Effect of cell-surface components and metabolites of lactic acid bacteria and probiotic organisms on cytokine production and induction of CD25 expression in human peripheral mononuclear cells

In the current study, the relative contribution of cell-surface components (CSC) and cell-free supernatants (CFS) in the immuno-modulatory properties of 17 strains of probiotic and lactic acid bacteria (LAB) was assessed. The production of pro- and antiinflammatory cytokines including IL-2, IL-4, IL-10, IL-12 p70, IFN-γ, tumor necrosis factor-α (TNF-α), and transforming growth factor-β was measured at different time points after stimulation of buffy coat derived-peripheral blood mononuclear cells (PBMC) from healthy donors with CSC and CFS of probiotic and LAB. Results showed that CSC of probiotic and LAB strains induced production of T helper 1 and 2 type cytokines. Transforming growth factor-β was stimulated at highest concentrations, followed by IL-10 and TNF-α. The CFS of all tested bacterial strains induced PBMC for significantly high levels of IL-10 secretion compared with unstimulated cells, but the values were less than lipopolysaccharide-stimulated cells. Cytokines due to CFS stimulation showed declined concentration for IL-2, TNF-α, and IL-4, and complete disappearance of IL-12, IFN-γ, and transforming growth factor-β in the cultured medium at 96 h of incubation. Results of cytokine data demonstrate proinflammatory TNF-α immune responses are mainly directed through cell-surface structures of probiotic and LAB, but antiinflammatory immune responses are mediated both by metabolites and cell-surfaces of these bacteria. The induction of CD4(+)CD25(+) regulatory T cells after stimulation of PBMC with CSC and CFS of probiotic and LAB showed regulatory T cell activity appeared to be influenced both by the CSC and metabolites, but was principally triggered by cell surfaces of probiotic and LAB strains.

A tandem liquid chromatography–mass spectrometry (LC–MS) method for profiling small molecules in complex samples

Liquid chromatography–mass spectrometry (LC–MS) methods using either aqueous normal phase (ANP) or reversed phase (RP) columns are routinely used in small molecule or metabolomic analyses. These stationary phases enable chromatographic fractionation of polar and non-polar compounds, respectively. The application of a single chromatographic stationary phase to a complex biological extract results in a significant proportion of compounds which elute in the non-retained fraction, where they are poorly detected because of a combination of ion suppression and the co-elution of isomeric compounds. Thus coverage of both polar and non-polar components of the metabolome generally involves multiple analyses of the same sample, increasing the analysis time and complexity. In this study we describe a novel tandem in-line LC–MS method, in which compounds from one injection are sequentially separated in a single run on both ANP and RP LC-columns. This method is simple, robust, and enables the use of independent gradients customized for both RP and ANP columns. The MS signal is acquired in a single chromatogram which reduces instrument time and operator and data analysis errors. This method has been used to analyze a range of biological extracts, from plant and animal tissues, human serum and urine, microbial cell and culture supernatants. Optimized sample preparation protocols are described for this method as well as a library containing the retention times and accurate masses of 127 compounds.