Segmental Challenge Research Articles

Expression of mRNA for IL-22 Binding Protein in the Broncheoalveolar Fluid after Inhaled Allergen Challenge in Subjects with Asthma

Background: T helper (Th)17/22 cells may play a role in allergic asthma. Recent findings showed increased mRNA expression for IL-22 in peripheral blood mononuclear cells (PBMC) from patients with asthma. Thereby, the role of the natural antagonist of IL-22, IL-22 binding protein (IL-22 BP), a soluble receptor for IL-22, remains to be elucidated. In this study, we investigate the expression of mRNA for IL-22 BP in individuals with asthma. Methods: We assessed the effect of allergen inhalation challenge on mRNA-expression for IL-22 and IL-22 BP, and IL-22 receptor in mononuclear cells of the broncheoalveolar fluid (BALF) from individuals with allergic asthmatic. Furthermore, in vitro we investigated mRNA expression for IL-22 and IL-22 BP, and IL 22 receptor after costimulation of peripheral blood mononuclear cells (PBMC) from individuals with asthma and normal controls with Th2 cytokines IL-4, IL-9, and GM-CSF in a time-dependent manner. Results: The expression of mRNA for IL-22-BP in PBMCs from healthy controls and individuals with asthma could be found after co-stimulation with IL-4 after a minimum of 12 hours, which hold on for 96 hours. The expression of mRNA for IL-22 BP in subjects with atopic asthma was discovered in mononuclear cells of the BALF after segmental allergen inhalation challenge. Our findings suggest a possible role for IL-22 BP in allergen-induced airway responses.

Neutrophils are a major source of the epithelial barrier disrupting cytokine oncostatin M in patients with mucosal airways disease

We have previously shown that oncostatin M (OSM) levels are increased in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS), as well as in bronchoalveolar lavage fluid, after segmental allergen challenge in allergic asthmatic patients. We also showed invitro that physiologic levels of OSM impair barrier function in differentiated airway epithelium. We sought to determine which hematopoietic or resident cell type or types were the source of the OSM expressed in patients with mucosal airways disease. Paraffin-embedded NP sections were stained with fluorescence-labeled specific antibodies against OSM, GM-CSF, and hematopoietic cell-specific markers. Live cells were isolated from NPs and matched blood samples for flow cytometric analysis. Neutrophils were isolated from whole blood and cultured with the known OSM inducers GM-CSF and follistatin-like 1, and OSM levels were measured in the supernatants. Bronchial biopsy sections from control subjects, patients with moderate asthma, and patients with severe asthma were stained for OSM and neutrophil elastase. OSM staining was observed in NPs, showed colocalization with neutrophil elastase (n=10), and did not colocalize with markers for eosinophils, macrophages, T cells, or B cells (n=3-5). Flow cytometric analysis of NPs (n=9) showed that 5.1%±2% of CD45+ cells were OSM+, and of the OSM+ cells, 56%±7% were CD16+Siglec-8-, indicating neutrophil lineage. Only 0.6 ±0.4% of CD45+ events from matched blood samples (n=5) were OSM+, suggesting that increased OSM levels in patients with CRS was locally stimulated and produced. Amajority of OSM+ neutrophils expressed arginase 1 (72.5%±12%), suggesting an N2 phenotype. GM-CSF levels were increased in NPs compared with those in control tissue and were sufficient to induce OSM production (P<.001) in peripheral blood neutrophils invitro. OSM+ neutrophils were also observed at increased levels in biopsy specimens from patients with severe asthma. Additionally, OSM protein levels were increased in induced sputum from asthmatic patients compared with that from control subjects (P<.05). Neutrophils are a major source of OSM-producing cells in patients with CRS and severe asthma.

Allergic asthma is distinguished by sensitivity of allergen-specific CD4+ T cells and airway structural cells to type 2 inflammation.

Despite systemic sensitization, not all allergic individuals develop asthma symptoms upon airborne allergen exposure. Determination of the factors that lead to the asthma phenotype in allergic individuals could guide treatment and identify novel therapeutic targets. We used segmental allergen challenge of allergic asthmatics (AA) and allergic nonasthmatic controls (AC) to determine whether there are differences in the airway immune response or airway structural cells that could drive the development of asthma. Both groups developed prominent allergic airway inflammation in response to allergen. However, asthmatic subjects had markedly higher levels of innate type 2 receptors on allergen-specific CD4+ T cells recruited into the airway. There were also increased levels of type 2 cytokines, increased total mucin, and increased mucin MUC5AC in response to allergen in the airways of AA subjects. Furthermore, type 2 cytokine levels correlated with the mucin response in AA but not AC subjects, suggesting differences in the airway epithelial response to inflammation. Finally, AA subjects had increased airway smooth muscle mass at baseline measured in vivo using novel orientation-resolved optical coherence tomography. Our data demonstrate that the development of allergic asthma is dependent on the responsiveness of allergen-specific CD4+ T cells to innate type 2 mediators as well as increased sensitivity of airway epithelial cells and smooth muscle to type 2 inflammation.

Eosinophil Peroxidase Catalyzed Protein Carbamylation Participates in Asthma

The biochemical mechanisms through which eosinophils contribute to asthma pathogenesis are unclear. Here we show eosinophil peroxidase (EPO), an abundant granule protein released by activated eosinophils, contributes to characteristic asthma-related phenotypes through oxidative posttranslational modification (PTM) of proteins in asthmatic airways through a process called carbamylation. Using a combination of studies we now show EPO uses plasma levels of the pseudohalide thiocyanate (SCN-) as substrate to catalyze protein carbamylation, as monitored by PTM of protein lysine residues into Nϵ-carbamyllysine (homocitrulline), and contributes to the pathophysiological sequelae of eosinophil activation. Studies using EPO-deficient mice confirm EPO serves as a major enzymatic source for protein carbamylation during eosinophilic inflammatory models, including aeroallergen challenge. Clinical studies similarly revealed significant enrichment in carbamylation of airway proteins recovered from atopic asthmatics versus healthy controls in response to segmental allergen challenge. Protein-bound homocitrulline is shown to be co-localized with EPO within human asthmatic airways. Moreover, pathophysiologically relevant levels of carbamylated protein either incubated with cultured human airway epithelial cells in vitro, or provided as an aerosolized exposure in non-sensitized mice, induced multiple asthma-associated phenotypes including induction of mucin, Th2 cytokines, IFNγ, TGFβ, and epithelial cell apoptosis. Studies with scavenger receptor-A1 null mice reveal reduced IL-13 generation following exposure to aerosolized carbamylated protein, but no changes in other asthma-related phenotypes. In summary, EPO-mediated protein carbamylation is promoted during allergen-induced asthma exacerbation, and can both modulate immune responses and trigger a cascade of many of the inflammatory signals present in asthma.

The Effects of Tumstatin on Vascularity, Airway Inflammation and Lung Function in an Experimental Sheep Model of Chronic Asthma

Tumstatin, a protein fragment of the alpha-3 chain of Collagen IV, is known to be significantly reduced in the airways of asthmatics. Further, there is evidence that suggests a link between the relatively low level of tumstatin and the induction of angiogenesis and inflammation in allergic airway disease. Here, we show that the intra-segmental administration of tumstatin can impede the development of vascular remodelling and allergic inflammatory responses that are induced in a segmental challenge model of experimental asthma in sheep. In particular, the administration of tumstatin to lung segments chronically exposed to house dust mite (HDM) resulted in a significant reduction of airway small blood vessels in the diameter range 10+–20 μm compared to controls. In tumstatin treated lung segments after HDM challenge, the number of eosinophils was significantly reduced in parenchymal and airway wall tissues, as well as in the bronchoalveolar lavage fluid. The expression of VEGF in airway smooth muscle was also significantly reduced in tumstatin-treated segments compared to control saline-treated segments. Allergic lung function responses were not attenuated by tumstatin administration in this model. The data are consistent with the concept that tumstatin can act to suppress vascular remodelling and inflammation in allergic airway disease.

Effect of GST variants on lung function following diesel exhaust and allergen co-exposure in a controlled human crossover study

BackgroundIsolated exposure to diesel exhaust (DE) or allergen can cause decrements in lung function that are impacted by the presence of genetic variants in the glutathione-S-transferase (GST) family but the effect of GST interactions with DE-allergen co-exposure on lung function is unknown. We aimed to assess the impact of DE and allergen co-exposure on lung function and the influence of GSTM1 or GSTT1 variation MethodsWe used a blinded crossover study design with 17 atopic subjects exposed to filtered air (FA; the control for DE) or DE for 2h. One hour following each exposure to DE or FA, bronchoscopy was performed to deliver a diluent-controlled segmental allergen challenge (SAC). Methacholine challenge and forced expiratory volume in 1s (FEV1) was performed pre-exposure (baseline airway responsiveness) and 24h post-exposure (effect of co-exposure). Additionally, FEV1 was performed hourly after DE/FA exposure and protein carbonyl content was measured in plasma as an oxidative stress marker. ResultsChanges in FEV1 from baseline were dependent on time following allergen exposure. DE, as opposed to FA, led to a significant change in FEV1 at 2h post-allergen exposure in GSTT1 variants only (24.5±19.6% reduction in GSTT1 null individuals vs. 9.2±7.3% reduction in GSTT1 present individuals). Moreover, plasma protein carbonyl level 4h after co-exposure was higher in the individuals who have the GSTT1 null genotype. ConclusionsThis suggests a gene-environment interaction that endangers susceptible populations co-exposed to DE and allergen.

Oncostatin M is produced by neutrophils, and promotes epithelial barrier dysfunction in mucosal airways disease

Abstract We have previously shown that Oncostatin M (OSM) was elevated in nasal polyp (NP) tissue from chronic rhinosinusitis (CRS) patients, and in BAL fluid following segmental allergen challenge (SAC) in allergic asthmatics (AA). Levels of OSM detected in vivo were sufficient to impair epithelial barrier function in ex vivo cultured airway epithelium. To determine which cell type(s) was producing OSM, NP sections were stained for OSM and hematopoietic cell specific markers. OSM showed co-localization with neutrophil elastase(n=10), but not with markers for eosinophils, macrophages, T cells or B cells (n=3–5). OSM mRNA correlated with mRNA for neutrophil markers CD16 (r=.61, p&amp;lt;.01) and cathepsin G (r=.56, p&amp;lt;.01) in mRNA from CRS patients and controls. OSM also correlated with the percent neutrophils in the BAL of AA following SAC (r=.50, p&amp;lt;.05), but did not correlate with total cell counts or eosinophil counts. Flow cytometric analysis of cells isolated from NP (n=10) showed that 14±6% of CD45+ cells were OSM+, and 85±6% of OSM+ cells were CD16+Siglec 8-, indicating neutrophil lineage. GM-CSF and FSTL-1 have been shown to induce OSM, and both were found to be elevated in NP. Additionally, GM-CSF, alone or in combination with FSTL-1, was sufficient to induce both OSM mRNA in ex vivo cultured blood neutrophils and OSM protein in the culture supernatants. These data suggest that neutrophils are the major producers of OSM in NP, and may potentially mediate epithelial barrier dysfunction in mucosal airways disease.

Controlled diesel exhaust and allergen coexposure modulates microRNA and gene expression in humans: Effects on inflammatory lung markers

Air pollution's association with asthma may be due to its augmentation of allergenic effects, but the role of microRNA (miRNA) and gene expression in this synergy is unknown. We sought to determine whether exposure to allergen, exposure to diesel exhaust (DE), or coexposures modulate miRNA, gene expression, or inflammatory pathways and whether these measurements are correlated. Fifteen participants with atopy completed this controlled study of 2hours of filtered air or DE (300μg PM2.5/m3) exposure, followed by saline-controlled segmental bronchial allergen challenge. Gene and miRNA expression in bronchial brushings and lung inflammatory markers were measured 48hours later, in study arms separated by approximately 4weeks. Expression of miRNAs, messenger RNAs, and inflammatory markers and their interrelationships were determined using regression. Robust linear models indicated that DE plus saline and DE plus allergen significantly modulated the highest number of miRNAs and messenger RNAs, respectively, relative to control (filtered air plus saline). In mixed models, allergen exposure modulated (q≤0.2) miRNAs including miR-183-5p, miR-324-5p, and miR-132-3p and genes including NFKBIZ and CDKN1A, but DE did not significantly modify this allergenic effect. Repression of CDKN1A by allergen-induced miR-132-3p may contribute to shedding of bronchial epithelial cells. Expression of specific miRNAs and genes associated with bronchial immune responses were significantly modulated by DE or allergen. However, DE did not augment the effect of allergen at 48hours, suggesting that adjuvancy may be transient or require higher or prolonged exposure. In silico analysis suggested a possible mechanism contributing to epithelial wall damage following allergen exposure.

Segmental allergen challenge increases levels of airway follistatin-like 1 in patients with asthma

Segmental allergen challenge induced increased levels of bronchoalveolar (BAL) follistatin like 1 (FSTL1). BAL macrophages both expressed FSTL1, and could be induced by FSTL1 to express MMP9 suggesting a potential role of FSTL1 in remodeling.

Human eosinophil activin A synthesis and mRNA stabilization are induced by the combination of IL-3 plus TNF.

Eosinophils contribute to immune regulation and wound healing/fibrosis in various diseases including asthma. Growing appreciation for the role of activin A in such processes led us to hypothesize that eosinophils are a source of this TGF-β superfamily member. TNFα (TNF) induces activin A by other cell types and is often present at the site of allergic inflammation along with the eosinophil activating common β (βc) chain-signaling cytokines (IL-5, IL-3, GM-CSF). Previously, we established that the combination of TNF plus a βc chain-signaling cytokine synergistically induces eosinophil synthesis of the remodeling enzyme MMP-9. Therefore, eosinophils were stimulated ex vivo by these cytokines and in vivo through an allergen-induced airway inflammatory response. In contrast to IL-5+TNF or GM-CSF+TNF, the combination of IL-3+TNF synergistically induced activin A synthesis and release by human blood eosinophils. IL-3+TNF enhanced activin A mRNA stability, which required sustained signaling of pathways downstream of p38 and ERK MAP kinases. In vivo, following segmental airway allergen challenge of subjects with mild allergic asthma, activin A mRNA was upregulated in airway eosinophils compared to circulating eosinophils, and ex vivo, circulating eosinophils tended to release activin A in response to IL-3+TNF. These data provide evidence that eosinophils release activin A and that this function is enhanced when eosinophils are present in an allergen-induced inflammatory environment. Moreover, these data provide the first evidence for post-transcriptional control of activin A mRNA. We propose that, an environment rich in IL-3+TNF will lead to eosinophil–derived activin A, which plays an important role in regulating inflammation and/or fibrosis.

Oxidant-antioxidant balance in asthma and the potential of antioxidant therapies

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Allergic Non-Asthmatic Adults Have Regional Pulmonary Responses to Segmental Allergen Challenge.

BackgroundAllergic non-asthmatic (ANA) adults experience upper airway symptoms of allergic disease such as rhinorrhea, congestion and sneezing without symptoms of asthma. The aim of this study was to utilize PET-CT functional imaging to determine whether allergen challenge elicits a pulmonary response in ANA subjects or whether their allergic disease is truly isolated to the upper airways.MethodsIn 6 ANA subjects, bronchoalveolar lavages (BAL) were performed at baseline and 24h after instillation of an allergen and a diluent in separate lung lobes. After instillation (10h), functional imaging was performed to quantify and compare regional perfusion, ventilation, fractional gas content (Fgas), and glucose uptake rate (Ki) between the baseline, diluent and allergen lobes. BAL cell counts were also compared.ResultsIn ANA subjects, compared to the baseline and diluent lobes, perfusion and ventilation were significantly lower in the allergen lobe (median [inter-quartile range], baseline vs. diluent vs. allergen: Mean-normalized perfusion; 0.87 [0.85–0.97] vs. 0.90 [0.86–0.98] vs. 0.59 [0.55–0.67]; p<0.05. Mean-normalized ventilation 0.89 [0.88–0.98] vs. 0.95 [0.89–1.02] vs. 0.63 [0.52–0.67], p<0.05). In contrast, no significant differences were found in Fgas between baseline, diluent and allergen lobes or in Ki. Total cell counts, eosinophil and neutrophil cell counts (cells/ml BAL) were significantly greater in the allergen lobe compared to the baseline lobe (all P<0.05).ConclusionsDespite having no clinical symptoms of a lower airway allergic response (cough and wheeze) allergic non-asthmatic subjects have a pulmonary response to allergen exposure which manifests as reduced ventilation and perfusion.

Morphometric analysis of inflammation in bronchial biopsies following exposure to inhaled diesel exhaust and allergen challenge in atopic subjects.

BackgroundAllergen exposure and air pollution are two risk factors for asthma development and airway inflammation that have been examined extensively in isolation. The impact of combined allergen and diesel exhaust exposure has received considerably less attention. Diesel exhaust (DE) is a major contributor to ambient particulate matter (PM) air pollution, which can act as an adjuvant to immune responses and augment allergic inflammation. We aimed to clarify whether DE increases allergen-induced inflammation and cellular immune response in the airways of atopic human subjects.MethodsTwelve atopic subjects were exposed to DE 300 μg.m−3 or filtered air for 2 h in a blinded crossover study design with a four-week washout period between arms. One hour following either filtered air or DE exposure, subjects were exposed to allergen or saline (vehicle control) via segmental challenge. Forty-eight hours post-allergen or control exposure, bronchial biopsies were collected. The study design generated 4 different conditions: filtered air + saline (FAS), DE + saline (DES), filtered air + allergen (FAA) and DE + allergen (DEA). Biopsies sections were immunostained for tryptase, eosinophil cationic protein (ECP), neutrophil elastase (NE), CD138, CD4 and interleukin (IL)-4. The percent positivity of positive cells were quantified in the bronchial submucosa.ResultsThe percent positivity for tryptase expression and ECP expression remained unchanged in the bronchial submucosa in all conditions. CD4 % positive staining in DEA (0.311 ± 0.060) was elevated relative to FAS (0.087 ± 0.018; p = 0.035). IL-4 % positive staining in DEA (0.548 ± 0.143) was elevated relative to FAS (0.127 ± 0.062; p = 0.034). CD138 % positive staining in DEA (0.120 ± 0.031) was elevated relative to FAS (0.017 ± 0.006; p = 0.015), DES (0.044 ± 0.024; p = 0.040), and FAA (0.044 ± 0.008; p = 0.037). CD138 % positive staining in FAA (0.044 ± 0.008) was elevated relative to FAS (0.017 ± 0.006; p = 0.049). NE percent positive staining in DEA (0.224 ± 0.047) was elevated relative to FAS (0.045 ± 0.014; p = 0.031).ConclusionsIn vivo allergen and DE co-exposure results in elevated CD4, IL-4, CD138 and NE in the respiratory submucosa of atopic subjects, while eosinophils and mast cells are not changed.Trial registrationURL: http://www.clinicaltrials.gov. Unique identifier: NCT01792232.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0114-z) contains supplementary material, which is available to authorized users.

Diesel exhaust augments allergen-induced lower airway inflammation in allergic individuals: a controlled human exposure study

RationaleTraffic-related air pollution has been shown to augment allergy and airway disease. However, the enhancement of allergenic effects by diesel exhaust in particular is unproven in vivo in the human...

Inter- and intrasubject variability of the inflammatory response to segmental endotoxin challenge in healthy volunteers

Segmental endotoxin challenge with lipopolysaccharide (LPS) can be used as a pharmacodynamic model to safely induce a transient airway inflammation in the peripheral lung of healthy subjects and to test the anti-inflammatory efficacy of investigational new drugs. In contrast to whole lung LPS challenge only a fraction of the dose is required that can be precisely administered to a specific lung region and a vehicle challenged segment as an intra-subject control can be included. The aim of this study was to assess the intra- and inter-individual variability of the response to segmental LPS challenge for the appropriate design and power calculation of future clinical trials.Two cohorts with 10 subjects each underwent two segmental LPS challenges within five weeks. The inflammatory response was evaluated in bronchoalveolar lavage (BAL) fluid at 6 (cohort 1) and 24 h (cohort 2) both in the LPS and in a vehicle challenged segment, as well as in plasma for up to 26 h post LPS challenge.While the cytokine response was more pronounced at 6 h, the influx of neutrophils and monocytes dominated at 24 h; e.g. neutrophils increased from a median (inter-quartile range, IQR) of 0.14 (0.16) and 0.09 (0.08)x104 cells/mL BAL fluid at baseline to 10.2 (17.1) and 19.3 (15.9)x104 cells/mL 24 h after the two separate challenges. The within-subject variability was higher than the between-subject variability for most of the markers. However, sample size estimations based on the variability of outcome variables found lower or equal numbers with cross-over designs compared to parallel group designs for cellular markers at 24 h and cytokine variables at 6 h.The segmental LPS challenge model was safe. Future study designs have to balance between burden to the study subjects (4 versus 2 bronchoscopies), variability (within-versus between-subject), and the desired outcome variable (cells versus chemo/cytokine).

Airway factor XIII associates with type 2 inflammation and airway obstruction in asthmatic patients

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. We sought to determine the association between FXIII and asthma pathobiology. 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. 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 (FEV1/forced vital capacity and residual volume/total lung capacity ratios) and greater reversibility to β-agonists. 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.

Oncostatin M promotes epithelial barrier dysfunction and is elevated in eosinophilic mucosal disease (HYP7P.255)

Abstract Epithelial barrier dysfunction is thought to play a role in many mucosal diseases including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis (EoE). Oncostatin M (OSM), was elevated in both mRNA (28.3 fold, p&amp;lt;.01, n=12) and protein (4.4 fold, p&amp;lt;.05, n=12-19) in the nasal polyps of CRS patients compared to control tissue. OSM protein was also elevated in bronchoalveolar lavage (BAL) of allergic asthma (AA) patients following segmental allergen challenge (SAC) vs. saline challenge (14 fold, p&amp;lt;.0001). Additionally, OSM mRNA was elevated in esophageal biopsies of EoE patients vs. controls, (3 fold, p&amp;lt;.01). OSM stimulation of differentiated airway epithelium induced barrier dysfunction, as measured by decreased transepithelial electrical resistance (63% reduction, p&amp;lt;.0001, n=11) and increased permeability to 10kD dextran (2.45 fold, p&amp;lt;.05, n=5). Staining for the tight junction protein, occludin showed that OSM stimulation of differentiated airway epithelium impaired the organization of the tight junctions compared to unstimulated controls (n=5). Levels of OSM protein in tissue lysates from CRS and controls correlated with levels of α2-macroglobulin, a marker of epithelial leak, in matched nasal secretions (r=.5055, p&amp;lt;.01). Additionally, OSM in the BAL following SAC in AA patients correlated with serum albumin, another marker of epithelial leak (r=.8062, p&amp;lt;.001). These results suggest that OSM may play a role in epithelial barrier dysfunction in mucosal disease.

Oncostatin M promotes mucosal epithelial barrier dysfunction, and its expression is increased in patients with eosinophilic mucosal disease

Epithelial barrier dysfunction is thought to play a role in many mucosal diseases, including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis. The objective of this study was to investigate the role of oncostatin M (OSM) in epithelial barrier dysfunction in human mucosal disease. OSM expression was measured in tissue extracts, nasal secretions, and bronchoalveolar lavage fluid. The effects of OSM stimulation on barrier function of normal human bronchial epithelial cells and nasal epithelial cells cultured at the air-liquid interface were assessed by using transepithelial electrical resistance and fluorescein isothiocyanate-dextran flux. Dual-color immunofluorescence was used to evaluate the integrity of tight junction structures in cultured epithelial cells. Analysis of samples from patients with CRS showed that OSM mRNA and protein levels were highly increased in nasal polyps compared with those seen in control uncinate tissue (P< .05). OSM levels were also increased in bronchoalveolar lavage fluid of allergic asthmatic patients after segmental allergen challenge and in esophageal biopsy specimens from patients with eosinophilic esophagitis. OSM stimulation of air-liquid interface cultures resulted in reduced barrier function, as measured by decreased transepithelial electrical resistance and increased fluorescein isothiocyanate-dextran flux (P< .05). Alterations in barrier function by OSM were reversible, and the viability of epithelial cells was unaffected. OSM levels in lysates of nasal polyps and uncinate tissue positively correlated with levels of α2-macroglobulin, a marker of epithelial leak, in localized nasal secretions (r= 0.4855, P< .05). These results suggest that OSM might play a role in epithelial barrier dysfunction in patients with CRS and other mucosal diseases.

A novel segmental challenge model for bleomycin-induced pulmonary fibrosis in sheep

ABSTRACTBackground: Idiopathic Pulmonary fibrosis (IPF) is a fatal respiratory disease, characterized by a progressive fibrosis and worsening lung function. While the outcomes of recent clinical trials have resulted in therapies to slow the progression of the disease, there is still a need to develop alternative therapies, which are able to prevent fibrosis. Aim: This study uses a segmental lung infusion of bleomycin (BLM) to investigate pulmonary fibrosis in a physiologically relevant large animal species. Methods: Two separate lung segments in eight sheep received two fortnightly challenges of either 3U or 30U BLM per segment, and a third segment received saline (control). Lung function was assessed using a wedged-bronchoscope procedure. Bronchoalveolar lavage fluid and lung tissue were assessed for inflammation, fibrosis and collagen content two weeks after the final dose of BLM. Results: Instillation of both BLM doses resulted in prominent fibrosis in the treated lobes. More diffuse fibrosis and loss of alveolar airspace was observed in high-dose BLM-treated segments, while multifocal fibrosis was seen in low-dose BLM-treated segments. Extensive and disorganised collagen deposition occurred in the BLM-treated lobes, compared to controls. Significant loss of lung compliance was also observed in the BLM-treated lobes, which did not occur in controls. Conclusions: Fibrosis comparable to IPF was induced into isolated lung segments, without compromising the respiratory functioning of the animal. This model may have potential for investigating novel therapies for IPF by allowing direct comparison of multiple treatments with internal controls, and sampling and drug delivery that are clinically relevant.

Glutathione S-transferase M1 modulates allergen-induced NF-κB activation in asthmatic airway epithelium.

Glutathione S-transferase M1 (GSTM1) is a phase II enzyme and regulator of inflammatory signaling in airway epithelial cells. We have found upregulation of neutrophilic airway inflammation in atopic asthmatics expressing GSTM1 gene (GSTM1+) compared to GSTM1null asthmatics. We hypothesized that GSTM1 modulates NF-κB activation in bronchial epithelium in atopic asthmatics. We determined regulation of allergen-induced NF-κB activation in bronchial epithelium by GSTM1 in human atopic asthmatics in vivo. Endobronchial biopsies and bronchoalveolar lavage fluid samples were collected from 13 GSTM1+ and 12 GSTM1null human atopic asthmatics at baseline and 24h after segmental allergen challenge. A quantitative analysis of NF-κB activation in airway epithelium was accomplished using a polyclonal antibody against the phosphorylated p65 component of NF-κB. Elastase-positive neutrophils in the bronchial wall were quantified. Postallergen neutrophilia in airway subepithelium and epithelial lining fluid was greater in GSTM1+ compared to GSTM1null asthmatics. Airway eosinophilia was similar in GSTM1+ and GSTM1null asthmatics. Allergen-provoked NF-κB induction in bronchial epithelium was significantly greater in GSTM1+ compared to GSTM1null asthmatics. Activation of NF-κB activation in airway epithelial cells correlated with interleukin-8 concentrations and absolute neutrophil numbers in bronchoalveolar lavage fluid in GSTM1+ but not GSTM1null asthmatics. Allergen-induced neutrophilic airway inflammation in GSTM1+ asthmatics is associated with NF-κB activation in airway epithelial cells in vivo. These novel data provide a potential mechanism of the genomic link between GSTM1 polymorphism and airway neutrophilia in atopic asthma.