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.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+)-ATPase,and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Microphytobenthic biomass assessment by pigment analysis: comparison of spectrophotometry and High Performance Liquid Chromatography methods

Samples of microphytobenthos from the Tagus estuary were analysed for photosynthetic pigments by spectrophotometry and High Performance Liquid Chromatography (HPLC). Chlorophyll a values obtained with HPLC and spectrophotometry methods presented a highly significant positive correlation for both spectrophotometric methods used (with and without the correction for pheopigments), but this relationship depended on the type of sediment. We concluded that spectrophotometric methods give reliable Chl-a values, being suited for routine analysis, when a vast number of replicates is needed. However, for the correct estimation of pheopigments, HPLC analysis is indispensable. In the literature, Chl-a estimations are expressed per content (μg g⁻¹) or concentration (mg m⁻²). We discuss the influence of sediment type on the results depending on the type of unit used, and propose a simple conversion factor based on sediment water content.     

Expression and subcellular localization of a 35-kDa carbonic anhydrase IV in a human pancreatic ductal cell line (Capan-1).

The high intraluminal concentrations of HCO(3)(-) in the human pancreatic ducts have suggested the existence of a membrane protein supplying the Cl(-)/HCO(3)(-) exchanger. Membrane-bound carbonic anhydrase IV (CA IV) is one of the potential candidates for this protein. The difficulties in isolating human pancreatic ducts have led the authors to study the molecular mechanisms of HCO(3)(-) secretion in cancerous cell lines. In this work, we have characterized the CA IV expressed in Capan-1 cells. A 35-kDa CA IV was detected in cell homogenates and purified plasma membranes. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase-C indicated that this CA IV was not anchored by a glycosylphosphatidylinositol (GPI). In contrast, its detection on purified plasma membranes by an antibody specifically directed against the carboxyl terminus of human immature GPI-anchored CA IV indicated that it was anchored by a C-terminal hydrophobic segment. Immunoelectron microscopy and double-labeling immunofluorescence revealed that this CA IV was present on apical plasma membranes, and in the rough endoplasmic reticulum, the endoplasmic reticulum-Golgi intermediate compartment, the Golgi complex, and secretory granules, suggesting its transport via the classical biosynthesis/secretory pathway. The expression in Capan-1 cells of a 35-kDa CA IV anchored in the apical plasma membrane through a hydrophobic segment, as is the case in the healthy human pancreas, should make the study of its role in pancreatic HCO(3)(-) secretion easier.     

Effect of vanadate of PHA-induced proliferation of human lymphocytes from young and old subjects

The effect of sodium orthovanadate on mitogen-induced proliferation of lymphocytes from young and old human subjects is reported. We found that vanadate is not mitogenic per se; it has an inhibitory effect during the first 3 days of culture, when both differentiation and proliferation take place; it enhances DNA synthesis, acting as a co-mitogen, in the following days of culture, when proliferation prevails. In spite of the fact that lymphocytes from the two groups differ in their responsiveness to PHA and in the activity of (Na+,K+)ATPase, no difference was found as for the effects of vanadate.     

Ritanserin, a serotonin-S2 receptor antagonist, does not prevent 5-hydroxytryptophan-induced beta-EP, beta-LPH and cortisol secretion

Ritanserin, a new serotonin antagonist selective upon S2 receptor subclass is available. Thus, in order to better define the positive control of serotoninergic pathway on proopiomelanocortin (POMC)-related peptide release, a group of 7 healthy male volunteers has been submitted to a 5-hydroxytryptophane (5-OH-TP) test (200 mg p.o.) before and after 4 days Ritanserin pretreatment. Plasma beta-endorphin (beta-EP), beta-lipotropin (beta-LPH) and cortisol levels were measured hourly for 4 h after each 5-OH-TP loading. Hormonal levels were measured by specific RIAs on extracted (cortisol) and chromatographed (beta-EP and beta-LPH) plasma samples. Basal plasma concentrations of the three hormones were unchanged by Ritanserin pretreatment. Similarly, the integrated areas of beta-LPH, beta-EP and cortisol release in response to 5-OH-TP remained unaffected by the receptor blockade. These data confirm that serotonin-acting drugs are able to stimulate POMC-related peptide release and indicate that such interaction is not mediated through S2 receptor subclass.     

Hydrolysis and protection from hydrolysis of enkephalins in human plasma

Seven groups of enkephalin-degrading enzymes and three groups of inhibitors active on these enzymes were separated from human plasma. The activity of the enzymes in hydrolyzing enkephalins and of the inhibitors in protecting enkephalins from proteolysis was measured. Results obtained with the endogenous inhibitors were compared to those relative to synthetic inhibitors. Data obtained indicate that all enkephalin-degrading enzymes found in plasma are significantly inhibited by the endogenous substances present in this tissue. The inhibition of the different classes of plasma enzymes by two of the three groups of endogenous substances is quite uniform, while one group of inhibitors appears specific to dipeptidylpeptidases. Results obtained are discussed in terms of the functional role of the inhibitory substances and of the possible pharmacological implication of their presence in human plasma.