the pathogenesis of infections due to c albicans and 
 related yeast is a complicated process that may involve a 
 constellation of virulence attributes including fungal 
 morphogenesis , secreted enzymes , biofilm formations , toxin 
 synthesis , and tissue adhesion . in addition , the ability 
 of mucosal pathogens to stably associate on epithelial surfaces 
 has been shown to be important for numerous microbial species that 
 infect mucosal sites . with respect to c albicans , even 
 adherence to epithelia may depend on a variety of cellular ligands 
 [ 26 ] . one feature of fungal surfaces related to 
 tissue binding is cell surface hydrophobicity   which we 
 previously explored through attachment of styrene microspheres to 
 yeast . our earlier studies exploited flow cytometry as a 
 means of evaluating the surface of a large number of yeast cells , 
 predicting on the basis of styrene bead binding , their 
 relative propensity for tissue adherence . these studies , however , 
 were limited in that actual tissue adherence was not measured by 
 the flow cytometry technique . we also found that several marketed 
 intravaginal products could alter the apparent surface 
 hydrophobicity of yeast   suggesting that they may be 
 useful as inhibitors of yeast adherence to epithelial cells . a 
 clear limitation of this in vitro was its lack of direct 
 measurement of yeast - epithelial interaction . the ability of 
 commercially available products to inhibit binding of styrene 
 beads to yeast would have more importance if binding to epithelial 
 cells could be demonstrated . the availability of consistent samples of human vaginal tissue for 
 use in fungal binding studies represents a serious limitation . the 
 use of buccal cells , though readily available , may not be a valid 
 model for vaginal yeast adherence . in addition , the use of 
 exfoliated vaginal or buccal cells with their extant microbial 
 flora would confound assays . recently , a continuous cell line of 
 vaginal epithelial cells ( vec )   has become available and 
 we elected to use these cultured vec to examine yeast cell binding 
 and explore the possibility that exposure of the vec to a variety 
 of compounds might mitigate yeast binding . previous studies involving yeast attachment to styrene particles 
  showed that several commercially available products could 
 interfere with this process . the c albicans strain used in this research was part of 
 our culture collection and was originally obtained as a clinical 
 isolate from vaginal samples . yeast strains in our collection are 
 maintained both as frozen stocks for long - term storage and by 
 propagation on sabouraud 's dextrose agar with the intermediate 
 storage at 4c and subculturing at 3-month intervals . we selected one strain , strain 397 , for use in these studies . 
 prior to use in adherence studies , the test organism was plated on 
 fresh sabouraud 's dextrose agar at 25c for 48 hours . previous studies indicated that these growth conditions produced 
 yeast cells that strongly bound styrene beads 
 [ 8 , 9 ] . using robinson et al 's work   as a guide , 
 fluorescein isothiocyanate ( fitc ) labeling of yeast cells was 
 carried out according to the protocol of cantinieaux et al 
  with modifications . briefly , yeast were harvested from 
 growth media and suspended in carbonate buffer ( 0.5 m 
 sodium carbonate / sodium bicarbonate , ph 9.5 ) and cell 
 concentration adjusted to an optical density of 0.74 au at 
 620 nm . fluorescein isothiocyanate ( sigma - aldrich , st 
 louis , mo ) was added to make the final concentration of 
 10 g / ml and was placed in the dark for 30 minutes at 
 room temperature . labeled yeast were harvested and suspended in 
 phosphate buffered saline and added to vaginal cells to assess 
 binding as described below . vaginal epithelial cells ( vec ) that were used in our study have 
 been described elsewhere . propagation employed 
 keratinocyte serum - free medium ( ksfm ) obtained from 
 gibco - invitrogen ( grand island , ny ) and supplemented with 
 manufacturer - supplied epidermal growth factor and bovine pituitary 
 extract . growth media also included penicillin ( 1000 u / ml ) , 
 streptomycin ( 1 g / ml ) , and fungizone 
 ( 0.0125 g / ml ) . vec were grown in 50 ml culture 
 flasks ( 25 cm surface area ) in 5% co2 
 and 100% humidity , and refed 3 times weekly until they 
 approached confluence . they were harvested by trypsinization for 
 5 minutes at room temperature followed by neutralization of the 
 trypsin by addition of dulbecco 's minimal essential median 
 containing 10% fetal bovine serum with gentamicin 
 ( 25 g / ml ) and vancomycin ( 250 g / ml ) . harvested cells were either diluted and placed in flasks for 
 continued growth or used in binding studies . vec used in this 
 study consistently showed greater than 95% viability by 
 trypan blue staining . estradiol 
 17 ( 1 , 3 , 5 , -estratriene-3 , 
 17-diol ) 
 was obtained from sigma - aldrich and dissolved in methanol as a 
 1 10 m stock solution and kept at 20c 
 until use . culture medium supplemented with estradiol contained 
 5 l estradiol stock in 5 ml of complete ksfm 
 ( 1 10 m final estradiol 
 concentration ) . the forward scatter channel was set 
 for logarithmic amplification at a range of 10 and the 
 side - scatter channel and fl-1h / channel employed logarithmic 
 amplification . because the assay used an excess of fluorescinated 
 yeast cells ( multiplicity of infection = 10 ) , the unbound yeast 
 cells were gated out and vec events were acquired with the gate 
 turned on , thereby eliminating most of the unbound yeast from 
 analyses . for most analyses , at least 5000 events , presumed to 
 be vec , were counted . inhibitors of yeast binding included several products marketed 
 under the brand name of k - y ( k - y jelly , 
 k - y liquid . k - y ultra , and k - y warming ) and were 
 donated by manufacturer . k - y jelly was diluted 1 : 10 weight 
 per volume prior to use in these studies because of its viscosity . data acquired from flow cytometry analysis included a 
 percentage of events that showed fluorescence greater than that 
 due   to vec autofluorescence ( percentage of m1 ) and mean fl1h 
 fluorescence . vec were cultured in ksfm medium with supplements as described in 
 the  methods . these cells grew rapidly and when growth 
 approached confluence provided about 1.2  10/50 ml 
 culture flask . cultured vec resembled basal cells as seen in 
 figure 1 and generally did not display fibroblast 
 morphology . because relatively few laboratories have worked with these cells , 
 we evaluated the behavior of these cells under a variety of 
 conditions that might relate to in vivo conditions . vec growth 
 with the addition of estradiol or with the addition of 10% 
 normal human serum was evaluated . estradiol promoted increased 
 cell proliferation , particularly during the first 24 hours after 
 passage of the cell line . cell counts in the presence of estrogen 
 were about 25% to 33% greater than in the absence of 
 estrogen at 24 hours but at 48 hours cell counts without 
 estradiol were 1.5 times greater than in cultures with 
 estradiol . growth of vec with 10% pooled normal human 
 serum did not enhance growth ; rather , vec adhered to culture 
 flasks poorly , cell counts were very low and detached cells 
 aggregated in the medium . as a result of this finding , subsequent 
 experiments employed vec cultivated without serum and without 
 added estradiol . in establishing the parameters for yeast - binding experiments , we 
  determined if vec , once 
 harvested , could be held overnight to allow additional experiments 
 to be performed on otherwise unused cells . we employed propidium 
 iodide uptake as a measure of cell integrity after harvest and 
 obtained the results shown in figure 2 . as revealed by 
 this study , the proportion of vec that took up propidium iodide 
 increased daily indicating that under conditions of storage , cell 
 integrity was decreased . from this study , we determined that we 
 would only use freshly harvested cells for this research . because the ultimate goal of this work was to quantitate yeast 
 binding to vec using flow cytometry , we established parameters for 
 cytometric analysis experimentally . as shown in 
 figure 3 , vec had higher forward and side scatter 
 signals than fluorescinated yeast . yeast cells alone fell into the 
 lower left quadrant of figure 3 , whereas the vec 
 occupied the other 3 quadrants . superimposing vec and yeast 
 plots indicated little overlap between these populations and also 
 suggested that we could create a gate that would exclude virtually 
 all unbound yeast cells . fluorescence from cells in the upper and 
 right quadrants would be attributed to vec with fluorescinated 
 yeast ( figure 3 ) . having established the flow cytometry parameters to be used in 
 this study , we next determined the ability of fluorescinated yeast 
 to bind to vec and to contribute to the fluorescence of the vec . as shown in figure 4 , vec alone showed a peak of 
 autofluorescence on the fl1h histogram . however , when 
 combined with labeled yeast , two fluorescent peaks were 
 identified , the second peak reflected the fluorescence of the 
 yeast attached to the vec . based on this pattern of fluorescence , 
 90% of vec appeared to bind yeast . because we were interested in the binding of yeast to vec 
 cultivated under conditions more reflective of in vivo conditions , 
 we examined overgrown cell cultures to determine if they showed 
 different binding characteristics compared to nonconfluent 
 cultures . in vivo , vaginal epithelial cells mature from the 
 metabolically active basal layer to become superficial , pyknotic 
 squames . this process is believed to be related to the superficial 
 layer of the vagina having migrated away from the underlying 
 basement membrane with attendant nutrient depletion and exposure 
 to environmental stress . we postulated that allowing cell cultures 
 to grow beyond the point at which we normally harvest them may 
 produce cells somewhat more like those that exist at the vaginal 
 lumen . consequently , we determined if growing cells to densities 
 greater than normal would alter binding properties . vec were 
 allowed to grow two days beyond the time at which we normally 
 harvested them . as shown by data in figure 5 , allowing vec to become overgrown actually increased the fluorescence due to 
 yeast binding nearly threefold . as indicated in the same figure , 
 addition of estradiol , which caused an initial increase in cell 
 proliferation , also seemed to support increased yeast binding . because our prior studies with styrene microspheres indicated that 
 several k - y products decreased the ability of yeast to bind the 
 hydrophobic beads , we determined if these same 
 intravaginal products affected binding of yeast to vec . as 
 illustrated in figure 6 , each of the products tested 
 altered the binding of yeast as indicated by diminished 
 fluorescence compared to controls . while yeast adherence was 
 decreased under these conditions , it was clearly not eliminated 
 entirely . investigation of fungal interactions with epithelium has been 
 methodologically challenging , with various methods being pursued 
 by different authors . one model system employs microscopic 
 evaluation of exfoliated vaginal cells [ 13 , 14 ] incubated 
 with microorganisms . buccal cells have been used as a surrogate 
 for vaginal cells although these may not be entirely comparable 
 , though they do have the advantage of being relatively 
 more available . variations on these techniques have included 
 examination of exfoliated cells from various sites   in a 
 range of women ( eg , pregnant , nonpregnant , diabetic ) as reported 
 by segal et al   or adherence of organisms to cells 
 derived from variant epithelial tissues ( normal versus cancerous ) 
 . to gain more consistency in observations , cultured hela cells , originally derived from cervical 
 cancer , have been employed in controlled adherence studies and can 
 be exposed to yeast cultivated under various conditions . the disadvantage of using hela or other similar cells is the 
 possibility of the cells changing over many thousands of passages 
 of culture . moreover , the fact that hela cells were explanted from 
 a neoplasm suggests another intrinsic difference from normal 
 vaginal epithelium . while the use of explanted or exfoliated cells 
 and tissues directly harvested from humans has at least the 
 appearance of being more physiologic , it also has the disadvantage 
 of being more difficult to employ in a controlled experiment 
 because of differences between donor cells . the present study 
 exploited the continuous culture of vec line that has not been 
 widely available previously . our laboratory has been engaged in developing flow cytometry 
 methods for characterizing microorganisms and their interactions 
 with surfaces [ 8 , 9 ] , because the techniques are facile and 
 generate significant amounts of data ( typical flow cytometry 
 readings evaluate 5,00010,000 cells for each sample tested ) . having 
 previously examined exfoliated buccal cells ( data not shown ) which 
 had a high level of endogenous fluorescence , we were uncertain if 
 vec would likewise show unacceptably high levels of fluorescence . 
 as shown in this report , we were able to appropriately adjust the 
 cytometer parameters such that unbound fluorescinated yeast cells 
 could be differentiated from unstained vaginal epithelial cells on 
 the basis of particle size and fluorescence and vaginal epithelial 
 cells fluorescently stained by the yeast attached to them . our method still suffered from the limitation that the cultured 
 vaginal epithelial cells were more like basal cells than the 
 squamous cells of the vaginal lumen . it is generally believed that 
 the maturation of vec from basal cells to luminal squames is the 
 result of the migration of these cells away from the basement 
 membrane with accompanying privation of nutrients and crowding 
 during the proliferative process . we attempted to introduce stress 
 into our cell cultures that might mimic epithelial proliferation 
 by allowing them to overgrow and by harvesting them later than we 
 normally would . we found that these presumably stressed cells 
 bound more fluorescinated yeast than did cells harvested on the 
 normal schedule . viability testing with trypan blue indicated that 
 the cells from overgrown cultures were still viable . this 
 experiment is admittedly an imperfect representation of the cell 
 maturation process that occurs in vivo and should probably be 
 followed up by the development of raft cultures . notably , introducing estrogen into this experiment 
 further increased yeast binding to vec and suggests an additional 
 variable that may be investigated in raft cultures . a practical result of the work identified several marketed 
 intravaginal products which were able to interfere with the 
 attachment of yeast to vec under the experimental conditions of 
 this study . while this knowledge is not presumed to provide a new 
 method of treating or preventing fungal vaginitis , it does suggest 
 that components of these marketed products may be useful as 
 vehicles for delivering topical antimycotics and could offer a 
 therapeutic advantage in such a context . previous research from 
 our laboratory indicated that bioadhesive vehicles containing an 
 active antifungal drug may influence the efficacy of the test drug 
  and suggests that the matrix for delivering topical 
 antifungals deserves more attention in order to improve currently 
 available products . vaginal epithelial cells 
 cultured at ksfm in polystyrene culture flasks . photograph taken 
 at 400 with phase contract microscopy . effect of storage at 
 4c on integrity of vec based on propidium iodide ( pi ) 
 staining . vec were harvested and stored in ksfm for 24 , 48 , 
 and 72 hours and were stained with propidium iodide 
 ( 0.5 mg / ml in pbs ) . shift of populations to 
 the right reflects greater pi staining which indicates loss of 
 cell integrity and increasing cell demise . the y - axis indicates 
 the number of events ( vec ) at each fluorescence level . pi staining 
 yielded a biphasic pattern with the second peak ( strong fl-2 
 fluorescence ) increased from 41.6% among fresh cells to 
 74.1% at cells stored for 72 hours supporting the use of 
 freshly harvested vec . cytometer setup that permits discrimination of yeast and 
 vaginal epithelial cells ( vec ) on the basis of cell geometry . upper panel shows that 99.7% of yeast cells are segregated into 
 the lower left quadrant . the fsc - ssc distribution of vec is 
 predominantly in the upper- and right - hand quadrants . we therefore 
 created a gate that includes the upper- and right - hand quadrants 
 in order to analyze the vec . when stained , 
 yeast become attached to vec providing a second fluorescent peak 
 at a level approximately one log greater than vec 
 autofluorescence . controls 
 consisted of vec incubated with fluorescenced yeast for 30 
 minutes before analysis . vec obtained from cultures that had grown 
 beyond the time they would ordinarily be harvested showed greater 
 relative fluorescence than did cells harvested on a normal 
 schedule . vec undergoing prolonged cultivation in the presence of 
 1.0  10 estradiol ( e2 ) showed the highest level of 
 fluorescence due to fluorescent yeast adherence . the amount of fl-1 
 fluorescence indicates the relative density of yeast binding to 
 vec . the 
 remaining bars indicate the binding of yeast to vec previously 
 treated with each of 4 k - y products . the lower fluorescence 
 after k - y treatment indicates reduced yeast binding to vec .
background . 
 pathogenesis of mucosal microorganisms 
 depends on adherence to the tissues they colonize and infect . for 
 candida albicans , 
 cell surface hydrophobicity may play a 
 significant role in tissue binding ability . 
 methods . a 
 continuous cell line of vaginal epithelial cells ( vec ) 
 was grown in keratinocyte serum - free medium ( ksfm ) with 
 supplements and harvested by trypsinization . 
 vec were combined 
 with yeast cells to evaluate adherence and inhibition of 
 adherence . in this experimental setup , 
 yeast stained with 
 fluorescein isothiocyanate were allowed to attach to vec and the 
 resulting fluorescent vec were detected by flow cytometry . 
 results . 
 vec were cultured and examined daily after 
 plating and showed morphology similar to basal epithelial cells . 
 culture media supplemented with estradiol showed increased vec 
 proliferation initially ( first 24 h ) but cell morphology was not 
 altered . 
 fluorescinated candida cells bound effectively 
 to the cultured vec . using fresh cells exposed to various 
 preparations of k 
 - y , we showed that all formulations of the 
 product reduced candida binding to vec by 25% to 
 50% . while vec were generally harvested for use in 
 experiments when they were near confluent growth , we allowed some 
 cultures to grow beyond that point and discovered that cells 
 allowed to become overgrown or stressed appeared to bind yeast 
 cells more effectively . 
 conclusion . 
 flow cytometry is a 
 useful method for evaluating binding of stained yeast cells to 
 cultured vec and has demonstrated that commercially available 
 products have the ability to interfere with the process of yeast 
 adherence to epithelial cells .