In some experiments the bacterial surface was desialylated with 13· In the earlier set of experiments that utilized neuraminidase, we noted that, following neuraminidase treatment, there were typically fewer cells in the dish, indicating that cells had lost cell-cell and/or cell-matrix adhesions. Sialic acid quantitation was carried out using the Sialic Acid (NANA) Assay kit from Biovision (Mountain View, CA) following the manufacturer’s protocol. For those who have almost any concerns regarding exactly where and also how to use sialic acid manufacturers (my company), you are able to call us at the site. Briefly, a 15-cm dish of HEK 293 cells (ca. 2 × 107) was transfected using the Superfect reagent (QIAGEN, Valencia, CA) according to manufacturer’s instructions with 6 μg of pAAV-CBA-luc, 12 μg of pXX6-80, and 10 μg of the AAV serotype helper plasmid. In PMVECs, staining for α(2,6)-linked sialic acids was barely detectable in control cells and was completely absent in neuraminidase-treated cells (Fig. 6A). These results support our earlier observations that PMVECs express little surficial α(2,6)-linked sialic acids. The phagocytosis of different pathogenic E. coli isolates was also enhanced by sialidase, which suggests that modifications on MDDC sialic acids may be considered in the development of MDDC-based antibacterial therapies. Hence, with particular relevance to DC-based therapies, the engineering of α2,6-sialic acid cell surface is a novel possibility to fine tune DC phagocytosis and immunological potency.
Dendritic cell maturation is characterized by profound phenotypic and physiological changes, including cytokine secretion, up-regulation of the expression of MHC and co-stimulatory molecules and down-regulation of further antigen internalization.2 These processes elevate the efficiency of bacterial antigen presentation to T lymphocytes, which is essential for the induction of specific responses to fight bacterial pathogens.3 Because of the pivotal role of DCs in the elicitation of adaptive immune responses, DC-based procedures are now being exploited as therapeutics to boost immunity against pathogens.4,5,6 Nevertheless, further studies are warranted to better elucidate DC-pathogen interactions and to improve the efficacy of these novel therapeutics. Dendritic cells (DCs) play an essential role in immunity against bacteria by phagocytosis and by eliciting adaptive immune responses. However, the relative role of these sialylated structures in phagocytosis of bacteria has not been reported. However, binding to sialic acid seems to be the major determinant of AAV6 transduction, since about 98% inhibition of transduction was observed following neuraminidase treatment (Fig. (Fig.2B2B). However, these concentrations of PPMP significantly inhibited bovine AAV infection, which requires gangliosides as receptors (36). Therefore, glycolipids seem not to be required for AAV1 and AAV6 transduction. For experiments, the adherent cell culture was kept till 80% confluency, then collected via 0.25% trypsin (Gibco), seeded into 4-chamber slides (Nunc Lab-Tek, Merck) in MEM containing limited serum levels (2% FCS) and then cultured for three further days to obtain Hepa-1c1c7 cells that are susceptible to human complement-mediated lysis.
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. Cells with the different samples were then divided into two tubes and either incubated for 1 h with mouse anti human factor P antibody (1:1000, Bio-Rad) or the respective control antibody. AAV2, which has been demonstrated to use integrin αVβ5, basic fibroblast growth factor receptor, or hepatocyte growth factor receptor as its coreceptor for cell entry (20, 30, 41), was much less sensitive to proteinase K treatment for transduction (Fig. (Fig.6).6). This report provides a comprehensive analysis of the market landscape, inspiring companies with the necessary information to make informed decisions regarding their business strategies and areas of potential growth. All other data supporting the findings of this study are available within the article and the supplemental information. This article contains supporting information. Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligated in the form desired to generate the plasmids required. To avoid compounding effects such as the competition for transcription factors between vector and competitor, a lambda phage DNA sequence instead of an expression cassette was packaged into AAV1, -2, and -6 capsids when used as competitors.
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. T lymphocytes primed by desialylated MDDCs expressed more interferon-γ when compared with priming by sialylated MDDCs. Inactivation of either the ST3Gal.1 or the ST6Gal.1 sialyltransferase resulted in a more mature phenotype and subsequent macropinocytosis down-regulation in murine DCs.19,22 Together, these observations suggest a tight correlation between DC maturation, macropinocytotic functions and the state of cell surface sialylation. Staining with anti-human CD14, BDCA-1 and HLA-DR antibodies (BioLegend, San Diego, CA) followed by flow cytometry analysis was used to monitor monocyte isolation, MDDC differentiation and maturation state. BLAST searches were initiated with RgNanOx/YjhC as queries followed by manual annotation of the ORFs going outward. To map complete sialometabolic pathways within individual microorganisms, BLAST searches were performed against all known Neu5Ac transporters (35) as well as for the Neu5Ac aldolase NanA and N-acetylmannosamine-6-phosphate epimerase NanE (using queries of different organismal origin).
