Questão agrária e políticas públicas em Minas Gerais : conflitos sociais e alternativas populares

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<i>Sporothrix brasiliensis</i> Infection Modulates Antimicrobial Peptides and Stress Management Gene Expression in the Invertebrate Biomodel <i>Galleria mellonella</i>

Sporothrix brasiliensis is the most pathogenic species, responsible for the Brazilian cat-transmitted sporotrichosis hyperendemic. In this scenario, an investigation of the pathogen–host interaction can provide relevant information for future treatment strategies. To this end, the invertebrate Galleria mellonella has proven to be a suitable alternative for evaluating the virulence of pathogenic fungi, since the insect immune system is similar to the mammalian innate immune response. The aim of this work was to investigate phenotypic and molecular aspects of the immune response of G. mellonella throughout the S. brasiliensis infection. Hemocyte density and the evolution of the fungal load were evaluated. In parallel, RT-qPCR expression analysis of genes encoding antimicrobial peptides (Gallerimycin and Galiomycin) and stress management genes (C7 Contig 15362 and C8 Contig 19101) was conducted. The fungal load and hemocyte densities increased simultaneously and proportionally to the deleterious morphological events and larvae mortality. Gallerimycin, C7 Contig 15362 and C8 Contig 19101 genes were positively regulated (p S. brasiliensis infection, characterizing a time-dependent and alternately modulated profile. Galiomycin gene expression remained unchanged. Our results contribute to the future proposal of potential alternative pathways for treating and consequently controlling S. brasiliensis zoonosis, a major public health issue in Latin America

Mimotope-Based Vaccines of <i>Leishmania infantum</i> Antigens and Their Protective Efficacy against Visceral Leishmaniasis

<div><p>Background</p><p>The development of cost-effective prophylactic strategies to prevent leishmaniasis has become a high-priority. The present study has used the phage display technology to identify new immunogens, which were evaluated as vaccines in the murine model of visceral leishmaniasis (VL). Epitope-based immunogens, represented by phage-fused peptides that mimic <i>Leishmania infantum</i> antigens, were selected according to their affinity to antibodies from asymptomatic and symptomatic VL dogs' sera.</p><p>Methodology/Main Findings</p><p>Twenty phage clones were selected after three selection cycles, and were evaluated by means of <i>in vitro</i> assays of the immune stimulation of spleen cells derived from naive and chronically infected with <i>L. infantum</i> BALB/c mice. Clones that were able to induce specific Th1 immune response, represented by high levels of IFN-γ and low levels of IL-4 were selected, and based on their selectivity and specificity, two clones, namely B10 and C01, were further employed in the vaccination protocols. BALB/c mice vaccinated with clones plus saponin showed both a high and specific production of IFN-γ, IL-12, and GM-CSF after <i>in vitro</i> stimulation with individual clones or <i>L. infantum</i> extracts. Additionally, these animals, when compared to control groups (saline, saponin, wild-type phage plus saponin, or non-relevant phage clone plus saponin), showed significant reductions in the parasite burden in the liver, spleen, bone marrow, and paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD8⁺ T cells, against parasite proteins. These animals also presented decreased parasite-mediated IL-4 and IL-10 responses, and increased levels of parasite-specific IgG2a antibodies.</p><p>Conclusions/Significance</p><p>This study describes two phage clones that mimic <i>L. infantum</i> antigens, which were directly used as immunogens in vaccines and presented Th1-type immune responses, and that significantly reduced the parasite burden. This is the first study that describes phage-displayed peptides as successful immunogens in vaccine formulations against VL.</p></div

Involvement of IL-12, CD4⁺ and CD8⁺ T cells in the IFN-γ production after <i>L. infantum</i> infection.

<p>Single-cells suspensions were obtained from the spleens of mice that were immunized with B10 clone plus saponin (<b>A</b>) or C01 clone plus saponin (<b>B</b>), 10 weeks after <i>L. infantum</i> infection. Levels of IFN-γ were measured in culture supernatants by capture ELISA of spleen cells cultures stimulated with SLA (20 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. Cultures were incubated in the absence (positive control) or in the presence of 5 µg mL⁻¹ of monoclonal antibodies (mAb) against mouse IL-12 (C017.8), CD4 (GK 1.5), or mouse CD8 (53-6.7). Statistically significant differences between non-treated control cells and cultures incubated with anti-CD4, anti-CD-8 and anti-IL-12 monoclonal antibodies were observed (***<i>P <</i>0.0001). Each bar represents the mean ± standard deviation (SD) of the IFN-γ levels of the different groups.</p

Amino acid sequences from target peptides present in the selected phage clones.

<p>Amino acid sequences from target peptides present in the selected phage clones.</p

Analysis of the cellular and humoral response in BALB/c mice after <i>L. infantum</i> challenge infection.

<p>BALB/c mice (n = 8, per group) received saline or were immunized subcutaneously in their left hind footpad with 25 µg of saponin, and with B10, C01 or wild-type phage clones (1×10¹¹ phages, each one), associated with saponin. Three doses were administered at 2-week intervals, and four weeks after the last immunization, the animals (n = 4) were infected subcutaneously in their right hind footpad with 1×10⁷ stationary promastigotes of <i>L. infantum</i>, and were followed for 10 weeks. Single-cells suspensions were obtained from the spleens of mice in this time, and were non-stimulated (medium; background control), or stimulated with SLA (20 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. Levels of IFN-γ, IL-12, GM-CSF, IL-4 and IL-10 were measured in culture supernatants by capture ELISA. Each bar represents the mean ± standard deviation (SD) of the cytokines levels of the different groups (<b>A</b>). Statistically significant differences between the B10 and C01 clones plus saponin and control groups (saline, saponin and wild-type phage plus saponin groups) were observed (***<i>P <</i>0.0001). The ratio between IL-12/IL-10 and IL-12/IL-4 levels (<b>B</b>), and between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>C</b>), are also showed. Statistically significant differences between the B10 and C01 clones plus saponin and control groups were observed (***<i>P</i> <0.0001). The ratio between SLA-specific IgG1 and IgG2a antibodies levels were calculated and statistically significant differences between the B10 and C01 clones groups and the saline and saponin were also observed (*<i>P</i> <0.005) (<b>D</b>).</p

Protection of BALB/c mice vaccinated with phage clones plus saponin against <i>L. infantum.</i>

<p>Mice inoculated with saline, saponin, wild-type phage (WTP), B10 or C01 phage clones plus saponin were subcutaneously infected with 1×10⁷ stationary phase promastigotes of <i>L. infantum.</i> The number of parasites in the liver (<b>A</b>), spleen (<b>B</b>), pawś draining lymph nodes (<b>C</b>), and bone marrow (<b>D</b>) was measured, 10 weeks after challenge by a limiting-dilution technique. Each bar represents the mean ± standard deviation (SD) of the groups. Statistically significant differences in the parasite load in all evaluated organs between the B10 and C01 clones plus saponin and control groups are showed (***<i>P</i> <0.0001). Data shown are representative of two independent experiments, which presented similar results.</p

Cellular and humoral response induced in BALB/c mice by immunization with B10 or C01 phage clones plus saponin.

<p>Single cells suspensions were obtained from the spleen of mice, four weeks after the last immunization. Cells were non-stimulated (medium; background control), or separately stimulated with individual clones (1×10¹¹ phages per mL, each one) for 48 h at 37°C, 5% CO₂. IFN-γ, IL-12, GM-CSF, IL-4, and IL-10 levels were measured in culture supernatants by capture ELISA (<b>A</b>). Each bar represents the mean ± standard deviation (SD) of the groups. Statistically significant differences in the IFN-γ and IL-12 levels between the B10 or C01 clone plus saponin groups and control mice (saline, saponin and wild-type phage plus saponin groups) were observed (***<i>P</i> <0.0001). The ratio between IL-12/IL-10 and IL-12/IL-4 levels (<b>B</b>), and between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>C</b>) are also showed. Statistically significant differences in the ratios between the B10 or C01 clone plus saponin groups and control groups were also observed (***<i>P</i> <0.0001). The ratio between the <i>Leishmania</i>-specific IgG1 and IgG2a antibodies was obtained from the different groups, and statistically significant differences between the B10 or C01 clone plus saponin and control groups were observed (***<i>P</i> <0.0001) (<b>D</b>).</p

Evaluation of the selectivity and specificity of the selected phage clones.

<p>Spleen cells suspensions were obtained from naive or chronically infected with <i>L. infantum</i> BALB/c mice. Cells were non-stimulated (medium; background control), or separately stimulated with each phage clone, as well as the wild-type and non-relevant clones (1×10¹⁰ phages, each one) for 48 h at 37°C, 5% CO₂. IFN-γ and IL-4 levels were measured in culture supernatants by capture ELISA. The black circles indicate the specificity of the phage clones, which was calculated by dividing the IFN-γ and IL-4 values obtained of each evaluated individual clone by respective values of these cytokines obtained after the wild-type phage stimulus, using spleen cells derived from naive mice. With the corrected values, the ratio between IFN-γ and IL-4 with these new values was calculated, and the specificity of the each clone was defined. The white circles indicate the selectivity, which was calculated by dividing the IFN-γ and IL-4 values obtained of each evaluated individual clone by respective values of these cytokines obtained after the non-relevant phage stimulus, using spleen cells derived from infected mice. With the corrected values, a ratio between IFN-γ and IL-4 using these new values was calculated, and the selectivity of each clone was defined.</p