Melanin particles isolated from the fungus Fonsecaea pedrosoi activates the human complement system.

BACKGROUND: Melanin production has been associated with virulence in various pathogenic fungi, including Fonsecaea pedrosoi, the major etiological agent for chromoblastomycosis, a subcutaneous fungal disease that occurs in South America. OBJECTIVE: The aim of this study was to evaluate the effects of acid-basic extracted F. pedrosoi melanin particles and fungal cell ghosts obtained by Novozym 234 treatment on their ability to activate the human complement system. METHODS: The ability of melanin particles and fungal cell ghosts to activate the human complement system was evaluated by complement consumption, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). FINDINGS: Unsensitised melanin particles and melanin ghosts presented complement consumption of 82.67 ± 2.08% and 96.04 ± 1.13%, respectively. Immunofluorescence assays revealed intense deposition of the C3 and C4 fragments on the surface of melanin particles and ghosts extracted from F. pedrosoi. Deposition of the C3, C4, and C5 fragments onto melanin samples and zymosan was confirmed by ELISA. Deposition of small amounts of C1q and C9 onto melanin samples and zymosan was detected by ELISA. CONCLUSION: Fonsecaea pedrosoi melanin particles and fungal cell ghosts activated the complement system mainly through an alternative pathway.

Assessment and characterization of biofilm formation among human isolates of Streptococcus dysgalactiae subsp. equisimilis.

The capacity to form biofilm is considered a protective mechanism that allows the bacteria to survive and proliferate in hostile environments, facilitating the maintenance of the infectious process. Recently, biofilm has become a topic of interest in the study of the human pathogen group A Streptococcus (GAS). Although GAS has not been associated with infection on medical implants, the presence of microcolonies embedded in an extracellular matrix on infected tissues has been reported. Despite the similarity between GAS and Streptococcus dysgalactiae subspecies equisimilis (SDSE), there are no studies in the literature describing the production of biofilm by SDSE. In this work, we assessed and characterized biofilm development among SDSE human isolates of group C. The in vitro data showed that 59.3% of the 118 isolates tested were able to form acid-induced biofilm on glass, and 28% formed it on polystyrene surfaces. More importantly, biofilm was also formed in a foreign body model in mice. The biofilm structure was analyzed by confocal laser scanning microscopy, transmission electron microscopy, and scanning electron microscopy. Long fibrillar-like structures were observed by scanning electron microscopy. Additionally, the expression of a pilus associated gene of SDSE was increased for in vitro sessile cells compared with planktonics, and when sessile cells were collected from biofilms formed in the animal model compared with that of in vitro model. Results obtained from the immunofluorescence microscopy indicated the biofilm was immunogenic. Our data also suggested a role for proteins, exopolysaccharide and extracellular DNA in the formation and accumulation of biofilm by SDSE.

Activation of the human complement system by pigmented and hypopigmented mycelia of the fungus Fonsecaea pedrosoi.

The action of the complement system on pigmented and hypopigmented mycelia of the fungus Fonsecaea pedrosoi, the major aetiological pathogen of the chromoblastomycosis is herein discussed. Fungi were grown in medium Czapeck-Dox at 37°C, for 14 days, without shaking to obtain pigmented mycelium. To obtain hypopigmented mycelium, the fungus was grown at the same conditions, but in the dark and with low oxygenation. Activation was measured by complement consumption and enzyme-linked immunosorbent assay. We also observed by immunofluorescence the deposition of C3, C4 fragments and C9 on the surface of the different forms studied. The results indicate that both forms were able to activate the complement system mainly by the alternative pathway. Pigmented mycelia had the highest consumption results, indicating that the pigment, melanin, may have influence in activation.

Spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides and their ability to activate human complement system in vitro.

Complement activation by spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides was studied using absorbed human serum in the presence or absence of chelators (EGTA or EDTA). We found that the spore caused full complement activation when incubated with EGTA-Mg2+ or without chelators, indicating that the alternative pathway is mainly responsible for this response. In order to compare activation profiles from each species, ELISAs for C3 and C4 fragments, mannan binding lectin (MBL), C-reactive protein (CRP) and IgG studies were carried out. All proteins were present on the species tested. Immunofluorescence tests demonstrated the presence of C3 fragments on the surface of all samples, which were confluent throughout fungal surfaces. The same profile of C3, C4, MBL, CRP and IgG deposition, observed in all species, suggests a similar activation behavior for these species.

Frequencies of feline blood types in the Rio de Janeiro area of Brazil.

BACKGROUND: The distribution and frequency of blood types in cat populations vary according to geographic region and breed. Frequencies of feline blood types in Rio de Janeiro city, as well as in other Brazilian areas, are unknown, and the risk of unmatched transfusions and neonatal isoerythrolysis has not been estimated. OBJECTIVES: The purpose of this study was to determine the frequency of feline blood types in the area of Rio de Janeiro, Brazil. METHODS: EDTA blood samples were obtained from 172 nonpedigreed domestic shorthair (DSH) cats (92 female, 80 male, 3 months-20 years old) in different sites of Rio de Janeiro city. Blood typing was performed by agglutination assays using Triticum vulgaris lectin and feline anti-A serum. The hemagglutination results for type B and AB cats were confirmed by high-performance thin-layer chromatography (HPTLC) of erythrocyte membrane gangliosides. RESULTS: The majority (163/172, 94.8%) of cats were type A, 2.9% were type B, and 2.3% were type AB. High-titer anti-A serum agglutinated RBCs from all cats in type A and type AB blood groups, with 3+ to 4+ agglutination. The probability that a type A cat would receive type B or AB blood in a first random transfusion was calculated as 2.25% and 2.20%, respectively. HPTLC analysis of glycolipids yielded a chromatographic profile characteristic of feline gangliosides for all blood groups. CONCLUSIONS: These results indicate a high prevalence of type A cats in Rio de Janeiro, Brazil, and a low frequency of type B and AB cats, consistent with what has been observed for DSH cats in other regions of the world.

Melanin particles isolated from the fungus Fonsecaea pedrosoi activates the human complement system

BACKGROUND Melanin production has been associated with virulence in various pathogenic fungi, including Fonsecaea pedrosoi, the major etiological agent for chromoblastomycosis, a subcutaneous fungal disease that occurs in South America. OBJECTIVE The aim of this study was to evaluate the effects of acid-basic extracted F. pedrosoi melanin particles and fungal cell ghosts obtained by Novozym 234 treatment on their ability to activate the human complement system. METHODS The ability of melanin particles and fungal cell ghosts to activate the human complement system was evaluated by complement consumption, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). FINDINGS Unsensitised melanin particles and melanin ghosts presented complement consumption of 82.67 ± 2.08% and 96.04 ± 1.13%, respectively. Immunofluorescence assays revealed intense deposition of the C3 and C4 fragments on the surface of melanin particles and ghosts extracted from F. pedrosoi. Deposition of the C3, C4, and C5 fragments onto melanin samples and zymosan was confirmed by ELISA. Deposition of small amounts of C1q and C9 onto melanin samples and zymosan was detected by ELISA. CONCLUSION Fonsecaea pedrosoi melanin particles and fungal cell ghosts activated the complement system mainly through an alternative pathway.

Migration inhibitory factor (MIF) released by macrophages upon recognition of immune complexes is critical to inflammation in Arthus reaction.

Deposition of immune complexes (IC) triggers Fc gamma R-dependent inflammation, leading to tissue damage in rheumatoid arthritis, systemic lupus erythematous, immune glomerulonephritis, and several immune vasculitides. Evidences support a role for macrophage migration inhibitory factor (MIF) in a number of inflammatory diseases, but the triggering of its secretion and its physiopathological role upon IC deposition remain elusive. Herein, we show that human macrophages secreted MIF after IC recognition, which in turn controlled the secretion of TNF. Macrophages from Mif-/- mice produced smaller amounts of TNF when stimulated with IgG-opsonized erythrocytes than wild-type (WT) cells. Using passive reverse Arthus reaction in the peritoneum and lungs as a model for IC-induced inflammation, we demonstrated that Mif-/- mice had a milder response, observed by reduced neutrophil recruitment, vascular leakage, and secretion of TNF, MIP-2, and keratinocyte-derived chemokine compared with WT controls. Adoptive transfer of alveolar macrophages from WT to Mif-/- mice rescued pulmonary neutrophil recruitment and TNF production upon passive reverse Arthus reaction. Our study indicates that Arthus inflammatory reaction is largely dependent on MIF and poses macrophages as a source of the MIF released upon IC recognition. These results give experimental support to the proposition that blockade of MIF might constitute an adjunctive, therapeutic approach to IC disease.

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.

Action of chlorogenic acid on the complement system

Previous research on plants used in folk medicine as antidotes against snake-bite revealed some constituents responsible for such protection. Chlorogenic acid (3-0-caffeoyl quinic acid) was one of these substances, studied with more attention. It has been shown that this substance binds to proteins through hydrophobic interactions and hydrogen bonds. This paper shows the preliminary results about the anti-complementary action of chlorogenic acid. Human and guinea pig sera, treated with chlorogenic acid, were added to the hemolytic system (sheep erythrocyte sensitized with hemolysin) to study its effect on the activation of the classical complement pathway. The action on the alternative pathway was studied with human serum treated with chlorogenic acid and zymosan. Our results show that chlorogenic acid presents anti-complementary action at the classical pathway, since the sera are not able to lysis the indicator system. The presence of C3b fragments on the surface of the yeast cells demonstrates that the alternative pathway was not affected.