1. Volume 131, Issue 6, Pages 1812-1825 (December 2006)
Saccharomyces boulardii Inhibits Inflammatory Bowel Disease by Trapping T Cells in Mesenteric Lymph Nodes 
Guillaume Dalmasso, Françoise Cottrez, Véronique Imbert, Patricia Lagadec, Jean-François Peyron, Patrick Rampal, Dorota Czerucka, Hervé Groux 
Gastroenterology 
Volume 131, Issue 6, Pages (December 2006)
DOI: /j.gastro
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2. Figure 1
Saccharomyces boulardii treatment inhibits wasting disease and inflammation in the lymphocyte transfer IBD model. IBD was induced in SCID mice by injection of naïve CD4+CD45RBhi T cells. Groups of 5 mice were then fed daily with ranging doses of S boulardii as indicated. Mice were forced fed with S boulardii solution in 200 μL PBS. (A) Weight was monitored every week during 5 weeks. Results are expressed as percentage weight loss over time as compared with the initial weight of mice. Mean of 4 representative experiments is shown. (B) Groups of 5 mice were treated with 100 μg S boulardii daily starting from day 0, 7, 17, or 21 as indicated. The control group was fed with PBS alone. Weight was monitored every week during 5 weeks. Mean of 2 representative experiments is shown. (C) H&E-stained sections (original magnification, 20×) of colons from SCID mice reconstituted with CD4+CD45RBhi T cells and fed during 5 weeks with different doses of S boulardii as indicated. (D) Colon sections were taken at 5 weeks or 8 weeks as indicated and graded relatively to their inflammatory status as detailed in the Materials and Methods section, *P < .02, Mann–Whitney U test. Mean ± SD of 4 representative experiments is shown. (E) Colon portions from SCID mice reconstituted with CD4+CD45RBhi T cells and fed or not 100 μg daily S boulardii solutions were digested with collagenase/dispase to collect infiltrating cells. Cells were counted and stained with anti-CD4-FITC and anti-CD3-PE mAb, and absolute number of CD4+ T cells was calculated. Results are expressed as millions of cells per mice. Mean ± SD of 3 representative experiments is shown. *P < .05, Mann–Whitney U test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

3. Figure 2
Saccharomyces boulardii treatment inhibits NF-κB activation in colon. (A) Immunohistochemistry analyses NF-κB activation using anti-p65 antibodies. Colon sections obtained from control SCID mice (SCID), CD4+CD45RBhi T-cell-reconstituted mice (CD4+CD45RBhi), CD4+CD45RBhi T-cell-reconstituted mice treated with 100 μg S boulardii (CD4+CD45RBhi+S boulardii) were fixed with cold acetone and stained by an anti-p65 Ab revealed by an ABC kit. In CD4+CD45RBhi T-cell-induced colitis, p65 was detected mainly in infiltrated cells’ nuclei (arrows) but not in S boulardii-treated mice. (original magnification, 100×; the second magnification was obtained by digital zooming). (B) EMSA for NF-κB (left panel) and supershift assays (right panel) in reconstituted SCID mice. EMSA assay shows that NF-κB-binding activity was dramatically increased in CD4+CD45RBhi T-cell-reconstituted SCID mice. S boulardii inhibited NF-κB in a dose-dependent manner. Supershift and competition with unlabelled probe, nuclear extracts from CD4+CD45RBhi T-cell-restored mice served as control. Excess of unlabelled NF-κB probe completely shifted the protein-DNA complexes. Incubation with antibodies against p50, p65 subunits completely abolished the NF-κB-DNA complex.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

4. Figure 3
Saccharomyces boulardii treatment inhibits cytokine gene expression in the colon. (A) SCID mice were transferred with CD4+CD45RBhi T cells (CD4+CD45RBhi line) and fed with S boulardii daily in a dose-dependent manner as indicated. Untreated SCID mice (SCID line) and mice transferred with both CD4+CD45RBhi and CD4+CD45RBlo (CD4+CD45RBhi+lo line) were used as control. RT-PCR was used to measure mRNA levels for TNF-α, IL-1β, IFN-γ, IL-6, and GAPDH. Amplification of GAPDH serves as a control for sample loading and integrity. (B) Real-time RT-PCR was performed on the same group of mice as indicated (S boulardii was used at 100 μg daily). Results are plotted against an arbitrary unit obtained by the ΔCt measurement against the SCID group used as control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

5. Figure 4
IFN-γ-producing Th1 cells accumulated in mLN after S boulardii treatment. (A) IFN-γ production of colon and mLN T cells from SCID mice reconstituted with CD4+CD45RBhi T cells and fed or not with S boulardii (100 μg daily) during 5 weeks. Cells were activated with coated anti-CD3 (10 μg/mL) + anti-CD28 (1 μg/mL) during 48 hours. IFN-γ production was measured by ELISA in culture supernatants. One experiment out of 3 is shown. *P < .05 Mann–Whitney U test. (B) IFN-γ gene expression was evaluated in colon and mLN cells by quantitative RT-PCR in mLN cells isolated from SCID mice reconstituted with CD4+CD45RBhi T cells and fed or not with S boulardii. *P < .05 Mann–Whitney U test. (C) CD4+ T-cell count of mLN and spleen isolated from SCID mice reconstituted with CD4+CD45RBhi T cells and fed or not with S boulardii solutions during 5 weeks. *P < .05 Mann–Whitney U test. (D) CXCR3 and CCR5 gene expression was evaluated by quantitative RT-PCR in mLN cells isolated from SCID mice reconstituted with CD4+CD45RBhi T cells and fed or not with S boulardii. *P < .05 Mann–Whitney U test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

6. Figure 5
Comprehensive analysis of gene expression in colon and lymph nodes of SCID mice with colitis treated with S boulardii. Control SCID mice or SCID mice transferred with CD4+CD45RBhi T cells and treated with either CD4+CD45RBlo T cells or S boulardii (100 μg daily) were used. After a week, mice were killed, and the gene expression profile of their mLN and colons (as indicated) was analyzed by comprehensive RT-PCR analysis. Some examples of highly differentially expressed genes among the different groups are shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

7. Figure 6
S boulardii induces a redistribution of T cells within mLN of BALB/c mice. (A) Total cell count of mLN isolated from BALB/c mice fed or not with S boulardii (200 μg daily) during 1 to 8 days. (B) Total cell count of mLN and spleen from BALB/c mice fed or not with S boulardii (200 μg daily) during 4 days. **P < .005, *P < .01, Mann–Whitney U test. (C) Absolute CD4+ and CD8+ T cell counts in the mLN and spleen from BALB/c mice fed or not with S boulardii (200 μg daily) during 4 days. One experiment out of 3 is shown. *P < .05, Mann–Whitney U test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

8. Figure 7
S boulardii induces migration of T cells in mLN. (A) In vivo migration assay. CD4+ T cells from BALB/c mice fed or not with S boulardii (200 μg daily for 4 days) were labeled with different fluorescent probes and coinjected with CD4+CD45RBhi T cells into SCID mice fed or not with yeast (100 μg daily for 4 weeks). Twenty-four hours after injection, the number of fluorescent cells that have migrated within the mLN of SCID mice was evaluated by flow cytometry. *P < .05, Mann–Whitney U test. (B) Constitutive chemokines CCL19, CCL21, and CXCL12 gene expression was evaluated by quantitative RT-PCR in mLN cells isolated from BALB/c mice and fed or not with S boulardii (200 μg daily) for 4 days. Expression of chemokines was analyzed by RT-PCR on cells isolated from mLN collected daily for 4 days. One experiment out of 2 is shown. (C) Constitutive chemokines CCL19, CCL21, and CXCL12 gene expression was evaluated by quantitative RT-PCR in mLN cells isolated from SCID mice reconstituted with CD4+CD45RBhi and fed or not with S boulardii (100 μg daily) for 4 days. Expression of chemokines was analyzed by RT-PCR on cells isolated from mLN collected weekly for 4 weeks. One experiment out of 2 is shown. (D) Inflammatory chemokines CXCL9, CXCL10, CXCL11, CCL4, and CXCL5 gene expression was evaluated by quantitative RT-PCR in mLN cells isolated from SCID mice reconstituted with CD4+CD45RBhi T cells and fed or not with S boulardii (100 μg daily) for 4 weeks.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions

9. Figure 8
S boulardii induces T-cell rolling on endothelial cells. (A and B) Total spleen leucocytes (A) or in vitro differentiated Th1 cells (B) were perfused in a flow chamber on a layer of untreated (gray bars), S boulardii supernatant (open bars), or S cerevisiae supernatant (light gray bars) pretreated mouse endothelial cells. Rolling cells were enumerated for a total of 9 fields. *P < .005, Mann–Whitney U test. (C) Firm adhesion of leucocytes on untreated or pre-treated with S.b. supernatant or S.c. supernatant, was numerated S boulardii supernatant or S cerevisiae supernatant pretreated mouse endothelial cells were numerated after 10 minutes in flow chamber adhesion assay. *P < .05 Mann–Whitney U test. One experiment out of 3 is shown. (D) Blocking experiment using anti-LFA-1, anti-L-selectin (L-Sel, MEL-14 antibody), and anti-PSGL-1. In vitro differentiated Th1 CD4+ T-cell populations were preincubated with indicated antibodies, washed, and perfused in a flow chamber on a layer of untreated (gray bars) or S boulardii supernatant pretreated mouse endothelial cells. Rolling cells were enumerated for a total of 9 fields; firm adhesion of leucocytes was numerated after 10 minutes in flow chamber adhesion assay. *P < .05 Mann–Whitney U test. One experiment out of 3 is shown. (E) Rolling events of CD4+ T cells untreated or preincubated with anti-PSGL-1 as indicated, on slides coated with recombinant mP-selectin, were counted after video recording on 9 different fields. *P < .05 Mann–Whitney U test. One experiment out of 3 is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 AGA Institute Terms and Conditions