33 research outputs found

    <i>Sod1</i> expression in mouse brain.

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    <p>Panel A and B. Quantification of <i>Sod1</i> mRNA expression in whole brain by real-time RT-PCR. N = 6 for all groups and samples were run in triplicate. All samples were duplexed for <i>Sod1</i> (Fam-label) and an endogenous control <i>GAPDH, β-actin</i> or <i>Thy-1</i> (Vic-label). Expression level is expressed in arbitrary units as normalised by the geometric mean of the quantity of the endogenous controls (<i>y</i>-axis). Error bars represent the standard deviation. (A) <i>Sod1</i> mRNA expression level for parental strain of the HS mice (except LP). (B) <i>Sod1</i> mRNA expression level grouped by allele (A/G) (A = A, BALB, C3H, C57; G = AKR, CBA, DBA). No significant difference was observed between the groups. Panels (C) and (D). Quantification of Sod1 protein in whole brain (10% homogenate, weight/volume) from n = 3 A allele mice (C57Bl/6) and G allele mice (FVB/N). FVB/N mice were used to represent a G allele strain rather than an HS parental strain due to availability of tissue. Samples were immunoblotted with rabbit polyclonal anti-human SOD1(Abcam) and detected with IRDye800CW (green) conjugated goat anti-rabbit IgG (Li-Cor). Anti-β-actin mouse monoclonal antibody (Sigma) was also included as a loading control and was detected using IRDye680 (red) conjugated goat anti-mouse IgG (Li-Cor). Fluorescence was visualised using an Odyssey infra-red imager (Li-Cor). (C) Uninoculated mice, (D) Terminally sick Chandler/RML prion inoculated mice.</p

    Kaplan-Meier survival curves.

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    <p>Data are shown as % of animals (female) surviving (y-axis) plotted against the number of days post inoculation (x-axis). (A) Transmission of Chandler/RML prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls (B) Transmission of ME7 prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT). (C) Transmission of MRC2 mouse adapted BSE prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT). A reduction in mean incubation time of 20%, 13%, and 24% was seen in A-C respectively. This reduction in survival was statistically significant for each transmission (P<0.001, Kaplan-Meier log-rank survival test).</p

    Western blots of PrP<sup>Sc</sup> from infected mouse brains.

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    <p>10% w/v brain homogenates (n = 3 per group) were digested with proteinase K and immunoblotted with anti-PrP monoclonal antibody ICSM35 (D-Gen Ltd, UK). (A) Transmission of Chandler/RML prions to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls. (B) Transmission of ME7 prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls. (C) Transmission of MRC2 mouse adapted BSE prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls. No differences were seen between the two groups regardless of prion strain.</p

    RML histology.

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    <p>Histological features of Chandler/RML prion transmission to <i>Sod1<sup>−/−</sup></i> (A–C) and wild type control (D–F) mice. Panels A and D show distribution of disease-associated PrP by immunohistochemistry using anti-PrP monoclonal antibody ICSM35. Panels B, C, E and F show detail from the hippocampus and are stained with haematoxylin and eosin (H&E) to visualise spongiform change and neuronal loss. There is almost no neuronal loss in the <i>Sod1<sup>−/−</sup></i> mice but mild neuronal loss is seen in the wild type animals. Overall, the pattern of spongiosis, gliosis and PrP distribution are similar between the two groups, however, the distribution of disease-associated PrP is patchier in the knockouts especially in the cortex. Scale bar corresponds to 3 mm (A, D), 660 µm (B, E) or 160 µm (C, F).</p

    Quantification of Sod and PrP<sup>C</sup>

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    <p>. 10% weight/volume brain homogenates immunoblotted with rabbit polyclonal anti-human SOD1 (Abcam) and detected with IRDye800CW (green) conjugated goat anti-rabbit IgG (Li-Cor). Anti-β-actin mouse monoclonal antibody (Sigma) was also included as a loading control and was detected using IRDye680 (red) conjugated goat anti-mouse IgG (Li-Cor). Fluorescence was visualised using an Odyssey infra-red imager (Li-Cor). (A) Brains from <i>Sod1<sup>+/+</sup></i> wild type mice inoculated with PBS compared with end stage Sod1<sup>+/+</sup> wild type mice inoculated with RML, ME7 and MRC2 prion strains. No differences were seen between the groups. (B) Uninfected mice. (C) Total SOD enzymatic activity in 10% (w/v) <i>Sod1<sup>+/+</sup></i> (WT) brains. Brains from terminally sick mice infected with RML, ME7 and MRC2 were compared with uninfected mice. Samples were run in triplicate with n = 6 for each group. Data are shown normalised by total protein content (µg/ml) as determined by a Bradford protein assay (mean ± standard deviation). No significant difference was seen between the groups. (D) PrP<sup>c</sup> levels in <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate control mice by ELISA. PrP<sup>c</sup> levels (µg/ml) were determined in triplicate using 10% (weight/volume) brain homogenate for <i>Sod1<sup>−/−</sup></i> (n = 3) and <i>Sod1<sup>+/+</sup></i> (n = 3) in a PrP specific ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054454#pone.0054454-Wadsworth2" target="_blank">[32]</a>. Data are shown normalised by total protein content (µg/ml and x 1000) as determined by a BCA assay (mean ± standard deviation). No significant difference was seen between the two groups.</p

    SNP genotyping in HS mice.

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    <p>Genomic location is given based on mouse genome assembly NCBI build 37. Details of the <i>Sod1</i> and <i>Il1-r1</i> SNPs are available from the Sanger Centre (<a href="http://www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl" target="_blank">www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl</a>). All polymorphisms were analysed by allele discrimination using a 7500 Fast real time PCR system (Applied Biosystems). For probe details see Table S1. For all genotypes, the statistical test used was the Kruskal-Wallis non-parametric ANOVA. The allelic test used was the Mann-Whitney test.</p

    Major strain distribution patterns genotyped for HS mice.

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    <p>The number of SNPs is taken from genomic sequence generated by the Sanger Institute (<a href="http://www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl" target="_blank">www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl</a>) and spans 5 kb upstream of the 5′UTR start site to the end of the 3′UTR (NCBI Build 37). Ambiguous SNPs have been excluded. The strain distribution pattern (BALB, CBA); (A, AKR, C3H, C57, DBA, LP) was also seen for <i>App</i> (239/978) but this was not genotyped in the HS mice. Other individual strain distribution patterns were seen ≤5 times.</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

    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

    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
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