2017-08-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/662703Background: Altered balance of the intestinal microbiota has been demonstrated in patient subsets with several GI disorders, including inflamatory bowel diseases (IBD). IBD are chronic diseases are believed to arise from the convergence of genetic, environmental, and microbial factors that trigger aberrant immune and tissue responses, resulting in intestinal inflammation. Recent evidence indicates that the intestinal microbiota playing a role in initiating, maintaining, and determining the phenotype of IBD. The main metabolic end products of undigested complex carbohydrates for bacterial fermentation in the colon are short-chain fatty acids (SCFAs). SCFAs are important energy sources and have diverse regulatory functions, including enhanced epithelial barrier function and immune tolerance. Some evidences showed it is beneficial treating patients with IBD by administration of SCFA or prebiotics that are known to enhance SCFA production. Much of the recent insight gained into host-microbiome interactions has been derived through the advancement of next generation sequencing approaches and metabolomics. A fecal sample provides the most direct information to study gut microbiota and its metabolites. However, inappropriate sample handling or storage will cause biases of studies. Rapid freezing to −80 °C, the commonly considered best-practice, is not feasible in many studies. There are no guidelines for fecal sample collection and storage on a systematic evaluation of the effect of time, storage temperature, storage duration in microbiomes studies. Aims: We want to evaluate whether OMNIgene.GUT kit or Stratec collection tube with stabilizer could be used in pre-analytic fecal samples storage for 16S rRNA analysis. We will evaluate fecal SCFAs level consistency when the samples are stored in different temperature and duration. We will apply the results to investigate the impact of gut microbiota composition and fecal SCFA on disease status and treatment response in patient with inflammatory bowel disease. Material and methods: This project is composed of four parts: Part 1. Fecal samples will be stored in OMNIgene.GUT kit, Stratec collection tube with stabilizer, and frozen in -80℃.With the exception of samples stored at −80°C, all samples were stored at ambient temperature for 72hrs or 7 days, and then frozen at − 20 °C until DNA extraction on day 14. We will assess faecal microbial composition by 16S rRNA sequencing. Part 2. Fecal samples will be subjected to a range of experimental conditions including four storage temperatures (Room temperature, 4 °C, −20 °C) and four storage durations at each of these temperatures (8 hr, 24 hr, 48hr). Another three aliquots stored in each experimental conditions will be stored at -80 °C for 1 week, 4 weeks and 12 weeks. Three fecal control aliquots will be immediately extracted. All samples will be sent for short chain fatty acids measurement and 16S rRNA sequencing. Part 3. We will recruit 50 fresh IBD patients or IBD patients with flare. All patients will provide fecal samples for fecal calprotectin, microbiota and SCFAs analyses at baseline, weeks 2 and 6 after the start of the therapy and every 12 weeks during the maintenance therapy. We will assess gut microbiome structure with 16S rRNA gene analysis and SCFAs level during (A) active disease and treatment-induced remission, and (B) during remission and diseases flare. Part 4. We will transplant the frozen adult human fecal microbiota (anti-TNF-α responder and anti-TNF-α non-responder) into germ-free C57BL/6J mice. The mice will be treated with dextran sodium sulfate (DSS) to induce colitis and we will evaluate the response of TNF-alpha inhibitors in mice.Gut microbiotashort chain fatty acidinflammatory bowel diseasestorage condition16S rRNA