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Preparation for Asthma Treatment: Ventolin Inhalers
Asthma has many well-known clinical manifestations, one of which is an accelerated decline in spirometric function. This occurs particularly in patients who have severe, longstanding, or adult-onset asthma ultimately leading to chronic, fixed airflow limitation of varying degree. This is thought to be secondary to structural changes in the airway, collectively referred to as remodeling, which involves changes in extracellular matrix, collagen, elastin, and airway smooth muscle. Accelerated decline in spirometric function also occurs in asthmatic subjects who report chronic cough and sputum production due to asthmatic mucus hypersecretion (AMH), but who do not have bronchiectasis, allergic bronchopulmonary aspergillosis, or a history of heavy smoking.
Matrix metalloproteinases (MMPs) play a role in tissue remodeling in diseases such as asthma, bronchiectasis, rheumatoid arthritis, and mesothelioma, and in wound healing. MMP-9 is a 92-kd collagenase that cleaves type IV collagen, which makes up a substantial portion of the basement membrane of airways and is regulated by the tissue inhibitor of metalloproteinase (TIMP)-1, which binds to MMPs in a 1:1 ratio. Its activity is increased in the bronchial biopsy specimens, blood, induced sputum, and BAL fluid (BALF) of patients who have severe or uncontrolled asthma. Ventolin Inhalers are worked out to stop the asthma attack, they are convenient in usage and may be ordered via buy-asthma-inhalers-online.
Because of the association of decline in lung function with AMH, severe asthma, and persistent airway inflammation, we hypothesized that MMP-9 activity would be increased in the blood and airway secretions in asthmatic patients who had severe disease and/or AMH (diagnosed at bronchoscopy), and that it would correlate with airway inflammation. Our aim was to compare MMP-9 activity and TIMP-1 expression in the serum and BALF in healthy subjects, patients with mild/moderate asthma, patients with severe asthma, and patients with AMH. In addition, we compared MMP-9 activity in serum and BALF.
Asthmatic and nonasthmatic subjects underwent clinical testing in the lung function laboratory and then underwent bronchoscopy within 2 weeks of laboratory testing. Asthmatic subjects were classified as having mucus hypersecretion if they had mucus causing occlusion of the subsegmental or larger airways visualized during bronchoscopy, as described by Thompson et al (bron-choscopists, G.G.K. and K.Y.). The study was approved by the Ethics Review Committee of the Central Sydney Area Health Service (reference X99-0217). Written informed consent was obtained from all subjects.
Seven nonasthmatic subjects and 22 asthmatic subjects were recruited from among patients and students at the Asthma Centre, Royal Prince Alfred Hospital, and from the University of Sydney. The diagnosis of asthma was based on the World Health Organization/National Heart, Lung, and Blood Institute guide-lines. Asthmatic subjects had clinically stable asthma, as defined by stable symptoms and medication usage over the previous 3 months, and had no symptoms of respiratory tract infection in the previous 6 weeks. None of the subjects were current smokers, ex-smokers of > 20 pack-years, or pregnant, or had coexistent chronic cardiac or pulmonary disease.
Clinical Assessment of Asthma
We administered a respiratory questionnaire- that included questions on the frequency and nature of asthma symptoms, medication use- and the presence of chronic cough that was productive of phlegm. The dose of inhaled corticosteroids was converted to equivalent doses of beclomethasone dipropionate, ventolin inhalers according to the following equation: 100 μg of beclomethasone dipropionate = 80 μg of budesonide = 50 μg of fluticasone. Skin prick tests were performed with a panel of 14 common aeroal-lergens applied to the forearm. Skin wheal sizes were considered to be positive if they were > 4 mm in mean diameter, and atopy was defined as the presence of one or more positive reactions.
Spirometry was measured in all subjects in accordance with American Thoracic Society criteria (VMAX; SensorMedics; Yorba Linda, CA), and the normal reference values used were those of Morris et al. Nonasthmatic subjects underwent methacholine challenge with doses ranging from 3 to 199 μmol using a nebulizer (model No. 646; DeVilbiss HealthCare; Somerset, PA) and a dosimeter (Rosenthal French; Baltimore, MD). Asthmatic subjects underwent methacholine challenge by the rapid method and received doses from 0.03 to 7.8 μmol administered with a hand-held nebulizer (model No. 45; DeVilbiss). The challenge was stopped in all subjects either after their FEV1 had decreased by > 20% of baseline values or after the final dose had been administered. Results were expressed as the log dose-response ratio (DRR).
Fiberoptic bronchoscopy (Olympus; Tokyo, Japan) was performed under IV sedation after premedication with IV atropine and local anesthesia of the upper airways half an hour before the procedure. BAL was performed in a subsegmental bronchus of the left lower lobe by infusing 50 mL of sterile 0.9% saline solution. BALF was separated into acellular and cellular components by centrifugation (400g for 10 min at4°C). The supernatant was further centrifuged (5,000g for 20 min at 4°C) to remove any debris and was stored at — 20°C in 1-mL aliquots for zymography.
The cell pellet was resuspended in phosphate-buffered saline (PBS) solution to its original BALF volume, filtered through nylon gauze (pore size, 60 |xm), then diluted 1:1 with 20 μL of Trypan blue for cell counting and viability assessment using a hemocytometer. To standardize cell numbers, after centrifugation (200g for 10 min at 20°C), PBS solution was added to the pellet to yield approximately 1 X 106 cells/mL. Seventy microliters of the cell suspension was added to a cytospin slide, spun at 5,000 revolutions per minute for 5 min and then stained (Diff Quick solution; Lab Aids; Sydney, NSW, Australia) for differential cell counting. At least 400 inflammatory cells were counted in each slide by two investigators (F.W.S.K. and C.D.) who were blinded to the clinical details of the subjects, and the average was used. The protein concentration of BALF was measured by calorimetric assay (DC protein Assay; Bio-Rad; Hercules, CA), with the spectrophotometer set at a wavelength of 655 nm (microplate reader model 450; Bio-Rad).
Zymography for MMP Activity
Gelatin zymography was used to assess the activity of MMP-9 in BALF as previously described. The supernatant was diluted two times with nonreducing loading buffer (400 mmol/L Tris-HCL, 5% sodium dodecyl sulfate, 20% glycerol, 0.006% bromo-phenol blue). Fifteen microliters of equal amounts of the sample (15 μL) were mixed with the loading buffer, and proteins were separated by polyacrylamide gel electrophoresis (0.75 mm; constant current 20 mA) consisting of an 8% sodium dodecyl sulfate solution with 1% gelatin (Bio-Rad). The gels were incubated in a renaturing buffer (2.5% Triton X-100 buffer) for 30 min to remove the sodium dodecyl sulfate. After rinsing, the gels were incubated (37°C for 20 h) in an enzyme activation buffer (50 mmol/L Tris-HCL [pH 7.3], 200 mmol/L NaCl, and 0.02% Tween 20). The gels were then stained with Coomassie brilliant blue R250 stain and destained (5% methanol, 7% acetic acid in PBS solution), and the gelatinolytic activity was detected as clear bands. The molecular weight of the gelatinolytic bands was estimated relative to the prestained molecular-weight markers (see BluePlus2 Prestained Standard; Invitrogen, Carlsbad, CA), and an MMP-9 standard (Quantikine; R&D Systems; Minneapolis, MN) was run in each gel as a positive control.
Zymograms were digitized (Kodak Digital Science Image Station 440; NEN Life Science Products, Inc; Boston, MA), and gelatinolytic activity (Fig 1) was measured using freeware (Image J, release p 4.0.2; Scion Corporation, Frederick, MD). The gelatinolytic activity was measured as the product of mean optical density multiplied by the area of digestion of the band inside a region of interest that was drawn around the clearly visible digested band (Fig 1). The mean of three measurements was used. Variations in Coomassie brilliant blue staining between different zymograms was standardized by using a single biological standard that had a high level of gelatinolytic activity and that was not from one of the study subjects (Fig 2). A 1:64 dilution of this sample was used in all zymographic assays as the common standard to adjust for small differences by reference to the standard curve in Figure 2 (closed circle).
Enzyme-Linked Immunosorbent Assay
Total MMP-9 and TIMP-1 were measured in BALF and serum by enzyme-linked immunosorbent assay (ELISA) [human MMP-9 and human TIMP-1; Quantikine; R&D Systems) according to the instructions of the manufacturer. The minimum detectable amounts of MMP-9 and TIMP-1 were 0.156 and 0.08 ng/mL, respectively.
Asthmatic patients were classified by an asthma severity score based on the National Asthma Council Guidelines (Table 1). For analyses, subjects were classified into the following four groups: AMH (defined by bronchoscopic appearance, regardless of asthma severity); severe asthma (severity score, 3); mild/moderate asthma (severity score, 1 to 2); or nonasthmatic. Zymographic data and BAL cell counts were reported as the median (interquartile range). The MMP-9 activity data were nonnormally distributed (Shapiro-Wilk coefficient, 0.68; p < 0.0001) due to the zero values; therefore, differences between clinical groupings were examined by Kruskal-Wallis test, and any significant differences were further analyzed by pair-wise comparisons using Mann-Whitney tests. The relationships between MMP activity and inflammatory cell counts, FEV1, and DRR were examined using the Spearman rank correlation test. All other data were presented as the mean (95% confidence intervals) with differences between groups examined using analysis of variance (ANOVA).
Figure 1. Zymograms showing gelatin digestion by pro-MMP-9 (92 kd) and active MMP-9 (85 kd). Lane 1, internal standard; lane 2, MMP-9; lane 3, normal; lane 4, mild asthma; lane 5, severe asthma; lane 6, AMH.
Figure 2. Standard curve for the standardization of image analyses between zymograms. The undiluted standard sample was assigned an arbitrary concentration of 100 U, and the percentage concentration was derived from the dilution (eg, 1:2 dilution = 50%). • = relative concentration used in all zymograms as the internal standard.
Table 1—Asthma Severity Score
Severity Score Symptoms Bronchodilator Use FEVj,% predicted Taking Oral Corticosteroids 1 < Once/mo < Once/mo > 80% No 2 a Once/mo but < daily > Once/mo but < 3 Times/d 60-80% No 3 Daily or nocturnal symptoms > once/wk a 3 Times/d < 60% Yes
