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Materials and methods Intestinal epithelial cell culture The porcine small intestine epithelial cell line IPI-2I (ECACC 93100622) was established from the ileum of an adult boar (SLAd/d haplotype) [16]. IPI-2I cells were maintained in DMEM (Invitrogen, Cergy Pontoise, France) supplemented with 10% FCS (Sigma-Aldrich,Saint-Quentin, France), 4 mM L-glutamine (Invitrogen),insulin 10 μg/mL (Sigma-Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen). In all experiments, cells were cultured in 6-well plates (Falcon) to confluence. Before the addition of pre/probiotics and/or infection, cells were washed three times to remove antibiotics and culture media was replaced by DMEM media (Invitrogen) containing 4 mM L-glutamine (Invitrogen) and insulin 10 μg/mL (Sigma-Aldrich). Cells were used between passages 30-50 and periodically tested to avoid Mycoplasma contamination (MycoAlert® Mycoplasma Detection Kit, Lonza). Probiotic and prebiotic preparation Lyophilized Saccharomyces cerevisiae var. Boulardii (Scb,Biocodex, Laboratoires Montrouge, France) was rehydrated with 10 mL of DMEM and incubated for 30 min at 30°C. Then, yeast was counted with a Neubauer cell counter and methyl blue to exclude non-viable yeast. The yeast was added to the selected wells (multiplicity of infection, MOI = 3) and incubated overnight at 37°C and 10% CO2. Commercially prebiotic Salmosan® (patent WO2009/144070 A2, licensed by Industrial Técnica Pecuaria, ITPSA, Barcelona, Spain) contains more than 98% of β-galactomannan (βGM) which is a β-(1-4)-mannose backbone with branched galactose molecules (ratio galactose: mannose 1:4) [17]. Commercially prebiotic Salmosan® also contain less than 1% of other polysaccharides such as xylose, fructose, arabinose, glucose, galactosamine and fucose derived from the carob bean gum and the seed of the Ceratonia silliqua tree. This is product is industrially treated to become soluble in gut. βGM is was diluted in DMEM (1 mg/mL), homogenized and incubated 30 min at 37°C. Immediately before the infection, βGM was added to each well at 10 μg/mL. Host cell-pathogen assay Two pathogenic E.coli K88 (ETEC) strains were used in the host-pathogen assays. Initially, ETEC 56190 F4+ (K88ad, O8:K87:H19, LT+ and STb+) from INRA collection was used for cytokine and chemokine mRNA assays. For adhesion assays and cytokine protein secretion we used strain ETEC GN1034 F4+ (K88+, LT+, STa+ and STb+) provided by Dr Ignacio Badiola (Centre de Recerca en Sanitat Animal,CReSA, IRTA-UAB, Spain) which was more adherent to IPI-2I cells [18]. Both ETEC strains were preserved frozen in glycerol 15% at -80°C until their use. Before infection, 50 μL of ETEC were added to 20 mL of Luria-Bertrani media (LB) and cultured for 3-4 h at 37°C with 180 rpm rotational agitation (Multitron HT, Infors). For the infection, ETEC was used at exponential growth phase by determination of absorbance at 600 nm. ETEC was used at MOI = 10, previously determined by cytotoxic lactate dehydrogenase activity assay (Cytotoxicity Detection Kit Plus LDH, Roche). In vitro challenge lasted 3 h for gene expression and bacterial adherence studies or 24 h for supernatant cytokine determination. After host cell-pathogen co-culture, cells or supernatants were respectively sampled and stored until their analysis. |
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