158 Chapter 7 Supporting information Methods Study population and screening procedures Screening consisted of a questionnaire, physical examination, spirometry to determine baseline lung function and airway hyperresponsiveness (PC20 defined as concentration methacholine bromide required to achieve 20% fall in Forced Expiratory Volume in 1 second, FEV1), allergy tests and routine blood and urine investigation as described [1]. Inclusion criteria were intermittent-to-mild asthma according to criteria of the Global Initiative for Asthma, sensitization tot HDM confirmed by a positive skin prick test and a positive radioallergosorbent test, FEV1 higher than 70% of the predicted value, PC20 between 1.2 – 9.6 mg/ml (corresponding with increased airway hyperresponsiveness), no clinically significant abnormalities during physical examination, hematological and biochemical screening, controlled asthma defined as Asthma Control Questionnaire score <1.5, age 18-45 years, no smoking for ≥1 year, ≤10 pack years of smoking and not pregnant [2]. We enquired about the use of antibiotics in the three months prior to study participation. Two patients in the control group had used antibiotics versus no patients in the antibiotic group. This study was part of the CAST-study (C1-inhibitor in Allergic ASThma Patients, ClinicalTrials.gov identifier NCT03051698), in which the effect of C1-inhibitor or microbiota depletion on lung inflammation in allergic asthma patients was investigated [1]. The Medical Ethics Committee of the Academic Medical Center, Amsterdam, approved the study. All patients gave written informed consent. Study design and materials All patients collected a baseline fecal sample, which was stored at -20°C at home and transported to the study center for storage at -80°C within 24 hours. Patients allocated to the antibiotics group received oral broad-spectrum antibiotics (ciprofloxacin 500 mg q12h, vancomycin 500 mg q8h and metronidazol 500 mg q8h) for seven days. After a 36-hour wash-out period of the antibiotics, patients in the antibiotics group collected a second fecal sample which was stored similarly. The 36-hour interval between antibiotics and HDM/LPS challenge was chosen to avoid direct interference of the antibiotics with any of the measurements. Patients allocated to the control group received no antibiotic treatment. Inhaled corticosteroids were withheld at least 24 hours prior to the study day and long-acting beta-agonists were withheld at least 14 days prior to the study day. The study day was initiated by a pulmonologist who performed a segmental challenge using a fiberoptic videobronchoscope to instill saline in one lung segment (serving as control) and a combination of HDM extract and lipopolysaccharide (LPS) in the contralateral segment. HDM extract (50 biological units, Dermatophagoides pteronyssinus origin; Allergopharma, Zeist, the Netherlands) was combined with LPS (75 ng, from Escherichia coli; Clinical Center Reference Endotoxin, kindly provided by Anthony Suffredini, National Institute of Health, Bethesda, MD), thereby mimicking environmental house dust exposure [3]. Seven hours later, bronchoalveolar lavage fluid (BALF) was collected by the same pulmonologist during a second bronchoscopy as previously described [4]. Instillation of HDM/LPS by bronchoscopy did not induce any severe clinical symptoms in either group. One participant in the control group experienced fever after completing the second bronchoscopy and was one day admitted for observation. The fever was most likely caused by translocation of nasopharyngeal
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