Additional file 1: of Downstream tests, treatments, and annual direct payments in older men cared for by primary care providers with high or low prostate-specific antigen screening rates using 100 percent Texas U.S. Medicare public insurance claims data: a retrospective cohort study

Flowchart for construction of primary care provider sample with high versus low prostate specific antigen test ordering rate, 100% Texas U.S. Medicare public insurance claims data. (DOC 31 kb

Additional file 2: of Downstream tests, treatments, and annual direct payments in older men cared for by primary care providers with high or low prostate-specific antigen screening rates using 100 percent Texas U.S. Medicare public insurance claims data: a retrospective cohort study

Flowchart for construction of patient sample for assignment to primary care providers with high versus low prostate specific antigen test ordering rate, 100% Texas U.S. Medicare public insurance claims data. (DOC 31 kb

TLR4 mediated the maturation of DCs induced by EV71 VLPs.

<p>(A) Human DCs were pre-incubated with 20<i> </i>µg/mL of anti-TLR2, anti-TLR4, or IgG2a (as an isotype control) separately for 1<i> </i>h and were then challenged with EV71 VLPs (10<i> </i>µg/mL) for 16<i> </i>h. The control was not treated with EV71 VLPs. Supernatants from the cell cultures were collected to determine levels of IL-12 p70, IL-12 p40, and IL-10. Significant differences in the levels of cytokines produced by DCs treated with Abs and those treated with IgG2a are indicated by asterisks (*<i>p</i><0.05). (B) Human DCs were pre-incubated with 20<i> </i>µg/mL of anti-TLR4 mAb or IgG2a (as an isotype control) separately for 1<i> </i>h and were then treated with 10<i> </i>µg/mL EV71 VLPs for 30<i> </i>min on ice to allow binding to the cell surface. Surface-bound EV71 VLPs were measured with flow cytometry through immunostaining with anti-EV71 monoclonal antibodies. The control cells (no VLPs; dotted line) were stained with the same primary antibody followed by a secondary antibody. The bar diagram shows the MFI with statistics for the anti-TLR4 mAb effect on binding of EV71 VLPs to DCs. (C) HEK293 cells were transfected with control plasmids, TLR4, or TLR4+MD2. The transfected cells were stimulated with medium alone, LPS (50<i> </i>ng/mL), or EV71 VLP (10<i> </i>µg/mL) for 24<i> </i>h, and IL-8 protein in the supernatants was measured by ELISA. This experiment was repeated three times with similar results. (D) Proteinase K-treated EV71 VLPs abrogated the production of IL-12 p70, IL-12 p40, and IL-10 by DCs. Human DCs were incubated with medium alone (Control), proteinase K (10<i> </i>ng/mL), EV71 VLPs (10<i> </i>µg/mL), proteinase K-treated EV71 VLPs, LPS (100<i> </i>ng/mL), or proteinase K-treated LPS for 24<i> </i>h. At the end of the incubation time, the production of IL-12 p70, IL-12 p40, and IL-10 was analyzed by ELISA. Representative data from three different donors with similar results are shown. Values represent the means ± SEs of triplicate samples, and asterisks indicate statistically significant differences (*, <i>P</i><0.05).</p

The maturation and activation of DCs induced by EV71 VLPs required TLR4 and NF-κB activity and enabled naïve T cells to initiate Th1-cell responses.

<p>The maturation and activation of DCs induced by EV71 VLPs required TLR4 and NF-κB activity and enabled naïve T cells to initiate Th1-cell responses.</p

Induction of IκBα degradation and NF-κB activation by incubation of human DCs with EV71 VLPs.

<p>(A) Human DCs were treated with EV71 VLPs (10<i> </i>µg/mL) for the indicated times. Western blotting was used to analyze cytosolic fractions for degradation of IκBα. The lower panel shows anti-α-tubulin staining of the same blot and confirmed equal loading of all samples. (B) Human monocyte-derived DCs were not treated (Cont) or were treated with LPS (100<i> </i>ng/mL) or EV71 VLPs (5 or 10<i> </i>µg/mL) for 2<i> </i>h, and nuclear fractions were prepared. An electrophoretic mobility shift assay was used to assay NF-κB binding activity in the nuclear fractions. To assess the specificity of the binding, a 100-fold excess of cold NF-κB probe (a double-stranded NF-κB oligonucleotide, 5′-AGTTGA<u>GG<b>G</b>GACTTTCCC</u>AGGC-3′) or mutant probe (a double-stranded mutated oligonucleotide, 5′-AGTTGA<b>GG<u>C</u>GACTTTCCC</b>AGGC-3′) was added to the EV71 VLP condition. A double-stranded mutated probe, 5′-AGTTGA<u>GG<b>C</b>GACTTTCCC</u>AGGC-3′, was used to examine the specificity of binding of NF-κB to DNA (the underlined sequence is identical to the κB consensus sequence except for a <b>G</b>-to-<b>C</b> substitution in the NF-κB DNA binding motif). This experiment was repeated three times (from three different donors) with similar results.</p

Effects of EV71 VLPs on the endocytic capacity of human monocyte-derived DCs.

<p>At day 5, immature DCs were stimulated with medium alone (Control), LPS (100<i> </i>ng/mL; LPS), or EV71 VLPs (10<i> </i>µg/mL; VLP) for 24<i> </i>h and then incubated with FITC-dextran for 1<i> </i>h at 4°C (dotted lines) or 37°C (solid lines). This experiment was repeated three times (from three different donors) with similar results. The values shown in the flow cytometry profiles are the mean fluorescence intensity indices.</p

EV71 VLPs enhanced T-cell responses.

<p>(A) Immature DCs were stimulated with medium alone (Control), LPS (100<i> </i>ng/mL), or EV71 VLPs (10 and 20<i> </i>µg/mL; VLP) for 24<i> </i>h. Proliferation of autologous T cells was measured after 5 days of coculture with DCs. (B) Supernatants were analyzed for IFN-γ and IL-5 produced by activated T cells after 2 days of culture. Representative data from three different donors with similar results are shown. Values represent the means ± SEs of triplicate samples, and asterisks indicate statistically significant differences (*, <i>P</i><0.05).</p

Binding of EV71 VLPs to immature human monocyte-derived DCs.

<p>For the analysis of concentration-dependent effects, human monocyte-derived DCs were incubated with 1 or 10<i> </i>µg/mL EV71 VLPs for 30<i> </i>min on ice to allow binding to the cell surface. For the analysis of time-dependent effects, DCs were incubated with 5<i> </i>µg/mL of EV71 VLPs for 30 or 120<i> </i>min on ice. Detection was facilitated using an anti-EV71 Ab and anti-mouse-IgG Ab (labeled with Alexa Fluor 488). Levels of fluorescence were determined using immunofluorescence confocal microscopy (A) and FACS analysis (B, C). (B) and (C) indicate the respective concentration-dependent and time-dependent effects of the binding of EV71 VLPs to immature human monocyte-derived DCs. The control cells (no VLPs; filled histogram) were stained with the same primary antibody (anti-EV71 Ab) followed by a secondary antibody. (D) and (E) indicate the bar diagram of mean fluorescence intensity (MFI) with statistics for concentration- and time-dependent binding of EV71 VLPs to DCs. Representative data from three different donors with similar results are shown. Values represent the means ± SEs of triplicate samples, and asterisks indicate statistically significant differences (*, <i>P</i><0.05).</p

EV71 VLPs increased cytokines production and enhanced the abundance of cell-surface molecules in human DCs.

<p>(A) Human DCs were cultured for 24<i> </i>h in the presence of 100<i> </i>ng/mL LPS or various concentrations of EV71 VLPs. At the end of the incubation time, the culture medium was collected, and IL-12 p70, IL-12 p40, and IL-10 levels were measured using ELISA. (B) Human DCs were incubated with EV71 VLPs (10<i> </i>µg/mL) for 16, 24, or 48<i> </i>h. At the end of the incubation time, levels of secreted IL-12 p70, IL-12 p40, and IL-10 were analyzed by ELISA. (C) Human DCs were treated with EV71 VLPs (10<i> </i>µg/mL; VLP), LPS (100<i> </i>ng/mL; LPS), or medium alone (Control) for 24<i> </i>h, and levels of surface markers were analyzed by flow cytometry (dotted line, isotype control; solid line, specific mAbs). The values shown in the flow cytometry profiles are the mean fluorescence intensity indices. Representative data from three different donors with similar results are shown. Values represent the means ± SEs of triplicate samples, and asterisks indicate statistically significant differences (*, <i>P</i><0.05).</p

Uptake of EV71 VLPs by immature human monocyte-derived DCs.

<p>For the analysis of concentration-dependent effects, DCs were collected and incubated with different concentrations of CFDA-labeled EV71 VLPs (0, 2, 5, 10, or 20<i> </i>µg/mL) for 120<i> </i>min at 37°C. For the analysis of time-dependent effects, DCs were incubated with 10<i> </i>µg/mL of CFDA-labeled EV71 VLPs for different time points (0, 15, 30, or 120<i> </i>min) at 37°C. At the end of the incubation time, fractions were collected and fixed before double staining with an allophycocyanin-labeled anti-CD1a Ab. Fluorescence was examined by immunofluorescence confocal microscopy (an enlargement of a single cell is shown) (A) and FACS analysis (B, C). (B) and (C) indicate the respective concentration-dependent and time-dependent uptake of EV71 VLPs to immature human monocyte-derived DCs. (D) and (E) show bar diagrams of MFIs with statistics for concentration- and time-dependent uptake of EV71 VLPs. Representative data from three different donors with similar results are shown. Values represent the means ± SEs of triplicate samples, and asterisks indicate statistically significant differences (*, <i>P</i><0.05).</p