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Clinical case series suggest beneficial effects of low-dose intermittent hypoxia in asthma. We tested cardiopulmonary effects of repetitive acute hypoxic preconditioning (RAHP) during allergic inflammation. Brown Norway rats were sensitized to house dust mites (HDM) and exposed to 4-week RAHP or normoxia (SHAM), concurrent with weekly HDM or saline (SAL) challenges. We assessed methacholine responses and lung HIF-1α expression at endpoint, and weekly blood pressure (BP). RAHP relative to SHAM: 1) in HDM-challenged rats, showed no protection against HDM-induced airway dysfunction and did not significantly impact BP (week 4 mean BP difference = 10.51 mmHg, p = 0.09) or HIF-1α expression; 2) in SAL-challenged rats, attenuated airway responses to methacholine, reduced BP (week 4 mean BP average difference = -8.72 mmHg, p = 0.04) and amplified HIF-1α expression (p = 0.0086). Four weeks of RAHP did not mitigate the allergen-induced lower airway dysfunction and may detrimentally affect BP. However, it elicited beneficial cardiopulmonary responses in SAL-challenged rats, concurrent with increased HIF-1α expression.

Background Despite advances in asthma care, disparities persist. Black patients are disproportionally affected by asthma and also have poorer outcomes compared with white patients. Objective We sought to determine associations between black and white patients and asthma-related health care use, accounting for complex relationships. Methods This study was completed as part of the National Heart, Lung, and Blood Institute's Severe Asthma Research Program, a prospective observational cohort. Between November 2012 and February 2015, it enrolled 579 participants 6 years and older with 1 year of observation time and complete data. Inverse probability of treatment weighting was used to balance racial groups with respect to community and family socioeconomic variables and environmental exposure variables. The primary outcome was emergency department (ED) use for asthma. Secondary outcomes included inhaled corticosteroid use, outpatient physician's office visits for asthma, and asthma–related hospitalization. Results Black patients had greater odds of ED use over 1 year (odds ratio, 2.19; 95% CI, 1.43-3.35) but also differed in the majority (>50%) of baseline variables measured. After statistical balancing of the racial groups, the difference between black and white patients with respect to ED use no longer reached the level of significance. Instead, in secondary analyses black patients were less likely to see an outpatient physician for asthma management (adjusted odds ratio, 0.57; 95% CI, 0.38-0.85). Conclusions The disparity in ED use was eliminated after consideration of multiple variables. Social and environmental policies and interventions tailored to black populations with a high burden of asthma are critical to reduction (or elimination) of these disparities.

Rationale and Objectives To determine lobar ventilation patterns in asthmatic lungs with hyperpolarized 3He magnetic resonance imaging (HP 3He MRI). Materials and Methods Eighty-two subjects (14 normal, 48 mild-to-moderate asthma, and 20 severe asthma) underwent HP 3He MRI, computed tomography (CT), and pulmonary function testing. After registering proton to 3He images, we segmented the lungs from proton MRI and further segmented the five lung lobes (right upper lobe [RUL], right middle lobe [RML], and right lower lobe [RLL]; left upper lobe and left lower lobe [LLL]) by referring to the lobar segmentation from CT. We classified the gas volume into four signal intensity levels as follows: ventilation defect percent (VDP), low ventilation percent, medium ventilation percent, and high ventilation percent. The local signal intensity variations in the ventilated volume were estimated using heterogeneity score (Hs). We compared each ventilation level and Hs measured in the whole lung and lobar regions across the three subject groups. Results In mild-to-moderate asthma, the RML and RUL showed significantly greater VDP than the two lower lobes (RLL and LLL) (P ≤ .047). In severe asthma, the pattern was more variable with the VDP in the RUL significantly greater than in the RLL (P = .026). In both asthma groups, the lower lobes (RLL and LLL) showed significantly higher high ventilation percent and Hs compared to the three upper lobes (all P ≤ .015). Conclusions In asthma, the RML and RUL showed greater ventilation abnormalities, and the RLL and LLL were more highly ventilated with greater local heterogeneity. These findings may facilitate guided bronchoscopic sampling and localized airway treatment in future studies.

Background Sex influences the risk of wheezing illnesses and the prevalence of asthma throughout childhood. Objective To better understand the mechanisms of these effects, we analyzed longitudinal relationships between sex, lung physiology, and asthma in the Childhood Origins of ASThma birth cohort study. Methods Childhood Origins of ASThma birth cohort study children were followed prospectively from birth and assessed annually. Results of spirometry, fractional exhaled nitric oxide (Feno), mannitol provocation testing, and 3 He gas magnetic resonance imaging were assessed by sex using multivariate models including age, asthma diagnosis, and wheezing histories. Results Girls had higher prebronchodilator forced expiratory volume in 0.5 seconds/forced vital capacity values than did boys (mean difference, 0.017; 95% CI, 0.000-0.034; P = .05) of equivalent age. Postbronchodilator findings were more pronounced, with boys demonstrating reduced forced expiratory volume in 0.5 seconds/forced vital capacity values than did girls of equivalent age (mean difference, 0.032; 95% CI, 0.014-0.049; P = .0005). Conversely, girls were noted to have higher ventilation defects on 3 He magnetic resonance imaging than did boys ( P = .01). No differences were noted in the rate of positive responses to mannitol provocation or Feno measurements. Conclusions Lower airflow values are present by spirometry for prepubertal boys than for age-matched girls; however, greater 3 He ventilation defects were noted in girls. This could represent a greater degree of subclinical air trapping in prepubertal girls because residual volumes are not detected on standard spirometric readings. No differences were noted between the 2 sexes with airway hyperresponsiveness (mannitol provocation testing) or inflammation (Feno). Prospective peripubertal follow-up will determine whether these differences persist or change with the de novo expression and remission of asthma based on sex and age.

Interstitial pulmonary fibrosis is caused by the excess production of extracellular matrix (ECM) by Fb in response to TGF-β1. Here, we show that the peptidyl-prolyl isomerase Pin1 modulates the production of many pro- and antifibrogenic cytokines and ECM. After acute, bleomycin injury, Pin1−/− mice showed reduced, pulmonary expression of collagens, tissue inhibitors of metalloproteinases, and fibrogenic cytokines but increased matrix metalloproteinases, compared with WT mice, despite similar levels of inflammation. In primary fibroblasts, Pin1 was required for TGF-β-induced phosphorylation, nuclear translocation, and transcriptional activity of Smad3. In Pin1−/− cells, inhibitory Smad6 was found in the cytoplasm rather than nucleus. Smad6 knockdown in Pin1−/− fibroblasts restored TGF-β-induced Smad3 activation, translocation, and target gene expression. Therefore, Pin1 is essential for normal Smad6 function and ECM production in response to injury or TGF-β and thus may be an attractive therapeutic target to prevent excess scarring in diverse lung diseases. Background: Interstitial pulmonary fibrosis is caused by the excess production of extracellular matrix (ECM) by Fb in response to TGF-β1. Results: The peptidyl-prolyl isomerase Pin1 modulates the production of many pro- and antifibrogenic cytokines and ECM after bleomycin injury. Conclusion: Pin1 controls Smad6 function. Significance: Pin1 may be a therapeutic target to prevent pathologic lung scarring.

Ventilation defect percent (VDP) measured in asthmatics with hyperpolarized helium-3 MRI was more strongly associated with ED visits and hospitalizations due to asthma exacerbation than were conventional biomarkers of lung function and inflammation.

Sensitivity to the fungus Alternaria is associated with asthma persistence and severity. Current therapeutic options for treating Alternaria-induced airway inflammation are limited. In this study, Brown Norway rats are used to study the effectiveness of a DNA-based vaccine delivered to the airway in attenuating the response to a major Alternaria allergen, rAlt a 2. Compared to untreated sensitized animals, or animals receiving an "out-of-frame" DNA-based vaccine, animals treated with "in-frame" DNA vaccine showed an attenuation in specific IgE antibody titers to rAlt a 2, an increase in IgG(2b) (a Th1 response), a reduction in spontaneous IL-13 release by peribronchial lymph node cell suspensions, and an attenuation in the decrease in total lung capacity 72 h post-allergen challenge. Further, histopathologic examination of the lung tissues revealed reduced pulmonary inflammation post-allergen challenge in the DNA-vaccine-treated compared to sensitized, untreated animals. We conclude that a DNA-based vaccine delivered to the airway significantly influences the immunologic, pulmonary physiologic, and histological alterations induced by challenge with a major Alternaria allergen, rAlt a 2, in sensitized animals.

Rationale and Objectives Although multiple detector computed tomography (MDCT) and hyperpolarized gas magnetic resonance imaging (HP MRI) have demonstrated ability to detect structural and ventilation abnormalities in asthma, few studies have sought to exploit or cross-validate the regional information provided by these techniques. The purpose of this work is to assess regional disease in asthma by evaluating the association of sites of ventilation defect on HP MRI with other regional markers of airway disease, including air trapping on MDCT and inflammatory markers on bronchoscopy. Materials and Methods Both HP MRI using helium-3 and MDCT were acquired in the same patients. Supervised segmentation of the lung lobes on MRI and MDCT facilitated regional comparisons of ventilation abnormalities in the lung parenchyma. The percentage of spatial overlap was evaluated between regions of ventilation defect on HP MRI and hyperlucency on MDCT to determine associations between obstruction and likely regions of gas trapping. Similarly, lung lobes with high defect volume were compared to lobes with low defect volume for differences in inflammatory cell number and percentage using bronchoscopic assessment. Results There was significant overlap between sites of ventilation defect on HP MRI and hyperlucency on MDCT suggesting that sites of airway obstruction and air trapping are associated in asthma. The percent (r = 0.68; P = .0039) and absolute (r = 0.61; P = .0125) number of neutrophils on bronchoalveolar lavage for the sampled lung lobe also directly correlated with increased defect volume. Conclusions These results show promise for using image guidance to assess specific regions of ventilation defect or air trapping in heterogeneous obstructive lung diseases such as asthma.

Viral infections are closely linked to wheezing in infancy, and those children with recurrent virus-induced wheezing episodes are at great risk for chronic childhood asthma. Infancy is a time of increased susceptibility to viral infections, and this stage is also characterized by pulmonary alveolar multiplication and extensive remodeling of the airways to accommodate growth. This coincidence, together with the observation that children with asthma can have structural lung changes and functional deficits at an early age, suggests that viral infections could adversely affect lung development. Inflammatory mediators induced by viral infection are known to have effects on the remodeling process, suggesting a plausible mechanism to support this theory. Furthermore, animal models of viral infection during lung growth and development suggest that developmental factors are important in determining the consequences of infection on long-term lung function. Greater understanding of the effects of viral infections on lung development and growth in early childhood might lead to the discovery of additional strategies for the prevention of recurrent wheezing and chronic asthma.

We describe a reversed-phase assay of hydroxyproline in rat lung tissue using sarcosine for the internal standard and pre-injection reaction with both o-phthalaldehyde (OPA) and 9-fluorenylmethylchloroformate (FMOC). Intra-assay variability in the concentration range of 25-500 microM hydroxyproline was less than 1%. Normal rat (left) lung was found to have a hydroxyproline content of 1.08+/-0.18 mg/lung. This ability to measure minute amounts of hydroxyproline is being applied to the measure of collagen and pathological fibrosis.

Respiratory viral infections have been associated with exacerbations of asthma in humans, and are known to produce airway obstruction and hyperresponsiveness in rats. Virus-induced airway dysfunction may result in part from inflammatory cells and their products, and agents that target these mechanisms might therefore attenuate viral airway injury. The 21-aminosteroid class of drugs has been reported to attenuate tissue injury in a variety of models, and we hypothesized that U-83836E, an orally-active aminosteroid, would prevent the development of airway dysfunction during acute viral illness. Adult rats were inoculated with either parainfluenza type 1 (Sendai) virus or sterile vehicle, treated with either U-83836E 20 mg/kg or water by oral gavage twice daily, and studied on postinoculation day 5, 6 or 7. Anesthetized, paralysed, mechanically ventilated rats were placed in a body plethysmograph for measurements of airway obstruction (resistance, dynamic compliance, eucapneic PaO2), and responsiveness to i.v. methacholine; lungs were lavaged to obtain inflammatory cells. The water-treated virus group was significantly different from the non-infected controls for all variables. Virus-induced hyperresponsiveness was attenuated (P = 0.027) by aminosteroid treatment, although airway obstruction and inflammation were not improved by the treatment. We conclude that 21-aminosteroids may protect airways from virus-induced hyperresponsiveness.

Background Coagulation Factor XIII (FXIII) plays an important role in wound healing by stabilizing fibrin clots and cross-linking extracellular matrix proteins. FXIII is expressed in cells of the monocyte/macrophage and dendritic cell lineages in response to type 2 cytokines. Objective We sought to determine the association between FXIII and asthma pathobiology. Methods We analyzed the expression of FXIII mRNA and protein levels in bronchoalveolar lavage samples obtained before and after segmental allergen challenge from patients with mild asthma and in induced sputum samples collected from patients with mild-to-moderate and severe asthma. Results FXIII mRNA and protein levels were highly upregulated in bronchoalveolar cells and fluid after allergen challenge and mRNA levels correlated with protein levels. In sputum of asthmatic patients, FXIII expression was positively correlated with type 2 immune response and dendritic cell markers (CD209 and CD207). FXIII expression was also associated with increased airflow limitation (FEV 1 /forced vital capacity and residual volume/total lung capacity ratios) and greater reversibility to β-agonists. Conclusions FXIII expression was upregulated in the airways of asthmatic patients after allergen exposure. Expression in the sputum of asthmatic patients correlated with the type 2 immune response and airflow limitation. Excessive activity of FXIII could contribute to the pathophysiology of airway obstruction in asthmatic patients.

After viral bronchiolitis at an early age, a chronic asthma-like syndrome develops in BN, but not F344, rats. We hypothesized that the BN strain is less effective at clearing virus from the involved tissues. Weanling BN and F344 rats were inoculated with Sendai virus, and lung and peribronchial lymph nodes were harvested from each strain at 5 to 84 days after infection; control tissues were obtained from noninfected rats. Lung viral titers were similar for the 2 strains, with no infectious virus detectable by day 10. However, viral RNA was detected consistently by means of RT-PCR analyses in lungs and lymph nodes of both strains from days 10 to 27 and was still present at day 84 in some of the tissues from each strain. In contrast, there were strain-related differences in immune responses because IL-13 levels remained increased in the lung secretions of BN rats at 4 weeks after inoculation. Thus although Sendai virus could persist for at least 3 months after an acute infection in rats, this did not differ with strain. The persistent increase in IL-13 suggests instead that the strain-related variability in virus-associated airway pathology might be determined by the host response to infection rather than by the intensity or duration of infection. (J Allergy Clin Immunol 2002;110:607-9.)

Background Obstructive sleep apnea (OSA) may worsen asthma, but large studies are lacking and the underlying mechanisms are unknown. Objective The objective of this study was to determine the prevalence of OSA risk among patients with asthma of different severity compared with normal controls (NC), and among asthmatics, to test the relationship of OSA risk with asthma burden and airway inflammation. Methods Subjects with severe (SA, n = 94) and nonsevere asthma (NSA, n = 161), and NC (n = 146) were recruited in an add-on substudy, to the observational Severe Asthma Research Program (SARP) II; subjects completed sleep quality, sleepiness and OSA risk (Sleep Apnea scale of the Sleep Disorders Questionnaire [SA-SDQ]) questionnaires, and clinical assessments. Sputum was induced in a subset of asthmatics. Results Relative to NC, despite similar sleep duration, the subjects with SA and NSA had worse sleep quality, were sleepier, and had higher SA-SDQ scores. Among asthmatics, higher SA-SDQ was associated with increased asthma symptoms, β-agonist use, health care utilization, and worse asthma quality of life. A significant association of SA-SDQ with sputum polymorphonuclear cells% was noted: each increase in SA-SDQ by its standard deviation (6.85 units) was associated with a rise in % sputum neutrophils of 7.78 (95% CI 2.33-13.22, P = .0006), independent of obesity and other confounders. Conclusions OSA symptoms are more prevalent among asthmatics, in whom they are associated with higher disease burden. OSA risk is associated with a neutrophilic airway inflammation in asthma, which suggests that OSA may be an important contributor to the neutrophilic asthma. Further studies are necessary to confirm these findings and better understand the mechanistic underpinnings of this relationship.

Background Magnetic resonance imaging (MRI) with 3 He does not require ionizing radiation and has been shown to detect regional abnormalities in lung ventilation and structure in adults with asthma, but the method has not been extended to children with asthma. Measurements of regional lung ventilation and microstructure in subjects with childhood asthma could advance our understanding of disease mechanisms. Objective We sought to determine whether 3 He MRI in children can identify abnormalities related to the diagnosis of asthma or prior history of respiratory illness. Methods Forty-four children aged 9 to 10 years were recruited from a birth cohort at increased risk of asthma and allergic diseases. For each subject, a time-resolved 3-dimensional image series and a 3-dimensional diffusion-weighted image were acquired in separate breathing maneuvers. The numbers and sizes of ventilation defects were scored, and regional maps and statistics of average 3 He diffusion lengths were calculated. Results Children with mild-to-moderate asthma had lower average root-mean-square diffusion length ( X r m s ¯ ) values ( P = .004), increased regional SD of diffusion length values ( P = .03), and higher defect scores ( P = .03) than those without asthma. Children with histories of wheezing illness with rhinovirus infection before the third birthday had lower X r m s ¯ values ( P = .01) and higher defect scores ( P = .05). Conclusion MRI with 3 He detected more and larger regions of ventilation defect and a greater degree of restricted gas diffusion in children with asthma compared with those seen in children without asthma. These measures are consistent with regional obstruction and smaller and more regionally variable dimensions of the peripheral airways and alveolar spaces.

and Viral Inflammatory Stimuli in Rats With the Chronic Postbronchiolitis Asthma-Like Phenotype R. L. SORKNESS1, L. A. ROSENTHAL2, T. PIER2, S. P. AMINEVA2, R. J. SZAKALY2, R. F. LEMANSKE, Jr.3; 1Sch of pharmacy, UNIVERSITY OF WISCONSIN, Madison, WI, 2Medicine, UNIVERSITY OF WISCONSIN, Madison, WI, 3Pediatrics, UNIVERSITY OF WISCONSIN, Madison, WI. RATIONALE: Airway inflammation in asthma may be due to enhanced vulnerability of airways to common inflammatory stimuli. We hypothesized that rats with chronic airway dysfunction would differ from normals in their acute pulmonary inflammatory responses. METHODS: Normal BN rats were compared with asthma-like BN rats that were >10wks post early age bronchiolitis. Acute allergic inflammation was induced by a single exposure to aerosolized ragweed pollen extract 2wks after sensitization, and evaluated at 1 or 7 days; viral inflammation was induced with a tracheal insufflation of attenuated mengovirus, and evaluated in 3 days. Airspace infiltrates were quantified with bronchoalveolar lavage (BAL), and airway wall eosinophils (eos) with staining of major basic protein (MBP) in lung sections. RESULTS: Unchallenged normal and asthma-like rats did not differ significantly with regard to BAL eos or airway MBP. However, the asthmalike rats had significantly greater airway wall MBP (p=0.001) and fewer BAL eos (p=0.02) 1 day after allergen challenge, and fewer BAL eos (p=0.04) with similar airway MBP 7 days after allergen. During viral infection a similar pattern occurred, the asthma-like rats having few eos and neutrophils in BAL (p

Rationale After Sendai virus-induced bronchiolitis as weanlings, BN, unlike F344, rats develop a postbronchiolitis asthma-like phenotype, which is prevented by IFN-γ therapy. Innate mechanisms of Sendai virus-induced IFN-γ production, which are dependent on NK cells and the production of IFN-γ-inducing cytokines (IL-12 and IL-18), are markedly attenuated in BN, compared with F344, weanlings. GM-CSF and Flt-3 ligand may enhance innate immunity by augmenting dendritic cell or dendritic and NK cell development, respectively. We hypothesized that treatment with GM-CSF and Flt-3 ligand would counteract the reduced capacity of BN weanlings to secrete IFN-γ and IFN-γ-inducing cytokines in response to virus. Methods Splenocytes from uninfected weanlings were preincubated for 16 h with or without GM-CSF or Flt-3 ligand and then for 24 h with or without Sendai virus. IFN-γ, IL-12, and IL-18 were measured in supernates by ELISA. Results GM-CSF and Flt-3 ligand triggered 12- and 8-fold increases, respectively, in virus-induced IFN-γ production by BN splenocytes ( p Conclusions GM-CSF-mediated enhancement of virus-induced IFN-γ production from BN splenocytes appeared to result from increases in IL-12 secretion, suggesting that GM-CSF augmented deficient dendritic cell activity. Flt-3 ligand-mediated, IL-12-independent increases in virus-induced IFN-γ production may arise from enhancement of deficient NK cell responses. These results suggest a basis for attenuated innate anti-viral responses in BN weanlings.

Rationale Animal studies involving administration of aerosolized drugs are complicated by the difficulties of generating and delivering the aerosol to the lungs effectively and reproducibly. A method utilizing a metered dose inhaler (MDI) would allow simplification and standardization of aerosol dosing. Methods 18 adult BN rats were sensitized to ragweed. A spacer chamber was modified with a latex face mask that allowed insertion of an awake rat's nose and mouth into the chamber opposite the MDI. After MDI actuation, rats breathed either placebo or fluticasone 220 aerosol from the chamber for 60s, once daily X7d prior to and 1d after aerosolized ragweed challenge. Others were treated with s.c. dexamethasone X3d for comparison. Body and thymus weights were indicators of systemic steroid effect, and lung volumes, forced expiratory maneuvers and BAL cells were indicators of pulmonary allergic responses at 48h after allergen challenge. Results Fluticasone MDI treatment caused body weight loss and thymus weight loss compared with placebo treatment, indicating sufficient pulmonary delivery to obtain systemic effects. Body weight loss was greater with systemic dexamethasone treatment. Fluticasone also protected the rats from pulmonary allergic responses, with preservation of total lung capacity, FEV 0.2 and FVC, and reduced BAL inflammatory cells, compared with placebo treatment (p 0.2 and BAL eosinophils, and both the fluticasone and dexamethasone groups showed small within-group variability. Conclusion This method of administering MDI aerosols to rats is simple, effective, and reproducible.

Rationale Allergens with intrinsic protease activity have been shown to enable Th2 priming, and allow the establishment of airway inflammation and airway hyperresponsiveness in Th1-skewed mice. We attempted to induce Th2 responses in the lung of Fisher (F344) rats, a Th1-skewed animal model, using a protease-containing crude Alternaria extract. Methods Four groups of F344 rats were evaluated over a six-week period. Group 1 was sensitized to crude Alternaria in AlOH 3 and nebulized with crude Alternaria biweekly. Group 2 was sensitized to crude Alternaria in AlOH 3 and exposed to crude Alternaria biweekly via direct intranasal inoculation. Group 3 was not sensitized and nebulized with crude Alternaria biweekly. Group 4 was not sensitized and nebulized with saline biweekly. At the end of six weeks, airway hyperresponsiveness was determined by methacholine challenge. Right-sided bronchoalveolar lavage was performed, and cell counts with differentials were obtained from the bronchoalveolar fluid (BALF). Protease levels in the crude Alternaria extract were confirmed using a protease assay kit. Results No differences in airway hyperresponsiveness or BALF cell counts with differentials were observed between the four groups (p>0.05). A correlation between the absolute eosinophil count in the BALF and PC20 was observed (r s =-0.57). The protease level in the crude Alternaria extract was 130ng/mg protein. Conclusion We were unable to induce airway hyperresponsiveness or evidence of Th2 responses in the BALF of F344 rats, despite the concentration of protease activity present in the crude Alternaria extract. Threshold doses of protease activity required to induce Th2 airway responses in rat models are currently unknown.

We studied the effects of passive, isocapnic changes in ventilation on tracheal smooth muscle tone in 3 awake and 6 anesthetized dogs. Ventilation was altered using mechanical ventilation under hyperoxic conditions, and tracheal tone was measured using the pressure within a water-filled balloon in an isolated segment of trachea. Hypocapnia was prevented at increased VA by increasing FICO2. When f and VT were changed reciprocally, keeping VA constant, tracheal tone did not change. However, when either f or VT was changed independently, tracheal tone decreased with an increase in VA. This effect was abolished following thoracic vagotomy. We conclude that tracheal tone is reflexly decreased during an increase in VA.