Table 2 Target genes and sequence of the primers used. Table 2
Related Figures (7)
Probiotic strains tested. Table 1 Fig. 1. Expression of a reporter gene under control of inducible NF-KB promoter in activated transgenic Caco-2 cells, inflammatory activated and treated by probiotics during 6 h. The results are expressed as the ratio between assay and control obtained with cells stimulated and untreated with probiotics. The results are given as average values of three differen experiments + standard deviation. *Different from control untreated by probiotics, P<0.05. None of the tested strains or GOS alone induced se cretion of IL-8 or NF-KB activation when they were co-cultured with HT-29 cells without any activation (data not shown). Table 3 shows the variation values of both secreted IL-8 and activated NF-KB in inflammatory stimulated HT-29 cells treated by probiotics in com parison with the untreated control. By considering the parameters monitored, different groups can be distinguished. Three of the five bifidobacteria (B. bifidum LMI 02 and LMI 20, B. pseudocatenulatum LMI 14), Lactobacillus helveticus ROO52 and L. lactis R1058 ind uced a dramatic reduction of both secretion of IL-8 and activation of NF-KB with a more reproducible effect on IL-8 secretion. The second group including Bifidobacterium breve ROO70, B. longum RO175, t hree lactobacilli (L. farciminis CIP 103136, Lactobacillus plantarum R1012, Lactobacillus When tested alone, GOS did not induce any significant variation of proliferation. 20% of inhibition of proliferation was considered as the limit for the effects to be considered significant because of the poor * Different from untreated by probiotics control, P<0.05. IL-8 and NF-kB relative quantification in HT-29 cells inflammatory activated and challenged with probiotics. The results are expressed as ratio between assay and control realized with cells inflammatory stimulated and untreated with probiotics. The results are given as average values of three different experiments + standard deviation. Fig. 2. Variations of HT-29 cell proliferation after a treatment by combined probiotics associated with GOS or glucose. The results are expressed as a ratio between assay and contre performed with untreated cells. The results are given as average values of three different experiments + standard deviation. “Different from negative control, P<0.05. Fig. 3. Dose range effect of MOI of B. breve ROO70 and L. lactis R1058 or OA concentration on HT-29 cell proliferation expressed as percentage of cell proliferation in comparison to untreated control. The results are given as average values of three different experiments + standard deviation. *Different from negative control, P<0.05. For further experiments, we focused on the B. breve/L. lactis/OA association because of the proliferation inhibition values and specific GOS effect, which could lead to synbiotic applications. In an attempt to determine whether bacteria and/or OA were involved in the anti- proliferative effects of the B. breve/L. lactis/OA association, dose range assays were conducted. Fig. 3 gives the HT-29 cell proliferation variations at different multiplicities of infection (MOI) of the combined bacteria in the presence of OA at 10 g/Lor at different OA concentrations in the presence of combined bacteria (MOI=100). For a constant concentration of OA at 10 g/L, inhibition of proliferation was signifi- cantly reduced for MOI 1000 and 100 (at MOI 1000 the observed effect may have been due to cytotoxic effects induced by bacterial acidification of the medium). Proliferation inhibition decreased from MOI 1000 to MOI 10 and no influence on proliferation was observed for MOI 1 and MOI 0.1. When OA was added at a final concentration of 10 g/L, the anti- proliferative effects were correlated to the MOI value with a loss of cell growth reduction for MOI<10. For a constant MOI=100 the anti- The strongest and most reproducible effect was observed with B. breve ROO70 and L. lactis R1058 in presence of OA, with an inhibition value of 66.31% +/— 2.05%. For the other probiotic mixtures, there were no significant differences between OA and OD for a given probiotic mixture. All the other probiotics/glucose or synbiotic combinations exerted lower and less reproducible proliferation inhibitions. The combination of B. breve ROO70, L. rhamnosus ROO11, L. lactis R1058 had lower but still significant effects for any carbon source. L. rhamnosus ROO11 and L. lactis R1058, when co-cultured with cells on glucose as the sole carbon source, reached the same level but with less reproducibility. B. breve ROO70 and L. rhamnosus ROO11 did not reduce HT-29 proliferation in presence of glucose and inhibited Fig. 4. Real time RT-PCR analyses of human intestinal alkaline phosphatase expression. The data are normalized to {$-actin expression and displayed relative to control. The results are given as average value of three different experiments + standard deviation. “Different from negative control, P<0.05. Legend: +++ inhibition of more than 50%, ++ inhibition of more than 20%, + inhibition reaching 20%, NS no significant result, ND not determined Summarised results from the inflammation and proliferation models. Table 4
