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Background: Asthma diagnosis may be challenging particularly in patients with mild symptoms without an obstructive pattern in spirometry. Detection of airway hyperresponsiveness (AHR) by a positive methacholine challenge (MCC) is still an important diagnostic tool to confirm the presence of asthma with reasonable certainty. However, it is time consuming and could be exhausting for patients. We aimed to identify the predictive factors for AHR in children with respiratory symptoms without obstructive pattern in spirometry. Methods: Data from children who had undergone MCC were analyzed retrospectively. The demographic features of patients along with laboratory results were collected. Results: A total of 123 children with a median age of 10.5 years were enrolled. AHR was detected in 81 children (65.8%). The age of the children with AHR was significantly younger. The prevalences of aeroallergen sensitization, nocturnal cough, wheezing, and a baseline forced expiratory flow at 75% of vital capacity (FEF75) 75 Conclusion: In children suspected of having asthma, but without an obstructive pattern in the spirometry, combining independent predictors, which can be easily obtained in clinical practice, might be used to identify children with AHR.

Background: It is still debatable whether dog ownership during early childhood is a risk factor for the development of allergic diseases. Objective: We investigated the association of dog ownership in early life with sensitization and asthma in childhood. Methods: Data from the Cohort for Childhood Origin of Asthma and Allergic diseases were used to investigate the association between dog ownership at any time from pregnancy to 1 year of age and sensitization to aeroallergens at 3 and 7 years old, bronchial hyperresponsiveness (BHR), and asthma at 7 years old. We analyzed the cytokine levels in cord blood (CB) and indoor environmental measurement concentrations in the mother’s residence obtained at 36 weeks of pregnancy. Results: Sensitization to dogs at age 3 and 7 did not differ between dog ownership and nonownership, but dog ownership during early life decreased the risk of sensitization to aeroallergens at age 7 (aOR = 0.44, 95% CI 0.21–0.90). Dog ownership significantly increased the risk of nonatopic BHR (aOR = 2.86; 95% CI 1.32–6.21). In addition, dog ownership was associated with asthma, especially nonatopic asthma at 7 years old (aOR = 2.73, 95% CI 1.02–7.32; aOR = 7.05, 95% CI 1.85–26.90, respectively). There were no significant differences in the concentrations of IL-13 or interferon-γ in CB or indoor environmental measurements according to dog ownership during pregnancy. Conclusion: Early-life dog exposure in this birth cohort has been shown to reduce atopy but increase the risk of nonatopic BHR and nonatopic asthma at 7 years old.

Background: Common variable immunodeficiency (CVID) is one of the most frequent primary immunodeficiencies and is characterized by disturbed immunoglobulin production and dysregulation of the immune system. Results of previous studies suggest a higher prevalence of bronchial asthma (BA) in CVID patients than in the general population. We initiated this study to evaluate lung functions and identify risk factors for BA and bronchial hyperresponsiveness (BHR) in patients with CVID. Methods: Twenty-three patients with CVID were included in this study. In all of them, spirometry and a metacholine bronchoprovocation test were performed. We also investigated the role of atopy, eosinophilic inflammation, and potential risk factors such as gender, age, or immunoglobulin levels at the time of diagnosis. Results: BHR was confirmed in 12 patients (52%), all of whom had normal FEV1 and FEV1/FVC. However, BHR-positive patients had significantly decreased MEF25. BHR-positive patients had also more symptoms related to bronchial obstruction, with 8 of them (35%) being suspected of having BA at the end of the study. A higher prevalence of BHR was found in females, with a relative risk of 2.89. Conclusions: An increased prevalence of BHR and BA was detected in CVID patients compared to the general population. BA may develop despite the disturbed immunoglobulin production, and the majority of patients display nonatopic and noneosinophilic properties. These results suggest a limited role of atopy and eosinophilic inflammation in the pathogenesis of BA in CVID patients.

Background: Lysophosphatidic acid (LPA), a prototypic member of a large family of lysophospholipids, has been recently shown to play a role in immune responses to respiratory diseases. The involvement of LPA in allergic airway inflammation has been reported, but the mechanism remains unclear. Object: We analyzed the biological activity of LPA in vitro and in vivo and investigated its role in allergic inflammation in mice using an LPA receptor 2 (LPA2) antagonist. Methods: We used a murine model with acute allergic inflammation, in which mice are sensitized and challenged with house dust mite, and analyzed airway hyperresponsiveness (AHR), pathological findings, Th2 cytokines, and IL-33 in bronchoalveolar lavage fluid (BALF) and lung homogenates. The effect of LPA on Th2 differentiation and cytokine production was examined in vitro using naive CD4+ T cells isolated from splenocytes. We also investigated in vivo the effects of LPA on intranasal administration in mice. Results: The LPA2 antagonist suppressed the increase of AHR, the number of total cells, and eosinophils in BALF and lung tissue. It also decreased the production of IL-13 in BALF and IL-33 and CCL2 in the lung. LPA promoted Th2 cell differentiation and IL-13 production by Th2 cells in vitro. Nasal administration of LPA significantly increased the number of total cells and IL-13 in BALF via regulating the production of IL-33 and CCL-2-derived infiltrating macrophages. Conclusion: These findings suggest that LPA plays an important role in allergic airway inflammation and that the blockade of LPA2 might have therapeutic potential for bronchial asthma.

Background: Vehicle-induced air pollution may increase the prevalence and severity of asthma. Pollens are important sources of outdoor allergens associated with asthma. Outdoor pollution may influence the structure of pollen grains, resulting in enhanced immune reactions. Objective: This study aims to investigate the impact that artemisia pollen extracts exposed to diesel emissions (APEDE) may induce – allergic airway inflammation, pulmonary pathology and immune imbalance – in mice. Methods: Sixty male Balb/c mice were equally randomized into 5 groups, sensitized with 30 μL artemisia pollen extracts (APE) or APEDE adsorbed on 2 mg aluminum hydroxide gel by intraperitoneal injection on day 0, 7, 14, and 22, and challenged intranasally once per day with 30 μL APE or APEDE from day 29 to 36. The controlling group used phosphate-buffered saline as control. Results: In mice immunized and challenged by APEDE, the clinical phenotype of eosinophils, neutrophils in bronchoalveolar lavage fluid (BALF), tracheal wall thickness, airway smooth muscle thickness and airway resistance increased significantly. Pathophysiological parameters such as interleukin (IL)-17A and tumour necrosis factor-α production in BALF and serum, and the ratio of Th17/Treg cells in CD4+ cells increased significantly, while IL-10 in BALF and serum and the ratio of Treg cells decreased significantly. It was further found that the expression of oxidative stress marker 3-nitrotyrosine (3-NT) and the activation of nuclear factor kappa B (NF-κB) were significantly increased. The correlation analysis showed that the expression of 3-NT was positively correlated with the activation of NF-κB. Conclusion: Our findings suggested that pollens exposed to diesel exhaust enhance allergic responses, which may contribute to an increased prevalence of allergic diseases in urban environments with serious exhaust emissions.

Background: Psychological stress is associated with the aggravation of asthma symptoms. Glucocorticoids (GC), which are stress hormones released upon exposure to stress, have the potential to shift immune responses towards a predominant Th2 response by priming antigen-presenting cells to produce lower levels of IL-12 as well as reducing the development of regulatory T cells. However, the involvement of GC in psychological stress-induced exacerbations of allergic asthma has not yet been clarified. Methods: Sensitized mice were exposed to restraint stress followed by forced swimming stress, during which a GC receptor antagonist or a GC synthesis inhibitor was administered, and then antigen was inhaled. Corticosterone levels in the blood were measured in stressed and nonstressed mice. After antigen inhalation, the airway responses to aerosolized methacholine, epithelial mucus secretion and airway inflammation were evaluated, and the IL-13 contents in bronchoalveolar lavage fluid were measured. Results: The exposure to stress significantly increased corticosterone levels. Allergic airway responses and the increase of IL-13 contents evoked by antigen inhalation were significantly higher in stressed mice than in nonstressed mice. The administration of a GC receptor antagonist and a GC synthesis inhibitor during stress exposure significantly reduced the exacerbation of the airway responses and the increase of IL-13 contents in stressed mice challenged with antigen. Conclusions: These results indicate that the increased release of GC upon exposure to stress has a priming effect on the aggravation of allergic airway responses following the exposure, suggesting a pathophysiological role for the neuroendocrine axis in linking psychological stress to asthma exacerbations.

Background: Allergic asthma is a chronic inflammatory airway disease caused by exposure to airborne allergens. In order to develop novel therapies for allergic asthma, models that are relevant to human disease are needed. Methods: Female BALB/c mice were presensitised subcutaneously with alum-adsorbed recombinant cat allergen Fel d 1, followed by intranasal challenges with cat dander extract spiked with recombinant Fel d 1 for 7 weeks. For reference, mice were presensitised and challenged with ovalbumin following the same protocol. Airway hyperresponsiveness, serum antibodies, airway inflammation and cell infiltration, and cytokines in lung tissue and bronchoalveolar lavage were measured. Results: Mice presensitised with recombinant Fel d 1 and challenged with cat dander extract or presensitised and challenged with ovalbumin showed airway hyperresponsiveness in response to metacholine. Mice of the cat allergen model showed influx of neutrophils, eosinophils and lymphocytes in bronchoalveolar lavage, combined with increased levels of IL-17a and increased IL-4 mRNA expression in lung tissue. In contrast, mice sensitised and challenged with ovalbumin showed a predominant influx of eosinophils in bronchoalveolar lavage and had an increased expression of IL-5 in lung tissue. Both protocols induced features of lung tissue remodelling and allergen-specific antibody responses. Conclusions: The presented mouse model for cat allergen-induced asthma exhibits hallmarks of chronic allergic asthma, like airway hyperresponsiveness, a mixed neutrophilic/eosinophilic infiltration in bronchoalveolar lavage, expression of IL-17a and signs of remodelling in lung tissue. The model will provide a relevant platform for the development of novel treatment strategies.

Background: Information about the biological properties of Blomia tropicalis allergens is scarce. It is predicted that Blo t 12, an allergen with two described isoforms, contains a chitin-binding domain, similar to that found in peritrophins. Th2 adjuvant properties have been described for chitin. Therefore, it is feasible that binding to this carbohydrate influences its allergenicity. We aimed to evaluate the chitin-binding activity of Blo t 12 isoallergens and its effect on airway inflammation and antibody responses in a murine model of allergen sensitization. Methods: Chitin-binding assays were conducted with the recombinant isoallergens Blo t 12.0101 and Blo t 12.0102. BALB/c mice were sensitized via i.p. with any of the two isoforms (alone, with chitin or alum) and then challenged intranasally. Methacholine-induced bronchial hyperreactivity was tested by whole-body plethysmography and lung sections were stained with hematoxylin and eosin and periodic-acid Schiff. Total IgE and allergen-specific IgE, IgG1 and IgG2 levels were measured by ELISA. Results: The two isoforms bound chitin, but Blo t 12.0101 showed a stronger binding capacity. Both isoforms induced total and allergen-specific IgE, airway hyperreactivity, bronchial inflammation and mucus secretion without any adjuvant; however, when administered with chitin, Blo t 12.0101 induced higher total IgE levels. The IgG1/IgG2a ratio was significantly higher in mice immunized with Blo t 12.0101 than those immunized with Blo t 12.0102. As peritrophins, Blo t 12 was detected in mite feces. Conclusions: Blo t 12 isoforms are chitin-binding proteins that induce airway inflammation and bronchial hyperreactivity. However, for Blo t 12.0101, chitin reinforces its effects on total IgE production.

Background: Bronchial asthma is a chronic inflammatory disease of the airway. Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, is activated by environmental stress and plays a crucial role in the induction of apoptosis and inflammation. To examine whether ASK1 is involved in the induction of bronchial asthma, we investigated the role of ASK1 using a genetic approach in the production of cytokines, as well as the development of airway hyperreactivity (AHR) and antibody responses using a murine airway inflammation model. Methods: ASK1-deficient (ASK1-/-) and control wild-type (WT) mice were immunized with ovalbumin (OVA) without alum intraperitoneally, followed by intranasal administration of OVA. Airway infiltration of inflammatory cells, cytokine production, AHR and antibody production were assayed. The asthmatic phenotype was assessed following intranasal administration of IL-13 or TNF-α. Results: ASK1-/- mice sensitized with OVA displayed an impaired inflammatory cell infiltration into airways and a decreased AHR relative to WT mice. Moreover, the production of OVA-specific IgE antibodies and proasthmatic cytokines (IL-5, IL-13 and TNF-α) was substantially reduced in OVA-stimulated ASK1-/- mice. Intranasal administration of IL-13 and OVA enhanced the accumulation of inflammatory cells in OVA-primed ASK1-/- mice. The OVA-induced AHR in response to methacholine was enhanced by IL-13 in WT mice but not ASK1-/- mice. Conclusions: The ASK1 signaling pathway regulates the OVA-induced asthmatic phenotype, specifically AHR sensitivity and cytokine production. Therefore, the ASK1 signaling pathway is a promising target for therapeutic intervention in some asthmatic patients.

Background: Asthma is characterized as a chronic inflammatory disorder of the airways associated with an enhanced TH2 response to inhaled allergens. CD4+ T regulatory (Treg) cells are controlled by the master transcription factor FoxP3 and strictly maintain peripheral immunotolerance. Epigenetic regulation of FoxP3 by DNA methyltransferase inhibitors, such as 5-azacytidine (Aza), can generate a steady supply of functional Treg cells. Therefore, we propose that Aza can augment Treg cells in vivo to prevent the pathogenesis of asthma. Methods: BALB/c mice were sensitized with chicken ovalbumin (OVA) and treated with different doses of Aza. Airway hyperresponsiveness to methacholine, eosinophilia in bronchoalveolar lavage fluid, circulating titers of OVA-specific IgG1 and IgE, and stimulating levels of TH2 cytokines from splenocytes were then determined. Cellular populations were examined by flow cytometry. PC61 antibody, which depletes CD25+ cells, was used to verify the role of CD25+ cells in Aza-induced tolerance. Results: Administration of Aza to OVA-sensitized mice diminished airway hyperreactivity, pulmonary eosinophilia, levels of OVA-specific IgG1 and IgE in serum, and secretion of TH2 cytokines from OVA-stimulated splenocytes in a dose-dependent manner. Percentages of CD25+ and FoxP3+ cells in the CD4+ cell population were notably increased in Aza-treated mice compared to sensitized control mice. Furthermore, the major symptoms of asthma were exacerbated by depleting CD25+ cells in Aza-treated mice. Conclusions: Aza may have applications as a novel clinical strategy to increase the production of Treg cells in order to modulate the airway inflammation associated with asthma.

Background: Allergic rhinitis frequently occurs as a comorbid condition in asthmatic people, suggesting that the upper and lower airways may be immunologically linked. Our research group has developed an experimental aeroallergen model of asthma in cats. We hypothesized that aeroallergen sensitization and challenge would induce morphologic changes in the nasal airways of cats that mimic those observed in the bronchial airways. Methods: Five mixed breed cats were sensitized to Bermuda grass allergen and then serially challenged with aerosolized Bermuda grass allergen to induce an asthmatic phenotype. Four control cats were similarly treated with saline vehicle. Nasal tissues and lungs were processed for histopathological and morphometric analyses. Results: Eosinophilic inflammation, epithelial hypertrophy and mucous cell metaplasia were observed along the pulmonary axial airway mucosa of allergen-sensitized (asthmatic) cats. Mild eosinophilic inflammation was observed in the nasal airways of asthmatic cats. This alteration was confined primarily to the anterior nasal cavity, resulting in an increase in tissue eosinophils at this site compared to controls (p < 0.05). A marked increase in tissue mast cells was observed throughout all regions of the nasal airways of asthmatic cats compared to control cats (p < 0.05). There was no difference in intraepithelial mucosubstances between the nasal airways of controls and asthmatic cats. There was no correlation between upper and lower airway eosinophils or mast cells. Conclusion: Cats with experimentally induced asthma exhibit morphologic changes in the nasal airways that are distinct from the alterations observed in the lungs. These results are similar to those observed in people with comorbid asthma and allergic rhinitis.

Background: Nonspecific airway hyperresponsiveness (AHR) is one of the cardinal features of bronchial asthma. Early AHR is caused by chemical mediators released from pulmonary mast cells activated in an IgE-dependent way. However, the mechanism of late AHR remains unclear. Methods: Features of airway allergic inflammation were analyzed, including antigen-induced AHR, using a murine model of asthma. The model was suitable for examining the sequential early molecular events occurring after the initial airway exposure to antigen. Results: AHR increased at 10–12 h after airway challenge, followed by the second-phase response, which was larger and broader in resistance at 18–30 h. Pretreatment of sensitized animals with anti-tumor necrosis factor (TNF) before airway challenge or induction of allergic asthma in TNF–/– mice resulted in abrogation of the first-phase late AHR. Intratracheal instillation of TNF induced a single peak of AHR at 10 h. IgE and IgG immune complexes induced the development of the first-phase late AHR by TNF production. Pretreatment with cytosolic phospholipase inhibitor and 5-lipoxygenase inhibitors abolished the first-phase late AHR as well as the leukotriene B4 levels in the airway. CpG-oligodeoxynucleotide (ODN) pretreatment reduced airway levels of Th2 cytokines, eosinophil infiltration and second-phase late AHR. However, CpG-ODN did not reduce TNF levels or the magnitude of first-phase late AHR. Conclusion: Biphasic late AHR occurs in a murine model of asthma. First- and second-phase late AHR is caused by TNF and Th2 response, respectively.

Background: The observation that asthma becomes more prevalent following puberty in females suggests estrogen potentiates the development of this disease. However, most studies examining the role of estrogen in rodent models of asthma are complicated by their reliance on ovariectomised mice in which hormones other than estrogen are also attenuated. Methods: We aimed to understand the influence of estrogen on allergic airway disease by using type I (tamoxifen) or type II (ICI 182,780) antagonists in female mice or delivering estradiol to male mice during aeroallergen challenge. Results: The antagonists showed that estrogen promoted both the mobilisation of bone marrow eosinophils and egression of eosinophils to the airway lumen. These findings were corroborated in male mice treated with estradiol, which increased eosinophil numbers in both blood and airways. Estrogen stimulated goblet cell hyperplasia and baseline lung resistance, but had little effect on the number of eosinophils in the bronchial submucosa or methacholine-induced airway hyperreactivity. Estrogen receptor α was expressed by CD4+ T cells from allergic mice, and estrogen promoted the production of IL-5 and IL-13, and suppressed the production of the eicosanoid 12-HETE by mediastinal lymph node cells. Conclusions: These data show that during aeroallergen challenge, estrogen stimulates Th2 cytokine production, which may be linked to its ability to suppress 12-HETE. Lung resistance at baseline, goblet cell hyperplasia and the compartmentalisation of eosinophils was also influenced by estrogen. However, estrogen does not play a major role in stimulating enhanced sensitivity to methacholine-induced lung resistance.

Background: Nonasthmatic eosinophilic bronchitis (NAEB) and cough variant asthma (CVA) are common causes of chronic cough. Both are characterized by eosinophilic inflammation in the airways. However, airway hyperresponsiveness, which is a characteristic feature of CVA, is not observed in NAEB. We hypothesized that endogenous bronchodilator S-nitrosothiol (SNO) levels are different between patients with NAEB and CVA. Methods: SNO concentrations in sputum supernatant were measured using a commercially available kit in 20 NAEB and 21 CVA patients. Results: The mean sputum eosinophil counts and exhaled nitric oxide values were similar in patients with NAEB (12.4 ± 2.3%, 80.6 ± 8.1 ppb) and CVA (15.3 ± 3.7%, 97.7 ± 9.2 ppb). By contrast, SNO levels in the airway lining fluid of NAEB patients were substantially higher than those of CVA patients (87.1 ± 9.8 vs. 46.8 ± 4.8 µM; p < 0.05). Conclusions: SNOs may be an important factor in determining the development of airway hyperresponsiveness in the presence of eosinophilic inflammation.

Background: Transient receptor potential vanilloid-1 (TRPV1) may modulate allergic airway inflammation because it is expressed not only on the nerve endings but also on several cells of the immune system. We wanted to know the characteristics of airway and systemic responses against sensitization and challenge with allergens in TRPV1 receptor gene knockout mice (TRPV1–/–). Methods: TRPV1–/– and their wild-type counterparts (TRPV1+/+) were sensitized with either house dust mite (HDM) or ovalbumin (OVA) via intranasal (i.n.) or intraperitoneal (i.p.) route before the final i.n. challenge with the corresponding allergen. One day after the final challenge, serum IgE levels, cytokine levels in the bronchoalveolar lavage fluid (BALF), and the number of BALF cells were examined after measuring bronchial hyperresponsiveness against methacholine. Results: Compared to TRPV1+/+, TRPV1–/– showed enhanced Th2-biased response after i.n. HDM or OVA sensitization, including increased levels of serum IgE, interleukin 4 (IL-4) and eosinophils in the BALF. By contrast, when sensitized via i.p. route, the response against OVA or HDM was almost similar between TRPV1+/+ and TRPV1–/–. Conclusion: TRPV1 receptor may downregulate Th2-biased immune response when sensitized via airways, although this was not the case when sensitized systemically.

Background: Little is known about the role of the cysteinyl leukotriene (cysLT) 2 receptor in the pathophysiology of asthma. The aim of this study is to investigate the effects of a cysLT1 receptor antagonist (montelukast) and a dual cysLT1/2 receptor antagonist (BAY-u9773) on airway hypersensitivity and airway inflammation induced by antigen challenge in ovalbumin (OVA)-sensitized guinea pigs. Methods: Male Hartley guinea pigs sensitized with OVA were intraperitoneally administered 0.1, 1, or 10 mg/kg of montelukast or 0.1 mg/kg of BAY-u9773 and then challenged with inhaled OVA. Airway reactivity to acetylcholine, inflammatory cells in bronchoalveolar lavage (BAL) fluid, and eosinophil infiltration in airway walls after OVA challenge were evaluated. Results: Pretreatment with 1 or 10 mg/kg, but not 0.1 mg/kg, of montelukast significantly suppressed airway hypersensitivity and eosinophil infiltration into the BAL fluid. Moreover, 0.1 mg/kg of BAY-u9773 significantly suppressed the development of these markers. The suppressive effects of BAY-u9773, although not significantly different, trended toward being greater than those of montelukast. Although all of the doses of montelukast tested and 0.1 mg/kg of BAY-u9773 significantly suppressed eosinophil infiltration in airway walls, the suppressive effect of BAY-u9773 was significantly greater than that of 0.1 mg/kg of montelukast. Conclusion: Signaling may contribute to the pathophysiology of asthma via the cysLT1/2 receptor.

Background: Although several mechanisms underlying the asthma-obesity connection have been proposed, debates still remain. This study was to determine whether overweight is associated with a higher prevalence of atopy, asthma symptoms, airway obstruction, bronchial hyperresponsiveness (BHR) or biomarkers of inflammation in a sample of Korean adolescents. Methods: We conducted a cross-sectional survey involving questionnaires, skin tests, spirometry and methacholine challenge tests among 717 adolescents from Seoul (South Korea). Overweight status was defined as a BMI greater than the local age- and gender-specific 85th percentile. Results: Overweight subjects more frequently reported ever having wheezing (24.6 vs. 14.0%, p = 0.001) and wheezing in the previous 12 months (11.5 vs. 6.3%, p = 0.02) than normal-weight subjects, especially in boys. Atopy was more common among overweight adolescents than among those of normal weight (61.5 vs. 49.2%, p = 0.002), especially in boys (65.0 vs. 52.8%, p = 0.005). Overweight subjects had higher total WBC counts and eosinophil counts, especially boys. The presence of BHR was more common only among overweight girls (32.8 vs. 18.0%, p = 0.028). Overweight status was a significant risk factor for the presence of atopy (odds ratio = 1.49; 95% CI 1.06–2.10), after adjusting for various confounders by logistic regression analysis. Conclusions: An association was found between overweight status and both atopy and an increased prevalence of wheezing in adolescent Korean boys. These findings suggest that being overweight in puberty may be one of several risk factors responsible for atopy, BHR, and asthma symptoms.

Background: Natural killer T (NKT) cells play an essential role in the development of airway hyperresponsiveness (AHR) in murine asthma. However, their role in the pathogenesis of human asthma has not been defined. The present study investigated whether NKT cells were associated with AHR or airway inflammation in human asthma. Methods: We measured the number of NKT cells in peripheral blood and induced sputum obtained from 61 asthmatics, 10 patients with eosinophilic bronchitis (EB) and 17 controls. EB was used as a disease control model to investigate the mechanisms of AHR in asthma outside the influence of eosinophilic airway inflammation. The quantities of CD3+CD56+ NKT cells, 6B11+ NKT cells and Vα24+ NKT cells were measured by flow cytometry. Results: The measurement of NKT cells did not differ among the 3 groups in peripheral blood. However, in sputum, CD3+CD56+ NKT cells and Vα24+ NKT cells were significantly increased in patients with asthma and EB compared to controls, and 6B11+ NKT cells were also increased in patients with asthma and EB, although not significantly. There was no difference in the measurements of sputum NKT cells between asthmatics and patients with EB. Sputum Vα24+ NKT cells were inversely associated with the degree of AHR in asthmatics. Conclusions: Sputum NKT cells might play an important role in the pathogenesis of eosinophilic airway inflammation in human chronic stable asthma. However, functional studies on airway NKT cells are needed to elucidate the association between NKT cells and AHR.

Background: The aim was to optimize antigen challenge for induction of airway hyperresponsiveness (AHR) and inflammation in BALB/c mice sensitized to ovalbumin (OVA). Comparisons were made between mice challenged with OVA either as an aerosol or intranasally. The protocol that induced maximal AHR in BALB/c mice was thereafter tested in C57BL/6 mice. Method: Methacholine responsiveness was measured using the flexiVent® system to assess AHR. Inflammatory responses were investigated by histology and cell counts in bronchoalveolar lavage (BAL) fluid. Results: 48 h after challenge with 1 or 6% OVA aerosols, there were similar increments in AHR and BAL cells, predominantly eosinophils. When comparing the effect of 1% OVA aerosol on AHR and cell infiltration at 24 and 48 h after challenge, the responses were similar. At 24 h, intranasal OVA administration (20–200 µg) caused a dose-dependent increase in AHR. BAL cells were increased by all intranasal OVA doses and to a greater extent than after 1% OVA aerosol challenge but without any dose dependency. Histological examination confirmed that there was an increase of eosinophils in lung tissue following either challenge. In C57BL/6 mice, baseline tissue elastance was the only functional outcome that was increased after intranasal OVA challenge. Even though the AHR response was negligible in C57BL/6 mice, a similar infiltration of BAL cells was observed in both strains. Conclusion: Intranasal challenge was more effective than aerosol challenge at inducing both AHR and airway inflammation in BALB/c mice. Although intranasal challenge caused airway inflammation in C57BL/6 mice, this strain is not optimal for studying AHR.

Background: Measurement of fractional exhaled nitric oxide (FENO) is useful in assessing eosinophilic airway inflammation. Smoking may modify airway inflammation and reduce FENO levels, compromising the diagnostic value of FENO in smokers. How smoking influences FENO in atopic versus nonatopic asthmatics is unknown. The aim of the present study was to compare FENO in atopic and nonatopic steroid-naive young asthmatic adults and in healthy subjects in terms of smoking. Methods: Forty-six (30 atopic) smoking and 70 (54 atopic) nonsmoking steroid-naive army conscripts (mean age 20 years) with current symptomatic asthma underwent FENO measurement, skin prick tests, spirometry with a bronchodilation test, bronchial histamine challenge, and a standardized exercise test. Ten healthy smokers and 9 healthy nonsmokers underwent FENO measurement, spirometry and bronchial histamine challenge. Results: Smokers with asthma showed significantly higher FENO than did healthy smokers and nonsmokers (p = 0.001, both comparisons). Among atopic asthmatics, FENO was lower in smokers than in nonsmokers (p = 0.002) whereas among nonatopic asthmatics no such difference was detectable (p = 0.89). However, even among nonatopic asthmatic smokers FENO was significantly higher than among healthy controls (p = 0.01). Conclusion: Smoking seems to attenuate the increase in FENO in atopic but not in nonatopic asthmatics. This finding suggests differences in biochemical mechanisms of NO formation in atopic and nonatopic asthma. However, FENO was significantly higher both in atopic and nonatopic asthmatic smokers than in healthy controls. This suggests that FENO can be applied for diagnostic purposes also in young adult smokers.

Background: Helper T (Th) cells are deeply involved in the pathophysiology of bronchial asthma, such as eosinophilic inflammation, bronchial hyperresponsiveness (BHR), airflow limitation and remodeling. It is still unclear whether Th cells contribute to BHR independently of eosinophilic inflammation. The double GATA (dblGATA) site is a high-affinity GATA-binding site in the GATA-1 promoter. dblGATA site-deficient (ΔdblGATA) mice lack eosinophils. Method: Ovalbumin (OVA)-reactive Th clones were transferred into ΔdblGATA and wild-type (WT) mice of BALB/c background. The number of eosinophils in the bronchoalveolar lavage fluid (BALF) and bronchial responsiveness to methacholine were examined after OVA challenge. Results: The number of BALF eosinophils was significantly increased in WT mice, but not detectable in ΔdblGATA mice. BHR was also induced in WT mice, but significantly attenuated in ΔdblGATA mice. Conclusion: Eosinophils are involved in T-cell-mediated BHR.

Background: CpG administration abolishes airway inflammation and remodeling in acute models of allergic airway disease. Methods: Herein, we investigated the therapeutic effect of CpG in a chronic fungal model of asthma. TLR9+/+ and TLR9–/– mice were sensitized to soluble Aspergillus fumigatus antigens and challenged with live A. fumigatus conidia. Mice were treated with intraperitoneal (IP) or intranasal (IN) CpG, or left untreated 14–28 days after conidium challenge. All features of allergic airway disease were attenuated in TLR9+/+ mice treated with IN CpG, including airway hyperresponsiveness (AHR), mucus production, and peribronchial fibrosis. Results: TLR9–/– mice treated with IN CpG exhibited attenuated airway remodeling but not AHR. Whole-lung IL-12 levels were significantly elevated in both TLR9+/+ and TLR9–/– mice receiving IN CpG but not in either group receiving IP CpG. Whole-lung IL-10 levels were significantly elevated in IN CpG-treated TLR9+/+ mice but not in TLR9–/– mice receiving IN CpG. Increased whole-lung transcript and protein levels of the scavenger receptors SR-A and MARCO were observed in TLR9–/– mice compared with TLR9+/+ mice, possibly accounting for the CpG responsiveness in the knockout group. Conclusions: Together, these data show that IN CpG has a therapeutic effect during established fungal asthma, which is TLR9 dependent and independent.

Background: Microbial exposures in both childhood and adult life are protective against atopy, allergic rhinitis and atopic asthma. In adults, this protective effect is paralleled by an increased prevalence of non-atopic asthma. This study was undertaken to investigate associations between occupational endotoxin exposure and atopic sensitization and bronchial hyperresponsiveness to methacholine (BHR) in agricultural workers. In addition, the role of atopy in endotoxin-related respiratory effects was studied. Methods: Data were available for 427 farmers and agricultural industry workers, for whom airborne endotoxin exposure levels were estimated by 249 personal exposure measurements. Atopy was assessed as specific serum IgE to common inhalant allergens, and respiratory symptoms and personal characteristics by standardized questionnaires. BHR was determined in a subset of 113 subjects. Associations were adjusted for age, sex, smoking and living on a farm during childhood. Results: Endotoxin exposure was positively associated with BHR and wheeze (p < 0.05). In contrast, endotoxin exposure was inversely associated with atopy and IgE to grass pollen (p < 0.001). The proportions of wheeze and BHR that were attributable to atopy were only 16.6 and 32.8%, respectively. Conclusions: High endotoxin exposure is a risk factor for BHR and wheeze, which were characterized by a predominantly non-atopic phenotype. At the same time, endotoxin exposure is related to a reduced risk of atopy and IgE to grass pollen in adults. It is unlikely that this is entirely a result of healthy worker selection, as significant inverse associations between endotoxin and IgE to grass pollen were found regardless of reported allergic symptoms.

Background: The migration of dendritic cells (DCs) from the lungs to the regional lymph nodes is necessary for the development of allergic airway disease. Following activation, mast cells release a variety of stored or de novo-produced inflammatory mediators, several of them being capable of activating DCs. In this study, the role of mast cells on DC migration from the lungs to the thoracic lymph nodes was investigated in sensitized mice. Methods: Mast cell-deficient mice (KitW-sh/W-sh) and their wild-type counterparts were sensitized intraperitoneally with ovalbumine (OVA) in saline and challenged by a single intranasal administration of OVA labeled with a fluorescent dye (OVA-Alexa). Results: Following challenge, the relative and absolute amount of OVA- Alexa-positive DCs was clearly increased in sensitized wild-type mice compared to nonsensitized mice. In contrast, sensitized KitW-sh/W-sh showed no increase in OVA-Alexa-positive DCs compared to nonsensitized mast cell-deficient animals. In sensitized KitW-sh/W-sh mice reconstituted with bone marrow-derived mast cells (BMMCs), the number of OVA- Alexa-positive DCs was comparable to that in sensitized wild-type animals. However, transfer of allergen-exposed BMMCs to sensitized mice prior to airway challenge augmented airway inflammation similarly in wild-type and mast cell-deficient mice. In line with this, sensitization with allergen-pulsed DCs induced allergic airway disease independently of mast cells. Conclusions: This study shows an interaction between mast cells and DCs following allergen challenge in sensitized hosts. However, the function of mast cells can be bypassed in models utilizing activated allergen-exposed DCs to initiate the development of allergic airway disease.

Background/Aim: Evidence demonstrating an important role of the intestinal microbiota in the incidence of allergic disorders has led to the concept of using probiotics as possible antiallergic therapy. This study aimed to select a bacterial strain with the best antiallergic treatment effects from a panel of 6 bacterial strains in a mouse model of ovalbumin(OVA)-allergic asthma. Methods: OVA-sensitized BALB/c mice were orally administered the bacterial strains Bifidobacterium breve M-16V, B. infantis NumRes251, B. animalis NumRes252 and NumRes253, Lactobacillus plantarum NumRes8 and L. rhamnosus NumRes6. After challenge by OVA inhalation in the lungs, the response to methacholine was measured. Pulmonary inflammation was assessed by analyzing bronchoalveolar lavage fluid for the presence of eosinophils, neutrophils, macrophages and lymphocytes and for interleukin 4, interleukin 5, interleukin 10 and interferon-γ. OVA-specific IgE, IgG1 and IgG2a were measured in serum. Next, the effect on acute allergic skin reaction was measured after treatment with B. breve M-16V and L. plantarum NumRes8. Results: Of the panel of 6 strains, B. breve M-16V and L. plantarum NumRes8 inhibited (1) the response to methacholine, (2) reduced the number of eosinophils in the bronchoalveolar lavage fluid, (3) reduced both OVA-specific IgE and (4) OVA-specific IgG1, whereas the other strains did not affect all these parameters simultaneously. B. breve M-16V but not L. plantarum NumRes8 reduced interleukin 4, interleukin 5 and interleukin 10. Furthermore, B. breve M-16V but not L. plantarum NumRes8 reduced acute allergic skin reactions to OVA. Conclusion: B. breve M-16V was identified as the most potent antiallergic strain.

Background: Asthma is a chronic respiratory disease, which needs a safer medication preferably in inhalation form. In view of this, we have evaluated the effect of inhaled carbenoxolone (CBX), a herbal-derived compound, on asthma in a mouse model. Methods: Mice were sensitized and challenged with ovalbumin (OVA) to develop certain characteristic features of asthma such as airway hyperreactivity (AHR), airway eosinophilia, lung inflammation and mucus hypersecretion. To evaluate the effect of CBX on the above asthmatic features, CBX (2.5, 5 and 10 mg/ml, 3 ml) or vehicle (water) was given by inhalation. AHR was determined using whole-body plethysmography. Infiltration of eosinophils was estimated by microscopy. Lung inflammation and mucus hypersecretion were assessed using hematoxylin and eosin, and periodic acid-Schiff staining, respectively. Th-2 cytokines, IL-4 and IL-5 were measured in bronchoalveolar lavage (BAL) fluid and IgE in sera. To identify the possible mode of CBX action, we measured corticosterone levels in the BAL fluid and 5-lipoxygenase (5-LO) expression in the lungs. Results: CBX (5 mg/ml) inhalation markedly alleviated AHR (p = 0.0032) and reduced lung inflammation and mucus hypersecretion. Also, it prevented the increase in IL-4 (p = 0.0192), IL-5 (p = 0.0116) and eosinophils (p < 0.0005) in the BAL fluid, and OVA-specific IgE levels (p = 0.00061) in sera. 5-LO expression was also markedly reduced. However, corticosterone levels were not affected. Conclusions: Inhaled CBX alleviates the asthmatic features in mice and could be a potent nebulized therapy in clinical asthma.

Background: Stat3, Socs3 and cytokines play an integral role in the coordination and persistence of inflammation. However, a clear understanding of the role played by the Stat3/IL-6 and Socs3 pathway in airway inflammation is lacking. We report the alteration in the status of expression and activation of Stat3 by ovalbumin (OVA), and establish its relationship with Socs3 and IL-6 in the lungs of mice with eosinophilic pulmonary inflammation and airway hyperresponsiveness. Methods: Alterations in the expression of Stat3, Socs3 and IL-6 were determined in a murine model of asthma, where Balb/c mice were sensitized and challenged with OVA (OVA/OVA) and compared with control mice sensitized and challenged with saline (SAL) (SAL/SAL) mice. The OVA/OVA mice were characterized by a moderate increase in methacholine-induced specific airway resistance, the presence of 150 μg/ml of OVA-specific IgG and 8.93 μg/ml OVA-specific IgE antibody and elevated levels of eosinophils and Th2 cytokines (IL-4 and IL-5) in the bronchoalveolar lavage fluid. In contrast SAL/SAL mice had low eosinophils, IL-4 and IL-5 and no OVA-specific IgG and IgE antibodies in the BALF. Stat3 and Socs3 expression profiles were monitored in OVA/OVA and Stat3- and Socs3-silenced OVA/OVA mice. Furthermore, expression of IL-6 in Stat3- and Socs3-silenced mice and the exogenous effect of IL-6 on Stat3 were studied. Results: The results show that expression and activation of Stat3 mRNA and proteins are significantly low in lung of OVA/OVA mice in comparison to SAL/SAL mice following OVA challenge. An increased pool of Socs3 mRNA is observed in OVA/OVA mice with or without OVA challenge and in SAL/SAL mice 24 h after OVA challenge. Transient in vivo blocking of Socs3 gene by Socs3 siRNA restores the expression of IL-6 mRNA and protein in OVA/OVA mice, and nasal administration of recombinant IL-6 to OVA/OVA mice enhanced Stat3 mRNA expression. Conclusions: Our data suggest that airway inflammation is associated with low expression of Stat3 and IL-6 and overexpression of Socs3 genes in a mouse model of asthma. Furthermore, IL-6 is under the influence of the Socs3 gene and may contribute to the negative regulation of Stat3 via IL-6 following a challenge with an allergen during the development of asthma.

Background: Exposure and sensitization to pet allergens are associated with allergic asthma in children. Conflicting data have emerged regarding the potential benefit of air cleaners with respect to a reduction of indoor pet allergens and bronchial hyperresponsiveness (BHR). Methods: In a randomized controlled trial 36 asthmatic children with sensitization to cat and/or dog and significant exposure to cat and/or dog allergen (Fel d 1 and/or Can f 1 >500 ng/g of carpet dust) were included in order to study the effect of high-efficiency particulate arresting (HEPA) air cleaners placed in the living room and bedroom compared with the effect of sham air cleaners. Patients were allocated to two groups: group 1 exposed to active filters and group 2 exposed to sham filters. At month 0, after 6 months and after 12 months, pulmonary function testing and cold air challenge were performed, serum eosinophil cationic protein (ECP) and specific IgE to seven aeroallergens were determined, and carpet dust samples and filters were collected. Major pet allergen concentrations (Fel d 1, Can f 1) were determined in filters and bulk dust samples. Results: Thirty patients completed the study. After 6 and 12 months, there was no significant change in delta FEV1 after cold air challenge, or in the use of medication and serum ECP levels. However, there was a trend in the active group towards an improvement of bronchial hyperresponsiveness, whereas the sham filter group showed a deterioration of BHR. Conclusion: Although HEPA air cleaners retained airborne pet allergens, no effect on disease activity or allergen concentrations in bulk dust samples was observed in this study.

Background: CpG oligodeoxynucleotides (CpG ODNs) are reported to protect against airway eosinophilia and hyperresponsiveness in animal models of asthma. However, little is known about the effects of CpG ODNs on house dust mites, one of the most common environmental allergens, causing allergic asthma. In the present study, we evaluated the immunomodulatory effects of CpG ODNs on the development of house dust mite-induced airway inflammation and remodeling in mice. Methods: Mice were instilled with Dermatophagoides farinae (Der f) into the trachea 8 times without any additional adjuvants. 48 h after the final allergen instillation, the airway responsiveness to acetylcholine (Ach) was measured, and bronchoalveolar lavage (BAL) and histopathological examination were carried out. CpG ODNs were instilled into the trachea mixed with Der f at the first allergen instillation. Results: Repeated instillation of Der f induced increases in airway responsiveness to Ach, the numbers of inflammatory cells, the levels of T-helper type 2 cytokines and transforming growth factor-β1 in the BAL fluid. Furthermore, goblet cell hyperplasia, the thickness of the epithelium and subepithelial fibrosis were observed. The simultaneous instillation of CpG ODNs with Der f at the first allergen instillation showed significant inhibition of these parameters dose dependently. Conclusions: Our results demonstrate that CpG ODNs have inhibitory effects on Der f-induced airway hyperresponsiveness and eosinophilia, as well as airway remodeling, and that CpG ODNs can be a therapeutic approach for the treatment of house dust mite-induced asthma.

Background: Eosinophils play a pivotal role in the generation of asthma inflammation. Interleukin (IL)-5 is the major activator of eosinophils. We hypothesize that modulating IL-5 activity could be an effective strategy for asthma therapy. In this study, we tested whether the plasmid encoding human IL-5 as a xenogeneic DNA vaccine could induce the production of autoantibodies, and be used for asthma treatment. Methods: A eukaryotic plasmid encoding the human IL-5 was constructed, and used as a DNA vaccine. A mouse model of asthma was established to observe its antiasthma activities. Eosinophils in tissue, blood and the bronchoalveolar lavage were stained and counted. Airway hyperresponsiveness (AHR) was determined by whole body plethysmography. Antibody characters and cytokines were detected with immunological methods. Results: Immunization with a plasmid encoding the human IL-5 as DNA vaccine reduced airway inflammation, reversed Th2 cytokines, and decreased AHR in mice. In addition, this immunization induced the production of polyclonal antibodies that were cross-reactive with native murine IL-5, and IgG1 and IgG2a were the major subclasses. Adoptive transfer of the purified antibodies from the sera of mice immunized with the plasmid encoding the human IL-5 resulted in similar antiasthma effects. Conclusions: Our results suggest that active vaccination against IL-5 may be a rational therapeutic approach for the treatment of asthma and potentially other eosinophilic disorders.

Background: Viral infection causes asthma exacerbations and airway hyperreactivity. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) of viral or synthetic origin in a fashion different from protein kinase R (PKR). The aim of this study was to examine the expression and function of TLR3 in human airway smooth muscle (ASM) cells. Methods: Expression of TLR3 and muscarinic receptor (MR), histamine receptor (HR), and cysteinyl leukotriene receptor (CysLTR) subtypes was analyzed by quantitative real-time PCR, flow cytometry, or Western blotting. It was assessed whether ASM cells respond to polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of dsRNA, with alterations in M2R, M3R, H1R, and CysLT1R expression. The function of these subtypes was evaluated by cholinergic regulation of forskolin-stimulated cyclic AMP accumulation or by mobilization of intracellular calcium upon stimulation. Results: ASM cells expressed TLR3 and PKR, and intracellular TLR3 expression was demonstrated. Poly I:C caused decreased M2R and increased M3R expression, without affecting H1R and CysLT1R expression. Poly I:C-treated cells showed decreased cholinergic inhibition of forskolin-stimulated cyclic AMP accumulation and enhanced calcium flux in response to acetylcholine, but not to histamine and LTD4. These modulating effects of poly I:C were reversed by chloroquine, but not by 2-aminopurine. Conclusions: The data indicate that poly I:C internalized by ASM cells differentially regulates M2R and M3R expression and function by interacting with TLR3 rather than with PKR, suggesting that these changes may contribute to airway hyperreactivity.

Asthma is an inflammatory lung disease, in which CD1d-restricted natural killer T (NKT) cells play an importantpathogenic role. Also, recent reports indicated that a cytokine, thymic stromal lymphopoietin (TSLP), is essential for the development of antigen-induced asthma. Here we examined the relationship between NKT cells and TSLP in a mouse model of asthma. NKT cells express TSLP receptor as well as IL-7 receptor α-chain. TSLP acts on NKT cells to preferentially increase their IL-13 production but not IFN-γ and IL-4. In an allergen-induced asthma model, the development of airway hyperreactivity, a cardinal feature of asthma, was increased in TSLP transgenic mice, whereas this effect was not observed in TSLP transgenic mice lacking NKT cells. Interestingly, in the NKT cell-lacking TSLP transgenic mice, pulmonary eosinophilia and increase in IgE did not improve. Pulmonary lymphocytes from the NKT cell-lacking TSLP transgenic mice produced much less IL-13 upon CD3 stimulation than those from NKT cell-competent TSLP transgenic mice. These resultssuggest that, in allergen-induced asthma, TSLP acts on NKT cells to enhance airway hyperreactivity by upregulating their IL-13 production, whereas eosinophilia and IgE production are not influenced.

Background: Eosinophils contribute to the early features of allergic lung inflammation through the generation and release of a plethora of mediators. Eosinophil peroxidase (EPO) is one of the eosinophil granule proteins involved in the early response, but its participation in airway remodeling is not established. The present study addressed this question comparing an EPO-deficient mouse strain (NZW) with BALB/c and C57Bl/c strains. Methods: Mice were immunized with ovalbumin/alum, challenged twice with ovalbumin aerosol, and lung responses were measured at day 22 or 28. Collagen, mucus and eosinophils were determined in lung sections stained with picrosirius, periodic acid-Schiff or hematoxylin-eosin; transforming growth factor-β and vascular endothelial growth factor were determined by ELISA, lipid bodies by enumeration in osmium-stained eosinophils, and airway reactivity to methacholine in isolated lung preparations. Results: NZW mice showed significantly less collagen around bronchi and blood vessels, less mucus and less eosinophils around bronchi. Eosinophil lipid body formation and airway hyperreactivity were comparable among strains. Levels of transforming growth factor-β were also comparable; however, the NZW mice showed much higher levels of vascular endothelial growth factor, even under basal conditions. Conclusions: In allergic lung inflammation, the combination of EPO deficiency and overexpression of VEGF found in NZW mice is associated with less collagen deposition, less mucus and reduced tissue eosinophilia. Eosinophil activation and airway hyperreactivity in NZW mice were similar to the other strains.

Background: Susceptibility to the development of bronchial asthma (BA) and other atopic diseases is known to be associated with genetic components. Objective: To evaluate the possible role of the polymorphisms in IL-4 gene promoters (C-33T, C-590T and G-1098T) in modulating allergic response and asthma in the Russian population. Methods: The polymorphism analysis was carried out by PCR-RFLP; IL-4 and total IgE concentrations were determined by ELISA. Results: In the case group, the T allele was found at frequencies of 74% (C-33T), 51% (C-590T) and 5% (G-1098T); in the control group the frequencies were 22, 42 and 8%, respectively. Only the C-33T polymorphism was associated with BA. The concentrations of total IgE and serum IL-4 were raised in the case group, while in the control group they were normal. Serum IL-4 level depended on C-33T polymorphism both in the case and control groups, the mutant T allele promoting its increase. The dependence on C-590T polymorphism was detected only in the case group. As for the total IgE level, in both cases it depended on the polymorphism in the case group rather than the control. G-1098T polymorphism did not demonstrate any correlations with total IgE or serum IL-4 levels. All 3 polymorphisms did not affect the severity of BA in the case group. On the basis of the computer analysis, we propose that the T-33C region is the CREB-binding site. Conclusions: Our data suggest that IL-4 promoter polymorphism in the Russian population might play a role both conferring susceptibility to BA and modulating the levels of serum IL-4 and total IgE.

Background: Natural rubber latex (NRL) has emerged as a major cause of respiratory allergy among specific exposed groups of individuals. Since latex allergens are dispersed in the environment it is conceivable that latex proteins are both inhaled and ingested. The mechanism of latex allergy and the immune responses following reexposure of latex allergens by the intranasal route was studied in a murine model of latex allergy developed by intragastric sensitization with NRL. Methods:BALB/c mice were sensitized intragastrically (‘ig’), intranasally (‘in’) or ‘ig’ followed by ‘in’ challenge with NRL allergens. The cellular and humoral immune responses, lung function and histological changes were determined. Results: Peripheral blood eosinophilia was observed in the ‘ig’ and ‘ig/in’-NRL-sensitized animals in comparison to normal controls (p < 0.05). The ‘ig’ group showed a marked increase over control mice in serum total IgE, NRL-specific IgG and IgG subclasses (p < 0.05). Increased levels of IL-4, IL-5, IL-10, and IL-13 were detected in ‘ig’-NRL-sensitized mice. Intranasal exposure with NRL after ‘ig’ sensitization further enhanced the cytokine levels. A tendency towards enhanced stimulation was determined in ‘ig’-sensitized mice; a significant difference was shown in the ‘ig/in’-group (p < 0.05). Increased airway hyperreactivity was found in ‘ig’-NRL-sensitized-mice (15.1 ± 2.5 vs. 8.9 ± 1.7 cm H2O·ml–1·s, p < 0.05). Mucus secretion from jejunal epithelium and eosinophilic infiltration into the jejunal lamina propria were observed in the ‘ig’-NRL-sensitized-mice. Conclusions:The results demonstrate that intragastric NRL sensitization did not induce specific tolerance, and additional intranasal exposure with latex allergens resulted in systemic allergic manifestations in the murine model.

Objective: We investigated the role of antioxidants in airway hyperresponsiveness to acetylcholine using young asthma model mice, which were sensitized and stimulated with ovalbumin. Methods: The mice had been fed either a normal diet, an α-tocopherol-supplemented diet or a probucol-supplemented diet 14 days before the first sensitization. They were immunized with antigen at intervals of 12 days and, starting from 10 days after the second immunization, they were exposed to antigen 3 times every 4th day using an ultrasonic nebulizer. Twenty-four hours after the last antigen inhalation, airway responsiveness to acetylcholine was measured and bronchoalveolar lavage fluid (BALF) was collected. A blood and lung tissue study was also carried out. Results: Twenty-four hours after the last antigen challenge, both IL-4 and IL-5 in the BALF of α-tocopherol-supplemented mice were significantly decreased. The IL-5 level in probucol-supplemented mice was also decreased, but there was no difference in IL-4 levels. The serum IgE level was decreased in probucol-supplemented mice. Differential cell rates of the fluid revealed a significant decrease in eosinophils due to antioxidant supplementation. Airway hyperresponsiveness to acetylcholine was also repressed in antioxidant-supplemented mice. In histological sections of lung tissue, inflammatory cells and mucus secretion were markedly reduced in antioxidant-supplemented mice. We investigated the antioxidant effect on our model mice by examining 8-isoprostane in BALF and lung tissue, and acrolein in BALF; however, our experiment gave us no evidence of the antioxidant properties of either α-tocopherol or probucol contributing to the reduction of airway inflammation. Conclusion: These findings indicate that α-tocopherol and probucol suppress allergic responses in asthma model mice, although these two drugs cause suppression in different ways that are unrelated to antioxidation.

Background: In patients with allergic rhinitis, bronchial hyperresponsiveness (BHR) and airway inflammation may increase during pollen exposure. BHR can be assessed by adenosine-5′-monophosphate (AMP) or methacholine challenge. It has been suggested that BHR to AMP is more closely related to airway inflammation than BHR to methacholine. Seasonal allergic rhinitis offers a dynamic model to study changes in BHR and airway inflammation during natural allergen exposure. Methods: We measured BHR [provocative concentration causing a 20% fall (PC20) in forced expiratory volume in 1 s (FEV1)] to AMP and methacholine, and induced sputum cells in 16 rhinitis patients before and during the 2001 grass pollen season. Results: There was a decrease in PC20 methacholine during pollen exposure (geometric mean PC20 from 3.22 to 1.73 mg/ml, p = 0.0023), whereas no reduction was observed for PC20 AMP (p = 0.11). There was no increase in sputum eosinophils [pre: 0.69% (95% confidence interval 0.22–2.07); during: 1.85 (0.55– 5.6), p = 0.31]. Although the correlation of log PC20 methacholine and log PC20 AMP at baseline was good (r = 0.76, p = 0.001), individual seasonal changes (doubling concentrations) in PC20 methacholine were not correlated with changes in PC20 AMP (ρ = 0.21, p = 0.44). There was no correlation between baseline log PC20 methacholine or seasonal changes in PC20 methacholine and sputum eosinophils (p > 0.12, all correlations). In contrast, there was a significant correlation between seasonal changes in PC20 AMP and changes in sputum eosinophils (ρ = –0.59, p = 0.025). Conclusions: These data show dissimilarity between seasonal changes in PC20 AMP and methacholine in patients with seasonal allergic rhinitis. Moreover, PC20 AMP seems to be more closely related to sputum eosinophils than PC20 methacholine. The clinical significance of this discrepancy is unclear.

Thus the pathophysiology of allergic asthma is traditionally explained by mast celland/or Th2 cell-mediated airway inflammation, which is a manifestation of the acquired immune response. On the other hand, recent studies have identified various other important molecules associated with phenotypes of asthma, including tumor necrosis factor (TNF)[2, 3] , thymic stromal lymphoprotein [4, 5] , interleukin (IL)-33 [6, 7] and IL-25 [8, 9] . TNFis produced mainly in macrophages and mast cells, and thymic stromal lymphoprotein, IL-33 and IL-25 are mainly from epithelial cells and various leukocytes. Those molecules are considered to play roles in the innate immune response. In order to understand novel pathophysiological mechanisms of asthma, a recent trend has been toward the generation of novel animal models, including asthma associated with both acquired and innate immunity, severe asthma such as steroid-resistant phenotypes [10–12] , neutrophilic asthma [13, 14] and the viral exacerbation of allergic inflammation [15, 16] . It is believed that these new asthma models will lead to the development of new strategies for asthma. In this issue of International Archives of Allergy and Immunology , Kim et al. [17] present a well-conducted murine asthma model of AHR, where part of the mechanism reported was TNF-mediated airway inflammation. They observed detailed time-course changes in the occurrence of AHR after antigen (ovalbumin) challenge in According to established knowledge, the mechanisms of allergic asthma are as follows: when an inhaled allergen penetrates the airway epithelium, it is detected by dendritic cells, which migrate to the lymph node and present the processed antigen to T cells, and B cells start to produce antigen-specific immunoglobulin E (IgE). IgE binds to Fc receptors on a variety of cell types including mast cells and basophils. When an allergic individual is subsequently exposed to the specific allergen, the allergen binds to the specific IgE on the cells, causing activation of mast cells and basophils to release chemical mediators, such as histamine, arachidonic acid metabolites and proteases into the airway tissue. These mediators cause an early asthmatic response, which is an airway obstruction induced within 30 min after allergen exposure. There is also a specific population of asthmatic subjects which exhibits a late asthmatic response, i.e. an airway obstruction triggered several hours after allergen challenge and persisting for a relatively long time. The late asthmatic response has been thought to be based on airway inflammation orchestrated by Th2 cells and eosinophils [1] . In addition to the early and late asthmatic responses, Th2-biased airway inflammation leads to airway hyperresponsiveness (AHR) to nonspecific stimuli and structural remodeling of airway tissues. Various experimental models reproduce these phenotypes of asthma. Published online: September 25, 2012

Background: Angiotensin II is a putative mediator in asthma, but the effect of topical administration of type 1 angiotensin II (AT1) receptor antagonists on allergic airway reactions is not known. Objective: To investigate the effect of intranasal administration of CV-11974, an AT1 receptor antagonist, and of PD123319, a type 2 angiotensin II (AT2) receptor antagonist, on antigen-induced airway reactions in guinea pigs. Methods: Thirty minutes after intranasal topical administration of CV-11974 (0.1 or 1.0 mg/ml) or PD123319 (10 mg/ml) into the airways, the animals were given an antigen challenge. Airway hyperresponsiveness and bronchoalveolar lavage fluid were analyzed 24 h after the antigen challenge. Results: Although these compounds did not inhibit antigen-induced early-phase bronchoconstriction or late-phase airway eosinophilia, intranasal administration of CV-11974 (but not PD123319) inhibited antigen-induced airway hyperresponsiveness in a dose-dependent manner 24 h after the antigen challenge. Conclusion: Intranasal administration of an AT1 receptor antagonist reduces antigen-induced airway hyperresponsiveness.

Background: Chronic exposure to fenoterol (FEN), a β2-adrenergic receptor (β2-AR) agonist, was shown to induce both airway hyperresponsiveness and airway remodeling in experimental animals. Objective: We wanted to know the effects of chronic exposure to procaterol (PRO), a β2-AR agonist, on airway function and structure, because this agent is widely used as a bronchodilator in Japan. For comparison, the effects of FEN were also examined. Methods: Aerosolized PRO (0.1 or 1 mg/ml), FEN (1 mg/ml) or vehicle (0.9% NaCl) was given to guinea pigs 3 times a day for 6 weeks. Sublaryngeal deposition of these agents was calculated using radioisotopes. At 72 h after the last inhalation of PRO, FEN or vehicle, the dose-response relationship between lung resistance (RL) and intravenously administered acetylcholine (ACh) was measured. After measuring RL, histological changes in noncartilaginous airway dimensions were evaluated. Results: The amount of sublaryngeal deposition of 0.1 mg/ml PRO in the present study was speculated to be 100 times larger than that of therapeutic dose. ACh concentrations causing 2-fold, 10-fold and maximal increases in RL were not different in 4 groups tested. In the smaller membranous airways (2-AR agonists than in control animals (23 and 25, and 96% higher in animals treated with 0.1 and 1 mg/ml PRO or 1 mg/ml FEN, respectively). The degree of the increase was significantly less in PRO-treated animals than in FEN-treated animals (p < 0.01). Conclusion: Our results did not provide any evidence that regular inhalation of PRO at the therapeutic dose might induce bronchial hyperresponsiveness. In addition, huge amounts of PRO only caused a mild thickening of the adventitial areas, suggesting that PRO may be a weak inducer of airway remodeling compared with FEN.

Background: In naive rodents, repeated exposure to aerosolized antigen induces suppression of the Th2 response to the antigen. We hypothesized that more prolonged exposure of established asthma model to antigen aerosols may downregulate asthmatic phenotype. Methods: After establishing an ovalbumin (OVA)-induced asthma model, mice were further exposed to OVA (prolonged exposure group) or phosphate-buffered saline (positive controls) 3 days per week for 6 weeks. During week 7, the mice of both groups were finally challenged with OVA. Results: Prolonged OVA exposure resulted in marked suppression of serum OVA-specific immunoglobulin E (IgE) antibody levels, eosinophilia of the airway, and airway hyperresponsiveness (AHR). However, airway remodeling characterized by goblet cell hyperplasia and airway fibrosis was observed to the same degree in both groups. These effects were accompanied by diminished production of Th2 cytokines such as interleukin-4 (IL-4), IL-5 and IL-13 in bronchoalveolar lavage fluid (BALF) and cultured supernatant of splenocytes. Furthermore, prolonged exposure markedly increased IL-12 levels in BALF. Conclusions: Prolonged antigen exposure has inhibitory effects on eosinophilic inflammation, AHR and IgE response to antigen, but not on airway remodeling, presumably via inhibition of Th2 cytokines and increased IL-12 production in the lungs.

Patients with cough variant asthma (CVA) and classic asthma are frequently among subjects who present at clinics complaining of a chronic persistent cough. To reveal the features of CVA, we examined the differences in the clinical appearance between CVA and classic asthma. Ten CVA subjects and 11 classic asthmatics were enrolled in the study; they were recruited among patients who presented at the National Minamiokayama Hospital complaining of a chronic cough. The number of eosinophils in peripheral blood was 256 ± 45.8/µl in CVA and 400 ± 123/µl in classic asthma. Eosinophils represented 67% of the cells of sputum in CVA and 82% in classic asthma. Bronchial responsiveness to methacholine was Dmin 1.37 ± 0.56 U in CVA and 0.71 ± 0.46 U in classic asthma. There was no significant difference in these three parameters. There was only a significant difference in v̇25 between CVA and classic asthma, 80.0 ± 6.9 and 52.2 ± 10.0%, respectively. Eosinophil inflammation was almost the same in both CVA and classic asthma.

Lung inflation has a beneficial effect on the airways of healthy subjects. It acts as a bronchoprotector, that is to prevent bronchoconstriction, and as a bronchodilator, in that it reverses bronchial obstruction. The bronchoprotective effect of deep inspiration is more potent than the bronchodilatory one, and the two phenomena appear to advocte different mechanisms. Asthmatics and rhinitics with airways hyperresponsiveness show an impairment in bronchoprotection induced by deep breaths, whereas the bronchodilatory effect, although reduced, is still effective. The lack of the bronchoprotective effect of deep inspiration may contribute to the development of airways hyperresponsiveness. The mechanisms through which lung inflation exerts its beneficial role in healthy subjects, and the factors impairing such an effect in those with airways hyperresponsiveness, are currently under investigation.

Background: The accumulation of eosinophils in the lung is a hallmark of asthma. In addition to cytokines such as IL-5 which are essential, chemokines have been implicated in the recruitment of eosinophils to the airway. In particular, eotaxin has been shown to be a selective and potent eosinophil chemoattractant, important in the pathogenesis of allergic disease. The goal of the present study was to define the role of eotaxin-1 in the development of allergen-induced eosinophilic airway inflammation and airway hyperresponsiveness (AHR) to inhaled methacholine (MCh). Methods: Eotaxin-1-deficient mice were sensitized and exposed to a single challenge with allergen. Airway function and airway and tissue as well as peripheral blood and bone marrow eosinophilia were examined 18 and 48 h after the last challenge. Results: Following allergen sensitization and challenge, eotaxin-1-deficient mice developed levels of AHR to inhaled MCh at 18 and 48 h comparable to controls. Further, levels of bronchoalveolar lavage (BAL) and tissue eosinophilia at the same time points were comparable in the two strains of mice. Tissue eosinophilia, assessed by quantitating major basic protein staining cells, preceded BAL eosinophilia in a similar manner. Bone marrow and peripheral blood eosinophilia were unimpaired in deficient mice. Conclusion: The results demonstrate that the major eotaxin, eotaxin-1 is not essential for the development of airway eosinophilia or AHR, implying that other chemokines, alone or in combination, can overcome this deficiency.

Eosinophils used to be thought of as regulators of allergic inflammation, but there is now evidence to the contrary; eosinophils have been found to be the major effector cells responsible for the late asthmatic response (LAR), airway hyperresponsiveness (AHR) and, at least in part, airway remodeling by releasing leukotrienes and highly basic and cytotoxic proteins such as major basic protein (MBP). However, a recent clinical trial using humanized anti-interleukin-5 monoclonal antibody found a failure to reduce AHR and the LAR, whereas the antibody entirely abolished tissue eosinophilia. In addition, abundant MBP has recently been found in mast cells as well as in eosinophils by our transcriptome (the whole transcripts that a cell expresses) screening of all leukocyte types. Eosinophils are indeed unlikely to be involved in the LAR and may not be unique cytotoxic cells in asthma. It is now necessary to determine the real role of eosinophils and whether early intervention to block eosinophil recruitment into the asthmatic lung does prevent airway remodeling and AHR.

Background: Docosahexaenoic acid (DHA) found in fish oil is known to depress inflammation-related mediators. We investigated a novel delivery method of tridocosahexaenoyl-glycerol (DHA-TG). Methods: BALB/c mice (6–8 weeks old) were primed intraperitoneally with ovalbumin (OVA) and Al(OH)3 on days 0 and 7, and with aerosolized OVA on day 7. Primed mice were challenged by repeated exposure to aerosolized OVA on days 15–17. Just before each exposure to aerosolized OVA, the mice were also exposed to an aerosol of emulsified DHA-TG or soybean oil, or saline (days 7 and 15–17). Bronchial hyperresponsiveness (BHR) to methacholine was measured, and bronchoalveolar lavage fluid was obtained 24 h after the last challenge (day 18). Lungs were histologically examined. Results: Bronchoalveolar lavage fluid of saline-treated mice showed an increased cellularity with predominant eosinophils. Exposure to DHA-TG significantly reduced the total cell number and the eosinophil percentage in lavage fluid, whereas soybean oil did not. Conclusion: DHA but not soybean oil exposure reduced BHR and cell infiltration to bronchovascular bundles. This type of DHA administration could be studied in clinical trials.

Background: Prominent eosinophil infiltration takes place in asthmatic bronchi, and damages bronchial epithelial cells. Aim: This study was designed to investigate whether induction of apoptosis in infiltrated cells in the airways is beneficial or harmful. Methods: A/J mice, which are genetically predisposed to be hyperresponsive to acetylcholine, were immunized with ovalbumin (OA) and alum. Thereafter, they were subjected to a 2-week regimen of OA inhalation, during which they were also administered either hamster anti-mouse Fas monoclonal antibody or hamster IgG (sham control) intranasally. Pulmonary function was then analyzed using whole-body plethysmography. Results: Inhalation of OA increased both airway responsiveness to acetylcholine and infiltration of eosinophils. Administration of anti-Fas antibody induced apoptosis in the infiltrating eosinophils and abolished the increase in airway responsiveness to acetylcholine. Conclusion: Induction of apoptosis in eosinophils infiltrating asthmatic bronchi has a beneficial effect on airway hyperresponsiveness.

Background: Mouse models have been established mirroring key features of human bronchial asthma including airway hyperresponsiveness (AHR). Acute airway obstruction in response to an allergen challenge, however, remains to be demonstrated in these models. Objective: A mouse model of allergic lung inflammation was employed to analyze the development of specific (allergen-induced) and nonspecific (methacholine-induced) airway obstruction. Methods: Mice were sensitized to ovalbumin (OVA) and challenged with OVA aerosol twice each week during four weeks. Changes in lung functions were determined by noninvasive head-out body plethysmography. The development of acute airway obstruction after OVA challenge and AHR after methacholine aerosol application were assessed by a decrease in the mid-expiratory flow rate (EF50). Results: Two airway challenges were sufficient to induce AHR (5.7 vs. 15 mg/ml methacholine). Further OVA challenges reduced the baseline EF50 from 1.85 to 1.20 ml/s (4th week) and induced acute airway obstruction. The OVA-induced obstruction was maximal in the 4th week (EF50 = 0.91 ml/s). Conclusion: The development of acute airway obstruction in allergen-sensitized mice was demonstrated by means of head-out body plethysmography. In our model, AHR was observed before the development of airway obstruction.

This review evaluates the role of IL–5 and IL–5–mediated eosinophil airway infiltration in the development of allergen–driven airway hyperresponsiveness. It discusses the structure and function of IL–5 and its receptor and the mechanisms of IL–5–triggered eosinophil accumulation and inflammation of the airways. New research data from murine models of airway inflammation and hyperresponsiveness utilizing different modes of sensitization to allergen and anti–IL–5 antibody or IL–5–deficient knock–out mice underscore the outstanding role of this cytokine in the pathogenesis of bronchial asthma. This review identifies possible directions for future treatment of airway hyperresponsiveness and concludes that targeting IL–5–driven inflammatory responses may be most beneficial for a novel therapy in bronchial asthma.

Background: Natural rubber latex has been reported as a major cause of allergy and asthma in a number of individuals. One of the occupational groups most affected by latex allergy are the health care workers who are frequently exposed to natural rubber latex products in their patient care activities. The immunopathogenesis of latex allergy is not well understood. In order to understand the immune mechanism in latex allergy, we have developed a mouse model of latex allergy. Methods: Both wild–type and IL–4 knockout BALB/c mice were challenged intranasally with latex proteins and their immune responses, lung pathology, and airway reactivity were evaluated. Results: The total serum IgE and latex specific IgE, IgG1, and peripheral blood and lung eosinophil levels in wild type BALB/c mice were enhanced by the latex exposure, while no IgE or eosinophil were detected in IL–4 knockout mice. Latex–specific IgG1 levels in the sera were lower in IL–4 knockout animals compared to wild mice. However, latex–specific IgG2a antibody was higher in all the IL–4 knockout mice compared to wild type mice. Both the wild type and IL–4 knockout animals developed increased airway resistance after antigen challenge when compared to control animals, although the airway resistance response of IL–4 knockout animals was attenuated compared to the wild–type animals. The histology of the lungs of these two groups of animals was similar. Conclusion: In spite of the differences in the immune responses in the two groups of mice, there were comparable lung inflammatory responses, suggesting a multifactorial pathogenetic mechanism.