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In 1968, Buckley and van Uden described the non-fermenting yeast Candida blankii (C. blankii) found in the organs of a mink1. Until recently, this microorganism had only been the subject of biotech...

Paracoccidioidomycosis is a systemic granulomatous disease caused by Paracoccidioides spp. A peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4(+) helper-1 immune response in mice and protects against intratracheal challenge with virulent P. brasiliensis. Previously, we evaluated the efficacy of the P10 peptide alone or combined with antifungal drugs in mice immunosuppressed and infected with virulent isolate of P. brasiliensis. In the present work, our data suggest that P10 immunization leads to an effective cellular immune response associated with an enhanced T cell proliferative response. P10-stimulated splenocytes increased nitric oxide (NO) production and induced high levels of IFN-γ, IL-1β and IL-12. Furthermore, significantly increased concentrations of pro-inflammatory cytokines were also observed in lung homogenates of immunized mice. P10 immunization was followed by minimal fibrosis in response to infection. Combined with antifungal drugs, P10 immunization most significantly improved survival of anergic infected mice. Administration of either itraconazole or sulfamethoxazole/trimethoprim together with P10 immunization resulted in 100 % survival up to 200 days post-infection, whereas untreated mice died within 80 days. Hence, our data show that P10 immunization promotes a strong specific immune response even in immunocompromised hosts and thus P10 treatment represents a powerful adjuvant therapy to chemotherapy.

Non-mammalian models have been used to investigate fungal virulence. In this work we have explored the use of Galleria mellonella as an infection model for the pathogenic dimorphic fungi Histoplasma capsulatum and Paracoccidioides lutzii. In mammalian models these fungi cause similar infections, and disease outcomes are influenced by the quantity of the infective inocula. We describe a similar aspect in a G. mellonella model and characterize the pathogenesis features in this system. Infection with P. lutzii or H. capsulatum, in all inoculum used, killed larvae at 25 and 37°C. However, there was a lack of correlation between the number of yeast cells used for infection and the time to larvae death, which may indicate that the fungi induce protective responses in a dynamic manner as the lowest concentrations of fungi induced the most rapid death. For both fungi, the degree of larvae melanization was directly proportional to the inocula size, and this effect was visibly more apparent at 37°C. Histological evaluation of the larvae showed a correlation between the inoculum and granuloma-like formation. Our results suggest that G. mellonella is a potentially useful model to study virulence of dimorphic fungi.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Human adenoviruses comprise an important group of etiologie agents that are responsible for various diseases in adults and children, such as respiratory, ocular, gastroenteric, and urinary infections. In immunocompromised and organ-transplanted individuals, these agents can cause generalized infections. Rapid diagnostic methods for detecting these infectious agents are not widely available.& para;& para;The aim of this work was to produce monoclonal and polyclonal anti-adenovirus antibodies to be used in a rapid diagnostic test for respiratory infections.& para;& para;Adenovirus hexons were satisfactorily purified by ultracentrifugation and chromatography. After virus purification, anti-hexon monoclonal antibodies were produced and characterized, following classical methods. Antibodies were specific for adenoviruses 2,3,5, and 41. The proposed immunochromatographic test was standardized using colloidal gold.& para;& para;The standardization of the rapid test was sufficient to detect adenovirus antigens (in nasopharyngeal lavage samples) with sensitivity of 100% and specificity of 85% when compared to direct immunofluorescence.& para;& para;The immunochromatographic assay prototype was sufficiently sensitive to detect B (3), C (2 and 5), and F (41) adenovirus samples. Although based on preliminary data, the test demonstrated the same performance as direct immunofluorescence, but with the advantage of being a point-of-care test. Further studies are still needed to confirm its effectiveness in clinical practice. (C) 2017 Sociedade Brasueira de Infectologia. Published by Elsevier Editera Ltda. Univ Fed São Paulo, Dept Med, Lab Virol, Disciplina Infectol, São Paulo, SP, Brazil Univ Fed São Paulo, Inst Biociencias, Dept Microbiol, Lab Adenovirus, São Paulo, SP, Brazil Inst Biociencias, Dept Microbiol, Lab Micol, São Paulo, SP, Brazil Inst Butantan, Lab Bacteriol, São Paulo, SP, Brazil Univ Fed São Paulo, Dept Med, Lab Virol, Disciplina Infectol, São Paulo, SP, Brazil Univ Fed São Paulo, Inst Biociencias, Dept Microbiol, Lab Adenovirus, São Paulo, SP, Brazil FAPESP: 2011/50100-6 Web of Science

Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs) from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-γ and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P. brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P. brasiliensis adding to other well-studied mediators of the immune response to this fungus.

Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America. PCM is primarily caused by Paracoccidioides brasiliensis and less frequently by the recently described, closely related species Paracoccidioides lutzii. Current treatment requires protracted administration of systemic antibiotics and relapses may frequently occur despite months of initial therapy. Hence, there is a need for innovative approaches to treatment. In the present study we analyzed the impact of two monoclonal antibodies (mAbs) generated against Heat Shock 60 (Hsp60) from Histoplasma capsulatum on the interactions of P. lutzii with macrophages and on the experimental P. lutzii infection. We demonstrated that the Hsp60-binding mAbs labeled P. lutzii yeast cells and enhanced their phagocytosis by macrophage cells. Treatment of mice with the mAbs to Hsp60 before infection reduced the pulmonary fungal burden as compared to mice treated with irrelevant mAb. Hence, mAbs raised to H. capsulatum Hsp60 are protective against P. lutzii, including mAb 7B6 which was non-protective against H. capsulatum, suggesting differences in their capacity to bind to these fungi and to be recognized by macrophages. These findings indicate that mAbs raised to one dimorphic fungus may be therapeutic against additional dimorphic fungi, but also suggests that biological differences in diseases may influence whether a mAb is beneficial or harmful.

Nesse trabalho avaliamos a proteção que o anticorpo monoclonal (AcM) contra CMH e contra Hsp60 fornecem aos animais infectados com as leveduras de Paracoccidioides brasiliensis e P. lutzii em modelo profilático. O tratamento com os AcMs de isotipos IgG2a e IgG2b foram protetores, induzindo a secreção das citocinas IL-12p70, IFN-g e TNF-a, padrão de resposta imune Th1. Nós mostramos por imunomarcação que moléculas de CMH e Hsp60 estão acessíveis na célula. E o anticorpo monoespecífico contra CMH e os anticorpos policlonais contra melanina, gerados nesse trabalho foram eficazes nos ensaios in vitro de proteção. Foram gerados anticorpos monoclonais contra glicoproteina extraída de Pb18, o AcM reconheceu, por imunomarcação, estruturas internas e a parede celular da levedura. Avaliamos um novo modelo de hospedeiro, com o uso da larva Galleria mellonella, o que pode servir de triagem e reduzir desta forma o uso excessivo de camundongos, as leveduras P. lutzii e H. capsulatum são letais para as larvas e evocam resposta celular que se organizam semelhante a um granuloma. In this work we evaluated the protection that the monoclonal antibody (MAb) against Hsp60 and against CMH provides the animals infected with the yeast Paracoccidioides brasiliensis and P. lutzii in prophylactic model. The treatment with the MAbs of IgG2a and IgG2b isotypes were protective, inducing the secretion of IL-12p70, IFN-g and TNF-a, Th1 pattern of immune response. We show by immunogold that Hsp60 and MHC molecules are accessible on the cell. And the monospecific antibody against HCM and polyclonal antibodies against melanin, generated in this study were effective in in vitro assays of protection. Monoclonal antibodies were generated glycoprotein extracted from Pb18, Mab recognized by immunogold, internal structures and cell wall material. Evaluated a new type of host, using the larvae Galleria mellonella, which can serve as a screening and reduce thus the overuse of mice, yeast P. lutzii and H. capsulatum are lethal to larvae and evoke cellular responses that are organized like a granuloma.

Made available in DSpace on 2016-03-22T17:26:12Z (GMT). No. of bitstreams: 1 lu.pdf: 261722 bytes, checksum: 1c11e1c34accfc4670f8f282ac24f62f (MD5) Previous issue date: 2008-11-18 Objetivo: Determinar a prevalência de Cardiopatia Congênita em portadores da Síndrome de Down da cidade de Pelotas, descrevendo os tipos mais freqüentes e avaliando os fatores associados. Métodos: Estudo transversal que incluiu as crianças nascidas e residentes em Pelotas (RS), no período de janeiro de 2000 até dezembro de 2005, portadoras de Síndrome de Down. Os dados foram obtidos através de entrevistas realizadas em domicílios dos pacientes com as mães ou com seus familiares legais. Para análise dos fatores relacionados à presença de Cardiopatia Congênita, foram realizadas análises univariada e bivariada. Resultados: Foram entrevistadas 47 mães de pacientes com Síndrome de Down, sendo que 26 (55,3%) pacientes apresentavam diagnóstico de Cardiopatia Congênita. Destes, 28% receberam avaliação cardiológica precoce, antes dos três meses de vida. A cardiopatia mais freqüente foi a Comunicação Interatrial (17%), O Defeito do Septo Átrio-Ventricular ocorreu em cinco pacientes. A análise bivariada entre o desfecho presença de cardiopatia congênita e as exposições: idade materna, idade paterna, cor dos pais e da criança, presença de outras malformações e sexo da criança mostraram que estas associações não foram estatisticamente significativas. Conclusões: A prevalência de Síndrome de Down e Cardiopatia Congênita em nossa região apresentam-se semelhantes às encontradas por outros autores, ressaltando a importância da suspeita diagnóstica e o encaminhamento precoce por parte dos pediatras para avaliação cardiológica. Destaca-se, também, o baixo número de pacientes que realizaram cariótipo. Além disso, o número de malformações associadas foi inferior ao encontrado por outros autores

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.

Ajoene has been described as an antithrombotic, anti-tumour, antifungal, antiparasitic and antibacterial agent. This study deals with the efficacy of ajoene to treat mice intratracheally infected with Paracoccidioides brasiliensis. The results indicate that ajoene therapy is effective in association with antifungal drugs (sulfametoxazol/trimethoprim), showing a positive additive effect. Ajoene-treated mice developed Th1-type cytokine responses producing higher levels of IFN-gamma and IL-12 when compared to the infected but untreated members of the control group. Antifungal activity of ajoene involves a direct effect on fungi and a protective pro-inflammatory immune response. Reduction of fungal load is additive to chemotherapy and therefore the combined treatment is mostly effective against experimental paracoccidioidomycosis.

A castanheira (Bertholletia excelsa Humb. & Bonpl.) é uma árvore nativa da região Amazônica muito valorizada por suas sementes, as castanhas-do-Brasil, que apresentam alto valor nutritivo e são uma rica fonte de selênio, um agente antioxidante. O Brasil está entre os países que mais produzem e exportam estas castanhas. As condições climáticas da região Amazônica, assim como as demais etapas da cadeia produtiva, podem favorecer a infecção fúngica neste substrato, principalmente por Aspergillus nomius, espécie extremamente relacionadas à produção de aflatoxinas. Esta micotoxina está associada ao desenvolvimento de tumores, imunossupressão e alterações hepáticas tornando-a um risco para a saúde pública. Sendo assim, a realização de estudos que forneçam informações adequadas sobre o comportamento de A. nomius é de extrema relevância, pois contribuem no conhecimento das condições propícias para a produção de aflatoxinas. Neste contexto, o objetivo deste estudo foi avaliar a resposta ecofisiológica de cepas de A. nomius isoladas de castanhas-do-Brasil (crescimento micelial, expressão gênica e produção de aflatoxinas) em diferentes temperaturas (25, 30 e 35 °C). O crescimento micelial foi mensurado diariamente a partir da inoculação de 8 cepas em ágar coco, mantidas no escuro até 7 dias. A partir destas colônias foi feita análise da expressão dos genes aflR, aflD e aflQ, envolvidos na biossíntese das aflatoxinas, com utilização de PCR em Tempo Real. Com as mesmas colônias também foi feita análise do potencial aflatoxigênico (B1, B2, G1 e G2) qualitativo (Cromatografia em Camada Delgada) e quantitativo (Cromatografia Líquida de Alta Eficiência). A temperatura ideal para crescimento micelial das cepas de A. nomius foi 30 °C. Esta condição foi a melhor para a expressão dos genes aflR, aflD e aflQ. Contudo, o gene aflQ também apresentou alta expressão a 25 °C e foi o gene mais expresso em todas as temperaturas avaliadas. Em relação ao potencial toxigênico das cepas, a maior produção ocorreu a 25 °C. Em todas as temperaturas avaliadas houve maior produção de aflatoxinas do grupo B do que do grupo G. Pôde-se observar que a temperatura que propiciou a maior produção destas toxinas coincide com as da região Amazônica, território nativo das castanheiras. Com base nos resultados reportados, este estudo poderá servir como ferramenta na elaboração de eficientes estratégias para o controle de A. nomius e aflatoxinas em castanhas-doBrasil. The Brazil nut tree (Bertholletia excelsa Humb. & Bonpl.) is an Amazonian native species that produce seeds with high nutritional value and rich source of selenium, an antioxidant agent. Brazil is one of the major producers and exporters of these nuts. The Amazon weather conditions in the production area and also on the productive chain play a critical role in the fungal infection, specially by Aspergillus nomius, important species associated to the aflatoxin contamination. This mycotoxin is related to the development of tumors, immunosuppression and liver alterations that becomes a risk to public health. Therefore, studies that provides adequate informations about the behavior of A. nomius are extremely relevant, contributing to better understand the favorable conditions to the aflatoxins production. The main goal of the present study was to evaluate the ecophysiological response of A. nomius strains isolated from Brazil nuts (mycelial growth, gene expression and aflatoxin production) at different temperatures (25 °C, 30 °C, and 35 °C). The mycelial growth of 8 strains was measured daily for 7 days in coconut agar. From these colonies, the expression of aflR, aflD, and aflQ genes, that are involved in aflatoxin biosynthesis was analyzed by using Real Time PCR. From the same colonies, the aflatoxigenic potential (B1, B2 , G1 and G2 ) were analyzed qualitative and quantitative by Thin Layer Chromatography and High Performance Liquid Chromatography, respectively. Mycelial growth assessment revealed that the optimal temperature for the radial growth rate and the average of final growth was at 30 °C. This was also the best condition for the expression of aflR, aflD, and aflQ genes. However, the aflQ also showed high expression at 25 °C and was the most expressed gene at all evaluated temperatures. The highest aflatoxin production occurred at 25 °C, with higher toxins production on group B than group G. It was possible to notice that the optimum temperature to aflatoxin production coincides with those in Amazon region, the most important producing area. These results also may contribute to enhance the management strategies of aflatroxin control in Brazil nuts.