Cytokine profiles of mice immunized with coated SmRho-chitosan nanoparticles.

<p>Splenocytes isolated from mice immunized with CH-Rho-Alg, CH-Rho-Cpg-Alg, and CH-Rho-Alg (i.m.) were assayed for IL-10 (A) and INF-γ (B) production in response to <i>in vitro</i> stimulation with SWAP (25 µg/ml), rSmRho (25 µg/ml), SEA (25 µg/ml) or medium alone as control. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001894#s3" target="_blank">Results</a> represent the mean ± SD of each group. *Statistically significant differences between cytokines produced after SWAP, rSmRho or SEA stimulation compared with unstimulated splenocytes (control) (p<0.05).</p

Expression and purification of rSmRho.

<p>(A) Western Blotting profile of the <i>E. coli</i> (BL21 pRARE) transformed with the pDEST42-SmRho. Lane: MW – Prestained protein molecular weight marker (Fermentas). Lanes 1 and 2 represent a clone before and after induction with 0.5 mM IPTG, respectively. Lanes 3 and 4: soluble and insoluble fraction after <i>E. coli</i> (BL21 pRARE) lysis, respectively. (B) SDS-PAGE 12% profile of Ni²⁺ chromatography. Lanes: MW – Unstained protein molecular weight marker (Fermentas); 1 - flow through; 2–5 fractions of rSmRho–6×HIS-tag fusion protein eluted after Ni2+ chromatography. Positions of molecular mass standards (kDa) are indicated. Arrows indicate the purified rSmRho.</p

Representative granulome histopathology of lungs from infected mice after 40 and 90 days of infection.

<p>BALB/c mice were euthanized 40 and 90 days after treatment. The lungs, spleens, and livers were excised, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. The sections were stained with hematoxylin–eosin and examined microscopically. Infected, group only infected with <i>P. brasiliensis</i>. Infected/T, same as Infected, but treated with fluconazole. rPb27/T, group infected and posteriorly immunized with rPb27 and treated with fluconazole. In each photo, the scale bar represents 25.9 µm.</p

SmRho loading efficiency on chitosan particles before and after coating with alginate.

<p>SmRho loading efficiency on chitosan particles before and after coating with alginate.</p

Release profile of protein SmRho from alginate-coated chitosan nanoparticles in SGF (A) and SIF (B) at 37°C (mean ± SD, n = 3).

<p>Release profile of protein SmRho from alginate-coated chitosan nanoparticles in SGF (A) and SIF (B) at 37°C (mean ± SD, n = 3).</p

Protective effect and liver granuloma size induced by C57BL/6 mice vaccination and challenged with 25 <i>S. mansoni</i> cercariae.

*<p> <b>Statistically significant compared with the control group (p<0.05).</b></p

IgG isotypes production against rPb27 by infected and immunized mice.

<p>Antibody response against rPb27 in mice infected and immunized with this recombinant protein associated or not with fluconazole chemotherapy was determined by ELISA assay after 40 (A) and 90 (B) days of treatment. Control, mice without any intervention. rPb27, group infected and posteriorly immunized with rPb27. rPb27/T, same as rPb27, but treated with fluconazole. Bars represent the means and standard deviations of optical density (O.D.) at 1∶400 serum dilution in each experimental group (n = 3). * significant (p<0,05) difference in relation to the control group. # significant (p<0,05) difference in relation to the rPb27 group.</p

Representative histopathology of lungs, livers and spleens from infected mice, after 40 days of treatment.

<p>BALB/c mice were euthanized 40 days after treatment. The lungs, spleens, and livers were excised, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. The sections were stained with hematoxylin–eosin and examined microscopically. Infected, group only infected with <i>P. brasiliensis</i>. Infected/T, same as Infected, but treated with fluconazole. rPb27, group infected and posteriorly immunized with rPb27. rPb27/T, same as rPb27, but treated with fluconazole. In each lung photos, the scale bar represents 427.3 µm, while in each liver and spleen photos, the scale bar represents 56.9 µm.</p

Representative histopathology of lungs, livers and spleens from infected mice, after 90 days of treatment.

<p>BALB/c mice were euthanized 90 days after treatment. The lungs, spleens, and livers were excised, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. The sections were stained with hematoxylin–eosin and examined microscopically. Infected, group only infected with <i>P. brasiliensis</i>. Infected/T, same as Infected, but treated with fluconazole. rPb27, group infected and posteriorly immunized with rPb27. rPb27/T, same as rPb27, but treated with fluconazole. In each lung photos, the scale bar represents 416.6 µm, while in each liver and spleen photos, the scale bar represents 55.6 µm.</p

Fungal recovery in lung, spleen and liver of infected mice.

<p>The CFUs were estimated 40 (A) and 90 days (B) post treatment in organs from mice infected intratracheally with 3×10⁵<i>P. brasiliensis</i> yeast cells and subjected to fluconazole treatment combined or not with rPb27 immunization. Control mice were only infected with <i>P. brasiliensis</i> (Infected), adjuvant mice were inoculated with <i>C. parvum</i>-Al(OH)₃ with fluconazole treatment (Adjuvant/T) or not (Adjuvant), and rPb27 mice were immunized with recombinant protein combined to fluconazole treatment (rPb27/T) or not (rPb27). All groups of mice were infected with the same number of yeast cells. Bars represent the Log₁₀(UFC/g) means and standard deviations from organs of 3 to 5 animals in each group. * significant (p<0,05) difference in relation to the group of mice only infected.</p