sporotrichosis is a chronic mycosis that affects skin and subcutaneous tissues , but it also can spread to other organs through the lymph vessels [ 14 ] .
it is acquired by traumatic implantation of the etiologic agent , the dimorphic fungus sporothrix schenckii .
the mycelial morphotype is found in soil , wood , and plants , while the yeast morphotype is found in host tissues [ 6 , 7 ] .
surface components of s. schenckii cell wall ( cw ) have a possible role in its pathogenicity .
accordingly , ergosterol peroxide helps the organism to evade the host 's immune response , promoting fungal infection .
also , a lipid antigen has been shown to decrease the production of proinflammatory cytokines such as tnf- , il-1 , and also il-12 and il-10 in experimental models of sporotrichosis [ 9 , 10 ] ; moreover , it is capable of inhibiting macrophage phagocytosis in in vitro assays .
one of the most studied cw components of s. schenckii is a peptide - polysaccharide known as peptide - rhamnomannan , a glycoconjugate molecular complex with a wide range of molecular weights composed of 33.5% rhamnose , 57% mannose , and 14.2% protein . in murine models of sporotrichosis , the peptide - rhamnomannan is involved in the anti - inflammatory response diminishing the production of il-1 and tnf- , and in in vitro lymphoproliferation assays have shown that it contains components with different mitogenic activities [ 1315 ] .
recently , a 70  kda glycoprotein ( gp70 ) isolated from the cw of the yeast morphotype of s. schenckii has become a relevant cell surface component .
this molecule has an isoelectric point ( ip ) of 4.1 and about 5.7% of its molecular weight ( mw ) corresponds to carbohydrate residues .
some important features of gp70 are its ability to adhere to extracellular matrix proteins   and to induce a specific humoral response in s. schenckii infected mice .
interestingly , administration of anti - gp70 monoclonal antibodies appears to have protective effect against fungal infection in mice .
in addition to the antigenic gp70 , s. schenckii strains also express a 60  kda immunodominant glycoprotein ( gp60 ) [ 19 , 20 ] , and both have been proposed as potential virulence factors since they are expressed by the most virulent strains of the sporothrix complex .
recent studies seem to indicate that gp70 and gp60 share the same peptide but differ in glycosylation pattern , ip , and mw . in order to shed light on the role of the gp60 during infection by s. schenckii
, the antigen was purified by isoelectric focusing and continuous elution electrophoresis , and its effect on the immune response in two mice strains with experimental cutaneous sporotrichosis was evaluated .
all procedures carried out in animals were approved by the animal ethics committee , facultad de medicina , universidad nacional autnoma de mxico , and followed the mexican official guide ( nom 062-zoo-1999 ) for the care and use of laboratory animals .
male c57bl/6 and balb / c mice , 810 weeks old , were purchased from harlan ( mexico city , mexico ) .
the animals were free of parasites or pathogens and were fed mouse chow ( purina de mxico , mxico ) and water ad libitum .
mice were housed in separate cages with wood shavings as nesting material , and five individuals were housed per cage .
they were maintained in a 12/12  h light / dark cycle in a room thermostatically maintained at 24  2c throughout the study .
groups of ten experimental animals by mice strain and each corresponding control group of five mice were conformed .
two 12-week - old male new zealand rabbits weighing 3.21 and 3.35  kg were used to obtain hyperimmune serum . 

conidia were obtained from the mycelial morphotype prepared from a 10-day - old culture grown at 28c in ypg medium [ 0.3%  ( w / v ) yeast extract , 1%  ( w / v ) peptone , and 2%  ( w / v ) glucose ] at ph 4.5 .
the yeast morphotype was obtained in ypg medium , ph 7.2 , inoculated with 5  10 conidia ml , and incubated for 10 days at 37c with shaking ( 120  rpm ) .
the harvested cells ( centrifugation at 7000  g for 10  min ) were washed twice with lysis buffer [ 50  mm tris - hcl , ph 7.5 , supplemented with 1  mm phenylmethylsulfonyl fluoride ( pmsf ) ] and maintained at 20c until used .
yeast cells were resuspended in lysis buffer and broken with glass beads ( 0.450.5  mm in diameter ) in an msk cell homogenizer ( braun melsungen , germany ) by alternate periods of breaking ( 40  s ) and cooling ( 60  s ) until all cells were broken . to isolate the cw ,
the cell homogenate was centrifuged at 1300  g for 15  min at 4c , and the pellet was washed thrice with lysis buffer to remove any intracellular component associated with the cw during the cell - breaking process .
cw proteins were extracted with hot 2%  ( w / v ) sodium dodecyl sulfate ( sds ) as described previously , precipitated with 70% ethanol for 2  h at 20c , and stored at 70c until use .
cw proteins were analyzed by 2d - page gels as described by ruiz - baca et al . .
briefly , the extracted proteins were cleaned with the readyprep 2d cleanup kit ( bio - rad ) following the manufacturer 's instructions .
samples of 160  g of protein were resuspended in hydration buffer [ 7  m urea , 2  m thiourea , 4%  ( w / v ) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ( chaps ) , 20  mm dithiothreitol ( dtt ) , 0.5%  ( w / v ) ampholytes , and 0.002%  ( w / v ) bromophenol blue ] and applied on immobilized ph 47 gradient strips ( 7  cm , bio - rad ) .
isoelectric focusing ( ief ) was performed in a protean ief system ( bio - rad ) using the following conditions : 250 volts ( v ) for 20  min , 4000  v for 2  h until 10000  v / h was reached .
after ief , the strips were incubated sequentially for 15  min in equilibrium buffer i [ 50  mm tris / hcl , ph 8.8 , 6  m urea , 30%  ( v / v ) glycerol , 2%  ( w / v ) sds , and 0.5% ( w / v ) dtt ] and equilibrium buffer ii [ 50  mm tris / hcl , ph 8.8 , 6  m urea , 30%  ( v / v ) glycerol , 2%  ( w / v ) sds , and 2%  ( w / v ) iodoacetamide ] under constant stirring . for the second dimension ,
the strips were mounted on 10% sds - page gels and run at 95  v for 2  h in a mini - protean 3 system ( bio - rad ) .
the gels were either stained by coomassie blue g-250 or transferred to nitrocellulose membranes . to this purpose , the same methodology reported by ruiz - baca et al .   was followed .
briefly , several samples of the gp60 antigen from 2d - page gels stained with coomassie blue were obtained .
the gel pieces were macerated with the help of a mortar and resuspended in sterile distilled water .
a dose was intramuscularly injected every 7 days for 4 weeks into male new zealand rabbits .
each dose contained approximately 12.5  g of gp60 suspended in a volume of 1  ml ( 50% distilled water and 50% adjuvant ) .
the first and three last doses contained complete and incomplete freund 's adjuvant ( sigma ) , respectively .
one week after the last dose , rabbits were sacrificed and bled to death , the serum was collected , and immunoglobulins were fractionated with 50% ammonium sulfate .
briefly , the membrane was blocked for 1  h with a skim milk solution ( 5% , w / v ) in phosphate - buffered saline ( pbs ) , ph 7.2 .
after washing thrice with pbs , membrane was incubated overnight with either anti - gp60 rabbit polyclonal antibody diluted 1  :  2000 or sera from nonimmunized or preimmunized mice with gp60 diluted 1  :  100 in pbs supplemented with 0.05% 
membrane was then washed thrice with the same buffer and incubated in anti - rabbit igg or anti - mouse igg , both goat horseradish peroxidase - conjugated secondary antibodies , diluted 1  :  1000 in pbs with gentle shaking for 2  h. after washing with pbs , enzyme activity was revealed with a solution containing 3 - 3-diaminobenzidine ( dab , 1  mg / ml ) and 0.01%  ( v / v ) h2o2 . as a positive control for the presence of anti - gp60 antibodies in mice ,
yeast cells were obtained from a 10-day - old culture , washed thrice with pbs , and centrifuged for 5  min at 7000  g. the resultant cell pellet was fixed for 30  min in 2% ( w / v ) paraformaldehyde diluted in pbs at 4c and washed four times with cold pbs .
fixed cells were incubated for 1  h at room temperature ( rt ) with the anti - gp60 polyclonal antibody diluted 1  :  100 in pbs solution containing 5% 
( w / v ) bovine serum albumin ( bsa ) . after washing thrice with pbs
, cells were incubated for 1  h at rt in the dark with a goat anti - rabbit antibody conjugated to fluorescein isothiocyanate ( fitc ) , diluted 1  :  50 in pbs solution containing 0.1%  ( w / v ) bsa .
finally , yeast cells were washed thrice with pbs and analyzed using the axio - observer z1 lsm 700 carl zeiss confocal microscope .
yeast cells processed as described above but incubated either with preimmune serum or with the secondary antibody conjugated to fitc only were used as control .
other cells fixed only in paraformaldehyde were used to assess the potential autofluorescence . for the purification of gp60 , cw proteins were extracted as described above .
approximately 6  mg of proteins was resuspended in 18  ml of hydration buffer at a concentration of 7%  ( v / v ) ampholytes with a ph gradient of 310 .
the suspension of proteins was loaded onto a rotofor preparative ief cell ( bio - rad ) and ran for 4  h at a constant power of 12 watts ( w ) using a powerpac universal power supply ( bio - rad ) .
subsequently , fractions enriched in gp60 were pooled and mixed with hydration buffer to reach 18  ml without further addition of ampholytes .
this sample was run on the same equipment for 2.5  h at a constant power of 12  w. fractions enriched in gp60 were precipitated with 70%  ( v / v ) ethanol at 20c for 24  h and centrifuged and the resulting pellets were resuspended in 2x buffer [ 0.125  m tris - hcl , ph 6.8 ; 4%  ( w / v ) sds , 20%  ( v / v ) glycerol , 200  mm -mercaptoethanol , and 0.002%  ( w / v ) bromophenol blue ] .
the samples were separated by continuous elution electrophoresis for 6  h at 1  w in a mini - prep cell ( bio - rad ) .
fractions enriched in gp60 were pooled and kept in elution buffer [ 0.3%  ( w / v ) tris - hcl , 1.4%  ( w / v ) glycine , and 1%  ( w / v ) sds ] .
the glycoprotein was monitored along the steps of purification by sds - page electrophoresis and western blot using anti - gp60 as primary antibody .
the gel pieces were washed and reduced with dtt and alkylated with iodoacetamide and in gel - digested with trypsin .
lc - ms / ms analysis was performed on an orbitrap fusion tribrid ( thermo scientific ) utilizing a nanospray ionization source .
an instrument method was used to collect full mass spectrum every three seconds and continuously fragment the most intense ions with 38% collision - induced dissociation ( cid ) and record the resulting ms / ms spectra .
raw tandem mass spectra were converted to mzxml files and then to peak list files ( pkl ) via the transproteomic pipeline ( seattle proteome center , seattle , wa ) .
pkl were searched using mascot ( matrix scientific , boston , ma ) against separate target and decoy databases containing dimorphic fungi proteins ( sporothrix , coccidioides , blastomyces , paracoccidioides , lacazia , and penicillium marneffei ) downloaded june 17 , 2014 , from the dimorphic fungal database from broad institute and national center for biotechnology information ( ncbi ) .
mascot settings were as follows : tryptic enzymatic cleavages allowing for up to 2 missed cleavages , peptide tolerance of 800  ppm , fragment ion tolerance of 0.8  da , fixed modification due to carboxyamidomethylation of cysteine ( + 57  da ) , and variable modifications of oxidation of methionine ( + 16  da ) and deamidation of asparagine or glutamine ( + 0.98  da ) .
statistically significant protein was determined at a 1% protein false discovery rate applied via proteoiq ( nusep , bogart , ga ) by loading mascot.dat target and decoy search files into the software program . to evaluate the effect of gp60
, balb / c and c57bl/6 mice strains were injected intramuscularly with 100  l ( 50% elution buffer and 50% adjuvant ) containing 2  g of gp60 every 7 days for three weeks ( gp60-preimmunized mice groups ) ; the first and subsequent doses were administered in complete and incomplete freund adjuvant , respectively .
animals of each strain received the same treatment protocol without adding gp60 ( nonimmunized mice groups ) .
one week after treatment completion , blood samples from each mouse were collected in 500  l tubes by facial vein phlebotomy to determine the anti - gp60 antibodies using the anti - gp60 polyclonal antibodies as a positive control .
thereafter , all mice were infected subcutaneously by injecting 100  l of pbs containing 5  10 conidia of s. schenckii .
mice were examined every three days during three weeks by evaluating the thickness of dorsoplantar inflammation with a mitutoyo micrometer and the presence of skin ulceration or scarring in foot dorsum . additionally ,
five animals of each strain neither infected nor treated with gp60 were used as control groups .
19 days after infection , the mice were bled to determine anti - gp60 antibodies and some cytokines of th1 and th2 responses .
the cytokines il-1 , tnf- , il-12p40 , il-12p70 , mip-2 , and ifn- from the th1 response and il-6 , il-4 , and il-10 from th2 response were determined using the luminex xmap milliplex analyst platform technology ( millipore kit ) according to the manufacturer 's instructions .
briefly , the serum samples were diluted in the assay buffer contained in the kit at 1  :  1  ( v / v ) ratio .
afterwards , a 96-well elisa plate was washed with washing buffer and 25  l of each standard , control , and sample was added to the respective wells followed by 25  l of assay buffer and 25  l of magnetic beads .
the plate was stirred at rt and after 2  h , it was washed twice with washing buffer and 25  l of detection antibody was added to each well . following this , the plate was stirred at rt and after 1  h , 25  l of streptavidin and phycoerythrin was added and incubated for 30  min at rt . finally , after two additional washes the plate was read on a magpix reader after adding 150  l of driving fluid to each well .
data from cytokine levels are expressed as mean  sd and analyzed by anova followed by tukey test .
anti - gp60 polyclonal antibodies were produced to monitor the reactivity of the yeast cell to this antigen and to track gp60 during the purification process .  
figure 1 shows the separation of the cw proteins in 2d - page gels , where at least six isoforms of the glycoprotein are seen in a ph range between 4.5 and 5.5 ( figure 1(a ) ) .
these isoforms were cut from different 2d - page gels and used to inoculate a rabbit intramuscularly .
one week after the last immunization dose , the rabbit was bled to obtain hyperimmune serum which was analyzed by western blot ( figure 1(b ) ) .
immunofluorescence and confocal microscopy confirmed the expression of gp60 in the yeast morphotype of the fungus .
this morphotype was observed with phase contrast microscopy ( figures 2(a ) and 2(c ) ) .
images indicate that gp60 is located on the cell surface of yeast cells ( figure 2(b ) ) .
controls incubated with preimmune sera and paraformaldehyde - associated autofluorescence were negative ( figure 2(d ) ) . in order to obtain sufficient amounts of purified gp60 to determine its effect on the immune response , we standardized a purification protocol based on liquid phase ief and continuous elution electrophoresis
. the protein was monitored through the various purification steps by western blot assays using the anti - gp60 antibodies .
results from liquid phase ief showed that most of the pollutant proteins were in fractions 3 and 4 , and despite the fact that gp60 was detected in every fraction , results showed a higher antigen concentration in fractions corresponding to lanes 5 through 11 ( figures 3(a ) and 3(b ) ) .
these fractions were pooled and again subjected to ief , which showed a higher concentration of gp60 in fractions corresponding to lanes 11 through 14 ( figures 3(c ) and 3(d ) ) .
subsequently , enriched fractions obtained after the second ief were pooled and the pool was purified by continuous elution electrophoresis .
this step yielded 5 fractions containing gp60 with a high degree of purity ( figures 3(e ) and 3(f ) , lanes 2 , 3 , 4 , 5 , and 6 ) with the different isoforms ranging from 55 to 65  kda .
the spots of the different gp60 isoforms separated in 2d - page gels stained with coomassie blue were cut from the gel and exposed to tryptic digestion .
the resulting peptides were analyzed by mass spectrometry ( lc - ms / ms ) and gp60 was identified as a carboxy - cis , cis - muconate cyclase of s. schenckii ( table 1 ) .
only gp60-preimmunized c57bl/6 mice showed the presence of anti - gp60 antibodies prior to infection , although detection was very low as can be seen in figure 4 ( lanes 3 and 4 ) .
nonimmunized groups of both strains did not produce anti - gp60 antibodies ( figure 4 , lanes 2 and 5 ) .
however , 21 days after infection , gp60-preimmunized mice of both strains , as well as those that were infected only ( nonimmunized mice groups ) , showed anti - gp60 antibodies .
a higher detection was shown by the groups that received gp60 before infection ( figure 5 , lanes 4 , 5 , 6 , 10 , 11 , and 12 ) compared to the nonimmunized groups ( figure 5 , lanes 1 , 2 , 3 , 7 , 8 , and 9 ) , particularly the c57bl/6 strain ( figure 5 , lanes 4 , 5 , and 6 ) .
nonimmunized mice of both strains seemed to heal faster than those preimmunized with gp60 prior to infection ( figure 6 ) ; however , nonimmunized c57bl/6 mice ( figure 6(a ) ) did not show significant differences in foot dorsum ulceration ( figure 7(a ) ) as compared to those of the same strain that received gp60 prior to infection ( figure 6(b ) ) , contrary to nonimmunized balb / c mice ( figure 6(c ) ) which showed significant differences ( figure 7(b ) ) compared to gp60-preimmunized mice of the same strain ( figure 6(d ) ) . a lower susceptibility to s. schenckii
was noted in c57bl/6 mice , since nonimmunized c57bl/6 mice showed lower ulceration than nonimmunized balb / c mice ( figure 7(c ) ) . also , a different effect induced by gp60 was noted between the strains since gp60-preimmunized c57bl/6 mice showed a significant lower ulceration than gp60-preimmunized balb / c mice ( figure 7(d ) ) .
no statistical differences in inflammation were detected between nonimmunized and gp60-preimmunized groups ( data not shown ) .
the cytokine profiles expressed by c57bl/6 and balb / c mice strains were different in the baseline levels and during infection with s. schenckii .
control c57bl/6 mice showed lower levels of cytokines such as tnf- , il-1 , and il-12 ( p70 ) compared with control group of balb / c ( figure 8) , which suggests a predisposition towards the inflammatory or th1 response in balb / c mice .
the nonimmunized c57bl/6 group showed significantly higher levels of tnf- and il-1 as compared to the control group .
likewise , the level of mip-2 was 5-fold higher in nonimmunized mice compared to the control group ( figure 8(a ) ) .
in the case of nonimmunized balb / c mice , the levels of tnf- and il-1 increased 45- and 8-fold over the control value , respectively ( figure 8(b ) ) , that is , much more than in nonimmunized c57bl/6 group . on the contrary ,
the levels of mip-2 increased about 2-fold over the baseline levels ( figure 8(b ) ) , which is about half the increase observed in nonimmunized c57bl/6 mice .
in nonimmunized c57bl/6 mice , the il-12 ( p70)/il-12 ( p40 ) ratio decreased as compared to the control group ( figure 9(a ) ) , contrary to the levels of ifn- , which increased significantly ( figure 8(c ) ) . in contrast to c57bl/6 , nonimmunized balb / c mice showed an increase of almost twice in the il-12 ( p70)/il-12 ( p40 ) ratio as compared to the control group ( figure 9(b ) ) , and the same occurred with ifn- ( figure 8(d ) ) .
regarding the cytokines of the th2 response , nonimmunized c57bl/6 mice showed a significant decrease in the levels of il-10 and il-4 with respect to the control group , but the levels of il-6 increased almost 5-fold ( figure 8(e ) ) .
in nonimmunized balb / c group , the levels of il-10 and il-6 increased almost twice , whereas the level of il-4 increased more than twice over the control group ( figure 8(f ) ) ; however , although the levels of the th2 response increased in nonimmunized balb / c mice , the increase was not as high as that of the cytokines characteristic of th1 . in the gp60-preimmunized c57bl/6 group ,
the levels of tnf- and il-1 decreased significantly when compared to the nonimmunized group ; accordingly , the amount of tnf- reached baseline levels in the control group , while that of il-1 was below the control value ( figure 8(a ) ) .
on the other hand , mip-2 remained in the same level as the nonimmunized group ; significant differences were observed only after comparing with the control group ( figure 8(a ) ) .
the levels of tnf- dropped more than 100-fold with respect to the nonimmunized group , falling even below the control levels , yet no significant differences were observed when compared with the control group .
the level of il-1 decreased almost 12-fold as compared to nonimmunized mice whereas the level of mip-2 was unaffected as compared to the nonimmunized group , remaining above the level of the control group ( figure 8(b ) ) .
it is noteworthy that in both strains the levels of mip-2 did not change in any of the gp60-preimmunized groups compared to the nonimmunized groups . in gp60-preimmunized c57bl/6 mice , the il-12 ( p70)/il-12 ( p40 ) ratio decreased compared to the nonimmunized and control groups ( figure 9(a ) ) .
ifn- also decreased significantly ( to control levels ) compared to nonimmunized group ( figure 8(c ) ) . in gp60-preimmunized balb / c mice , the il-12 ( p70)/il-12 ( p40 ) ratio decreased 4-fold and 2-fold compared to the nonimmunized and control groups , respectively ( figure 9(b ) ) , and the level of ifn- also decreased , though not significantly compared to nonimmunized group ( figure 8(d ) ) .
although a slight decrease in ifn- was observed in gp60-preimmunized mice , gp60 appears to have a greater effect on the il-12 ( p70)/il-12 ( p40 ) ratio , decreasing the heterodimeric form and increasing the homodimeric one .
furthermore , the levels of il-10 decreased significantly in gp60-preimmunized c57bl/6 mice compared to nonimmunized group , falling even below that control level .
the level of il-4 also decreased significantly compared to nonimmunized and control groups ( figure 8(e ) ) .
it is worth noting that although the levels of both th1 and th2 cytokines decreased in gp60-preimmunized c57bl/6 mice , they were not very affected in terms of ulceration ( figures 6(b ) and 7(a ) ) being as damaged as nonimmunized c57bl/6 mice ( figure 6(a ) ) . in gp60-preimmunized balb / c mice ,
the level of il-10 decreased over 17-fold as compared to nonimmunized group , reaching levels below baseline values .
the levels of il-6 and il-4 also decreased to the control level ( il-6 ) and below the control level ( il-4 ) ( figure 8(f ) ) .
in the present work , gp60 from cw of s. schenckii was purified by isoelectric focusing and continuous elution electrophoresis , and its effects over the immune response in an experimental subacute sporotrichosis model in two mice strains were evaluated . to isolate this glycoprotein as a single antigenic peptide , a purification protocol which takes advantage of
the purified gp60 was sequenced by tandem mass spectrometry , yielding a sequence homologous to secreted gp70 , a carboxy - cis , cis - muconate cyclase involved in the -ketoadipate pathway required for the catabolism of aromatic compounds .
however , unlike gp60 , which was sequenced from the form present in the cell surface , the sequence of gp70 was obtained from the secreted , extracellular protein , which might confer differences in the immune mechanisms induced by each antigen , as it has been demonstrated with the bad-1 protein of blastomyces dermatitidis ; accordingly , the cw form induces regulatory mechanisms of the immune response through the production of tgf- , whereas the secreted counterpart independently regulates from this cytokine .
however , several cw enzymes such as the -1,3-glucosyltransferase of coccidioides posadasii   and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) from paracoccidioides brasiliensis , altogether with other enzymes of pathways such as glycolysis and the krebs and glyoxylate cycles , are classified as moonlight or multifunctional proteins due to their participation in pathogenic processes independently of their metabolic activities .
confocal microscopy , using a rabbit polyclonal antibody against gp60 ( figure 1(b ) ) , demonstrated that this protein is distributed along the cell surface of the yeast morphotype ( figure 2(b ) ) .
location of gp60 implicates that it could be involved in different potential roles as protective , immunosuppressive , immunostimulatory , or adhesive proteins .
the 60  kda heat shock protein ( hsp60 ) of histoplasma capsulatum has been associated with protection against stress conditions , since it is expressed when the yeast is subjected to thermal stress , acting as chaperone of proteins with essential functions .
the 120  kda protein present on the surface of b. dermatitidis has been proposed as a modulator of the immune response , since it is capable of inhibiting the inflammatory response through the production of tgf- , contrary to the glycoprotein of the outer wall of the spherules of c. immitis and c. posadasii , which acts as an immunodominant antigen capable of inducing cellular and humoral responses during parasitic infections [ 30 , 31 ] .
p. brasiliensis gapdh and a 30  kda protein act as adhesins and have an important role in the interaction between the fungus and the host cells [ 26 , 32 ] . in the same line ,
gp70 of s. schenckii has been reported to have a role as an adhesin associated with the virulence of s. schenckii strains [ 16 , 33 ] .
however , an increased expression of this glycoprotein has been associated with less virulent strains of s. brasiliensis .
therefore , the presence of gp60 on the cell surface and the homology with gp70 suggest a role in immune and adhesion mechanisms . in the present work ,
their analysis in nonimmunized , gp60-preimmunized , and control mice groups demonstrated that levels of th1 and th2 response cytokines in c57bl/6 mice remained low compared to those of balb / c mice , except for mip-2 which was slightly higher in c57bl/6 mice .
the importance of mip-2 lies in its ability to attract neutrophils to the infected areas in the early stages of the immune response , regardless of the type of pathogen .
moreover , it is known that the levels of mip-2 change as a proportional function of il-6 , with a direct relationship between them .
the concentration of il-6 noticeably increased in nonimmunized c57bl/6 mice compared to the controls . besides being associated with the th1 response
, il-6 promotes the growth and differentiation of b lymphocytes stimulating the humoral response , which suggests a slight tendency of the c57bl/6 mice to the th2 response , which probably explains the stronger antibody response in this strain .
il-6 is also involved in the activation of the th17 response , characterized by neutrophil recruitment and inflammation ; thus , it is not entirely clear what type of immune response in c57bl/6 mice is relevant to confront infection by s. schenckii . on the other hand , nonimmunized balb / c mice produced higher amounts of proinflammatory cytokines or th1 response , as indicated by the levels of tnf- , il-1 , and il-12p70 .
in fact , increased levels of tnf- have been associated with the severity of sporotrichosis skin lesions in balb / c mice .
moreover , more severe skin lesions in balb / c than in c57bl/6 mice correlate with higher il-4 levels observed in balb / c mice , according to models of p. brasiliensis infection in which elevated levels of il-4 are related to a worsening of the infection .
this is probably due to a decreased fungicide capacity of lung phagocytes from normal mice compared with that of phagocytes from il-4-deficient mice .
the low levels of il-10 observed in nonimmunized c57bl/6 mice compared to nonimmunized balb / c may explain why lower tissue damage was noted in c57bl/6 mice , as has been reported in infections caused by p. brasiliensis where il-10 deficiency leads to increased immunity and regressive infection without enhancing tissue pathology . in this sense , it is worth noting that the amount of specific antibodies against gp60 was higher in the c57bl/6 strain than in the balb / c strain .
accordingly , sera from patients with sporotrichosis contain antibodies recognizing s. schenckii antigens in a range of 4070  kda .
furthermore , a seroprevalence of the 6070  kda antigen exists , and patients with this mycosis contain serum antibodies against such a protein .
this immunodominant 6070  kda antigen also has been demonstrated in feline sporotrichosis , suggesting its potential as a marker for diagnosis or as a candidate for the development of therapeutic vaccines .
humoral response in mice infected with species from the s. schenckii complex shows an antibody production pattern similar to human and cat patterns , where 6070  kda recognition becomes a constant .
this suggests a convergent humoral response between the three mammals species hosts that may increase our understanding of the coevolution of these hosts with s. schenckii species .
whereas the th2 response is associated with fungal susceptibility , it is known that antibodies can affect the balance of cytokines and the induction of regulatory t cells that help to reduce tissue damage caused by exacerbated inflammatory responses .
this fact should be considered in the interpretation of mice immune response , along with the route of infection , the concentration and virulence of the inoculum , the mouse strain used , and its genetic background .
the mannose receptor is the one that recognizes the conidial morphotype , so a different expression of this receptor in each mouse strain may determine a difference in immune response , even though this receptor can activate both inflammatory and regulatory pathways .
furthermore , the magnitude of the response may also vary according to the morphotype , as yeasts coincubated with mast cells have been unable to induce degranulation in in vitro assays , contrary to what occurs in conidia , which supports the increased immunological activity of the conidial morphotype .
results of the present study emphasize the lack of a detailed knowledge of the mechanisms of pathogenicity of the different species leading to the development of sporotrichosis .
preimmunization of c57bl/6 and balb / c strains with gp60 resulted in a downregulation effect on both strains of mice whose cytokine levels were lower as compared to the nonimmunized , particularly in the case of balb / c .
these results could explain why monoclonal antibodies directed against other s. schenckii antigens such as gp70 have an immunoprotective effect in murine models .
noteworthy , antibodies against gp70 are not the only ones which have a protective role against s. schenckii infection , since passive transference of sera from mice containing antibodies against a 44  kda hydrolase and a 47  kda enolase has shown a protective role during murine sporotrichosis .
it is worth noting that gp60-preimmunized c57bl/6 mice seemed to be able to resolve the infection as nonimmunized mice .
it has been demonstrated that dendritic cells from c57bl/6 mice incubated with s. schenckii promote combinations of th1 and th17 responses .
a recent in vitro assay suggests that th1/th17 combined responses against s. schenckii depend on il-23 .
notably , il-17 is involved in neutrophil recruitment and in the production of mip-2 ; a cytokine markedly increased in nonimmunized and gp60-preimmunized c57bl/6 mice .
thus , gp60 does not seem to have an effect on the th17 response . indirectly
, we may infer that c57bl/6 mice tend to assemble th17 response after infection with s. schenckii .
dendritic cells increase il-23 production after ligation of -glucans by the dectin-1 receptor regulating fungal pathogenicity via th-17 responses .
furthermore , vaccination with attenuated yeast cells of primary pathogenic fungi such as b. dermatitidis , c. posadasii , and h. capsulatum induces protective th17 responses against a lethal infection .
galectin-3 receptor - deficient mice express higher levels of th17 response cytokines as tgf-1 , il-23 , il-17 , and il-6 , in a model of h. capsulatum infection .
thus , it could be hypothesized that c57bl/6 immunity tendency towards th17 response , as it was observed in our study , might be related to recognition of s. schenckii conidia through the differential expression between c57bl/6 and balb / c mice of the galectin-3 receptor and its ligand .
previous reports indicate that balb / c mice infected with s. schenckii express a th1 response mediated by ligation of tlr-2 and tlr-4 receptors [ 9 , 10 , 53 , 54 ] .
probably , preimmunization with gp60 blocks one of these receptors and prevents activation signaling pathways that involve nf - kb transcription factor .
additionally , gp60 may have interfered with caspase-1-dependent signaling pathways activation , which has proven to be important for the production of th1 cytokines during experimental sporotrichosis [ 55 , 56 ] .
resistance to paracoccidioidomycosis in mice is related to paracoccin tlrs activation that triggers a balanced th1 immunity .
furthermore , during s. schenckii antigens recognition , the lack of tlr-2 and tlr-4 receptors , combined with the presence of these surface antigens , stimulates tgf--mediated regulatory responses with inhibition of th1 response [ 53 , 54 ] . decreased levels of th1 cytokines observed in gp60-preimmunized mice could be related to these mechanisms whose balance determines the efficiency of th1 immune response mediated by cytokines .
the il-12-p70/il-12-p40 ratio decreased in gp60-preimmunized mice , mainly in the balb / c strain , indicating the presence of a downregulation effect compared with nonimmunized mice since il-12 p70 is considered the bioactive one .
this cytokine is produced only after dendritic cells are primed with microbial stimuli that upregulate il-12 p40 , which stimulates cd40 expression .
the relevance of il-12 as a protective cytokine has been demonstrated in gerbils infected with s. schenckii   but also as linker between innate and adaptive responses mediated by dendritic cells after s. schenckii phagocytosis .
thus , gp60 might block one of the steps that lead the production of the bioactive form of this cytokine , avoiding the development of a full th1 response .
finally , it seems that gp60 has a greater downregulation effect on the th1 response , since c57bl/6 , a strain that probably depends on a different response like th17 to fight this fungus , was not as affected as balb / c strain , which appears to be dependent on th1 response in order to clear the infection with s. schenckii .
 cell wall ( cw ) components of fungus sporothrix schenckii are the major inductors antigens of immune responses . 
 the immunodominant 60  kda glycoprotein ( gp60 ) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown . in this work 
 , the immunological effects of cw - purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized c57bl/6 and balb / c mice strains which were then infected with s. schenckii conidia . 
 results showed that both mice strains use different cytokine profiles in order to fight s. schenckii infection ; c57bl/6 mice seem to use a th17 response while balb / c mice tend to depend on a th1 profile . 
 preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains . 
 there were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains . 
 however , skin lesions due to the infection in gp60-preimmunized mice were more severe in balb / c than in c57bl/6 mice , suggesting that the antigen exerts a higher downregulatory effect on the th1 response . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion