Allosteric modulator potentiates ß2AR agonist-promoted bronchoprotection in asthma models.

Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled ß2-adrenergic receptor (ß2AR) agonists (ß2-agonists) promote - with limited efficacy - bronchodilation in asthma. All ß2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a ß2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of G-protein coupled receptor allosteric ligands, we investigated the impact of Cmpd-6 on ß2AR-mediated bronchoprotection. Consistent with our findings using human ß2ARs, Cmpd-6 allosterically potentiated ß2-agonist binding to guinea pig ß2ARs and downstream signaling of ß2ARs. In contrast, Cmpd-6 had no such effect on murine ß2ARs, which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced ß2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but - in line with the binding studies - not in mice. Moreover, Cmpd-6 robustly potentiated ß2 agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced ß2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of ß2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.

Polypharmacy Interactions Impacting Methacholine Challenge Testing for Asthma Assessment in Older People.

Objective To investigate potential reasons for unusually high incidence of negative Methacholine Challenge Tests (MCT), following standardized MCT medication-hold protocol, in older people with physician-diagnosed asthma. Design An analysis of a longitudinal observational parent study of asthma. Setting Community-dwelling participants were evaluated in an outpatient clinic and at home. Participants Screening inclusion criteria for the parent study included 60 years of age or older, physician diagnosis of asthma, and a positive response to at least one of six asthma screening questions. Participants were enrolled in the study if they also demonstrate either: (1) a postbronchodilator administration response showing an increase of at least 12% and 200 mL in forced expiratory volume or an increase of at least 12% and 200 mL in forced vital capacity, or (2) an MCT result of PC20 ≤ 16 mg/mL (indicating bronchial hyper-responsiveness, MCT positive). Exclusion criteria included diagnosis of cognitive impairment or dementia, residing in a long-term care facility, more than 20 pack/ year smoking history or a history of smoking within the previous five years, inability to perform pulmonary function testing maneuvers, and a Prognostic Index score of greater than 10. Interventions Analysis of participant data for non-medication- and medication-exposure factors for association with negative MCT results. Results Anticholinergic burden and statin use were positively associated with negative MCT. Conclusion Medications not accounted for in medication-hold protocols, and concurrently in use, may impact clinical tests and outcomes.

Steady-state estradiol triggers a unique innate immune response to allergen resulting in increased airway resistance.

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.

Inhibition of Full Smooth Muscle Contraction in Isolated Human Detrusor Tissues by Mirabegron Is Limited to Off-Target Inhibition of Neurogenic Contractions.

Mirabegron is used for treatment of storage symptoms in overactive bladder (OAB) caused by spontaneous bladder smooth muscle contractions. However, owing to limitations in available studies using human tissues, central questions are still unresolved, including mechanisms underlying improvements by mirabegron and its anticontractile effects in the detrusor. Here, we assessed concentration-dependent mirabegron effects on contractions of human detrusor tissues in frequency-response curves and concentration-response curves for different cholinergic and noncholinergic agonists. Detrusor tissues were sampled from patients undergoing radical cystectomy. Contractions were induced by electric field stimulation (EFS) and by cumulative concentrations of cholinergic agonists, endothelin-1, and the thromboxane A2 analog U46619. EFS-induced contractions were inhibited using 10 µM mirabegron, but not using 1 µM. Inhibition by 10 µM mirabegron was resistant to the ß 3-adrenergic antagonist L-748,337. Concentration-dependent contractions by carbachol were not inhibited by 1 µM or 10 µM mirabegron. Concentration-response curves for methacholine were slightly right-shifted by 10 µM, but not 1 µM mirabegron. Concentration-dependent contractions by endothelin-1 or U46619 were not changed by mirabegron. In contrast, the muscarinic antagonist tolterodine right-shifted concentration-response curves for carbachol and methacholine and inhibited EFS-induced contractions. In conclusion, inhibition of neurogenic contractions in isolated detrusor tissues by mirabegron requires concentrations highly exceeding known plasma levels during standard dosing and the known binding constant (Ki values) for ß 3-adrenoceptors. Full contractions by cholinergic agonists, endothelin-1, and U46619 are not affected by therapeutic concentrations of mirabegron. Improvements of storage symptoms are most likely not imparted by inhibition of ß 3-adrenoceptors in the bladder wall itself. SIGNIFICANCE STATEMENT: Mirabegron is used for overactive bladder (OAB) treatment, but the underlying mechanisms are unclear, and preclinical and clinical findings are controversial due to limitations in available studies. Our findings suggest that inhibition of detrusor contractions by mirabegron is limited to neurogenic contractions, which requires unphysiologic concentrations and does not involve ß 3-adrenoceptors. Mechanisms accounting for improvements of OAB by mirabegron are located outside the urinary bladder.

Bronchial hyperresponsiveness to methacholine/AMP and the bronchodilator response in asthmatic children.

Bronchodilator response (BDR) is assessed to estimate the reversibility of airflow obstruction. Bronchial hyperresponsiveness (BHR) is a characteristic feature of asthma and is usually measured by means of bronchial challenges using direct or indirect stimuli. The aim of the present study was to compare BHR to methacholine (direct) and that to adenosine 5'-monophosphate (AMP) (indirect) with regard to their relationships to BDR in asthmatic children. Methacholine and AMP challenge tests were performed on 138 children with mild-to-moderate asthma, and the provocative concentration causing a 20% decline in forced expiratory volume in 1 s (FEV1) (PC20) was determined for each challenge. BDR was calculated as the change in FEV(1), expressed as a percentage of the initial value, after inhalation of 400 µg salbutamol. Methacholine PC20 correlated significantly but weakly with BDR (r = -0.254; p = 0.003). However, there was a significant and strong correlation between AMP PC20 and BDR (r = -0.489; p = 0.000). For AMP PC20, the relationship was closer than for methacholine PC20 (p = 0.024 for comparison between correlation coefficients). The same figures were observed when BDR was expressed as a percentage of the predicted value. A stronger correlation of BDR with AMP PC20 than with methacholine PC20 suggests that BDR may be better reflected by BHR as assessed by AMP challenge than by methacholine challenge.

Relationship between airway responsiveness to mannitol and to methacholine and markers of airway inflammation, peak flow variability and quality of life in asthma patients.

BACKGROUND: Airway hyperresponsiveness (AHR) to stimuli that cause bronchial smooth muscle (BSM) contraction indirectly through the release of endogenous mediators is thought to reflect airway inflammation more closely compared with AHR measured by stimuli that act directly on BSM. METHODS: Fifty-three adult non-smoking asthmatics (28 females, 18-56 years) who were not taking inhaled steroids were challenged with mannitol (up to 635 mg) and methacholine (up to 8 mumol). Induced sputum eosinophils, exhaled nitric oxide (eNO), peak flow variation and clinical severity of asthma according to the Global Initiative for Asthma guidelines were measured in addition to the health-related quality-of-life score using the Juniper asthma quality-of-life questionnaire. FINDINGS: Both AHR to mannitol as well as to methacholine was associated with elevated markers of airway inflammation: in 83% of asthma patients with AHR to mannitol, and in 88% of asthma patients with AHR to methacholine, the eNO level was >20 p.p.b. Sputum% eosinophils >1% was measured in 70% of asthma patients with AHR to mannitol and in 77% of asthma patients with AHR to methacholine. In asthma patients without AHR, 15% had an eNO level >20 p.p.b., but none had sputum% eosinophils >1%. AHR to mannitol was more closely associated with the percentage of sputum eosinophils (PD(15) to mannitol vs. sputum% eosinophils: r: -0.52, P<0.05), compared with AHR to methacholine (PD(20) to methacholine vs. sputum% eosinophils: r: -0.28, NS). Furthermore, there was a stronger correlation between AHR to mannitol and the level of eNO [PD(15) to mannitol vs. eNO (p.p.b.): r: -0.63, P<0.001], compared with AHR to methacholine [PD(20) to methacholine vs. eNO (p.p.b.): r: -0.43, P<0.05]. INTERPRETATION: In asthma patients not being treated with steroids, AHR to mannitol and to methacholine indicated the presence of airway inflammation. AHR to mannitol reflected the degree of airway inflammation more closely when compared with methacholine.

Effect of adenosine A2A receptor activation in murine models of respiratory disorders.

Activation of the adenosine A(2A) receptor has been postulated as a possible treatment for lung inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). In this report, we have studied the anti-inflammatory properties of the reference A(2A) agonist CGS-21680, given intranasally at doses of 10 and 100 microg/kg, in a variety of murine models of asthma and COPD. After an acute ovalbumin challenge of sensitized mice, prophylactic administration of CGS-21680 inhibited the bronchoalveolar lavage fluid inflammatory cell influx but not the airway hyperreactivity to aerosolized methacholine. After repeated ovalbumin challenges, CGS-21680 given therapeutically inhibited the bronchoalveolar lavage fluid inflammatory cell influx but had no effect on the allergen-induced bronchoconstriction, the airway hyperreactivity, or the bronchoalveolar lavage fluid mucin levels. As a comparator, budesonide given intranasally at doses of 0.1-1 mg/kg fully inhibited all the parameters measured in the latter model. In a lipopolysaccharide-driven model, CGS-21680 had no effect on the bronchoalveolar lavage fluid inflammatory cell influx or TNF-alpha, keratinocyte chemoattractant, and macrophage inflammatory protein-2 levels, but potently inhibited neutrophil activation, as measured by bronchoalveolar lavage fluid elastase levels. With the use of a cigarette smoke model of lung inflammation, CGS-21680 did not significantly inhibit bronchoalveolar lavage fluid neutrophil infiltration but reversed the cigarette smoke-induced decrease in macrophage number. Together, these results suggest that activation of the A(2A) receptor would have a beneficial effect by inhibiting inflammatory cell influx and downregulating inflammatory cell activation in asthma and COPD, respectively.

Low-dose theophylline does not exert its anti-inflammatory effects in mild asthma through upregulation of interleukin-10 in alveolar macrophages.

BACKGROUND: There is accumulating evidence that theophylline has anti-inflammatory or immunomodulatory effects. This may be, in part, mediated via an upregulation in the production of the anti-inflammatory cytokine interleukin (IL)-10. We determined whether low-dose theophylline (LDT) would increase the production of IL-10, and attenuate the production of proinflammatory cytokines by alveolar macrophages. METHODS: In a double-blind, placebo-controlled, crossover study involving 15 steroid-free patients with mild asthma, fiberoptic bronchoscopy and bronchoalveolar lavage (BAL) were performed at the end of the treatment and placebo periods. Alveolar macrophages were cultured in vitro, and we measured their release of IL-10, GM-CSF, and TNF-alpha. We also measured IL-10 production in whole blood together with the number of monocytes and T cells expressing intracellular IL-10 by flow cytometry. RESULTS: LDT did not increase the production of IL-10, or attenuate the production of GM-CSF or TNF-alpha by alveolar macrophages. However, after theophylline treatment, there was a significant reduction in mean (SD) (95% CI) BAL eosinophil number from 3.4 (1.7)% (95% CI 2.4-4.4) to 1.7 (1.0)% (95% CI 1.1-2.3) compared with placebo (P<0.05). Similarly, there was no increase in whole-blood IL-10 release or in the number of monocytes and T cells expressing intracellular IL-10 after treatment. CONCLUSIONS: LDT has an anti-inflammatory effect in asthma; however, this effect is not mediated via the production of IL-10 or the attenuation of GM-CSF or TNF-alpha. The mechanisms of theophylline activity remain to be determined.

Alergia a los animales de laboratorio en bioterios de la Universidad Central de Venezuela: factores de riesgo / Allergy to laboratory animals at the Central University of Venezuela: risk factors

Los trabajadores de laboratorios de animales tienen un alto riesgo de desarrollar alergias ocupacionales. En muchos casos, la severidad de los síntomas impide seguir trabajando con los animales. Realizamos este estudio para estimar la frecuencia de síntomas de alergia en un grupo de ocho trabajadores de laboratorios de animales procedentes de dos bioterios de la Universidad Central de Venezuela, a fin de determinar su asociación con varios factores del huésped. Todos los participantes completaron un cuestionario y se les realizaron pruebas de hipersensibilidad inmediata a alergenos comunes, pruebas de provocación con metacolina y determinación de IgE sérica total. Los síntomas de alergia se presentaron en 50 por ciento de los pacientes y estaban relacionados con el tiempo de exposición laboral, pruebas de piel positivas e IgE sérica elevada. Por tanto, una historia personal de atopía, pruebas de piel a alergenos comunes positiva, y un valor IgE sérica elevado parecieran ser factores de riesgo importantes para el desarrollo de alergia a a los animales de laboratorio

Time course of the recovery from bronchoconstriction induced by inhaled methacholin in asthmatic and normal subjects

OBJETIVOS: Determinar o tempo de recuperaçäo e os mecanismos envolvidos na recuperaçäo da constriçäo brônquica induzida pela metacolina inalada em indivíduos asmáticos e hígidos. PACIENTES E MÉTODOS: No primeiro estudo, seis asmáticos e três näo-asmáticos, todos näo fumantes, foram estudados em três dias separados. Em cada dia os indivíduos inalaram doses de metacolina que iam sendo duplicadas (com uma concentraçäo máxima de até 256mg/ml) até que houvesse uma queda de 2o por cento no seu volume expiratório forçado no primeiro segundo...

The semi-proportional hazards model revisited: practical reparametrizations.

Reformulations of the semi-proportional hazards model are outlined making estimation and testing of stratum-covariate interaction effects easily accessible within the framework of the stratified Cox proportional hazards model. The method is illustrated by a practical analysis of variables influencing bronchial responsiveness with data from the Hordaland study of obstructive lung disease.

Bronchial responsiveness of aged asthmatic patients to bronchodilator and methacholine.

Geriatric asthma is characterized by prolonged illness, lower remission rate, poor response to therapy and higher mortality rate. We studied bronchodilator response and methacholine challenge in 25 aged non-smoking asthmatic patients; thirty-two young asthmatic patients were included as control. The elderly patients had poorer baseline pulmonary function and were more responsive to a bronchodilator than the younger patients. The response to bronchoprovocation did not show any difference between the two groups. Our findings suggested that the airways of elderly asthmatics are as sensitive as those of younger patients and should not be under-treated.