BACKGROUND
drouhet  <cit>  described the existence of over  <dig>  species of fungi widespread in nature, and more than  <dig> may be associated with human mycoses.
in the last two decades, it was observed a dramatic raise in mortality of immunosupressed individuals associated with fungal infection.
although antifungal therapies have been successful and selective, the outbreaks of resistant strains, together with an increase on fungal tolerance levels to currently available antifungal, were described by several reports  <cit> .
therefore, a compelling search for novel antifungal therapies has been greatly stimulated.
studies carried out during the 1990s demonstrated that many species of fungi are vulnerable to inhibitors of enzymes of the sphingolipid biosynthesis pathway, such as inositol phosphorylceramide  synthase  <cit> .
this particular enzyme transfers myo-inositol-1-phosphate from phosphatidylinositol to ceramide, the first and an essential step for the biosynthesis of glycoinositol phosphorylceramides , a class of complex anionic glycosphingolipids  widely distributed among fungal species  <cit> .
in this manner, gipcs synthesis are highly susceptible to ipc synthase inhibitors, which in turn are remarkably toxic to many mycopathogens, but exhibit low toxicity in man, since the ipc or ipc-synthase gene are absent in mammals  <cit> .
the detailed characterization of gipcs from a variety of fungi revealed an extensive structural diversity.
based on further studies, more than  <dig> distinct gipc structures have been identified to date, which may present one of the  <dig> well-confirmed core structures distinguishable at the monoglycosyl level and absent in mammals  <cit> .
some of these gipcs have antigenic glycoside determinants, such as terminal β-d-galactofuranose residues, which are recognized by human sera, suggesting their potential as targets for immunodiagnostic and the possibility of therapy based on stimulation of mammalian humoral response  <cit> .
it should be emphasized that the expression of these gipcs is considerably dependent on species, and at least for some mycopathogens, strongly regulated during morphogenesis  <cit> .
in this context, to investigate the role of gsls in differentiation and colony formation of paracoccidioides brasiliensis, histoplasma capsulatum, and sporothrix schenckii, we used three monoclonal antibodies  raised to fungal gsls: a) mab mest- <dig> directed to terminal galfβ1→3/6manp  <cit> , b) mab mest- <dig> directed to β-glucosylceramide  <cit> , and c) mab mest- <dig> directed to terminal manpα1→3manpα1→2ins .
table  <dig> summarizes the reactivity of mabs mest- <dig>  - <dig> and -3: i) to lipids extracted from yeast and mycelium forms, which were analyzed by high performance thin layer chromatography  immunostaining, and ii) to yeast and mycelium forms of fungi used in this work, that were analyzed by indirect immunofluorescence .
as shown in this paper, the availability of mabs specifically directed to different gsl structures may be used as effective tools to a more accurate understanding of the organizational pattern and the biological role of gsls of different fungi.
reactivity of mabs mest- <dig>  - <dig> and - <dig>  with fungal glycolipids by hptlc immunostaining ; and with fixed fungi by indirect immunofluorescence .
pb = p. brasiliensis; ss = s. schenckii; hc = h. capsulatum; y = yeast; m = mycelium; mest- <dig> recognizes epitope galfβ1→3/6manp; mest- <dig> recognizes fungal glucosylceramide ; mest- <dig> recognizes epitope manpα1→3manpα1→2ins; "+" indicates positive staining; "-" indicates negative staining, and "np" indicates epitope not present.
<cit> .
RESULTS
characterization of mab mest-3
aiming to study the biological role of gipcs, and since expression of these glycoconjugates with terminal galactofuranose residues, which are recognized by mest- <dig>  is restricted to p. brasiliensis , h. capsulatum  and a. fumigatus , we decided to develop a mab directed to gipc pb- <dig>  from p. brasiliensis, which structure manpα1→3manpα1→2ipc is expressed in a wide variety of fungi, and therefore a mab directed to such structure would be highly desirable to detect a large number of pathogenic fungi.
among a few clones showing reactivity with gipc pb- <dig>  a clone secreting an igg2a monoclonal antibody was established, and termed mest- <dig>  by hptlc-immunostaining  it was observed that mest- <dig> reacts with pb- <dig> from yeast and mycelium forms of p. brasiliensis, and other gipcs containing the same structure as pb- <dig>  such as hc-y <dig> from yeasts of h. capsulatum , ss-y <dig> from yeasts of s. schenckii , af- <dig> from hyphae of a. fumigatus , and an- <dig> from hyphae of a. nidulans .
moreover, lanes  <dig> and  <dig> of figure 1a-b confirm that mycelium forms of h. capsulatum and s. schenckii do not express gipcs bearing the epitope recognized by mest- <dig>  as described before  <cit> .
also, by solid-phase radioimmunoassay , it was verified that mab mest- <dig> was able to detect as low as  <dig> ng of purified pb- <dig>  hc-y <dig>  ss-y <dig> and af- <dig> .
conversely, no reactivity of mest- <dig> with gipcs, presenting the structures manp manpipc ; manα1→2ipc  and manα1→3manα1→6ipc , was detected by hptlc-immunostaining or ria.
treatment of pb- <dig> with sodium m-periodate led to a decrease of 82% of mab mest- <dig> binding to this gipc, indicating that mest- <dig> recognizes the carbohydrate moiety of pb- <dig> , the structural features of the glycoepitope, recognized by mest- <dig>  was analyzed by inhibition assays on solid-phase ria carried on 96-well plates pre-coated with purified pb- <dig> antigen using different methyl-glycosides, disaccharides and glycosylinositols derived from gipcs.
as shown in figure  <dig>  methyl-α-d mannopyranoside, manα1→2man and manα1→6man did not inhibit mest- <dig> binding to pb- <dig>  whereas disaccharide manα1→3man and glycosylinositol manα1→3manα1→2ins, at a concentration of  <dig> mm, were able to inhibit by 80% the binding of mest- <dig> to pb- <dig> antigen.
in addition, glycosylinositol manα1→3manα1→6ins, derived from ss-m <dig> of mycelium forms of s. schenckii, was not able to inhibit mest- <dig> binding to pb- <dig>  taking together, these data indicate that the epitope recognized by mest- <dig> is not restricted to the terminal residue of mannose, but also includes the subterminal residues of mannose and myo-inositol .
therefore, these results clearly indicate that mest- <dig> recognizes specifically gipcs presenting the linear structure manpα1→3manpα1→2myo-inositol.
indirect immunofluorescence with mest-3
as shown in figure  <dig>  indirect immunofluorescence using mest- <dig> showed that yeast forms of p. brasiliensis and h. capsulatum present homogenous surface labeling, whereas yeast forms of s. schenckii present an interspersed labeling at cell surface.
by hptlc immunostaining or ria, mab mest- <dig> showed reactivity with gipcs isolated from mycelium forms of p. brasiliensis and hyphae of a. fumigatus and a. nidulans , but it is noteworthy that no fluorescence was observed with mycelium forms of p. brasiliensis and hyphae of a. fumigatus and a. nidulans .
as expected, by immunostaining and ria , no reactivity of mest- <dig> was observed with mycelium forms of s. schenckii and h. capsulatum.
negative controls using an irrelevant mab showed no fluorescence .
effect of monoclonal antibodies on fungal growth
by counting the total number of colony forming units , the effect of mabs mest- <dig>  - <dig> and - <dig> at different concentrations on fungal growth was analyzed.
under the conditions described in methods, it was determined for p. brasiliensis, h. capsulatum and s. schenckii, a total of  <dig> ±  <dig>   <dig> ±  <dig> and  <dig> ±  <dig> cfus, respectively.
as shown in figure 4a, mabs mest- <dig> and - <dig> were effective in inhibiting p. brasiliensis and h. capsulatum cfus in a dose-dependent manner.
mab mest- <dig> was able to inhibit p. brasiliensis and h. capsulatum cfu by about 38% and 45%, respectively, while mest- <dig> inhibited p. brasiliensis, h. capsulatum and s. schenckii cfus by about 30%, 55% and 65%, respectively .
conversely, as expected, mest- <dig> was not able to inhibit s. schenckii cfu, since this fungus does not present glycolipids containing terminal residues of β-d-galactofuranose  <cit> .
it should be noted that mest- <dig> did not present significant cfu inhibitory activity in none of the three fungi used in this study.
confirming these results, p. brasiliensis, h. capsulatum and s. schenckii were grown in media containing mabs for  <dig> h, after that, mtt {3-- <dig> -diphenyltetrazolium bromide} was added to measure the growth rate.
as observed in figure 4b, mest- <dig> and - <dig> inhibited significantly the growth of p. brasiliensis and h. capsulatum, whereas for s. schenckii, only mest- <dig> was able to inhibit fungal growth.
effect of monoclonal antibodies on fungal dimorphism
in order to analyze the effect of mabs mest- <dig>  - <dig> and - <dig> on yeast to mycelium transformation of p. brasiliensis, h. capsulatum and s. schenckii, at first, yeast forms were incubated with these mabs for  <dig> h at 25°c, which is the optimum temperature for mycelia growth.
as observed for cfu, mabs mest- <dig> and - <dig> were also able to inhibit in a dose-dependent manner the yeast to mycelia differentiation of p. brasiliensis and h. capsulatum .
in these experiments,  <dig> μg/ml of mest- <dig> was able to inhibit the conversion of approximately 50% of p. brasiliensis, and 55% of h. capsulatum from yeast to mycelia.
under the same condition, mest- <dig> was not able to inhibit the conversion from yeast to mycelia in s. schenckii .
moreover, mab mest- <dig> was able to inhibit the conversion of yeast to mycelia of approximately 30% of p. brasiliensis, 55% for h. capsulatum and 50% for s. schenckii .
furthermore, considering the relative proportion of yeast and mycelia forms as well the hyphal length, it was verified that mab mest- <dig>  and mest- <dig>  were able to inhibit p. brasiliensis and h. capsulatum yeast to mycelia differentiation as early as  <dig> h after mab incubation.
additionally, only mest- <dig>  was able to inhibit s. schenckii yeast to mycelium differentiation.
in contrast, no inhibition of yeast to mycelium differentiation was observed upon incubation of these fungal species with mest- <dig> .
parallel experiments showed that after washing and replacing medium containing mabs by antibody-free medium; the fungi tested were able to restore their growth and/or transformation, indicating that mabs mest- <dig>  - <dig> and - <dig> present a fungistatic effect .
in another set of experiments, it was evaluated the effect of mab mest- <dig> and - <dig> on p. brasiliensis mycelium to yeast transformations, and as expected, it was not observed a significant inhibition, since these antibodies do not react or react weakly with mycelium forms.
thus,  <dig> μg/ml of mest- <dig> and mest- <dig> inhibited, respectively, 6% and 9% the transition from mycelium to yeast of p. brasiliensis.
figure  <dig> shows the differentiation of p. brasiliensis mycelia forms grown in presence  or not  of mest- <dig> for  <dig> h at 37°c.
in order to illustrate the differentiation inhibition, but not picturing the real inhibition percentage, figure 7b pictured a field with high concentration of hyphae form.
discussion
mab mest- <dig> specificity
in this paper, we describe the characterization of mest- <dig>  an igg2a monoclonal antibody directed to the structure  from gipc pb- <dig> of p. brasiliensis.
among different methyl-glycosides, disaccharides and glycosylinositols, only manpα1→3manp and manpα1→3manpα1→2ins inhibited mest- <dig> binding to pb- <dig> in solid-phase ria.
furthermore, mest- <dig> was unable to recognize, by solid-phase ria or hptlc-immunostaining, the intact gipc ss-m <dig> , thus suggesting that α1→ <dig> linkage of the subterminal mannose unit to inositol represents a sterical hindrance for antigen recognition by mest- <dig>  therefore, the minimum epitope required for optimum binding of mest- <dig> to pb- <dig> and similar gipcs, would comprise the two linear mannose residues in specific linkage and the myo-inositol residue as follows: manpα1→3manpα1→2ins.
by indirect immunofluorescence, it was observed that mest- <dig> is reactive only with yeast forms of p. brasiliensis, h. capsulatum and s. schenckii, which is in agreement with previous data describing the crypticity of gipc pb- <dig> and glccer in mycelium forms of p. brasiliensis  <cit> .
accordingly, despite the detection of the gipc pb- <dig> extracted from hyphae of p. brasiliensis by hptlc and hptlc immunostaining with mab mest- <dig>  it should be noted the complete lack of mest- <dig> reactivity by immunofluorescence with fixed mycelia forms.
taking together, these data suggest that a particular organization of gsls and/or cell wall structure, distinct from that found in yeast forms, is required for mab mest- <dig> binding to fungal gipc pb- <dig>  in this context, the access of antibodies directed to gsls of mycelium forms seems to be strongly affected by organizational or structural aspects that do not favor the interaction antigen-antibody.
growth and dimorphism inhibition by anti-glycosphingolipid mabs
there are several reports in the literature showing the importance of neutral glycosphingolipids, such as cerebrosides, on fungal growth and morphological transition  <cit> .
rodrigues et al.
<cit>  described that the addition of purified human antibodies, directed to glccer from cryptococcus neoformans, inhibited cell budding and growth of this fungus.
therefore, the effects of three mabs , directed to different fungal gsls, were analyzed on colony formation  of pathogenic dimorphic fungi .
experiments using mab mest- <dig>  directed to fungal glccer, showed no significant inhibition of cfu or effect in dimorphism of the fungi studied.
these data do not corroborate the results from rodrigues et al.
<cit> .
possible explanations for these results may be related to the source of the antibodies, human and murine, in our case, or fungal species, since this effect was only observed in c. neoformans.
our results using mab mest- <dig>  directed to pb- <dig> and hc-y <dig>  showed significant inhibition of fungal growth and differentiation of p. brasiliensis and h. capsulatum from yeast to mycelia.
as expected, no inhibition with mest- <dig> was observed for s. schenckii, since this specie does not express galactofuranose-bearing gsls.
on the other hand, mest- <dig> was able to inhibit cfu, fungal growth and differentiation of all three fungi studied.
mest- <dig> was able to cause higher inhibition of cfu and differentiation for h. capsulatum and s. schenckii than for p. brasiliensis.
this lower degree of inhibition showed by p. brasiliensis could be attributed to the low gipc pb- <dig> concentration in yeast forms of this fungus  <cit> .
on the other hand, gipcs hc-y <dig> and ss-y <dig>  which bear the same structure as pb- <dig>  represent about 30% and 20% of acidic glycolipid fraction from h. capsulatum and s. schenckii yeast forms respectively  <cit> .
conversely, results observed in the mycelium to yeast transformation, were not straightforward, a possible explanation could be related to the non-reactivity of mabs mest- <dig>  - <dig> and - <dig>  with mycelia forms, as observed by immunofluorescence assay .
moreover, in h. capsulatum and s. schenckii, the transformation of mycelium to yeast takes at least three weeks in normal conditions, and the mycelium web hinders clear yeast observation and quantification.
it is now well established that the precise build up of lipid rafts is necessary to efficiently guide signal transduction through cell membrane  <cit> , some new evidences indicate that in fungi, these constructions are also necessary for fungal survival and maintenance of the infection  <cit> .
two attractive hypotheses for the inhibitory activity of mabs mest- <dig> and mest- <dig> on the yeast growth and differentiation would be: i) the association mab-gipcs sterically inhibits the formation of functional glycosphingolipid-enriched microdomains , thus blocking the action of key enzymes responsible for cell wall synthesis  <cit> , and ii) an already existing microdomain to which a mab-gipc association takes place is not functional, hindering essential signal transduction to fungal growth and/or dimorphism.
confocal microscopy, with p. brasiliensis cell wall stained with calcofluor white indicates two places of mest- <dig> binding, i) extensive and internal to calcofluor labeling, i.e.
plasma membrane, and ii) discrete external as well as co-labeling with calcofluor, these preliminary data led us to suggest a new conceptual model of gsl arrangements in yeast forms where microdomain-like regions containing gipcs could also be located at the external surface of the cell wall.
further work to substantiate this concept model is under investigation in our laboratory aiming an extensive comprehension of fungal glycosphingolipid enriched microdomains regarding their composition, surface localization, role in signaling processes and possible role in host cell binding and infection.
this study and others have shown that specific gipcs are found in a large variety of pathogenic fungi  <cit> .
in some cases, those gipcs are recognized by sera from patients with paracoccidioidomycosis, histoplasmosis or aspergillosis  <cit> , indicating that gipcs are immunogenic and able to induce the production of human antibodies during fungal infections.
the broad distribution of gipcs in pathogenic fungi and the antifungal activity of monoclonal antibodies directed to gipcs indicate that these molecules may represent potential targets for the development of new therapeutical approaches based on induction of protective antibodies.
CONCLUSIONS
the fine specificity of mest- <dig> was assessed by inhibition assays using different methyl-glycosides, disaccharides and oligosaccharides.
only manα1→3man and the glycoinositol manα1→3manα1→2ins, from pb- <dig>  were able to inhibit, by about 95%, mest- <dig> binding to pb- <dig> antigen of p. brasiliensis.
the epitope recognized by mest- <dig> was defined as manα1→3manα1→2ins; this structure was already described in a variety of pathogenic fungi  <cit> .
studies using mabs mest- <dig> and mest- <dig>  as fungal growth inhibitors showed that anti-gipcs mabs presented a strong inhibitory activity on growth, differentiation and colony formation of p. brasiliensis, h. capsulatum, and s. schenckii.
on the other hand, no statistically significant inhibition was observed with anti-glccer .
these results strongly suggest that mabs directed to particular glycosphingolipids are able to interfere on fungal growth and differentiation.
an attractive hypothesis is that, by hindering the formation of functional lipid rafts by association with mabs, a possible dependence of surface organization of glycosphingolipids in fungi is essential for different fungal processes, such as growth, morphological transition, and infectivity.

 drouhet described the existence of over species of fungi widespread in nature, and more than may be associated with human mycoses. in the last two decades, it was observed a dramatic raise in mortality of immunosupressed individuals associated with fungal infection. although antifungal therapies have been successful and selective, the outbreaks of resistant strains, together with an increase on fungal tolerance levels to currently available antifungal, were described by several reports. therefore, a compelling search for novel antifungal therapies has been greatly stimulated. studies carried out during the 1990s demonstrated that many species of fungi are vulnerable to inhibitors of enzymes of the sphingolipid biosynthesis pathway, such as inositol phosphorylceramide synthase. this particular enzyme transfers myo-inositol-1-phosphate from phosphatidylinositol to ceramide, the first and an essential step for the biosynthesis of glycoinositol phosphorylceramides, a class of complex anionic glycosphingolipids widely distributed among fungal species. in this manner, gipcs synthesis are highly susceptible to ipc synthase inhibitors, which in turn are remarkably toxic to many mycopathogens, but exhibit low toxicity in man, since the ipc or ipc-synthase gene are absent in mammals. the detailed characterization of gipcs from a variety of fungi revealed an extensive structural diversity. based on further studies, more than distinct gipc structures have been identified to date, which may present one of the well-confirmed core structures distinguishable at the monoglycosyl level and absent in mammals. some of these gipcs have antigenic glycoside determinants, such as terminal β-d-galactofuranose residues, which are recognized by human sera, suggesting their potential as targets for immunodiagnostic and the possibility of therapy based on stimulation of mammalian humoral response. it should be emphasized that the expression of these gipcs is considerably dependent on species, and at least for some mycopathogens, strongly regulated during morphogenesis. in this context, to investigate the role of gsls in differentiation and colony formation of paracoccidioides brasiliensis, histoplasma capsulatum, and sporothrix schenckii, we used three monoclonal antibodies raised to fungal gsls: a) mab mest- directed to terminal galfβ1→3/6manp, b) mab mest- directed to β-glucosylceramide, and c) mab mest- directed to terminal manpα1→3manpα1→2ins. table summarizes the reactivity of mabs mest- - and -3: i) to lipids extracted from yeast and mycelium forms, which were analyzed by high performance thin layer chromatography immunostaining, and ii) to yeast and mycelium forms of fungi used in this work, that were analyzed by indirect immunofluorescence. as shown in this paper, the availability of mabs specifically directed to different gsl structures may be used as effective tools to a more accurate understanding of the organizational pattern and the biological role of gsls of different fungi. reactivity of mabs mest- - and - with fungal glycolipids by hptlc immunostaining; and with fixed fungi by indirect immunofluorescence. pb = p. brasiliensis; ss = s. schenckii; hc = h. capsulatum; y = yeast; m = mycelium; mest- recognizes epitope galfβ1→3/6manp; mest- recognizes fungal glucosylceramide; mest- recognizes epitope manpα1→3manpα1→2ins; "+" indicates positive staining; "-" indicates negative staining, and "np" indicates epitope not present..  characterization of mab mest-3 aiming to study the biological role of gipcs, and since expression of these glycoconjugates with terminal galactofuranose residues, which are recognized by mest- is restricted to p. brasiliensis, h. capsulatum and a. fumigatus, we decided to develop a mab directed to gipc pb- from p. brasiliensis, which structure manpα1→3manpα1→2ipc is expressed in a wide variety of fungi, and therefore a mab directed to such structure would be highly desirable to detect a large number of pathogenic fungi. among a few clones showing reactivity with gipc pb- a clone secreting an igg2a monoclonal antibody was established, and termed mest- by hptlc-immunostaining it was observed that mest- reacts with pb- from yeast and mycelium forms of p. brasiliensis, and other gipcs containing the same structure as pb- such as hc-y from yeasts of h. capsulatum, ss-y from yeasts of s. schenckii, af- from hyphae of a. fumigatus, and an- from hyphae of a. nidulans. moreover, lanes and of figure 1a-b confirm that mycelium forms of h. capsulatum and s. schenckii do not express gipcs bearing the epitope recognized by mest- as described before. also, by solid-phase radioimmunoassay, it was verified that mab mest- was able to detect as low as ng of purified pb- hc-y ss-y and af-. conversely, no reactivity of mest- with gipcs, presenting the structures manp manpipc; manα1→2ipc and manα1→3manα1→6ipc, was detected by hptlc-immunostaining or ria. treatment of pb- with sodium m-periodate led to a decrease of 82% of mab mest- binding to this gipc, indicating that mest- recognizes the carbohydrate moiety of pb-, the structural features of the glycoepitope, recognized by mest- was analyzed by inhibition assays on solid-phase ria carried on 96-well plates pre-coated with purified pb- antigen using different methyl-glycosides, disaccharides and glycosylinositols derived from gipcs. as shown in figure methyl-α-d mannopyranoside, manα1→2man and manα1→6man did not inhibit mest- binding to pb- whereas disaccharide manα1→3man and glycosylinositol manα1→3manα1→2ins, at a concentration of mm, were able to inhibit by 80% the binding of mest- to pb- antigen. in addition, glycosylinositol manα1→3manα1→6ins, derived from ss-m of mycelium forms of s. schenckii, was not able to inhibit mest- binding to pb- taking together, these data indicate that the epitope recognized by mest- is not restricted to the terminal residue of mannose, but also includes the subterminal residues of mannose and myo-inositol. therefore, these results clearly indicate that mest- recognizes specifically gipcs presenting the linear structure manpα1→3manpα1→2myo-inositol. indirect immunofluorescence with mest-3 as shown in figure indirect immunofluorescence using mest- showed that yeast forms of p. brasiliensis and h. capsulatum present homogenous surface labeling, whereas yeast forms of s. schenckii present an interspersed labeling at cell surface. by hptlc immunostaining or ria, mab mest- showed reactivity with gipcs isolated from mycelium forms of p. brasiliensis and hyphae of a. fumigatus and a. nidulans, but it is noteworthy that no fluorescence was observed with mycelium forms of p. brasiliensis and hyphae of a. fumigatus and a. nidulans. as expected, by immunostaining and ria, no reactivity of mest- was observed with mycelium forms of s. schenckii and h. capsulatum. negative controls using an irrelevant mab showed no fluorescence. effect of monoclonal antibodies on fungal growth by counting the total number of colony forming units, the effect of mabs mest- - and - at different concentrations on fungal growth was analyzed. under the conditions described in methods, it was determined for p. brasiliensis, h. capsulatum and s. schenckii, a total of ± ± and ± cfus, respectively. as shown in figure 4a, mabs mest- and - were effective in inhibiting p. brasiliensis and h. capsulatum cfus in a dose-dependent manner. mab mest- was able to inhibit p. brasiliensis and h. capsulatum cfu by about 38% and 45%, respectively, while mest- inhibited p. brasiliensis, h. capsulatum and s. schenckii cfus by about 30%, 55% and 65%, respectively. conversely, as expected, mest- was not able to inhibit s. schenckii cfu, since this fungus does not present glycolipids containing terminal residues of β-d-galactofuranose. it should be noted that mest- did not present significant cfu inhibitory activity in none of the three fungi used in this study. confirming these results, p. brasiliensis, h. capsulatum and s. schenckii were grown in media containing mabs for h, after that, mtt {3-- -diphenyltetrazolium bromide} was added to measure the growth rate. as observed in figure 4b, mest- and - inhibited significantly the growth of p. brasiliensis and h. capsulatum, whereas for s. schenckii, only mest- was able to inhibit fungal growth. effect of monoclonal antibodies on fungal dimorphism in order to analyze the effect of mabs mest- - and - on yeast to mycelium transformation of p. brasiliensis, h. capsulatum and s. schenckii, at first, yeast forms were incubated with these mabs for h at 25°c, which is the optimum temperature for mycelia growth. as observed for cfu, mabs mest- and - were also able to inhibit in a dose-dependent manner the yeast to mycelia differentiation of p. brasiliensis and h. capsulatum. in these experiments, μg/ml of mest- was able to inhibit the conversion of approximately 50% of p. brasiliensis, and 55% of h. capsulatum from yeast to mycelia. under the same condition, mest- was not able to inhibit the conversion from yeast to mycelia in s. schenckii. moreover, mab mest- was able to inhibit the conversion of yeast to mycelia of approximately 30% of p. brasiliensis, 55% for h. capsulatum and 50% for s. schenckii. furthermore, considering the relative proportion of yeast and mycelia forms as well the hyphal length, it was verified that mab mest- and mest- were able to inhibit p. brasiliensis and h. capsulatum yeast to mycelia differentiation as early as h after mab incubation. additionally, only mest- was able to inhibit s. schenckii yeast to mycelium differentiation. in contrast, no inhibition of yeast to mycelium differentiation was observed upon incubation of these fungal species with mest-. parallel experiments showed that after washing and replacing medium containing mabs by antibody-free medium; the fungi tested were able to restore their growth and/or transformation, indicating that mabs mest- - and - present a fungistatic effect. in another set of experiments, it was evaluated the effect of mab mest- and - on p. brasiliensis mycelium to yeast transformations, and as expected, it was not observed a significant inhibition, since these antibodies do not react or react weakly with mycelium forms. thus, μg/ml of mest- and mest- inhibited, respectively, 6% and 9% the transition from mycelium to yeast of p. brasiliensis. figure shows the differentiation of p. brasiliensis mycelia forms grown in presence or not of mest- for h at 37°c. in order to illustrate the differentiation inhibition, but not picturing the real inhibition percentage, figure 7b pictured a field with high concentration of hyphae form. discussion mab mest- specificity in this paper, we describe the characterization of mest- an igg2a monoclonal antibody directed to the structure from gipc pb- of p. brasiliensis. among different methyl-glycosides, disaccharides and glycosylinositols, only manpα1→3manp and manpα1→3manpα1→2ins inhibited mest- binding to pb- in solid-phase ria. furthermore, mest- was unable to recognize, by solid-phase ria or hptlc-immunostaining, the intact gipc ss-m, thus suggesting that α1→ linkage of the subterminal mannose unit to inositol represents a sterical hindrance for antigen recognition by mest- therefore, the minimum epitope required for optimum binding of mest- to pb- and similar gipcs, would comprise the two linear mannose residues in specific linkage and the myo-inositol residue as follows: manpα1→3manpα1→2ins. by indirect immunofluorescence, it was observed that mest- is reactive only with yeast forms of p. brasiliensis, h. capsulatum and s. schenckii, which is in agreement with previous data describing the crypticity of gipc pb- and glccer in mycelium forms of p. brasiliensis. accordingly, despite the detection of the gipc pb- extracted from hyphae of p. brasiliensis by hptlc and hptlc immunostaining with mab mest- it should be noted the complete lack of mest- reactivity by immunofluorescence with fixed mycelia forms. taking together, these data suggest that a particular organization of gsls and/or cell wall structure, distinct from that found in yeast forms, is required for mab mest- binding to fungal gipc pb- in this context, the access of antibodies directed to gsls of mycelium forms seems to be strongly affected by organizational or structural aspects that do not favor the interaction antigen-antibody. growth and dimorphism inhibition by anti-glycosphingolipid mabs there are several reports in the literature showing the importance of neutral glycosphingolipids, such as cerebrosides, on fungal growth and morphological transition. rodrigues et al. described that the addition of purified human antibodies, directed to glccer from cryptococcus neoformans, inhibited cell budding and growth of this fungus. therefore, the effects of three mabs, directed to different fungal gsls, were analyzed on colony formation of pathogenic dimorphic fungi. experiments using mab mest- directed to fungal glccer, showed no significant inhibition of cfu or effect in dimorphism of the fungi studied. these data do not corroborate the results from rodrigues et al.. possible explanations for these results may be related to the source of the antibodies, human and murine, in our case, or fungal species, since this effect was only observed in c. neoformans. our results using mab mest- directed to pb- and hc-y showed significant inhibition of fungal growth and differentiation of p. brasiliensis and h. capsulatum from yeast to mycelia. as expected, no inhibition with mest- was observed for s. schenckii, since this specie does not express galactofuranose-bearing gsls. on the other hand, mest- was able to inhibit cfu, fungal growth and differentiation of all three fungi studied. mest- was able to cause higher inhibition of cfu and differentiation for h. capsulatum and s. schenckii than for p. brasiliensis. this lower degree of inhibition showed by p. brasiliensis could be attributed to the low gipc pb- concentration in yeast forms of this fungus. on the other hand, gipcs hc-y and ss-y which bear the same structure as pb- represent about 30% and 20% of acidic glycolipid fraction from h. capsulatum and s. schenckii yeast forms respectively. conversely, results observed in the mycelium to yeast transformation, were not straightforward, a possible explanation could be related to the non-reactivity of mabs mest- - and - with mycelia forms, as observed by immunofluorescence assay. moreover, in h. capsulatum and s. schenckii, the transformation of mycelium to yeast takes at least three weeks in normal conditions, and the mycelium web hinders clear yeast observation and quantification. it is now well established that the precise build up of lipid rafts is necessary to efficiently guide signal transduction through cell membrane, some new evidences indicate that in fungi, these constructions are also necessary for fungal survival and maintenance of the infection. two attractive hypotheses for the inhibitory activity of mabs mest- and mest- on the yeast growth and differentiation would be: i) the association mab-gipcs sterically inhibits the formation of functional glycosphingolipid-enriched microdomains, thus blocking the action of key enzymes responsible for cell wall synthesis, and ii) an already existing microdomain to which a mab-gipc association takes place is not functional, hindering essential signal transduction to fungal growth and/or dimorphism. confocal microscopy, with p. brasiliensis cell wall stained with calcofluor white indicates two places of mest- binding, i) extensive and internal to calcofluor labeling, i.e. plasma membrane, and ii) discrete external as well as co-labeling with calcofluor, these preliminary data led us to suggest a new conceptual model of gsl arrangements in yeast forms where microdomain-like regions containing gipcs could also be located at the external surface of the cell wall. further work to substantiate this concept model is under investigation in our laboratory aiming an extensive comprehension of fungal glycosphingolipid enriched microdomains regarding their composition, surface localization, role in signaling processes and possible role in host cell binding and infection. this study and others have shown that specific gipcs are found in a large variety of pathogenic fungi. in some cases, those gipcs are recognized by sera from patients with paracoccidioidomycosis, histoplasmosis or aspergillosis, indicating that gipcs are immunogenic and able to induce the production of human antibodies during fungal infections. the broad distribution of gipcs in pathogenic fungi and the antifungal activity of monoclonal antibodies directed to gipcs indicate that these molecules may represent potential targets for the development of new therapeutical approaches based on induction of protective antibodies.  the fine specificity of mest- was assessed by inhibition assays using different methyl-glycosides, disaccharides and oligosaccharides. only manα1→3man and the glycoinositol manα1→3manα1→2ins, from pb- were able to inhibit, by about 95%, mest- binding to pb- antigen of p. brasiliensis. the epitope recognized by mest- was defined as manα1→3manα1→2ins; this structure was already described in a variety of pathogenic fungi. studies using mabs mest- and mest- as fungal growth inhibitors showed that anti-gipcs mabs presented a strong inhibitory activity on growth, differentiation and colony formation of p. brasiliensis, h. capsulatum, and s. schenckii. on the other hand, no statistically significant inhibition was observed with anti-glccer. these results strongly suggest that mabs directed to particular glycosphingolipids are able to interfere on fungal growth and differentiation. an attractive hypothesis is that, by hindering the formation of functional lipid rafts by association with mabs, a possible dependence of surface organization of glycosphingolipids in fungi is essential for different fungal processes, such as growth, morphological transition, and infectivity. 
it was also observed that fungal gipcs were able to inhibit t lymphocyte proliferation "in vitro", and studies regarding the importance of these molecules to fungal survival showed that many species of fungi are vulnerable to inhibitors of sphingolipid biosynthesis. in this paper, we describe a detailed characterization of an igg2a monoclonal antibody, termed mest- directed to the paracoccidioides brasiliensis glycolipid antigen pb-. studies performed on fungal cultures clearly showed the strong inhibitory activity of mest- on differentiation and colony formation of paracoccidioides brasiliensis, histoplasma capsulatum and sporothrix schenckii. on the other hand, mab mest- specifically directed to fungal glucosylceramide was able to promote only a weak inhibition on fungal differentiation and colony formation. these results strongly suggest that mabs directed to specific glycosphingolipids are able to interfere on fungal growth and differentiation. thus, studies on surface distribution of gipcs in yeast and mycelium forms of fungi may yield valuable information regarding the relevance of glycosphingolipids in processes of fungal growth, morphological transition and infectivity. similar inhibitory results were observed when these fungi where incubated with a different mab, which recognizes gipcs bearing terminal residues of β-d-galactofuranose linked to mannose.
 studies carried out during the 1990's demonstrated the presence of fungal glycoinositol phosphorylceramides with unique structures, some of them showed reactivity with sera of patients with histoplasmosis, paracoccidioidomycosis or aspergillosis. it was also observed that fungal gipcs were able to inhibit t lymphocyte proliferation "in vitro", and studies regarding the importance of these molecules to fungal survival showed that many species of fungi are vulnerable to inhibitors of sphingolipid biosynthesis.  in this paper, we describe a detailed characterization of an igg2a monoclonal antibody, termed mest- directed to the paracoccidioides brasiliensis glycolipid antigen pb-. mab mest- also recognizes gipcs bearing the same structure in other fungi. studies performed on fungal cultures clearly showed the strong inhibitory activity of mest- on differentiation and colony formation of paracoccidioides brasiliensis, histoplasma capsulatum and sporothrix schenckii. similar inhibitory results were observed when these fungi where incubated with a different mab, which recognizes gipcs bearing terminal residues of β-d-galactofuranose linked to mannose. on the other hand, mab mest- specifically directed to fungal glucosylceramide was able to promote only a weak inhibition on fungal differentiation and colony formation.  these results strongly suggest that mabs directed to specific glycosphingolipids are able to interfere on fungal growth and differentiation. thus, studies on surface distribution of gipcs in yeast and mycelium forms of fungi may yield valuable information regarding the relevance of glycosphingolipids in processes of fungal growth, morphological transition and infectivity. 