Biomedical subjects

Marcio L Rodrigues

Publications and source records attributed to Marcio L Rodrigues.

At least 19 recordsLinked to original sources

PubMed2006

Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport.

The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment.

Animals↗

PubMed2006

Binding of glucuronoxylomannan to the CD14 receptor in human A549 alveolar cells induces interleukin-8 production.

Glucuronoxylomannan (GXM) is the major capsular polysaccharide of Cryptococcus neoformans. GXM receptors have been characterized in phagocytes and endothelial cells, but epithelial molecules recognizing the polysaccharide remain unknown. In the current study, we demonstrate that GXM binds to the CD14 receptor in human type II alveolar epithelial cells, resulting in the production of the proinflammatory chemokine interleukin-8.

Cryptococcus neoformans↗

PubMed2006

Characterization of an ecto-ATPase activity in Fonsecaea pedrosoi.

In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 84.6 +/- 11.3 nmol Pi h(-1) mg(-1) mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 +/- 27.6 nmol Pi h(-1) mg(-1)) in the presence of 5 mM MgCl2, with values of Vmax and apparent Km for Mg-ATP(2-) corresponding to 541.9 +/- 48.6 nmol Pi h(-1) mg(-1) cellular dry weight and 1.9 +/- 0.2 mM, respectively. The Mg2+-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A1(V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg2+ -stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P2 purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.

Adenosine Triphosphatases↗

PubMed2006

An ectophosphatase activity in Cryptococcus neoformans.

There is increasing evidence in the literature showing that fungal pathogens express biologically active ectoenzymes. The expression of surface phosphatases at the cell surface of Cryptococcus neoformans, the etiologic agent of cryptococcosis, was evaluated in the present study. Different isolates of C. neoformans express ectophosphatase activity, which is not influenced by capsule size or serotype. The cryptococcal enzyme is an acid phosphatase, inhibited by classic inhibitors of ectophosphatases, including ammonium molybdate and sodium salts of fluoride and orthovanadate. Only the inhibition of enzyme activity caused by sodium orthovanadate has been shown to be irreversible. The cryptococcal ectoenzyme is also inhibited by Zn2+ and inorganic phosphate, the final product of reactions catalyzed by phosphatases. The ectophosphatase from C. neoformans efficiently releases phosphate groups from different phosphorylated amino acids, giving a higher rate of phosphate removal when phosphothreonine is used as a substrate. Yeast cells with irreversibly inhibited ectophosphatases are less capable of adhering to animal epithelial cells than fungi fully expressing enzyme activity, suggesting that ectoenzyme expression can contribute to the pathogenesis of C. neoformans.

Cryptococcus neoformans↗

PubMed2005

Glucuronoxylomannan-mediated interaction of Cryptococcus neoformans with human alveolar cells results in fungal internalization and host cell damage.

Infection by Cryptococcus neoformans begins with inhalation of infectious propagules. Fungi reach the lung tissue and interact with epithelial cells in a crucial but poorly understood process. In this study, the interaction of C. neoformans with the human alveolar epithelial cell lineage A549 was investigated, focusing on the relevance of the capsular polysaccharide in this process. The association of encapsulated strains with A549 cells was significantly inhibited by a monoclonal antibody to glucuronoxylomannan (GXM), a major component of the cryptococcal capsule. A purified preparation of GXM produced similar results, suggesting the occurrence of surface receptors for this polysaccharide on the surface of alveolar cells. A549 cells were in fact able to bind soluble GXM, as confirmed by indirect immunofluorescence analysis using the anti-polysaccharide antibody. C. neoformans is internalized after GXM-mediated interaction with A549 cells in a process that culminates with death of host cells. Our results suggest that C. neoformans can use GXM for attachment to alveolar epithelia, allowing the fungus to reach the intracellular environment and damage host cells through still uncharacterized mechanisms.

Cell Line↗

PubMed2005

The multitude of targets for the immune system and drug therapy in the fungal cell wall.

Recent studies on fungi revealed that several cytosolic and membrane components migrate to the cell wall together with secreted proteins and biosynthetic polysaccharides to build a dynamic immunoreactive structure. New aspects of fungal cell wall assembly and biosynthesis, focusing on the potential of glycolipids, melanin, heat-shock proteins, histone and surface antigens as targets of drugs and antifungal antibodies are discussed.

Antifungal Agents↗

PubMed2005

Ceramide glycosylation and fatty acid hydroxylation influence serological reactivity in Trypanosoma cruzi glycosphingolipids.

Ceramide mono (CMH) or dihexoside (CDH) fractions from Trypanosoma cruzi (Dm28c clone) were identified as glucosyl and lactosylceramides containing non-hydroxylated fatty acids. The di-glycosylated form was much more efficiently recognized by sera from T. cruzi-immunized rabbits, indicating that glycosylation influences antigenicity. Fatty acid hydroxylation was also a determinant of serological reactivity, since an alpha-hydroxylated CMH, only present at the Y clone, was recognized by the hyperimmune sera. In summary, these data indicate that T. cruzi CMHs with non-hydroxylated fatty acids are unable to induce antibody responses in animal hosts, which is reverted by the addition of a sugar residue or an alpha-hydroxyl group.

Animals↗

PubMed2005

Characterization of an ecto-ATPase activity in Cryptococcus neoformans.

Cryptococcus neoformans is the causative agent of pulmonary cryptococcosis and cryptococcal meningoencephalitis, which are major clinical manifestations in immunosuppressed patients. In the present study, a surface ATPase (ecto-ATPase) was identified in C. neoformans yeast cells. Intact yeasts hydrolyzed adenosine-5'-triphosphate (ATP) at a rate of 29.36+/-3.36nmol Pi/hx10(8) cells. In the presence of 5 mM MgCl(2), this activity was enhanced around 70 times, and an apparent K(m) for Mg-ATP corresponding to 0.61mM was determined. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases, V-ATPases, Na(+)-ATPases or P-ATPases had no effect on the cryptococcal ATPase, but extracellular impermeant compounds reduced enzyme activity in living cells. ATP was the best substrate for the cryptococcal ecto-enzyme, but it also efficiently hydrolyzed inosine 5'-triphosphate (ITP), cytidine 5'-triphosphate (CTP), guanosine 5'-triphosphate (GTP) and uridine-5'-triphosphate (UTP). In the presence of ATP, C. neoformans became less susceptible to the antifungal action of fluconazole. Our results are indicative of the occurrence of a C. neoformans ecto-ATPase that may have a role in fungal physiology.

Adenosine Triphosphatases↗

PubMed2005

Structure, cellular distribution, antigenicity, and biological functions of Fonsecaea pedrosoi ceramide monohexosides.

Monohexosylceramides (CMHs, or cerebrosides) have been reported as membrane and cell wall constituents of both pathogenic and nonpathogenic fungi, presenting remarkable differences in their ceramide moiety compared to mammalian CMHs. Current evidence suggests that CMHs are involved in fungal differentiation and growth and contribute to host immune response. Here we describe a structural diversity between cerebrosides obtained from different forms of the human pathogen Fonsecaea pedrosoi. The major CMH species produced by conidial forms displayed the same structure previously demonstrated by our group for mycelia, an N-2'-hydroxyhexadecanoyl-1-beta-d-glucopyranosyl-9-methyl-4,8-sphingadienine. However, the major cerebroside species purified from sclerotic cells carries an additional hydroxyl group, bound to its long-chain base. The structural difference between cerebrosides from mycelial and sclerotic cells was apparently not relevant for their antigenicity, since they were both recognized at similar levels by sera from individuals with chromoblastomycosis and a monoclonal antibody to a conserved cerebroside structure. Preincubation of fungal cells with anti-CMH monoclonal antibodies had no effect on the interaction of F. pedrosoi sclerotic cells with murine macrophages. In contrast to what has been described for other fungal species, sclerotic bodies are resistant to the antifungal action of anti-CMH antibodies. Immunofluorescence analysis showed that recognition of sclerotic cells by these antibodies only occurs at cell wall regions in which melanization is not evident. Accordingly, melanin removal with alkali results in an increased reaction of fungal cells with anti-CMH antibodies. Our results indicate that cerebroside expression in F. pedrosoi cells is associated with dimorphism and melanin assembly on the fungal cell wall.

Animals↗

PubMed2005

Exposure of human leukemic cells to direct electric current: generation of toxic compounds inducing cell death by different mechanisms.

Treatment with direct electric current (DC) influences the growth of several cancer cells. In this work, we evaluated the effects of DC treatment on the human leukemic cell line HL60. Human cells were separately treated in the presence of the cathode or the anode or without contact with the electrodes. In all systems, DC-treated cells presented an impaired ability to proliferate. Growth inhibition was dependent on the generation of soluble products of electrolysis. Cathodic treatment of HL60 cells predominantly induced lysis, whereas treatment without contact with electrodes did not induce alterations in cell viability. In contrast, cell stimulation by the anode resulted in irreversible membrane damage, as demonstrated by trypan blue and 7-aminoactinomycin staining. Analysis of these cells by transmission electron microscopy indicated that necrosis is a major mechanism inducing cell death. In addition, apoptotic-like cells were observed under light microscopy after anodic treatment. Accordingly, DNA from anodic-treated cells presented a typical pattern of apoptosis. Apoptotic cell death was only generated after the treatment of HL60 cells in conditions in which the generation of chloride-derived compounds was favored. These results indicate that the nature of the products from cathodic or anodic reactions differently influences the mechanisms of cell death induced by DC-derived toxic compounds.

Cell Death↗

PubMed2004

Glucosylceramides in Colletotrichum gloeosporioides are involved in the differentiation of conidia into mycelial cells.

Glucosylceramides (GlcCer) were extracted from the plant pathogen Colletotrichum gloeosporioides and purified by several chromatographic steps. By using electrospray ionization mass spectrometry and nuclear magnetic resonance, GlcCer from C. gloeosporioides were identified as N-2'-hydroxyoctadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and N-2'-hydroxyoctadecenoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against these structures were produced and used as tools for the evaluation of the role of GlcCer in the morphological transition of C. gloeosporioides. In the presence of antibodies to GlcCer, the differentiation of conidia into mycelia was blocked. Since GlcCer is present in several plant pathogens, the inhibitory activity of external ligands recognizing these structures may be applicable in other models of fungal infections.

Antibodies, Monoclonal↗

PubMed2004

Structure and biological functions of fungal cerebrosides.

Ceramide monohexosides (CMHs, cerebrosides) are glycosphingolipids composed of a hydrophobic ceramide linked to one sugar unit. In fungal cells, CMHs are very conserved molecules consisting of a ceramide moiety containing 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids, and a carbohydrate portion consisting of one residue of glucose or galactose. 9-Methyl 4,8-sphingadienine-containing ceramides are usually glycosylated to form fungal cerebrosides, but the recent description of a ceramide dihexoside (CDH) presenting phytosphingosine in Magnaporthe grisea suggests the existence of alternative pathways of ceramide glycosylation in fungal cells. Along with their unique structural characteristics, fungal CMHs have a peculiar subcellular distribution and striking biological properties. In Pseudallescheria boydii, Candida albicans, Cryptococcus neoformans, Aspergillus nidulans, A. fumigatus, and Schizophyllum commune, CMHs are apparently involved in morphological transitions and fungal growth. The elucidation of structural and functional aspects of fungal cerebrosides may therefore contribute to the design of new antifungal agents inhibiting growth and differentiation of pathogenic species.

Cerebrosides↗

PubMed2004

Differential expression of sialylglycoconjugates and sialidase activity in distinct morphological stages of Fonsecaea pedrosoi.

The expression of sialoglycoconjugates in Fonsecaea pedrosoi conidia, mycelia, and sclerotic cells was analyzed using influenza A and C virus strains, sialidase treatment, and lectin binding. Conidium and mycelium whole cells were recognized by Limax flavus (LFA), Maackia amurensis (MAA), and Sambucus nigra (SNA) lectins, denoting the presence of surface sialoglycoconjugates containing alpha 2,3- and alpha 2,6-sialylgalactosyl sequences. Sialidase-treated conidia reacted more intensively with peanut agglutinin (PNA), confirming the occurrence of sialyl-galactosyl linkages. Conidial cells agglutinated in the presence of influenza A and C virus strains, which confirmed the results obtained from lectin-binding experiments and revealed the presence of sialoglycoconjugates bearing 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) surface structures. Western blotting analysis with peroxidase-labeled LFA demonstrated the occurrence of sialylglycoproteins in protein extracts from conidia and mycelia, with molecular masses corresponding to 56 and 40 kDa. An additional band of 77 kDa was detected in conidial extracts, suggesting an association between sialic acid expression and morphogenesis. Synthesis of sialic acids was correlated with sialidase expression, since both conidial and mycelial morphological stages presented secreted and cell-associated enzyme activity. Sialoglycoconjugates were not detected in F. pedrosoi sclerotic cells from in vitro and in vivo sources, which also do not express sialidase activity. The surface sialyl residues in F. pedrosoi are apparently involved in the fungal interaction with immune effector cells, since sialidase-treated conidia were less resistant to phagocytosis by human neutrophils from healthy individuals. These findings suggest that sialic acid expression in F. pedrosoi varies according to the morphological transition and may protect infecting propagules against immune destruction by host cells.

Ascomycota↗

PubMed2004

Antinociceptive and free radical scavenging activities of Cocos nucifera L. (Palmae) husk fiber aqueous extract.

In the current study, the analgesic and free radical scavenging properties of an aqueous extract from the husk fiber of Cocos nucifera L. (Palmae) were demonstrated by the use of in vivo and in vitro models. The orally administered Cocos nucifera aqueous extract (200 or 400 mg/kg) inhibited the acetic acid-induced writhing response in mice. Tail flick and hot plate assays demonstrated that treatment of animals with this plant extract at 200 mg/kg induced attenuation in the response to a heat stimulus. A LD(50) of 2.30 g/kg was obtained in acute toxicity tests. Topic treatment of rabbits with the Cocos nucifera extract indicated that it does not induce any significant dermic or ocular irritation. In vitro experiments using the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) photometric assay demonstrated that this plant extract also possesses free radical scavenging properties.

Administration, Oral↗

PubMed2004

A monoclonal antibody to glucosylceramide inhibits the growth of Fonsecaea pedrosoi and enhances the antifungal action of mouse macrophages.

Fungal glucosylceramides (GlcCer) are conserved lipid components in a large variety of pathogenic and non-pathogenic fungal species, but their biological functions are still unclear. Recent studies demonstrate that GlcCer are immunologically active components inducing the production of antifungal antibodies. In this work, a major GlcCer was purified and characterized from mycelial forms of Fonsecaea pedrosoi, the most frequent causative agent of chromoblastomycosis. As determined by fast atom bombardment mass spectrometry (FAB-MS) analysis, the purified molecule was identified as the conserved structure N-2'-hydroxyhexadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. A monoclonal antibody (Mab) against this structure was used in indirect immunofluorescence with the different morphological stages of F. pedrosoi. Only the surface of young dividing cells was recognized by the antibody. Treatment of F. pedrosoi conidia with the Mab against GlcCer resulted in a clear reduction in fungal growth. In addition, the Mab also enhanced phagocytosis and killing of F. pedrosoi by murine cells. These results suggest that, in F. pedrosoi, GlcCer seem to be cell wall components targeted by antifungal antibodies that directly inhibit fungal development and also enhance macrophage function, supporting the use of monoclonal antibodies to GlcCer as potential tools in antifungal immunotherapy.

Animals↗

PubMed2004

Ectophosphatase activity in conidial forms of Fonsecaea pedrosoi is modulated by exogenous phosphate and influences fungal adhesion to mammalian cells.

A cell-wall-associated phosphatase in hyphae of Fonsecaea pedrosoi, a fungal pathogen causing chromoblastomycosis, was previously characterized by the authors. In the present work, the expression of an acidic ectophosphatase activity in F. pedrosoi conidial forms was investigated. The surface phosphatase activity in F. pedrosoi is associated with the cell wall, as demonstrated by transmission electron microscopy. This enzyme activity was strongly inhibited by exogenous inorganic phosphate (P(i)). Accordingly, removal of P(i) from the culture medium of F. pedrosoi resulted in a marked (130-fold) increase of ectophosphatase activity. With the artificial phosphatase substrate p-nitrophenyl phosphate, a Km value of 0.63+/-0.04 mM was estimated for the phosphatase activity of fungal cells strongly expressing the enzyme activity. This enzyme activity was not modulated by cations. Conidia with greater ectophosphatase activity showed greater adherence to mammalian cells than did fungi cultivated in the presence of P(i) (low phosphatase activity). Surface phosphatase activity was apparently involved in the adhesion to host cells, since the enhanced attachment of F. pedrosoi to host cells was reversed by pre-treatment of conidia with phosphatase inhibitor. Since conidial forms are the putative infectious propagules in chromoblastomycosis, the expression and activity of acidic surface phosphatases in these cells may contribute to the early mechanisms required for disease establishment.

Animals↗

PubMed2004

Melanin from Fonsecaea pedrosoi induces production of human antifungal antibodies and enhances the antimicrobial efficacy of phagocytes.

Fonsecaea pedrosoi is a fungal pathogen that produces melanin. The functions of melanin and its possible influence in the protective immunological response during infection by F. pedrosoi are not known. In this work, treatment of F. pedrosoi mycelia with proteases and glycosidases followed by a denaturing agent and hot concentrated acid left a black residue. Scanning electron microscopy demonstrated that this processed melanized residue resembled very closely the intact mycelium in shape and size. Melanin particles were also isolated from culture fluids of conidia or sclerotic forms of F. pedrosoi. Secreted melanins were reactive with sera from infected human patients, suggesting that F. pedrosoi synthesizes melanin in vivo. The antibodies against melanin were purified from patients' sera and analyzed by indirect immunofluorescence. They reacted with sclerotic cells from patients' lesions as well as with sclerotic bodies cultivated in vitro, conidia, mycelia, and digested residues. Treatment of F. pedrosoi with purified antibodies against melanin inhibited fungal growth in vitro. The interaction of F. pedrosoi with phagocytes in the presence of melanin resulted in higher levels of fungal internalization and destruction by host cells, which was accompanied by greater degrees of oxidative burst. Taken together, these results indicate that melanin from F. pedrosoi is an immunologically active fungal structure that activates humoral and cellular responses that could help the control of chromoblastomycosis by host defenses.

Animals↗

PubMed2003

Differentiation of Fonsecaea pedrosoi mycelial forms into sclerotic cells is induced by platelet-activating factor.

Platelet-activating factor (PAF) has been shown to induce the differentiation of several cell types. In this work, we evaluated the effects of PAF on the formation of sclerotic cells of Fonsecaea pedrosoi, the major causative agent of chromoblastomycosis. Cell differentiation was evaluated by light and electron microscopy, which showed that treatment of mycelial forms with PAF results in the generation of sclerotic bodies with typical morphological characteristics. Biochemical features of PAF-induced sclerotic cells were also analyzed and compared with those from sclerotic forms induced by propranolol, a previously described differentiating agent of F. pedrosoi. Chemical analyses of lipid and carbohydrate components from PAF- or propranolol-induced sclerotic bodies revealed that palmitic, stearic, oleic and linoleic acids were the major fatty acid components, while glucose, mannose, galactose and rhamnose were detected as the principal sugar constituents in these cells. Surface carbohydrate components of PAF- and propranolol-induced sclerotic cells were also evaluated, by flow cytometry analysis with twelve different lectins. The profile of reactivity of PAF- or propranolol-induced fungal cells with lectins was also very similar. Hydrolysis of the synthetic substrate p-nitrophenylphosphate by fungal cells demonstrated that the addition of PAF or propranolol to the mycelial cultures similarly promotes a significant increase in ecto-phosphatase activity. These results indicate that the differentiation of F. pedrosoi mycelial cells induced by PAF generates authentic sclerotic forms, as confirmed by the analysis of morphological and biochemical attributes. Since PAF is synthesized in normal conditions by the human host, these observations may have a correlation with the differentiation of F. pedrosoi in vivo.

Ascomycota↗