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During the magister project the student has studied the symbiotic combination between Cellobiose a dietary fiber and two probiotic candidates (published data: Amar et al, 2012). The influence of this potential prebiotic on the bacterial growth, secretion of inhibiting substances and exopolysaccharides (EPSs) production has been assessed. Data revealed that cellobiose exerted selective growth stimulation on tested probiotic strains, and reduced on the other hand pathogens development. The mechanism of antagonistic activity was strain dependent, however in the presence of the prebiotic the production of both acidic secretions and bacteriocins were increased compared to the control resulting in more efficient anti-microbial activity. We also observed a significant increase in the probiotic exopolysaccahrides production by this fiber, which may contribute to ameliorate their adherence properties. Data indicated that cellobiose might be used as prebiotic or symbiotic in association with probiotic micro-organisms. In a subsequent study the possibility of improving adhesion properties of probiotic strains using various carbon sources has been evaluated (Submitted paper). Results showed that the nature of saccharidic substrate strongly influenced the adhesion and aggregation abilities of four potential probiotics. We observed also an association between adherence and auto-aggregation in the presence of pectin, glucose, lactose and saccharose. Data indicated also that carbon substrate exerts a major influence on the exopolysaccharides production. The highest EPSs levels obtained with S.thermophilus in the presence of lactose (807mg/L) and L. rhamnosus in the presence of saccharose (680mg/L) were associated with an elevated adhesion capacity (86.21% and 96.74% respectively). At the light of this study it appears possible to improve the adhesion properties of probiotic strains through a pre-incubation with a suitable carbon source. During the PhD project, the applicant has studied the anti-inflammatory activity of Lactobacillus. reuteri strains on monocyte derived dendritic cells in response to various bacterial stimuli (L. acidophilus NCFM, S. aureus 43300, E. coli 10536, P. aeruginosa 25853 and LPS) (published data: Amar et al, 2015). The immune modulatory effect was assessed in terms of maturation markers (CD80, CD83 and CD86), cytokine production (IL-10, IL-12) and signal transduction (p38, ERK1/2). Interestingly, when weakly IL-12 inducing L.reuteri 5289 and IL-12 inducing strains were mixed; this probiotic efficiently inhibited strong IL-12 production in DCs, with a concomitant increase in IL-10. L.acidophilus NCFM induced almost the same amount of IL-10 when seeded with DCs alone or in combination with L.reuteri while IL-12 production was only abrogated in the presence of the probiotic. Therefore, it seems that IL-10 is not the only element responsible of the down regulation of IL-12 production. Data revealed that Gram negatives and LPS were higher inducer of maturation markers with no correlation with cytokine production. However, the tested probiotic was only able to down regulate the expression of CD83 in parallel stimulation of DCs with LPS. Concerning signal transduction, our data showed that, upon L. reuteri 5289 stimulation, p38expression was earlier down regulated compared to L. acidophilus stimulation, while ERK1/2 pathway phosphorylation lasts longer when L. reuteri 5289 was added to L. acidophilus, suggesting that it might be responsible for the inhibition of IL12 production. There-fore, our data suggest that IL-12 production by L. acidophilus NCFM could be sustained by prolonged p38 signalization, while ERK 1/2pathway activation is associated to IL-12 inhibition mediated by L.reuteri 5289. Image stream analysis revealed that tested probiotic possesses high adherence capacity to DCs surface compared to other tested bacteria. This higher affinity could be favorable for a faster interaction with DCs enabling to switch the immune balance toward an anti-inflammatory response when seeding the probiotic with pro-inflammatory strains. Given the observed immunosuppressive effects of L. reuteri 5289 in co-culture with either Gram positive or Gram negative bacteria, L. reuteri 5289 might represent a potential treatment effective to modulate the pro-inflammatory intestinal milieu such as the one observed in Crohn pathology. Improved understanding of the immune regulatory mechanisms evoked by probiotics is crucial for a more precise design of efficient bacteria-mediated interventions in inflammatory bowel diseases or other immune-mediated diseases as well as for their employment as adjuvant to enhance the effectiveness of DC vaccines in treating cancer. Further studies are needed to identify the active bacterial molecules beyond the observed inhibitory effects induced by L. reuteri. This work has been supported by an Erasmus Munsdus Nostrum Eumare grant (204195-EM-1-2011-1-ES-ERAMUNDUS-EMA21).
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