Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection.

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We studied 52 patients with disseminated histoplasmosis, 30 with the acquired immunodeficiency syndrome (AIDS) (cohort 1) and 22 not co-infected with the human immunodeficiency virus (cohort 2). Demographic, clinical, laboratory, mycologic findings, as well as antifungal therapy and highly active antiretroviral (HAART), were analyzed. Skin lesions were significantly higher in cohort 1 than in cohort 2 (P = 0.001). Anemia, leukopenia, and an elevated erythrocyte sedimentation rate were also more pronounced in cohort 1 than in cohort 2 (P < 0.001). Histoplasma capsulatum was isolated more often in cohort 1 than in cohort 2 (P < 0.05) patients, but antibodies to H. capsulatum were detected more frequently in cohort 2 than in cohort 1 (P < 0.05). Itraconazole treatment was less effective in cohort 1 than in cohort 2 (P = 0.012). In cohort 1 patients, HAART improved response to antifungals when compared with individuals not given HAART (P = 0.003), who exhibited higher mortality rates (P = 0.025). Cohort 1 patients who were given dual antifungal and anti-retroviral therapies responded as well as the non-HIV patients in cohort 2, who were treated only with itraconazole. These results indicate the need to promote restoration of the immune system in patients with AIDS and histoplasmosis.

We have developed a murine model of pulmonary infection byParacoccidioides brasiliensis in which resistance was associated with immunological activities governed by gamma interferon (IFN-γ). To better characterize this model, we measured type 1 and type 2 cytokines in the lungs and investigated the effect of endogenous IFN-γ depletion by monoclonal antibodies in the course of infection of susceptible (B10.A) and resistant (A/Sn) mice. At weeks 4 and 8 after infection, lungs from susceptible animals presented levels of IFN-γ, interleukin-4 (IL-4), IL-5, and IL-10 higher than those in resistant mice. In both mouse strains, neutralization of endogenous IFN-γ induced exacerbation of the pulmonary infection, earlier fungal dissemination to the liver and spleen, impairment of the specific cellular immune response resulting in significantly lower delayed-type hypersensitivity reactions, and increased levels of immunoglobulin G1 (IgG1)- and IgG2b-specific antibodies. Histopathological analysis demonstrated that depletion of IFN-γ changes the focal granulomatous lesions found in the lungs of B10.A and A/Sn mice into coalescent granulomata which destroy the pulmonary architecture. These results suggest that irrespective of the mouse strain, IFN-γ plays a protective role and that this cytokine is one major mediator of resistance against P. brasiliensis infection in mice.

Itraconazole effectively controls active paracoccidioidomycosis but appears not to hinder lung fibrosis. Clinical records and chest radiographs from 47 itraconazole-treated patients with prolonged posttherapy follow-up (mean follow-up period, 5.6 years) were analyzed; the radiographs were interpreted following pneumoconiosis standards that consider the lungs as 6 fields and grade damage according to the number of fields involved. Infiltrative lesions were observed at diagnosis in 93.6% of the patients. Fibrosis was observed in 31.8% of the patients at diagnosis and had not cleared at the end of the observation period in any of these patients. Fibrosis also developed de novo in 11 patients (25%), so that by the end of the follow-up period it was seen in 53.2% of patients overall. Fibrosis correlated with severity of infiltrates at diagnosis: fibrosis was present in 83% of patients with very severe infiltration and in 12.5% of patients with minor infiltration. Among patients with severe infiltration, fibrosis was present in 30%; this increased (to 75%) when bullae were concomitantly present at diagnosis. Prompt initiation of treatment is necessary to avoid the development of fibrosis.

Using the intraperitoneal route of infection, we demonstrated previously that A/Sn mice are resistant and B10.A mice are susceptible to Paracoccidioides brasiliensis infection. Since paracoccidioidomycosis is a deep systemic granulomatous disorder that involves primarily the lungs and then disseminates to other organs and systems, we herein investigated the course of the infection and the resulting immune responses developed by A/Sn and B10.A mice after intratracheal infection with P. brasiliensis yeast cells. It was observed that A/Sn mice develop a chronic benign pulmonary-restricted infection, whereas B10.A mice present a chronic progressive disseminated disease. A/Sn animals were able to restrict fungal infection to the lungs despite the increased fungal load at the beginning of the infection. This behavior was associated with low mortality rates, the presence of adequate and persistent delayed-type hypersensitivity reactions, oxidative burst by bronchoalveolar cells, and production of high levels of specific antibodies in which immunoglobulin G2a (IgG2a) and IgG3 isotype titers were significantly higher than those observed in the susceptible mice. In contrast, B10.A animals showed a constant pulmonary fungal load and dissemination to the liver and spleen. This infection pattern resulted in high mortality rates, discrete delayed-type hypersensitivity reactivity, poorly activated or nonactivated bronchoalveolar cells, and production of specific IgG2b isotype titers significantly higher than those observed in the resistant mice at week 4 of infection. Thus, A/Sn and B10.A mice maintain the same resistance patterns as those observed previously with the intraperitoneal route of infection. Furthermore, the obtained results suggest that resistance to paracoccidioidomycosis is associated with T-cell, macrophage, and B-cell activities that are known to be mediated by gamma interferon.

Evidence that disease due to the thermally dimorphic fungus Paracoccidioides brasiliensis occurs postpuberty predominantly in males led us to hypothesize that hormonal factors critically affect its pathogenesis. We show here that estrogens inhibit mycelialto yeast-form transformation of P. brasiliensis in vitro. Transformation of three isolates was inhibited to 71, 33, and 19% of the control values in the presence of 10-10, 10-8, and 10-6 M 17i-estradiol, respectively. The synthetic estrogen diethylstilbestrol was active but less potent than estradiol, whereas testosterone, 17a-estradiol, tamoxifen, and corticosterone were inactive. This function was specifically inhibited, since yeast-to-mycelium transformation, yeast growth, and yeast reproduction by budding were unaffected by 171-estradiol. Of note is the fact that mycelium-to-yeast transformation occurs as the first step in vivo in the establishment of infection. The cytosol of the three isolates studied possesses a steroid-binding protein which has high affinity for 17j3-estradiol. We believe that this binding protein represents a P. brasiliensis hormone receptor which can also recognize mammalian estrogens. We hypothesize that the ability of estrogen to decrease or delay mycelium-to-yeast transformation at the initial site of infection contributes to or is responsible for the marked resistance of females, and that the binder described is the molecular site of action.

Patients with paracoccidioidomycosis often present pulmonary fibrosis and exhibit important respiratory limitations. Based on an already established animal model, the contribution of viable and non-viable P. brasiliensis propagules to the development of fibrosis was investigated. BALB/c male mice, 4-6 weeks old were inoculated intranasally either with 4x10(6) viable conidia (Group I), or 6. 5x10(6) fragmented yeast cells (Group II). Control animals received PBS. Six mice per period were sacrificed at 24, 48, 72h (initial) and 1, 2, 4, 8, 12 and 16 weeks post-challenge (late). Paraffin embedded lungs were sectioned and stained with H&E, trichromic (Masson), reticulin and Grocot&tacute;s. During the initial period PMNs influx was important in both groups and acute inflammation involving 34% to 45% of the lungs was noticed. Later on, mononuclear cells predominated. In group I, the inflammation progressed and granulomas were formed and by the 12th week they fussed and became loose. Thick collagen I fibers were observed in 66.6% and 83.3% of the animals at 8 and 12 weeks, respectively. Collagen III, thick fibers became apparent in some animals at 4 weeks and by 12 weeks, 83% of them exhibited alterations in the organization and thickness of these elements. In group II mice, this pattern was different with stepwise decrease in the number of inflammatory foci and lack of granulomas. Although initially most animals in this group had minor alterations in thin collagen I fibers, they disappeared by the 4th week. Results indicate that tissue response to fragmented yeast cells was transitory while viable conidia evoked a progressive inflammatory reaction leading to granuloma formation and to excess production and/or disarrangement of collagens I and III; the latter led to fibrosis.