High interleukin-4 expression and interleukin-4 gene polymorphisms are associated with susceptibility to human paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is caused by dimorphic fungi from theParacoccidioides brasiliensis complex. Previous studies have demonstrated that the severity of disease is associated with a T-helper 2 immune response characterised by high interleukin (IL)-4 production. In the present study we analysed two polymorphisms in the IL-4gene (-590 C/T and intron-3 microsatellite) in 76 patients with PCM and 73 control subjects from an endemic area. The production of IL-4 by peripheral blood mononuclear cells after antigen or phytohaemagglutinin stimulation was determined by ELISA. A significant correlation was observed between the RP2/RP2 intron-3 genotype and infection with Paracoccidioides sp.(p = 0.011), whereas the RP1/RP1 genotype was correlated with resistance. No significant correlation was observed for the IL-4promoter polymorphism. Furthermore, the low IL-4 expression observed in the control group compared with patients was associated with the RP1/RP1 genotype. These results suggest that IL-4polymorphisms might be associated with the ability of the host to control Paracoccidioides sp.infection. The relevance of this polymorphism is supported by the observation that patients with disease produce high levels of IL-4 following mitogen or antigen stimulation. The IL-4gene is located in the cytokine cluster region of chromosome 5 where other polymorphisms have also been described.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Constituintes químicos de Hyptidendron canum (Pohl ex Benth.) R. Harley (Lamiaceae)

Chemical investigation of Hyptidendron canum stems resulted in the isolation of betulinic, ursolic and euscaphic acids. From the leaves were isolated 3β-O-β-galactopiranosilsitosterol, ursolic aldehyde, and mixtures of maslinic acid and 2α-hydroxyursolic acid, α and β-amyrin, uvaol and erythrodiol, sitosterol and stigmasterol, spathulenol and globulol. Hexane and chloroform leave fractions as well as ursolic and betulinic acids showed antifungal activities against the yeast form of Paracoccidioides brasiliensis.

Adaptação de espécies arbóreas nativas em um sistema agrossilvicultural, submetidas a extremos climáticos de geada na região de Florianópolis

O principal objetivo deste trabalho foi avaliar a resposta de cinco espécies arbóreas nativas submetidas a extremos climáticos de geada em um sistema agroflorestal, na região de Florianópolis, Santa Catarina (latitude de 27º35' S , longitude 48º34' W e altitude de 1,84 m). O solo é do tipo Neossolo quartzarênico hidromórfico distrófico, textura arenosa, com elevada flutuação do lençol freático. Foram tomadas sete parcelas de dez árvores, seguindo o delineamento estatístico inteiramente casualizado. Os parâmetros analisados foram altura total, número de folhas, incremento médio em altura total e número de folhas de cada árvore (quantificados a cada quatro meses), porcentagem de árvores com folhas danificadas pela geada e sobrevivência de cada planta após a geada. Os parâmetros estatísticos analisados foram a média e o desvio-padrão. Para analisar os resultados utilizou-se o teste de Tukey, a 5% de probabilidade. Os ingás (Inga uruguensis e Inga sessilis) apresentaram tolerância à geada e um alto potencial para implantação em SAFs nas condições edafoclimáticas em estudo. A espécie tucaneira (Citharexylium myrianthum) apresentou pouca tolerância à geada, porém mostrou alta taxa de rebrota. As espécies corticeira (Erythrina falcata), olandi (Calophyllum brasilienses) e licurana (Hieronyma alchorneoides) apresentaram alta mortalidade em razão da geada, não se mostrando indicadas para compor um SAF na região em estudo.

Mastite por Paracoccidioidomicose: Relato de Caso

A paracoccidioidomicose é uma importante micose sistêmica, endêmica na América Latina. A infecção é usualmente adquirida por inalação das partículas do micélio. Na sua maioria são infecções assintomáticas e estão associadas a vários fatores do hospedeiro, como sexo, idade, fatores genéticos, bem como às características do agente infeccioso e sua virulência. Apresentamos um caso de mastite por paracoccidioidomicose, com o objetivo de demonstrar que pacientes idosas e com abcessos na mama devem ser submetidas à biópsia.

Intoxicação experimental aguda por Senecio brasiliensis em ovinos e indução de resistência à intoxicação

Ovinos são mais resistentes à intoxicação por Senecio spp. que bovinos e equinos. Para determinar se essa resistência é induzida pela ingestão de pequenas e repetidas doses da planta e se essa resistência é duradoura, foram realizados três experimentos com folhas e talos verdes de Senecio brasilienses. Para determinar a dose mínima que causa intoxicação aguda (experimento 1), foram administradas doses únicas de 60, 80, 90, 100 e 100g/kg de peso corporal (pc) a cinco ovinos, respectivamente. Os animais que receberam 60 e 80 g/kg de pc de S. brasiliensis não adoeceram, porém o ovino que recebeu 80g/kg de pc apresentava fibrose e megalocitose discretas nas biópsias realizadas aos 90, 120 e 150 dias do término da administração da planta. Os ovinos que receberam 90 e 100g/kg de pc apresentaram anorexia, prostração, movimentos de pedalagem, dor abdominal e morte 12-48 horas após o aparecimento dos sinais clínicos. Na necropsia havia ascite, petéquias disseminadas e acentuação do padrão lobular hepático. Histologicamente havia necrose hemorrágica centro-lobular. No Experimento 2 a dose capaz de causar a intoxicação aguda foi fracionada e administrada em duas, cinco e 10 doses diárias para 3 ovinos, respectivamente. A dose tóxica fracionada não provocou sinais clínicos de intoxicação em nenhum dos ovinos, porém havia fibrose periportal e megalocitose moderadas nas biopsias realizadas aos 60 dias do término da administração da planta, as quais não evoluíram. O ovino que recebeu a dose fracionada em 10 administrações não apresentou lesões histológicas nas biópsias. Para determinar se os ovinos tornam-se resistentes à forma aguda da intoxicação (experimento 3), foram administradas doses diárias de 15g/kg de pc por 30 dias e 30g/kg de pc por 10 dias a quatro ovinos. No dia seguinte à última administração dois ovinos receberam a dose única de 100g/kg de pc de S. brasiliensis, mas não adoeceram nem apresentaram lesões em biópsias realizadas 15 e 30 dias após o desafio. Entretanto, dois ovinos que receberam essa dose, 15 e 45 dias após a última administração da planta, respectivamente, apresentaram anorexia, dor abdominal, movimentos de pedalagem e morreram 12 horas após o início dos sinais clínicos. As lesões macro e microscópicas eram semelhantes às observadas nos ovinos do Experimento 1. Os resultados do presente trabalho demonstraram que, experimentalmente, ovinos intoxicam-se de forma aguda com doses únicas de 90-100g/kg de pc, mas são capazes de suportar essas doses após a ingestão contínua de pequenas quantidades da planta, no entanto, essa suposta resistência é perdida se os ovinos deixam de ingerir a mesma. Pode-se sugerir também que a intoxicação aguda natural, provavelmente, não ocorre devido à improbabilidade de ingestão, por um ovino, da quantidade necessária de planta em um período curto de tempo.

Lung tissue mechanics in the early stages of induced paracoccidioidomycosis in rats

Pulmonary dysfunction represents the most important cause of death in patients with paracoccidioidomycosis (PBM). In order to investigate the functional changes of the lungs in the early stages of PBM, a model of benign disease was developed by intratracheal challenge of 12-week old isogenic Wistar rats with 1 x 106 yeast forms of Paracoccidioides brasiliensis. Animals were studied 30 and 60 days after infection, when fully developed granulomas were demonstrable in the lungs. Measurements of airway resistance, lung elastance and tissue hysteresis were made during sinusoidal deformations (100 breaths/min, tidal volume = 2 ml) with direct measurement of alveolar pressure using the alveolar capsule technique. Infection caused a significant increase in hysteresis (infected: 1.69, N = 13; control: 1.13, N = 12, P = 0.024, ANOVA), with no alterations in airway resistance or lung elastance. Histopathological analysis revealed the presence of fully developed granulomas located in the axial compartment of the lung interstitial space. These results suggest that alterations of tissue mechanics represent an early event in experimental PBM

Immunohistochemical demonstration of TGF-ß and decorin in paracoccidioidal granulomas

Different patterns of granulomas have been observed in 6- to 8-week-old mice after ip inoculation with 5 x 10(6) yeast cells of Paracoccidioides brasiliensis. Transforming growth factor-ß (TGF-ß) is a cytokine that has been shown to participate in fibrosis and granuloma formation; its activities seem to be modulated by the small proteoglycan decorin. In the present study, TGF-ß and decorin expression in epiploon granulomas was assessed by immunohistochemistry in susceptible (B10.A) and resistant (A/J) mice after 15, 30, 120 and 150 days of P. brasiliensis ip infection. The epiploon was collected, fixed in Methacarn solution and embedded in paraffin, and 5-µm thick sections were used for immunohistochemical analysis employing the streptavidin-biotin-peroxidase technique. The former mouse strain developed fatal disease with many disseminated lesions increasing in size and number during the infection and the latter developed mild disease with the presence of encapsulated granulomas. In the epiploon, TGF-ß was present on macrophages, giant cells, lymphocytes and fibroblasts, and absent on neutrophils. It was also detected in areas of fibrosis and necrosis, as well as disperse in amorphous extracellular matrix, mostly in resistant mice. Decorin was present circumscribing macrophages and giant cells containing fungi, but absent on these cells. In both mouse strains, decorin was found at the periphery of the lesions, and markedly in milky spot granulomas. In resistant mice, positivity was found around fibrotic and necrotic areas of encapsulated and residual lesions containing lysed fungi. Decorin was found associated with thick fibers around encapsulated lesions. In susceptible mice, the size and number of lesions increased with the progression of the disease and were correlated with the weaker expression of decorin. We suggest an association of decorin with the fibrogenic process observed in paracoccidioidal granulomas.

The left lung is preferentially targeted during experimental paracoccidioidomycosis in C57BL/6 mice

Paracoccidioidomycosis (PCM) is a chronic systemic mycosis caused by the inhalation of the thermally dimorphic fungus Paracoccidioides brasiliensis as well as the recently described P. lutzii. Because the primary infection occurs in the lungs, we investigated the differential involvement of the right and left lungs in experimental P. brasiliensis infection. Lungs were collected from C57BL/6 mice at 70 days after intravenous infection with 1×106 yeast cells of a virulent strain of P. brasiliensis (Pb18). The left lung, which in mice is smaller and has fewer lobes than the right lung, yielded increased fungal recovery associated with a predominant interleukin-4 response and diminished synthesis of interferon-γ and nitric oxide compared with the right lung. Our data indicate differential involvement of the right and left lungs during experimental PCM. This knowledge emphasizes the need for an accurate, standardized protocol for tissue collection during studies of experimental P. brasiliensis infection, since experiments using the same lungs favor the collection of comparable data among different mice.