Volume 157, Issue 3, Pages e4 (September 2019)

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Volume 157, Issue 3, Pages 637-646.e4 (September 2019) Bifidobacterium breve Bif195 Protects Against Small-Intestinal Damage Caused by Acetylsalicylic Acid in Healthy Volunteers  Brynjulf Mortensen, Clodagh Murphy, John O’Grady, Mary Lucey, Gafer Elsafi, Lillian Barry, Vibeke Westphal, Anja Wellejus, Oksana Lukjancenko, Aron C. Eklund, Henrik Bjørn Nielsen, Adam Baker, Anders Damholt, Johan E.T. van Hylckama Vlieg, Fergus Shanahan, Martin Buckley  Gastroenterology  Volume 157, Issue 3, Pages 637-646.e4 (September 2019) DOI: 10.1053/j.gastro.2019.05.008 Copyright © 2019 AGA Institute Terms and Conditions

Gastroenterology 2019 157, 637-646. e4DOI: (10. 1053/j. gastro. 2019 Copyright © 2019 AGA Institute Terms and Conditions

Figure 1 Primary and secondary end points. (A) Mean Lewis score per visit. (B) The primary end point mean Lewis score AUC ± standard error of the mean per treatment arm. (C) Median number of ulcers per visit. (D) The secondary end point ulcer number AUC ± standard error of the mean per treatment arm. *P < .05. Effects sizes were (B) 30% lower AUC in the Bif195 arm and (D) 33% lower AUC in the Bif195 arm. Gastroenterology 2019 157, 637-646.e4DOI: (10.1053/j.gastro.2019.05.008) Copyright © 2019 AGA Institute Terms and Conditions

Figure 2 Tertile stratification of ulceration. (A, C, and E) Median ulcer numbers per visit and (B, D, and F) mean ulcer number AUC ± standard error of the mean from VCE stratified on small intestine tertiles (thirds of small intestine). ∗P < .05. Gastroenterology 2019 157, 637-646.e4DOI: (10.1053/j.gastro.2019.05.008) Copyright © 2019 AGA Institute Terms and Conditions

Figure 3 (A) Mean serum concentrations of PGE2 per visit and (B) AUC ± standard error of the mean. Mean serum concentrations of (C) TXB2 per visit and (D) AUC ± standard error of the mean. Gastroenterology 2019 157, 637-646.e4DOI: (10.1053/j.gastro.2019.05.008) Copyright © 2019 AGA Institute Terms and Conditions

Figure 4 Other secondary end points measured in the trial. AUC ± standard error of the mean (SEM) of the (A) pain module and (B) total score from in the GSRS questionnaire. (C) AUC ± SEM of blood I-FABP, (D) AUC ± SEM of red spots from VCE, and (E, F) AUC ± SEM of (E) fecal and (F) blood calprotectin. ∗P < .05. Gastroenterology 2019 157, 637-646.e4DOI: (10.1053/j.gastro.2019.05.008) Copyright © 2019 AGA Institute Terms and Conditions

Supplementary Figure 1 Enrollment and randomization of subjects according to the CONSORT Flow Diagram. Gastroenterology 2019 157, 637-646.e4DOI: (10.1053/j.gastro.2019.05.008) Copyright © 2019 AGA Institute Terms and Conditions

Supplementary Figure 2 Representative VCE data. Representative images obtained by VCE from 1 participant throughout the intervention period. All images were obtained from the first tertile of the small intestine. The pictures show (A) visit 2 with normal intestinal mucosa, (B) visit 3 with normal intestinal mucosa, (C) visit 4 with ulcer highlighted by blue circle, (D) visit 4 with villous edema, (E) visit 5 with ulcer highlighted by blue circle, (F) visit 5 with villous edema highlighted by blue circle, (G) visit 6 with ulcer highlighted by blue circle, and (H) visit 7 with normal intestinal mucosa. Gastroenterology 2019 157, 637-646.e4DOI: (10.1053/j.gastro.2019.05.008) Copyright © 2019 AGA Institute Terms and Conditions

Supplementary Figure 3 Boxplot showing the relative abundances of B breve in stool at visits 2 through 7. The box extends from the first quartile (Q1) to the third quartile (Q3), and the line within the box shows the median value. The lower whisker extends to the smallest value within Q1 – 1.5 × interquartile range (IQR), and the upper whisker extends to the largest value within Q3 + 1.5 × IQR. Values outside the whiskers are shown as circles. After unblinding, a post hoc laboratory and bioinformatic analysis was performed on DNA extracted from all obtained fecal samples by using a NucleoSpin 96 Soil kit (Macherey-Nagel, Düren, Germany) and randomly sheared into 350–base pair fragments. Libraries were constructed using NEBNext Ultra Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA) and sequenced to at least 30 million read pairs per sample (2 × 150–base pair paired-end Illumina sequencing). Sequencing reads were filtered to remove human and low-quality reads, mapped to the Clinical Microbiomics Human Gut 22M gene catalog, and summarized as a taxonomic relative abundance table, as described previously.21 The involved parties were kept blinded to the intervention during analyses. Changes in relative abundances of taxa between visit 2 and the integral of later time points was tested using the Wilcoxon rank-sum test and corrected for multiple comparison using a Bonferroni correction. Similarly, the Bray–Curtis distance between visit 2 and later time points were compared between the 2 arms (t test). V, visit. Gastroenterology 2019 157, 637-646.e4DOI: (10.1053/j.gastro.2019.05.008) Copyright © 2019 AGA Institute Terms and Conditions

Supplementary Figure 4 Boxplot showing the Bray–Curtis dissimilarity of stool microbial composition, comparing visit 2 with later visits (V3–V7). The box extends from the first quartile (Q1) to the third quartile (Q3), and the line within the box shows the median value. The lower whisker extends to the smallest value within Q1 – 1.5 × inter-quartile range (IQR), and the upper whisker extends to the largest value within Q3 + 1.5 × IQR. Values outside the whiskers are shown as circles. V, visit. Gastroenterology 2019 157, 637-646.e4DOI: (10.1053/j.gastro.2019.05.008) Copyright © 2019 AGA Institute Terms and Conditions