Protein expression profiles for CD55, CD14, and CD11b following addition of LPS (1 µg/ml) to THP-1 cells pretreated with or without 100 nM 1,25-D3 (72 h).

<p>A multiplexed fluorescence-based immunoblot approach was used to determine corresponding protein expression profiles from cells whose mRNA profiles are shown in Fig. 2. A, the temporal protein induction pattern of CD55, CD11b and β-actin following a LPS-challenge (without pretreatment). CD14 levels are not shown because the apparent 50-fold induction remained below the threshold of detection (nd). B, the relative band intensities for CD55 and CD11b, normalized to β-actin, are graphically presented as the average fold-change (relative to time zero) of three independent experiments (± SD). In some instances the SD are covered by the data point symbol. C and D, the multiplex immunoblot of LPS-induced protein expression in cells pretreated for 72 h with 1,25-D3 is shown in panel C; the fold induction is graphically represented in panel D as the average fold-change (relative to time zero) of three independent experiments (± SD). In some instances the SD are covered by the data point symbol. The apparent 100-fold induction of CD14 following 1,25-D3 pretreatment but before LPS (time = 0) was within the immunoblot detection limits.</p

NF-κB activation was required for LPS-induced CD55 expression in 1,25-D3 pretreated THP-1 monocytes.

<p>As illustrated under panel A, at the end of the 72 h pretreatment phase with 100 nM 1,25-D3, either vehicle or one of two NF-kB inhibitors [50 µM MG132 (MG) or 30 µm parthenolide (Pa)] was added and the incubation continued for an additional 1 h. Then LPS (1 µg/ml) or vehicle was added. Cells were ultimately harvested at 2 h (mRNA) or 24 h (protein) post addition of LPS or vehicle. Quantitative PCR was used to determine CD55 (A) and CD11b (D) mRNA levels assessed at 2 h. Differences between mRNA pool sizes were determined using a two tailed student’s t test. Asterisks (Fig. 5A) indicate statistical differences between indicated treatments; *P<0.05, **P<0.01. CD55 and CD11b protein expression was assessed at 24 h following a vehicle or LPS challenge by immunoblotting (B) and quantification (C and E), respectively. Because of the semi-qualitative nature of the protein determinations, a statistical analysis was not performed on these data. Values represent means +/− SD (n = 3).</p

CD55 mRNA and protein expression was not altered during the early response phase (0–24 h) following a 1,25-D3 preincubation period of 72 h with human THP-1 monocytes.

<p>THP-1 cells were cultured in complete media in the absence or presence of 100 nM 1,25-D3 (added at time = 0) and harvested at the indicated times. A, CD55, CD14 and CD11b mRNA pool sizes were determined by quantitative PCR analysis using 18S rRNA as a reference. Fold-induction is expressed relative to vehicle treated controls. Values shown are the mean +/− SD of 3 independent experiments. In some instances, the SD bars are covered by the data point symbol. Shaded data points indicate statistical differences from control values. P<0.05 was considered significant. B, protein expression was analyzed in cells at various times following addition of 1,25-D3, using a fluorescence-based immunoblot approach. Corresponding controls showed no difference over the same time period.</p

Pretreatment with 1,25-D3 for 72 h sustained the subsequent LPS-induced expression of CD55 mRNA in THP-1 cells, compared to cells pretreated with vehicle for 72 h.

<p>THP-1 cells were first preincubated in complete culture media with vehicle or with 100 nM 1,25-D3. Seventy-two h later (t = 0 on A–C), either vehicle or 1 µg/ml LPS was added and the incubation continued for an additional 72 h. Cells were harvested at the indicated times and quantitative PCR (n = 3) was used to determine CD55 (A), CD14 (B) and CD11b (C) mRNA pool sizes using the absolute copy number method. Mean levels are expressed in arbitrary units normalized to 18S rRNA (± SD). Differences in mRNA pool size were determined using a two tailed student’s t test. Shaded data points indicate statistical differences from corresponding controls.</p

CD55 expression was enhanced on the surface of essentially all THP-1 cells at 24 h, following addition of LPS to 1,25-D3 pretreated cells.

<p>THP-1 cells were grown on coverslips, pretreated with 100 nM 1,25-D3 for 72 h and then stimulated with either vehicle or LPS for 24 h, as indicated. Serial 0.5 µm optical sections were obtained by confocal microscopy (40 × magnification). A, sections containing either a central (internal Z-stack) or the apical region of the cells (cell surface Z-stack) and the entire 3D image of LPS treated cells are shown. White represents CD55 staining; nucleic acid staining was omitted for clarity. B, shows a graphical representation of mean (±SD) CD55 expression levels (normalized to nucleic acid content) for three independent experiments with two fields quantified per experiment (n = 6).</p

Analysis of the phylogenetic group determination and the profile of virulence factors among the <i>E</i>. <i>coli</i> strains isolated from patients with chronic inflammation of the maxillary sinuses.

<p>Analysis of the phylogenetic group determination and the profile of virulence factors among the <i>E</i>. <i>coli</i> strains isolated from patients with chronic inflammation of the maxillary sinuses.</p