156 Materials and Methods Study population Patients were included based on their participation in the String of Pearls initiative, a Dutch nationwide project for which detailed phenotypic information and biological materials are collected from patients with IBD.16 Patients were recruited from the outpatient clinic of the Department of Gastroenterology in theUniversityMedical Center Groningen (UMCG) and enrolledbetween 20102015. For this study, frozen fecal and serum samples were collected from patients with Crohn’s disease (CD) (n=34), ulcerative colitis (UC) (n=17) or inflammatory bowel disease unclassified (IBD-U) (n=4). For each patient, demographic and clinical information was collected, including age, sex, medication use (including biologicals), the Montreal disease classification, surgical history, and disease activity, all of which was assessed at time of sampling. Clinical disease activity was assessed using the Harvey-Bradshaw Index (HBI) for patients with CD and the Simple Clinical Colitis Activity Index (SCCAI) for patients with UC or IBD-U. The Montreal disease classification was recorded from their most recent visit to the outpatient clinic. Patients provided written informed consent for their participation in the study. This study was approved by the Institutional Review Board (IRB) of the UMCG (registered as no. 08/338) and was performed in accordance with the principles of the Declaration of Helsinki (2013). As controls, a cohort of 55 age- and gendermatched healthy individuals was included from the Northern Dutch Lifelines cohort study, from which only data on age and gender were recorded and who had self-proclaimed gastro-intestinal health. From these individuals, stool and serum samples were analyzed in a similar fashion as for those of patients with IBD. Sample preparation Fecal samples were diluted 50-fold by adding 0.25 grams of fecal sample to 12.25 ml filtered phosphate-buffered saline (PBS). Homogenized samples were centrifuged at 700 xg for 5 min to remove large particulates. The supernatant was stored in 1 ml aliquots at -20°C until further analysis. For 16S rRNA gene sequencing, DNA was extracted from fecal samples using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and following the exact same procedure as described previously.17 In addition, a bead beating cell lysis step was performed using a Precellys 24 homogenizer (Bertin Technologies, Aix-en-Provence, France) and glass beads (0.1 mm) at 5.5 m/s in three rounds of 1 min at room temperature. Magnetic-activated cell sorting (MACS) A 1 ml fecal sample aliquot (50-fold diluted) was centrifuged at 16,000 xg for 5 min, after which the bacterial pellet was dissolved in 1 ml PBS and centrifuged again. Subsequently, the bacterial pellet was resuspended in 100 μl autologous serum (100-fold diluted in PBS) and incubated on ice for 30 min. This suspension was added to 1 ml PBS/EDTA and centrifuged at 16,000 xg for 5 min. Next, the resulting bacterial pellet was resuspended in 500 μl Anti-Human IgG (γ-chain specific)- Biotin antibody (Sigma-Aldrich, St. Louis, MO, USA) which was 100-fold diluted in PBS/EDTA and incubated on ice for 30min. Opsonized bacteria were sorted using a kit for magnetic-activated cell sorting (MACS) (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) as follows: 1 ml PBS/EDTA Chapter 5
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