Paracoccidioides brasiliensis Interferes on Dendritic Cells Maturation by Inhibiting PGE2 Production

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE₂, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE₂ inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-α. Assays using exogenous PGE₂ confirmed an association between PGE₂ inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.     

High levels of expression of full length human pro-alpha 2(V) collagen cDNA in pro-alpha 2(V)-deficient hamster cells

A full length cDNA encoding human pro-alpha 2(V) collagen was constructed. Partial sequencing of the cDNA and primer extension analysis of mRNA from fibroblasts found that pro-alpha 2(V) mRNA differs from the mRNAs of other fibrillar collagens in the increased length of its 5'-untranslated region. The pro-alpha 2(V) cDNA was placed downstream of the human cytomegalovirus immediate early promoter/regulatory sequences for expression studies in cultured Chinese hamster lung cells. These cells have been shown previously to synthesize large quantities of pro-alpha 1(V) homotrimers as their only collagenous product. Transfection resulted in a number of clonal cell lines that express human alpha 2(V) RNA at levels comparable to, and in some cases greater than, levels found in normal human skin fibroblasts. Pro-alpha 2(V) chains produced in the majority of clonal lines were of sufficient quantity to complex all available endogenous pro-alpha 1(V) chains. Chimeric heterotrimers, composed of hamster alpha 1(V) and human alpha 2(V) chains in a 2:1 ratio, were stable to pepsin digestion and were found predominantly associated with the cell layer. Surprisingly, pro-alpha 2(V) chains, in excess to pro-alpha 1(V) chains, were found in the extracellular matrix and, in much greater abundance, in media. These chains were pepsin sensitive, indicating that pro-alpha 2(V) chains can be secreted as nonstable homotrimers or as free chains.