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The role of excessive airway constriction in the hyperresponsiveness to nebulized methacholine in mice with experimental asthma is still contentious. Yet, there have been very few studies investigating whether the increased in vivo response to methacholine caused by experimental asthma is associated with a corresponding increase in ex vivo airway constriction. Herein, the responses to nebulized methacholine in vivo and airway constriction in lung slices ex vivo were studied in 8‐ to 10‐week‐old male mice of two strains, BALB/c and C57BL/6. Experimental asthma was induced by administering house dust mites (HDM) intranasally, once daily, for 10 consecutive days. Complementary ex vivo studies were conducted with excised tracheas to measure and compare isometric force. As expected, the in vivo response to methacholine, and especially the hyperresponsiveness caused by HDM, was greater in BALB/c than in C57BL/6 mice. In contrast, there were no differences in maximal airway constriction between mouse strains, and the hyperresponsiveness to nebulized methacholine caused by HDM in both mouse strains was not associated with a corresponding increase in ex vivo airway constriction. The experiments with excised tracheas demonstrated no differences in isometric force between strains and between mice with and without experimental asthma. It is concluded that the hyperresponsiveness to nebulized methacholine in an acute mouse model of asthma induced by repeated HDM exposures is not associated with excessive airway constriction ex vivo. What is the central question of this study? In this study, we investigated the association between the in vivo response of the respiratory system to nebulized methacholine and the ex vivo responsiveness of airways in two mouse strains with and without experimental asthma induced by repetitive intranasal exposures to house dust mites. The ex vivo assays included measurements of airway constriction in lung slices and measurements of isometric force with excised tracheas. What is the main finding and its importance? Although striking differences in the in vivo response to methacholine were observed between mouse strains and between mice with and without experimental asthma, these changes were not matched by corresponding changes in ex vivo airway responsiveness.

This study investigated allergy immunotherapy potential of Lactobacillus paracasei L9 to prevent or mitigate the particulate matter 2.5 (PM2.5) enhanced pre-existing asthma in mice. Firstly, we used a mouse model of asthma (a 21-day ovalbumin (OVA) sensitization and challenge model) followed by PM2.5 exposure twice on the same day of the last challenge. PM2.5 was collected from the urban area of Beijing and underwent analysis for metals and polycyclic aromatic hydrocarbon contents. The results showed that PM2.5 exposure enhanced airway hyper-responsiveness (AHR) and lead to a mixed Th2/ IL-17 response in asthmatic mice. Secondly, the PM2.5 exposed asthmatic mice were orally administered with L9 (4×107, 4×109 CFU/mouse, day) from the day of first sensitization to the endpoint, for 20 days, to investigate the potential mitigative effect of L9 on asthma. The results showed that L9 ameliorated PM2.5 exposure enhanced AHR with an approximate 50% decrease in total airway resistance response to methacholine (48 mg/ml). L9 also prevented the exacerbated eosinophil and neutrophil infiltration in bronchoalveolar lavage fluid (BALF), and decreased the serum level of total IgE and OVA-specific IgG1 by 0.44-fold and 0.3-fold, respectively. Additionally, cytokine production showed that L9 significantly decreased T-helper cell type 2 (Th2)-related cytokines (IL-4, -5, -13) and elevated levels of Th1 related IFN-γ in BALF. L9 also reduced the level of IL-17A and increased the level of TGF-β. Taken together, these results indicate that L9 may exert the anti-allergic benefit, possibly through rebalancing Th1/Th2 immune response and modulating IL-17 pro-inflammatory immune response. Thus, L9 is a promising candidate for preventing PM exposure enhanced pre-existing asthma.

Background: Preliminary data in a randomly selected pediatric cohort study in 8-year-olds suggested a rate of positivity to a methacholine challenge test that was unexpectedly high, roughly 30%. The current recommendation for a negative methacholine test is a 20% decrease in the forced expiratory volume in one second at a dose greater than 400 μg. This was derived from studies in adults using the obsolete English Wright nebulizer. One explanation for the high incidence of positivity in the study in 8-year-olds could be that children deposit more methacholine on a μg/kg basis than adults, due to differences in their breathing patterns. The purpose of this study was to determine if pediatric breathing patterns could result in a higher dose of methacholine depositing in the lungs of children based on μg/kg body weight compared with adults. Methods: An AeroEclipse Breath Actuated nebulizer delivered methacholine aerosol, generated from a 16 mg/mL solution, for one minute, using age-appropriate breathing patterns for a 70 kg adult and a 30 and 50 kg child produced by a breathing simulator. Predicted lung deposition was calculated from the collected dose of methacholine on a filter placed at the nebulizer outport, multiplied by the fraction of the aerosol mass contained in particles ≤5 μm. The dose of methacholine on the inspiratory filter was assayed by high performance liquid chromatography (HPLC). Particle size was measured using laser diffraction technology. Results: The mean (95% confidence intervals) predicted pulmonary dose of methacholine was 46.1 (45.4, 46.8), 48.6 (45.3, 51.9), and 36.1 (34.2, 37.9) μg/kg body weight for the 30 kg child, 50 kg child, and 70 kg adult, respectively. Conclusions: On a μg/kg body weight, the predicted pulmonary dose of methacholine was greater with the pediatric breathing patterns than with the adult pattern.

Abstract Background: Asthma is widely treated using inhaled corticosteroid/long-acting beta agonist (LABA) combinations, for example, fluticasone propionate/salmeterol (FPS) dry powder inhaler, marketed as Advair® Diskus®. Some regulators require generics to demonstrate local (lung) therapeutic equivalence (LTE) for each component of the FPS reference, ideally with a dose-response within the approved FPS dose range. We sought to develop a methacholine challenge (MeCh) LTE methodology for assessing the LABA (salmeterol) component of FPS. Methods: Forty-six patients with asthma received single doses of albuterol (active control; 90 or 180 μg), FPS (100/50 or 200/100 μg), and placebo on 5 separate study days. Spirometry and MeCh were performed 1, 6, and 10 hours after study drug inhalation. Primary endpoint was provocative concentration of methacholine producing a 20% fall in forced expiratory volume in 1 second (PC20). Study entry required screening PC20 ≤8 mg/mL, with a greater than fourfold increase (and PC20 ≤128 mg/mL) after 180 μg albuterol. Results: Both albuterol (90 and 180 μg) and FPS (100/50 and 200/100 μg) significantly increased PC20 compared with placebo (sustained 6 and 10 hours postdose with FPS but not albuterol). The dose-response slopes (95% confidence interval) estimated 1 hour after treatment were 0.374 (−0.068 to 0.815) and 0.310 (−0.135 to 0.754) between low and high doses of albuterol and FPS, respectively, both nonsignificant. Slopes were shallower than those available in the literature for albuterol and formoterol, but similar to those for salmeterol. Conclusions: These data confirm that the bronchoprotective effect of FPS lasts longer than that of albuterol. The shallow dose-response slope we observed for albuterol is contrary to previous reports, probably due to the measurement of PC20 beginning at 1 hour postdose. The results suggest that use of MeCh to assess LTE for salmeterol formulations may be more difficult to accomplish than it is for albuterol and formoterol products.

Abstract Rationale The American Thoracic Society guidelines (1999) for methacholine challenge tests (MCTs) using the 2-minute tidal breathing protocol were developed for the now-obsolete English-Wright (EW) nebulizer. In addition, the guideline recommendation to use the provocative concentration of methacholine causing a 20% drop in FEV1 (PC20) rather than the provocative dose of methacholine causing a 20% drop in FEV1 (PD20) for determining the level of bronchial hyperresponsiveness has been challenged. Objectives To determine if cumulative dose or concentration of methacholine delivered to the airways is the determinant for airway responsiveness and to validate use of the AeroEclipse* II BAN (Aero; Trudell Medical International, London, ON, Canada) nebulizer compared with use of the reference standard EW nebulizer. Methods Subjects with asthma (10–18 yr old) participated in randomized, controlled cross-over experiments comparing four MCT protocols using standard methacholine concentrations, but varying: (1) methacholine starting concentration (testing for cumulative effect); (2) nebulizer (EW versus Aero); and (3) inhalation time. PD20 was calculated using nebulizer output rate, inhalation time, and preceding doses delivered. ANOVA analyses were used to compare geometric means of PC20 and PD20 between protocols. Results A total of 32 subjects (17 male) participated. PC20 differed when starting concentration varied (0.46 vs. 0.80 mg/ml; P < 0.0001), whereas PD20 did not (0.06 vs. 0.08 mg). PC20 differed with the EW versus the Aero nebulzer with 30-second inhalation (1.19 vs. 0.43 mg/ml; P = 0.0006) and the EW versus the Aero nebulizer with 20-second inhalation (1.91 vs. 0.89 mg/ml; P = 0.0027), whereas PD20 did not (0.07 vs. 0.06 mg and 0.11 vs. 0.09 mg, respectively). Conclusions In MCTs, the cumulative dose (PD20), not the PC20, determines bronchial responsiveness. Modern nebulizers may be used for the test if clinical interpretation is based on PD20. Clinical trial registered with www.clinicaltrials.gov (NCT01288482).

PUFA modulate immune function and have been associated with the risk of childhood atopy and asthma. We investigated the effect of maternal fat intake in mice on PUFA status, elongase and desaturase gene expression, inflammatory markers and lung function in the offspring. C57BL/6J mice (n 32) were fed either standard chow (C, 20·4 % energy as fat) or a high-fat diet (HFD, 39·9 % energy as fat) for 4 weeks prior to conception and during gestation and lactation. At 21 d of age, offspring were weaned onto either the HFD or C, generating four experimental groups: C/C, C/HF, HF/C and HF/HF. Plasma and liver fatty acid composition were measured by GC and gene expression by quantitative PCR. Lung resistance to methacholine was assessed. Arachidonic acid concentrations in offspring plasma and liver phospholipids were increased by HFD; this effect was greater in the post-natal HFD group. DHA concentration in offspring liver phospholipids was increased in response to HFD and was higher in the post-natal HFD group. Post-natal HFD increased hepatic fatty acid desaturase (FADS) 2 and elongation of very long-chain fatty acid 5 expression in male offspring, whereas maternal HFD elevated expression of FADS1 and FADS2 in female offspring compared with males. Post-natal HFD increased expression of IL-6 and C-C motif chemokine ligand 2 (CCL2) in perivascular adipose tissue. The HFD lowered lung resistance to methacholine. Excessive maternal fat intake during development modifies hepatic PUFA status in offspring through regulation of gene expression of enzymes that are involved in PUFA biosynthesis and modifies the development of the offspring lungs leading to respiratory dysfunction.

The gastrointestinal microbiota influences immune function throughout the body. The gut-lung axis refers to the concept that alterations of gut commensal microorganisms can have a distant effect on immune function in the lung. Overgrowth of intestinal Candida albicans has been previously observed to exacerbate allergic airways disease in mice, but whether subtler changes in intestinal fungal microbiota can affect allergic airways disease is less clear. In this study we have investigated the effects of the population expansion of commensal fungus Wallemia mellicola without overgrowth of the total fungal community. Wallemia spp. are commonly found as a minor component of the commensal gastrointestinal mycobiota in both humans and mice. Mice with an unaltered gut microbiota community resist population expansion when gavaged with W. mellicola; however, transient antibiotic depletion of gut microbiota creates a window of opportunity for expansion of W. mellicola following delivery of live spores to the gastrointestinal tract. This phenomenon is not universal as other commensal fungi (Aspergillus amstelodami, Epicoccum nigrum) do not expand when delivered to mice with antibiotic-depleted microbiota. Mice with Wallemia-expanded gut mycobiota experienced altered pulmonary immune responses to inhaled aeroallergens. Specifically, after induction of allergic airways disease with intratracheal house dust mite (HDM) antigen, mice demonstrated enhanced eosinophilic airway infiltration, airway hyperresponsiveness (AHR) to methacholine challenge, goblet cell hyperplasia, elevated bronchoalveolar lavage IL-5, and enhanced serum HDM IgG1. This phenomenon occurred with no detectable Wallemia in the lung. Targeted amplicon sequencing analysis of the gastrointestinal mycobiota revealed that expansion of W. mellicola in the gut was associated with additional alterations of bacterial and fungal commensal communities. We therefore colonized fungus-free Altered Schaedler Flora (ASF) mice with W. mellicola. ASF mice colonized with W. mellicola experienced enhanced severity of allergic airways disease compared to fungus-free control ASF mice without changes in bacterial community composition.

Background There are few studies comparing diagnostic accuracy of different lung function parameters evaluating dose–response characteristics of methacholine (MCH) challenge tests (MCT) as quantitative outcome of airway hyperreactivity (AHR) in asthmatic patients. The aim of this retrospectively analysis of our database (Clinic Barmelweid, Switzerland) was, to assess diagnostic accuracy of several lung function parameters quantitating AHR by dose–response characteristics. Methods Changes in effective specific airway conductance (sG eff ) as estimate of the degree of bronchial obstruction were compared with concomitantly measured forced expiratory volume in 1 s (FEV 1 ) and forced expiratory flows at 50% forced vital capacity (FEF 50 ). According to the GINA Guidelines the patients ( n  = 484) were classified into asthmatic patients ( n  = 337) and non-asthmatic subjects ( n  = 147). Whole-body plethysmography (CareFusion, Würzburg, Germany) was performed using ATS-ERS criteria, and for the MCTs a standardised computer controlled protocol with 3 consecutive cumulative provocation doses (PD 1 : 0.2 mg; PD 2 : 1.0 mg; PD 3 : 2.2 mg) was used. Break off criterion for the MCTs were when a decrease in FEV 1 of 20% was reached or respiratory symptoms occurred. Results In the assessment of AHR, whole-body plethysmography offers in addition to spirometry indices of airways conductance and thoracic lung volumes, which are incorporated in the parameter sG eff , derived from spontaneous tidal breathing. The cumulative percent dose-responses at each provocation step were at the 1 st level step (0.2 mg MCH) 3.7 times, at the 2 nd level step (1 mg MCH) 2.4 times, and at the 3 rd level step (2.2 mg MCH) 2.0 times more pronounced for sG eff , compared to FEV 1 . A much better diagnostic odds ratio of sG eff (7.855) over FEV 1 (6.893) and FEF 50 (4.001) could be found. Moreover, the so-called dysanapsis, and changes of end-expiratory lung volume were found to be important determinants of AHR. Conclusions Applying plethysmographic tidal breathing analysis in addition to spirometry in MCTs provides relevant advantages. The absence of deep and maximal inhalations and forced expiratory manoeuvres improve the subject’s cooperation and coordination, and provide sensitive and differentiated test results, improving diagnostic accuracy. Moreover, by the combined assessment, pulmonary hyperinflation and dysanapsis can be respected in the differentiation between “asthmatics” and “non-asthmatics”.

IL-17 is associated with asthma, and T H H17 cells are found in the airways of individuals with asthma. Dean Sheppard and his colleagues now report that IL-17A (but not IL-17F) directly enhances contractile responses in airway smooth muscle cells. Mice lacking T H 17 cells in the lungs exhibit reduced airway hyper-responsiveness in response to allergen challenge. Emerging evidence suggests that the T helper 17 (T H 17) subset of αβ T cells contributes to the development of allergic asthma. In this study, we found that mice lacking the αvβ8 integrin on dendritic cells did not generate T H 17 cells in the lung and were protected from airway hyper-responsiveness in response to house dust mite and ovalbumin sensitization and challenge. Because loss of T H 17 cells inhibited airway narrowing without any obvious effects on airway inflammation or epithelial morphology, we examined the direct effects of T H 17 cytokines on mouse and human airway smooth muscle function. Interleukin-17A (IL-17A), but not IL-17F or IL-22, enhanced contractile force generation of airway smooth muscle through an IL-17 receptor A (IL-17RA)–IL-17RC, nuclear factor κ light-chain enhancer of activated B cells (NF-κB)–ras homolog gene family, member A (RhoA)–Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling cascade. Mice lacking integrin αvβ8 on dendritic cells showed impaired activation of this pathway after ovalbumin sensitization and challenge, and the diminished contraction of the tracheal rings in these mice was reversed by IL-17A. These data indicate that the IL-17A produced by T H 17 cells contributes to allergen-induced airway hyper-responsiveness through direct effects on airway smooth muscle.

Rationale Asthma is a chronic airway condition that occurs more often in women than men during reproductive years. Population studies have collectively shown that long-term use of oral contraceptives decreased the onset of asthma in women of reproductive age. In the current study, we hypothesized that steady-state levels of estrogen would reduce airway inflammation and airway hyperresponsiveness to methacholine challenge. Methods Ovariectomized BALB/c mice (Ovx) were implanted with subcutaneous hormone pellets (estrogen, OVX-E2) that deliver consistent levels of estrogen [68 ± 2 pg/mL], or placebo pellets (OVX-Placebo), followed by ovalbumin sensitization and challenge. In conjunction with methacholine challenge, immune phenotyping was performed to correlate inflammatory proteins and immune populations with better or worse pulmonary outcomes measured by invasive pulmonary mechanics techniques. Results Histologic analysis showed an increase in total cell infiltration and mucus staining around the airways leading to an increased inflammatory score in ovarectomized (OVX) animals with steady-state estrogen pellets (OVX-E2-OVA) as compared to other groups including female-sham operated (F-INTACT-OVA) and OVX implanted with a placebo pellet (OVX-Pl-OVA). Airway resistance (Rrs) and lung elastance (Ers) were increased in OVX-E2-OVA in comparison to F-INTACT-OVA following aerosolized intratracheal methacholine challenges. Immune phenotyping revealed that steady-state estrogen reduced CD3+ T cells, CD19+ B cells, ILC2 and eosinophils in the BAL across all experiments. While these commonly described allergic cells were reduced in the BAL, or airways, we found no changes in neutrophils, CD3+ T cells or CD19+ B cells in the remaining lung tissue. Similarly, inflammatory cytokines (IL-5 and IL-13) were also decreased in OVX-E2-OVA-treated animals in comparison to Female-INTACT-OVA mice in the BAL, but in the lung tissue IL-5, IL-13 and IL-33 were comparable in OVX-E2-OVA and F-INTACT OVA mice. ILC2 were sorted from the lungs and stimulated with exogenous IL-33. These ILC2 had reduced cytokine and chemokine expression when they were isolated from OVX-E2-OVA animals, indicating that steady-state estrogen suppresses IL-33-mediated activation of ILC2. Conclusions Therapeutically targeting estrogen receptors may have a limiting effect on eosinophils, ILC2 and potentially other immune populations that may improve asthma symptoms in those females that experience perimenstrual worsening of asthma, with the caveat, that long-term use of estrogens or hormone receptor modulators may be detrimental to the lung microenvironment over time. Highlights Steady levels of estrogen are associated with changes in allergic inflammation. When E2 is maintained at a level comparable to the mid-follicular phase of ovulation mucus production and airway hyperreactivity are increased. Estrogen reduces eosinophils and ILC2 following allergen challenges. Neutrophils and CD19+ B cells were unaffected in the lung tissues by estrogen treatment.