1B), a Cyclopamine research buy finding confirming the absence of LPS contamination in the His-OprF preparation. Interestingly, levels of IL-12p70 production were higher and those of IL-10 and IL-6 lower in DCs stimulated with the recombinant porin as compared to the native porin (Fig. 1C), a finding suggesting the superior capacity of the recombinant OprF to activate DCs for Th1 priming. Figure 1 Activation of murine dendritic find more cells by OprF. Purified splenic dendritic cells (DCs) were pulsed with LPS (10 μg/ml), native (n) or recombinant (His) OprF at different concentrations for 18 hrs before the assessment of costimulatory molecule
expression (A) and cytokine production (B and C). FACS analysis was done by staining with FITC and PE-conjugated mAbs to costimulatory molecules. Number represent percent of positive cells. Cytokine levels were determined in the culture supernatants by cytokine-specific ELISA. * Indicates P < .05 (cytokine production by LPS- or porin-pulsed versus unpulsed (-) DCs). ** Indicate P < .05 (cytokine production by n-OprF-pulsed tlr4 -/- DCs versus n-OprF-pulsed WT DCs only and His-OprF-pulsed DCs versus
n-OprF-pulsed DCs). OprF-pulsed DCs protect mice from PAO1 infection Based on these results, we assessed the capacity of DCs pulsed with 3-deazaneplanocin A order either porin to immunize mice against P. aeruginosa lung infection. To this purpose, porin-pulsed DCs were administered to mice a week before the intranasal infection with the PAO1 strain. Mice were monitored for bacterial growth, lung inflammatory pathology and cytokine production locally in the lung (at 4 days after the infection) or in the thoracic lymph nodes (TLNs, at 7 days after infection). The results (Fig. 2A) showed that the adoptive transfer of DCs pulsed with n-OprF exerted significant protection in terms of reduced bacterial growth, both at 4 and 7 days after the infection. No effects on bacterial clearance was observed upon adoptive transfer of unpulsed DCs.Interestingly, an even higher bacterial clearance was observed upon adoptive transfer of mafosfamide DCs pulsed with His-OprF, being the bacterial
growth dramatically reduced as early as 4 days after the infection. Figure 2 OprF-pulsed DCs protect mice from infection with the PAO1 strain. Splenic 105 dendritic cells (DCs), either unpulsed (-) or pulsed as in legend to figure 1, were administered into recipient mice intraperitoneally a week before the intranasal injection of 3 × 107 P. aeruginosa PAO1 strain. (A) Resistance to infection was assessed in terms of CFU at different days after the infection and (B) cytokine production in lung homogenates and culture supernatants of total cells from TLNs stimulated with plate bound anti-CD3e (2 μg/ml) and anti-CD28 (2 μg/ml) for 72 hours. Results are expressed as mean ± SE. * Indicates P < .05, mice receiving pulsed versus unpulsed (-) DCs. In C, – and + alone indicate uninfected and infected mice, respectively.