6 research outputs found

    Exosomes are released from scrapie-infected B cells <i>ex vivo</i>.

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    <p>Spleens were dissected from 129/Sv×C57BL/6 mice 30 days after i.p. inoculation with 1% (w/v) RML I6200. MACS-isolated B lymphocytes were cultured under passive leakage (A) and basal (B) conditions essentially as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-t004" target="_blank">Table 4</a> and tissue culture supernatants were isolated by sequential centrifugation (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#s4" target="_blank">Materials and Methods</a>). After centrifugation at 100,000× g for 2 h pellets were resuspended in PBS, absorbed onto carbon-coated grids and negatively stained with 1% uranyl acetate. Cup-shaped exosome-like membrane particles of different sizes (see arrows) are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1B</a>. Twenty randomly recorded images (surface area: 2.82 µm<sup>2</sup>) from each condition were counted and the number of exosome-like particles (1.7±1.2 (A) and 22.8±6.5 (B) per surface area, p≪0.001) determined in a blinded manner. Scale bar: 0.2 µm.</p

    Isolation of splenic cell types by magnetic-activated cell sorting.

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    <p><b>A</b>: Schematic representation for the isolation of specific splenic cell types from mice. Splenocytes were released by repeated collagenase digestion from freshly dissected spleens, followed by removal of erythrocytes and purification of splenocytes on Lympholyte M gradients. Splenic cell types are isolated by positive selection with magnetic beads coated with cell type-specific mAbs as specified. <b>B</b>: The purities of MACS-isolated cells were analysed by FACS using cell-type specific mAbs and isotype controls as specified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#s4" target="_blank">Materials and Methods</a>. One representative out of three experiments is shown. (Bv) CD11<sup>low</sup> B220<sup>+</sup> pDCs, isolated with murine plasmacytoid dendritic antigen-1 (mPDCA-1) showed a purity of about 90% in three independent experiments. (Bvi) The macrophage population, isolated with CD11b microbeads after depletion of CD11c<sup>+</sup> cells was contaminated with CD11c<sup>+</sup> CD11b<sup>+</sup> mDCs. Macrophages were therefore isolated by FACS instead (<b>C</b>). <b>C</b>: Splenocytes labelled with mAbs against anti-CD11b (M1/70) and anti-CD11c (HL3) were isolated by FACS using a DAKO cell sorter.</p

    Infectious titers of MACS-isolated cells after homogenization by sonication and ribolyzation.

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    ‡<p>Inputs of infectious cell homogenates are expressed as cell number equivalents. Aliquots of 30 µl were inoculated i.c. into groups of six Tga20 mice for mouse bioassay and 300 µl aliquots were layered onto prion-susceptible cells per well for SCEPA, respectively.</p>†<p>Infectious titers were calculated according to the Spearman-Karber method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat.1002538-Dougherty1" target="_blank">[55]</a> and are expressed as log LD<sub>50</sub>/g ± SE for bioassay and log TCIU/g ± SE for SCEPA.</p>$<p>Infectious titers were calculated using a GLM with binomial family complementary log-log link and expressed as mean log TCIU/g ± SE of two independent experiments with six technical repeats each.</p>#<p>Controls represent MACS-isolated pDCs and B lymphocytes from spleens 129/Sv×C57BL/6 mice inoculated with 1% (w/v) uninfected CD1 brain homogenates and sacrificed at 30 dpi.</p>*<p>Level of significance for maximum likelihood estimates (GLM) between infectious titers of ribolyzed versus sonicated pDCs as determined by SCEPA.</p><p>Four 129/Sv×C57BL/6 mice were inoculated i.p. with 100 µl of 1% (w/v) RML and 1% (w/v) uninfected CD1 brain homogenate (control), respectively. At 30 d.p.i spleens were dissected and pDCs and B cells isolated by MACS according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1</a>. Aliquots of 1×10<sup>7</sup> cells/ml OFCS, supplemented with protease inhibitors were homogenized by sonication <i>(A)</i> or ribolyzation <i>(B)</i> according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#s4" target="_blank">Materials and Methods</a>. To determine infectious titers the cell homogenates were serially diluted 1∶10 and inoculated intracerebrally into Tga20 mice or transferred onto layers of susceptible PK1-2 cells in parallel experiments. Infectious titers were determined by non-parametric statistical analysis for bioassay (Spearman and Karber) and GLM for SCEPA and expressed as log LD50 units/10<sup>6</sup> cells and log TCIU/10<sup>6</sup> cells, respectively. A 10<sup>−2</sup> dilution of cell homogenates corresponds to 2×10<sup>5</sup> cell equivalents/ml or 6×10<sup>3</sup> cell equivalents per 30 µl inoculum for mouse bioassay and 6×10<sup>4</sup> cell equivalents per 300 µl per well for SCEPA, respectively. Infectious titers represent log mean values ± SE of six independent experiments for SCEPA and log mean values ± SE of a single experiment for mouse bioassay.</p

    Sensitivity for prion detection of SCEPA and mouse bioassay.

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    *<p>The ratio between PrP<sup>Sc</sup>-positive and total wells is shown for one representative out of eight independent experiments.</p>†<p>Infectious titers were calculated with the Spearman-Karber formula <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat.1002538-Dougherty1" target="_blank">[55]</a> and expressed as tissue culture infectious units (TCIU)/g brain for SCEPA and LD<sub>50</sub> units/g brain for mouse bioassay, respectively.</p>$<p>Infectious titers were calculated using a GLM with binomial family complementary log-log link and expressed as mean log TCIU/g brain ± SE of 8 independent experiments for SCEPA and mean log LD50 units/g ± SE for two independent bioassays.</p>‡<p>Infectious titers were estimated for the combined two bioassays using a GLM regression with complementary log-log link function and expressed as log LD50 units/g brain.</p><p>The sensitivity for prion detection of SCEPA and mouse bioassay was determined by endpoint titration using RML mouse brain homogenate I6200. Aliquots of I6200 (10% (w/v), 9.3 log LD<sub>50</sub> units/g brain <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat.1002538-Cronier1" target="_blank">[7]</a>) were serially diluted 1∶10 into uninfected CD1 brain homogenate (10% w/v) in a range between 10<sup>−4</sup> and 10<sup>−10</sup>. For mouse bioassay, groups of six Tga20 mice were inoculated intracerebrally with 30 µl of 1% (w/v) RML homogenates and attack rates and scrapie incubation times (Inc. time) were determined. In parallel experiments brain homogenates were diluted 1∶1000 into OFCS and cell layers of highly prion susceptible N<sub>2</sub>aPK1-2 cells were infected with 300 µl aliquots. The input of prion infectivity for bioassay and SCEPA is expressed as mouse ic LD<sub>50</sub> units. A 10<sup>−7</sup> dilution of I6200 corresponds to 200 LD<sub>50</sub> units/ml or 6 LD<sub>50</sub> units per 30 µl inoculum for the mouse bioassay and 60 LD<sub>50</sub> units per 300 µl per well for SCEPA, respectively. Infectious titers for SCEPA, expressed as TCIU/g brain represent mean values ± SE of 8 independent experiments. For mouse bioassay, two independent experiments are shown and titers are expressed as LD<sub>50</sub>/g brain ± SE.</p

    Time-dependent accumulation of prion infectivity in isolated splenic cell types.

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    ‡<p>Myeloid cells were isolated with CD11b magnetic beads after partial depletion of DCs and contain CD11c<sup>−</sup> CD11b<sup>+</sup> macrophages and CD11c<sup>+</sup> CD11b<sup>+</sup> mDCs (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1B</a>).</p>†<p>CD11b<sup>+</sup> macrophages devoid of mDCs contaminants were isolated by FACS (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1C</a>).</p>#<p>Infectious titers are represented as mean values ± SE, and as lower and upper limits of 95% confidence intervals (conf.int).</p><p>Groups of ten 129/Sv×C57BL/6 mice, inoculated i.p. with 100 µl aliquots of 1% (w/v) RML brain homogenate I6200 were culled at various time points after inoculation as specified above and splenocytes and splenic cell types were serially isolated by MACS after Collagenase digestion and Lympholyte purification according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1</a>. Infectious titers were determined by SCEPA using a GLM as specified above. Mean values ± SE and 95% confidence intervals for at least three independent experiments are shown at 14 and 30 dpi. Data from a single experiment are shown where no SE is reported. The detection limit of the assay for splenic cell types was 0.15 TCIU/Mio, for MACS-isolated cells from whole blood 5 TCIU/Mio.</p

    Prions are released from scrapie-infected splenic cell cultures <i>ex vivo.</i>

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    †<p>Control incubations were performed in basal medium at atmospheric (atm.) CO<sub>2</sub> and 37°C. For dendritic cell cultures Triton X-100 was added to a final concentration of 0.01% in basal medium.</p><p>Fifteen 129Sv×C57BL/6 were inoculated i.p. with 100 µl RML I6200 and culled at 60 d.p.i. Splenic cell types were isolated by Collagenase perfusion according to the experimental procedure depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1</a>. The levels of cellular infectivity were determined after MACS isolation. MACS-isolated B and T lymphocytes were then cultured at a concentration of 1×10<sup>6</sup>/ml in basal medium (IMDM medium, 10% FBS) in absence or presence of LPS (50 µg/ml) and IL-4 (10 ng/ml). DCs were cultured in basal medium, supplemented with 200 ng GM-CSF. To remove cells and debris the conditioned medium was collected after 36 h of culture, centrifuged at 300× g for 10 min, 5,000× g for 15 min and 10,000× g for 30 min and the supernatant was collected at each of the sequential centrifugations. The supernatant was then centrifuged for 2 h at 100,000× g and resuspended in PBS, serially diluted and infectious titers were determined using SCEPA. The detection limit for SCEPA was 0.1 TCIU/Mio cells. Mean values ± SE of three independent experiments are shown. Data from two independent experiments are shown for the release of infectivity from DCs.</p