Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungusParacoccidioidomycosis (PCM) is a granulomatous disease caused by the thermodimorphic fungus Paracoccidioides brasiliensis, which is prevalent in Brazil and other Latin American countries (5, 6, 33). Some individuals develop one of the two main clinical forms of PCM. The acute form is characterized by impaired cellular immunity, negative delayed-type hypersensitivity reactions, increased systemic proliferation of the fungus, and high mortality rate. The chronic form, ranging from mild to severe chronic disease, shows exacerbated host cellular immune responses and formation of granulomas containing fungal cells and may evolve to develop extensive sequelae, including fibrotic lesions and impairment of lung function (33, 39).Vaccines against PCM are still not available for human use, but promising formulations have been experimentally tested during the last few years. Irradiated P. brasiliensis or cellular antigens fractionated by anion-exchange chromatography conferred partial protection against fungal proliferation in the murine model (11). The extracellular gp43 glycoprotein, the major diagnostic antigen of P. brasiliensis, is the most intensively studied component aimed at a vaccine for PCM control. Previous reports have shown that mice immunized with the purified protein, DNA, or anti-idiotypic monoclonal antibody were partially protected against challenges by P. brasiliensis (28,36,37,40). A 15-amino-acid peptide (QTLIAIHTLAIRY AN), designated P10, contains the gp43 immunodominant CD4 ϩ T-cell-specific epitope presented by major histocompatibility complex class II molecules from three different mouse haplotypes (37) and most human HLA-DR alleles (17, 18). Indeed, parenteral immunization with P10 in complete Freund adjuvant (CFA), or in the form of a truncated multiple-antigen peptide (MAP) complex, induced protective Th1 cellular immune responses in mice against intratracheal (i.t.) challenge with a virulent P. brasiliensis isolate (37,38,41).The rational use of vaccines has been significantly improved after elucidation of innate immune mechanisms in mammalian cells. The recognition of distinct pathogen-associated molecular patterns by members of the Toll-like receptor (TLR) family initiates a signaling cascade mediated by adaptor proteins, including MyD88 and interleukin-1 (IL-1) receptor-associated kinase, that culminates in the production of proinflammatory cytokines, such as tumor necrosis factor alpha and IL-12, and increased expression of cell surface molecules involved in epitope presentation by antigen-presenting cells (APC) (1,19). Proper APC activation by TLR agonists represents a key step for an effective adaptive immune response induced by pathogens or vaccines and explains, at least in part, the marked adjuvant effects of several bacterial molecules, including lipopolysaccharides, lipoproteins, peptidoglycan fragments, and flagellins (2).Flagellin, the structural subunit of bacterial flagellum, is a

Lomentospora prolificans is an emerging opportunistic fungus with a high resistance to antifungal agents and it can cause localized infections in immunocompetent patients and disseminated infections with a high mortality rate in immunosuppressed patients. Glucosylceramides (GlcCer) are synthetized in the majority of known fungal pathogens. They are bioactive molecules presenting different functions, such as involvement in fungal growth and morphological transitions in several fungi. The elucidation of the primary structure of the fungal surface glycoconjugates could contribute for the understanding of the mechanisms of pathogenicity. In this work, GlcCer species were isolated from mycelium and conidia forms of L. prolificans and their chemical structures were elucidated by mass spectrometry (ESI-MS). GlcCer purified from both forms presented a major species at m/z 750 that corresponds to N -2-hydroxyhexadecanoyl-1-β- D -glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against GlcCer could recognize L. prolificans GlcCer species from mycelium and conidia, suggesting a conserved epitope in fungal GlcCer. In addition, in vivo assays showed that purified GlcCer species from both forms was able to induce a high secretion of pro-inflammatory cytokines by splenocytes. GlcCer species also promote the recruitment of polymorphonuclear, eosinophils, small peritoneal macrophage (SPM) and mononuclear cells to the peritoneal cavity. GlcCer species were also able to induce the oxidative burst by peritoneal macrophages with NO and superoxide radicals production, and to increase the killing of L. prolificans conidia by peritoneal macrophages. These results indicate that GlcCer species from L. prolificans are a potent immune response activator.

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