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SummaryWe earlier demonstrated that nitric oxide (NO) is a fungicidal molecule against Sporothrix schenckii in vitro. In the present study we used mice deficient in inducible nitric oxide synthase (iNOS -/-) and C57BL/6 wildtype (WT) mice treated with Nx-nitro-arginine (Nitro-Arg-treated mice), an NOS inhibitor, both defective in the production of reactive nitrogen intermediates, to investigate the role of endogenous NO during systemic sporotrichosis. When inoculated with yeast cells of S. schenckii, WT mice presented T-cell suppression and high tissue fungal dissemination, succumbing to infection. Furthermore, susceptibility of mice seems to be related to apoptosis and high interleukin-10 and tumour necrosis factor-a production by spleen cells. In addition, fungicidal activity and NO production by interferon-c (IFN-c) and lipopolysaccharide-activated macrophages from WT mice were abolished after fungal infection. Strikingly, iNOS -/-and Nitro-Arg-treated mice presented fungal resistance, controlling fungal load in tissues and restoring T-cell activity, as well as producing high amounts of IFN-c Interestingly, macrophages from these groups of mice presented fungicidal activity after in vitro stimulation with higher doses of IFN-c. Herein, these results suggest that although NO was an essential mediator to the in vitro killing of S. schenckii by macrophages, the activation of NO system in vivo contributes to the immunosuppression and cytokine balance during early phases of infection with S. schenckii.

Adhesive interactions play important roles in coordinating T cell migration and activation, which are mediated by binding of integrins to RGD motif found on extracellular matrix proteins. Disintegrins, isolated from snake venoms, contain the RGD sequence that confers selectivity to integrin interaction. We have investigated the ability of three RGD-disintegrins, ligands of alpha(5)beta(1) and alpha(v)beta(3), Flavoridin (Fl), Kistrin (Kr) and Echistatin (Ech), in modulating the activation of human T lymphocyte. The disintegrins induced T cell proliferation and CD69 expression. This activation parallels with actin cytoskeleton reorganization and tyrosine phosphorylation. Furthermore, the peptides induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Finally, RGD-disintegrins were capable of driving NF-kappaB nuclear translocation and c-Fos expression, in a PI3K and ERK1/2 activities dependent manner. This report is the first to show that RGD-disintegrins interact with integrins on human T lymphocyte surface, modulating cell proliferation and activation of specific pathways coupled to integrin receptor.

Lipoxins (LX) and 15-epi-LX are lipids with a potent inhibitory effect on angiogenesis, in different models in vivo and in vitro. ATL-1, a synthetic analog of 15-epi-LXA4, inhibits various actions stimulated by vascular endothelial growth factor (VEGF). However, LX actions on endothelial cells (EC) in tumor-related contexts are still unknown. Here, we investigated the modulation of EC by ATL-1, in a model that mimics tumor extravasation. We observed that the analog inhibited endothelial permeability induced by VEGF, through the stabilization of VE-cadherin/β-catenin-dependent adherens junctions. We tested the ability of MV3 cells, a highly metastatic melanoma cell line, to transmigrate across unchallenged EC monolayers for 18 h, as compared to NGM normal melanocytes. ATL-1 was able to inhibit only melanoma extravasation. MV3 cells secrete large amounts of VEGF and we observed that ATL-1 per se did not alter this ability. Melanoma cells skills to crossing endothelial monolayers were due to the steady accumulation of tumor-derived VEGF. When endothelial cells were challenged with exogenous VEGF, added at levels comparable to those secreted by MV3 cells over 18 h, and a short-term (4h) transendothelial migration assay was performed, both melanoma and melanocyte cells were able to extravasate, and ATL-1 was able to block the passage of both cells. These results indicate that ATL-1 has a potent inhibitory effect on the permeability induced by VEGF, and that this pharmacological effect could be used to block tumor extravasation across endothelial barriers, with a possible prospect of reducing the haematogenic spread of cancer cells.