Fungicidal activity of human peripheral blood leukocytes against Paracoccidioides brasiliensis yeast cells.

Although epidemiological findings suggest that normal humans are resistant to Paracoccidioides brasiliensis infection, the host defense mechanisms against this fungus have not been fully understood. Here we examined human leukocytes for antifungal activity against yeast cells of this fungus, using an improved mycological culture medium with high plating efficiency for the yeast cell. In an attempt to minimize the impairment of leukocyte activities during the isolation process, leukocytes removed by centrifugation from a buffy coat of peripheral blood were used in the antifungal assay without further fractionation. The leukocytes thus prepared effectively killed P. brasiliensis yeast cells within the first 4 h of co-culture. Adding interferon-gamma (37 ng/ml), granulocyte-macrophage colony-stimulating factor (5 ng/ml), interleukin (IL)-1beta (12 ng/ml), or IL-4 (12 ng/ml) to the assay system enhanced the leukocyte antifungal (growth inhibitory) activity by 48 h. By contrast, addition of IL-8 (50 ng/ml) impaired the leukocyte activity. Tumor-necrosis factor-alpha (50 ng/ml) or IL-10 (25 ng/ml) had no effect in this respect. Dexamethasone (1 micromol/l) reduced the antifungal activity of leukocytes. This is the first demonstration that human leukocytes are able to effectively kill yeast cells of P. brasiliensis.

Synergistic antifungal effect of fluconazole and human polymorphonuclear leukocytes on Paracoccidioides brasiliensis: effect of interferon-gamma and granulocyte-macrophage colony-stimulating factor.

To better understand the in vivo efficacy of fluconazole (FCZ), we investigated the possible synergy of fungistatic FCZ with human polymorphonuclear leukocytes (PMN) against Paracoccidioides brasiliensis (Pb). The effect of interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in this system was also studied. For this purpose, FCZ, PMN, PMN + FCZ, PMN + IFN-gamma, PMN + IFN-gamma + FCZ, PMN + GM-CSF and PMN + GM-CSF + FCZ were co-cultured with Pb and the cfu of Pb was measured. The antifungal effect of FCZ on yeast cells of Pb was concentration-dependent. At 0.1 microg ml(-1), FCZ had no effect on the growth of Pb. At 0.2 microg ml(-1) FCZ showed a growth-inhibitory effect on three isolates of Pb in a long-term (120 h) assay, and at 0.6 microg ml(-1) or higher FCZ was fungicidal. Fungistatic concentration of FCZ (0.4 microg ml(-1)) acted synergistically with fungistatic PMN for killing isolate Bt-4 during the first 24 h of co-culture. Moreover, IFN-gamma and GM-CSF substantially enhanced the synergistic antifungal effect of PMN and FCZ. These findings provide a better understanding of why FCZ is more efficacious in in vivo models of paracoccidioidomycosis than is predicted by in vitro susceptibility tests.

Inhibitory effect of deferoxamine or macrophage activation on transformation of Paracoccidioides brasiliensis conidia ingested by macrophages: reversal by holotransferrin.

Conidia of P. brasiliensis ingested by murine macrophages at 37 degrees C showed enhanced transformation to yeast cells and further intracellular growth compared with conidia in culture medium alone. Treatment of macrophages with the iron chelator deferoxamine inhibited the intracellular conidium-to-yeast transformation. Cytokine-activated macrophages could also exert this inhibitory effect. Holotransferrin reversed the inhibitory effect of either deferoxamine or activated macrophages on intracellular conidium-to-yeast transformation. These results indicate that iron restriction is one of the mechanisms by which activated macrophages control the intracellular transformation of ingested conidia and growth of yeast cells.

Support of Paracoccidioides brasiliensis multiplication by human monocytes or macrophages: inhibition by activated phagocytes.

The interaction of human monocytes or monocyte-derived macrophages and yeast-form Paracoccidioides brasiliensis was studied in vitro. Yeast cells were readily ingested by adherent monocytes or macrophages. Multiplication of P. brasiliensis, measured by growth as colony forming units (cfu) on a supplemented medium with good plating efficiency, was greater in monocyte co-cultures compared to the number of cfu obtained from complete tissue-culture medium (CTCM). Multiplication increased with time in macrophage co-cultures, e.g., from two-six-fold in 24 h to nine-fold in 72 h. Microscopic observations indicated that ingested yeast cells multiplied inside macrophages. When monocytes were treated with supernate cytokines (CK) from concanavalin-A-stimulated mononuclear cells, then co-cultured with P. brasiliensis, multiplication was significantly inhibited compared with control monocyte co-cultures. Treatment of macrophages--derived from monocytes by culture in vitro for 3 days--for a further 3 days with CK resulted in maximal inhibition of multiplication over the subsequent 72 h. Similarly, when monocyte-derived macrophages (after culture for 7 days) were treated for 3 days with recombinant human gamma-interferon (IFN; 300 U/ml) or CK they restricted multiplication of P. brasiliensis by 65% and 95%, respectively, compared with control macrophages. Antibody to IFN abrogated the effect of IFN or CK treatment. These findings show that ingested P. brasiliensis can multiply in human monocytes or macrophages and that this multiplication can be restricted by activated monocytes or macrophages.

[Sulindac and indomethacin effects on lamb fetuses chronically monitored]. / Effectos del sulindaco e indometacina en fetos de ovejas monitorizados crónicamente.

Sulindac and indometacin, both prostaglandins inhibitors, have a similar tocolytic effectiveness, in contrast their fetal effects seem to be different. For specify these differences we compared the effects of the infusion of sulindac with known effects of indometacin. We operated 6 fetal lambs, in which polyvinyl catheters were inserted chronically for continuous measure of heart rate, blood pressure, incidence of fetal breathing movements and arterial pH and gas tension. The fetuses were infused in a 2 hour control period with vehicle, and then with 150 mg of either sulindac or indometacin (in alternate days) for a 6 hour period. There was no change in any variable when sulindac was given, but there was a significant increase in fetal breathing movements with indometacin. The reason for this difference is not clear, and requires further studies. These results added to previous reports supports the hypothesis that sulindac could be a first choice tocolytic drug for treatment of premature labor when no fetal effect is desired.

[Effect of arginine-vasopressin infusion on the secretion of tracheal fluid in sheep fetuses]. / Efecto de la infusión de arginina-vasopresina en la secreción de liquido traqueal en fetos de oveja.

Timely evacuation of alveolar fluid, release of surfactant and the beginning of continuous breathing, are key processes for an adequate adaptation of the fetus to the extrauterine life. Fetal vasopressin increases during labor and inhibit the secretion of tracheal fluid through a mechanism still unknown. The aim of this study was to elucidate the mechanism whereby vasopressin inhibit the secretion of lung fluid. We used fetal sheep chronically catheterized and infused either with vasopressin, vasopressin agonist (V2; dDAVP) or vasopressin antagonist (V1). Tracheal flow was measured during basal and infusions periods of 2 hours, monitoring fetal blood pressure, heart rate and blood pH and gases. Vasopressin and the V1 vasopressin antagonist caused a significant reduction in tracheal fluid flow, effect that was potentiated when both peptides were infused together. The V2 vasopressin agonist had no effect on the secretion of lung fluid. We concluded that vasopressin causes a significant inhibition of lung liquid secretion through a mechanism different to the activation of V1 and V2 receptors, and we propose the existence of other (s) kind of receptors (or receptors) for vasopressin that is (are) active during fetal life.

Fate of conidia of Paracoccidioides brasiliensis after ingestion by resident macrophages or cytokine-treated macrophages.

Conidia ingested by resident macrophages had an enhanced percentage of transformation to yeast cells compared with those in culture medium without macrophages. The yeast cells subsequently grew intracellularly by budding. Macrophages treated with cytokines from antigen-stimulated spleen cells from immunized mice significantly inhibited transformation of ingested conidia.

Synergistic effect of cortisol and thyrotrophin releasing hormone on lung maturation in the fetal sheep entails connective tissue maturation.

Pressure-volume relationships and collagen and elastin contents were measured in the lungs of fetal sheep infused either with saline (n = 4), thyrotrophin-releasing hormone (TRH; n = 6), cortisol (n = 9) or TRH plus cortisol (n = 10) at 128 days of gestation (term = 149 days) for 7 days. Lung distensibility (V40 = 1.8 +/- 0.1 ml/g wet wt; mean +/- SD) and stability (V5 = 0.6 +/- 0.1) increased along with collagen (C) (10.1 +/- 2.7 micrograms/mg) and elastin (E) contents (128 +/- 35 ng/mg) in the animals infused with TRH plus cortisol and were significantly higher (p < 0.05) than those observed in TRH (V40 0.62 +/- 0.07; V5 0.32 +/- 0.04; C 3.53 +/- 1.3; E 38.2 +/- 8.3), cortisol (V4 0.66 +/- 0.6; V5 0.27 +/- 0.03; C 4.27 +/- 0.8; E 41.02 +/- 12.7) or saline infused fetuses (V40 0.40 +/- 0.1; V5 0.20 +/- 0.06; C 3.28 +/- 0.9; E 31.5 +/- 9.2). Plasma concentrations of prolactin (PRL), triiodothyronine (T3) and cortisol (F) were also higher in the group of fetuses infused with both hormones in comparison with the other groups. In fetuses treated with TRH plus cortisol, PRL (32 +/- 8.3 ng/ml) and T3 (308.3 +/- 36 micrograms/dl) were significantly higher than in those infused with cortisol alone (PRL 3.7 +/- 2.3; T3 128 +/- 30) or with saline (PRL 4.2 +/- 1.6; T3 < 5 micrograms/dl). In the group treated with TRH alone, PRL also increased significantly (37 +/- 6.4), but T3 increased only slightly (18 +/- 3.4).(ABSTRACT TRUNCATED AT 250 WORDS)

Killing of Paracoccidioides brasiliensis conidia by pulmonary macrophages and the effect of cytokines.

The ability of conidia, the infectious form of the dimorphic fungus Paracoccidioides brasiliensis, to be killed in vitro by murine pulmonary macrophages was studied. Mice were immunized by intravenous injection of killed conidia, which resulted in cellular immunity demonstrated by delayed type hypersensitivity in vivo and macrophage migration inhibition factor production in vitro. Resident pulmonary macrophages from non-immune mice were able to significantly kill the conidia (28%). Such macrophages treated with supernatants (cytokines) from antigen-stimulated immune mononuclears had a markedly enhanced ability to kill conidia (73%). These results show that activated pulmonary macrophages are potent killers of conidia of P. brasiliensis and that immune mononuclears play a role in activation of macrophages. Activated macrophages may be important for pulmonary defense against the initial stages of infection with this fungus.

Ultrastructure of phagocytosed Paracoccidioides brasiliensis in nonactivated or activated macrophages.

Transmission electron microscopy was used to study ultrastructures in Paracoccidioides brasiliensis yeast cells after ingestion by nonactivated or cytokine-activated murine peritoneal macrophages. Yeast cells ingested by nonactivated macrophages had typical bi- and trilayered cell walls, plasma membranes, mitochondria, nuclei, vacuoles, etc., which remained intact for 24 h of coculture. In contrast, yeast cells ingested by activated macrophages exhibited abnormal mitochondrial ultrastructures within 4 h of interaction. Subsequent events that occurred were the formation of several clear vacuoles per cell, disintegration of the cytoplasm, and development of empty cells with intact walls. These findings provide, for the first time, insights into stepwise damage to fungal cells by activated macrophages (of particular interest in this instance because of prior evidence that the damage is due to nonoxidative mechanisms) and give possible clues regarding fungicidal mechanisms.

Virulence of Paracoccidiodes brasiliensis: the influence of in vitro passage and storage.

Stability of virulence in P. brasiliensis isolates was studied with respect to the in vitro culture history and methods used for storage. Virulence in yeast-form P. brasiliensis isolates was tested in a chronic pulmonary murine model of paracoccidiodomycosis where progression of disease was quantitated in terms of colony forming units recoverable from lungs. Four isolates of P. brasiliensis, including recently isolated form patients or experimental animals, caused chronic progressive disease. Two isolates with a history of subculturing showed attenuation by causing resolving but chronic disease. An attenuated isolate became avirulent subsequent to 15 more years of subculturing. These findings suggest that virulence of P. brasiliensis can be attenuated or lost subsequent to cycles of subculturing over long periods. Our data suggest that the use of fresh P. brasiliensis isolates may be needed to provide reproducible virulence for experimental systems.

Intracellular multiplication of Paracoccidioides brasiliensis in macrophages: killing and restriction of multiplication by activated macrophages.

The effect of coculturing yeast-form Paracoccidioides brasiliensis with murine cells was studied. Coculture of resident peritoneal or pulmonary macrophages with P. brasiliensis for 72 h dramatically enhanced fungal multiplication 19.3 +/- 2.4- and 4.7 +/- 0.8-fold, respectively, compared with cocultures with lymph node cells or complete tissue culture medium alone. Support of P. brasiliensis multiplication by resident peritoneal macrophages was macrophage dose dependent. Lysates of macrophages, supernatants from macrophage cultures, or McVeigh-Morton broth, like complete tissue culture medium, did not support multiplication of P. brasiliensis in 72-h cultures. Time course microscopic studies of cocultures in slide wells showed that macrophages ingested P. brasiliensis cells and that the ingested cells multiplied intracellularly. In sharp contrast to resident macrophages, lymphokine-activated peritoneal and pulmonary macrophages not only prevented multiplication but reduced inoculum CFU by 96 and 100%, respectively, in 72 h. Microscopic studies confirmed killing and digestion of P. brasiliensis ingested by activated macrophages in 48 h. These findings indicate that resident macrophages are permissive for intracellular multiplication of P. brasiliensis and that this could be a factor in pathogenicity. By contrast, activated macrophages are fungicidal for P. brasiliensis.

In vivo and in vitro activation of pulmonary macrophages by IFN-gamma for enhanced killing of Paracoccidioides brasiliensis or Blastomyces dermatitidis.

The fungicidal capacity of murine pulmonary macrophages (PuM) activated in vitro with IFN or lymphokines or in vivo with IFN was studied. PuM treated overnight with IFN (1000 U/ml), Con A-stimulated spleen cell culture supernatants, or lymph node cells plus Con A significantly killed yeast cells of the Gar w isolate of Paracoccidioides brasiliensis 45.5 +/- 2.1%, 72.0 +/- 4.2%, and 51.5 +/- 0.7% respectively. Two other isolates of P. brasiliensis (Ru and LA) were also killed (45 and 34%) by PuM activated by lymph node cells plus Con A. Control PuM had lesser but significant capacity for killing of P. brasiliensis isolates, ranging from 15 to 22%. Killing of P. brasiliensis by PuM activated by Con A-stimulated spleen cell culture supernatants could not be significantly inhibited by superoxide dismutase, catalase, or azide. When mice were treated in vivo with 4 X 10(5) IFN U i.p. and PuM isolated 24 h later, the PuM had significantly enhanced ability to kill P. brasiliensis (47.0 +/- 6.3%) compared with PuM from control mice (25.0 +/- 4.2%). PuM thus activated also showed enhanced killing (43%) of a second isolate compared with control PuM (22%). PuM from IFN-treated mice were able to significantly kill Blastomyces dermatitidis (37.5 +/- 0.7%) compared with control PuM (4.5 +/- 6.3%). These results show that PuM can be activated in vitro and in vivo by IFN for enhanced fungicidal activity against two pulmonary fungal pathogens and suggests that immunologic production of IFN could be an important factor in host defenses against these diseases.

A culture medium for Paracoccidioides brasiliensis with high plating efficiency, and the effect of siderophores.

The plating efficiency of Paracoccidioides brasiliensis on standard mycological media is poor, impairing its isolation and recovery from various sources, particularly infected tissues. We describe a medium that markedly improves P. brasiliensis plating efficiency. It consists of a synthetic medium (modified McVeigh-Morton) supplemented with 4% (v:v) horse serum and 5% (v:v) culture filtrate from stationary phase P. brasiliensis cultures. A commercially available medium (brain-heart infusion), ordinarily inferior to unsupplemented McVeigh-Morton medium, is at least as efficacious as supplemented McVeigh-Morton medium when supplemented in this manner. We show that plating efficiency varies among P. brasiliensis isolates and can even vary with the isolate's history of passage in culture. In contrast, all isolates studied could produce the growth enhancing factors present in culture filtrate. Some siderophores produced by other fungi can be substituted for the culture filtrate, whereas others can be substituted for both the filtrate and serum. The enhancing effect of filtrate and/or serum could be removed by chelating iron. P. brasiliensis-produced siderophores are likely to be the growth enhancing moiety in culture filtrates.

Gamma-interferon activation of macrophages for killing of Paracoccidioides brasiliensis and evidence for nonoxidative mechanisms.

Fungicidal activity of murine peritoneal macrophages for the yeast form of the dimorphic fungal pathogen P. brasiliensis was studied. Killing was assessed by reduction of colony forming units (CFU) using a new medium which has a good plating efficiency. Resident peritoneal macrophages phagocytosed but did not kill P. brasiliensis. Macrophages treated overnight with recombinant gamma-interferon (IFN), lymph node cells plus concanavalin A (Con A) or Con A-stimulated spleen cell culture supernatants (Con A Sup) reproducibly killed three different isolates of P. brasiliensis (35 - 55%, P less than 0.05 - P less than 0.001). This is the first demonstration of killing of this organism by macrophages. Activated macrophages did not show enhanced phagocytosis of P. brasiliensis. Activation of macrophages for killing by IFN was dose-dependent and, varying with the isolate, 100 - 10,000 U/ml was required for inducing significant fungicidal effects against P. brasiliensis. Activation of macrophages by IFN or Con A Sup was abrogated by anti-IFN antibody. These results suggest that immune modulation may be an approach to therapy of paracoccidioidomycosis. Killing was not significantly inhibited in the presence of superoxide dismutase (450 U/ml), catalase (20,000 U/ml), dimethylsulfoxide (300 mM) or azide (1 mM). This indicated that killing mechanism(s) did not depend upon products of the oxidative burst. These results show that P. brasiliensis can be significantly killed by activated macrophages without products of the oxidative burst.

Effect of murine polymorphonuclear leukocytes on the yeast form of Paracoccidioides brasiliensis [corrected].

The fungicidal activity of murine polymorphonuclear neutrophils from the peripheral blood or elicited intraperitoneally with thioglycollate or with antigen in Paracoccidioides brasiliensis-sensitized [corrected] or nonsensitized mice was studied. Although peripheral blood, thioglycollate-elicited, and antigen-elicited neutrophils from normal mice or thioglycollate-elicited neutrophils from P. brasiliensis-sensitized [corrected] mice killed Candida albicans (57% to 84%), they failed to significantly reduce inoculum colony forming units of P. brasiliensis [corrected] (0% to 13%). In contrast, antigen-elicited neutrophils from sensitized mice reduced colony forming units of P. brasiliensis [corrected] by 40%, and exhibited significantly enhanced candidacidal activity compared to thioglycollate-elicited neutrophils from normal or sensitized mice but not peripheral blood neutrophils from normal mice. Fresh serum, but not specific antibody, was required for optimal killing of P. brasiliensis [corrected], presumably representing an essential role for complement. Killing of P. brasiliensis [corrected] by antigen-elicited neutrophils from sensitized mice correlated with their ability to produce an enhanced oxidative burst, as measured by luminol-assisted chemiluminescence, when interacting with killed P. brasiliensis [corrected] cells. These results indicate that in P. brasiliensis-sensitized [corrected] hosts an inflammatory reaction to P. brasiliensis [corrected] results in activation of neutrophils for significant killing of the pathogen.

An evaluation of the enzyme-linked immunoabsorbent assay (ELISA) for quantitation of antibodies to Paracoccidioides brasiliensis.

The ELISA procedure was adapted for quantitation of antibodies against Paracoccidioides brasiliensis. Using a yeast cytoplasmic antigen and sera from patients with proven paracoccidioidomycosis, we found that 66% of sera reacted at titers greater than or equal to 1:128. Titers of this magnitude were obtained only for 4-5% of sera from healthy blood donors, tuberculosis patients and patients with other systemic mycoses. The exception was sera from patients with histoplasmosis (36% had titers greater than or equal to 1:128). Follow-up of 10 paracoccidioidomycosis patients during the course of therapy indicated a gradual decrease in antibody titers. Because of the technical advantages of the ELISA procedure in comparison with the standard complement fixation test, the ELISA test has potential utility for the quantitative determination of antibodies in patients with paracoccidioidomycosis.

Toxic effect of products of oxidative metabolism on the yeast form of Paracoccidioides brasiliensis.

The effectiveness of toxic oxygen metabolites in killing the yeast form of Paracoccidioides brasiliensis (the form that occurs in host tissues) was studied with a fluorescence method in vitro. The two isolates studied were similar in susceptibility and H2O2 alone was lethal with an LD50 of 15-25 mM. The addition of halide (5 X 10(-4) M) augmented the lethality of H2O2 and in that setting H2O2 was c.90% lethal at 5 X 10(-5) M. Killing was most effective in the presence of peroxidase, when only 5 X 10(-6) M H2O2 (a concentration attainable in vivo by phagocytes) was required for a 95% kill. Kinetic studies revealed that toxic concentrations of H2O2 alone or of the H2O2-halide-peroxidase (PPH) system produced significant killing in 1 min; killing was maximal in 15 min. The PPH system was the more rapid in action. The dependence of the PPH killing system on H2O2 was demonstrated by an absence of killing in the presence of catalase. The susceptibility of P. brasiliensis to H2O2 and the PPH system appeared different in some respects from that noted for other dimorphic fungal pathogens. These studies suggest that toxic oxygen metabolites are important in host defence against P. brasiliensis.

Chronic murine paracoccidioidomycosis: effect of ketoconazole on clearance of Paracoccidioides brasiliensis and immune response.

In a murine model of chronic pulmonary and disseminated paracoccidioidomycosis, ketoconazole (100 mg kg-1 in 0.3% agar) given by gavage twice daily for 1 or 2 months enabled all mice to clear disseminated Paracoccidioides brasiliensis from the spleen. Clearance of P. brasiliensis from the lungs was more difficult, and was achieved in 60% of the mice treated for 2 months. Sera from agar-treated control mice at days 77 and 103 post-infection demonstrated precipitating antibodies to P. brasiliensis antigens, but sera from ketoconazole-treated mice were precipitin-negative, indicating a favorable prognosis. Delayed hypersensitivity reactions to P. brasiliensis antigens in ketoconazole-treated mice were not significantly greater than in controls; consequently this test correlated less well with response than levels of serum antibody. This is the first use of this animal model of paracoccidioidomycosis to study the effect of antifungal drug protocols on the resolution of the disease. It also demonstrates the utility of this model in addressing clinically relevant questions about this disease and its treatment.

Comparison of various techniques for determining viability of Paracoccidioides brasiliensis yeast-form cells.

The viability of Paracoccidioides brasiliensis yeast-form cells was determined by colony-forming units, direct fluorescent staining, and production of germ tubes in slide culture. The first procedure was unreliable and time consuming; the latter two showed better correlation with hemacytometer total cell counts and required significantly less time.