19 research outputs found

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

    No full text
    †<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

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

    No full text
    ‡<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

    An additional human chromosome 21 causes suppression of neural fate of pluripotent mouse embryonic stem cells in a teratoma model-3

    No full text
    <p><b>Copyright information:</b></p><p>Taken from "An additional human chromosome 21 causes suppression of neural fate of pluripotent mouse embryonic stem cells in a teratoma model"</p><p>http://www.biomedcentral.com/1471-213X/7/131</p><p>BMC Developmental Biology 2007;7():131-131.</p><p>Published online 29 Nov 2007</p><p>PMCID:PMC2211317.</p><p></p> tissue types in all tumours, and multiple HSA21 PCR markers were negative in the deleted regions, deletions must have occurred before any differentiation of ES cells in the tumours took place. The regions of the HSA21 gene map which are trisomic in some of the current segmental trisomy mouse models are indicated on the right by vertical lines, for comparison

    Limb-clasping and body tone abnormalities in <i>Tardbp<sup>+/Q101X</sup></i> mice.

    No full text
    <p>(<b>A</b>) <i>Tardbp<sup>+/Q101X</sup></i> mice develop hindlimb clasping. Plots show onset of hindlimb-clasping. The Y-axis is percentage of mice <i>not</i> showing the phenotype. At 7 weeks of age no mice showed limb clasping. Female mice (left panel): <i>Tardbp<sup>+/+</sup></i> n = 12, <i>Tardbp<sup>+/Q101X</sup></i> n = 15; Male mice (right panel): <i>Tardbp<sup>+/+</sup></i> n = 16, <i>Tardbp<sup>+/Q101X</sup></i> n = 16. Overall difference between <i>Tardbp<sup>+/+</sup></i> and <i>Tardbp<sup>+/Q101X</sup></i> p = 0.002 (Kaplan Meier log rank statistic), with a significant difference also noted between male and female <i>Tardbp<sup>+/Q101X</sup></i> mice up to one year (p = 0.046). (<b>B</b>) Progressive development of abnormal body tone in male and female <i>Tardbp<sup>+/Q101X</sup></i> mice. No mice displayed softer, abnormal body tone at 7 weeks of age. Male mice: <i>Tardbp<sup>+/+</sup></i> n = 16, <i>Tardbp<sup>+/Q101X</sup></i> n = 16. Female mice <i>Tardbp<sup>+/+</sup></i> n = 12, <i>Tardbp<sup>+/Q101X</sup></i> n = 15. <i>Tardbp<sup>+/+</sup></i> versus <i>Tardbp<sup>+/Q101X</sup></i> p<0.0001 (Kaplan Meier log rank statistic), with a trend in sex differences of <i>Tardbp<sup>+/Q101X</sup></i> genotype up to one year of age, p = 0.07 (Kaplan Meier log rank statistic).</p