Cezar Mendes de Assis

Immunochemical study of a Paracoccidioides brasiliensis polysaccharide-like antigen

The polysaccharide antigen from P. brasiliensis has been largely employed in serologic tests ,as well as in skin tests, to evaluate cellular immunity. SDS-PAGE analysis of this antigen has revealed a variability in the number of bands exhibited by isolates SN, 265, 339, 113, and 18 (7 to 16 bands). The antigens obtained from isolates 2, PTL, 192 and Adel showed two or three bands. Glycoprotein analysis demonstrated a broad region between 50 and 90 kDa. Major bands of 48 and 30 kDa were present in almost all antigens. Optimal complement fixing dilution appears to be unaffected by the number of bands presented by different antigens. The immunoblot analysis revealed that the 90 and 30 kDa bands were mainly recognized by sera from paracoccidioidomycosis patients. Bands of high molecular weight were also recognized by most of the sera studied. Sera from histoplasmosis recognized the 94 kDa band. In conclusion, although the isolates exhibit quantitative variability in the number of fractions, it is possible to use only one or two samples given the greatest frequency of reactivity is seen in the 30 and 90 kDa fractions.

Paracoccidioides brasiliensis: a mycologic and immunochemical study of a sample isolated from an armadillo (Dasipus novencinctus)

A sample of P. brasiliensis isolated from the spleen and the liver of an armadillo (Dasipus novencinctus) has been analysed under a mycological and immunochemical viewpoint. The armadillo was captured in an area of Tucuruí (State of Pará, Brazil), the animal being already established as an enzootic reservoir of P. brasiliensis at that region of the country. This sample maintained in the fungal collection of the Tropical Medicine Institute of São Paulo (Brazil) numbered 135, has got all the characteristics of P. brasiliensis, with a strong antigenic power and low virulence for guinea-pigs and Wistar rats. The specific exoantigen of P. brasiliensis--the glycoprotein with a molecular weight of 43 kDa--was easily demonstrated with double immunodiffusion, immunoelectrophoresis, SDS-PAGE and immunobloting techniques.

Obtenção de exoantígenos de Histoplasma capsulatum em meio de neopeptona, glicose, tiamina e asparagina (NGTA)

The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1,2 and 3 months, at 36oC and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta - USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoc-cidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1, 2 and 3 months, at 36 degrees C and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta--USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoccidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.O presente trabalho teve como objetivo a producao de exoantigenos H e M das amostras 58, B-679, A-811 e O187 de Histoplasma capsulatum, utilizando o meio NGTA (neopeptona, glicose, tiamina e asparagina) em periodos de cultivo de 1, 2 e 3 meses, a 36oC, sob agitacao constante (50 v.p.m.). Os antigenos brutos foram avaliados contra anti-soro e antigeno de Histoplasma capsulatum de referencia (Center for Disease Control), 4 soros de pacientes portadores de paracoccidioidomicose, 7 de histoplasmose e soro hiperimune anti-H. capsulatum produzido em coelhos, atraves da reacao de imunodifusao dupla. Verificou-se que, com excecao de B-679 com 1 mes de crescimento, todos os demais exoantigenos apresentaram as fracoes H e M de precipitacao. Os exoantigenos obtidos de A-811 apresentaram so a banda H. Excetuando-se os exoantigenos 58 e B-679 com 1 mes de crescimento, todos os demais exoantigenos reagiram contra soros de pacientes com histoplasmose. Em relacao aos soros de pacientes com paracoccidioidomicose, somente os exoantigenos 58 e O187 nao apresentaram reacao cruzada. Todos os exoantigenos reagiram frente ao soro hiperimune de coelho anti-H. capsulatum. Para obtencao de exoantigenos de H. capsulatum, sugerimos que as amostras sejam cultivadas sob as condicoes anteriormente descritas, adotando-se o periodo de 3 meses de crescimento, utilizando-se exoantigenos de referencia como controles da reacao.

Obtençäo e avaliaçäo de antígenos de Aspergillus fumigatus

Antigens from three strains of Aspergillus fumigatus (354, 356, and JIG) and an antiserum against the mixing of these antigens have been produced, and evaluated immunochemically. The antigens were obtained through a modified Coleman & Kaufman technique (culture filtrate concentrated by acetone). Analysis by the immunodiffusion test (ID) against homologous serum has yielded 100% sensitivity (with the studied sera). Concerning heterologous sera we found reactivity with a serum of a patient of candidiasis and another with histoplasmosis. The same result was obtained with a reference antigen in immunodiffusion, showing similar standards of response. Titration of the antiserum by ID and counterimmunoelectrophoresis showed a title of 1:32, and by complement fixation (micro-technique) a title of 1:128. Using immunoelectrophoresis (IEF), the produced antiserum yielded 8 lines of precipitation (5 in the anodic pole and 3 in the cathodic one). In SDS-PAGE at 12.5% the antigen has presented a rather complex electro-phoretic profile (26 proteic subunits with a molecular weight ranging from 18 a > 100 kDa). Immunogenicity of the antigen was observed in all fractions of SDS-PAGE when the immu-noblotting against the antiserum was carried out.

In vitro action of some disinfectants on Paracoccidioides brasiliensis yeast forms

The fungicidal action of sodium hypochlorite (0.3, 1, 2.5, 5 and 10%); formaldehyde (2, 5, and 10%); and ethyl alcohol (70%) on yeast forms of Paracoccidioides brasiliensis Pb 18 and a newly-isolated Goiana strain was described. Contact between the fungus and the disinfectants was maintained for 1, 2, 24, 48 and 72 hours at room temperature. Viability was evaluated by the fluorescein diacetate-ethidium bromide treatment, culture in solid and liquid media (36 degrees C and 26 degrees C); yeast to mycelial germination at room temperature; and radiometric study of metabolic activity. All concentrations of disinfectants were found to be effective in inactivating Pb 18 and Goiana strains, except for the 1-hour contact with 2% formaldehyde, in which fluorescein diacetate-ethidium bromide treatment was found to reveal 40 and 27% of viable cells, respectively. The yeast to mycelial germination method was considered to reveal faster and similar results as compared to culture in solid and liquid media.

Histoplasma capsulatum exocellular antigens. Obtention in neopeptone, glucose, thiamine and asparagine medium (NGTA)

The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1,2 and 3 months, at 36oC and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta - USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoc-cidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1, 2 and 3 months, at 36 degrees C and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta--USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoccidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.O presente trabalho teve como objetivo a producao de exoantigenos H e M das amostras 58, B-679, A-811 e O187 de Histoplasma capsulatum, utilizando o meio NGTA (neopeptona, glicose, tiamina e asparagina) em periodos de cultivo de 1, 2 e 3 meses, a 36oC, sob agitacao constante (50 v.p.m.). Os antigenos brutos foram avaliados contra anti-soro e antigeno de Histoplasma capsulatum de referencia (Center for Disease Control), 4 soros de pacientes portadores de paracoccidioidomicose, 7 de histoplasmose e soro hiperimune anti-H. capsulatum produzido em coelhos, atraves da reacao de imunodifusao dupla. Verificou-se que, com excecao de B-679 com 1 mes de crescimento, todos os demais exoantigenos apresentaram as fracoes H e M de precipitacao. Os exoantigenos obtidos de A-811 apresentaram so a banda H. Excetuando-se os exoantigenos 58 e B-679 com 1 mes de crescimento, todos os demais exoantigenos reagiram contra soros de pacientes com histoplasmose. Em relacao aos soros de pacientes com paracoccidioidomicose, somente os exoantigenos 58 e O187 nao apresentaram reacao cruzada. Todos os exoantigenos reagiram frente ao soro hiperimune de coelho anti-H. capsulatum. Para obtencao de exoantigenos de H. capsulatum, sugerimos que as amostras sejam cultivadas sob as condicoes anteriormente descritas, adotando-se o periodo de 3 meses de crescimento, utilizando-se exoantigenos de referencia como controles da reacao.