Selection criteria for enrolment in the study were vaginal delivery at term and uncomplicated perinatal period. Questionnaires were collected with data on the Staurosporine cell line parents, including demography, smoking and asthma.

Data of the child on demography, respiratory symptoms and risk factors for asthma were collected by postal questionnaires sent every 6 months starting at the age of 3 weeks until the age of 36 months. The question on the presence of wheezing referred to the period between two questionnaires, e.g. the presence of wheezing in the questionnaire at 6 months referred to the time period between 3 weeks and 6 months. The study protocol was approved by the medical ethics committees of the participating institutes. All parents gave written informed consent. Symptoms of wheeze were assessed JAK activation by International Study of Asthma and Allergies in Childhood core questions [9]. Information about doctor’s diagnosed parental asthma was collected

by the following question: ”Did a doctor ever diagnose asthma?”. Based on the longitudinal questionnaire data on wheeze symptoms in the first 3 years of life, children were classified according to the ‘loose’ Asthma Predictive Index (API) into an API positive and an API negative group. According to the ‘loose’ index a positive API included wheezing during the first three years of life and eczema or parental history of asthma [10]. Approximately 2 g of stools was collected into a sterile recipient by the parents at 3 weeks of age. The sample was sent to the laboratory under anaerobic conditions where it was stored immediately at -70°C until analysis. DNA was extracted from fecal samples based on the method of selleck compound Pitcher et al. [11] modified by Vanhoutte et al. [5]. A saline suspension of feces was made by diluting Mirabegron 1 g of wet feces in 10 ml of sterile saline solution and homogenized using a stomacher. Of this fecal sample suspension, 1 ml was centrifuged at 20,000 g for

5 min. After removal of the supernatant, the pellet was resuspended in 1 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and was again centrifuged at 20,000 g for 5 min. The pellet was resuspended in 150 μl enzyme solution (6 mg lysozyme powder [Serva] and 40 μl mutanolysine [Sigma] dissolved in 110 μl TE (1 ×) per sample) followed by incubation at 37°C for 40 min. Next, 500 μl GES reagent (Guanidiumthiocyanate-EDTA-Sarkosyl; 600 g l-1 guanidiumthiocyanate [Sigma], 200 ml l-1 0.5 M EDTA, 10 g l-1 sarkosyl) was added to complete all lysis, after which the solution was put on ice for 10 min. In the following step, 250 μl ammonium acetate (7.5 M) was added and the mixture was put on ice for 10 min. Subsequently, two chloroform-iso-amylalcohol extractions were performed with 500 μl chloroform/iso-amylalcohol solution (24/1). Finally, DNA was precipitated by adding 0.54 volumes of ice-cold isopropanol.