Membrane and extracellular antigens of Paracoccidioides brasiliensis (Mexo): identification of a 28-kDa protein suitable for immunodiagnosis of paracoccidioidomycosis.

In this work, we analyzed serological responses of paracoccidioidomycosis (PCM) patients to membrane and extracellular antigens (Mexo) of Paracoccidioides brasiliensis by ELISA, immunoblot technique and immunofluorescence assays to identify a specific antigen profile. Among 140 PCM serum samples analyzed, a homogeneous IgG response to Mexo was observed. The specificity of this antigen was 96.6% in relation to control sera and 81.2% to sera from patients with diverse infections. Patients undergoing treatment for more than 1 year showed a reduced antibody response against Mexo. These results suggest that the presence of anti-Mexo antibodies might be an indicator of active disease. A protein from Mexo with a molecular weight of 28 kDa (Pb28) was the most specific antigen in humoral immune responses to PCM, since it reacted with 100% of patient sera and did not react with heterologous serum samples tested. This protein was purified by molecular filtration chromatography in FPLC system and, when tested by immunoblotting, it maintained its reactivity and specificity of 100% with PCM sera. The Pb28 N-terminal amino acid sequence comparison analysis in the non-redundant GenBank database at NCBI revealed no significant homology to known PCM proteins or to other fungal proteins of known function. Since the 28-kDa protein of P. brasiliensis seems to be specific for PCM, it can be used as an alternative antigen in immunoblotting diagnostic methods.

Modulation of CD28 and CD86 expression in patients with paracoccidioidomycosis in different periods of treatment.

Paracoccidioides brasiliensis, a thermo-dimorphic fungus, is the ethiologic agent of paracoccidioidomycosis (PCM). The recidive is the greatest obstacle of this disease, because the yeast usually returns after the long treatment period. In the present work, we have investigated the cellular immune response of cells from peripheral blood drawn from patients with different duration of PCM. The classification of patients ranged from nontreated to those with long-standing disease over 5 years. Unstimulated as well as cells stimulated with phytohemaglutinin or two different antigen preparations, secreted (MEXO) or somatic (PbAg) of P. brasiliensis, were characterized. We found that cells from patients with disease proliferate considerably upon stimulation with the antigen preparations and that cells from patients with disease of long duration does not proliferate that vigorously as from patients with more recent diagnosis. Both interferon (IFN)-gamma and interleukin (IL)-4 appear to be increased in patients, but IFN-gamma tended to increase upon treatment while IL-4-secretion decreased. With respect to CD28 and CD86, we found that the subset of CD28 positive CD8 cells are decreased in all stages of the disease as compared to control individuals. A subset of CD86 positive CD19 cells appeared to be considerably increased compared to the controls. Indeed, our results demonstrated that the treatment of PCM patients promoted a regulation of IFN-gamma, IL-4 levels and CD28, CD86 expression bringing new insight to the cellular immune response in PCM.