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008 -CCFC STUDENT PRIZE N Godwin, L Hyde, A Mackenzie, DR Mack Intestinal epithelial cell (IEC) homeostasis is maintained by protective mechanisms under genetic control in the gastrointestinal tract. We have previously reported that Lactobacillus rhamnosus GG upregulates IEC mucin expression, a supra-mucosal protective factor and this same strain is also reported to prevent cytokine-induced apoptosis. Probiotic modulation of mucin expression is not strain specific and other probiotic strains such as Lactobacillus plantarum 299v (Lp299v) also upregulate IEC mucin expression. The aim of this study is to assess whether Lp299v prevents apoptosis in vitro when faced with cytokine challenge. HT-29 cells were grown in glucose-free, galactose-containing medium to differentiate along a small intestinal lineage. Cells were incubated with Lp299v for one hour or Lactobacillus plantarum adh- a derivative of Lp299v that does not induce mucin expression. Following incubation, cells were washed then challenged with cytokine mixture (TNF-alpha, IFN-gamma, and IL-1a) for 15 minutes. To assess for apoptosis, we evaluated both TUNEL analysis and a caspase activity assay. Enumeration by fluorescent microscopy was used to determine effect, with results expressed as percentage of 200 cells counted in a chosen microscopic field. Results of the TUNEL assay demonstrate a marked decrease in apoptosis noted in cell populations treated with Lp299v prior to cytokine exposure (16% versus 93% cell apoptosis when cells are subjected to cytokine treatment in the absence of Lp299v pre-incubation). Consistent with this are counts deriving from caspase activity assays which parallel this improvement in IEC viability (8% apoptosis when cells are incubated with Lp299v and cytokines versus 46% cell death when in the presence of cytokines alone). L. plantarum adh- decreases the amount of apoptosis inflicted upon cells by cytokine challenge, as detected by both TUNEL (29%) and caspase assay (15%) however not as dramatically as Lp299v. Taken together, select probiotic strains modulate IECs to genetically determine innate protection through multiple mechanisms such as supra-cellular elaboration of protective compounds (i.e. mucins) and IEC homeostasis through inhibition of apoptosis. Effects of toxic inflammatory responses may be moderated by probiotics to enhance survival of mucosal cells and may provide enhanced mucosal barrier function.
Children's Hospital of Eastern Ontario Research Institute, University of Ottawa, Ottawa, Ontario
Partial funding received from Probi AB and Lallemand Inc