(MRS) broth medium (Oxoid, Basingstoke, United Kingdom) at 37uC under anaerobic conditions for 18–24 hours. To obtain the CFS, the probiotic strain was grown anaerobi- cally at 37uC in MRS for 24 hours. The supernatant was obtained by centrifugation at 12,0006g for 10 min, neutralised to pH 7.0 with NaOH (1 N) and concentrated tenfold by lyophilization. Supernatants were sterilized by filtering through a 0.22-mm-pore- size filter (Minisart hydrophilic syringe filter; Sartorius Stedim Biotech GmbH, Goettingen, Germany) and storage at 220uC until use. The supernatant was added 7% v/v. The pathogenic strain was provided by the Spanish Type Culture Collection (CECT; Burjassot, Spain). The pathogen used in the current study was S. typhi CECT 725. The culture was propagated aerobically in tryptone soy broth (Panreac Quimica, Barcelona, Spain). For the experiments, the pathogen was cultured for 8 hours at 37uC in tryptone soy broth and then subcultured 1:500 in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) containing 10% foetal bovine serum (FBS; Gibco Invitrogen, Paisley, United Kingdom) at 37uC overnight. Cell Preparation DCs generated from umbilical cord blood CD34+ progenitor cells (haematopoietic stem cells) were supplied by the MatTek Corporation (Ashland, MA). These cells were seeded in 24-well plates in DC maintenance medium (DC-MM; MatTek) containing cytokines and antibiotics. Bacterial Co-culture and DC Stimulation Cell cultures were seeded in 24-well plates at a final concen- tration of 26105 DCs/well. For incubations, DC-MM was replaced with RPMI-1640 media. DCs were directly challenged by the addition of the probiotic live bacteria (107 CFU/ml) or CFS, the pathogen (106 CFU/ml) or both. Escherichia coli lipopolysaccharide (LPS; Sigma-Aldrich) at 20 ng/ml was used as a positive control. Negative-control cultures contained un- stimulated DCs. All of the plates were incubated at 37uC in a 5% CO2/95% air atmosphere for 4 hours. After incubation, the medium was removed and replaced with fresh DC-MM containing cytokines and antibiotics. After 20 hours, all of the plates were centrifuged at 2006g, and the culture supernatants were collected for cytokine analysis. The cells were collected for RNA extraction. The results shown are the mean 6 SEM of three independent experiments. Cytokine and Chemokine Quantification in Culture Supernatants IL-1b, IL-6, IL-8, IL-10, IL-12(p40), IL-12(p70), TNF-a, IFNc, MCP-1/CCL2, MIP-1a/CCL3, RANTES/CCL5, MDC/ CCL22 and TGF-b were measured with MILLIplexTM immu- noassays (Linco Research Inc., MO) using the Luminex 200 References 1. FAO/WHO (2001) Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. 2. Collado MC, Isolauri E, Salminen S, Sanz Y (2009) The impact of probiotic on gut health. Curr. Drug. Metab. 10: 68–78. 3. Lebeer S, Vanderleyden J, De Keersmaecker SC (2010) Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens. Nat. Rev. Microbiol. 8: 171–84. 4. Bongaerts GP, Severijnen RS (2005) Preventive and curative effects of probiotics in atopic patients. Med. Hypotheses. 64: 1089–92. 5. Ouwehand A, Nermes M, Collado MC, Rautonen N, Salminen S, et al. (2009) Specific probiotics alleviate allergic rhinitis during the birch pollen season. World. J. Gastroenterol. 15: 3261–8. Modulation of Human DCs by a Lactobacillus Strain system according to the manufacturer’s instructions. The results shown are the mean 6 SEM of three independent experiments. Reverse Transcriptase (RT) Reaction and Polymerase Chain Reaction (PCR) As previously described, DCs were stimulated with bacteria for 4 hours and collected 20 hours later. The DCs were lysed and total RNA was extracted using the RNAqueous Kit (Ambion, Paisley, United Kingdom) and treated with Turbo DNase (Ambion) according to the manufacturer’s recommendations. The RNA quality was verified with a Model 2100 Bioanalyser (Agilent, Santa Clara, USA) and the RNA concentration was determined using a RediPlate 96 RiboGreen RNA Quantitation Kit (Gibco, Invitrogen). Real-time RT-PCR analysis of the samples was performed using a Human TLR Signaling Pathway PCR Array (SABiosciences Corporation, Frederick, MD), which includes primer pairs specific for 20 genes involved in TLR- mediated signalling pathways: TLR1, TLR2, TLR3, TLR4, TLR5, TLR9, MYD88, TNF, IRAK-1, IRAK-4, TOLLIP, CASP8, IL-10, TAK-1, JNK, NFKBIA, NFKB-1, TBK-1, MAPK14 and IRF-3. The housekeeping gene GAPDH was used as a control. Briefly, cDNA was synthesised from total RNA with an RT2 First-Strand Kit (SABiosciences). The cDNA was then subjected to real-time PCR with an RT2 Real-time PCR SYBR Green/ROX Kit (SABiosciences) on an ABI Prism 7500 sequence detector (Applied Biosystems, Foster City, CA). The PCR conditions were 1 cycle of 95uC for 10 min followed by 40 cycles of 95uC for 15 s and 60uC for 1 min. The expression levels of the target genes were normalised to those of untreated DCs (control). The expression level of each gene was analysed with RT2 Profiler PCR Array Data Analysis software (version 3.4; SABiosciences). The changes in expression or activity levels were expressed as fold changes (Fc). These results reflect the fold increase relative to the control samples (untreated DCs). Statistical Analysis NCSS 2007 software (Kaysville, UT) was used for the statistical analysis. The differences in cytokine expression levels and gene expression between treatments were evaluated with the Mann- Whitney U-test. The results are presented as the mean + SEM of three independent experiments. All P values,0.05 were consid- ered to be significant and are indicated in the text with an asterisk compared to the controls. In addition, differences between DCs treated with Salmonella and Salmonella and the probiotic/CFS were also evaluated. P values,0.05 were considered to be significant and are indicated with a pound sign (#). Author Contributions Conceived and designed the experiments: AG CGLL. Performed the experiments: MBB SMQ. Analyzed the data: MBB. Contributed reagents/ materials/analysis tools: EM MJB FR. Wrote the paper: MBB AG. 6. Huang JS, Bousvaros A, Lee JW, Diaz A, Davidson EJ (2002) Efficacy of probiotic use in acute diarrhea in children: a meta-analysis. Dig. Dis. Sci. 47: 2625–34. 7. Borchers AT, Selmi C, Meyers FJ, Keen CL, Gershwin ME (2009) Probiotics and immunity. J. Gastroenterol. 44: 26–46. 8. Ohnmacht C, Pullner A, King SB, Drexler I, Meier S, et al. (2009) Constitutive ablation of dendritic cells breaks self-tolerance of CD4 T cells and results in spontaneous fatal autoimmunity. J. Exp. Med. 206: 549–59. 9. Kushwah R, Hu J (2011) Role of dendritic cells in the induction of regulatory T cells. Cell. Biosci. 1: 20. 10. Rizzello V, Bonaccorsi I, Dongarra` ML, Fink LN, Ferlazzo G (2011) Role of natural killer and dendritic cell crosstalk in immunomodulation by commensal bacteria probiotics. J. Biomed. Biotechnol. 2011: 473097. PLOS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e43197