Here, we show that treatment with a sialidase significantly improved the capacity of both immature and mature MDDCs to phagocytose Escherichia coli. Namely, neuraminidase-treated DCs show increased gene transcription of proinflammatory and Th1-promoting cytokines and induce greater proliferative responses of T lymphocytes. Dendritic cells (DCs) play an essential role in immunity against bacteria by phagocytosis and by eliciting adaptive immune responses. Previously, we demonstrated that human monocyte-derived DCs (MDDCs) express a high content of cell surface α2,6-sialylated glycans. Mock-up vaccines contain an active ingredient for an influenza virus that has not circulated recently in human populations and thus mimics the novelty of a pandemic virus. Monocytes were cultured in complete RPMI-1640 medium supplemented with 1000 U/ml of recombinant human IL-4 (rhIL-4) and rhGM-CSF, for 6 days, to give rise to immature moDC. For purification of these recombinant proteins, the corresponding expression plasmids were transformed into BL21(DE3) pLysS, and single colonies were grown overnight in 10 ml of lysogeny broth with Cm15 Kan25.
To assay for oxidoreductase activity, the purified recombinant proteins were incubated in 100-μl reactions at 37 °C overnight with 1 mg/ml 2,7-anhydro-Neu5Ac or Neu5Ac in 20 mm sodium phosphate buffer, pH 7.5, in the presence 500 μm NADH. The clarified lysate was run through an immobilized metal affinity chromatography column to elute the C-terminal His6-tagged proteins, which eluted in sharp peaks with a single 500 mm imidazole step. These were further purified by size-exclusion chromatography with elution in 50 mm KPi, 200 mm NaCl, pH 7.8, dialyzed against assay buffer (20 mm Tris, 150 mm NaCl, 2 mm tris(2-carboxyethyl)phosphine, pH 7. For more information regarding sialic acid manufacturers take a look at the web page. 5), and finally concentrated to 0.5-1 mm for storage at 4 °C. After harvest, pellets were resuspended in equilibration buffer (50 mm KPi, 200 mm NaCl, 20% glycerol, 40 mm imidazole, pH 7.8) and disrupted with sonication. The structure was acquired from a crystal grown in the JCSG Plus screen (100 mm sodium citrate, pH 5.5, 20% PEG 3000). The diffraction experiment was performed on the I04 beamline at Diamond Light Source Ltd.
To characterize the reaction intermediate, a sample containing 3 mm 2,7-anhydro-Neu5Ac, 100 μm NADH, 15 μm RgNanOx, and 1 mm DSS-d6 in PBS/D2O was prepared and analyzed at 293 K. A full set of 2D NMR experiments, including HSQC (hsqcetgpsi), COSY (cosygpqf), TOCSY (mlevph) and TOCSY-HSQC (hsqcdietgpsi), was acquired to fully assign 1H and 13C signals of the intermediate. All 1D NMR experiments were performed using a Bruker Advance I 500-MHz spectrometer with a 5-mm PATXI 1H/D-13C/15N Z-GRD probe at 293 K. To follow the kinetics of the reaction and assess the position of deuteration, two samples containing 2 mm 2,7-anhydro-Neu5Ac, 100 μm NADH, and 60 μm RgNanOx were used, one in deuterated PBS buffer (PBS/D2O) and one in standard PBS buffer (PBS/H2O, containing 10% D2O for locking purposes). The resulting supernatants were loaded onto an AmaZon Speed ETD (Bruker) mass spectrometer and analyzed by direct injection in negative mode. Negative controls were included for each component of the experiment individually as well as a dye-only control well. In these cases, the negative fractions (CD14− PBMCs), obtained after monocyte isolation, were maintained in culture until mixed lymphocyte cultures with autologous moDCs. This allows maximization of the level of sialic acid produced in the culture medium.
In the above method, micro-organism can be culture in conditions comprising an exponential growth phase which starts with the inoculation of the fermenter and last until exhaustion of the carbon substrate (for example glucose at 17.5 g.L−1). It has not yet been possible to obtain well-diffracting crystals of any substrate analog complex of the protein. An expression cassette can be constructed for production of more than one protein. Desialylated MDDCs had a significantly more mature phenotype, with higher expression of MHC molecules and interleukin (IL)-12, tumour necrosis factor-α, IL-6 and IL-10 cytokines, and nuclear factor-κB activation. The relative mRNA levels were normalized against the arithmetic mean of the β-actin and GAPDH expression and calculated by the adapted formula 2−ΔCt × 1000, which infers the number of mRNA molecules of the gene of interest per 1000 molecules of the endogenous controls.31 ΔCt represents the difference between the cycle threshold of the target gene and that of the endogenous control genes. Twenty-four hours after transduction, cells were analyzed for luciferase expression with a luminometer. Cells were then cultured in RPMI-Glutamax-I™ supplemented with 5% (volume/volume) FBS, 50 μg/ml gentamicin sulphate (Cellgro, Mediatech Inc., Manassas, VA), 50 μm 2-mercaptoethanol (Invitrogen) and 10 ng/ml murine GM-CSF (R&D Systems) for 7 days.