Hydro-ethanolic extract of Curcuma longa affects tracheal responsiveness and lung pathology in ovalbumin-sensitized rats.

Anti-inflammatory effect of Curcuma longa (C. longa) was shown previously. In the present study, the effect of the plant on tracheal responsiveness and lung pathological features in ovalbumin-sensitized rats was evaluated. Six groups of rats including control (C), ovalbumin (OVA)-sensitized (S), S groups treated with C. longa (CL; 0.75, 1.50, and 3.00 mg/ml equal to 150, 300 and 600 mg/kg/day) and dexamethasone (D; 1.25 µg/ml) were studied (n=8 in each group). The extract of C. longa and dexamethasone were administered with daily drinking water of animals during sensitization period (for 21 days). Following the treatment period, tracheal responsiveness to methacholine and ovalbumin and lung pathological features was investigated. Tracheal responsiveness to methacholine and OVA and lung pathological scores were increased in group S compared to controls (p<0.01 to p<0.001); however, these parameters in groups treated with dexamethasone and two higher concentrations of C. longa were significantly decreased compared to group S (p<0.05 to p<0.001). Tracheal responsiveness to methacholine was decreased from 50 to 400% due to the extract treatment. All concentrations of C. longa significantly decreased interstitial fibrosis compared to group S (p<0.05 to p<0.001). Treatment with the extract resulted to improvement of pathological changes from 20 to 70%. These results showed a preventive effect for C. longa extract on tracheal responsiveness and lung pathological insults in sensitized rats which were similar or even more than those of dexamethasone at used concentrations.

Maintenance of contractile function of isolated airway smooth muscle after cryopreservation.

Isolated human airway smooth muscle (ASM) tissue contractility studies are essential for understanding the role of ASM in respiratory disease, but limited availability and cost render storage options necessary for optimal use. However, to our knowledge, no comprehensive study of cryopreservation protocols for isolated ASM has been performed to date. We tested several cryostorage protocols on equine trachealis ASM using different cryostorage media [1.8 M dimethyl sulfoxide and fetal bovine serum (FBS) or Krebs-Henseleit (KH)] and different degrees of dissection (with or without epithelium and connective tissues attached) before storage. We measured methacholine (MCh), histamine, and isoproterenol (Iso) dose-responses and electrical field stimulation (EFS) and MCh force-velocity curves. We confirmed our findings in human trachealis ASM stored undissected in FBS. Maximal stress response to MCh was decreased more in dissected than undissected equine tissues. EFS force was decreased in all equine but not in human cryostored tissues. Furthermore, in human cryostored tissues, EFS maximal shortening velocity was decreased, and Iso response was potentiated after cryostorage. Overnight incubation with 0.5 or 10% FBS did not recover contractility in the equine tissues but potentiated Iso response. Overnight incubation with 10% FBS in human tissues showed maximal stress recovery and maintenance of other contractile parameters. ASM tissues can be cryostored while maintaining most contractile function. We propose an optimal protocol for cryostorage of ASM as undissected tissues in FBS or KH solution followed by dissection of the ASM bundles and a 24-h incubation with 10% FBS before mechanics measurements.

Eight-year follow-up of airway hyperresponsiveness in patients with primary Sjögren's syndrome.

OBJECTIVE: To evaluate in a longitudinal study the influence of airway hyperresponsiveness (AHR) on lung function in patients with primary Sjögren's syndrome (pSS). METHODS: Lung function was studied over an eight-year period in 15 patients who fulfilled the Copenhagen criteria for primary Sjögren's syndrome and who were covered in our earlier published study on AHR in patients with Sjögren's syndrome. Standard spirometry and measurements of lung volumes, diffusing capacity (DLCO), and AHR to methacholine were performed. RESULTS: A significant decline over time was found in total lung capacity (TLC), vital capacity (VC), forced vital capacity (FVC), functional residual capacity (FRC), and expiratory midflows (FEF50). A sign of small airway obstruction (decrease in FEF50) at entry correlated with VC at follow-up (r = .8, P < .003), and the individual change in FEF50 during the observation period correlated with the individual change in VC (r = .6, P < .05). Six patients had increased AHR, and three of them had decreased DLCO. Six of the patients progressively reduced DLCO over time, and five of them had spirometric signs of increased small airway obstruction. CONCLUSIONS: During this eight-year follow-up we observed that one-third of the patients with pSS developed a significant reduction in lung function. Our findings suggest that small airways obstruction and AHR are associated with reduction of VC and development of impaired DLCO as a sign of interstitial lung disease in this group of patients.

Long-term effects of recurrent intermittent hypoxia and hyperoxia on respiratory system mechanics in neonatal mice.

BACKGROUND: Premature infants are at increased risk for wheezing disorders. Clinically, these neonates experience recurrent episodes of apnea and desaturation often treated by increasing the fraction of inspired oxygen (FIO2). We developed a novel paradigm of neonatal intermittent hypoxia with subsequent hyperoxia overshoots (CIHO/E) and hypothesized that CIHO/E elicits long-term changes on pulmonary mechanics in mice. METHODS: Neonatal C57BL/6 mice received CIHO/E, which consisted of 10% O2 (1 min) followed by a transient exposure to 50% FIO2, on 10-min repeating cycles 24 h/d from birth to P7. Baseline respiratory mechanics, methacholine challenge, RT-PCR for pro and antioxidants, radial alveolar counts, and airway smooth muscle actin were assessed at P21 after 2-wk room air recovery. Control groups were mice exposed to normoxia, chronic intermittent hyperoxia (CIHE), and chronic intermittent hypoxia (CIHO). RESULTS: CIHO/E and CIHE increased airway resistance at higher doses of methacholine and decreased baseline compliance compared with normoxia mice. Lung mRNA for NOX2 was increased by CIHO/E. Radial alveolar counts and airway smooth muscle actin was not different between groups. CONCLUSION: Neonatal intermittent hypoxia/hyperoxia exposure results in long-term changes in respiratory mechanics. We speculate that recurrent desaturation with hyperoxia overshoot may increase oxidative stress and contribute to wheezing in former preterm infants.

Effects of antenatal lipopolysaccharide and postnatal hyperoxia on airway reactivity and remodeling in a neonatal mouse model.

BACKGROUND: Antenatal inflammation and preterm birth are associated with the development of airway diseases such as wheezing and asthma. Utilizing a newborn mouse model, we assessed the effects of maternal inflammation and postnatal hyperoxia on the neonatal airway. METHODS: Pregnant C57/Bl6 dams were injected with lipopolysaccharide (LPS) or saline on embryonic day 16. Offspring were placed in room air or hyperoxia (50% O2) for 7 d and then returned to normoxia. Airway mechanics, histology, and laser capture micro-dissection (LCM) were performed. RESULTS: At postnatal day 21, maternal LPS- and 50% O2-exposed pups exhibited increased resistance and decreased compliance compared to 21% O2 pups; however their effects were not synergistic. LPS and hyperoxia each increased the thickness of airway smooth muscle (ASM), but not the airway epithelial layer. Structural changes were largely limited to the conducting airways. Upregulation of inflammatory markers in the lung was observed at birth. LCM revealed increased collagen-3, transforming growth factor ß, and connective tissue growth factor expression with LPS and hyperoxia within the ASM layer. CONCLUSION: These novel studies provide functional, structural, and molecular evidence that antenatal inflammation is detrimental to the developing airway. Exposure to moderate hyperoxia does not exacerbate LPS effects on the airway.

The polymeric mucin Muc5ac is required for allergic airway hyperreactivity.

In asthma, airflow obstruction is thought to result primarily from inflammation-triggered airway smooth muscle (ASM) contraction. However, anti-inflammatory and smooth muscle-relaxing treatments are often temporary or ineffective. Overproduction of the mucin MUC5AC is an additional disease feature that, while strongly associated pathologically, is poorly understood functionally. Here we show that Muc5ac is a central effector of allergic inflammation that is required for airway hyperreactivity (AHR) to methacholine (MCh). In mice bred on two well-characterized strain backgrounds (C57BL/6 and BALB/c) and exposed to two separate allergic stimuli (ovalbumin and Aspergillus extract), genetic removal of Muc5ac abolishes AHR. Residual MCh responses are identical to unchallenged controls, and although inflammation remains intact, heterogeneous mucous occlusion decreases by 74%. Thus, whereas inflammatory effects on ASM alone are insufficient for AHR, Muc5ac-mediated plugging is an essential mechanism. Inhibiting MUC5AC may be effective for treating asthma and other lung diseases where it is also overproduced.

Prenatal retinoid deficiency leads to airway hyperresponsiveness in adult mice.

There is increasing evidence that vitamin A deficiency in utero correlates with abnormal airway smooth muscle (SM) function in postnatal life. The bioactive vitamin A metabolite retinoic acid (RA) is essential for formation of the lung primordium; however, little is known about the impact of early fetal RA deficiency on postnatal lung structure and function. Here, we provide evidence that during murine lung development, endogenous RA has a key role in restricting the airway SM differentiation program during airway formation. Using murine models of pharmacological, genetic, and dietary vitamin A/RA deficiency, we found that disruption of RA signaling during embryonic development consistently resulted in an altered airway SM phenotype with markedly increased expression of SM markers. The aberrant phenotype persisted postnatally regardless of the adult vitamin A status and manifested as structural changes in the bronchial SM and hyperresponsiveness of the airway without evidence of inflammation. Our data reveal a role for endogenous RA signaling in restricting SM differentiation and preventing precocious and excessive SM differentiation when airways are forming.

The effects of particle size on measurement of airway hyperresponsiveness to methacholine.

BACKGROUND: The effect of particle size on methacholine provocation concentration causing a decrease in forced expiratory volume of 1 second (FEV1) of 20% (PC20) is debatable. OBJECTIVE: To evaluate the functional effects of 3 different particle size nebulizers on methacholine PC20. METHODS: Participants were randomly assigned to have 3 methacholine challenges on 3 separate days. Nebulizer mass median aerodynamic diameter (MMAD) was provided by manufacturers. The Wright nebulizer (MMAD, 1.0 µm), Aeroneb (MMAD, 3 µm), and Aeroneb (MMAD, 5 µm) were calibrated, and the nebulizer outputs were calculated to administer 0.26 mL of methacholine over 120, 112, and 83 seconds, respectively. After each inhalation, spirometry was performed and the test was terminated when the PC20 was achieved. RESULTS: Eight nonsmoking patients with mild asthma (4 male and 4 female) completed the study. The mean (SD) age was 25 (13.9) years, and the mean (SD) baseline FEV1 was 88% (11.3%). Patients using the Aeroneb (MMAD, 5 µm) nebulizer had the lowest PC20 (bronchoconstricted at lowest methacholine concentration), with a PC20 geometric mean of 0.62 mg/mL compared with patients using the Aeroneb (MMAD, 3.0 µm), who had a PC20 of 1.76 mg/mL, and patients using the Wright nebulizer (MMAD, 1.0 µm), who had a PC20 of 6.32 mg/mL. There was a significant difference in PC20 across all particle sizes (P < .001). The pairwise differences revealed a P < .001 between 3 µm and 1 µm and between 5 µm and 1 µm and a P = .008 between 5 µm and 3 µm. CONCLUSION: Our results reveal a variability in methacholine PC20 using 3 different nebulizers, despite adjusting the nebulizers' outputs. Our results are consistent with the previous reports, which recommended using larger particle size nebulizers in the assessment of airway hyperresponsiveness in asthma. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00529477.

Dampness and moulds in workplace buildings: associations with incidence and remission of sick building syndrome (SBS) and biomarkers of inflammation in a 10 year follow-up study.

There are few longitudinal studies on health effects of dampness and moulds in workplace buildings. We studied associations between dampness and indoor moulds in workplace buildings and selected biomarkers as well as incidence and remission of sick building syndrome (SBS). The study was based on a ten-year prospective study (1992-2002) in a random sample of adults (N=429) from the Uppsala part of the European Community Respiratory Health Survey (ECRHS). The 10-year incidence (onset) of general, mucosal, dermal symptoms and any symptom improved when away from the workplace (work-related symptoms) was 7.2%, 11.6%, 6.4% and 9.4% respectively. The 10-year remission of general, mucosal, dermal symptoms and work-related symptoms was 71.4%, 57.1%, 70.4% and 72.2% respectively. Signs of dampness in the floor construction in any workplace building during follow up (cumulative exposure) was associated with incidence of mucosal symptoms (OR=2.43). Cumulative exposure to moldy odor was associated with incidence of work-related symptoms (OR=2.69). Cumulative exposure to dampness or moulds was associated with decreased remission of work-related symptoms (OR=0.20 for water leakage, OR=0.17 for floor dampness, and OR=0.17 for visible indoor mould growth). Working in a building repaired because of dampness (repaired building) or mould was associated with decreased remission of work-related symptoms (OR=0.32). Any dampness or moulds at baseline in the workplace building was associated with increased bronchial responsiveness (BR) and higher levels of Eosinphilic Cationic Protein (ECP) in serum and Eosinophilic counts in blood at baseline. Cumulative exposure to dampness and moulds, and work in a repaired building, was associated with increased BR at follow-up. In general, dampness and moulds in the workplace building is associated with increased incidence and decreased remission of SBS, as well as increased bronchial responsiveness and eosinophilic inflammation.

A linear parametric approach for analysis of mouse respiratory impedance.

Assessment of the lung mechanics is crucial in lung function studies. Commonly lung mechanics is achieved through measurement of the input impedance of the lung where the experimental data is ideal for the application of system identification techniques. This study proposes a new approach for investigating the severity of lung conditions and also evaluating the treatment progression. The proposed method is established based on linear parametric identification of lung input impedance in mice and is applied to normal and asthmatic models (including acute, tolerant and chronic asthma) as well as a pharmacological intervention model. Experimental findings confirm the effectiveness of the analysis technique applied here. We discuss the potential application of this method to analyses of human lung mechanics.

Methacholine dry powder inhaler as a new tool for bronchial challenge test.

BACKGROUND: The methacholine (MCH) challenge test is performed to detect bronchial hyperresponsiveness in subjects suffering from asthma. It is conducted by inhaling spasmogen substances at increasing doses and measuring FEV1-PD20 variation following the bronchoconstriction evoked. AIM: This paper describes a new method for MCH challenge test using pre-metered respirable powders of MCH at different doses for facilitating test execution. The availability of a series of pre-metered doses gives higher control over aerosolized dose and fine particle fraction (respirable dose), improving the accuracy and repeatability of the test. Dosimetric tests with MCH solution and pre-dosed powder challenge tests were clinically compared. METHODS AND MATERIALS: The inhalation powders were prepared by spray drying of solutions of methacholine, mannitol and hydroxypropylmethylcellulose in which different concentrations of MCH were included. The methacholine powders prepared were carefully characterized in terms of aerodynamic properties. RESULTS: Inhalation powders containing methacholine from 12.5 to 200 microg per metered dose, having a fine particle fraction between 40 and 60%, were prepared using mannitol and cellulose polymer. Eighteen subjects (12 hyperresponsive and six normal) were subjected to both the MCH solution and powder tests in random sequence. No significant differences in FEV1 and PD20 values were found between the challenge tests performed with liquid and powder formulations of methacholine. CONCLUSIONS: Powders of MCH having high respirability of the delivered doses can be prepared by spray drying. They allow for the performance of a challenge test using a dry powder inhaler. The powder dose series can be an alternative to the current dosimetric test with MCH solutions.

Single cell analysis and temporal profiling of agonist-mediated inositol 1,4,5-trisphosphate, Ca2+, diacylglycerol, and protein kinase C signaling using fluorescent biosensors.

The magnitude and temporal nature of intracellular signaling cascades can now be visualized directly in single cells by the use of protein domains tagged with enhanced green fluorescent protein (eGFP). In this study, signaling downstream of G protein-coupled receptor-mediated phospholipase C (PLC) activation has been investigated in a cell line coexpressing recombinant M(3) muscarinic acetylcholine and alpha(1B) -adrenergic receptors. Confocal measurements of changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)), using the pleckstrin homology domain of PLCdelta1 tagged to eGFP (eGFP-PH(PLCdelta)), and 1,2-diacylglycerol (DAG), using the C1 domain of protein kinase Cgamma (PKCgamma) (eGFP-C1(2)-PKCgamma), demonstrated clear translocation responses to methacholine and noradrenaline. Single cell EC(50) values calculated for each agonist indicated that responses to downstream signaling targets (Ca(2+) mobilization and PKC activation) were approximately 10-fold lower compared with respective Ins(1,4,5)P(3) and DAG EC(50) values. Examining the temporal profile of second messenger responses to sub-EC(50) concentrations of noradrenaline revealed oscillatory Ins(1,4,5)P(3), DAG, and Ca(2+) responses. Oscillatory recruitments of conventional (PKCbetaII) and novel (PKCepsilon) PKC isoenzymes were also observed which were synchronous with the Ca(2+) response measured simultaneously in the same cell. However, oscillatory PKC activity (as determined by translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate protein) required oscillatory DAG production. We suggest a model that uses regenerative Ca(2+) release via Ins(1,4,5)P(3) receptors to initiate oscillatory second messenger production through a positive feedback effect on PLC. By acting on various components of the PLC signaling pathway the frequency-encoded Ca(2+) response is able to maintain signal specificity at a level downstream of PKC activation.

Conformation of ligands bound to the muscarinic acetylcholine receptor.

Many biogenic amines evoke a variety of physiological responses by acting on G protein-coupled receptors. We have determined the conformation of two acetylcholine analogs, (S)-methacholine and (2S,4R,5S)-muscarine, bound to the M(2) muscarinic acetylcholine receptor (M(2) mAChR) by NMR spectroscopy. The analysis of the transferred nuclear Overhauser effect indicated that the receptor selectively recognized the conformers of (S)-methacholine and (2S,4R,5S)-muscarine with the gauche O-C2-C1-N dihedral angle at +60 degrees. This is distinct from the predominant conformations of these ligands in solution with O-C2-C1-N dihedral angle (+80 to approximately 85 degrees ) in the absence of the M(2) mAChR, as assessed by analyses of the coupling constants and nuclear Overhauser effect spectroscopy. We have also built a molecular model of the M(2) mAChR-(S)-methacholine complex, based on the X-ray crystallographic structure of rhodopsin. This model indicated that the conformation with the gauche O-C2-C1-N dihedral angle at +55.5 degrees, which is similar to the one determined by NMR measurement, is energetically favored in the binding of (S)-methacholine to the receptor. We suggest that this conformation represents the binding of the agonist to the M(2) mAChR in the absence of G protein.

Stability of frozen methacholine solutions in unit-dose syringes for bronchoprovocation.

OBJECTIVE: Methacholine solutions < 0.25 mg/mL must be prepared fresh daily, while concentrations > or = 0.25 mg/mL must be prepared at 2-week intervals according to US Food and Drug Administration-required labeling. The purpose of this report was to determine whether freezing methacholine solutions in unit-dose syringes would allow less frequent preparation. DESIGN: Diluent containing 0.5% sodium chloride, 0.275% sodium bicarbonate, and 0.4% phenol was used to prepare 11 concentrations of methacholine ranging from 0.031 to 32.0 mg/mL. Three milliliters of each dilution was placed into 5-mL polypropylene syringes and immediately frozen. Methacholine concentrations were determined using a validated high-performance liquid chromatography assay after preparation (time zero) and at 0.5, 1, 1.5, 2, 3, 4, 5, and 6 months. On the day of analysis, the samples were allowed to thaw to room temperature. An additional set of each dilution was stored at room temperature for 24 h after thawing and then analyzed for methacholine. RESULTS: Samples > or = 0.062 mg/mL analyzed immediately after thawing retained > or = 90% of labeled potency for at least 6 months, while the 0.031-mg/mL sample retained 90% potency for 4 months. Most samples analyzed 24 h after thawing lost potency. CONCLUSION: If prepared and stored in unit-dose syringes frozen, methacholine solutions containing 0.062 to 32.0 mg/mL can be prepared at 6-month intervals, and solutions containing 0.031 mg/mL can be prepared at 4-month intervals. Once thawed, unused methacholine solutions should be discarded.

Selective activity of butyrylcholinesterase in serum by a chemiluminescent assay.

In a previous study, we showed that purified commercial esterase activity can be detected in a chemiluminescent assay based on the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) to 2-methyl-1-propenol, which is subsequently oxidized by the horseradish peroxidase (HRP)-H(2)O(2) system. The purpose of this study was to verify the applicability of this assay to human serum. The existence of an esterase activity capable of hydrolysing MPB is indicated by the fact that the MPB-serum-HRP-H(2)O(2) system consumes oxygen and emits light. Both signals were abolished by prior serum heat inactivation and were preserved when serum was stored at < or =4 degrees C. Addition of aliesterase inhibitors, such as fluoride ion and trichlorfon or the cholinesterase inhibitor eserine, totally prevents light emission. The butyrylcholinesterase-specific substrate benzoylcholine causes a delay in both O(2) uptake and light emission, while the specific acetylcholinesterase substrate, acetyl-beta-methylcholine, had practically no effect. Purified butyrylcholinesterase, but not acetylcholinesterase, triggered light emission. The finding that butyrylcholinesterase is responsible for the hydrolysis of MPB in serum should serve as the basis for the development of a specific chemiluminescent assay for this enzyme.

Stability of stored methacholine solutions: study of hydrolysis kinetic by IP-LC.

Methacholine chloride is a powerful cholinergic bronchoconstrictor agent used during bronchial airway hyper-responsiveness diagnosis. Methacholine is susceptible to hydrolysis in aqueous solutions in acetic acid and beta-methylcholine. In the present work, kinetics of hydrolysis with different solvents (water and phosphate-buffered saline (PBS) pH 7.4) at different temperatures have been studied using a newly developed high-performance liquid chromatography. At 4 degrees C, kinetic determination of hydrolysis in methacholine chloride solutions (50 mg/ml) shows no hydrolysis in either aqueous or phosphate-buffered solutions over a 40-day period. At 30 degrees C, concentration of unbuffered methacholine chloride solutions remained unchanged, but buffered methacholine chloride solutions have degradation up to 5.5% over a 40-day period. At 40 degrees C, concentration of unbuffered methacholine chloride has degradation up to 5% and buffered methacholine chloride solutions have degradation up to 10% over a 40-day period. Methacholine chloride solutions are susceptibly to be used in hospital pharmacy at different concentrations. We have studied pH and osmolality for methacholine solutions prepared with different diluents potentially used in hospital pharmacies, i.e. deionized water, 0.9% NaCl and PBS pH 7.4. We have demonstrated that methacholine solutions prepared with deionized water at 50 mg/ml and diluted with PBS pH 7.4 from 5 to 40 mg/ml are isoosmotic and potentially available for inhalation tests to measure non-specific bronchial hyper-responsiveness.

[Pharmaceutical contribution to the non specific challenge in bronchial provocation tests]. / Contribution pharmaceutique a l'exploration de l'hyperréactivité bronchique non spécifique.

Aerosols of methacholine chloride are used in inhalation tests to measure non-specific bronchial responsiveness. Inhalation challenge solutions are not commercially available neither by pharmaceutical industry nor in France by the PCH-APHP. To reply to doctor's request, dilute concentrations of methacholine must be prepared properly in order to protect the patient and to assure doctor of test reproductibility. Osmolarity, pH and degradation of different dilute concentration of methacholine chloride have been compared with two diluents. This study allowed us to validate our preparation of solutions.

Stability of methacholine chloride in isotonic sodium chloride using a capillary electrophoresis assay.

OBJECTIVE: To investigate the stability of methacholine chloride in 0.9% sodium chloride solutions. METHOD: Methacholine powder was mixed with diluent to a final concentration of 5 and 10 mg/ml. Duplicates of each admixture were divided and stored in glass vials at 25 degrees C, 4 degrees C and -20 degrees C for 12 months. At appropriate times intervals, samples were removed from solutions and analysed. Methacholine concentrations were measured using a high performance capillary electrophoresis assay. RESULTS: No colour or other visual changes were seen in any sample. However, an additional peak was observed in some samples. CONCLUSION: Methacholine chloride solutions 5 mg/ml were stable in isotonic sodium chloride after refrigeration or freezing over a period of one year; methacholine chloride solutions 10 mg/ml were stable for one year after freezing. The solutions stored at ambient temperature were stable for 35 days and for less than 14 days, respectively, for the 5 and the 10 mg/ml solutions.