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Respiratory viral infections have been associated with airway obstruction and hyperresponsiveness, and exacerbations of asthma. Although virus-induced asthma is thought to be precipitated by airway inflammation, the clinical efficacy and rationale for using antiinflammatory treatment during such exacerbations remains controversial. The purpose of this study was to use a well characterized animal model of respiratory viral illness to test the hypothesis that the inflammatory response to viral infection is responsible for the development of airway dysfunction. Adult rats were inoculated with either Sendai virus or sterile vehicle and treated with daily injections of dexamethasone or saline. At postinoculation d 4, 5, or 6, rats were evaluated for airway obstruction, hyperresponsivenes, inflammation, and lung viral titers. Saline-treated infected rats had significant airway obstruction (increased resistance, decreased dynamic compliance), hyperresponsiveness (i.v. methacholine), and inflammation (increased bronchoalveolar lavage leukocytes) compared with noninfected controls. In contrast, dexamethasone-treated infected rats had no increase in bronchoalveolar lavage leukocytes and significantly smaller changes in airway physiology, but had increased lung viral titers compared with saline-treated infected rats. We conclude that glucocorticoid suppression of the inflammatory response to respiratory viral infection largely prevents virus-associated airway dysfunction.

Rationale: Air trapping and ventilation defects on imaging are characteristics of asthma. Airway wall thickening occurs in asthma and is associated with increased bronchial vascularity and vascular permeability. Vascular endothelial cell products have not been explored as a surrogate to mark structural airway changes in asthma. Objectives: Determine whether reporters of vascular endothelial cell perturbation correlate with airway imaging metrics in patients with asthma of varying severity. Methods: Plasma from Severe Asthma Research Program subjects was analyzed by ELISAs for soluble von Willebrand factor mature protein (VWF:Ag) and propeptide (VWFpp), P-selectin, and platelet factor 4. Additional subjects were analyzed over 48 hours after whole-lung antigen challenge. We calculated ventilation defect volume by hyperpolarized helium-3 magnetic resonance imaging and areas of low signal density by multidetector computed tomography (less than −856 Hounsfield units [HU] at functional residual capacity and −950 HU at total lung capacity [TLC]). Measurements and Main Results: VWFpp and VWFpp/Ag ratio correlated with and predicted greater percentage defect volume on hyperpolarized helium-3 magnetic resonance imaging. P-selectin correlated with and predicted greater area of low density on chest multidetector computed tomography less than −950 HU at TLC. Platelet factor 4 did not correlate. Following whole-lung antigen challenge, variation in VWFpp, VWFpp/Ag, and P-selectin among time-points was less than that among subjects, indicating stability and repeatability of the measurements. Conclusions: Plasma VWFpp and P-selectin may be useful as surrogates of functional and structural defects that are evident on imaging. The results raise important questions about why VWFpp and P-selectin are associated specifically with different imaging abnormalities.

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.

Studies with isolated cells are important to the understanding of mechanisms by which eosinophils participate in allergic inflammation. Due to species variability, isolation techniques and cell biology need to be defined for each source. We developed methods to obtain rat eosinophils with purity and viability exceeding 90%, characterized the superoxide anion production of these cells in response to standard activators, and compared these results with those previously obtained in our laboratories with the use of human eosinophils. Rat eosinophils responded vigorously to phorbol myristate acetate and poorly to platelet-activating factor and to N-formyl-methionyl-leucyl-phenylalanine, parallel to the responses of human eosinophils. In contrast, rat eosinophils responded unlike human eosinophils to other activators, having a larger response to calcium ionophore A23187, a smaller response to serum-treated or serum-opsonized zymosan, and a negative rather than positive modulatory effect of cytochalasin B. We conclude that rat eosinophils can be obtained in high purity and with intact responsiveness to a number of different activators.

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.

To investigate the utility of hyperpolarized He-3 MRI for detecting regional lung ventilated volume (VV) changes in response to exercise challenge and leukotriene inhibitor montelukast, human subjects with exercise induced bronchoconstriction (EIB) were recruited. This condition is described by airway constriction following exercise leading to reduced forced expiratory volume in 1 second (FEV1) coinciding with ventilation defects on hyperpolarized He-3 MRI.Thirteen EIB subjects underwent spirometry and He-3 MRI at baseline, postexercise, and postrecovery at multiple visits. On one visit montelukast was given and on two visits placebo was given. Regional VV was calculated in the apical/basilar dimension, in the anterior/posterior dimension, and for the entire lung volume. The whole lung VV was used as an end-point and compared with spirometry.Postchallenge FEV1 dropped with placebo but not with treatment, while postchallenge VV dropped more with placebo than treatment. Sources of variability for VV included region (anterior/posterior), scan, and treatment. VV correlated with FEV1/ forced vital capacity (FVC) and forced expiratory flow between 25 and 75% of FVC and showed gravitational dependence after exercise challenge.A paradigm testing the response of ventilation to montelukast revealed both a whole-lung and regional response to exercise challenge and therapy in EIB subjects.

Background Outcomes of pulmonary physiology have a central place in asthma clinical research. Objective At the request of National Institutes of Health (NIH) institutes and other federal agencies, an expert group was convened to provide recommendations on the use of pulmonary function measures as asthma outcomes that should be assessed in a standardized fashion in future asthma clinical trials and studies to allow for cross-study comparisons. Methods Our subcommittee conducted a comprehensive search of PubMed to identify studies that focused on the validation of various airway response tests used in asthma clinical research. The subcommittee classified the instruments as core (to be required in future studies), supplemental (to be used according to study aims and in a standardized fashion), or emerging (requiring validation and standardization). This work was discussed at an NIH-organized workshop in March 2010 and finalized in September 2011. Results A list of pulmonary physiology outcomes that applies to both adults and children older than 6 years was created. These outcomes were then categorized into core, supplemental, and emerging. Spirometric outcomes (FEV 1 , forced vital capacity, and FEV 1 /forced vital capacity ratio) are proposed as core outcomes for study population characterization, for observational studies, and for prospective clinical trials. Bronchodilator reversibility and prebronchodilator and postbronchodilator FEV 1 also are core outcomes for study population characterization and observational studies. Conclusions The subcommittee considers pulmonary physiology outcomes of central importance in asthma and proposes spirometric outcomes as core outcomes for all future NIH-initiated asthma clinical research.

Infections with the picornavirus, human rhinovirus (HRV), are a major cause of wheezing illnesses and asthma exacerbations. In developing a murine model of picornaviral airway infection, we noted the absence of murine rhinoviruses and that mice are not natural hosts for HRV. The picornavirus, mengovirus, induces lethal systemic infections in its natural murine hosts, but small genetic differences can profoundly affect picornaviral tropism and virulence. We demonstrate that inhalation of a genetically attenuated mengovirus, vMC(0), induces lower respiratory tract infections in mice. After intranasal vMC(0) inoculation, lung viral titers increased, peaking at 24 h postinoculation with viral shedding persisting for 5 days, whereas HRV-A01a lung viral titers decreased and were undetectable 24 h after intranasal inoculation. Inhalation of vMC(0), but not vehicle or UV-inactivated vMC(0), induced an acute respiratory illness, with body weight loss and lower airway inflammation, characterized by increased numbers of airway neutrophils and lymphocytes and elevated pulmonary expression of neutrophil chemoattractant CXCR2 ligands (CXCL1, CXCL2, CXCL5) and interleukin-17A. Mice inoculated with vMC(0), compared with those inoculated with vehicle or UV-inactivated vMC(0), exhibited increased pulmonary expression of interferon (IFN-α, IFN-β, IFN-λ), viral RNA sensors [toll-like receptor (TLR)3, TLR7, nucleotide-binding oligomerization domain containing 2 (NOD2)], and chemokines associated with HRV infection in humans (CXCL10, CCL2). Inhalation of vMC(0), but not vehicle or UV-inactivated vMC(0), was accompanied by increased airway fluid myeloperoxidase levels, an indicator of neutrophil activation, increased MUC5B gene expression, and lung edema, a sign of infection-related lung injury. Consistent with experimental HRV inoculations of nonallergic, nonasthmatic human subjects, there were no effects on airway hyperresponsiveness after inhalation of vMC(0) by healthy mice. This novel murine model of picornaviral airway infection and inflammation should be useful for defining mechanisms of HRV pathogenesis in humans.

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.)

To evaluate the association of various leukocytes with pulmonary resistance and methacholine responsiveness, we induced pulmonary eosinophil-rich inflammation in IgE-sensitized (ovalbumin) Sprague Dawley rats. Sensitized rats were challenged with either relevant (OA) or irrelevant antigen by tracheal insufflation a) with no other treatment, b) in conjunction with intravenous Sephadex beads pretreatment, or c) with antigen coupled covalently to Sepharose beads. About 24 h after antigen challenge, respiratory system resistance (Rrs), response to aerosolized methacholine, and pulmonary histopathology were evaluated. Challenge with OA, insufflation with Sepharose, and treatment with i.v. Sephadex all independently increased inflammatory cell infiltrates, but the combination of OA with the other agents did not significantly enhance the inflammatory response over OA alone. Interactive stepwise regression techniques were utilized to identify correlates for Rrs and methacholine responsiveness. Mononuclear cell score was a significant predictor (p < .01) for Rrs, and insufflation of Sepharose had a significant independent effect on Rrs (p = .01) above that predicted by mononuclear cell infiltrates. Conversely, eosinophil score and neutrophil score were not significant predictors for Rrs, and challenges with antigen or Sephadex had no significant independent effect on Rrs beyond that predicted from mononuclear cell infiltrates. Eosinophil score was the only significant histological predictor for methacholine responsiveness (p < .0001). Challenges with Sephadex, antigen and Sepharose did not significantly change methacholine responsiveness independently of the changes associated with eosinophil infiltrates. These findings suggest that mononuclear cells and eosinophils contribute to increases in airway resistance and responsiveness, respectively, following the induction of pulmonary inflammation by both allergic and non-allergic stimuli.

In this cross-sectional evaluation of 77 severe and 71 non-severe asthmatics ages 6–17, boys, unlike girls with severe asthma, exhibited adult-like patterns of air-trapping and post-bronchodilator airflow limitation, apparently coincident with the onset of the severe asthma phenotype.

Complementary and alternative medicine is used commonly for respiratory diseases. This review summarizes data that identify potential links between dietary intake and asthma, and results of interventional trials of herbal substances for the treatment of asthma, chronic obstructive pulmonary disease, and acute bronchitis.

Viral bronchiolitis in human infants has been associated with permanent changes in small airways and gas exchange and an increased incidence of hyperresponsive airways later in life. Respiratory infection by Sendai virus in neonatal rats also has been reported to cause permanent changes in lung morphology and increased numbers of bronchiolar mast cells and eosinophils. We evaluated pulmonary mechanics, gas exchange, and airway responsiveness in rats at 7 and 13–16 wk after neonatal Sendai virus infection. Rats from the virus group had lower arterial PO2 and increased total lung resistance compared with controls. There were no significant differences between groups for arterial PCO2, dynamic lung compliance, quasi-static respiratory system compliance, or vital capacity. Rats from the infected group were significantly more sensitive to aerosolized methacholine than were controls, although both virus and control groups became less sensitive with age. We conclude that neonatal Sendai virus infection in rats results in persistent alterations in lung function and airway responsiveness. This phenomenon may be valuable for the study of the relationships among airway inflammation, lung morphology, and airway hyperresponsiveness, and it may be relevant to human airway disease.

Background Rhinovirus infections are frequent causes of asthma exacerbations. Objective This study was conducted to test whether subjects with and without allergic asthma have different responses to infection and to identify baseline patient risk factors that predict cold outcomes. Methods Twenty subjects with mild persistent allergic asthma and 18 healthy subjects were experimentally inoculated with rhinovirus-16. Subjects were evaluated at baseline, during the acute infection, and during recovery for asthma and cold symptoms by using a validated questionnaire. Sputum and nasal lavage fluid were evaluated for viral shedding, cytokines, and cellular inflammation. Results There were no group-specific significant differences in peak cold symptom scores (10.0 ± 5.8 vs 11.1 ± 6.2, asthmatic vs healthy subjects), peak nasal viral titers (log 10 4.3 ± 0.8 vs 3.7 ± 1.4 50% tissue culture infective dose/mL, respectively), or changes in peak flow during the study (10% ± 10% vs 8% ± 6%, respectively). Rhinovirus-16 infection increased peak asthma index values in the asthmatic group (median, 6 → 13; P = .003) but only marginally in the healthy group (median, 4 → 7; P = .09). More asthmatic subjects had detectable eosinophils in nasal lavage and sputum samples at baseline and during infection, but otherwise, cellular and cytokine responses were similar. Baseline sputum eosinophilia and CXCL8 (IL-8) levels were positively associated with cold symptoms, whereas CCL2 (monocyte chemotactic protein 1) levels were inversely associated with nasal viral shedding. Conclusions These findings suggest that subjects with mild allergic asthma and healthy subjects have similar cold symptoms and inflammatory and antiviral responses. In addition, eosinophilia and other selective baseline measures of airway inflammation in subjects with or without asthma might predict respiratory outcomes with rhinovirus infection.

Eosinophilic inflammation is a cornerstone of chronic asthma that often culminates in subepithelial fibrosis with variable airway obstruction. Pulmonary eosinophils (Eos) are a predominant source of TGF-beta1, which drives fibroblast proliferation and extracellular matrix deposition. We investigated the regulation of TGF-beta1 and show here that the peptidyl-prolyl isomerase (PPIase) Pin1 promoted the stability of TGF-beta1 mRNA in human Eos. In addition, Pin1 regulated cytokine production by both in vitro and in vivo activated human Eos. We found that Pin1 interacted with both PKC-alpha and protein phosphatase 2A, which together control Pin1 isomerase activity. Pharmacologic blockade of Pin1 in a rat asthma model selectively reduced eosinophilic pulmonary inflammation, TGF-beta1 and collagen expression, and airway remodeling. Furthermore, chronically challenged Pin1(-/-) mice showed reduced peribronchiolar collagen deposition compared with wild-type controls. These data suggest that pharmacologic suppression of Pin1 may be a novel therapeutic option to prevent airway fibrosis in individuals with chronic asthma.

Purpose To image respiratory dynamics and three-dimensional (3D) ventilation during inhalation, breath-hold, and exhalation for evaluation of obstructive lung disease using a single dose of hyperpolarized (HP) He-3 during MRI. Materials and Methods A single 2D-3D projections inside Z encoding (PRIZE)-2D acquisition was performed that consisted of a rapid 2D radial acquisition phase during inhalation of the HP He-3, a 3D acquisition phase during a breath-hold interval, and finally the same 2D radial acquisition during a forced exhalation maneuver followed by tidal breathing. The 3D PRIZE acquisition was comprised of radial sampling in the coronal plane and Fourier encoding in the patient's anterior–posterior direction. Nine patients with mild/moderate to severe asthma were studied (two individuals were studied twice) using this technique. Results Breath-hold and dynamic imaging results showed physiological abnormalities and were compared with results from standard spirometry, body plethysmography, and computed tomography (CT). Dynamic images depicted regions of differential gas clearance and trapping observed during and after forced exhalation that were corroborated as regions of air trapping on CT imaging. Conclusion The 2D-3D PRIZE-2D acquisition allowed for 3D depiction of ventilation during a breath-hold, as well as detection of gas trapping. Imaging results were confirmed with spirometry, body plethysmography, and CT. J. Magn. Reson. Imaging 2007. © 2007 Wiley-Liss, Inc.

The noninvasive assessment of lung function using imaging is increasingly of interest for the study of lung diseases, including chronic obstructive pulmonary disease (COPD) and asthma. Hyperpolarized gas MRI (HP MRI) has demonstrated the ability to detect changes in ventilation, perfusion, and lung microstructure that appear to be associated with both normal lung development and disease progression. The physical characteristics of HP gases and their application to MRI are presented with an emphasis on current applications. Clinical investigations using HP MRI to study asthma, COPD, cystic fibrosis, pediatric chronic lung disease, and lung transplant are reviewed. Recent advances in polarization, pulse sequence development for imaging with Xe-129, and prototype low magnetic field systems dedicated to lung imaging are highlighted as areas of future development for this rapidly evolving technology.

Background Infiltration, accumulation, and degranulation of eosinophils in the lung are hallmarks of active allergic asthma. The pulmonary response to inhaled allergen triggers the secretion of eosinophil chemoattractants and antiapoptotic cytokines, including GM-CSF, IL-3, IL-4, IL-5, and eotaxin, among others. We recently showed that in vitro Pin1 regulated eosinophil production of and response to GM-CSF. Objective We sought to determine the effect of Pin1 inhibition on pulmonary eosinophilia after allergen challenge. Methods The Pin1 inhibitor juglone (5-hydroxy-1,4-naphthoquinone) was administered to allergen-sensitized and allergen-challenged Brown Norway rats. Bronchoalveolar lavage fluid and lungs were assessed for inflammation, cytokine expression, and Pin1 activity. Results Juglone-treated rats showed a dramatic reduction (approximately 75%) in bronchoalveolar lavage fluid and pulmonary eosinophilia but no change in lymphocyte, monocyte/macrophage, or neutrophil numbers. GM-CSF and IL-5 expression were also significantly reduced, whereas Pin1-independent cytokines, such as eotaxin or IL-4, as well as housekeeping mRNAs and proteins, including actin, were unaffected by juglone. The eosinophils present in the lung in juglone-treated rats showed significantly greater apoptosis. Conclusion These data suggest that in vivo Pin1 blockade attenuates GM-CSF and IL-5 production and can selectively reduce eosinophilic allergic inflammation. Clinical implications Eosinophils can be selectively reduced by Pin1 blockade, despite allergen challenge.

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.

Although both asthmatics and allergic rhinitics develop an acute inflammatory response to lower airway allergen challenge, only asthmatics experience airway obstruction resulting from chronic environmental allergen exposure. Hypothesizing that asthmatic airways have an altered response to chronic allergic inflammation, we compared the effects of repeated low-level exposures to inhaled Alternaria extract in sensitized rats with preexisting chronic postbronchiolitis airway dysfunction versus sensitized controls with normal airways. Measurements of air space (bronchoalveolar lavage) inflammatory cells, airway goblet cells, airway wall collagen, airway wall eosinophils, airway alveolar attachments, and pulmonary physiology were conducted after six weekly exposures to aerosolized saline or Alternaria extract. Postbronchiolitis rats, but not those starting with normal airways, had persistent increases in airway wall eosinophils, goblet cell hyperplasia in small airways, and loss of lung elastic recoil after repeated exposure to aerosolized Alternaria extract. Despite having elevated airway wall eosinophils, the postbronchiolitis rats had no eosinophils in bronchoalveolar lavage at 5 days after the last allergen exposure, suggesting altered egression of tissue eosinophils into the air space. In conclusion, rats with preexisting airway pathology had altered eosinophil trafficking and allergen-induced changes in airway epithelium and lung mechanics that were absent in sensitized control rats that had normal airways before the allergen exposures.

Early life respiratory viral infections and atopic characteristics are significant risk factors for the development of childhood asthma. It is hypothesized that repeated respiratory viral infections might induce structural remodeling by interfering with the normal process of lung maturation; however, the specific molecular processes that underlie these pathological changes are not understood. To investigate the molecular basis for these changes, we used an established Sendai virus infection model in weanling rats to compare the post-infection transcriptomes of an atopic asthma susceptible strain, Brown Norway, and a non-atopic asthma resistant strain, Fischer 344. Specific to this weanling infection model and not described in adult infection models, Sendai virus in the susceptible, but not the resistant strain, results in morphological abnormalities in distal airways that persist into adulthood. Gene expression data from infected and control lungs across five time points indicated that specific features of the immune response following viral infection were heightened and prolonged in lungs from Brown Norway rats compared with Fischer 344 rats. These features included an increase in macrophage cell number and related gene expression, which then transitioned to an increase in mast cell number and related gene expression. In contrast, infected Fischer F344 lungs exhibited more efficient restoration of the airway epithelial morphology, with transient appearance of basal cell pods near distal airways. Together, these findings indicate that the pronounced macrophage and mast cell responses and abnormal re-epithelialization precede the structural defects that developed and persisted in Brown Norway, but not Fischer 344 lungs.

Background Metered-dose inhalers (MDIs) are convenient, simple, inexpensive, and reproducible devices for administering aerosolized drugs through the pulmonary route, but methods have not been available for use of these devices in small animals. Objective We sought to test the efficacy of delivery of fluticasone through an MDI to rats with a rodent-adapted spacer chamber and to compare this treatment with systemic dexamethasone for the acute pulmonary allergic inflammatory response. Methods Changes in body and thymus weights were used as indicators for systemic steroid effects. Rats were sensitized to ragweed pollen extract 2 weeks before the experiment, and pulmonary allergic responses were evaluated 48 hours after a single aerosolized antigen challenge on the basis of bronchoalveolar leukocytes, lung tissue sections, total lung capacity, and forced expiratory volumes. Results Inhaled fluticasone caused dose-related systemic effects, indicating successful pulmonary drug delivery. Inhaled fluticasone was more effective than placebo but less effective than systemic dexamethasone in attenuating the increase in lung eosinophils and inflammatory infiltrates and the decrease in total lung capacity associated with the allergic inflammatory response. Inhaled fluticasone prevented airway obstruction and proximal inflammation, as did dexamethasone, but it appeared to have less effect in areas of lung served by the most distal airways. Conclusion This is an effective method for use of MDIs to deliver inhaled drugs to small laboratory animals, and it should be valuable for investigations of treatment effects, as well as for in vivo testing of delivery devices.

After Sendai virus (SeV)-induced bronchiolitis as weanlings, BN, but not F344, rats develop a postbronchiolitis asthma-like phenotype, which can be prevented by supplemental interferon (IFN)-gamma treatment. We have shown that splenocytes from BN weanlings, compared with those from F344 weanlings, have a markedly reduced capacity for IFN-gamma production. We hypothesized that SeV-induced IFN-gamma production occurs via innate mechanisms that are attenuated in BN weanlings. Therefore, we investigated potential mechanisms of SeV-induced IFN-gamma production in BN and F344 weanlings. SeV-stimulated splenocytes secreted the IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18. BN splenocytes produced significantly less IL-12 (P = 0.001) and IL-18 (P0.001) than did F344 splenocytes. Depletion studies demonstrated that natural killer cells were the primary source of SeV-induced IFN-gamma production. Anti-IL-12 antibody, IL-12 p40 homodimer, and IL-18 binding protein each inhibited SeV-induced IFN-gamma production by 82-94%, and the combination of IL-12 p40 homodimer and IL-18 binding protein abolished SeV-induced IFN-gamma production, demonstrating synergism between IL-12 and IL-18. Therefore, SeV-induced IFN-gamma production occurred via innate IL-12-, IL-18-, and natural killer cell-dependent mechanisms, which were attenuated in BN weanlings. Attenuation of innate IFN-gamma-producing responses to SeV in BN weanlings may be a critical factor in their susceptibility to postbronchiolitis chronic airway dysfunction.

Analogous to childhood-onset asthma in humans, rats may develop a chronic asthmalike phenotype, depending on their genetic background and the age at which they experience a viral airway injury. Brown Norway rats develop a postbronchiolitis asthmalike phenotype that may be prevented with supplements of interferon-gamma (IFN-gamma); we hypothesized that the normally resistant F344 rat strain would develop the asthmalike phenotype if the IFN-gamma response were suppressed during viral illness. Weanling F344 rats were pretreated with anti-IFN-gamma or control antibody, and inoculated with Sendai virus or vehicle. Anti-IFN-gamma treatment reduced lung IFN-gamma and increased IL-4 mRNA during the infection. Physiologic studies performed 8 wk later revealed premature airway closure (p = 0.03) and elevated specific pulmonary resistance (p0.01) in the postbronchiolitis anti-IFN-gamma group compared with noninfected controls and untreated postbronchiolitis rats. However, unlike the postbronchiolitis asthmalike phenotype in Brown Norway rats, bronchiolar inflammation and fibrosis were absent in the F344 rats. Lung elastic recoil and alveolar surface density also were unchanged compared with noninfected control rats. We conclude that there is an interactive effect of a weak IFN-gamma response and viral bronchiolitis at an early age that may result in persistent postbronchiolitis airway dysfunction. The presence of premature airway closure that is independent of airway wall inflammation or changes in lung elastic recoil suggests peripheral airway instability as a mechanism for the airway obstruction.

After parainfluenza type 1 (Sendai) virus infection as weanlings, Brown Norway (BN), unlike Fischer 344 (F344), rats develop an asthma-like phenotype. Reduced postinfection interferon (IFN)-gamma levels in bronchoalveolar lavage fluid from BN weanlings and the prevention of chronic airway sequelae in BN rats by IFN-gamma treatment led to the hypothesis that cells from BN weanlings have a reduced ability to secrete IFN-gamma. After stimulation with Sendai virus or interleukin (IL)-12, splenocytes from uninfected BN weanlings secreted significantly less IFN-gamma than did splenocytes from F344 weanlings (P0.005), as determined by enzyme-linked immunosorbent assay. Because levels of potential IFN-gamma-secreting cells in the spleen differed between the strains, natural killer (NK) cells, an important IFN-gamma source during early antiviral responses, were purified from spleens of uninfected weanlings. When stimulated with IL-12, BN NK cells secreted significantly less IFN-gamma than did F344 NK cells (P0.001). Incubation of NK cells from either strain with IL-12 and IL-18 resulted in synergistic increases in IFN-gamma production, but BN cells still secreted significantly less IFN-gamma than did F344 cells (P0.05). Similarly, after incubation with either IFN-alpha or IFN-alpha plus IL-18, BN NK cells secreted significantly less IFN-gamma than did F344 NK cells (P0.05). Therefore, reduced IFN-gamma secretion by NK cells in BN weanlings may play a role in the development of postviral chronic airway dysfunction.

After viral bronchiolitis at an early age, Brown Norway (BN) rats develop chronic airway dysfunction consisting of inflammation, remodeling, episodic reversible obstruction, and hyperresponsiveness. We hypothesized that supplementation of interferon gamma (IFN-gamma) during viral illness would alter the inflammatory response and attenuate the postviral sequelae. Weanling rats were treated daily with aerosolized interferon gamma (IFN-gamma), from 2 d prior through 7 d after inoculation, and compared with saline-treated infected rats and with noninfected control rats. The IFN-gamma treatment had no significant effect on viral titers, growth retardation, or total bronchoalveolar leukocytes, but there was a slight decrease in lung interleukin-4 (IL-4) mRNA (p = 0.015) during the first week. Despite having minimal effects on the acute illness, the IFN-gamma had marked effects on postviral sequelae, the IFN-gamma group having less bronchiolar inflammation (p = 0.025) and fibrosis (p = 0.01), and lacking abnormalities in pulmonary resistance (p = 0.028) and dynamic compliance (p = 0.006) compared with the untreated postviral group. We conclude that IFN-gamma modulated the inflammatory response to viral illness, such that acute airway injury did not evolve into chronic airway dysfunction. If similar processes contribute to the development of human asthma, it may be possible to interrupt the progression of airway dysfunction with an early immunomodulatory intervention.

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.

to the editor: We thank Drs. Macklem and Irvin ([3][1]) for their comments, and for their insights regarding the 1996 paper coauthored by Dr. Macklem ([2][2]). As noted in our paper ([4][3]), the study by Gibbons et al. ([2][2]) was the first to introduce the concept of partitioning the forced

Previous studies suggested that although rats that were passively sensitized [monoclonal murine immunoglobulin E (IgE)] would respond to pulmonary antigen challenge with an immediate increase in resistance, they exhibited no late increases in resistance, unlike late changes in rats actively sensitized to preferentially produce IgE antibody. We hypothesized that passively sensitized rats also would not develop antigen-induced pulmonary inflammation. In a blinded protocol we compared immediate responses and pulmonary resistance and inflammation at 8, 19 and 24 h after challenge with placebo antigen, with dinitrophenol-bovine serum albumin (DNP-BSA) to elicit a passively sensitized response, or with ovalbumin (OA) to elicit an actively sensitized response. Despite similar immediate responses to OA and DNP-BSA, only the rats challenged with OA had marked inflammatory changes and a significant incidence of late elevations in resistance. Inflammation scores and lung resistance were significantly correlated only in the OA group. We also observed that anesthesia with fentanyl/droperidol significantly attenuated the immediate but not the late responses to antigen challenge, compared with rats anesthetized with ketamine. We conclude that IgE-mediated immediate responses to pulmonary antigen challenge are insufficient, and may be unnecessary, to initiate antigen-induced late inflammatory changes.

The pathogenesis of the late asthmatic response has been of interest due to the fact that its development has been associated with airway obstruction, airway inflammation, and airway hyperresponsiveness: all features of chronic asthma. In order to study more precisely late responses, a number of animal models have been developed. Previous reports have described alterations in airway mechanics at both 3 to 6 and 17 h after antigen challenge in sensitized guinea pigs. In these experiments, however, animals were challenged through the nose with aerosolized antigen, and airway conductance was measured using whole body plethysmography while the animals breathed through the upper airways. In rats similarly challenged, the predominant immediate change in resistance occurs in the upper airways. The purpose of this study, therefore, was to evaluate the contribution made by both the upper and lower airways to late changes after allergen challenge in guinea pigs. Outbred female Hartley guinea pigs were actively or passively sensitized (IgG1 antibody response) to three different antigens and challenged either by direct tracheal insufflation (sheep gamma globulin [SGG] or oxazolone-human serum albumin [OX-HSA]) or by aerosolization (ovalbumin [OA]). Lung resistance (RL) and dynamic compliance (Cdyn) were measured in anesthetized guinea pigs through a tracheostomy tube, and specific airway conductance (SGaw) was measured in unanesthetized, nose-breathing guinea pigs using a whole body plethysmograph. Late responses were defined as a RL greater than the mean + 2 SD RL or as a decrease in SGaw from baseline greater than 2 SD from the placebo-challenged (bovine serum albumin) group.(ABSTRACT TRUNCATED AT 250 WORDS)

to the editor: In his Viewpoint, Dr. Permutt ([5][1]) offers the intriguing hypothesis that in asthma, narrowing of large airways causes dynamic hyperinflation with an increase in total lung capacity (TLC), which in turn limits a further adaptive increase in TLC during air trapping associated with

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