Julio Croce

Allergenic evaluation of Malassezia furfur crude extracts.

Crude extracts of the lipophilic yeast Malassezia furfur were obtained from 2, 6, 10 and 28 day old cultures. The in vitro cultivation periods corresponded, respectively, to the lag phase, middle of the log phase, end of log phase and the decline phase of the growth curve, which was based on viable cell counts obtained with a fluorescent viability test. Biochemical analyses showed that the protein and carbohydrate contents were greater in day 10 extracts. Seventy patients with different allergic manifestations and 30 healthy volunteers were skin prick tested using the extracts. Of these, thirteen (18.57%) patients gave positive responses. SDS PAGE gradient electrophoretic profiles of the preparations indicated that the 28 day extracts contained the greatest number of protein bands with molecular weights ranging mostly between 30 and 94 kDa. Immunoblots incubated with individual patient sera showed that four IgE binding M. furfur allergens of approximately 88, 61, 52 and 39 kDa were present in the 28 day extracts. The components identified could be used for detecting IgE mediated responses to M. furfur among individuals affected with different allergic conditions.

Biochemical, antigenic and allergenic characterization of crude extracts ofDrechslera (Helminthosporium) monoceras

In a previous study with airborne mould extracts we verified thatDrechslera (Helminthosporium) monoceras presented stronger reactions than those presented by 42 other moulds isolated in São Paulo city. In the present study, we evaluated the biochemical composition and the antigenicity of crude extracts obtained from vegetative and conidial stage ofD. monoceras using Czapeck broth (CB) modified and tris-HCl for extraction. The maximum values of total proteins and lipids were verified in the crude extract obtained in the 28th day of growth, and maximum values of carbohydrates were observed in the extracts of the 16th, 22nd and 26th days. The fractionated proteins by SDS-PAGE presented bands with molecular weights between 14.4 to 67 Kd; the 28th day extract showed a larger number of bands. The carbohydrates and amino acids were characterized by thin-layer chromatography. The antigenicity of the crude extracts was verified by immunodiffusion reaction in agar against rabbit hyperimmune sera. Precipitation lines were observed in all studied extracts and common antigenic molecular populations. Based on the above results, the 28th day extract was selected to verify the induction of IgE antibody responses in immunizations of Balb/c and cAF-1 mice, and titer by passive cutaneous anaphylaxis test using Wistar rats. The maximum titers obtained were 160 in cAF-1 mice and 1.280 in Balb/c mice. The results suggest that the 28th day extract contains allergenic fractions and should be chosen for future studies related to fractionation, characterization and standardization in diagnostic methods and immunotherapy.

Candidin: comparison of two antigens for cutaneous delayed hypersensitivity testing

A candidin, which is a suspension of killed yeast cells, is commonly used for intradermal tests of delayed hypersensitivity, to evaluate the immunological cellular competence of the patient, when the test is applied along with other similar tests. When working with a cellular antigen, the histopathology of positive skin tests reveals a cellular infiltrate which not only presents a characteristic hypersensitivity reaction but also a neutrophilic abscess in the central part. This research presents the results of a comparison between the yeast cell suspension and the polysaccharide antigens, both obtained from the same strains of Candida albicans. The results obtained by skin tests in one hundred individuals were 61.0% with the polysaccharide antigen and 69.0% with the yeast cell suspension antigen. Concordant results concerning the two antigens were observed in 82.0% of the individuals. The discussion section presents an assumption to explain the differences of positivity obtained with the two antigens. We conclude that the polysaccharide antigen can be utilized in the intradermal test of delayed hypersensitivity to Candida albicans.