Fig 1.
Production of recombinant Sm29 protein from Schistosoma mansoni functionalized with gold nanoparticles.
(A) Recombinant Sm29 was expressed in a heterologous system and purified by affinity chromatography. Purity was assessed by SDS-PAGE 15% and Coomassie blue staining. The red arrow indicates 1 µg of pure Sm29 with approximately 17 KDa, as expected. KDa represents the molecular ladder. (B) UV-VIS assessed gold nanoparticles functionalized with cysteamine (AuNPs-Cys) at 400-1000 nm, and three representative spectra are displayed. (C) The AuNP-Cys-Sm29 complex concentration was determined by ELISA using anti-His tag antibodies against the protein (1:5000), and the absorbance detected was interpolated with a Sm29 standard curve. Four representative results of each batch yield are displayed in µg/mL. For the experiments, gel concentrations were adjusted to 5 or 10 µg/mL before use.
Fig 2.
Topical AuNP-Cys-Sm29 reduces infection in mice infected with L. braziliensis.
Mice (n = 6) were infected with L. braziliensis in the ear dermis, and three weeks later, mice were treated with topical AuNP-Cys-Sm29 (at 5 or 10μg/ml) for four weeks (boxed area). Control mice were left untreated (n = 4) or were treated with vehicle (AuNP-Cys, n = 6), and lesion development was measured weekly. Data are from a representative experiment performed with six mice per group. *p = 0.0381, AuNPs-Cys-Sm29 (5 µg/mL) vs Untreated. #p = 0.0216, AuNPs-Cys-Sm29 (5 µg/mL) vs AuNPs-Cys. (B) Area under curves (AUC) shown in (A). *p = 0.0469, AuNPs-Cys-Sm29 (5 µg/mL) vs Untreated. (C) Pictures depict lesions at 6 weeks post-infection. Data are from a representative experiment.
Fig 3.
Combination treatment of topical AuNP-Cys-Sm29 + Sbv reduces lesion development and parasite load in vivo.
Mice (n = 9–10) were infected with L. braziliensis in the ear dermis, and three weeks later, mice were treated with topical AuNP-Cys-Sm29 (5μg/ml) alone or in combination with Sbv (50 mg/kg/day) for four weeks (boxed area). Control mice (n = 9–10) were treated with vehicle (AuNP-Cys) alone or in combination with Sbv. (A) The course of lesion development was measured weekly. *p = 0.0281, AuNP-Cys-Sm29 + Sbv vs. AuNP-Cys + Sbv (week 5 post infection); #p = 0.0002, AuNP-Cys-Sm29 + Sbv vs. vehicle (AuNP-Cys) alone (weeks 5 and 6 post infection). Six weeks post-infection, mice (n = 3–4) treated with AuNP-Cys-Sm29 + Sbv or with AuNP-Cys + Sbv were euthanized, and ears were stained with H&E and analyzed by optical microscopy under 20X magnification. Representative sections are shown. (B) Remaining mice were followed up until week 12 for the measurement of ear thickness and calculation of the disease burden (AUC). *p = 0.0299; AuNPs-Cys-Sm29 + Sbv vs. vehicle (AuNP-Cys) alone. Alternatively, six weeks post infection, mice were euthanized (n = 6) and parasite load was determined at the inoculation site (C) and the dLN (D), 6 weeks post-infection, by limiting dilution analysis. *p = 0.0249; AuNP-Cys-Sm29 + Sbv vs. AuNP-Cys alone; *p = 0.0167, AuNPs-Cys-Sm29 + Sbv vs. AuNP-Cys + Sbv; *p = 0.0249, AuNPs-Cys-Sm29 + Sbv vs. AuNP-Cys + Sm29. Data are from a representative experiment.
Fig 4.
Combination treatment of topical AuNP-Cys-Sm29 + Sbv modulates cytokine production.
Mice (n = 6) were infected with L. braziliensis in the ear dermis, and three weeks later, mice were treated with topical AuNP-Cys-Sm29 (5μg/ml) alone or in combination with + Sbv (50 mg/kg/day) for four weeks. Control mice (n = 6) were treated with vehicle (AuNP-Cys) alone or in combination with Sbv. Mice were euthanized six weeks after parasite inoculation, and cells from draining lymph nodes were restimulated in vitro. Levels of cytokines were determined in culture supernatants. (A) IFN-γ: *p = 0.0317, AuNPs-Cys-Sm29 + Sbv vs. AuNP-Cys + Sm29; (B) TNF: *p = 0.0159, AuNPs-Cys-Sm29 + Sbv vs. AuNP-Cys + Sm29; *p = 0.0159, AuNPs-Cys-Sm29 + Sbv vs. AuNP-Cys, and (C) IL-10, *p = 0.0476; AuNPs-Cys-Sm29 + Sbv vs. AuNP-Cys + Sm29. Data are from a representative experiment.
Fig 5.
Frequency of cytokine-producing CD4 + T cells in mice treated with topical AuNP-Cys-Sm29 + Sbv.
Mice (n = 6) were infected with L. braziliensis in the ear dermis, and three weeks later, mice were treated with topical AuNP-Cys-Sm29 (5μg/ml) + Sbv (50 mg/kg/day) for four weeks. Control mice (n = 6) were treated with vehicle (AuNP-Cys) + Sbv. Mice were euthanized six weeks after parasite inoculation, and cells from the lesion site (ear) were stained for IFN-γ or TNF. (A) Representative contour plots, frequency of CD4+ T cells expressing IFN-γ or TNF. (B) Percentage of CD4+IFN-γ+ or CD4+TNF+ cells in mice treated with AuNP-Cys-Sm29 + Sbv or AuNP-Cys + Sbv. (C) Number of CD4+IFN-γ+ or CD4+TNF+ cells in mice treated with AuNP-Cys-Sm29 + Sbv or vehicle (AuNP-Cys) + Sbv; *p = 0.0286. Cell populations were pregated on Singlets/Live/CD45+/TCRb+/CD4+. Data are from a representative experiment.