Segmental Allergen Challenge Research Articles

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.

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...

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.

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.

Noninvasive quantification of airway inflammation following segmental allergen challenge with functional MR imaging: a proof of concept study.

To evaluate oxygen-enhanced T1-mapping magnetic resonance (MR) imaging as a noninvasive method for visualization and quantification of regional inflammation after segmental allergen challenge in asthmatic patients compared with control subjects. After institutional review board approval, nine asthmatic and four healthy individuals gave written informed consent. MR imaging (1.5 T) was performed by using an inversion-recovery snapshot fast low-angle shot sequence before (0 hours) and 6 hours and 24 hours after segmental allergen challenge by using either normal- or low-dose allergen or saline. The volume of lung tissue with increased relaxation times was determined by using a threshold-based method. As a biomarker for oxygen transfer from the lungs into the blood, the oxygen transfer function ( OTF oxygen transfer function ) was calculated. After the third MR imaging examination, eosinophils in bronchoalveolar lavage fluid were counted. Differences between times and segments were analyzed with nonparametric Wilcoxon matched-pairs test and Spearman correlation. In lung segments treated with the standard dose of allergen, the OTF oxygen transfer function was decreased at 6 hours in asthmatic patients, compared with saline-treated segments (P = .0078). In asthmatic patients at 24 hours, the volume over threshold was significantly increased in normal allergen dose-treated segments compared with saline-treated segments (P = .004). In corresponding lung segments, the volume over threshold at 24 hours in the asthmatic group showed a positive correlation (r = 0.65, P = .0001) and the OTF oxygen transfer function at 6 hours showed an inverse correlation (r = -0.67, P = .0001) with the percentage of eosinophils in the bronchoalveolar lavage fluid. OTF oxygen transfer function and volume over threshold are noninvasive MR imaging-derived parameters to visualize and quantify the regional allergic reaction after segmental endobronchial allergen challenge.

Phase-contrast MRI for Detection of Mild Systemic Hemodynamic Response after Segmental Allergen Challenge in Asthmatic Patients

Detection of a systemic hemodynamic response in patients suffering from allergic asthma after segmental endobronchial allergen challenge using phase-contrast magnetic resonance imaging (MRI). Nine asthma patients and four healthy volunteers were examined using MRI (1.5T) before (0 hour), 6 hours, and 24 hours after segmental allergen challenge. Two-dimensional phase-contrast MRI measurements were performed in the aorta (AO) and in the pulmonary artery (PA). In addition, short-axis balanced steady state free precession cardiac cine MRI was performed. Maximum systolic flow, maximum flow acceleration, acceleration volume, acceleration time, distensibility, ejection fraction, stroke volume, end-systolic/diastolic volume, cardiac mass, heart rate (HR), and cardiac output (CO) were determined. Spirometry and bronchoalveolar lavage were also performed. In patients with asthma, maximal systolic flow and maximal flow acceleration increased 6 hours after provocation in the AO (112.3% and 118.9%, respectively) and PA (113.9% and 116.0%, respectively) compared to baseline (100%, P<.05). HR and CO increased significantly at 6 hours (115% and 118%, respectively) compared to baseline (100%, P=.003). In healthy subjects, almost all MRI-derived hemodynamic parameters did not significantly change at 6 hours and were significantly lower than baseline values at 24 hours (P<.02). Twenty-four hours after allergen challenge, all MRI-derived flow parameters were significantly lower in the control group compared to the asthma group (P<.05). HR, CO, and cardiac function parameters measured at 24 hours showed no significant difference comparing the two groups (P>.05). In asthmatic patients, MRI-derived hemodynamic parameters using phase-contrast MRI are slightly altered after segmental allergen provocation compared to normal controls indicating a mild systemic reaction to local allergen challenge.

Vitamin D binding protein and vitamin D in human allergen-induced endobronchial inflammation.

Allergic asthma is a chronic disease of the airways associated with airway hyperresponsiveness, a variable degree of airflow obstruction, airway remodelling and a characteristic airway inflammation. Factors of the vitamin D axis, which include vitamin D metabolites and vitamin D binding protein (VDBP), have been linked to asthma, but only few data exist about their regulation in the lung during acute allergen-induced airway inflammation. Therefore, we analysed the regulation of factors of the vitamin D axis during the early- and late-phase reaction of allergic asthma. Fifteen patients with mild allergic asthma underwent segmental allergen challenge. VDBP was analysed in bronchoalveolar lavage fluid (BALF) and serum using the enzyme-linked immunosorbent assay (ELISA) technique. 25-hydroxyvitamin D(3)[25(OH)D(3)] and 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)] were analysed by a commercial laboratory using the liquid chromatography-mass spectrometry (LC/MS) technique. VDBP (median 2·3, range 0·2-7·1 μg/ml), 25(OH)D(3) (median 0·060, range < 0·002-3·210 ng/ml) and 1,25(OH)(2)D(3) (median < 0·1, range < 0·1-2·8 pg/ml) were significantly elevated in BALF 24 h but not 10 min after allergen challenge. After correction for plasma leakage using the plasma marker protein albumin, VDBP and 25(OH)D(3) were still increased significantly while 1,25(OH)(2)D(3) was not. VDBP and 25(OH)D(3) were correlated with each other and with the inflammatory response 24 h after allergen challenge. Serum concentrations of all three factors were not influenced by allergen challenge. In conclusion, we report a significant increase in VDBP and 25(OH)D(3) in human BALF 24 h after allergen challenge, suggesting a role for these factors in the asthmatic late-phase reaction.

Identification and quantification of basophils in the airways of asthmatics following segmental allergen challenge.

During asthma attacks, allergens activate sensitized basophils in the lung, thereby aggravating symptoms. Due to the paucity of basophils in bronchial lavage fluid and the lack of specific basophil detection and quantification methods, basophil-directed research in these samples was hampered in the past. This study aimed to establish and validate a flow cytometry-based basophil detection and quantification method for human basophils from bronchoalveolar lavage (BAL) and blood as a prerequisite for a better understanding of their pathogenic contribution and subtyping of asthma phenotypes. BAL and blood leukocytes from seasonal asthmatics were analyzed by flow cytometry. Chipcytometry, a highly sensitive single-cell analysis method, was used to validate the staining panel for basophils. Cell differentials of May-Grünwald-Giemsa-stained cytospins were used to compare basophil percentages. BAL basophils are identifiable as CD123(+) HLA-DR(-) CD3(-) CD14(-) CD19(-) CD20(-) CD56(-) cells in flow cytometrical analysis. Their identity was validated by Chipcytometry. CD203c was highly expressed by BAL basophils, whereas it was expressed at variable levels on blood basophils. The two quantification methods correlated, although more basophils were detected by flow cytometry. Furthermore, the increase in basophil percentages in the lung correlated with the decrease in the basophil percentages in the blood after allergen challenge. We here validated a reliable basophil quantification method, which is independent of the cell's activation and degranulation state. The results obtained with this method indicate that basophils are directly recruited from the blood circulation to the airway lumen.

Quantification of Pulmonary Inflammation after Segmental Allergen Challenge Using Turbo-Inversion Recovery-Magnitude Magnetic Resonance Imaging

There is a need to develop novel noninvasive imaging biomarkers that help to evaluate antiinflammatory asthma treatments. To investigate whether the extent of the segmental lung edema measured noninvasively using turbo-inversion recovery-magnitude magnetic resonance imaging (TIRM MRI) corresponds to the severity of the regional allergic reaction determined by the percentage of eosinophils in bronchoalveolar lavage fluid (BAL) 24 hours after segmental allergen challenge in patients with asthma compared with normal control subjects. Eleven volunteers with allergic asthma and five healthy volunteers underwent segmental challenges with different allergen doses by two bronchoscopies 24 hours apart. They had lung MRI at baseline and 6 and 24 hours after segmental challenge. MRI TIRM scores were correlated with the eosinophilic response at 24 hours. In patients with asthma, there were significant differences of eosinophil percentages in BAL at 24 hours from segments given standard-dose, low-dose, or no allergen (saline) (P < 0.001). Correspondingly significant differences between the TIRM score in allergen standard-dose, low-dose, and saline-treated segments were observed at 24 hours post-challenge (P < 0.001). With increasing TIRM score at 24 hours the percent eosinophils per segment 24 hours post-challenge also increased accordingly (P < 0.001). There was interobserver agreement for TIRM score grading (kappa = 0.72 for 24-h time point). The MRI-based noninvasive TIRM score is a promising biomarker for the noninvasive detection of the inflammatory response after segmental allergen challenge in patients with asthma and may serve to monitor the therapeutic effectiveness of novel antiinflammatory drugs in future human trials.

A detailed phenotypic analysis of immune cell populations in the bronchoalveolar lavage fluid of atopic asthmatics after segmental allergen challenge

BackgroundAtopic asthma is characterized by intermittent exacerbations triggered by exposure to allergen. Exacerbations are characterized by an acute inflammatory reaction in the airways, with recruitment of both innate and adaptive immune cells. These cell populations as well as soluble factors are critical for initiating and controlling the inflammatory processes in allergic asthma. Detailed data on the numbers and types of cells recruited following allergen challenge is lacking. In this paper we present an extensive phenotypic analysis of the inflammatory cell infiltrate present in the bronchoalveolar lavage (BAL) fluid following bronchoscopically directed allergen challenge in mild atopic asthmatics.MethodsA re-analysis of pooled data obtained prior to intervention in our randomized, placebo controlled, double blinded study (costimulation inhibition in asthma trial [CIA]) was performed. Twenty-four subjects underwent bronchoscopically directed segmental allergen challenge followed by BAL collection 48 hours later. The BAL fluid was analyzed by multi-color flow cytometry for immune cell populations and multi-plex ELISA for cytokine detection.ResultsAllergen instillation induced pro-inflammatory cytokines (IL-6) and immune modulating cytokines (IL-2, IFN-γ, and IL-10) along with an increase in lymphocytes and suppressor cells (Tregs and MDSC). Interestingly, membrane expression of CD30 was identified on lymphocytes, especially Tregs, but not eosinophils. Soluble CD30 was also detected in the BAL fluid after allergen challenge in adult atopic asthmatics.ConclusionsAfter segmental allergen challenge of adult atopic asthmatics, cell types associated with a pro-inflammatory as well as an anti-inflammatory response are detected within the BAL fluid of the lung.

Compartmentalized chemokine-dependent regulatory T-cell inhibition of allergic pulmonary inflammation

Induction of endogenous regulatory T (Treg) cells represents an exciting new potential modality for treating allergic diseases, such as asthma. Treg cells have been implicated in the regulation of asthma, but the anatomic location in which they exert their regulatory function and the mechanisms controlling the migration necessary for their suppressive function in asthma are not known. Understanding these aspects of Treg cell biology will be important for harnessing their power in the clinic. We sought to determine the anatomic location at which Treg cells exert their regulatory function in the sensitization and effector phases of allergic asthma and to determine the chemokine receptors that control the migration of Treg cells to these sites in vivo in both mice and human subjects. The clinical efficacy and anatomic location of adoptively transferred chemokine receptor-deficient CD4(+)CD25(+) forkhead box protein 3-positive Treg cells was determined in the sensitization and effector phases of allergic airway inflammation in mice. The chemokine receptor expression profile was determined on Treg cells recruited into the human airway after bronchoscopic segmental allergen challenge of asthmatic patients. We show that CCR7, but not CCR4, is required on Treg cells to suppress allergic airway inflammation during the sensitization phase. In contrast, CCR4, but not CCR7, is required on Treg cells to suppress allergic airway inflammation during the effector phase. Consistent with our murine studies, human subjects with allergic asthma had an increase in CCR4-expressing functional Treg cells in the lungs after segmental allergen challenge. The location of Treg cell function differs during allergic sensitization and allergen-induced recall responses in the lung, and this differential localization is critically dependent on differential chemokine function.

Segmental allergen challenge enhances chitinase activity and levels of CCL18 in mild atopic asthma.

Allergic airway inflammation contributes to the airway remodelling that has been linked to increased obstruction and morbidity in asthma. However, the mechanisms by which allergens contribute to airway remodelling in humans are not fully established. CCL18, chitotriosidase (CHIT1) and YKL-40 are readily detectable in the lungs and contribute to remodelling in other fibrotic diseases, but their involvement in allergic asthma is unclear. We hypothesized that CCL18, YKL-40 and CHIT1 bioactivity are enhanced in allergic asthma subjects after segmental allergen challenge and are related to increased pro-fibrotic and Th2-associated mediators in the lungs. Levels of CCL18 and YKL-40 protein and chitotriosidase (CHIT1) bioactivity in bronchoalveolar lavage (BAL) fluid, as well as CCL18, YKL-40 and CHIT1 mRNA levels in BAL cells were evaluated in patients with asthma at baseline and 48h after segmental allergen challenge. We also examined the correlation between CCL18 and YKL-40 levels and CHIT1 activity with the levels of other pro-fibrotic factors and chemokines previously shown to be up-regulated after allergen challenge. Chitotriosidase activity and YKL-40 and CCL18 levels were elevated after segmental allergen challenge and these levels correlated with those of other pro-fibrotic factors, T cell chemokines, and inflammatory cells after allergen challenge. CCL18 and YKL-40 mRNA levels also increased in BAL cells after allergen challenge. Our results suggest that CCL18 and YKL-40 levels and CHIT1 activity are enhanced in allergic airway inflammation and thus may contribute to airway remodelling in asthma.

A Randomized Controlled Trial to Evaluate Inhibition of T-Cell Costimulation in Allergen-induced Airway Inflammation

T lymphocytes are important in the pathogenesis of allergic asthma. Costimulation through CD28 is critical for optimal activation of T cells, and inhibition of this pathway with CTLA4Ig has been shown to be effective in preventing airway inflammation and hyperresponsiveness in animal models of asthma. Abatacept, a humanized version of CTLA4Ig, has been approved for treatment of rheumatoid arthritis, providing the opportunity to test whether inhibition of costimulation is an effective strategy to treat people with asthma. To determine if 3 months of treatment with abatacept reduced allergen-induced airway inflammation in people with mild atopic asthma. Randomized, placebo-controlled, double-blinded study. Bronchoscopically directed segmental allergen challenge was performed on 24 subjects followed by bronchoalveolar lavage 48 hours later. Subjects were randomized 1:1 to receive abatacept or placebo, followed by a second allergen challenge protocol after 3 months of study drug. There was no significant reduction in allergen-induced eosinophilic inflammation in the abatacept-treated group compared with placebo (17.71% ± 17.25% vs. 46.39% ± 29.21%; P = 0.26). In addition, we did not detect an effect of abatacept on FEV1, provocative concentration of methacholine sufficient to induce a 20% decline in FEV1, or asthma symptoms. Subjects treated with abatacept had an increased percentage of naive and a corresponding decrease in memory CD4(+) T cells in the blood compared with placebo. Inhibition of CD28-mediated costimulation with abatacept does not seem to alter the inflammatory response to segmental allergen challenge or clinical measures of asthma symptoms in people with mild atopic asthma. Clinical trial registered with ClinicalTrials.gov (NCT 00784459).

A sensitive high throughput ELISA for human eosinophil peroxidase: A specific assay to quantify eosinophil degranulation from patient-derived sources

Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.

Chemokine-mediated Treg trafficking is required to suppress allergic airway inflammation (175.12)

Abstract Research has implicated a role for regulatory T cells (Tregs) in preventing asthma; however, where Tregs exert their regulatory function and the mechanisms that localize Tregs to their functional location have not been addressed. We hypothesized that chemokine receptors expressed on Tregs play an important role in directing these cells to the allergic lung and/or the reactive lymph node. We developed two murine models of allergic airway to address this hypothesis. In the sensitization model, naïve mice are injected with antigen-specific Tregs prior to immunization and aerosol challenge. In the effector model, antigen-specific Tregs are injected into mice after immunization but prior to aerosol antigen challenge. Transferred wildtype Tregs are capable of suppressing allergic airway inflammation in both models. We have found that CCR7 is required in the sensitization model and CCR4 is required in the effector model, suggesting Treg trafficking into lymph nodes is required to suppress allergic airway inflammation during the allergen sensitization phase while Treg trafficking into the lung is required to suppress allergic airway inflammation during the recall response to allergen. Using a segmental allergen challenge model in human allergic asthma subjects we further show an important role for CCR4+ Tregs in human allergic asthma. These results support our mouse data and demonstrate that CCR4 may play an important role in Treg recruitment into the allergic airways of humans.

Natural‐source d‐α‐tocopheryl acetate inhibits oxidant stress and modulates atopic asthma in humans in vivo

Asthma is associated with oxidant stress and diminished antioxidant defenses. Yet, the mechanistic role of oxidant stress and antioxidant supplementation in human asthmatics remains uncertain. We determined the effect of high doses of the antioxidant natural-source d-α-tocopheryl acetate for 16 weeks on allergen-induced airway oxidant stress, inflammation, and bronchial responsiveness to methacholine and allergen in atopic asthmatics in vivo. Thirty-three mild atopic asthmatics underwent bronchoscopy with baseline bronchoalveolar lavage and segmental allergen challenge. The allergen-challenged airway was lavaged 24 h later. At least 3 weeks later, patients underwent inhaled challenges with methacholine and specific allergen. Volunteers took 1500 IU of natural-source d-α-tocopheryl acetate daily for at least 16 weeks. At the end of the treatment, the two bronchoscopies and inhaled methacholine and allergen challenges were repeated. F(2)-isoprostanes, specific markers of oxidant stress, and selected Th1 and Th2 cytokines were analyzed in the lavage fluid. Following supplementation of natural-source d-α-tocopheryl acetate, plasma concentrations of α-tocopherol increased and γ-tocopherol decreased. Both baseline and allergen-induced F(2)-isoprostanes significantly decreased, providing biochemical evidence for an antioxidant effect. Natural-source d-α-tocopheryl acetate reduced allergen-provoked concentrations of interleukin 3 and interleukin 4 and augmented levels of interleukin 12 in bronchoalveolar lavage fluid. Natural-source d-α-tocopheryl acetate improved airway responsiveness to methacholine but did not alter airway reactivity to specific allergen. Inhibition of oxidant stress by natural-source d-α-tocopheryl acetate modulates allergic inflammation and airway hyperresponsiveness in human atopic asthmatics in vivo. These results need to be confirmed by a randomized placebo-controlled trial.

125 The Association between Mast Cells and Remodelling of the Small Airways in Chronic Asthma

BackgroundRepeated airway challenges with House Dust Mite (HDM) allergen results in marked remodelling and mast cell hyperplasia in the small airways of allergic sheep. We now examine mast cell activation and its association with small airway function and remodelling in these sheep using a novel segmental allergen challenge approach.MethodsEight allergic sheep received weekly intra-lung challenges of HDM to the left caudal lung for 24 weeks. Eight separate sheep were used as controls. Baseline lung function was assessed in the left caudal segments of all sheep throughout the challenge regime using a wedged-bronchoscope technique. Airway tissue was collected from challenged segments from all sheep, 7 days following the final intra-lung challenge. The airway tissues were immunohistochemically labelled for chymase-mast cells and eosinophils. Collagen and airway smooth muscle content were assessed on Masson's Trichrome stained sections.ResultsResting lung function in the left caudal segment is elevated in 4 out of 8 sheep at the end of the repeated allergen challenge regime. Chymase mast cell density was significantly increased in the small bronchial walls of the HDM-challenged group compared to the control group (52 ± 8 vs 8 ± 4; P < 0.01). There were significant increases in bronchial collagen deposition in HDM-exposed segments compared to control segments (0.17 ± 0.02 vs 0.11 ± 0.02 mm2/BM, P < 0.05). A correlation analysis of individual sheep data showed that there was a trend for a direct association between the increases in bronchial collagen deposition and the density of chymase-labelled mast cells (rs = 0.71, P = 0.088). Eosinophil density in the small bronchial walls of HDM-challenged segments was also significantly increased compared to controls (65 ± 19 vs 11 ± 3 cells/mm2, P < 0.001), but not associated with collagen content. The bronchial smooth muscle content was not different between HDM-challenged and unexposed control segments.ConclusionsThe results show that repeated exposure to allergen results in significant increases in density of chymase-labelled mast cells, together with increased levels of collagen content in the small airways. The segmental challenge protocol allows for a novel approach to characterise the progressive remodelling events occurring in the small airways in chronic asthma.