Differential immune-stimulatory effects of LTAs from different lactic acid bacteria via MAPK signaling pathway in RAW 264.7 cells.

OBJECTIVE: Lipoteichoic acid (LTA) is an immune-stimulatory component found in the cell wall of lactic acid bacteria, which are a major group of Gram-positive bacteria known to have beneficial health effects in humans. In this study, we evaluated the stimulatory effects of LTAs isolated from different lactobacilli species with mouse macrophage RAW 264.7 cells. METHODS: RAW 264.7 cells were stimulated with pLTA (isolated from Lactobacillus plantarum K8), rLTA (isolated from Lactobacillus rhamnosus), dLTA (isolated from Lactobacillus delbreukii), and sLTA (isolated from Lactobacillus sakei K101). Tumor necrosis factor (TNF)-α and interleukin (IL)-10 production were examined by ELISA, and nitric oxide (NO) production was assayed using Griess reaction. The mRNA and protein expression levels of inducible nitric oxide synthase (iNOS) was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Signaling molecules were also examined by Western blotting. RESULTS: pLTA and rLTA moderately induced TNF-α, IL-10, and NO production compared with stimulation of RAW 264.7 cells with dLTA and sLTA. Similar results were obtained for the mRNA and protein expression levels of iNOS. Western blot analysis showed that treatment of cells with pLTA or rLTA resulted in minimal phosphorylation of ERK, JNK and p38 MAPK while, dLTA and sLTA were strong activators of MAPK signaling. In addition, the glycolipid structure of LTAs was found to be composed of different fatty acid chain groups and lengths. Taken together, these results suggest that the differential immuno-stimulatory effects of LTAs isolated from different lactobacillus species may be related to their different ability to activate the MAPK signaling pathway, which are modulated by a unique glycolipid structure of LTA.

Qualitative and quantitative comparison of N-glycans between pig endothelial and islet cells by high-performance liquid chromatography and mass spectrometry-based strategy.

N-glycan structures released from miniature pig endothelial and islet cells were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), negative ion electrospray ionization (ESI) MS/MS and normal-phase high performance liquid chromatography (NP-HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N-glycans including 129 sialylated and 18 alpha-galactosylated glycans from pig endothelial cells and 80 N-glycans including 41 sialylated and one alpha-galactosylated glycans from pig islet cells. The quantity of the alpha-galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc-terminated N-glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using alpha 1,3-galactosyltransferase gene-knockout (GalT-KO) pig.