Relação da expressão de fatores de crescimento celular (IGF-1) e (SCF) com fatores prognósticos e o alvo da rapamicina em mamíferos (m-TOR) em mastocitomas cutâneos caninos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modulatory effect of prostaglandins on human monocyte activation for killing of high- and low-virulence strains of Paracoccidioides brasiliensis

The effect of indomethacin (Indo), a cyclo-oxygenase inhibitor, on the monocyte-mediated killing of a low-(Pb265) and a high-(Pb18) virulence strain of Paracoccidioides brasiliensis was examined. The Pb18 strain was not killed by either non-activated or interferon-gamma (IFN-gamma)-activated human monocytes but these cells did show fungicidal activity if pretreated with Indo. In contrast with IFN-gamma tumour necrosis factor-alpha (TNF-alpha) was very effective at stimulating the fungicidal activity of monocytes. While the low-virulence strain, Pb265, could not be killed by monocytes, cells preincubated with IFN-gamma demonstrated fungicidal activity. The killing of this strain was also induced by pretreatment of monocytes with Indo. The results suggest a negative role for prostaglandins, which are synthesized via the cyclo-oxygenase pathway, in the regulation of monocyte-mediated killing of virulent and avirulent strains of P. brasiliensis and that TNF-alpha generation during the fungus-monocyte interaction is more important in the killing of Pb265 than Pb18.

Paracoccidioides brasifiensis killing by IFN-gamma, TNF-alpha and GM-CSF activated human neutrophils: role for oxygen metabolites

Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against the fungus and several papers show the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, the studies focusing on polymorphonuclear neutrophils (PMNs) antifungal functions are scarcer. Thus, the objective of the present paper was to assess the capacity of human PMNs to kill virulent P brasiliensis strain in vitro, before and after priming with different cytokines. Moreover, the involvement of oxygen metabolites in this activity was evaluated. Nonactivated cells failed to exhibit antifungal activity. However, when these cells were IFN-gamma, TNF-alpha or GM-CSF activated, a significative fungicidal activity was detected. This process was significantly inhibited when P brasiliensis challenge occurred in presence of catalase (CAT - a scavenger of H2O2) and superoxide dismutase (SOD - a scavenger of superoxide anion). From these results it is concluded that cytokines activation is required for P brasiliensis killing by human PMNs, and that H2O2 and Superoxide anion participate as effectors molecules in this process.

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interleukin-15 increases Paracoccidioides brasiliensis killing by human neutrophils

Interleukin-15 is a pro-inflammatory cytokine produced by a wide range of different cell types, especially monocytes and macrophages, in response to infective agents, playing a crucial and modulatory role in innate and adaptive immune response. Infections by intracellular microorganisms such as some bacteria, protozoa and fungi point out the role of IL-15 in the activation of monocytes/macrophages and neutrophils, a process that represents an important defense mechanism in early periods of infection during the development of innate immune response. The aims of the present study were to evaluate the effects of IL-15 on human neutrophil fungicidal activity against a high virulent Paracoccidioides brasiliensis strain ( Pb18) and to verify whether this activity was mediated by oxidative metabolism such as the production of superoxide anion and H2O2 and if it was associated with an alteration of cytokine ( IL-8 and TNF-alpha) levels. Neutrophils from peripheral blood of healthy individuals were incubated in the presence and absence of IL-15 ( 12.5 - 250ng/ml) for 18h, at 37 degrees C, under tension of 5% CO2, then infected with Pb18 for 4h and evaluated for fungicidal activity, production of superoxide anion and H2O2, and quantification of cytokines IL-8 and TNF-a in the supernatant. Preincubation of neutrophils with IL-15 induced a significant increase in the fungicidal activity of such cells in a dose-dependent manner. After activation, there was an increase in the production of superoxide anion and H2O2 by these cells, suggesting participation of such metabolites in fungicidal activity. Catalase inhibits fungicidal activity, confirming the role of H2O2 in fungus killing. However, the levels of TNF-alpha and IL-8 were not modified after incubation with IL-15, which suggests that its role is not mediated by those cytokines. Taken together, results showed that IL-15 had a modulatory effect on human neutrophils infected in vitro with a high virulent strain of P. brasiliensis, which was characterized by an increased fungicidal activity mediated by a dependent mechanism of oxidative metabolism.

Production of prostaglandins and leukotrienes by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids, including prostaglandins and leukotrienes, during fungal infections is theorized to play a critical role on fungal survival and/or growth as well as on host immune response modulation. Host cells are one source of these mediators; however another potential source may be the fungus itself. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, PbBT79, Pb192) produce both, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)). Moreover, we asked if P. brasiliensis can use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase (COX) and lipoxygenase (5-LO) enzymes, for PGE(2) and LTB(4) production, respectively. Finally, a possible association between these eicosanoids and fungus viability was assessed. We demonstrated, using ELISA assays, that all P. brasiliensis strains, independently of their virulence, produce high PGE(2) and LTB(4) levels after a 4-hour culture, which were reduced after 8 hours. However, in both culture times, higher eicosanoids levels were detected when culture medium was supplemented with exogenous AA. Differently, treatment with indomethacin, a COX inhibitor, or MK886, a 5-LO inhibitor, induces a reduction on PGE(2) and LTB(4) levels, respectively, as well as in fungus viability. The data provide evidence that P. brasiliensis is able to metabolize either endogenous or exogenous AA by pathways that depend on COX and 5-LO enzymes for producing, respectively, PGE(2) and LTB(4) that are critical for its viability.

Killing of Paracoccidioides brasiliensis yeast cells by IFN-gamma and TNF-alpha activated murine peritoneal macrophages: evidence of H(2)O(2) and NO effector mechanisms

Paracoccidioidomycosis is a deep mycosis, endemic in Latin America, caused by Paracoccidioides brasiliensis. Macrophage activation by cytokines is the major effector mechanism against this fungus. This work aimed at a better understanding of the interaction between yeast cells-murine peritoneal macrophages and the cytokine signals required for the effective killing of high virulence yeast-form of P. brasiliensis. In addition, the killing effector mechanisms dependent on the generation of reactive oxygen or nitrogen intermediates were investigated. Cell preincubation with IFN-gamma or TNF-alpha, at adequate doses, resulted in effective yeast killing as demonstrated in short-term (4-h) assays. Both, IFN-gamma and TNF-alpha activation were associated with higher levels of H(2)O(2) and NO when compared to nonactivation. Treatment with catalase (CAT), a H(2)O(2) scavenger, and N(G)-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase inhibitor, reverted the killing effect of activated cells. Taken together, these results suggest that both oxygen and L-arginine-nitric oxide pathways play a role in the killing of highly virulent P. brasiliensis.

Interleukin-6 treatment enhances human monocyte permissiveness for Paracoccidioides brasiliensis growth by modulating cytokine production

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A sinalização do enfermeiro entre os papéis de familiares visitantes e acompanhante de adulto e idoso

Trata-se de um estudo qualitativo, utilizando-se, como referencial teórico, o Interacionismo Simbólico e, como referencial metodológico, a Grounded Theory, visando: compreender a experiência interacional de familiares visitantes e acompanhantes de adultos e idosos hospitalizados, há mais de sete dias, em um Hospital Universitário de grande porte do Estado de São Paulo, e desenvolver um modelo teórico representativo dessa experiência. As estratégias para obtenção dos dados foram a observação e a entrevista. Dos resultados, emergiram dois fenômenos: vivendo a expectativa pela internação no Hospital Universitário e assumindo o papel de familiar visitante ou de familiar acompanhante. A compreensão da experiência nos permitiu ampliar o conhecimento, referente ao movimento que eles empreenderam na vivência denominada como movendo-se perante a sinalização do enfermeiro entre os papéis de familiar visitante e familiar acompanhante: compartilhando uma experiência de poucos prazeres em solidariedade ao adulto e ao idoso hospitalizados.

Comparison of the sequences of the internal transcribed Spacer regions and PbGP43 genes of Paracoccidioides brasiliensis from patients and armadillos (Dasypus novemcinctus)

Paracoccidioides brasiliensis isolates from 10 nine-banded armadillos (Dasypus novemcinctus) were comparable with 19 clinical isolates by sequence analysis of the PbGP43 gene and ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and by random amplified polymorphic DNA. In this original ITS study, eight isolates differed by one or three sites among five total substitution sites.

Virulence profiles of ten Paracoccidioides brasiliensis isolates obtained from armadillos (Dasypus novemcinctus)

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis ( PCM), the most important systemic mycosis in Latin America. The armadillo, Dasypus novemcinctus, has been confirmed as the primary natural reservoir of this fungus. Its geographic distribution is similar to that of human PCM. In this study, virulence profiles of 10 P. brasiliensis isolates from different armadillos and of two clinical isolates were tested in an experimental hamster model. Pathogenicity was evaluated by counting cfu and performing histopathological analysis in the testis, liver, spleen and lung. Circulating specific antibodies were measured using enzyme- linked immunosorbent assay ( ELISA). All isolates from armadillos were virulent in the model, with dissemination to many organs. The clinical isolates, which had long been stored in cultured collections, were less virulent. The isolates were classified into four virulence categories according to number of cfu per gram of tissue: very high, high, intermediate and low. This study confirms that armadillos harbor pathogenic genotypes of P. brasiliensis, probably the same ones that infect humans.

Comparison between human and armadillo Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis

Sixty-three Paracoccidioides brasiliensis isolates obtained from three nine-banded armadillos (Dasypus novem-cinctus), one Amazonian armadillo's and 19 clinical isolates were compared by random amplified polymorphic DNA analysis with the primer OPG-19. The isolates were divided into three major clusters, I, II and III. Coincidences between human and armadillo isolates were observed in clusters I and II. Cluster III consisted only of armadillos' isolates. The results suggested that (I) humans may acquire P. brasiliensis infection by contact with armadillo's environment, (II) there may be P. brasiliensis genotypes peculiar to the animal, and (III) individual armadillos may be infected with P brasiliensis cells with different genotypes.

Prostaglandin E-2 production by high and low virulent strains of Paracoccidioides brasiliensis

The production of prostaglandins (PGs) during fungal infections could be an important suppressor factor of host immune response. Host cells are one source of prostaglandin E-2 (PGE(2)); however another potential source of PGE(2) is the fungal pathogen itself. Thus, both host and fungal PGE2 production is theorized to play a role in pathogenesis, being critical for growth of the fungus and to modulate the host immune response. The purpose of this work was to investigate if high and low virulent strains of Paracoccidioides brasiliensis have the capacity to produce PGE(2) in vitro, and if this production was related to the fungal growth. The results demonstrated that both strains of P. brasiliensis produce high levels of PGE(2) and the treatment with indomethacin, a cyclooxygenase inhibitor, significantly reduced the production of this mediator, as well as the viability of the fungus. Thus, our data indicate that PGE(2) is produced by P. brasiliensis by a cyclooxygenase-dependent metabolic pathway, and its production is required for fungal survival. This discovery reveals an important factor that has potentially great implications for understanding the mechanisms of immune deviation during infection.

A critical analysis of isolation of Paracoccidioides brasiliensis from soil

The present review is a critical analysis of positive and negative reports of the isolation of Paracoccidioides brasiliensis from soil. The strains isolated from soil or soil-contaminated material (dogfood, penguin feces) by Batista et al. in Recife, Brazil, Negroni in the Argentinian Chaco, Albornoz in rural Venezuela, Silva-Vergara et al, in Ibia, Brazil, Ferreira et al, in Uberlandia, Brazil, and Gezuele et al. at the Uruguayan base in the Antarctic region, presented mycological characteristics consistent with P. brasiliensis. In most of these studies, morphological characterization was complemented with an evaluation of virulence and antigenicity, and biochemical or molecular analysis. These isolations, therefore, can be considered true, supporting the concept of soil as an important element in the ecology of the pathogen. The large number of negative reports in attempts involving soil samples and the low repeatability of isolation of the fungus from the same area indicate that the specific conditions supporting growth of the pathogen in soil have not been fully clarified.

High frequency of Paracoccidioides brasiliensis infection in armadillos (Dasypus novemcinctus): an ecological study

The fungus Paracoccidioides brasiliensis has been isolated from nine-banded armadillos (Dasypus novemcinctus) in different regions where paracoccidiodomycosis (PCM) is endemic. The link between PCM and these animals has provided the first valuable clue in the effort to elucidate the ecological niche of P. brasiliensis. The present study was aimed at correlating P. brasiliensis infection in armadillos with local ecological features and, if possible, the presence of the fungus in the soil in the Botucatu hyperendemic area of PCM. In this region the mean temperature ranges from 14.8 to 25.8degreesC and the annual average precipitation is 1520 mm. The sites where 10 infected animals (positive group) were collected were studied and compared with the sites where five uninfected animals were found. The occurrence of the fungus in soil samples collected from the positive armadillos' burrows and foraging sites was investigated by the indirect method of animal inoculation. Environmental data from the sites of animal capture, such as temperature, rainfall, altitude, vegetation, soil composition, presence of water and proximity of urban areas, were recorded. All 37 soil samples collected from the sites had negative fungal cultures. Positive animals were found much more frequently in sites with disturbed vegetation, such as riparian forests and artificial Eucalyptus Or Pinus forests, in altitudes below 800 m, near water sources. The soil type of the sites of positive animals was mainly sandy, with medium to low concentrations of organic matter. The pH was mainly acidic at all the sites, although the concentrations of aluminum cations (H+Al) were lower at the sites where positive animals were found. Positive armadillos were also captured in sites very close to urban areas. Our data and previous studies indicate that P. brasiliensis occurs preferentially in humid and shady disturbed forests in a strong association with armadillos.