Nitric oxide production (ng/ml) by J774A.1 macrophages upon treatment with LieIF/IFN-γ at late (A, B) and early (C, D) time points after <i>L. donovani</i> infection.

<p>J774A.1 cells were treated pre-infection (A, C) and post-infection (B, D) with recombinant LieIF (10 µg/ml) and recombinant IFN-γ (1 ng/ml). Nitrite accumulation in the medium was measured by addition of Griess reagent, absorbance reading of the reaction product at 570 nm. All data are presented as the mean ± S.D of at least three independent experiments. Asterisks indicate statistically significant production of NO (p≤0.05).</p

Effect of pre-infection treatment of J774A.1 macrophages with LieIF/IFN-γ on intracellular <i>L. donovani</i> growth inhibition.

<p>(A) at early (4 h) and (B) late (72 h) time points after infection. J774A.1 cells were stimulated with recombinant LieIF (10 µg/ml) and recombinant ΙFN-γ (1 ng/ml). <i>L. donovani</i> growth inhibition at the late time point (72 h) is associated with reduction of the infection rate (C) and the parasite load (D). The parasite growth inhibition was determined by the colorimetric method alamarBlue and OD was measured at 570/630 nm. Infection rate and parasite load were determined by microscopic observation upon Giemsa's staining of slides to count the mean number of infected macrophages considering 200 macrophages and the mean number of intracellular amastigotes in 200 infected macrophages, respectively. Data are presented as the mean ± S.D of at least three independent experiments. Asterisks indicate statistically significant differences (p≤0.05).</p

Correlation of TNF-α production with generation of ROS in parasitized J774A.1 cells.

<p>The effect of TNF-α on ROS production in J774A.1 cells pre- and post- infection treated (A and B, respectively) with recombinant LieIF/IFN-γ (10 µg/ml and 1 ng/ml respectively), with or without anti-mouse TNF-α monoclonal antibody (4 µg/ml), was determined at the late time point (72 h) after <i>L. donovani</i> infection. Cells were analyzed for intracellular ROS with FACS Calibur. All data are presented as the mean ± S.D. of three independent experiments. Data are expressed by the formula: Geo Mean  =  Geo Mean_{(J774+LieIF+MON2+IFN-γ)} – Geo Mean_{(J774+MON2+IFN-γ).}</p

Expression and purification of the recombinant LieIF protein.

<p>(A) An aliquot of the purified LeIF was resolved by SDS polyacrylamide gel and stained with Coomassie brilliant blue. The positions of the Bio-Rad prestained marker (in kDa) are indicated at the left. (B) The identity of the protein LeIF was verified by using rabbit anti-LieIF primary polyclonal antibodies (1/1000 dilution).</p

ROS generation by J774A.1 macrophages pre- and post- infection treated (A and B, respectively) with recombinant LieIF (10 µg/ml) and recombinant IFN-γ (1 ng/ml).

<p>ROS generation was determined at the late time point (72 h) after <i>L. donovani</i> infection. Cells were labelled with 5 µM H₂DCFDA fluorescent probe and fluorescence of cells reacting with the probe was estimated by FACS analysis. All data are presented as the mean ± S.D of at least three independent experiments. Asterisks indicate statistically significant production of ROS (p≤0.05) compared to the control group of infected J774A.1 cells stimulated with IFN-γ (J774 + MON2 + IFN-γ). NL: for medium only; MON2: for the MHOM/IN/1996/THAK35 <i>L. donovani</i> strain used.</p

Effect of post-infection treatment of J774A.1 macrophages with LieIF/IFN-γ in intracellular <i>L. donovani</i> growth inhibition.

<p>(A) at early (19 h) and (B) late (72 h) time points after infection. J774A.1 cells were stimulated with 10 µg/ml recombinant LieIF and 1 ng/ml recombinant ΙFN-γ. <i>L. donovani</i> growth inhibition was determined by the colorimetric method alamarBlue and OD was measured at 570/630 nm. Data are presented as the mean ± S.D of at least three independent experiments. Asterisks indicate statistically significant inhibition of parasite growth (p≤0.05).</p

Ordonnance du roi, portant création de deux sous-aides majors dans le régiment de ses Gardes suisses, & de deux aides-majors dans chacun des régimens suisses & grisons qui sont à son service . Du 22 mars 1762

[Acte royal. 1762-03-22. Versailles]Avec mode text

Ordonnance du roi, pour régler l'établissement des recrues des troupes françoises, le prix des engagemens, la forme desdits engagemens, & celle des congés . Du 1er février 1763

[Acte royal. 1763-02-01. Versailles]Avec mode text

Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia

<div><p>A gp63PCR method was evaluated for the detection and characterization of <i>Leishmania (Leishmania)</i> (<i>L.</i>) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a <i>L. infantum</i> specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different <i>L. infantum</i> endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to <i>L. infantum</i> species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of <i>L. infantum</i> infections in dogs in Tunisia.</p></div

Gp63PCR-RFLP patterns of <i>Leishmania</i> parasites obtained from dog biopsies.

<p>A: digestion with <i>Msc</i>I restriction enzyme; B: digestion with <i>Sal</i>I restriction enzyme. 1: <i>L. donovani</i>, 2: <i>L. infantum</i>, 3: LN112, 4: LN129, 5: LN26, 6: LN11, 7: LN80, 8: LN2, 9: LN39, 10: LN77, 11: LN102, 12: LN110, 13: J1, 14: J3, 15: J5, 16: J6, 17: J7. All sizes are indicated in bp.</p