at 25weeks of age. Independent of exposure, peripheral blood cell counts fluctuated with chronologic age of animals, while IFNγ and IL-4 mRNA levels increased over time in a linear fashion. At 12weeks of age, total WBC, lymphocyte numbers, FoxP3 mRNA and IL-12 mRNA were dramatically reduced relative to earlier time points, but increased to a steady state with age. Exposure effects were observed for monocyte numbers, as well as CCR3, FoxP3, and IL-12 mRNA levels in peripheral blood. Significant differences in cell surface marker and cytokine expression were detected following in vitro HDM or PMA/ionomycin stimulation of PBMC isolated from animals exposed to either HDM or ozone. Lavage revealed a mixed immune phenotype of FoxP3, IFNγ and eosinophilia in association with combined HDM plus ozone exposure, which was not observed in blood. Collectively, our findings show that airborne challenges during postnatal development elicit measureable cell and cytokine changes in peripheral blood over time, but exposure-induced immune profiles are not mirrored in the lung.

Early Life Wildfire Smoke Exposure Is Associated with Immune Dysregulation and Lung Function Decrements in Adolescence.

The long-term health effects of wildfire smoke exposure in pediatric populations are not known. The objectives of this study were to determine if early life exposure to wildfire smoke can affect parameters of immunity and airway physiology that are detectable with maturity. We studied a mixed-sex cohort of rhesus macaque monkeys that were exposed as infants to ambient wood smoke from a series of Northern California wildfires in the summer of 2008. Peripheral blood mononuclear cells (PBMCs) and pulmonary function measures were obtained when animals were approximately 3 years of age. PBMCs were cultured with either LPS or flagellin, followed by measurement of secreted IL-8 and IL-6 protein. PBMCs from a subset of female animals were also evaluated by Toll-like receptor (TLR) pathway mRNA analysis. Induction of IL-8 protein synthesis with either LPS or flagellin was significantly reduced in PBMC cultures from wildfire smoke-exposed female monkeys. In contrast, LPS- or flagellin-induced IL-6 protein synthesis was significantly reduced in PBMC cultures from wildfire smoke-exposed male monkeys. Baseline and TLR ligand-induced expression of the transcription factor, RelB, was globally modulated in PBMCs from wildfire smoke-exposed monkeys, with additional TLR pathway genes affected in a ligand-dependent manner. Wildfire smoke-exposed monkeys displayed significantly reduced inspiratory capacity, residual volume, vital capacity, functional residual capacity, and total lung capacity per unit of body weight relative to control animals. Our findings suggest that ambient wildfire smoke exposure during infancy results in sex-dependent attenuation of systemic TLR responses and reduced lung volume in adolescence.

SMAD Signaling in the Airways of Healthy Rhesus Macaques versus Rhesus Macaques with Asthma Highlights a Relationship Between Inflammation and Bone Morphogenetic Proteins.

Bone morphogenetic protein (BMP) signaling is important for correct lung morphogenesis, and there is evidence of BMP signaling reactivation in lung diseases. However, little is known about BMP signaling patterns in healthy airway homeostasis and inflammatory airway disease and during epithelial repair. In this study, a rhesus macaque (Macaca mulatta) model of allergic airway disease was used to investigate BMP signaling throughout the airways in health, disease, and regeneration. Stereologic quantification of immunofluorescent images was used to determine the expression of BMP receptor (BMPR) Ia and phosphorylated SMAD (pSMAD) 1/5/8 in the airway epithelium. A pSMAD 1/5/8 expression gradient was found along the airways of healthy juvenile rhesus macaques (n = 3, P < 0.005). Membrane-localized BMPRIa expression was also present in the epithelium of the healthy animals. After exposure to house dust mite allergen and ozone, significant down-regulation of nuclear pSMAD 1/5/8 occurs in the epithelium. When the animals were provided with a recovery period in filtered air, proliferating cell nuclear antigen, pSMAD 1/5/8, and membrane-localized BMPRIa expression were significantly increased in the epithelium of conducting airways (P < 0.005). Furthermore, in the asthmatic airways, altered BMPRIa localization was evident. Because of the elevated eosinophil presence in these airways, we investigated the effect of eosinophil-derived proteins on BMPRIa trafficking in epithelial cells. Eosinophil-derived proteins (eosinophil-derived neurotoxin, eosinophil peroxidase, and major basic protein) induced transient nuclear translocation of membrane-bound BMPRIa. This work mapping SMAD signaling in the airways of nonhuman primates highlights a potential mechanistic relationship between inflammatory mediators and BMP signaling and provides evidence that basal expression of the BMP signaling pathway may be important for maintaining healthy airways.

Exhaled volatile organic compounds in individuals with a history of high altitude pulmonary edema and varying hypoxia-induced responses.

With ascent to altitude, certain individuals are susceptible to high altitude pulmonary edema (HAPE), which in turn can cause disability and even death. The ability to identify individuals at risk of HAPE prior to ascent is poor. The present study examined the profile of volatile organic compounds (VOC) in exhaled breath condensate (EBC) and pulmonary artery systolic pressures (PASP) before and after exposure to normobaric hypoxia (12% O2) in healthy males with and without a history of HAPE (Hx HAPE, n = 5; Control, n = 11). In addition, hypoxic ventilatory response (HVR), and PASP response to normoxic exercise were also measured. Auto-regression/partial least square regression of whole gas chromatography/mass spectrometry (GC/MS) data and binary logistic regression (BLR) of individual GC peaks and physiologic parameters resulted in models that separate individual subjects into their groups with variable success. The result of BLR analysis highlights HVR, PASP response to hypoxia and the amount of benzyl alcohol and dimethylbenzaldehyde dimethyl in expired breath as markers of HAPE history. These findings indicate the utility of EBC VOC analysis to discriminate between individuals with and without a history of HAPE and identified potential novel biomarkers that correlated with physiological responses to hypoxia.

Ozone-induced airway epithelial cell death, the neurokinin-1 receptor pathway, and the postnatal developing lung.

Children are uniquely susceptible to ozone because airway and lung growth continue for an extensive period after birth. Early-life exposure of the rhesus monkey to repeated ozone cycles results in region-specific disrupted airway/lung growth, but the mediators and mechanisms are poorly understood. Substance P (SP), neurokinin-1 receptor (NK-1R); and nuclear receptor Nur77 (NR4A1) are signaling pathway components involved in ozone-induced cell death. We hypothesize that acute ozone (AO) exposure during postnatal airway development disrupts SP/NK-1R/Nur77 pathway expression and that these changes correlate with increased ozone-induced cell death. Our objectives were to 1) spatially define the normal development of the SP/NK-1R/Nur77 pathway in conducting airways; 2) compare how postnatal age modulates responses to AO exposure; and 3) determine how concomitant, episodic ozone exposure modifies age-specific acute responses. Male infant rhesus monkeys were assigned at age 1 mo to two age groups, 2 or 6 mo, and then to one of three exposure subgroups: filtered air (FA), FA+AO (AO: 8 h/day × 2 days), or episodic biweekly ozone exposure cycles (EAO: 8 h/day × 5 days/14-day cycle+AO). O3 = 0.5 ppm. We found that 1) ozone increases SP/NK-1R/Nur77 pathway expression in conducting airways, 2) an ozone exposure cycle (5 days/cycle) delivered early at age 2 mo resulted in an airway that was hypersensitive to AO exposure at the end of 2 mo, and 3) continued episodic exposure (11 cycles) resulted in an airway that was hyposensitive to AO exposure at 6 mo. These observations collectively associate with greater overall inflammation and epithelial cell death, particularly in early postnatal (2 mo), distal airways.

Diverse forms of pulmonary hypertension remodel the arterial tree to a high shear phenotype.

Pulmonary hypertension (PH) is associated with progressive changes in arterial network complexity. An allometric model is derived that integrates diameter branching complexity between pulmonary arterioles of generation n and the main pulmonary artery (MPA) via a power-law exponent (X) in dn = dMPA2(-n/X) and the arterial area ratio ß = 2(1-2/X). Our hypothesis is that diverse forms of PH demonstrate early decrements in X independent of etiology and pathogenesis, which alters the arteriolar shear stress load from a low-shear stress (X > 2, ß > 1) to a high-shear stress phenotype (X < 2, ß < 1). Model assessment was accomplished by comparing theoretical predictions to retrospective morphometric and hemodynamic measurements made available from a total of 221 PH-free and PH subjects diagnosed with diverse forms (World Health Organization; WHO groups I-IV) of PH: mitral stenosis, congenital heart disease, chronic obstructive pulmonary lung disease, chronic thromboembolism, idiopathic pulmonary arterial hypertension (IPAH), familial (FPAH), collagen vascular disease, and methamphetamine exposure. X was calculated from pulmonary artery pressure (PPA), cardiac output (Q) and body weight (M), utilizing an allometric power-law prediction of X relative to a PH-free state. Comparisons of X between PAH-free and PAH subjects indicates a characteristic reduction in area that elevates arteriolar shear stress, which may contribute to mechanisms of endothelial dysfunction and injury before clinically defined thresholds of pulmonary vascular resistance and PH. We conclude that the evaluation of X may be of use in identifying reversible and irreversible phases of PH in the early course of the disease process.

Analysis of volatile compounds in exhaled breath condensate in patients with severe pulmonary arterial hypertension.

BACKGROUND: An important challenge to pulmonary arterial hypertension (PAH) diagnosis and treatment is early detection of occult pulmonary vascular pathology. Symptoms are frequently confused with other disease entities that lead to inappropriate interventions and allow for progression to advanced states of disease. There is a significant need to develop new markers for early disease detection and management of PAH. METHODOLGY AND FINDINGS: Exhaled breath condensate (EBC) samples were compared from 30 age-matched normal healthy individuals and 27 New York Heart Association functional class III and IV idiopathic pulmonary arterial hypertenion (IPAH) patients, a subgroup of PAH. Volatile organic compounds (VOC) in EBC samples were analyzed using gas chromatography/mass spectrometry (GC/MS). Individual peaks in GC profiles were identified in both groups and correlated with pulmonary hemodynamic and clinical endpoints in the IPAH group. Additionally, GC/MS data were analyzed using autoregression followed by partial least squares regression (AR/PLSR) analysis to discriminate between the IPAH and control groups. After correcting for medicaitons, there were 62 unique compounds in the control group, 32 unique compounds in the IPAH group, and 14 in-common compounds between groups. Peak-by-peak analysis of GC profiles of IPAH group EBC samples identified 6 compounds significantly correlated with pulmonary hemodynamic variables important in IPAH diagnosis. AR/PLSR analysis of GC/MS data resulted in a distinct and identifiable metabolic signature for IPAH patients. CONCLUSIONS: These findings indicate the utility of EBC VOC analysis to discriminate between severe IPAH and a healthy population; additionally, we identified potential novel biomarkers that correlated with IPAH pulmonary hemodynamic variables that may be important in screening for less severe forms IPAH.

Persistence of serotonergic enhancement of airway response in a model of childhood asthma.

The persistence of airway hyperresponsiveness (AHR) and serotonergic enhancement of airway smooth muscle (ASM) contraction induced by ozone (O3) plus allergen has not been evaluated. If this mechanism persists after a prolonged recovery, it would indicate that early-life exposure to O3 plus allergen induces functional changes predisposing allergic individuals to asthma-related symptoms throughout life, even in the absence of environmental insult. A persistent serotonergic mechanism in asthma exacerbations may offer a novel therapeutic target, widening treatment options for patients with asthma. The objective of this study was to determine if previously documented AHR and serotonin-enhanced ASM contraction in allergic monkeys exposed to O3 plus house dust mite allergen (HDMA) persist after prolonged recovery. Infant rhesus monkeys sensitized to HDMA were exposed to filtered air (FA) (n = 6) or HDMA plus O3 (n = 6) for 5 months. Monkeys were then housed in a FA environment for 30 months. At 3 years, airway responsiveness was assessed. Airway rings were then harvested, and ASM contraction was evaluated using electrical field stimulation with and without exogenous serotonin and serotonin-subtype receptor antagonists. Animals exposed to O3 plus HDMA exhibited persistent AHR. Serotonin exacerbated the ASM contraction in the exposure group but not in the FA group. Serotonin subtype receptors 2, 3, and 4 appear to drive the response. Our study shows that AHR and serotonin-dependent exacerbation of cholinergic-mediated ASM contraction induced by early-life exposure to O3 plus allergen persist for at least 2.5 years and may contribute to a persistent asthma phenotype.

Behavioral inhibition in rhesus monkeys (Macaca mulatta) is related to the airways response, but not immune measures, commonly associated with asthma.

Behavioral inhibition reflects a disposition to react warily to novel situations, and has been associated with atopic diseases such as asthma. Retrospective work established the relationship between behavioral inhibition in rhesus monkeys (Macaca mulatta) and airway hyperresponsiveness, but not atopy, and the suggestion was made that behavioral inhibition might index components of asthma that are not immune-related. In the present study, we prospectively examined the relationship between behavioral inhibition and airway hyperresponsiveness, and whether hormonal and immune measures often associated with asthma were associated with behavioral inhibition and/or airway hyperresponsiveness. In a sample of 49 yearling rhesus monkeys (mean=1.25 years, n=24 behaviorally inhibited animals), we measured in vitro cytokine levels (IL-4, IL-10, IL-12, IFN-γ) in response to stimulation, as well as peripheral blood cell percentages, cortisol levels, and percentage of regulatory T-cells (CD3+CD4+CD25+FOXP3+). Airway reactivity was assessed using an inhaled methacholine challenge. Bronchoalveolar lavage was performed and the proportion of immune cells was determined. Behaviorally inhibited monkeys had airway hyperresponsiveness as indicated by the methacholine challenge (p=0.031), confirming our earlier retrospective result. Airway hyperresponsiveness was also associated with lower lymphocyte percentages in lavage fluid and marginally lower plasma cortisol concentrations. However, none of the tested measures was significantly related to both behavioral inhibition and airway hyperresponsiveness, and so could not mediate their relationship. Airway hyperresponsiveness is common to atopic and non-atopic asthma and behavioral inhibition has been related to altered autonomic activity in other studies. Our results suggest that behavioral inhibition might index an autonomically mediated reactive airway phenotype, and that a variety of stimuli (including inflammation within lung tissue that is not specifically associated with behavioral inhibition) may trigger the airways response.

Ozone exposure alters serotonin and serotonin receptor expression in the developing lung.

Ozone, a pervasive environmental pollutant, adversely affects functional lung growth in children. Animal studies demonstrate that altered lung development is associated with modified signaling within the airway epithelial mesenchymal trophic unit, including mediators that can change nerve growth. We hypothesized that ozone exposure alters the normal pattern of serotonin, its transporter (5-HTT), and two key receptors (5-HT2A and 5-HT4), a pathway involved in postnatal airway neural, epithelial, and immune processes. We exposed monkeys to acute or episodic ozone during the first 2 or 6 months of life. There were three exposure groups/age: (1) filtered air, (2) acute ozone challenge, and (3) episodic ozone + acute ozone challenge. Lungs were prepared for compartment-specific qRT-PCR, immunohistochemistry, and stereology. Airway epithelial serotonin immunopositive staining increased in all exposure groups with the most prominent in 2-month midlevel and 6-month distal airways. Gene expression of 5-HTT, 5-HT2AR, and 5-HT4R increased in an age-dependent manner. Overall expression was greater in distal compared with midlevel airways. Ozone exposure disrupted both 5-HT2AR and 5-HT4R protein expression in airways and enhanced immunopositive staining for 5-HT2AR (2 months) and 5-HT4R (6 months) on smooth muscle. Ozone exposure increases serotonin in airway epithelium regardless of airway level, age, and exposure history and changes the spatial pattern of serotonin receptor protein (5-HT2A and 5-HT4) and 5-HTT gene expression depending on compartment, age, and exposure history. Understanding how serotonin modulates components of reversible airway obstruction exacerbated by ozone exposure sets the foundation for developing clinically relevant therapies for airway disease.

Simvastatin inhibits smoke-induced airway epithelial injury: implications for COPD therapy.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death. The statin drugs may have therapeutic potential in respiratory diseases such as COPD, but whether they prevent bronchial epithelial injury is unknown. We hypothesised that simvastatin attenuates acute tobacco smoke-induced neutrophilic lung inflammation and airway epithelial injury. Spontaneously hypertensive rats were given simvastatin (20 mg·kg(-1) i.p.) daily for either 7 days prior to tobacco smoke exposure and during 3 days of smoke exposure, or only during tobacco smoke exposure. Pretreatment with simvastatin prior to and continued throughout smoke exposure reduced the total influx of leukocytes, neutrophils and macrophages into the lung and airways. Simvastatin attenuated tobacco smoke-induced cellular infiltration into lung parenchymal and airway subepithelial and interstitial spaces. 1 week of simvastatin pretreatment almost completely prevented smoke-induced denudation of the airway epithelial layer, while simvastatin given only concurrently with the smoke exposure had no effect. Simvastatin may be a novel adjunctive therapy for smoke-induced lung diseases, such as COPD. Given the need for statin pretreatment there may be a critical process of conditioning that is necessary for statins' anti-inflammatory effects. Future work is needed to elucidate the mechanisms of this statin protective effect.

Postnatal exposure history and airways: oxidant stress responses in airway explants.

Postnatally, the lung continues to grow and differentiate while interacting with the environment. Exposure to ozone (O(3)) and allergens during postnatal lung development alters structural elements of conducting airways, including innervation and neurokinin abundance. These changes have been linked with development of asthma in a rhesus monkey model. We hypothesized that O(3) exposure resets the ability of the airways to respond to oxidant stress and that this is mediated by changes in the neurokinin-1 receptor (NK-1R). Infant rhesus monkeys received episodic exposure to O(3) biweekly with or without house dust mite antigen (HDMA) from 6 to 12 months of age. Age-matched monkeys were exposed to filtered air (FA). Microdissected airway explants from midlevel airways (intrapulmonary generations 5-8) for four to six animals in each of four groups (FA, O(3), HDMA, and HDMA+O(3)) were tested for NK-1R gene responses to acute oxidant stress using exposure to hydrogen peroxide (1.2 mM), a lipid ozonide (10 µM), or sham treatment for 4 hours in vitro. Airway responses were measured using real-time quantitative RT-PCR of NK-1R and IL-8 gene expression. Basal NK-1R gene expression levels were not different between the exposure groups. Treatment with ozonide or hydrogen peroxide did not change NK-1R gene expression in animals exposed to FA, HDMA, or HDMA+O(3). However, treatment in vitro with lipid ozonide significantly increased NK-1R gene expression in explants from O(3)-exposed animals. We conclude that a history of prior O(3) exposure resets the steady state of the airways to increase the NK-1R response to subsequent acute oxidant stresses.

Ozone exposure during the early postnatal period alters the timing and pattern of alveolar growth and development in nonhuman primates.

Exposure to oxidant air pollutants in early childhood, with ozone as the key oxidant, has been linked to significant decrements in pulmonary function in young adults and exacerbation of airway remodeling in asthma. Development of lung parenchyma in rhesus monkeys is rapid during the first 2 years of life (comparable to the first 6 years in humans). Our hypothesis is that ozone inhalation during infancy alters alveolar morphogenesis. We exposed infant rhesus monkeys biweekly to 5, 8 hr/day, cycles of 0.5 ppm ozone with or without house dust mite allergen from 1 to 3 or 1 to 6 months of age. Monkeys were necropsied at 3 and 6 months of age. A morphometric approach was used to quantify changes in alveolar volume and number, the distribution of alveolar size, and capillary surface density per alveolar septa. Quantitative real time PCR was used to measure the relative difference in gene expression over time. Monkeys exposed to ozone alone or ozone combined with allergen had statistically larger alveoli that were less in number at 3 months of age. Alveolar capillary surface density was also decreased in the ozone exposed groups at 3 months of age. At 6 months of age, the alveolar number was similar between treatment groups and was associated with a significant rise in alveolar number from 3 to 6 months of age in the ozone exposed groups. This increase in alveolar number was not associated with any significant increase in microvascular growth as measured by morphometry or changes in angiogenic gene expression. Inhalation of ozone during infancy alters the appearance and timing of alveolar growth and maturation. Understanding the mechanism involved with this altered alveolar growth may provide insight into the parenchymal injury and repair process that is involved with chronic lung diseases such as severe asthma and COPD.

Prediction of lung function response for populations exposed to a wide range of ozone conditions.

CONTEXT: A human exposure-response (E-R) model previously demonstrated to accurately predict population mean FEV1 response to ozone exposure has been proposed as the foundation for future risk assessments for ambient ozone. OBJECTIVE: Fit the original and related models to a larger data set with a wider range of exposure conditions and assess agreement between observed and population mean predicted values. MATERIALS AND METHODS: Existing individual E-R data for 23 human controlled ozone exposure studies with a wide range of concentrations, activity levels, and exposure patterns have been obtained. The data were fit to the original model and to a version of the model that contains a threshold below which no response occurs using a statistical program for fitting nonlinear mixed models. RESULTS: Mean predicted FEV1 responses and the predicted proportions of individuals experiencing FEV1 responses greater than 10, 15, and 20% were found to be in agreement with observed responses across a wide range of exposure conditions for both models. The threshold model, however, provided a better fit to the data than the original, particularly at the lowest levels of exposure. CONCLUSION: The models identified in this manuscript predict population FEV1 response characteristics for 18-35-year-old individuals exposed to ozone over a wide range of conditions and represent a substantial improvement over earlier E-R models. Because of its better fit to the data, particularly at low levels of exposure, the threshold model is likely to provide more accurate estimates of risk in future risk assessments of ozone-induced FEV1 effects.