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Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.

Interleukin (IL)-15 is a pleiotropic cytokine that regulates the proliferation and survival of many cell types. IL-15 is produced by monocytes and macrophages against infectious agents and plays a pivotal role in innate and adaptive immune responses. This study analyzed the effect of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with Paracoccidioides brasiliensis (Pb18), the agent of paracoccidioidomycosis. Peripheral blood monocytes were pre-incubated with IL-15 and then challenged with Pb18. Fungicidal activity was assessed by viable fungi recovery from cultures after plating on brain-heart infusion-agar. Superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), tumour necrosis factor-alpha (TNF-α), IL-6, IL-15 and IL-10 production by monocytes were also determined. IL-15 enhanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by addition of anti-IL-15 monoclonal antibody. A significant stimulatory effect of IL-15 on O₂⁻ and H₂O₂ release suggests that fungicidal activity was dependent on the activation of oxidative metabolism. Pre-treatment of monocytes with IL-15 induced significantly higher levels of TNF-α, IL-10 and IL-15 production by cells challenged with the fungus. These results suggest a modulatory effect of IL-15 on pro and anti-inflammatory cytokine production, oxidative metabolism and fungicidal activity of monocytes during Pb18 infection.

Aims: Silibinin is the major active component of silymarin, a polyphenolic plant flavonoid that has anti-inflammatory effects. The modulatory effect of silibinin on monocyte function against Paracoccidioides brasiliensis (Pb18) has not yet been demonstrated. The present study investigated whether the effect of silibinin on nuclear factor-kappa B (NF-kappaB) pathways may affect the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), transforming growth factor beta (TGF-beta1), prostaglandin E(2) (PGE(2)), nitric oxide (NO) and fungicidal activity of human monocytes challenged in vitro with Pb18., Main Methods: Peripheral blood monocytes from healthy individuals were treated with silibinin and challenged with Pb18 for 18h. TNF-alpha, IL-10, TGF-beta1 and PGE(2) expression were determined by immunoenzymatic assay (ELISA) and NO release was determined by the accumulation of nitrite in culture supernatants. Fungicidal activity of monocytes was analyzed after treatment with interferon-gamma plus silibinin and challenge with Pb18. NF-kappaB activation in cultured monocytes was evaluated by flow cytometry and ELISA., Key Findings: Silibinin partially inhibited p65NF-kappaB activation as the number of cells expressing this factor was reduced and the concentration of nuclear p65NF-kappaB was low, compared to untreated controls. The addition of silibinin also resulted in suppression of TNF-alpha, IL-10, TGF-beta1, PGE(2) and NO production but did not affect the fungicidal activity of monocytes against Pb18., Significance: Silibinin exerts anti-inflammatory and anti-fibrotic effects on CD14+/- human monocytes challenged by Pb18 by partial inhibition of p65NF-kappaB activation.

The effect of interleukin (IL)-6 on cytokine production was evaluated in human monocyte cultures infected with the virulent strain of Paracoccidioides brasiliensis (Pb18). Peripheral blood monocytes from healthy individuals were preincubated for 24 h with or without human recombinant IL-6, and then challenged with Pb18 for 4 h and 18 h. P. brasiliensis growth was assessed by viable fungi recovery from co-cultures after plating on BHI-agar. Moreover, tumor necrosis factor-alpha (TNF-alpha), IL-6 and IL-10 production in supernatant cultures was determined by enzyme immunoassay (ELISA). Monocyte preincubation with IL-6, and challenged with Pb18 for 4 h, led to significantly higher fungi recovery compared to non-treated co-cultures. The pretreatment of monocytes with IL-6 induced an inhibitory effect on TNF-alpha and IL-10 production during 18 h fungal infection. Otherwise, an autocrine stimulatory effect on IL-6 production was detected at 4 h and 18 h as represented by an elevation in IL-6 levels. The reduction in TNF-alpha levels and stimulation of IL-6 production induced by previous IL-6 treatment might be responsible for a significant increase in fungal growth in human monocytes. The results suggest that IL-6, by exerting a modulatory effect on cytokines production, makes monocyte more permissive for fungal growth.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production.

Paracoccidioidomycosis is a deep mycosis, endemic in Latin America, caused by Paracoccidioides brasiliensis. Macrophage activation by cytokines is the major effector mechanism against this fungus. This work aimed at a better understanding of the interaction between yeast cells-murine peritoneal macrophages and the cytokine signals required for the effective killing of high virulence yeast-form of P. brasiliensis. In addition, the killing effector mechanisms dependent on the generation of reactive oxygen or nitrogen intermediates were investigated. Cell preincubation with IFN-gamma or TNF-alpha, at adequate doses, resulted in effective yeast killing as demonstrated in short-term (4-h) assays. Both, IFN-gamma and TNF-alpha activation were associated with higher levels of H(2)O(2) and NO when compared to nonactivation. Treatment with catalase (CAT), a H(2)O(2 )scavenger, and N(G)-monomethyl-L: -arginine (L: -NMMA), a nitric oxide synthase inhibitor, reverted the killing effect of activated cells. Taken together, these results suggest that both oxygen and L: -arginine-nitric oxide pathways play a role in the killing of highly virulent P. brasiliensis.

Multinucleated giant cells (MGC) are characteristic cells in granulomatous disorders such as paracoccidioidomycosis (PCM) and also are formed in vitro from peripheral blood mononuclear cells by several stimuli. In this study, the authors investigated in vitro formation of MGC derived from monocytes of healthy individuals, stimulated with Paracoccidioides brasiliensis antigen (PbAg), compared with other stimuli such as IFN-gamma and supernatant of Con-A-stimulated peripheral blood mononuclear cells (CM-ConA). Besides, the fungicidal activity of monocytes and monocyte-derived MGC challenged with P. brasiliensis were compared, at a ratio of one fungus per 50 monocytes. Results demonstrated that PbAg, IFN-gamma, and CM-ConA stimuli were able to induce MGC generation, with fusion indices significantly higher than control cultures. Striking results were observed when MGC induced by PbAg and IFN-gamma presented higher fungicidal activity than monocytes, submitted to the same stimuli, showing a better capacity of these cells to kill P. brasiliensis. In summary, the results suggest that PbAg is able to induce MGC generation, and these cells presented higher fungicidal activity against P. brasiliensis than monocytes.

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM) that is one of the most prevalent systemic human mycoses in Latin America. Armadillos show a high incidence of PCM infection and could, therefore, be a natural reservoir for this fungus. In this study were compared the virulence profiles of isolates obtained from nine-banded armadillos (Dasypus novemcinctus) (PbT1 and PbT4) and isolates from PCM patients (Pb265 and Bt83). Pathogenicity was evaluated by fungal load and analysis of colony morphology. Immunity against the fungus was tested by delayed type hypersensitivity test (DTH) and antibody quantification by ELISA. The higher virulence of PbT1 and PbT4 was suggested by higher fungal load in spleen and lungs. Armadillo isolates and Bt83 presented a cotton-like surface contrasting with the cerebriform appearance of Pb265. All isolates induced cellular and humoral immune responses in infected BALB/c mice. DTH reactions were similarly induced by the four isolates, however, a great variability was observed in specific antibody levels, being the highest ones induced by Bt83 and PbT4. The present work confirms that armadillos harbor P. brasiliensis, whose multiplication and induced immunity in experimentally infected mice are heterogeneous, resembling the behavior of isolates from human PCM. This study reinforces the possibility that armadillos play an important role in the biological cycle of this pathogen.

Interleukin-15 is a cytokine produced by a wide range of different cell types, including macrophages, in response to lipopolysaccharide or microbial infection. This cytokine may play a crucial role in the activation of phagocytic cells against pathogens, especially during innate immune response. The effects of IL-15 on human polymorphonuclear leukocyte fungicidal activity against a highly virulent Paracoccidioides brasiliensis strain were investigated. Pretreatment of human neutrophils from healthy individuals with IL-15 for 18 hours increased cell fungicidal activity in a dose-dependent manner. In addition, the exposure to IL-15 induced an increase in neutrophil oxidative burst as evaluated by superoxide anion and H(2)O(2) release. Catalase inhibited fungicidal activity supporting a role for H(2)O(2) in fungus killing. In contrast, IL-8 and TNF-alpha levels were not affected by IL-15 suggesting that its effects were not mediated by these cytokines. Together, these results show that IL-15 is a potent stimulant of antifungal activities in human neutrophils, at least in part by a mechanism dependent on oxidative metabolism.

Chloroquine, due to its basic properties, has been shown to prevent the release of iron from holotransferrin, thereby interfering with normal iron metabolism in a variety of cell types. We have studied the effects of chloroquine on the evolution of experimental paracoccidioidomycosis by evaluating the viable fungal recovery from lung, liver and spleen from infected mice and H(2)O(2), NO production, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, IL-10 levels and transferrin receptor (TfR) expression from uninfected and infected peritoneal macrophages. Chloroquine caused a significant decrease in the viable fungal recovery from all organs tested, during all periods of evaluation. Peritoneal macrophages from chloroquine-treated infected mice showed higher H(2)O(2) production and TfR expression, and decreased levels of NO, endogenous and stimulated-TNF-alpha, IL-6 and IL-10 during the three evaluated periods. However, despite its suppressor effects on the macrophage function, the chloroquine therapeutic effect upon murine paracoccidioidomycosis was probably due to its effect on iron metabolism, blocking iron uptake by cells, and consequently restricting iron to fungus growth and survival.

Human monocytes lacked fungicidal activity against high virulence strain of Paracoccidioides brasiliensis, even after IFN-gamma activation. However, monocytes treated with indomethacin exhibited an effective killing against this fungus, suggesting a role of prostaglandin E2 (PGE2) in the inhibition process. Thus, the purpose of this work was to determine whether the effect of PGE2 in fungicidal activity was related with decrease on H(2)O(2) release, the metabolite involved in P. brasiliensis killing, and changes in the levels of TNF-alpha, IL-6 and IL-10. Human monocytes challenged with the fungus produced high PGE2 levels, which in turn inhibited the fungicidal activity of these cells by reducing H(2)O(2) and TNF-alpha production.

The production of prostaglandins (PGs) during fungal infections could be an important suppressor factor of host immune response. Host cells are one source of prostaglandin E(2) (PGE(2)); however another potential source of PGE(2) is the fungal pathogen itself. Thus, both host and fungal PGE2 production is theorized to play a role in pathogenesis, being critical for growth of the fungus and to modulate the host immune response. The purpose of this work was to investigate if high and low virulent strains of Paracoccidioides brasiliensis have the capacity to produce PGE(2) in vitro, and if this production was related to the fungal growth. The results demonstrated that both strains of P. brasiliensis produce high levels of PGE(2) and the treatment with indomethacin, a cyclooxygenase inhibitor, significantly reduced the production of this mediator, as well as the viability of the fungus. Thus, our data indicate that PGE(2) is produced by P. brasiliensis by a cyclooxygenase-dependent metabolic pathway, and its production is required for fungal survival. This discovery reveals an important factor that has potentially great implications for understanding the mechanisms of immune deviation during infection.

Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against the fungus and several papers show the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, the studies focusing on polymorphonuclear neutrophils (PMNs) antifungal functions are scarcer. Thus, the objective of the present paper was to assess the capacity of human PMNs to kill virulent P. brasiliensis strain in vitro, before and after priming with different cytokines. Moreover, the involvement of oxygen metabolites in this activity was evaluated. Nonactivated cells failed to exhibit antifungal activity. However, when these cells were IFN-gamma, TNF-alpha or GM-CSF activated, a significative fungicidal activity was detected. This process was significantly inhibited when P. brasiliensis challenge occurred in presence of catalase (CAT - a scavenger of H2O2) and superoxide dismutase (SOD - a scavenger of superoxide anion). From these results it is concluded that cytokines activation is required for P. brasiliensis killing by human PMNs, and that H2O2 and superoxide anion participate as effectors molecules in this process.

Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against this fungus, and several studies have shown the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, studies on polymorphonuclear neutrophils (PMNs), that are the first cells recruited to the infection sites, are scarcer. Thus, the objective of this paper was to assess whether interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is able to block the activity of IFN-gamma-activated human PMNs upon P. brasiliensis intracellular killing, in vitro. The results showed that IFN-gamma-activated PMNs have an effective fungicidal activity against the fungus. This activity was associated with the release of high levels of H(2)O(2), the metabolite involved in phagocytic cells antifungal activities. However, the concomitant incubation of these cells with IFN-gamma and IL-10 significantly blocked IFN-gamma activation. As a consequence, PMNs killing activity and H(2)O(2) release were inhibited. Together, our results show the importance of PMNs exposure to activator or suppressor cytokines in the early stages of paracoccidioidomycosis infection.

Monocytes and macrophages play a central role in innate and adaptive immune response against systemic fungal infections. Imbalances in suppressor or stimulatory cytokine secretion caused by these cells may influence disease development, microorganism death, and the nature of the adaptive immune response. This study analyzed the monocyte cytokine profiles of healthy individuals challenged with high and low virulent strains of P. brasiliensis and mRNA cytokine expression kinetics by reverse transcription polymerase chain reaction (RT-PCR). Peripheral blood monocytes from healthy volunteers were cultured in vitro with and without virulent (Pb18) or low virulence (Pb265) strains from P. brasiliensis viable yeast cells. Interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta1) were measured in culture supernatants by enzyme immunoassay (ELISA), and mRNA cytokine expression was determined by RT-PCR at 0, 4, 8, 12, 18 and 48 hr. Both P. brasiliensis strains induced monocyte production of IL-1beta, IL-6, IL-10 and TNF-alpha. Pb18 induced higher levels of IL-1beta, IL-6, and IL-10 than Pb265. IL-8 and TGF-beta1 levels were not significantly different from those cultured without stimulus. The mRNA cytokine expression was similar to supernatant cytokines measured by ELISA. In vitro monocyte challenge with virulent P. brasiliensis strain induces earlier and higher levels of pro- and anti-inflammatory cytokines than low virulence strain.

Human monocytes activated by recombinant tumor necrosis factor alpha (TNF-alpha) exhibited significant fungicidal activity on the yeast cells of a highly virulent strain of Paracoccidioides brasiliensis. This process was significantly inhibited in the presence of catalase (CAT - a scavenger of H2O2), but not in the presence of superoxide-dismutase (SOD - a scavenger of superoxide anion) or NG-monomethyl-L-arginine (NG-MMLA - a nitric oxide inhibitor). Furthermore, there was a direct association between the intracellular killing of the fungus and the production of H2O2 by activated cells. These results strongly suggest a role for H2O2 in the killing of highly virulent strains of P. brasiliensis by TNF-alpha-activated human monocytes.

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features.

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.

Ten isolates of Paracoccidioides brasiliensis were examined for differences in virulence in outbred mice intravenously inoculated with the fungus, associated with mycelial morphology, and genetic patterns measured by random amplified polymorphic DNA (RAPD). Virulence was evaluated by viable yeast cell recovery from lungs and demonstration of histopathologic lesions in different organs. The results showed that the isolates presented four virulence degrees: high virulence, intermediate, low and non-virulence. RAPD clustered the isolates studied in two main groups with 56% of genetic similarity. Strains with low virulence, Pb265 or the non-virulent, Pb192, showed glabrous/cerebriform morphology and high genetic similarity (98.7%) when compared to the other isolates studied. The same was observed with Bt79 and Bt83 that shared 96% genetic similarity, cottony colonies and high virulence. The RAPD technique could only discriminate P. brasiliensis isolates according to glabrous/cerebriform or cottony colonies, and also high from low virulence strains. Isolates with intermediate virulence such as Pb18, Pb18B6, Bt32 and Bt56 showed variability in their similarity coefficient suggesting that RAPD was able to detect genetic variability in this fungal species. Virulence profile of P. brasiliensis demonstrated that both mycelial morphologic extreme phenotypes may be associated with fungal virulence and their in vitro subculture time. Thus, RAPD technique analysis employed in association with virulence, morphologic and immunologic aspects might prove adequate to detect differences between P. brasiliensis isolates.

Monocytes and macrophages can produce a large repertoire of cytokines and participate in the pathogenesis of granulomatous diseases. We investigated the production of pro- and anti-inflammatory cytokines by monocytes from patients with active paracoccidioidomycosis. Peripheral blood monocytes from 37 patients and 29 healthy controls were cultivated with or without 10 microg/ml of lipopolysaccharide (LPS) for 18 h at 37 degrees C, and the cytokine levels were determined in the culture supernatants by enzyme immunoassay. The results showed that the endogenous levels of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-8, IL-10 and transforming growth factor beta detected in the supernatant of patient monocytes cultivated without stimulus were significantly higher than those produced by healthy controls. These data demonstrated that monocytes from patients with active paracoccidioidomycosis produce high levels of cytokines with both inflammatory and anti-inflammatory activities. However, patient monocytes produced significantly lower TNF-alpha and IL-6 levels in response to LPS when compared to normal subjects, suggesting an impairment in their capacity to produce these cytokines after LPS stimulation. Concentrations of IL-1beta, IL-8 and IL-10 in cultures stimulated with LPS were higher in patients than in controls. These results suggest that an imbalance in the production of pro- and anti-inflammatory cytokines might be associated with the pathogenesis of paracoccidioidomycosis.

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro. Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.

The polysaccharide fraction of Paracoccidioides brasiliensis mycelial cell wall (F1 fraction), the active component of which is composed of beta-glucan, was investigated in regard to the activation of human monocytes for fungal killing. The cells were primed with interferon-gamma (IFN-gamma) or F1 (100 and 200 microg ml(-1)) or F1 (100 and 200 microg ml(-1)) plus IFN-gamma for 24 h and then evaluated for H2O2 release. In other experiments, the cells were pretreated with the same stimuli, challenged with a virulent strain of P. brasiliensis and evaluated for fungicidal activity and levels of tumor necrosis factor (TNF-alpha) in the supernatants. F1 increased the levels of H2O2 in a similar manner to IFN-gamma. However, a synergistic effect between these two activators was not detected. On the contrary, a significant fungicidal activity was only obtained after priming with IFN-gamma plus F1. This higher activity was associated with high levels of TNF-alpha in the supernatants of the cocultures. Overall, P. brasiliensis F1 fraction induced human monocytes to release relatively high levels of TNF-alpha, which, in combination with IFN-gamma, is responsible for the activation of human monocytes for effective killing of P. brasiliensis.

We studied the correlation among cellular immune response, the pattern of lung granulomatous lesions and alterations in spleen lymphoid structure in Swiss mice inoculated intravenously with Paracoccidioides brasiliensis strain 18. The animals were evaluated at 24, 48 and 96 h after infection and further studied weekly for 18 weeks by: (i) the macrophage migration inhibition test with phytohemagglutinin (PHA) and P. brasiliensis antigen (PbAg); and (ii) histopathology of the lung and spleen lesions. One group of animals was gamma-irradiated (8 Gy), infected under the same conditions and evaluated for the pattern of lung granulomatous lesions and spleen lymphoid structure at 24, 48 and 96 h after infection. During the first week of infection, the non-irradiated animals presented a positive response to PHA and PbAg, compact granulomas in the lungs and a typical hyperplasia of the spleen white pulp. However, from weeks 2 to 5, a depression of the cell-mediated immunity (CMI) response to PHA and PbAg was observed in association with granulomas presenting only large mononuclear cells and lacking both giant cells and a peripheral halo of small mononuclear cells. This pattern of granuloma formation was similar to that seen in gamma-irradiated animals, whose cells involved in CMI were absent. After week 7, the non-irradiated animals showed granulomas characterized by the presence of giant cells and a peripheral halo of small mononuclear cells. This type of granuloma was formed concomitantly with recovery of the CMI and of the lymphoid structure of the spleen. The results showed a correlation among granulomas composed of large mononuclear cells, hypoplasia of the splenic tissue and impaired CMI. This correlation indicated that although granuloma morphogenesis per se does not depend on the activation of CMI, this response is important at later stages during modulation of the cellular composition of the granulomas.

Phagocytic cells play an important role in nonspecific resistance to fungal infection by mediating an inflammatory response and by a direct fungicidal action. In this study, the functional activity of peritoneal macrophages obtained from hamsters experimentally infected with strain Pb18 of Paracoccidioides brasiliensis was evaluated during 16 weeks of infection. The results showed that macrophages had a higher spreading ability associated with increased production of tumor necrosis factor alpha (TNF-alpha) and enhanced fungicidal activity during the early periods of infection. TNF-alpha levels remained elevated during all periods studied, while low levels of interleukin-1 beta (IL-1 beta) were produced during the infection. A necrotic area with dead fungi was observed at the inoculation site and the infection disseminated only to liver and lymph nodes in a few animals. These results suggest that during the early stages of infection with P. brasiliensis, macrophage activation by the high levels of TNF-alpha limited fungal dissemination. In contrast, in the later stages of infection, high levels of TNF-alpha were observed while the fungicidal activity of macrophages was lower and the animals presented loss of vitality resulting in their death. These observations suggest a complex role of TNF-alpha in experimental paracoccidioidomycosis of Syrian hamsters, involving not only resistance but also pathogenesis.

Isolates of Paracoccidioides brasiliensis may vary in virulence according to time of in vitro subcultivation. The present study compared the morphology and pathogenicity to hamsters of two P. brasiliensis isolates: one obtained from human lesions and maintained in the laboratory for several years (Pb-18) and the other isolate recovered from hamsters inoculated with organ homogenates from armadillos (Pb-T). The microscopic morphology of Pb-18 and Pb-T showed yeast cells with similar diameter. However, Pb-T produced a significantly higher number of buds per mother cell than Pb-18. Besides, the mycelial form of Pb-T developed abundant sporulation during 8 weeks of culture which was absent in the Pb-18 isolate. Virulence studies demonstrated that mortality rates, antibody levels, fungal load and extent of lesions in the organs were significantly higher in animals infected with Pb-T. The results demonstrated that Pb-T recently isolated from an animal was more virulent than Pb-18. These differences between the two P. brasiliensis isolates may be indicators of virulence attenuation in this fungal species.

Metabolites produced by pathogenic fungi may be involved in the pathogenesis of fungal infections consequently altering the defence mechanisms of the host. In this study the levels of Paracoccidioides brasiliensis antigens detected in the plasma of patients with paracoccidioidomycosis correlated with the suppression index detected by the low mitogenic response of peripheral blood mononuclear cells (PBMC) to phytohaemaglutinin (PHA). This inhibitory effect on lymphoproliferation was observed in the plasma of 58% of the patients, suggesting the presence of inhibitory factors. Plasma samples from paracoccidioidomycosis patients having or not having inhibitory factors showed no significant effect on chromosomes of lymphocytes from healthy individuals. However, these plasmas had a suppressive activity on the blastogenic response of these lymphocytes stimulated with PHA, that was independent of a cytotoxic effect. P. brasiliensis antigens added to the proliferative response of PBMC from healthy individuals stimulated or not stimulated with PHA showed a dose-dependent suppressor effect, reproducing the inhibitory effect of patients' plasma. We suggest that the antigens of P. brasiliensis present in the plasma of patients, even at low concentrations, can play an important role in the reduction of the cellular immune response and in the genesis of the immunoregulatory disturbances observed in paracoccidioidomycosis.

The effect of dialysable leukocyte extracts (DLE) obtained from hamsters immunized with Paracoccidioides brasiliensis (immune DLE) and from non-immunized hamsters (non-immune DLE) was studied in hamsters inoculated with P. brasiliensis by the intratesticular route. Treatment with immune or non-immune DLE was started during the third week of infection and was repeated at 7, 11, 15 and 19 weeks. A group of untreated infected animals was used as control. Animals were submitted to the delayed hypersensitivity skin test to P. brasiliensis antigen (PbAg) in vivo and assayed in vitro by the macrophage migration inhibition test in the presence of Phytohemagglutinin (PHA) and PbAg and by immunodiffusion for specific antibody. The animals were sacrificed at 4, 8, 12, 16 and 20 weeks. The morphology and extension of the lesions were studied at the inoculation site, and in lymph nodes, lungs, liver, spleen and kidneys. In contrast to the controls, animals treated with both DLEs maintained a positive cell-mediated immune response throughout the experiment and developed less extensive infection with a significantly lower number of fungi in the lesions. The results suggest that immune and non-immune DLE preparations modified the evolution of experimental paracoccidioidomycosis with equal efficiency. This similarity may be explained by the immunoregulatory activities of both extracts.

Dialysable leukocyte extracts (DLE) were obtained from lymph nodes and spleen cells of hamsters immunized with Paracoccidioides brasiliensis (immune DLE) or from non-immunized hamsters (non-immune DLE). Sensitivity to P. brasiliensis antigen (PbAg) was transferred by immune DLE to normal recipient hamsters and could be detected by the macrophage migration inhibition (MIF) assay on day three and by a positive skin test to PbAg on day seven after DLE inoculation. This reactivity persisted for 120 days. The specificity of DLE was studied by inoculation of immune or non-immune DLE into normal hamsters and transfer was evaluated 7 days later by the MIF assay and by skin tests to PbAg, Candida albicans antigen (CaAg), sonically treated bacille Calmette-Guérin (BCG) (SBCG) and ovalbumin (OVA). The immune DLE recipients showed a reactivity to PbAg that was significantly stronger than that of the nonimmune DLE recipients although both groups of recipient animals showed reactivity to CaAg and SBCG. The results suggest that both DLE preparations contain a non-specific antigen augmenting factor. This factor may stimulate the minimal background response to the antigens in nonimmunized recipient hamsters.