A C16:0 moiety. c Lowering terminal Y-type LA fragment ions of intact LA. As an example, fragment ions of LA composed of GlcN-GlcN (two amides) are presented.JOURNAL OF BIOLOGICAL CHEMISTRYC. jejuni LOS-TLR4 InteractionsFIGURE 3. Effect of C. jejuni LOS sialylation, amide linkages, and phosphorylation on TNF induction in THP-1 cells. THP-1 cells have been stimulated with ten ng/ml LOS for 20 h, and TNF levels have been assessed by ELISA. TNF levels have been compared using the amount of sialylation with the LOS from individual strains (A), levels from neuraminidase-treated LOS (N-treated; B), the relative abundance of 4 amide linkages (C), as well as the relative abundances of three or four phosphoryl moieties based on the locations in the respective LA peaks for every molecule (D). Information points represent imply values for an individual strain from a minimum of four independent experiments. A, one-way analysis of variance (*, p 0.05). B, unpaired Student’s t test evaluation (ns, not considerable). C and D, linear regression analyses have been performed.DPLA with a single PEA moiety for strain 33106 (calculated m/z 2004.6) with an extra fragmentation causing loss of H3PO4 (98 Da; calculated m/z 1906.six; Fig. 2H). For KJCattle8, the most abundant peaks had been indicative of DPLA lacking PEA (calculated m/z 1881.6 with additional loss of H3PO4 at m/z 1783.six Da; Fig. 2I). Minor peaks in other spectra included these consistent with fragment ions for two PEA-substituted DPLA at a calculated m/z of 2127.7 with ions observed for more loss of H3PO4 at m/z 2029.ETZ 7 and 3 PEA-modified DPLA at m/z 2250.SP-13786 7 with ions for added loss of H3PO4 at m/z 2152.7 (data not shown). As illustrated within the intact LOS spectrum from strain KJCattle8 (Fig. 2I), fragment ion peaks have been constant with PEA DPLA with 1 hydroxylaurate acyl group rather of a hydroxymyristate ( 28 Da; observed m/z 1973.six). We confirmed the presence or absence of variation in acyl chain length by evaluation from the O-deacylated spectra when assigning LA composition to the similar ions: DPLA (calculated m/z 1881.six) and C14 PEA DPLA with loss of H3PO4 (calculated m/z 1878.two). To evaluate the level of phosphorylation between the diverse strains, the abundance of tri- (2P PEA) or tetraphosphorylated (2P 2PEA) LA fragment ions was calculated relative towards the fragment ions of all the LA species for each and every strain (Fig. 2J). While wide diversity within the degree of phosphorylation was noted (percentage of tri- or tetraphosphorylated LA ranged amongst 26.PMID:24456950 three and 87.5 ; mean, 65.four ; normal deviation, 19.0 ), this did not correlate with the phylogenetic cluster.Moreover, the clinical presentation of an individual strain did not correlate with all the phosphorylation status (information not shown). TNF Expression by THP-1 Cells and Major Human Monocytes–To assess the influence of LOS modifications on innate immunity, TNF expression was selected as a marker for LOS-mediated TLR4 activation. TNF is actually a effectively characterized proinflammatory cytokine developed by monocytes in response to LOS/LPS via TLR4-MyD88-dependent signaling and serves as an accurate marker for the induction of other proinflammatory cytokines including IL-1 , IL-6, and MIP-1 in response to TLR4 activation (24, 25, 30, 31). Phorbol 12-myristate 13-acetate-differentiated THP-1 cells have been stimulated with purified LOS (ten ng/ml) for 20 h prior to cytokine analysis. Important TNF induction in response to all purified LOSs was observed. Importantly, a statistically substantial cor.
