Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to alpha 3 and alpha 5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, alpha 3 and alpha 5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-beta-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with alpha 3 and alpha 5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.Brazilian agencies: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Univ Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilFAPESP: 2011/22773-6FAPESP: 2012/11792-2Web of Scienc

W3 Sons: a estética da radiorreportagem

W3 Sons: a estética da radiorreportagem é uma série de cinco reportagens sobre a W3 Sul, uma avenida de Brasília. Este trabalho de conclusão de curso – projeto experimental na modalidade produto de comunicação – busca explorar os recursos da linguagem radiofônica para o gênero em questão. O objetivo é retratar o lugar como era, é e pode vir a ser a partir da memória e opinião de oito entrevistados e, assim, refletir sobre as possibilidades estéticas da radiorreportagem

Reactivity of MEST-1 (antigalactofuranose) with Trypanosoma cruzi, glycosylinositol phosphorylceramides (GIPCs): Immunolocalization of GIPCs in acidic vesicles of epimastigotes

Using confocal microscopy, MEST-1-positive immunofluorescence was observed within various Trypanosoma cruzi forms, except in cell-derived trypomastigotes. Glycosylinositol phosphorylceramides were identified by thin-layer chromatography immunostaining as the antigens recognized by MEST-1 in these parasites. in epimastigotes, labeling of MEST-1 coincided with acidic vesicles, indicating an internal localization of these glycoconjugates.Universidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023900 São Paulo, BrazilWeb of Scienc

Bone morphogenetic protein-2 functions as a negative regulator in the differentiation of myoblasts, but not as an inducer for the formations of cartilage and bone in mouse embryonic tongue

<p>Abstract</p> <p>Background</p> <p>In vitro studies using the myogenic cell line C2C12 demonstrate that bone morphogenetic protein-2 (BMP-2) converts the developmental pathway of C2C12 from a myogenic cell lineage to an osteoblastic cell lineage. Further, in vivo studies using null mutation mice demonstrate that BMPs inhibit the specification of the developmental fate of myogenic progenitor cells. However, the roles of BMPs in the phases of differentiation and maturation in skeletal muscles have yet to be determined. The present study attempts to define the function of BMP-2 in the final stage of differentiation of mouse tongue myoblast.</p> <p>Results</p> <p>Recombinant BMP-2 inhibited the expressions of markers for the differentiation of skeletal muscle cells, such as myogenin, muscle creatine kinase (MCK), and fast myosin heavy chain (fMyHC), whereas BMP-2 siRNA stimulated such markers. Neither the recombinant BMP-2 nor BMP-2 siRNA altered the expressions of markers for the formation of cartilage and bone, such as osteocalcin, alkaline phosphatase (ALP), collagen II, and collagen X. Further, no formation of cartilage and bone was observed in the recombinant BMP-2-treated tongues based on Alizarin red and Alcian blue stainings. Neither recombinant BMP-2 nor BMP-2 siRNA affected the expression of inhibitor of DNA binding/differentiation 1 (Id1). The ratios of chondrogenic and osteogenic markers relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a house keeping gene) were approximately 1000-fold lower than those of myogenic markers in the cultured tongue.</p> <p>Conclusions</p> <p>BMP-2 functions as a negative regulator for the final differentiation of tongue myoblasts, but not as an inducer for the formation of cartilage and bone in cultured tongue, probably because the genes related to myogenesis are in an activation mode, while the genes related to chondrogenesis and osteogenesis are in a silencing mode.</p

Horizontal Plasmid Transfer by Transformation in Escherichia coli: Environmental Factors and Possible Mechanisms

Transformation is one mode of horizontal gene transfer (HGT) in bacteria, wherein extracellular naked DNA is taken up by cells that have developed genetic competence. Sensitivity to DNase, which degrades naked DNA, is the key to distinguishing transformation from the DNase-resistant HGT mechanisms. In general, Escherichia coli is not believed to be naturally transformable; it develops high competence only under artificial conditions, including exposure to high Ca2+ concentrations. However, E. coli can reportedly express modest competence under certain conditions that are feasible in natural environments outside laboratory. In addition, recent data suggest that environmental factors influence multiple routes of transformation. In this mini review, we (1) summarize our studies on transformation-based HGT using E. coli experimental systems and (2) discuss the possible occurrence of transformation via multiple mechanisms in the environment and its possible impact on the spread of antibiotic resistance genes

Effect of anti-glycosphingolipid monoclonal antibodies in pathogenic fungal growth and differentiation. Characterization of monoclonal antibody MEST-3 directed to Manpα1→3Manpα1→2IPC

<p>Abstract</p> <p>Background</p> <p>Studies carried out during the 1990's demonstrated the presence of fungal glycoinositol phosphorylceramides (GIPCs) with unique structures, some of them showed reactivity with sera of patients with histoplasmosis, paracoccidioidomycosis or aspergillosis. It was also observed that fungal GIPCs were able to inhibit T lymphocyte proliferation "in vitro", and studies regarding the importance of these molecules to fungal survival showed that many species of fungi are vulnerable to inhibitors of sphingolipid biosynthesis.</p> <p>Results</p> <p>In this paper, we describe a detailed characterization of an IgG2a monoclonal antibody (mAb), termed MEST-3, directed to the <it>Paracoccidioides brasiliensis </it>glycolipid antigen Pb-2 (Man<it>p</it>α1→3Man<it>p</it>α1→2IPC). mAb MEST-3 also recognizes GIPCs bearing the same structure in other fungi. Studies performed on fungal cultures clearly showed the strong inhibitory activity of MEST-3 on differentiation and colony formation of <it>Paracoccidioides brasiliensis</it>, <it>Histoplasma capsulatum </it>and <it>Sporothrix schenckii</it>. Similar inhibitory results were observed when these fungi where incubated with a different mAb, which recognizes GIPCs bearing terminal residues of β-D-galactofuranose linked to mannose (mAb MEST-1). On the other hand, mAb MEST-2 specifically directed to fungal glucosylceramide (GlcCer) was able to promote only a weak inhibition on fungal differentiation and colony formation.</p> <p>Conclusions</p> <p>These results strongly suggest that mAbs directed to specific glycosphingolipids are able to interfere on fungal growth and differentiation. Thus, studies on surface distribution of GIPCs in yeast and mycelium forms of fungi may yield valuable information regarding the relevance of glycosphingolipids in processes of fungal growth, morphological transition and infectivity.</p

Membrane microdomain components of Histoplasma capsulatum yeast forms, and their role in alveolar macrophage infectivity

Analysis of membrane lipids of Histoplasma capsulatum showed that similar to 40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4 degrees C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30 kDa laminin-binding protein, and a 50 kDa protein recognized by anti-alpha 5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (m beta CD). Removal of ergosterol by m beta CD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30 kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. m beta CD treatment did not displace/remove the 50 kDa alpha 5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with m beta CD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30 kDa laminin-binding protein; ergosterol and/or the 30 kDa protein may be involved in macrophage infectivity. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Div Glycoconjugate Immunochem, Dept Biochem, Escola Paulista Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Div Glycoconjugate Immunochem, Dept Biochem, Escola Paulista Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023900 São Paulo, BrazilWeb of Scienc

Removal of accidentally ingested large foreign object via the anus after watchful waiting

One of the commonest complaints, for which a patient arrives in hospitals, is the presence of foreign body. It could be due to&nbsp;accidental ingestion or any other cause which leads to presences of a foreign body in the gastrointestinal tract. It is believed that&nbsp;foreign objects larger than 5–6 cm in size are unlikely to pass through the duodenum. Here, we describe a case wherein the patient&nbsp;accidentally swallowed a 7-cm-sized mouthguard that could not be removed by emergency upper gastrointestinal endoscopy but was&nbsp;subsequently removed via the anus after a period of watchful waiting