Cytokines.

<p>BALB/c mice (seven/group) were infected with 1x10⁶ of <i>P</i>. <i>brasiliensis</i> yeast. On 7 and 14 days, the animals were treateds with pMAC/PS-scFv. As control, the BALB/c mice was treateds with PBS (50μL) or empty vector. After seven days of last burst, the lung (A) and lynph nodes (B) cells were cultivated in vitro for 24 hours and the IFN-γ, IL-12 and IL-4 were measured by ELISA. *p<0.05 e **p<0,001. Results are representative of three independent experiments.</p

Phenotype of lung cells.

<p>BALB/c mice (seven/group) were infected with <i>P</i>. <i>brasiliensis yeast</i> (1x10⁶). After that, the animals received twice treatments with PBS (50μL), DC (1x10⁶), DC-pMAC/PS (1x10⁶ cells plus 20ng/mL DNA) and DC-pMAC/PS-scFv (1x10⁶ cells plus 20ng/mL DNA). After seven days from the last treatment, the lung cells were stained with anti-CD3e/CD4 (A), anti-CD3e/CD8 (B) and anti-CD40/CCR7 (C). As control of the infection, BALB/c mice received only PBS (50μL) by intratracheal pathway. The data was analyzed by flow cytometer. Flow cytometry graphs: one representative experiment is shown.</p

CD4⁺ and CD8⁺ T cells activation.

<p>Lymph node cells from seven BALB/c mice per group were obtained after intramuscular immunization with PBS, pMAC/PS or pMAC/PS-scFv. 3x10⁵ cells were cultivated with 20 μg/mL of gp43 antigen. After that, the cells were stained with CFSE and after 72 hours, lymphoproliferation of CD4⁺ (A) and CD8⁺ (B) was analyzed by flow cytometry. As a positive control, we used concanavalin mitogen (ConA). *p<0.05. Results are representative of three independent experiments.</p

Humoral response.

<p>BALB/c mice (seven/group) were infected previously with <i>P</i>. <i>Brasiliensis</i> yeast (1x10⁶) by intratracheal pathway. The animals were treateds twice with pMAC/PS-scFv and total IgG was measured (A). The ratio between IgG2a/IgG1 was analyse (B) *p<0.05. Results are representative of three independent experiments.</p

DCs molecule expression.

<p>DCs were transfected with pMAC/PS-scFv. After 48 hours the total DCs were stained with anti-MHCII/CD11c, anti-CD86, anti-CD40 and anti-CD80. The data were analyzed by flow cytometer. Results are representative of three independent experiments.</p

Phenotype of DCs and T cells.

<p>BALB/c mice (seven/group) were immunized via intramuscular injection of PBS, pMAC/PS or pMAC/PS-scFv. After 7 days the lymph nodes were obtained and the DCs CD11c⁺/CD8⁺, CD11c⁺/CD40 and CD11c⁺/DEC205 (A) and CD4⁺ (B) and CD8⁺ (C) T cells were analyzed by flow cytometer. *p<0.05. A. Flow cytometry graph: one representative experiment is shown. B and C: Data are expressed as the number of cells obtained with specific Ab. Results are representative of three independent experiments.</p

Transcriptional response of murine macrophages upon infection with opsonized Paracoccidioides brasiliensis yeast cells

Paracoccidioides brasiliensis is the etiologic agent of the Paracoccidioidomycosis the most common systemic mycosis in Latin America. Little is known about the regulation of genes involved in the innate immune host response to P. brasiliensis. We therefore examined the kinetic profile of gene expression of peritoneal macrophage infected with P. brasiliensis. Total RNA from macrophages at 6, 24 and 48 h was extracted, hybridized onto nylon membranes and analyzed. An increase in the transcription of a number of pro-inflammatory molecules encoding membrane proteins, metalloproteases, involved in adhesion and phagocytosis, are described. We observed also the differential expression of genes whose products may cause apoptotic events induced at 24 h. In addition, considering the simultaneous analyses of differential gene expression for the pathogen reported before by our group, at six hours post infection, we propose a model at molecular level for the P. brasiliensis-macrophage early interaction. In this regard, P. brasiliensis regulates genes specially related to stress and macrophages, at the same time point, up-regulate genes related to inflammation and phagocytosis, probably as an effort to counteract infection by the fungus. (c) 2007 Elsevier Masson SAS. All fights reserved