Antibody response to the 43 kDa glycoprotein of Paracoccidioides brasiliensis as a marker for the evaluation of patients under treatment

Sera of patients with paracoccidioidomycosis contained IgG-, IgA-, and IgM-specific antibodies to a 43 kDa antigen contained in the filtrate of a culture of Paracoccidioides brasiliensis. IgG- and IgA-specific antibodies were present in all observed patients. The IgM response was more frequent in acute cases, and the mean titers of IgG- and IgM-specific antibodies were higher in the acute forms. By the fourth month of chemotherapy, there was a decay of IgG, IgA, and IgM antibody titers to this antigen in acute cases, correlating with clinical improvement. The detection of IgG and IgA antibodies and the sequential determination of antibodies to the 43 kDa glycoprotein may be useful tools for serodiagnosis and evaluation of therapeutic efficacy.

Cell wall fractions from Paracoccidioides brasiliensis induce hypergammaglobulinemia

The antibody response against the antigen sheep red blood cells (SRBC) was investigated in mice pre-treated with formalin-killed Paracoccidioides brasiliensis or with cell wall fractions of the fungus. Pre-treatment with P. brasiliensis, as well as with the F1 fraction and beta-glucan significantly increased the anti-SRBC antibody response in the experimental groups as compared to the control group that received only SRBC. This immunomodulatory effect varied with the different doses employed and with pre-treatment time. We conclude that the cell wall fractions of P. brasiliensis might play an important role in the hypergammaglobulinemia associated with Paracoccidioidomycosis. © 1993 Kluwer Academic Publishers.

Infection and apparent invasion of Vero cells by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis probably uses many different mechanisms to establish itself in the host and cause disease. In this work, we assess an in vitro model system which uses cultured mammalian cells to investigate the virulence factors of P. brasiliensis. We were able to demonstrate an invasion process of the yeast form of this fungus in Vero cell cultures. We deduced that the overall invasive process involved three steps: adhesion, followed by invasion of individual epithelial cells and spread to adjacent cells.

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.

Distribution of exoantigens and a 43-kDa glycoprotein (gp43) in the yeast and mycelial forms of Paracoccidioides brasiliensis

Objective. Paracoccidioides brasiliensis antigens (strain 113) were located at ultrastructural level in both yeast and mycelial forms of the fungus. The reactivity of the sera employed was analysed. Materials and methods. Immunofluorescence and ultrastructural protein A-gold immunolabelling techniques were performed using two polyclonal antisera: one against P. brasiliensis exoantigens and the other against a 43-kDa glycoprotein (gp43). Immunoblotting assays were employed to define reactivity of these antisera with somatic and metabolic antigens of both forms of the fungus. Results. The techniques employed revealed in both yeast and mycelial forms of P. brasiliensis a similar antigenic distribution. The antigens deposits were seen within the cytoplasm, and over the cell wall of the fungus. The anti-exoantigen serum recognized several bands in both forms of the fungus. The anti-gp43 serum reacted strongly with the 43-kDa fraction and weakly with few other fractions. Conclusions. Immunocytochemical techniques suggest a protein synthesis within the cytoplasm followed by excretion through the cell wall. Similar results employing both polyclonal antisera were obtained.

Evaluation of Paracoccidioides brasiliensis exoantigen in the detection of delayed dermal hypersensitivity in experimental and human paracoccidioidomycosis

The exoantigen of Paracoccidioides brasiliensis standardized by Camargo et al. [1] (AgR) was used to evaluate the in vivo and in vitro cell immune response of experimental animals and of patients with paracoccidioidomycosis (PBM). Fava Netto antigen (AgF) was tested in parallel as a control antigen. The study was conducted with mice and guinea pigs infected with P. brasiliensis or immunized with its fungal antigens, on patients with PBM and on their respective control groups. The cell immune response was analysed by skin tests, and by the macrophage and leucocyte migration inhibition tests (MMIT and LMIT) in the animals and in the patients, respectively. The skin test with AgR as paracoccidioidin was positive in infected or immunized mice and guinea pigs and negative in control animals. The skin tests with AgR (24 h) showed 96.7% positivity in patients with PBM and were negative in control individuals. Histopathological study of the in vivo tests in the different experimental models was consistent with a delayed hypersensitivity response (DHR). Immunohistochemical study of the skin tests of PBM patients demonstrated a predominance of T lymphocytes, confirming the nature of a DHR to the fungal antigens. The in vitro cell immune response showed variable results for the various experimental models, i.e. significant rates of MMIT in immunized mice, a tendency to positivity in infected guinea pigs, and the absence of migration inhibition in PBM patients. Taken together, the data indicate that the AgR is efficient as paracoccidioidin in the evaluation of DHR in PBM, with an optimum time of reading the test of 24 h.

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment 1), we analyzed two antigen lots of two human isolates (Bt1 and Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh and Morton medium) in SDS-PAGE and in two immunological tests (immunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment 2), we compared the antigenic profile of three antigen hatches from three human isolates (Bt1, Bt2 and Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment 1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment 2, the reference system for P brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.

Growth-promoting factors for yeast cells of Paracoccidioides brasiliensis

Low-density seedings of yeast cells of Paracoccidioides brasiliensis give poor growth (as assessed by plating efficiency test) on conventional mycological agar media, and therefore growth-promoting factors for this fungus were sought. Water-extracts of yeast cells of six P. brasiliensis isolates were all considerably effective in promoting the growth of low-density seedings of P. brasiliensis isolates Pb-18 and Hachisuga, but had little effect on isolate Bt-4. Horse serum, at a concentration range of 2-4%, moderately or considerably promoted the growth of these P. brasiliensis isolates. Combinations of the fungus cell extracts with horse serum were highly effective in promoting the growth of all of the fungal isolates. The fungus cell extracts showed siderophore (microbial iron carrier) activity. An iron-chelator, ethylenediaminetetraacetic acid, at a concentration of 100 μM also highly promoted the growth of the fungal isolates in the presence of horse serum, and ferric ion added to culture medium was considerably effective in the growth promotion. These results suggest that deficient utilization of external iron by the fungus cell is one of the growth-limiting processes for low-density seedings of yeast cells of P. brasiliensis on conventional mycological agar media.

Paracoccidioides brasiliensis-gp43 used as paracoccidioidin

A purified glycoprotein of 43 000 daltons from Paracoccidioides brasiliensis (gp43) was tested as paracoccidioidin in delayed-type hypersensitivity (DTH) tests in both experimental animals (guinea pig and mice) and patients with paracoccidioidomycosis (PCM). The gp43 paracoccidioidin was compared with the traditional Fava Netto antigen (AgFN). In guinea pigs, the intradermal injection of 2 μg of gp43 showed a similar response to those obtained with AgFN, showing in histological sections a population of lymphoid cells that participate in DTH. In mice, gp43 at a dose of 3.75μg showed positive DTH response. The use of gp43 as paracoccidioidin in humans showed that this molecule can be used to evaluate the DTH response in patients with PCM. Of 25 PCM patients studied, 48% were positive to gp43 while only 28% were positive to AgFN; 12 PCM patients were completely anergic to both antigens. Considering only those 13 PCM patients who were responsive to gp43 and/or to AgFN, 92.3% reacted against gp43 and 53.8% reacted against AgFN (P < 0.05). Gp43 skin test responses (13.67 ± 9.56 mm) were significantly larger than those obtained with AgFN (8.43 ±3.69 mm). Immunohistochemical study of the human skin showed a perivascular inflammatory response constituted predominantly by T lymphocytes, macrophages and polymorphonuclear leukocytes. © 1996 ISHAM.

Cycloactive, aneugenic and clastogenic effects of Paracoccidioides brasiliensis exoantigen in human lymphocyte cultures

The in vitro effect of Paracoccidioides brasiliensis exoantigen on the human lymphocytes cell cycle and chromosomes was studied. Human peripheral blood lymphocyte cultures from ten healthy, white, non-smoking, non-related adult males (mean age 31·3 ± 8·2 years) were studied. Blood cultures were treated with three exoantigen concentrations (0·25, 2·50 and 10·00 μg ml -1). At least 1000 metaphases were analysed at each concentration, for evaluation of numerical and structural chromosome aberrations (cA) and 30 000 for mitotic index (MI). Among the treated cultures, statistically significant differences in the frequencies of MI and cA were not observed. Nevertheless, when compared with control cultures, they all showed a significantly lower frequency of MI and higher frequency of cA. It is suggested that the detected alterations were caused by the exoantigen, its fractions or its metabolites. © 1996 Informa UK Ltd All rights reserved.

Laminin-binding epitope on gp43 from Paracoccidioides brasiliensis is recognized by a monoclonal antibody raised against Staphylococcus aureus laminin receptor

Adhesion is regarded as an important step in the pathogenesis of several microorganisms. Thus, the ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. Studying the already characterized laminin-binding protein of Paracoccidioides brasiliensis, the 43 kDa glycoprotein (gp43), we evaluated whether MAb 1.H12, raised against the laminin-binding protein from Staphylococcus aureus, cross-reacts with that fungal protein. By immunoblot analysis we show that MAb 1.H12 recognizes gp43. This interaction is able to inhibit the laminin-mediated adhesion to epithelial cells as well as the P. brasiliensis infection in vivo. Moreover, through immunoenzymatic assays, we show that MAb 1.H12 recognizes gp43 in solid phase and that this interaction is partially inhibited by the addition of anti-gp43 MAbs. These results show that MAb 1.H12 recognizes the gp43, suggesting the presence of an epitope similar to those found in the other laminin-binding proteins from phylogenetically very distant cells. These findings reinforce the possibility of evolutionary conservation of such epitopes.

Human chromosome aberrations induced in vitro by Paracoccidioides brasiliensis glycoproteic component (GP 43)

The in vitro cytogenetic effects of the 43-kDa molecular mass exocellular glycoproteic component (GP 43) from Paracoccidioides brasiliensis were studied in cultures from human lymphocytes. The sample included 10 healthy, white, non-smoking, non-related males (mean age of 31.3 ± 8.2 years). Besides the control, three concentrations of GP 43 (0.125, 1.25 and 5 μg/ml) were used. In each group, around 1000 cells were examined in search of chromosome aberrations, and 30,000 metaphases were analysed for the determination of the Mitotic Index. The authors conclude that GP 43 most probably causes inhibition of the cell cycle and aneugenic and clastogenic effects.

Detection of circulating Paracoccidioides brasiliensis antigen in urine of paracoccidioidomycosis patients before and during treatment

For the diagnosis and follow-up of paracoccidioidomycosis patients undergoing therapy, we evaluated two methods (immunoblotting and competition enzyme immunoassay) for the detection of circulating antigen in urine samples. A complex pattern of reactivity was observed in the immunoblot test. Bands of 70 and 43 kDa were detected more often in urine samples from patients before treatment. The immunoblot method detected gp43 and gp70 separately or concurrently in 11 (91.7%) of 12 patients, whereas the competition enzyme immunoassay detected antigenuria in 9 (75%) of 12 patients. Both tests appeared to be highly specific (100%), considering that neither fraction detectable by immunoblotting was present in urine samples from the control group. gp43 remained present in the urine samples collected during the treatment period, with a significant decrease in reactivity in samples collected during clinical recovery and increased reactivity in samples collected during relapses. Reactivity of some bands was also detected in urine specimens from patients with 'apparent cure.' The detection of Paracoccidioides brasiliensis antigens in urine appears to be a promising method for diagnosing infection, for evaluating the efficacy of treatment, and for detecting relapse.

Involvement of the major glycoprotein (gp43) of Paracoccidioides brasiliensis in attachment to macrophages

The yeast form of Paracoccidioides brasiliensis, the causative agent of a deep mycosis in humans, is known to be phagocytized by, and to multiply inside, macrophages. In this work we describe the involvement of gp43, a major antigenic protein of P. brasiliensis, in the initial steps of attachment of the fungus to macrophages. Anti-gp43 F(ab) polyclonal fragments were capable of inhibiting phagocytosis in a concentrationdependent manner. Sheep red blood cells sensitized with purified gp43 were more endocytized than SRBC alone, and this process was also inhibited by anti-gp43 F(ab) fragments. Inhibition tests indicated the involvement of fucose and mannose residues in the phagocytosis of the fungus and of SRBC-gp43 by macrophages. Taken together, these results suggest that gp43 may be involved in the adherence and uptake of the fungus by murine peritoneal macrophages, and that this binding may be dependent on monosaccharide residues that are part of the gp43 glycoprotein. © 1998 ISHAM.

Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.