Fungal isolates and growth conditions
Paracoccidioides brasiliensis strain Pb18 was provided by Dr Z.P. Camargo, São Paulo, SP, Brazil. Yeast and mycelia forms of P. brasiliensis were grown at 37°C and 25°C, respectively, in PGY (peptone 5 g/L, glucose 15 g/L, yeast extract 5 g/L) using 2.5 L Fernbach flasks in a shaker at 100 rpm . Histoplasma capsulatum strain 496 from human pulmonary lesion  and Sporothrix schenckii strain 65 from human foot cutaneous lesion [22, 23], were kindly provided by Dr O. Gompertz, São Paulo, SP, Brazil. Yeast and mycelia forms of both fungi were grown in Brain Heart Infusion (BHI) (37 g/L) at 37°C and 25°C, respectively. After 5-7 days both yeast and mycelia forms of the various fungi were inactivated with 0.1% of thimerosal, and after an additional 48 h the fungi were collected by filtration on Whatman n° 1 filter paper, except for yeast forms of S. schenckii and H. capsulatum, which were harvested by centrifugation at 5,200 × g for 20 minutes.
Extraction and purification of glycosphingolipids (GSLs)
GSLs were extracted by homogenizing yeast or mycelia forms (~ 30 g) in an Omni-mixer (Sorvall Inc. Wilmington, DE), three times with 200 ml of isopropanol/hexane/water (IHW, 55:20:25, v/v/v, upper phase discarded), and twice with 200 ml of chloroform/methanol (CM, 2:1, v/v). The five extracts were pooled, dried on rotary evaporator, dialyzed against water and lyophilized. Neutral and acidic GSLs were separated in a DEAE-Sephadex A-25 column as described by Yu and Ledeen . Fractions containing GIPCs, were assessed by HPTLC on silica gel 60 plates (E. Merck, Darmstadt, Germany) using solvent A: chloroform/methanol/CaCl2 0.02%, (60:40:9; v/v/v), and stained with orcinol/H2SO4. For preparative-scale HPTLC separated GSL bands were visualized under UV light after spraying with primulin 0.01% in 80% aqueous acetone . GSLs were isolated from silica gel scraped from the plates by repeated sonication in IHW, as described .
Production of hybridomas
About 600 μg of GIPC Pb-2 purified from mycelia forms of P. brasiliensis were dissolved in 1.5 ml of distilled water and mixed with 1.5 mg of acid-treated heat-inactivated Salmonella minnesota. Aliquots (100 μl) of this suspension containing 40 μg of the antigen were used to immunize six weeks old BALB/c mice, by i.v. route, through the caudal vein once a week, over 4 weeks. After a rest period of 30 days, the immune response was boosted with 200 μl of the immunogenic complex. Three days later, the mice were sacrificed and their spleen removed. The lymphocytes were fused with NS-1 myeloma cells and placed in 96-well plates. Solid-phase RIA detected hybrids secreting immunoglobulins reacting with Pb-2. Only clones showing strong reactivity with Pb-2 of mycelia and yeast forms of P. brasiliensis were cloned by limited dilution as described [13, 24, 37]. Research ethical approval (CEP 0023/06) was granted by the Ethical Research Committee Boards of Universidade Federal de São Paulo.
Various GSLs were adsorbed on 96-well plates (Falcon Microtest III flexible assay plates, Oxnard, CA). Solutions (25 μl/well, 100 ng/first well) in ethanol of different GSLs were serially diluted, dried at 37°C and wells blocked with 1% bovine serum albumin (BSA) in 0.01 M phosphate-buffered saline (PBS), pH 7.2 (200 μl) for 2 h, and sequentially incubated with mAb MEST-3 (100 μl) overnight at 4°C, rabbit anti-mouse IgG (50 μl) for 2 h, and with 50 μl of 125I-labeled protein A in PBS with 1% of BSA (about 105 cpm/well) for 1 h. The amount of mAb MEST-3 bound to Pb-2 was determined by measuring the radioactivity in each well in a gamma counter .
Release of glycosylinositols by ammonolysis
Ammonolysis of GIPCs was performed as described by Barr and Lester  and Levery et al. . Briefly, 100 μg of GIPCs Pb-2 and Ss-Y2 were heated in a Teflon-lined screw-capped test tube with 10 N NH3.H20 (~ 1 mL) for 18 h at 150°C. The solution was cooled and evaporated under N2 stream at 37°C; this process was repeated after addition of a few drops of 2-propanol. The residue was sonicated in 1 mL of water and the lipophilic components were removed by passage of this solution through a small C18-silica solid-phase extraction cartridge, washing twice with 1 ml of water. The combined aqueous fraction containing free glycosylinositol was lyophilized and used for inhibition of antibody binding to GIPCs Pb-2.
Inhibition of antibody binding by different methyl glycosides, disaccharides and glycosylinositols
Initially, 75 μl of a 200 mM solution of several methyl-α- and β-D-glycosides (glucopyranoside, galactopyranoside and mannopyranoside), disaccharides (Manα1→2Man, Manα1→3Man and Manα1→6Man), purchased from Sigma (MO, USA), and the glycosylinositols (Manα1→3Manα1→2Ins and Manα1→3Manα1→6Ins, described above), were serially diluted with PBS in a 96-well plate. Each glycoside solution was incubated with 75 μl of MEST-3 at room temperature . After 2 h, aliquots of 100 μl were taken and incubated overnight at 4°C in 96-well plates pre-coated with the GIPC Pb-2 (100 ng/well) essentially as described under Binding assay.
Ninety-six-well plates were coated with different concentrations (100 ng to 5 pg) of GIPC Pb-2 and treated with 5 and 20 mM of sodium m-periodate in PBS (0.1 M, pH 7.0) at room temperature for 30 min . The plates were washed with PBS, reduced with NaBH4 (50 mM in PBS) during 30 min, blocked with 5% of BSA in PBS for 1 h, and incubated overnight with mAb MEST-3, and processed as described in Binding Assay.
High performance thin layer chromatography (HPTLC) immunostaining
GIPCs purified from different fungi were separated by HPTLC, and the immunostaining of the plates was performed by the procedure of Magnani et al. , modified by Zuolo et al.  and Takahashi et al. . GSLs (3 μg) were separated in solvent A: C:M:CaCl2 0.02% (60:40:9; v/v/v), after development, the plates were dried, soaked in 0.5% polymethacrylate in hexane, dried, and blocked for 2 h with 1% of BSA in PBS. Plates were then incubated with mAb MEST-3 overnight followed by sequential incubations with rabbit anti-mouse IgG and 125I-labeled protein A (2 × 107 cpm/50 ml of BSA/PBS).
Fungi were fixed with 1% formaldehyde in PBS for 10 min. Cells were washed, suspended in 1 ml of PBS, and 20 μl of the solution was added to a coverslip pre-treated with poly-L-lysine 0.1% during 1 h. Air dried preparations were soaked for 1 h in PBS containing 5% of BSA, and incubated subsequently with culture supernatant of mAb MEST-3 (2 h), biotin-conjugated goat anti-mouse IgG (1 h), and with avidin-conjugated fluorescein (1 h). After each incubation the coverslips were washed five times with PBS. The coverslips were examined with an epifluorescence microscope . Control experiments for each fungus were carried out, in the presence of an irrelevant monoclonal antibody, and no fluorescence was observed.
To evaluate the influence of mAbs directed to GSLs on the growth of different fungi, yeasts (104/ml) were incubated in 96-well plate in the presence of mAbs MEST-1, -2, or -3 for 24 h at 37°C, in concentration ranging from 2.5 to 50 μg/ml. The growth rate was evaluated by two procedures; 1) viable CFU were evaluated by plating 100 μl of the samples onto BHI or PGY agar plates, followed by incubation for 2 days at 37°C, and colony counting; or 2) 5 μl of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml MTT in phosphate-buffered saline, pH 7,4) were added to each well and the plates were further incubated at 37°C, for 3 h, after incubation the medium containing MTT was partially removed, and dimethyl sulfoxide (100 μl) was added to solubilize the MTT formazan product . The absorbance of each well was measured at 580 nm by a microtiter ELISA plate reader. Control systems were similarly treated with an irrelevant immunoglobulin (normal mouse total Ig) and plated. All experiments were repeated three times in triplicates, and the results shown are a representative of these experiments.
Fungal differentiation - yeast to mycelium
104 viable yeasts were suspended in 1 ml of PGY (P. brasiliensis) or BHI (H. capsulatum and S. schenckii) medium. The suspension was incubated in a 24-well plate and supplemented with mAb MEST-1, -2, or -3 (at a concentration of 2.5, 10, 25 or 50 μg/ml), after one hour at 37°C, 24-well plate was transferred to a 24°C incubator and kept for 2 days. The number of yeast showing hyphae growth was counted, and presented as percentage of those incubated with irrelevant immunoglobulins (normal mouse total Ig). In control experiment 100% of yeast showed hyphae formation. For each mAb concentration, 300 viable fungi (yeast or transforming-yeast) were counted, yeasts differentiating into mycelia forms were observed and evaluated considering hyphae formation. Control experiments were performed identically, with the addition of irrelevant immunoglobulins. Experiments were performed in triplicate sets and representative results are shown in Figure 5.
Fungal differentiation - mycelium to yeast
A 5 days old culture containing hyphae, was washed and combined in a tube with sterile PBS and 5 mm glass beads, this suspension was agitated in vortex (3 × 5 min), to broke the web mycelia in small hyphae. After decantation, the supernatant containing short lengths of hyphae was centrifuged and the hyphae suspended in 1 ml of PGY medium. The suspension was incubated in a 24-well plate and supplemented with mAb MEST-1, -2, or -3 (at a concentration of 2.5, 10, 25 or 50 μg/ml), at 37°C. After 48 h and 96 h of incubation cultures were analyzed under inverted microscopy. Controls experiments were performed identically, with the substitution of mAb to irrelevant immunoglobulins (normal mouse total Ig).