Immunohistochemical characterization of mononuclear cells in delayed hypersensitivity reactions to Paracoccidiodes brasiliensis (paracoccidioidin test)

The density and distribution of T cells, T helper cells, macrophages and B cells at the site of skin tests with a cytoplasmic Paracoccidioides brasiliensis antigen (paracoccidioidin) was studied at 24 and 48 h post-challenge in 10 patients with the chronic form of paracoccidioidomycosis and in 5 noninfected individuals. The in situ study was carried out using immunoperoxidase techniques and monoclonal antibodies. The controls showed negative skin test. In the patients, the great majority of the cells in the perivascular foci were T cells (CD43-positive cells) making up 47% and 48.6% of the total number of cells at 24 and 48 h respectively. Most of the T cells showed a T helper phenotype (CD45RO-positive cells). Approximately 25% of the cells were macrophages (CD68-positive cells) and there were very few B lymphocytes (CD20-positive cells). The present data on the microanatomy of paracoccidioidin skin test sites were consistent with a delayed type hypersensitivity pattern. Our results were comparable to those reported on skin tests for other granulomatous chronic diseases.

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.

Cell-free antigens from precocious Paracoccidioides brasiliensis culture induce a typical delayed-type hypersensitivity reaction

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Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu, SP, Brazil

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 degrees C (yeast form) and as cottonous colonies at 25 degrees C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population.

Experimental pulmonary paracoccidioidomycosis in the Syrian hamster: morphology and correlation of lesions with the immune response.

A model for pulmonary paracoccidioidomycosis in the hamster is described. The disease was induced by intratracheal inoculation of 1.7 x 10(5) viable yeast forms of P. brasiliensis. Lung histopathology, dissemination lesions and humoral and cellular immune responses were investigated at intervals up to 24 weeks after infection. Humoral immunity was studied by immunodiffusion and complement fixation tests. Cell-mediated immunity was evaluated in vitro by the macrophage migration inhibition test in the presence of phytohaemagglutinin and P. brasiliensis soluble antigen, and in vivo by the paracoccidioidin test. Thirty out of 35 infected animals (85.7%) developed pulmonary paracoccidioidomycosis. Dissemination lesions were observed in regional lymph nodes (82.8%), liver (8.5%) and spleen (5.7%). Lung involvement was mainly around bronchi and vessels. Regional lymph nodes were severely involved from the fourth week on, acquiring a pseudotumoral aspect at later stages. Specific antibodies were detected from the fourth week on, with titres increasing progressively. The cellular immune response to phytohaemagglutinin was intact throughout the experiment and the response to P. brasiliensis antigen was already detectable by the second week and remained positive to the end of the experiment. The skin test became positive from the fourth week on. Inoculation by the intratracheal route represents a highly effective way of infecting hamsters with P. brasiliensis, with the induction of localized disease, good antibody production and intact cell immunity.

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.

Fenotipagem das populações celulares e detecção in situ de citocinas em biópsias de pacientes com paracoccidioidomicose

Pós-graduação em Patologia - FMB