Subjects
This randomized, parallel group proof of concept study was approved and monitored by the UAB Institutional Review Board. Subjects were recruited from the University of Alabama at Birmingham (UAB) Lung Health Center’s Asthma Clinical Research Database from March 2009 through June 2011. Adults with mild-moderate persistent asthma (as defined by the NAEPP guidelines [13]) with at least a 12% FEV1 reversibility were eligible for enrollment. A physician diagnosis of asthma and documentation of reversible airflow obstruction was utilized to exclude patients with other causes of dyspnea. Individuals who underwent aerobic exercise regularly (3 or more times per week for more than 20 minutes at a time) during any of the six months prior to the study were not eligible for enrollment in order to facilitate the examination of exercise-mediated effects on asthmatic responses. In addition, individuals who smoked within 6 months from the start of the exercise protocol or with greater than a 10 pack year smoking history were excluded in order to exclude patients with chronic obstructive pulmonary disease (COPD). Individuals with other major illnesses, including coronary artery disease, congestive heart failure, stroke, severe hypertension, immunodeficiency states, or other conditions that would have interfered with participation in the study or with the proposed measurements were not eligible. In order to facilitate high adherence and data collection rates, individuals who were unable or unwilling to provide consent, perform the exercise protocol, provide pre- and post-study measurements, be contacted via telephone, or who intended to move out of the area within 6 months from the start of the study were excluded. Figure 1 illustrates the number of subjects screened and enrolled in the study.
Interventions
Subjects were recruited by the study coordinator and randomly assigned to either usual care or usual care with moderate intensity aerobic exercise treatment groups (Figure 1). Permuted variable size block randomization developed by the biostatistician was used to allocate subjects to the two study arms. The variable block size prevented exact knowledge of the next randomization assignment while maintaining equal allocation of subjects to the study arms throughout the study. All subjects were provided with two zippered pillow encasements and one mattress protector (Royal Heritage). These items were membrane free materials with a pore size less than 5% to reduce subject exposure to dust mite and dander in their bedding. In addition, study subjects randomized to the moderate intensity aerobic exercise group received a 3 month free membership to a local exercise facility at the time of the initial visit. This allowed the study subject a secure and safe environment in which to perform the exercise protocol. This strategy was designed to reduce adverse events associated with allergen exposure as well as prevent drop outs due to difficulty obtaining a location within which to perform the exercise protocol.
Usual care asthma education
All subjects received a brief (approximately 30 minute) coordinator-led educational intervention at the UAB Lung Health Center. Educational content focused on: i) the role of inflammation in asthma, ii) allergens that can trigger airway inflammation, iii) tips for avoiding or reducing exposure to triggers categorized as allergens (dust, bedding, furniture, pollens, food allergies, animal dander, mold, cockroaches), iv) caring for pillow and mattress covers, and v) good health practice (getting eight hours of sleep a day, drinking plenty of fluids, relaxing, eating a balanced diet and reducing stress).
Moderate intensity aerobic exercise
Those randomized to the moderate intensity aerobic exercise group completed a 12-week exercise training program at a frequency of 3 times per week, 30 minutes each session, at a steady-state intensity that achieved 60 – 75% of maximum heart rate (HRmax). In order to determine each subject’s HRmax and fitness level, subjects performed a mandated graded treadmill test to volitional fatigue using a modified version of the Bruce protocol [14]; this test was performed at the UAB Clinical Exercise Facility. Subjects’ fitness levels were measured in the same manner at the conclusion of the 12-week intervention. Because subjects were using medications which may have influenced heart rate (such as bronchodilators), we utilized the graded treadmill test to allow us to measure subjects’ true maximum heart rates while taking their usual medications; Ratings of Perceived Exertion were recorded throughout the treadmill test. Maximal oxygen uptake in one minute (VO2max), as measured with a metabolic cart, was accepted as accurate if at least 2 of 3 physiologic criteria were met: leveling off of VO2 with increasing workload, respiratory exchange ratio (RER) > 1.15, and heart rate equal to age-predicted maximum. The target heart rate range was then calculated for each subject.
Recommended exercise prescription included a 5 minute warm-up, 30 minutes of steady state exercise via walking, and a 5 min cool-down; thus total exercise time was 40 minutes per exercise bout. Compliance with this prescription was verified via heart rate monitor readings as described below. The exercise program was performed at the UAB Recreational Center and was completed in conjunction with standard patient education described above.
Subject visits
Subjects made three clinic visits to the UAB Lung Health Center. At the initial visit and prior to the start of the exercise protocol, all subjects underwent a complete physical with a board certified pulmonologist to ensure that the subjects were able to tolerate the exercise regimen. In addition, subjects completed health history and physical activity history questionnaires and documented asthma exacerbations at the initial visit. ECGs (12-lead) were used to permit safety monitoring of any previously un-diagnosed heart ailments and as part of the exercise testing for the subjects randomized to the exercise group. Lung function measurements and sample collection procedures (described below) were conducted at pre- and post-study intervals.
Exercise monitoring
Subjects randomized to the exercise treatment group were monitored for adherence to the exercise prescription. Throughout the study, aerobic exercise subjects were asked to wear a Polar Heart Rate Monitor (model 625X), which stores relevant exercise history information, including heart rate target zones, exercise duration in target zones, average heart rate, maximum heart rate, and total exercise time. Staff instructed subjects in the use of the heart rate monitor at the initial visit. Stored information, including length of the exercise session and average target heart rate during the exercise session, was downloaded onto a computer at the post-study visits. In addition, subjects kept a weekly exercise diary, which included the frequency of exercise, asthma-related symptoms and exacerbations, the use of pillow and mattress covers, and good health practices. Subjects submitted the exercise logs to the study coordinator at the post-study visits. Sign-in logs from the participating fitness center were monitored weekly in order to verify physical activity logs of subjects randomized to the exercise group. Subjects were called each week to ensure they were recording the exercise activity and to encourage adherence. In addition, phone calls permitted investigator evaluation of any increases in asthma symptoms, other health problems that interfered with their exercise prescription, or problems with heart rate monitors. It also provided direct feedback about non-compliance.
It is possible that subjects in the exercise group may have exhibited improved asthmatic responses as a consequence of increased interaction with or attention from individuals at the fitness center. In order to control for this interaction / attention within the exercise group, individuals in the usual care group also received weekly phone calls from the study coordinator. During these brief phone calls, the study coordinator asked the subject how he/she was doing and if there was anything related to his/her respective program with which he/she needed assistance.
Outcome indicators
The primary outcome indicator for this study was serum ECP. ECP is a marker of eosinophil activation found in both lavage fluids and serum of asthmatics. It has been demonstrated to correlate with asthma exacerbations and worsening as well as the effectiveness of asthma-related therapies [1, 2]. Secondary outcome indicators included asthma control scores, airway and peripheral blood inflammatory markers (nasal lavage ECP, serum cytokines, peripheral blood immune cell populations), lung function parameters (FEV1, FEV1/FVC), and fitness measures (VO2 peak, HR peak, RER, total treadmill-time).
Sample collection
Subjects provided blood and nasal lavage samples at the pre- and post-study visits. The post-study visit was conducted approximately 24 hours after the last session of exercise and at the same time of day in order to minimize effects of circadian rhythms on sample content [4].
Blood draw
Peripheral blood (15 ml at each visit) was collected in apyrogenic, heparinized tubes (Vacutainer, Becton Dickinson). Serum was separated and peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Ficoll-Paque (Pharmacia). Serum samples were quick frozen in a dry ice bath and stored at −80°C until analysis; peripheral blood immune cells underwent immediate analyses.
Nasal lavage
Nasal lavage was performed with a disposable metered-dose pump filled with isotonic saline solution at room temperature [15]. Excessive mucus was first cleared by one spray of saline followed by a forceful exsufflation through the nostril. The same nostril was lavaged with 6 ml of the saline solution, which remained in the nasal cavity for approximately one minute and was then removed. Nasal lavage fluid (NLF) was then centrifuged to remove particulate matter and stored immediately at −80°C.
Pro-inflammatory mediator analyses
Cytokine (ECP, IL-1β, IL-4, IL-5, IL-6, IL-13, TNFα) and total IgE content in serum and NLF were measured via enzyme linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (BioSource).
Cell differential analyses
Differential cell counts were performed on cells derived from peripheral blood as described previously [4]. Cell viability was determined via trypan blue exclusion and cell types were differentiated using Wright-Giemsa stain (Dade Behring Inc.). Cell differentials were determined from at least 500 leukocytes using standard hematological criteria.
Asthma control
Subjects completed the Juniper Asthma Control Questionnaire (ACQ) at initial and post-intervention study visits. Asthma control was determined by the score on the ACQ [16]. This instrument integrates common indicators of asthma control, including use of bronchodilators, nocturnal symptoms, cough, activity level, and pulmonary function. It assesses the full range of clinical impairment that patients with asthma experience and is highly sensitive to small changes in asthma control that are clinically significant. Scores range from 0 to 6. Lower scores reflect better control, and a difference of greater than 0.5 between the pre-study score and the post-study score is considered clinically significant. Scores greater than or equal to 1.5 indicate poorly-controlled asthma with a positive predictive value of 0.88 [16].
Pulmonary function analyses
Lung function was evaluated via spirometry using a portable Multispiro spirometer (Creative Biometrics) according to ATS/ERS guidelines [17]. Three forced vital capacity (FVC) maneuvers were performed for each subject and predicted values (FEV1, FVC) were determined.
Statistical analysis
Baseline characteristics between sedentary and moderate intensity groups were compared. Given the small sample size of the pilot study, paired comparisons were made using Fisher’s exact test for nominal characteristics (gender, smoking, race) and Wilcoxon Rank Sum for continuous measures (age, FEV1, etc.). Given the longitudinal nature of the study and the repeated outcome measures per subject, repeated measures analysis of variance techniques were applied to examine changes over time and to determine if the changes in outcomes over time differed by groups. Because repeated measures analysis of variance assumes normally distributed outcomes, distributional properties of the residuals from the repeated measures analysis of variance models were examined. Across all outcomes, only minor deviations from the normality assumption were observed.