Fig. 4

PCR detection of segmented filamentous bacteria in tissue biopsies of patients with Inflammatory bowel diseases. A Human cohorts screened for the presence of SFB.The analyzed samples included mucosal biopsies collected from three IBD patient cohorts enrolled in Barcelona, Paris and Munich. The Spanish (HSCT) cohort included patients enrolled in follow-up for CD following treatment with autologous hematopoietic stem cell transplantation (AHSCT). Ileal (n=44) and colonic biopsies (n=86) from CD patients were included in the analysis. The French (Biotherapy) cohort included longitudinally collected samples from CD and UC patients who are treated with anti-TNF and other biotherapy drugs and recruited in Paris. Ileal (n=43) and colonic biopsies (n=143) from CD and UC patients were included in the analysis. The German pediatric cohort is a longitudinal prospective study comprising children with IBD and were recruited in Munich. Ileal (n=72) and colonic biopsies (n=80) were included in the analysis. A positive control of DNA extracted from a sample positive for SFB positive based on 16S sequencing (Mali, provided by Pamela Schnupf) B Quantitative analysis of SFB abundance in tissue biopsies using two primer sets (published by Jonsson [29] and by Snel [54] and the newly designed primer set; unpublished data, Pamela Schnupf). Quantification of SFB in human tissue biopsies was performed using the MasterMix SensiFAST™ SYBR (BIOLINE) and 3 different primer sets: for_5‘-TGTGGGTTGTGAATAGCAAT-3‘, rev_5‘-GCGAGCTTCCCTCATTACAAGG-3’, for_5‘-AGGAGGAGTCTGCGGCACATTAGC-3‘ ,rev_5‘ TCCCCACTGCTGCCTCCCGTAG-3‘  and for,_ 5’-TGTAGGTTGTGAAGAACAAT-3’, rev_ 5’-GCGAGCTTCCCTCATTACAAGG-3’ ( modified primer sequences, unpublished data, Pamela Schnupf). D Gel electrophoresis showing  expected 200 bP PCR products in a selected subset of samples and no bands in the negative control.