Induction and secretion of elastinolytic and proteolytic activity in cultures of Paracoccidioides brasiliensis

P. brasiliensis parasitizes various human tissues and proteinases exported by this fungus may allow it to metabolize and invade host tissues. The influence of the culture medium on the production of proteinases by P. brasiliensis isolates was studied and the export of these enzymes was followed as a function of culture time. The fungus was grown in neopeptone, BSA, elastin orcollagen medium. The culture medium was assayed for azocollytic, elastinolytic and caseinolytic activity. Proteolytic activity was also analysed by electrophoresis of the culture medium on gelatin and casein substrate gels. P. brasiliensis expressed relatively high levels of azocoll, elastin and casein degrading activity in all types of medium, except in neopeptone medium. Generally, expression of azocollytic activity peaked during the third week of culture and caseinolytic activity during the fourth week of culture. Azocollytic activity was highest at pH 4.0 and caseinolytic activity at pH 8.0. Elastinolytic activity was also highest at pH 8.0. This activity, as well as the others, may provide the fungus with a source of carbon and nitrogen and may alsobe responsible for the invasion of host tissues, such as pulmonary elastic fiber, by P. brasiliensis.

Epithelial cells treated with genistein inhibit adhesion and endocytosis of Paracoccidioides brasiliensis.

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus' transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.

Cryptococcosis outbreak in psittacine birds in Brazil.

An outbreak of cryptococcosis occurred in a breeding aviary in São Paulo, Brazil. Seven psittacine birds (of species Charmosyna papou, Lorius lory, Trichoglossus goldiei, Psittacula krameri and Psittacus erithacus) died of disseminated cryptococcosis. Incoordination, progressive paralysis and difficulty in flying were seen in five birds, whereas superficial lesions coincident with respiratory alterations were seen in two birds. Encapsulated yeasts suggestive of Cryptococcus sp. were seen in faecal smears stained with India ink in two cases. Histological examination of the birds showed cryptococcal cells in various tissues, including the beak, choana, sinus, lungs, air sacs, heart, liver, spleen, kidneys, intestines and central nervous system. High titres of cryptococcal antigen were observed in the serum of an affected bird. In this case, titres increased during treatment and the bird eventually died. Yeasts were isolated from the nasal mass, faeces and liver of one bird. Cryptococcus neoformans var. gattii serovar B was identified based on biochemical, physiological and serological tests. These strains were resistant (minimum inhibitory concentration 64 microg/ml) to fluconazole. This is the first report of C. neoformans var. gattii occurring in psittacine birds in Brazil.

Epidemiologia das dermatofitoses em instituições públicas da região de Araraquara - SP / Epidemiology of the dermatophytosis in public institutions of the region of Araraquara city - SP

As dermatofitoses são processos infecciosos de pele, pêlo e unhas muito comuns no mundo inteiro. Com o intuito de avaliar a epidemiologia das infecções causadas por estes fungos em Instituições Públicas de Araraquara, 105 amostras de indivíduos com suspeita clínica de dermatofitose foram examinadas no Laboratório de Micologia da Faculdade de Ciências Farmacêuticas, de agosto a dezembro de 2001 e, destas, 47 foram positivas para dermatófitos. Trichophyton rubrum foi a espécie prevalente (56,9%), seguida por Microsporum canis (17%), T. tonsurans (10,6%), T. mentagrophytes (8,5%) e Epidermophyton floccosum (4,3%). T. rubrum foi mais frequente nas lesões dos interdígitos (81,5%) e M. canis foi o principal envolvido nas lesões do couro cabeludo (58,3%). Portanto, houve predomínio de fungos antropofílicos e zoofílicos, respectivamente, dado este que está de acordo com as estatísticas dos estados brasileiros da região Sudeste e Sul, bem como de outras regiões do mundo em que estes fungos foram os mais frequentes isolados de tinha dos pés e do couro cabeludo. Neste estudo, foi também verificada elevada percentagem de T. tonsurans (41,7%) em tinha do couro cabeludo, dado este inédito na região Sudeste

Microbiota fúngica de um prédio com laboratórios da região de Araraquara - SP / Environment fungi of laboratory buildings in Araraquara - SP region

De setembro de 2000 a janeiro de 2001, foram coletadas amostras de 21 ambientes internos de um prédio com laboratórios didáticos, de pesquisa e de atendimento à comunidade, bem como da área externa, em Araraquara, estado de São Paulo. Foi utilizado aparelho que funciona de acordo com o princípio descrito por Andersen, o MAS-100R (MERCK), de simples estágio, que utiliza placas de Petri de 90mm de diâmetro contendo meio de agar Sabouraud-cloranfenicol. Após cinco dias de incubação à temperatura de 25ºC, as colônias foram contadas, reisoladas e identificadas resultando na descrição de 21 taxa. Clodophialophora spp. foi o fungo mais isolado, tanto em ambientes internos como externos, seguido pelos gêneros Penicillium spp. e Mycelia sterilia. De acordo com resolução nº 9, de janeiro de 2003 da ANVISA, fungos considerados inaceitáveis foram encontrados em nove ambientes internos e um dos ambientes apresentou quantidade de fungos (em unidades formadoras de colônia por meio cúbico - UFC/m3) acima do limite aceitável. Entre os fungos isolados, 16 foram relatados como fungos oportunistas, nove relacionados a doenças de plantas e sete associados a problemas alérgicos

Epidemiologia de candidíase hospitalar: importância da identificação específica / Epidemiology of nosocomial candidiasis: the importance of specific identification

Infecções fúngicas sistêmicas são hoje importante causa de morbidade e mortalidade em pacientes imunossuprimidos ou com outras condições predisponentes. Candida albicans e as não-albicans são importante causa de infecções nosocomiais e vários destes agentes são menos suscetíveis às drogas antifúngicas, principalmente os azólicos, um fato que tem significado no tratamento destes pacientes. O moderno laboratório de micologia tem importante papel no esclarecimento destas infecções, incluindo sua detecção , identificação e a sensibilidade a drogas antifúngicas, bem como a análise epidemiológica. Neste estudo, foi comparada a distribuição de espécies de Candida relacionadas a fungemias e outras fontes, em quatro hospitais do Estado de São Paulo. Das 40 leveduras identificadas, C. albicans, C. parapsilosis e C. tropicalis foram isoladas, respectivamente, em 35 por cento, 50 por cento e 15 por cento, revelando uma tendência de ser maior a freqüência de espécies não-albicans. As fungemias foram causadas por C. parapsilosis (45,4 por cento). C. albicans (36,4 por cento) e C. tropicalis (18,2 por cento), o que revela um aumento de espécies não-albicans em relação a séries históricas. As três diferentes espécies foram incluídas em 6,3 e 4 biótipos diferentes, respectivamente para C.albicans, C.parapsilosis e C.tropicalis. Este estudo enfatiza a importância da avaliação de espécies de Candida especialmente em centros hospitalares com pacientes de risco.

Immunosuppression in paracoccidioidomycosis: T cell hyporesponsiveness to two Paracoccidioides brasiliensis glycoproteins that elicit strong humoral immune response.

To assess human cellular immune response to paracoccidioidomycosis (PCM), lymphocyte proliferative responses to purified antigens from Paracoccidioides brasiliensis were determined in healthy persons previously infected by the fungus (positive donors), in healthy noninfected persons (controls), and in PCM patients. Affinity-purified gp70 and gp43, the two major antigens in humoral immune responses, were used. Both induced lymphocyte proliferation (gp43 species-specific) in positive donors but not in controls; healthy persons previously infected by Histoplasma capsulatum reacted to gp70 and not to gp43. A similar cross-reactivity in antibody response to gp70 was previously reported; however, antibody response to gp43 has been considered specific. Lymphocytes from PCM patients, who, unlike positive donors, have high levels of anti-gp43 and anti-gp70 antibodies, proliferated poorly with gp70 and gp43 but better with other stimuli. This dichotomy between humoral and cellular antigen-specific responses suggests a Th2 immune response in PCM, which may be related to failure to control the infection.

Heteroarotinoids. Synthesis, characterization, and biological activity in terms of an assessment of these systems to inhibit the induction of ornithine decarboxylase activity and to induce terminal differentiation of HL-60 cells.

The synthesis of certain heteroarotinoids has been achieved, namely the systems (2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimethyl-6 -thiochromanyl)-2,4,6-heptatrienoic acid (1a), ethyl (2E,4E,6E)-3,7-dimethyl-7- (1,2,3,4-teterahydro-4,4-dimethyl-6-thiochromanyl)-2,4,6- heptatrienoate (1b), (2E,4E,6E)-3,7-dimethyl-7-(1,2,3,4-tetrahydro-4,4-dimethyl-6 -chromanyl)-2,4,6-heptatrienoic acid (1c), 2-phthalimidoethyl 3,7-dimethyl-7-(1,2,3,4-tetrahydro-4 4-dimethyl-6-thiochromanyl)-2,4,6-heptatrienoate (1d), methyl (E)-p-[2-(4,4- dimethyl-6-chromanyl)-1-propenyl]benzoate (2a), (E)-p-[2-(4,4-dimethyl-6-chromanyl)-1-propenyl]benzyl alcohol (2b), (E)-p-[2-(4,4-dimethyl-6-chromanyl)-1-propenyl]benzonitrile (2c), (E)-p-[2-(4,4-dimethyl-6-chromanyl)-1-propenyl]benzaldehyde (2d), methyl 4-[2-(2,3-dihydro-3,3-dimethyl-5-benzofuranyl)-1-propenyl] benzoate (3a), and (E)-p-[2-(2,3-dihydro-3,3-dimethyl-5-benzofuranyl)-1- propenyl]benzoic acid (3b). Characterization via elemental, IR, 1H NMR, and 13C NMR analyses was completed for these heterocycles. The biological activity of these heteroarotinoids was assayed by either the suppression of the 12-O-tetradecanoylphorbol 13-acetate (TPA) induced synthesis of ornithine decarboxylase (ODC) in mouse skin or the induction of differentiation of human (HL-60) promyelocytic cells. In the ODC assay, systems 1a-c exhibited strong activity (within 10% of or less than the control) whereas alcohols 2b and 3a showed good activity (within 50% of the control) as compared to either 13-cis-retinoic acid or trans-retinoic acid. Moderate activity was observed with 2a and 2b while 1d and 2c were essentially inactive. With the HL-60 assay, 1a and 1c were approximately 2- and 5-fold less active, respectively, than trans-retinoic acid. In contrast, 2a, 3a, and 3b induced differentiation of only a very small percentage of the cells. Acids 1a and 1c were the most active heteroarotinoids in the two biological assays. Consequently, the presence of the heteroatom does not eradicate the activity of the heteroarotinoids and thus they may have potential as chemotherapeutic agents.