(1 to 5 mice per experiment) treated with PBS. In a second set of experiments, injected SCID mice were treated with Nissle WT, Nissle ΔclbA or Nissle ΔclbA+clbA every 3 d for 35 d (n = 9 per group, one experiment). Body weight and survival rate were measured daily after the induction of colitis. Immediately after sacrifice, the extent of colitis damage was assessed before tissue sampling. The macroscopic parameters analyzed were (ranging from 0 to 7): stool consistency (0 to 2 = formed to severe diarrhea), tissue adhe- sions (0 to 2 = absence to severe adhesions) and colonic damage (0 to 3 = normal to severe). A total score of 8 was assigned when animals died from colitis. Samples of distal colon were stored at -80°C until assayed for cytokine expression measurement. For RNA extraction, frozen colonic samples were homogenized and disrupted in buffer RLT (Qiagen) using Precellys beads kit (CK-MIX beads; Bertin Technologies). Tubes were shaken in a Precellys® 24 bead apparatus (Bertin Technologies) at 6,000 rpm for 2 cycles of 10 sec with interval of 10 sec. RNA was then extracted by using Qiagen kit, as recommended by the manu- facturer. RNA (1μg) was treated with DNase (Sigma) prior to cDNA conversion using iSript cDNA synthesis kit (Bio-rad). qRT-PCR assays (see Table S2) was performed using IQ SYBR Green Supermix (Bio-rad). Reactions were performed on a Bio- Rad CFX96TM. For cytokine analysis, samples were normalized to the housekeeping gene hypoxanthine guanine phosphoribo- syl transferase 1 (HPRT) for intersample variation. Normalized fold expressions analyzed using CFX-manager software (Bio-rad) were done to PBS-treated colitis mice. Histology. Rat or mouse colonic tissues fixed in Duboscq- Brazil were embedded in paraffin, sectioned (7-μm slices) and stained with Hematoxylin and Eosin (H&E). Colonic damage were graded semi quantitatively in blinded fashion by one or two of the authors (I.M.; C.C.). C.C. was blinded to the experimental protocol used. Each of 5 histological parameters was scored as absent (0), mild (1), moderate (2), or severe (3): density of inflammatory cells, epithelial injury, edema, crypt abscess and epithelial hyperplasia. Histological score was the sum of these parameters. Statistical analysis. Results are expressed as mean ± SEM or median unless otherwise stated. For in vivo experiments, N refers to the number of animals per group used for each experi- ment. Statistical significance of differences between experimen- tal groups was performed using one-way or two-way analysis of variance (ANOVA) with Bonferroni multiple-comparison post- test, or Kruskal-Wallis non-parametric ANOVA with Dunn’s multiple-comparison post-test as apppropriate (Prism 5, Graph Pad Software). Two-side analyses were used throughout and p values < 0.05 were considered statistically significant. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Acknowledgments We thank the staff of the Cytometry platform of the Centre de Physiopathologie de Toulouse-Purpan for excellent technical help. This research was supported by grants from the non-profit Association F. Aupetit (AFA) and the French National Research Agency (ANR-09-MIEN-005-01). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Supplemental Materials Supplemental materials may be found here: ht tp : //w w w.la ndesbioscience.com /jou rna ls /g ut microbes / article/21737/ References 1. FAO/WHO. Food and Agricultural Organization / World Health Organization. Report -Health and Nutritional Properties of Probiotics in Food Including PowderMilk with Live Lactic Acid Bacteria. Cordoba, Argentina 2001. 2. Sanders ME, Levy DD. The science and regulations of probiotic food and supplement product label- ing. 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