12 research outputs found
Protection against bubonic and pneumonic plague of mice vaccinated with VTnF1.
<p>Mice having received a single oral dose of VTnF1 (10<sup>8</sup> CFU) were challenged via i.n. or s.c. routes, with various doses of bacteria as indicated. (A-D) Animals were challenged four weeks after vaccination by injection of (A, B) <i>Y</i>. <i>pestis</i> CO92, or (C, D) the un-encapsulated CO92Δ<i>caf</i>. (E) Vaccinated mice were challenged six months after vaccination. Mouse survival was recorded daily for 21 days. The number of mice surviving / number of animals tested is indicated above the corresponding bar for each condition. The Fisher Exact test was used for statistical analysis: *: p≤0.05; ***: p<0.001.</p
Cellular immune response of vaccinated mice.
<p>Splenocytes isolated from mice vaccinated orally with VTnF1 (10<sup>8</sup> CFU) 30 days (dark red boxes), or 180 days (black boxes) earlier, or from unvaccinated (naive) mice (blue boxes), were stimulated in vitro with 5 μg/ml of either an antigenic preparation of <i>Y</i>. <i>pestis</i> CO92Δ<i>caf</i> (noted <i>Y</i>.<i>p</i>.F1<sup>-</sup>) or purified F1 antigen, or the mitogen Concanavalin A (ConA, 1 μg/ml) as positive control. Supernatants taken three days after stimulation were tested for the presence of IFNγ (A), IL-17 (B), and IL-10 (C) by ELISA. Shown are the mean ± s.e.m. of 14 mice per condition (two pooled experiments). The unpaired Mann Whitney test was used for statistical analysis: *: p<0.05, **: p<0.01, ***: p<0.001, ns: not significant. Comparable results were obtained in two other experiments using naive and 30-days vaccinated mice (16 per group).</p
Humoral immune response induced by vaccination.
<p>(A) The antibody production induced by VTnF1 vaccination (10<sup>8</sup> CFU) was quantified at various days post vaccination using an ELISA measuring IgG directed against purified F1 antigen. Shown are the means ± sem of titers observed in two experiments performed during the first month post-vaccination (Exp.1, 7 mice) and during the six-months post-vaccination period (Exp.2, 14 mice). Mean IgG titers at each time point were compared statistically to that on day zero using the Mann-Whitney test, and all were significantly higher (p<0.001), except those on day 1. (B) The different Ig mouse isotypes present in sera were analyzed at sequential times. (C) The IgG response against antigens other than F1 was analyzed by ELISA against a <i>Y</i>. <i>pestis</i> CO92Δ<i>caf</i> sonicate, and (D) by western blotting. The <i>Y</i>. <i>pestis</i> CO92 wild type or CO92Δ<i>caf</i> (noted F1-) were used as source of blotted antigens and sera pooled either from naive mice or mice vaccinated 30 days earlier (noted VTnF1). The Caf1 band is indicated by an asterisk and the Caf1M band by a triangle. (E) The response against purified Yops was also analyzed by ELISA. Shown are results from 14 individual animals (dots), and group medians (horizontal line). The Mann-Whitney test was used for statistical analysis: ***: p<0.001.</p
Vaccination with VTnF1 prevents <i>Y</i>. <i>pestis</i> dissemination from the site of infection.
<p>(A) Mice vaccinated with VTnF1 (10<sup>8</sup> CFU i.g.) were infected four weeks later by s.c. injection of 10<sup>3</sup> CFU of the bioluminescent <i>Y</i>. <i>pestis</i> CO92::Tn7-P<i>ail</i>-<i>lux</i> in the ventral skin. Whole body bioluminescence of the animals was recorded at regular time intervals using an IVIS camera. Shown are white light photographs merged with the bioluminescence signal, scaled in the right margin. (B) The bioluminescence intensity emitted at the site of injection was measured for each animal and shown are means ± s.e.m. for each group. Animals missing at time point 92h died from infection before this bioluminescence recording. The paired Mann-Whitney test was used for statistical analysis: **: p<0.01.</p
In vivo dissemination of VTnF1 after oral vaccination.
<p>Groups of mice were inoculated orally with the VTnF1 vaccine (10<sup>8</sup> CFU) and were sacrificed at the indicated times to evaluate the VTnF1 loads in: (A) feces (two pellets/mouse), (B) Peyer’s patches (two patches/mouse), (C) the spleen (whole organ), (D) the liver (whole organ), and (E) mesenteric lymph nodes (all). Samples were minced and dilutions were plated on selective agar plates containing kanamycin to count colonies, with a detection limit of 10 CFU/sample. Shown are individual values from 7–14 mice per condition. The horizontal line indicates the median. The Mann Whitney test was used for statistical analysis: *: p ≤0.05, **: p <0.01, ***: p<0.001.</p
Effect of ShRNA-mediated <i>Sprn</i> knockdown on embryo resorption at E11.5.
*<p>p<0.05 (x<sup>2</sup> test) when compared to any of the FoxL2 results and to LS2 on P10 and FVB/N genetic backgrounds.</p><p>These data are a compilation of at least 2 independent experiments. No statistically significant variability was observed between the analyzed litters (3 for P10, more than 4 for the other lentiviral infections).</p
Histological analysis of E7.5 embryos.
<p>E7.5 embryos were fixed and stained by hematoxylin, eosin, and saffron. Top left: schematic representation of a mouse E7.5 embryo. 1,2: FVB/N <i>Prnp<sup>KO</sup></i> embryos. 3,4: FG12-injected FVB/N <i>Prnp<sup>KO</sup></i> embryos. 5–8: LS2-injected FVB/N <i>Prnp<sup>KO</sup></i> embryos. 3–8: embryos that were injected at the zygotic stage. 9: LS2-infected FVB/N <i>Prnp<sup>KO</sup></i> embryos. *: infection performed at the blastocyst stage. Interesting features include i) the size differences between injected and non-injected embryos, ii) the relatively important hemorrhagic tissue that is totally surrounding the LS2-injected FVB/N <i>Prnp<sup>KO</sup></i> embryos 5, 6 and 8, iii) the developmental defect of the ectoplacental cone (area surrounded using a dashed line ) of all the LS2-injected FVB/N <i>Prnp<sup>KO</sup></i> embryos (5–8) that even leads to its nearly complete disappearance in embryo 6 and iv) the important developmental delay and the total disorganization of the extra-embryonic ectoderm and of the ectoplacental cone of embryo 9. Scale: 250 µm. Sho: <i>Sprn</i>.</p
Prion Protein and Shadoo Are Involved in Overlapping Embryonic Pathways and Trophoblastic Development
<div><p>The potential requirement of either the Prion or Shadoo protein for early mouse embryogenesis was recently suggested. However, the current data did not allow to precise the developmental process that was affected in the absence of both proteins and that led to the observed early lethal phenotype. In the present study, using various <em>Prnp</em> transgenic mouse lines and lentiviral vectors expressing shRNAs that target the Shadoo-encoding mRNA, we further demonstrate the specific requirement of at least one of these two PrP-related proteins at early developmental stages. Histological analysis reveals developmental defect of the ectoplacental cone and important hemorrhage surrounding the <em>Prnp</em>-knockout-<em>Sprn</em>-knockdown E7.5 embryos. By restricting the RNA interference to the trophoblastic cell lineages, the observed lethal phenotype could be attributed to the sole role of these proteins in this trophectoderm-derived compartment. RNAseq analysis performed on early embryos of various <em>Prnp</em> and <em>Sprn</em> genotypes indicated that the simultaneous down-regulation of these two proteins affects cell-adhesion and inflammatory pathways as well as the expression of ectoplacental-specific genes. Overall, our data provide biological clues in favor of a crucial and complementary embryonic role of the prion protein family in <em>Eutherians</em> and emphasizes the need to further evaluate its implication in normal and pathological human placenta biology.</p> </div
Schematic representation of the embryonic phenotype induced by the lack of PrP and Shadoo.
<p>The lethal consequence of the absence of PrP and Shadoo during early mouse embryogenesis is indicated.</p
Effect of trophoblastic-restricted ShRNA- mediated <i>Sprn</i> knockdown on embryo resorption at E13.5.
*<p>p<0.05 (x<sup>2</sup> test) when compared to any of the other results.</p><p>These data are a compilation of at least 2 independent experiments. No statistically significant variability was observed between the analyzed litters (more than 4 for each lentiviral infections).</p