Bronchoconstrictor Response Research Articles

Bronchodilatory effects of the aqueous extract of Gynostemma pentaphyllum and gypenosides III and VIII in anaesthetized guinea-pigs

The bronchodilatory activity of the aqueous extract of Gynostemma pentaphyllum Makino leaves was investigated in anaesthetized guinea-pigs and compared with two of its isolated gypenosides (III and VIII). The results showed that the intravenous administration of the decoction of G. pentaphyllum (2.5, 5 or 10 mg kg(-1)) decreased bronchial resistance in basal conditions and significantly (P < 0.01) reduced (68% inhibition) the bronchoconstrictor action of histamine. Furthermore, the extract antagonized (80% inhibition) the bronchoconstrictor response induced by the antigen in sensitized guinea-pigs. Gypenosides III (0.7 mg kg(-1), i.v.) and VIII (0.3 mg kg(-1), i.v.) caused a similar protective effect in both experimental models used; however, the duration and the intensity of the action was less than that of the extract containing corresponding quantities of gypenosides III and VIII. This study confirmed the validity of the traditional use of this plant in the treatment of asthma and other respiratory disorders.

Comparative Effects of Caffeine and Albuterol on the Bronchoconstrictor Response to Exercise in Asthmatic Athletes

The main aim of this study was to evaluate the comparative and additive effects of caffeine and albuterol (short-acting beta (2)-agonist) on the severity of EIB. Ten asthmatic subjects with EIB (exercise-induced bronchoconstriction) participated in a randomized, double-blind, double-dummy crossover study. One hour before an exercise challenge, each subject was given 0, 3, 6, or 9 mg/kg of caffeine or placebo mixed in a flavored sugar drink. Fifteen minutes before the exercise bout, an inhaler containing either albuterol (180 microg) or placebo was administered to each subject. Pulmonary function tests were conducted pre- and post-exercise. Caffeine at a dose of 6 and 9 mg/kg significantly reduced (p<0.05) the mean maximum % fall in post-exercise FEV (1) to -9.0+/-9.2% and -6.8+/-6.5% respectively compared to the double-placebo (-14.3+/-11.1%) and baseline (-18.4+/-7.2%). There was no significant difference (p>0.05) in the post-exercise % fall in FEV (1) between albuterol ( PLUS CAFFEINE PLACEBO) (-4.0+/-5.2%) and the 9 mg/kg dose of caffeine (-6.8+/-6.5%). Interestingly, there was no significant difference (p>0.05) in the post-exercise % fall in FEV (1) between albuterol ( PLUS CAFFEINE PLACEBO) (-4.0+/-5.2%) and albuterol with 3, 6 or 9 mg/kg of caffeine (-4.4+/-3.8, -6.8+/-5.6, -4.4+/-6.0% respectively). Similar changes were observed for the post-exercise % fall in FVC, FEF (25-75%) and PEF. These data indicate that moderate (6 mg/kg) to high doses (9 mg/kg) of caffeine provide a significant protective effect against EIB. It is feasible that the negative effects of daily use of short-acting beta (2)-agonists by asthmatic athletes could be reduced simply by increasing caffeine consumption prior to exercise.

Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways

Cholinergic bronchoconstriction is mediated by M(2) and M(3) muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-β-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R(-/-) and M3R(-/-) mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.

Respiratory syncytial virus induces airway insensitivity to β-agonists in BALB/c mice

beta-Adrenergic agonists (beta-agonists) are commonly used to treat respiratory syncytial virus (RSV) bronchiolitis but are generally ineffective for unknown reasons. We have previously shown that RSV strain A2 inhibits bronchoalveolar epithelial responses to beta-agonists in a BALB/c mouse model by inducing heterologous keratinocyte cytokine (KC)/CXCR2-mediated desensitization of epithelial beta(2)-adrenergic receptors. The aim of the current study was to determine whether RSV also induces airway insensitivity to beta-agonists. Total lung resistance (R) was measured in anesthetized female BALB/c mice undergoing mechanical ventilation on a flexiVent computer-controlled piston ventilator. Data were analyzed using the single-compartment model. Infection with RSV A2 did not induce airway hyperresponsiveness to increasing doses of the nebulized cholinergic agonist methacholine (MCh) at any time point after RSV infection. Prenebulization with the beta-agonist terbutaline (100 muM) significantly attenuated bronchoconstrictive responses to 20 and 50 mg/ml MCh in uninfected mice and in mice infected with RSV 4-8 days postinfection (d.p.i.). However, in mice infected with replication-competent, but not UV-inactivated, RSV for 2 days, significant terbutaline insensitivity was found. Terbutaline insensitivity at 2 d.p.i. could be reversed by systemic preinfection treatment with neutralizing anti-CXCR2 antibodies, which reduced bronchoalveolar lavage (BAL) neutrophil counts but did not alter viral replication, BAL KC levels, or lung edema. Terbutaline insensitivity was also reversed by postinfection nebulization with neutralizing anti-KC or anti-CXCR2 antibodies and could be replicated in normal, uninfected mice by nebulization with recombinant KC. These data suggest that KC/CXCR2-mediated airway insensitivity to beta-agonists may underlie the modest utility of these drugs as bronchodilators in therapy for acute RSV bronchiolitis.

Safety of Binodenoson, a Selective Adenosine A 2A Receptor Agonist Vasodilator Pharmacological Stress Agent, in Healthy Subjects With Mild Intermittent Asthma

The pharmacological stress agents adenosine and dipyridamole are contraindicated in asthma patients because of the risk of adenosine receptor-mediated bronchospasm. Binodenoson, a selective adenosine A(2A) receptor agonist, produces maximal coronary hyperemia during pharmacological stress testing yet has a low affinity for the adenosine A(1), A(2B), and A(3) receptors that are probably responsible for bronchospasm. This study was conducted to assess the safety of binodenoson in 87 healthy young adult volunteers with documented mild, intermittent asthma. This study consisted of a dose-escalating, single-blinded phase and a placebo-controlled, double-blinded phase conducted in healthy, young adults with documented mild, intermittent, asthma. In the single-blinded phase, 3 sequential cohorts of 8 subjects received intravenous binodenoson (0.5, 1.0, and 1.5 microg/kg). In the double-blinded phase, commenced after medical review of results from the single-blinded phase, subjects were randomly assigned 2:1 to either binodenoson 1.5 microg/kg (n=41) or placebo (n=22). The primary end point was clinically significant bronchoconstriction, defined as a decrease in forced expiratory volume in 1 second of >/=20% from the preinjection measure. Secondary safety end points were changes from preinjection measure in forced expiratory volume in 1 second, forced vital capacity, and forced expiratory flow during the middle 50% of the forced vital capacity; vital signs; pulse oximetry; and adverse events. Binodenoson caused no clinically significant bronchoconstriction or alterations in pulmonary function parameters and transiently increased heart rate and systolic blood pressure. The most common treatment-emergent adverse events were tachycardia, dizziness, and flushing. Binodenoson was safe, well tolerated, and caused no clinically significant bronchoconstriction or pulmonary responses in a small population of healthy subjects with mild, intermittent asthma.

Inhaled montelukast inhibits cysteinyl-leukotriene-induced bronchoconstriction in ovalbumin-sensitized guinea-pigs: The potential as a new asthma medication

Oral cysteinyl-leukotriene (LT) receptor antagonists such as montelukast are used for reducing airway inflammation and exacerbations. However, inhaled therapy using LT receptor antagonists has not been studied. In the present study, the effect of inhaled montelukast was investigated on airway hyperresponsiveness measured by cysteinyl-LT induced bronchoconstriction in an animal model of asthma. Bronchoconstriction responses were induced by inhaled LTC4 and LTD4 (0.2 µg/ml each) or three doses of intravenous LTC4 and LTD4 (0.3, 1, 3 µg/kg) in ovalbumin (OVA)-sensitized Hartley male guinea-pigs. The response was measured by the change in peak pressure of airway opening (Pao). The effect of montelukast was evaluated by the comparison of bronchoconstriction responses between the groups of animals pre-treated with 15-min inhalation of 10 mg/ml montelukast and saline. To evaluate the tissue injury which might be caused by montelukast inhalation, lung tissues were examined for the histology. The broncoconstriction responses induced by inhaled LTC4 and LTD4 were enhanced by OVA sensitization in the guinea-pigs. In sensitized animals, the significant increases in peak Pao were 18.5 ± 2.1 cmH 2O by LTC4 inhalation and 25.0 ± 1.6 cmH 2O by LTD4 inhalation on average. Prior treatment of inhaled montelukast potently suppressed the peak Pao increases induced by both inhaled and intravenous LTC4 and LTD4 (all P < 0.01 vs. saline control). Moreover, the suppression of inhaled montelukast against LTD4-induced bronchoconstriction was observed for at least up to 24 h. According to the histological examination, montelukast inhalation produced no injury to the lung tissue. Inhaled montelukast, a cysteinyl-LT receptor antagonist, was effective in inhibiting cysteinyl-LT-induced acute bronchoconstriction, and may have the potential for clinical use as a new asthma drug.

Repeatability And Validity Of IUATLD Respiratory Questionnaire Responses As A Measure Of Asthma In An Ethiopian Population

To assess the repeatability and validity of the IUATLD respiratory symptoms questionnaire in relation to exercise-induced bronchoconstriction or bronchodilator responses in a community in southern Ethiopia. A case-control study. Rural and small town setting in southern Ethiopia, April to May 2006. Two hundred and forty seven adults and children who previously reported wheeze in the past year, and 174 who did not. Administered IUATLD bronchial symptoms questionnaire; standardised free-running exercise test or (for those with airflow obstruction) assessment of bronchodilator response to inhaled salbutamol. Kappa values for four-week repeatability for the wheeze and asthma questions were 0.61 (95% CI 0.52 to 0.70) and 0.75 (0.63 to 0.87), respectively. Of the 58 people who reported wheeze in 2003 and in April 2006, only five had a positive exercise test or bronchodilator challenge (Positive Predictive Value (PPV) 0.09, 95% CI 0.01 to 0.22). Of the 12 who reported asthma in 2003 and April 2006, three had a positive result to either to exercise test or bronchodilator challenge test (PPV 0.25, 95% CI 0.05 to 0.50). Our findings suggest that self-reported wheeze and asthma have good short-term repeatability, but do not closely reflect exercise-induced bronchospasm or bronchodilator responsiveness. The validity of questionnaire methods of studying asthma epidemiology in developing countries needs further investigation.

Changes in lung volumes and airway responsiveness following haematopoietic stem cell transplantation

Changes in lung volume occur following haematopoietic stem cell transplantation (HSCT); airway hyperresponsiveness was occasionally reported, without mechanistic explanation. The present authors studied 17 patients by standard methacholine (MCh) challenge before and then 3 and 12 months after HSCT (n = 16 and n = 13, respectively). Another 6 patients were challenged before and 3 months after HSCT using a modified challenge to investigate the effect of deep inhalations. No patient developed bronchiolitis obliterans or bronchiolitis obliterans organising pneumonia. At 3 months, forced vital capacity (FVC) was significantly reduced by 0.33+/-0.55 L, forced expiratory volume in one second (FEV(1)) by 0.31+/-0.50 L, total lung capacity (TLC) by 0.39+/-0.37 L and single-breath diffusing capacity of the lung for carbon monoxide (D(L,CO)) by 15+/-12%. At 12 months, TLC decreased by 0.43+/-0.36 L and D(L,CO )by 8+/-8%. With standard challenge, no significant changes in FEV(1) response to MCh were observed after HSCT but FVC decreased significantly less after HSCT compared with prior to HSCT, suggesting less air trapping. With modified challenge, deep inhalations reversed the MCh-induced decrease in partial expiratory flow more after HSCT compared with before HSCT and this correlated with TLC decrements. In conclusion, an increase in airway responsiveness is unlikely after haematopoietic stem cell transplantation, at least in patients without pulmonary complications, and mechanisms opposing airway narrowing may blunt the bronchoconstrictor response.

CONTINUOUS MEASURES OF BREATHING AND PHASE ANGLE IN CONSCIOUS CYNOMOLGUS MONKEYS

We evaluated an inductance plethysmography (IP) system in 3 allergic cynomolgus monkeys for measurement of breathing and phase angle (Φ) as a surrogate for airway resistance. Initial studies in anesthetized, intubated animals compared a standard pneumotachograph measure of ventilation with IP to estimate the accuracy of the measurement. Data showed excellent agreement between IP and the standard approach. 24 hours of baseline unanesthetized breathing was then recorded. After 24 hours, monkeys were re‐anesthetized, exposed to an aerosolized ascaris suum antigen and immediately returned to their cages. A subsequent 24 hours of recording provided data from each animal during and following a bronchoconstrictor response. Data indicate that ventilatory pattern is distinctly altered for a period of at least 60 minutes post challenge at which point breathing recovers to normal values. These data agree with our AUC measures in anesthetized animals after antigen exposure. The Φ for each breath was also calculated. During bronchoconstriction, a distinct different angle was measurable. Upon recovery, Φ returned to normal and remained stable, regardless breathing pattern. We conclude that this system is a useful tool to measure breathing in unencumbered, conscious animals for a long period and may be useful in evaluating bronchoconstrictor events.

PROXIMAL AND DISTAL LUNG MECHANICS IN ANTIGEN CHALLENGED ALLERGIC CYNOMOLGUS MONKEYS

We used the forced oscillation technique (FOT) to measure impedance in the pulmonary system and the effects of antigen challenge on the proximal and distal lung. In addition, we treated animals with two oral doses of placebo or betamethasone (1 mg/kg) 18 and 2 hours prior to antigen challenge. FOT provides indices of proximal (Newtonian airway resistance RN) and distal airway status (tissue damping (G) and elastance (H)). Ascaris suum antigen challenge caused a significant increase in RN, G and H. Pretreatment with betamethasone significantly reduced the peak increase in RN and the area under the curve of the FOT response over 60 minutes. G and H responses were unaffected by steroid treatment. These data indicate that, using the FOT, we can dissociate the response of proximal and distal airways to an antigen challenge. Moreover, steroid pre‐treatment can reduce the bronchoconstrictor response to inhaled antigen but this effect is primarily via effects on proximal airways with little effect on the distal airways and parenchymal component of pulmonary impedance. These data may help to explain the apparent disconnect between the empirical and subjective measures of efficacy for some asthma treatments.

Selective Deficiency of PGE2 Inhibits Goblet Cell Metaplasia in a Murine Model of Asthma

RationaleInhalation of Prostaglandin E2 (PGE2) inhibits the early and late phase bronchoconstrictor response to allergen in patients with asthma. Nonselective inhibition of prostaglandin synthesis augments inflammation in mouse models of airway disease, but the responsible prostaglandins have not been clarified. We investigated the role of PGE2 in a model of airways inflammation using mice deficient in a critical PGE synthase, microsomal PGE synthase-1 (mPGES-1).MethodsWe treated mPGES-1 null mice with a C57BL/6 background and wild-type controls with intranasal saline or Dermatophagoides farinea-1 (D. farinae) extract twice weekly for 3 weeks. At the end of the protocol, we analyzed histologic, physiologic, and transcriptional responses.ResultsmPGES-1 null mice treated with D. farinae exhibited significantly increased airway hyperreactivity (AHR), slightly augmented pulmonary inflammation, and normal BAL fluid eosinophilia compared to similarly treated wild-type controls. Unexpectedly, mPGES-1 null mice demonstrated 80% decrease in goblet cell metaplasia and mucus-related gene transcripts.ConclusionsLack of mPGES-1 abrogates the development of goblet cell metaplasia, despite enhanced D. farinae-induced AHR. RationaleInhalation of Prostaglandin E2 (PGE2) inhibits the early and late phase bronchoconstrictor response to allergen in patients with asthma. Nonselective inhibition of prostaglandin synthesis augments inflammation in mouse models of airway disease, but the responsible prostaglandins have not been clarified. We investigated the role of PGE2 in a model of airways inflammation using mice deficient in a critical PGE synthase, microsomal PGE synthase-1 (mPGES-1). Inhalation of Prostaglandin E2 (PGE2) inhibits the early and late phase bronchoconstrictor response to allergen in patients with asthma. Nonselective inhibition of prostaglandin synthesis augments inflammation in mouse models of airway disease, but the responsible prostaglandins have not been clarified. We investigated the role of PGE2 in a model of airways inflammation using mice deficient in a critical PGE synthase, microsomal PGE synthase-1 (mPGES-1). MethodsWe treated mPGES-1 null mice with a C57BL/6 background and wild-type controls with intranasal saline or Dermatophagoides farinea-1 (D. farinae) extract twice weekly for 3 weeks. At the end of the protocol, we analyzed histologic, physiologic, and transcriptional responses. We treated mPGES-1 null mice with a C57BL/6 background and wild-type controls with intranasal saline or Dermatophagoides farinea-1 (D. farinae) extract twice weekly for 3 weeks. At the end of the protocol, we analyzed histologic, physiologic, and transcriptional responses. ResultsmPGES-1 null mice treated with D. farinae exhibited significantly increased airway hyperreactivity (AHR), slightly augmented pulmonary inflammation, and normal BAL fluid eosinophilia compared to similarly treated wild-type controls. Unexpectedly, mPGES-1 null mice demonstrated 80% decrease in goblet cell metaplasia and mucus-related gene transcripts. mPGES-1 null mice treated with D. farinae exhibited significantly increased airway hyperreactivity (AHR), slightly augmented pulmonary inflammation, and normal BAL fluid eosinophilia compared to similarly treated wild-type controls. Unexpectedly, mPGES-1 null mice demonstrated 80% decrease in goblet cell metaplasia and mucus-related gene transcripts. ConclusionsLack of mPGES-1 abrogates the development of goblet cell metaplasia, despite enhanced D. farinae-induced AHR. Lack of mPGES-1 abrogates the development of goblet cell metaplasia, despite enhanced D. farinae-induced AHR.

Frequencies of circulating CD4+CD25+CD127low cells in atopics are altered by bronchial allergen challenge

Recent studies in rodents revealed that regulatory T cells (T reg cells) with CD4+CD25+ phenotype can exert suppressive effects on experimentally-induced allergic airway inflammation and airway hyper-reactivity. It is unclear however, whether modulations of bronchoconstriction responses in human subjects might be related to T reg cells. We report here for the first time the changes in frequency of circulating lymphocytes with putative T reg cell phenotype (CD4+CD25+CD127low) in relation to bronchoconstriction phenotype following an intrabronchial allergen challenge. Thirty-one house dust mite sensitive patients were challenged with Dermatophagoides pteronyssinus extract (Dp). Eleven isolated early responders (IER) were compared with nine dual (early and late) responders (DR) and to 11 non-responders (NR). Frequencies of peripheral blood CD4+CD25+CD127low lymphocytes were assessed in all groups of patients by using three-parameter flow cytometry before, and six and 24 h, after allergen inhalation. At baseline, frequencies of CD4+CD25+CD127low lymphocytes were not statistically different among NR, IER and DR. When all individuals were analyzed together, a statistically significant decrease in frequency of CD4+CD25+CD127low lymphocytes was observed 6 h after the bronchial challenge. Interestingly, such a pattern was found consistently only in NR, while IER and DR displayed varying responses resulting in a trend similar to that of NR. Twenty-four hours after the bronchial challenge, frequencies of CD4+CD25+CD127low lymphocytes in all groups tended to return to baseline values. Our data indicate that bronchial allergen inhalation in sensitive patients (predominantly in those who did not develop significant bronchoconstriction) is associated with a decrease of proportion of peripheral lymphocytes with regulatory T cell phenotype.

ORAI and Store-Operated Calcium Influx in Human Airway Smooth Muscle Cells

The initial bronchoconstrictor response of the asthmatic airway depends on airway smooth muscle (ASM) contraction. Intracellular calcium is a key signaling molecule, mediating a number of responses, including proliferation, gene expression, and contraction of ASM. Ca(2+) influx through receptor-operated calcium (ROC) or store-operated calcium (SOC) channels is believed to mediate longer term signals. The mechanisms of SOC activation in ASM remain to be elucidated. Recent literature has identified the STIM and ORAI proteins as key signaling players in the activation of the SOC subtype; calcium release-activated channel current (I(CRAC)) in a number of inflammatory cell types. However, the role for these proteins in activation of SOC in smooth muscle is unclear. We have previously demonstrated a role for STIM1 in SOC channel activation in human ASM. The aim of this study was to investigate the expression and define the potential roles of the ORAI proteins in SOC-associated Ca(2+) influx in human ASM cells. Here we show that knockdown of ORAI1 by siRNA resulted in reduced thapsigargin- or cyclopiazonic acid (CPA)-induced Ca(2+) influx, without affecting Ca(2+) release from stores or basal levels. CPA-induced inward currents were also reduced in the ORAI1 knockdown cells. We propose that ORAI1 together with STIM1 are important contributors to SOC entry in ASM cells. These data extend the major tissue types in which these proteins appear to be major determinants of SOC influx, and suggest that modulation of these pathways may prove useful in the treatment of bronchoconstriction.

Comparative Effects of a High-Intensity Interval Warm-Up and Salbutamol on the Bronchoconstrictor Response to Exercise in Asthmatic Athletes

Comparative Effects of a High-Intensity Interval Warm-Up and Salbutamol on the Bronchoconstrictor Response to Exercise in Asthmatic Athletes

Differential Effects of Dexamethasone on the Proximal and Distal Lung Response to Antigen Challenge in Allergic Cynomolgus Monkeys

The proximal and distal portions of the lungs may respond differently to antigen challenge and bronchodilator treatment. This difference may contribute to differences in actual and perceived efficacy of therapies. In this study we used the forced oscillation technique (FOT) to measure impedance in the pulmonary system and discern the effects of antigen challenge on proximal (large airway) and distal (small airway and lung parenchyma) portions of the lung. In addition we treated the animals with two i.m. injections of either a saline control or dexamethasone (0.5 mg/kg) 18 and 1 hour(s) before the antigen challenge. The FOT technique was used to measure indices of proximal airway status, Newtonian airway resistance (RN), and distal airway status, including tissue damping (G) and tissue elastance (H). Challenging the animals with Ascaris Suum antigen caused a significant increase in both the proximal and distal lung measures. Pretreatment with dexamethasone significantly reduced the peak increase in RN but not G or H. In addition, the area under the curve (AUC) of the FOT response over 60 minutes was significantly reduced for the RN but again, G and H were not significantly reduced. These data indicate that, using the FOT, we can dissociate the response of proximal and distal airways to an antigen challenge. Moreover, steroid pre-treatment can reduce the bronchoconstrictor response to inhaled antigen but this effect is primarily via effects on the proximal airways with little effect on the distal airways and parenchymal component of pulmonary impedance. These data may help to provide a mechanism for evaluation of novel therapies for small airway dysfunction.

Gene-environment interactions in a mutant mouse kindred with native airway constrictor hyperresponsiveness

We mutagenized male BTBR mice with N-ethyl-N-nitrosourea and screened 1315 of their G3 offspring for airway hyperresponsiveness. A phenovariant G3 mouse with exaggerated methacholine bronchoconstrictor response was identified and his progeny bred in a nonspecific-pathogen-free (SPF) facility where sentinels tested positive for minute virus of mice and mouse parvovirus and where softwood bedding was used. The mutant phenotype was inherited through G11 as a single autosomal semidominant mutation with marked gender restriction, with males exhibiting almost full penetrance and very few females phenotypically abnormal. Between G11 and G12, facility infection eradication was undertaken and bedding was changed to hardwood. We could no longer detect airway hyperresponsiveness in more than 37 G12 offspring of 26 hyperresponsive G11 males. Also, we could not identify the mutant phenotype among offspring of hyperresponsive G8-G10 sires rederived into an SPF facility despite 21 attempts. These two observations suggest that both genetic and environmental factors were needed for phenotype expression. We suspect that rederivation into an SPF facility or altered exposure to pathogens or other unidentified substances modified environmental interactions with the mutant allele, and so resulted in disappearance of the hyperresponsive phenotype. Our experience suggests that future searches for genes that confer susceptibility for airway hyperresponsiveness might not be able to identify some genes that confer susceptibility if the searches are performed in SPF facilities. Experimenters are advised to arrange for multigeneration constancy of mouse care in order to clone mutant genes. Indeed, we were not able to map the mutation before losing the phenotype.

Elevated expression of adenosine A1 receptor in bronchial biopsy specimens from asthmatic subjects

Asthmatics, unlike healthy subjects, experience bronchoconstriction in response to inhaled adenosine, and extracellular adenosine concentrations are elevated in the bronchoalveolar lavage fluid and exhaled breath condensate of asthmatic subjects. However, little is known about the location and expression of adenosine receptors in asthmatic airways. The aim of the present study was to investigate the distribution of adenosine A(1) receptors in bronchial biopsy specimens from mildly asthmatic steroid-naïve subjects and then compare the degree of expression with that of healthy subjects. Biopsy sections were immunostained using an adenosine A(1) receptor antibody, the selectivity of which was validated in specific experiments. Image analysis was then performed in order to determine differences in immunostaining intensity. Immunostaining of biopsy sections from the asthmatic subjects revealed strong expression of the A(1) receptor, located predominantly in the bronchial epithelium and bronchial smooth muscle. In comparison, very weak immunostaining was observed in biopsy specimens obtained from healthy subjects. Image analysis revealed that the intensity of positive staining of the asthmatic bronchial epithelium and smooth muscle regions was significantly greater than that observed for the healthy epithelium and smooth muscle. In conclusion, the sensitivity of asthmatics to inhaled adenosine coupled with increased adenosine A(1) receptor expression implies that these receptors play a role in the pathophysiology of this disease.

Montelukast inhibits inflammatory responses in small airways of the Guinea-pig

Increased resistance in the small airways is a major contributor of airway obstruction in asthma. The role of leukotrienes (LT) in determining inflammation and obstruction of small size bronchi is not completely understood. Here, we have examined the effect of the cysteinyl-leukotriene ( CysLT 1) receptor antagonist, montelukast, against the bronchoconstriction and inflammatory responses induced by exogenous leukotriene and by allergen challenge in small size (⩽1 mm) Guinea-pig bronchi. Montelukast potently (pA 2 8.3) inhibited the contraction induced by LTD 4 in small bronchi taken from naïve Guinea-pigs. Furthermore, montelukast reduced the contraction produced by in vitro ovalbumin (OVA) challenge in small size bronchi from sensitized Guinea-pigs. Montelukast (10 μg kg −1) also blocked plasma protein extravasation and accumulation of inflammatory cells (eosinophils) induced by OVA challenge in small intra-parenchymal bronchi of OVA sensitized animals. These findings provide additional evidence that CysLT 1 receptor antagonism reduces allergic reactions that cause contractile and inflammatory responses in Guinea-pig small airways during OVA challenge. If the anti-bronchospastic and anti-inflammatory actions of the CysLT 1 receptor antagonists observed in the small airways of Guinea-pigs occur also in man these effects may contribute to the beneficial effects of montelukast in asthmatic patients.

Responsiveness to montelukast is associated with bronchial hyperresponsiveness and total immunoglobulin E but not polymorphisms in the leukotriene C4 synthase and cysteinyl leukotriene receptor 1 genes in Korean children with exercise‐induced asthma (EIA)

As previous studies have shown that cysteinyl leukotrienes are important mediators in exercise-induced bronchoconstriction (EIB), and leukotriene receptor antagonists (LTRAs) such as montelukast have been shown to improve post-exercise bronchoconstrictor responses, we herein investigated whether clinical responsiveness to montelukast was associated with polymorphisms in the genes encoding leukotriene C4 synthase (LTC4S) and cysteinyl leukotriene receptor 1 (CysLTR1) and/or clinical parameters in Korean asthmatic children with EIB. The study population consisted of 100 asthmatic children with EIB. The individuals studied were given exercise challenge tests before and after receiving montelukast (5 mg/day) for 8 weeks. Responders were defined as children showing>10% post-treatment improvement in forced expiratory volume in 1 s (FEV1). The LTC4S A(-444)C and CysLTR1 T(+927)C polymorphisms were genotyped by PCR-restriction fragment length polymorphism analysis. Of 100 enrolled children, 68 were classified as responders and 32 were classified as non-responders. No significant association was observed between montelukast responsiveness and LTC4S or CysLTR1 genotype, either alone or in combination. In contrast, montelukast-induced improvement in FEV(1) after exercise was correlated with higher pre-treatment PC20 (methacholine) values (r=0.210, P=0.036) and lower total IgE levels (r=-0.216, P=0.031). The LTC4S A(-444)C and CysLTR1 T(+927)C genotypes do not appear to be useful for predicting clinical responsiveness to montelukast, whereas bronchial hyperresponsiveness and total IgE appear to predict the degree of montelukast responsiveness in Korean asthmatic children with EIB.

Activation of protease‐activated receptor‐2 reduces airways inflammation in experimental allergic asthma

Proteinase-activated receptors (PAR)-2 are members of the family of G-protein-coupled receptors activated by proteases. These receptors are widely expressed in several tissues and in virtually all cells involved in rhinitis and asthma. In particular, proteinases activating PAR-2 may affect airway functions and play a role in human diseases. Assessment of the role of PAR-2 in bronchoconstriction, airway responsiveness and immune response after allergic challenge, in rabbits sensitized to Par j 1, the major allergen of Parietaria judaica pollen. Evaluation of antigen challenge in rabbits treated with PAR-2-activating peptide (PAR-2AP) (SLIGRL) or the scrambled peptide LSIGRL or vehicle immediately before allergen exposure measuring airway responsiveness. Characterization of bronchoalveolar lavage (BAL) following histamine challenge and phenotype analysis of cells by flow cytometry and analysis of cytokine production by quantitative PCR. PAR-2AP pre-treatment, but not the scrambled peptide, was able to significantly inhibit bronchoconstriction, airway hyper-responsiveness and to modulate the immune response induced by allergic challenge in sensitized rabbits. The phenotype analysis of the cells recovered from BAL showed an increase in RLA-DR-positive cells while RTLA-positive cells were unchanged. IFN-gamma and IL-2 production were inhibited, with a concomitant increase in IL-10 of about 10-fold over the control values. In this experimental model, PAR-2 modulates bronchoconstriction interfering with antigen challenge-induced immune response in rabbits sensitized and challenged to Par j 1.