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 & Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh & 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 batches from three human isolates (Bt1, Bt2 & 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.

Optimization of a mouse immunization protocol with Paracoccidioides brasiliensis antigens.

The objectives of the present study were to optimize the protocol of mouse immunization with Paracoccidioides brasiliensis antigens (Rifkind's protocol) and to test the modulation effect of cyclophosphamide (Cy) on the delayed hypersensitivity response (DHR) of immunized animals. Experiments were carried out using one to four immunizing doses of either crude particulate P. brasiliensis antigen or yeast-cell antigen, followed by DHR test four or seven days after the last immunizing dose. The data demonstrated that an immunizing dose already elicited response; higher DHR indices were obtained with two or three immunizing doses; there were no differences between DHR indices of animals challenged four or seven days after the last dose. Overall the inoculation of two or three doses of the yeast-cell antigen, which is easier to prepare, and DHR test at day 4 simplify the original Rifkind's immunization protocol and shorten the duration of the experiments. The modulation effect of Cy on DHR was assayed with administration of 2.5, 20 and 100 mg/kg weight at seven day intervals starting from day 4 prior to the first immunizing dose. Only the treatment with 2.5 mg Cy increased the DHR indices. Treatment with 100 mg Cy inhibited the DHR, whereas 20 mg Cy did not affect the DHR indices. Results suggest an immunostimulating effect of low dose of Cy on the DHR of mice immunized with P. brasiliensis antigens.

The use of a mixture of somatic and culture filtrate antigens in the evaluation of the immune response to Paracoccidioides brasiliensis.

Three Paracoccidioides brasiliensis antigens, namely a culture filtrate preparation, a somatic antigen and a mixture of equal parts of the two, were tested by two serological techniques against sera from patients with paracoccidioidomycosis, and in an in vivo delayed hypersensitivity model in mice. The antigen mixture was more sensitive than the two individual antigens for the evaluation of the humoral and cellular immune response to P. brasiliensis, both in man and in experimental animals.

Reticulin antibodies: relationship with endocardium-vessel-interstitium antibodies and heterophilic antibodies.

1. Indirect immunofluorescence was used to compare reticulin antibodies, endocardium-vessel-interstitium (EVI) antibodies and heterophilic antibodies in serum samples from patients with degenerative and inflammatory diseases, Chagas' disease, or paracoccidioidomycosis and from healthy blood donors. 2. The antigen substrates used were rat, mouse and human liver for reticulin antibodies, mouse and human heart and skeletal muscle for EVI factor, and rat heart and rat, mouse and human kidney and stomach for heterophilic antibodies. 3. The three antibodies produced extremely similar fluorescent patterns and were present simultaneously in a large proportion of reticulin-positive serum samples. 4. The three antibodies were significantly absorbed by heterophilic antigens such as sheep and rat red blood cells and guinea pig kidney, by Trypanosoma cruzi (an antigen associated with EVI factor) and by Paracoccidioides brasiliensis. However, they did not react with several human tissues. 5. These results suggest that reticulin and EVI antibodies can be considered to be heterophilic antibodies.

Reticulin antibodies: relationship with endocardium-vessel-interstitium antibodies and heterophilic antibodies

Indirect immunofluorescence was used to compare reticulin antibodies, endocardium-vessel-interstitium (EVI) antibodies and heterophilic antibodies in serum samples from patients with degenerative and inflammatory diseases, Chagas' disease, or paracoccidioidomycosis and from healthy blood donors. The antigen substrates used were rat, mouse and human liver for reticulin antibodies, mouse and human heart and skeletal muscle for EVI factor, and rat heart and rat, mouse and human kidney and stomach for heterophilic antibodies. The three antibodies produced extremely similar fluorescent patterns and were present simultaneously in a large proportion of reticulin-positive serum samples. The three antibodies were significantly absorbed by heterophilic antigens such as sheep and rat red blood cells and guinea pig kidney, by Trypanosoma cruzi (an antigen associated with EVI factor) and by Paracoccidioides brasiliensis. However, they did not react with several human tissues. These results suggest that reticulin and EVI antibodies can be considered to be heterophilic antibodies

[Agar gel microdiffusion reaction in the serologic diagnosis of paracoccidioidomycosis]. / Reação de microimunodifusão em gel de ágar no diagnóstico sorológico da paracoccidioidomicose.

We used the micro- and macroimmunodiffusion test for the qualitative and quantitative measurement of anti--P. brasiliensis antibodies in serum of patients with paracoccidioidomycosis. All 103 paracoccidioidomycosis sera (100%) were positive in the microtest versus 87% positivity index in the macrotest. All 83 control sera from patients with other diseases were negative in both tests. Titers of the positive sera tended to be higher in the microtest, which revealed sharper and easier to read precipiting bands. Microimmunodiffusion is simple to be performed, requires a minimum amount of reagents and allows the simultaneous testing of 102 sera. It may replace the macrotest specially in laboratories dealing with great serologic routine.

Serology of paracoccidioidomycosis. I. Evaluation of the indirect immunofluorescent test.

The purposes of the present work were: i) to study the positivity indices and compare titers obtained with the indirect immunofluorescence (II), tube precipitation (TP), complement fixation (CF) and double immunodiffusion on agar gel (ID) tests in the sera of 196 patients with paracoccidioidomycosis before treatment, and ii) to compare the initial titers of II with those obtained 1 year or more after treatment. II was the most sensitive serologic reaction (85.2%), and the positivity indices for CF, ID and TP were 67.7%, 66.0% and 50.0%, respectively. The sera tended to show parallel mean titers in II, CF and TP tests. One year after treatment there was a fall in titers of II in 66.2% of patients. The data, taken as a whole, demonstrate the usefulness of the indirect immunofluorescent test and the importance of using 2 or more serologic tests for the diagnosis and monitoring of patients with paracoccidioidomycosis.

Serology of paracoccidioidomycosis. II. Correlation between class-specific antibodies and clinical forms of the disease.

Untreated and previously treated patients with paracoccidioidomycosis were studied for: (i) serum levels of total IgG, IgM and IgA immunoglobulins, by radial immunodiffusion and Paracoccidioides brasiliensis (Pb) antibodies, by indirect immunofluorescence; (ii) correlation between their levels with the clinical forms of the disease; (iii) correlation between the serum titres obtained by tube precipitin with those of anti-Pb IgG, IgM and IgA. In the untreated group, serum IgG levels were significantly increased in patients with the more systemic forms of the disease, especially the acute progressive form. Serum IgA levels were significantly increased in all patients with no statistical difference between clinical forms. Serum IgM levels were normal in all patients. Anti-Pb IgG, IgA and IgM were detected in 97.5%, 32.5% and 45.0% of all cases, respectively. There was a sharp tendency towards higher levels of anti-Pb IgG among those with the acute progressive form (83.4%) in relation to the chronic, more localized forms, mixed form (68.0%) and isolated organic form (55.5%). In the untreated and previously treated group sera, there was positive correlation between the level of anti-Pb IgG and positivity for the tube precipitin test, suggesting that the precipitin-type antibodies are of the IgG class. Broadly, the present data demonstrate a polyclonal activation of the humoral immune system in paracoccidioidomycosis, with a positive relationship between serological results and severity of the disease.

Indirect fluorescent test for detection of anti-Paracoccidioides brasiliensis antibodies using coated bentonite particles as antigen.

An indirect fluorescent test was developed for detecting antibodies to Paracoccidioides brasiliensis using bentonite particles as antigen (Bent-IF). The bentonite particles were coated with P. brasiliensis polysaccharide antigen and tested with sera from paracoccidioidomycosis patients (36 sera), normal blood donors (32 sera) and patients with non-mycotic diseases (29 sera). The titres given by the positive sera were compared with those of complement fixation (CF), immunodiffusion (ID) and immunofluorescent test using yeast forms of the fungus as antigen (conventional-IF). All normal blood donors' sera gave a negative Bent-IF, conventional-IF, ID and CF tests. All paracoccidioidomycosis sera were reactive in conventional-IF and gave concordant results in Bent-IF. There was no correlation between CF and Bent-IF titres. 27.6% of sera from patients with non-mycotic diseases gave weak titres in both IF-tests. The present data indicate that the Bent-IF is a sensitive and simple serodiagnostic technique comparable with the conventional P. brasiliensis antibody test.