Evaluación de la actividad antifúngica de extractos liquénicos e identificación de sus metabolitos / Evaluation of the fungicide activity from lichens extracts and identification their metabolites

Los líquenes son los hongos que establecen una relación simbiótica con un alga o cianobacteria. En esta simbiosis se producen por parte del hongo, una serie de metabolitos secundarios conocidos como sustancias liquénicas; las cuales presentan una marcada actividad antibiótica. En Cuba no se tienen antecedentes sobre estudios de metabolitos liquénicos por lo que se propone; evaluar el efecto fungicida de extractos liquénicos producidos por especies cubanas así como identificar sus metabolitos. Se emplearon líquenes de diferentes zonas del país (Parmotrema dilatatum, P. tinctorum, P. praesorediosum P. cristiferum, Ramalina americana, Cladonia ceratophylla y Cladonia portentosa spp. pacífica), a los cuales se les extrajo con acetona, las sustancias liquénicas almacenadas en el talo. Los extractos fueron probados contra los hongos fitopatógenos Rhizoctonia solani y Phythophtora nicotianae; por el método de envenenamiento del medio de cultivo agar papa dextrosa a concentraciones de: 0,01 por ciento; 0,03 por ciento y 0,07 por ciento. Se utilizó un control negativo de dimetilsufóxido al 0,07 por ciento y se determinaron los porcentajes de inhibición, cuyos resultados fueron analizados estadísticamente. Los metabolitos secundarios presentes en los extractos se identificaron por cromatografía de capa fina (TLC). Exceptuando el extracto liquénico de P. cristiferum, todos los demás mostraron más de un 50 por ciento de inhibición del crecimiento de ambos hongos a la concentración de 0,07 por ciento, mientras que a las restantes concentraciones los valores fueron variados con diferencias significativas con respecto al control. Se lograron identificar tres metabolitos liquénicos: metil 2‘-O- metilmicrofilinato, 4-O-Demetilmicrofilinico y el ácido ramaniloico.

Lichens are fungi that establish a symbiotic relationship with an alga or cyanobacterium. This symbiosis produced by the fungus, a series of secondary metabolites known as lichen substances; which show a strong antibiotic activity. In Cuba there is no background on the studies above lichen metabolites so it is proposed; evaluate the fungicidal activity of lichen extracts produced by Cuban species and to identify metabolites. Lichens from different areas of the country (Parmotrema dilatatum, P. tinctorum, P.praesorediosum, P. cristiferum, Ramalina americana, Cladonia ceratophylla and Cladonia portentosa spp. pacífica), to which it extracted with acetone, the lichen substances stored in tallus. The extracts were tested against the fungal pathogens Rhizoctonia solani and Phythophtora nicotianae; poisoning by the method of culture medium potato dextrose agar at concentrations of 0.01 percent; 0.03 percent and 0.07 percent. A negative control to 0.07 percent dimethylsulfoxide was used and the percentage of inhibition, the results were analyzed statistically determined. Secondary metabolites present in the extracts were identified by thin layer chromatography (TLC). Except P. cristiferum lichen extract, all others showed more than 50 percent growth inhibition of both fungi at concentration of 0.07 percent, while the remaining concentrations were varied values with significant differences from the control. Was made to identify three lichen metabolites metilmicrofilinato 2‘ methyl-O, 4-ODemetilmicrofilinico and ramaniloico acid.

Assessment of indoor air fungi in Western-Anatolia, Turkey.

This study was conducted to determine fungal spores in the indoor air of the houses in the city of Afyon, Western-Anatolia, Turkey. We investigated the seasonal properties of mould spores in 10 houses of Afyon over a period of one year. Viable moulds were recovered from all 10 houses. Twenty seven different moulds were isolated and identified from the indoor air of the houses. The most common genus was Cladosporium spp. (31.9%), followed by Aspergillus spp. (18.6%), Penicillium spp. (15.5%), Altemaria spp. (13.0%) and other species (21.0%). The mould concentration was higher in the kitchens than in other parts of the houses such as the living rooms and bedrooms (p < 0.05). The fungal flora of the air in the Afyon city region has a seasonal variation. All fungal species had their highest prevalence in summer and their lowest in winter, but only Aspergillus spp. had a significant seasonal variation (p = 0.012). Viable moulds are common in the houses of Afyon. Reducing these indoor fungi is necessary to improve the health of individuals with fungal-induced diseases like asthma.

Detection of fungi spectrum in industrial and home bakeries and determinated fungal allergy with skin prick test.

Airborne fungal pathogens such as Penicillium, Aspergillus, Cladosporium, Trichophyton, and Alternaria may cause health problems. In this research, the fungal flora at different bakeries and their potential allergenic effects on the workers were investigated. We investigated 148 workers at 17 industrial type bakeries and 62 workers at 17 home type bakeries in Afyon. Our study was performed in two different seasons and climates, between January 2004 and June 2004. Fungal flora was detected by using Petri-dish method. In the winter, Penicillium was the dominant genus, while Cladosporium was the dominant genus during the summer, in both types of bakeries. The allergenic properties of dominant culturable fungi on workers involved in the bakeries were determined with the skin-prick test. It was found that with workers in the industrial type bakeries, the most common skin test positivity was caused by Penicillium. In the other hand, the skin test positivity, performed on workers in the home type bakeries, was equally caused by Penicillium, Trichophyton and Aspergillus.

Enzyme-linked immunosorbent assay for detection and quantification of Cryptococcus neoformans antigen

An enzyme-linked immunosorbent assay was standardized for the detection of cryptococcal antigen in serum and cerebrospinal fluid. The system was evaluated in clinical samples from patients infected by human immunodeficiency virus with and without previous cryptococcosis diagnosis. The evaluated system is highly sensitive and specific, and when it was compared with latex agglutination there were not significant differences. A standard curve with purified Cryptococcus neoformans antigen was settled down for the antigen quantification in positive samples

Growth and production phases of pycnoporus sanguineus

Foi observado que na cultura de Pycnoporus sanguineus MIP 89007, a síntese de substâncias com atividade antimicrobiana ocorreu principalmente entre o 18§. e o 23§. dia de incubaçäo. Além disso, foi também constatado que a substância produzida foi rapidamente degradada quando permaneceu no caldo de cultura após ter cessado a síntese e que os extratos obtidos a partir do fungo somente retiveram a atividade quando mantidos à vácuo

Biochemical analysis of the methylic antigen of Paracoccidioides brasiliensis

Yeast forms of five strains of Paracoccidioides brasiliensis (SN, 2, 18, 192 and JT-1) were cultured in a synthetic medium for obtaining methylic antigens. These antigens were lyophilized and studied for each strain, to determine their partial biochemical composition, through measurements of total lipid, protein and carbohydrate contents. Lipids of methylic antigens were purified and analysed for sterols, phospholipids, glycolipids, lipoproteins, and partial characterization of sterols. Significant differences were found among antigenic preparations derived from distinct P. brasiliensis strains, in relation to the quantitative determinations. On the other hand, sterol analysis revealed the presence of ergosterol, lanosterol and squalene in all samples. The diversity verified in the biochemical characteristics of antigens derived from different P. brasiliensis strains, confirm the need of using a pool of fungal samples in order to produce antigen preparations for serological procedures without hampering their sensitivity

Esporotricosis experimental en ratas / Sporotrichosis experimental on rats

Ratas endocriadas se infectaron por vía intraperitoneal y en la cola con una suspensión de conidios de Sporothrix schenckii. Los frotis coloreados con May Grunwald-Giemsa y Gram Nicolle, así como los cultivos de macerados de los órganos que se biopsiaron: hígado, bazo, pulmón y glándula suprarrenal y de la cola manifestaron a los l5 días de inoculación una diseminación del hongo; no así a los 30 días de infección, excepto el material correspondiente a la cola que presentó en ese tiempo elementos de S.schenckii. Los estudios inmunológicos realizados permitieron la estandarización del exoantígeno preparado, se detectaron anticuerpos en los sueros de las ratas a los 15 y 30 días posteriores a la inoculación. Las intradermorreacciones realizadas en los animales provocaron una buena respuesta frente al exoantígeno preparado. De esta forma, se comprueba que la rata es un animal adecuado para la infección experimental y la estandarización de antígenos de S.schenckii

Evaluation of different immunization protocols with P. brasiliensis antigens in guinea pigs

The objective of the present study was to develop an efficient and reproducible protocol of immunization of guinea pigs with P. brasiliensis antigens as an animal model for future studies of protective immunity mechanisms. We tested three different antigens (particulate, soluble and combined) and six protocols in the presence and absence of Freund's complete adjuvant and with different numbers of immunizing doses and variable length of time between the last immunizing dose and challenge. The efficacy of the immunizing protocol was evaluated by measuring the humoral and cellular anti-P. brasiliensis immune response of the animals, using immunodiffusion, skin test and macrophage migration inhibition test. It was observed that: 1. Three immunizing doses of the antigens induced a more marked response than two doses; 2. The highest immune response was obtained with the use of Freund's complete adjuvant; 3. Animals challenged a long time (week 6) after the last immunizing dose showed good anti-P. brasiliensis immune response; 4. The particulate antigen induced the lowest immune response. The soluble and the combined antigens were equally efficient in raising good humoral and cellular anti-P. brasiliensis immune response

Caracterización de antígenos Paracoccidioides brasiliensis: inmunodiagnóstico serológico mediante western blotting / Characterization of Paracoccidioides brasiliensis antigens. Serological immunodiagnosis using western blotting

The electrophoretic characterization on SDS-PAGE gels of Paracoccidioides brasiliensis antigens, strains 2511 and 6688; and the additional use of immunoblotting has permitted in this study to identify the immunogenic, sensitive and specific antigen fractions for the diagnosis of Paracoccidioidomycosis. The antigenic preparations showed differences depending on the morphologic form of the fungus and the method utilized. The procedure revealed heterogeneity in the humoral immune response of the patients studied and permitted us to establish indices of activity of Paracoccidioidomycosis by the sequential analysis of sera obtained during different stages of the disease. The high sensitivity of this method makes it useful as an additional technique for the serologic immunodiagnosis of Paracoccidioidomycosis

Serologic characterization of Paracoccidioides brasiliensis E2 antigen

1. The behavior of the specific E2 antigen of paracoccidioides brasiliensis was studied by agarose gel counterimmonoelectrophoresis. When immunoelectrophoresis. When the gel was read immediately after the electrophoretic run no precipitation band was visible. Visualization of the complex was possible only after incubation of the gel at room temperature overnight. 2. At alkaline pH, the, the E2 antigen migrates in the direvtion of the cathodem as do the immunoglobulins. The higher sensitivity of counterimmunoelectrophoresis when compared to double immunodiffusion for the diagnosis of paracoccidioidomycosis is due to the presence of antibodies directed agaisnt antigens which migrate to the anode. 3. The use of specific antiserum to E2 antigen reference permits the double immunodiffusion method to be a very sensitive test for the specific serodiagnosis of paracoccidioidomycosis

Estudio de un modelo de candidiasis sistemica experimental en ratones rockland / Model of experimental systemic candidosis in Rockland mice

Se presentan los resultados obtenidos en el estudio de un modelo de candidiasis sistémica en ratones rockland. Un total de 65 animales fueron inoculados por vía intracardíaca con 7.5 x 10.5 células de C.albicans. La mitad de los animales murieron en la primera semana y el 80% habían muerto al completar la segunda semana post-infección. En los animales que sobrevivieron hasta la cuarta semana se comprobó la existencia de lesiones renales activas con cultivos positivos para C.albicans. El tratamiento con anfotericina B por vía intraperitoneal modificó el curso de la infección, ya que la totalidad de los 15 animales tratados sobrevivió cuatro semanas y 5 exhibieron cultivos renales negativos. Las pruebas de contrainmunoelectroforesis con antígeno citoplasmático de C.albicans solo dieron resultados positivos en 7/18 ratones que vivieron hasta la cuarta semana. No hubo correlación entre los resultados de estas pruebas serológicas y la gravedad de las lesiones comprobadas en la autopsia.

Fractionation of culture filtrate antigens of Aspergillus fumigatus.

Polyacrylamide disc gel electrophoresis (PAGE) and two-dimensional crossed immunoelectrophoresis (2D-CIE) of crude culture filtrate antigens prepared from three clinical isolates of Aspergillus fumigatus revealed a striking similarity in the number and nature of their antigenic components, notwithstanding the wide differences in their total protein contents. The antigenic components, which are largely proteins and glycoproteins, varied from 9-10 in number depending on the isolate. A prominent glycoprotein band, common to all the three isolates, merits further purification and detailed study. The serodiagnostic value of culture filtrate antigens of A. fumigatus vis-a-vis mycelial antigens, has been discussed.

Soros hiperimunes anti-Paracoccidioides brasiliensis

Quinze soros hiperimunes, contra 5 amostras de Paracoccidioides brasiliensis, foram produzidos em coelhos brancos da raça Nova Zelândia, sendo utilizados 3 coelhos para cada amostra do fungo. O imunógeno era constituído por mistura de 0,3ml de lisado, 0,2ml de filtrado, 0,5ml de suspensäo de células leveduriformes vivas e 0,1ml de adjuvante de Freund completo. Inoculaçöes semanais, durante 10 semanas, de 0,5ml da mistura, foram aplicadas, por via subcutânea nas regiöes axilares dos coelhos. Os anti-soros obtidos foram experimetnados em reaçöes de fixaçäo do complemento, precipitaçäo em meio líquido, dupla difusäo em gel de ágar, imunoeletroforese e imunoeletrosmoforese. Os resultados obtidos justificam esta publicaçäo

Estudio de un antígeno de dermatofitos / Antigen of dermatohytes

El objeto del presente trabajo es la obtención y estudio de un antígeno de dermatofitos útil para la realización de pruebas serológicas y cutáneas en pacientes com dermatofitias. Este reactivo se preparó mediante una modificación de la técnica de exoantígenos de Kaufman y Standard y se le dosó el contenido proteico que fue de 48 mg/ml. Prueba en animales de experimentación: Se utilizaron 2 lotes de cobayos; al primero (16 animales) se les produjo una tiña experimental en el flanco rasurado con un cultivo de T. mentagrophytes var. yesosa. Todos los animales presentaban pruebas cutáneas positivas con el antígeno 1/10 y 12/16 cuando se lo utilizó diluido 1/100. Sólo un cobayo presentó anticuerpos demostrables por CIE. El 2§ lote (18 cobayos) fue sensibilizado mediante 4 inyecciones semanales por vía subcutánea, de una suspensión densa de micelio de dermatofitos. La intradermorreacción con el antígeno 1/100 fue positiva en 14 dudosa en 4 y todos mostraron hipersensibilidad retardada frente al antígeno 1/10. Once animales mostraron anticuerpos por CIE y a 8 de ellos se les efectuó fijación de complemento que fue positiva en todas con títulos entre 1/16 y 1/512. Individuos control: Se efectuaron pruebas intradérmicas en 315 personas (211 hombres y 104 mujeres) internadas en distintas salas del Hospital Muñiz; 216 fueron negativas y 99 positivas. Grupo de pacientes: Se estudiaron 204 enfermos con distintas formas clínicas de dermatofitas. A todos se les efectuó 204 enfermos se les efectuó examen micológico y prueba cutánea y a 131 de ellos se les buscó anticuerpos por CIE. 82/204 presentaron reacción inmediata a la dermatofina y 95/199 hipersensibilidad retardada. Sólo 8 de las 131 CIE mostraron bandas de preciptación. Conclusiones: 1. El antígeno es útil para pruebas cutáneas...

Determinación de la inmunidad a Thermoactinomyces thalpophilus en Jujuy / Determination of the immunity to Thermoactinomyces thalpophilus in Jujuy, Argentina

De los actinomicetos termófilos aislados en una localidad de la zona azucarera de Jujuy, el más común fue Thermoactinomyces thalpophilus. Se preparó un antígeno metabólico, según el método de doble diálisis modificado. Se hicieron pruebas cutáneas con la dilución 1:10 en 114 personas de las cuales 19 tuvieron una reacción positiva inmediata. Se observaron 1 ó 2 líneas de preciptación frente al antígeno concentrado, con seis sueros, al hacer la prueba de doble difusión en gel de agar con fosfatos a pH 7,4. Es evidente que la población de la localidad está expuesta a los agentes causantes de la pneumonitis por hipersensibilidad llamda bagazosis y es posible la presencia de esta patología

Purificación de antigenos somaticos del Paracoccidioides Brasiliensis: estudio preliminar / Purification of somatic antigens of Paracoccidioides brasilienses. Preliminary study

Se describen los procedimientos de purificación empleados para la separación de las facciones antigénicas a partir de un material somático obtenido por rotura de células levaduras completas de P. brasiliensis. Dichas fracciones mostraron ser proteínas con pesos moleculares de 66 y 85 Kd; la primera de ellas reaccionó con sueros específicos produciendo una banda de precipitado idéntica a una de las 3 desarrolladas por el antígeno total. Los resultados señalan la posibilidad de obtener antígenos purificados, químicamente identificados y cuyo uso pudiera, en el futuro, representar ventajas para el diagnóstico serológico de la paracoccidioidomicosis, permitiendo separar, repetidamente, solo aquel componente reconocidamente activo