Mycopathologia (2006) 162: 2532 DOI 10.

1007/s11046-006-0036-7

Springer 2006

Candida-induced oral epithelial cell responses

E.A. Lilly1,4, J.E. Leigh2,4, S.H. Joseph3, & P.L. Fidel Jr.1,4
Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center and School of Dentistry, New Orleans, LA, USA; 2Department of General Dentistry, Louisiana State University Health Sciences Center and School of Dentistry, New Orleans, LA, USA; 3Department of Medicine, Section of Infectious Disease, Louisiana State University Health Sciences Center and School of Dentistry, New Orleans, LA, USA; 4Center of Excellence in Oral and Craniofacial Biology, Louisiana State University Health Sciences Center and School of Dentistry, 100 Florida Ave, New Orleans, LA, 70119, USA
Received 2 Feburary 2006; accepted in revised form 22 May 2006
1

Abstract Objective. Oropharyngeal candidiasis (OPC), caused by Candida albicans, is the most common oral infection in HIV+ persons. Oral epithelial cells are considered important for innate host defense against OPC with production of cytokines in response to C. albicans and the ability to inhibit Candida growth in vitro. The purpose of this study was to determine if Candida similarly induces cytokines by oral epithelial cells from HIV+ persons, including those with OPC, as well as to determine if cytokines can inuence the oral epithelial cell anti-Candida activity. Methods. Supernatants from oral epithelial cells from HIV+ persons with and without OPC cultured with Candida were evaluated for cytokines by ELISA, or cytokines were added to the standard growth inhibition assay using epithelial cells from HIV) persons. Results. Results showed low Candida-induced epithelial cell cytokine production from HIV+ persons, but with some elevated proinammatory cytokines (TNF-a, IL-6) in those with OPC compared to those without OPC. The addition of specic proinammatory or Th cytokines had no eect on oral epithelial cell anti-Candida activity in healthy HIV) persons. Conclusion. These results suggest that oral epithelial cells from HIV+ persons can contribute at some level to the oral cytokine milieu in response to Candida during OPC, but that cytokines do not appear to inuence oral epithelial cell anti-Candida activity. Key words: Candida, cytokines, human immunodeciency virus, oral epithelial cells, oropharyngeal candidiasis

Introduction Candida albicans is a commensal organism of the gastrointestinal and reproductive tracts as well as the primary pathogen of oropharyngeal candidiasis (OPC). OPC is the most common oral infection in persons infected with human immunodeciency virus (HIV) and occurs most frequently when CD4+ T cells are reduced below 200 cells/ll. While cell-mediated immunity (CMI) by Th1-type CD4+ T cells is considered the predominant host defense mechanism against mucosal C. albicans infections [13], several local mechanisms also

appear important. Among these are locally present cytokines that bathe the tissue and inuence immune reactivity [4, 5], and oral epithelial cells that have antifungal activity in vitro [69]. Cytokines have a strong inuence on host responses to pathogenic insults. In the oral cavity, an evaluation of T helper (Th) cytokines in the saliva of HIV+ subjects with and without OPC revealed that, in contrast to the Th0/Th1-type prole in HIV) subjects, a Th2-type prole, associated with susceptibility to candidal infections [10], predominated in HIV+ subjects and was more profound in those with OPC [4, 5]. In addition, an

26 analysis of tissue-associated cytokine expression showed that mRNA for several proinammatory, T helper, and CD8+ T cell-associated cytokines and chemokines were increased in OPC lesions [11]. Epithelial cells are now considered to play a role in host defense, including antigen presentation [1214], antimicrobial activity [1518], and the production of cytokines/chemokines in response to Candida and other microorganisms [19, 20]. Indeed, a study from our laboratory showed that oral epithelial cells collected from saliva of healthy volunteers produced several proinammatory cytokines both constitutively and in response to C. albicans, while Th2-type cytokines and chemokines were produced at low to undetectable concentrations [21]. As mentioned, epithelial cells also have antifungal activity. Specically, oral epithelial cells collected from saliva inhibit 6090% of Candida growth in vitro at relatively low eector to target ratios [6]. Vaginal epithelial cells have similar activity, albeit weaker [22]. The epithelial cells are active against both morphological forms of C. albicans as well as other Candida species [6]. The mechanism includes a strict requirement for cell contact with no role for soluble factors [7], is fungistatic rather than fungicidal [8], and is noninammatory. The latest data suggest an acid-labile mechanism that does not require live epithelial cells [9]. Clinically, this activity has been found to be reduced in HIV+ persons with OPC [6], indicating that epithelial cells may represent an important innate host defense mechanism against C. albicans at the oral mucosa. The purpose of the present study was to determine if proinammatory cytokines induced by C. albicans are similarly produced by oral epithelial cells from HIV+ individuals, and if so, whether there is a dierence between those with and without OPC, and also to determine if the presence of specic cytokines could inuence the anti-Candida activity of oral epithelial cells. (LSUHSC) HIV Outpatient Program (HOP) of the Medical Center of Louisiana at New Orleans and the Charity Hospital Dental Clinic. Informed consent was obtained from each participant, and all procedures were conducted in accordance with the Institutional Review Board at LSUHSC, New Orleans and the Research Committee of the HOP. Subjects were part of an established cohort consisting of HIV) and HIV+ persons with and without OPC. HIV+ persons without OPC were positive for oral Candida colonization. A subset of this cohort was chosen for this analysis, including HIV+ OPC) (n = 6) and HIV+ OPC+ (n = 5) subjects. Of these, 5 OPC) and 3 OPC+ patients were on highly active antiretroviral therapy (HAART). HAART was dened as receiving three or more antiretroviral drugs, while monotherapy or dual therapy without a protease inhibitor was classied as non-HAART. All patients were cigarette smokers. In addition, 9 HIV) healthy volunteers were enrolled. Diagnosis of oropharyngeal candidiasis and detection of oral yeast colonization The diagnosis of OPC was made on the clinical appearance of white curd-like plaques (pseudomembranous) or red, atrophic areas (erythematous) in the oral cavity as described previously [2]. Oral swabs from both infected and uninfected buccal mucosa (BM) were plated on Sabouraud-dextrose agar (SAB; Becton Dickinson Microbiology Systems) and Chromagar (CHROMagar Microbiology) and incubated for 48 h at 34 and 37 C, respectively. Diagnosis of OPC was conrmed by hyphae present on a smear made from the swab with potassium hydroxide (KOH) and a positive swab culture with characteristic colony morphology. Asymptomatic colonization was assigned based on the presence of colonies following plate culture, but without any signs of infection or positive KOH smear. Initial speciation was assigned according to colony color on Chromagar. Green colonies were processed for germ tube formation (incubation in fetal bovine serum [FBS] for 2 h at 37 C), with those forming germ tubes classied as C. albicans. Non-green colonies were speciated using API biochemical tests (API 20 AUX; BioMerieux).

Materials and methods Subjects HIV+ subjects were recruited and evaluated at the Louisiana State University Health Sciences Center

27 Oral epithelial cell isolation Oral epithelial cells were collected as previously described (Steele et al., 2000). Briey, unstimulated saliva (10 ml) from each participant was expectorated into a single polypropylene test tube and centrifuged at 800 g for 5 min, and the cell pellet was washed twice with sterile phosphate-buered saline (PBS), resuspended in Hanks Balanced Salt Solution (HBSS) (Gibco, Gaithersburg, MD), and passed over a 20-lm sterile nylon membrane (Spectrum, Rancho Dominguez, CA). The resulting enriched epithelial cells ($95% pure) were frozen in cryopreservative solution (50% fetal bovine serum [FBS], 25% RPMI 1640 tissue culture medium, 15% dimethyl sulfoxide), and stored at )70 C until use. At the time of use, the cells were thawed, washed, and enumerated by trypan blue dye exclusion. Viability was consistently between 60 and 85% before and after freezing. Stimulator/target cells C. albicans 3153A from the National Collection of Pathogenic Fungi (London, United Kingdom) was grown on Sabouraud dextrose agar (Becton Dickinson, Sparks, MD) at 34 C. One colony was used to inoculate 10 ml of phytone-peptone (PP) broth (Becton Dickinson) supplemented with 0.1% glucose for 1218 h at 25 C in a shaking water bath. The blastoconidia were collected, washed with PBS, and enumerated on a hemacytometer using trypan blue dye exclusion. Epithelial cell cytokine production To simulate an infectious condition, oral epithelial cells from HIV+ subjects with and without OPC were cocultured with Candida at a ratio of 1:100 in separate wells for 48 and 96 h in a total volume of 2 ml of Phytone Peptone broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with 10% fetal bovine serum and 1% penicillin (100 U/ml) and streptomycin (100 lg/ml; Gibco) in 24-well tissue culture plates (Costar, Corning, NY) as determined optimally and described previously [21]. Controls included epithelial cells cultured in medium alone (constitutive production) in separate wells and C. albicans cultured alone (negative control). At each time point, the epithelial cell-Candida coculture and the respective control cultures were aspirated from a dierent set of individual wells and centrifuged for 5 min at 800 g. The supernatants were collected and stored at )70 C until assayed. Cocultures consisted of 6 HIV+ OPC) and 5 HIV+ OPC+ subjects examined individually. Cytokine analysis Culture supernatants were assayed for proinammatory (TNF-a, IL-1a, and IL-6), Th1 (IFN-c and IL-2), and Th2-type (IL-10) cytokines by enzymelinked immunosorbent assay (ELISA) using commercially available capture and biotinylated detection antibodies (BD Pharmingen, San Diego, CA). Standard curves were generated by using the respective recombinant human cytokines described above. The absorbance values and concentrations of each cytokine were determined using an FL600 uorescence plate reader (Bio-Tek Corp., Winooski, VT) and KC4 software (Bio-Tek). Constitutive production (medium alone) was subtracted out and data was illustrated as specic pg/ml. Cytokine eect on growth inhibition assay Epithelial cells from HIV) healthy volunteers were cocultured with Candida in PP broth supplemented with 10% FBS and 1% penicillin (100 U/ml) and streptomycin (100 lg/ml) at an optimal eector to target ratio of 5:1 in triplicate wells of a 96-well microtiter plate (Costar, Cambridge, MA) as previously described [6]. Recombinant human cytokine (IL-1a, TNF-a, IFN-c, IL-10) (R&D Systems, Minneapolis, MN) without preservatives was added at concentrations of 1, 10, and 100 pg/ml to epithelial cell/Candida coculture wells in a volume of 50 ll of PP broth. Controls included the eector and target cell coculture without cytokines and eector and target cells cultured independently in PP broth with and without cytokine. The cultures were incubated for 9 h at 37 C in 5% CO2 in the presence of 1 lCi [3H]-glucose (ICN, Costa Mesa, CA). Following the incubation, 100 ll of sodium hypochlorite solution (bleach) was added to all wells and left for 5 min, and the cell extracts were harvested onto glass ber lters using a PHD cell harvester (Cambridge Technologies, Watertown, MA). The incorporated [3H]-glucose was measured by liquid scintillation. The incorporation of glucose by Candida and epithelial cells during the 9 h

28 incubation generally ranged from 15,000 to 30,000 cpm and 1,000 to 4,000 cpm, respectively. The percent growth inhibition was calculated as follows: % growth inhibition = 1 ) [(mean experimental cpm) ) (mean eector cpm)/mean Candida cpm] 100. Growth inhibition calculations using experimental and control wells with cytokine was conducted independently of those without cytokines and compared. Statistics The Students t test was applied. Signicant dierences were dened as P < 0.05 using a two-tailed test. All statistics were performed using GraphPad Prism (GraphPad Software, San Diego, CA). Results Candida-induced cytokine production by oral epithelial cells in HIV+ persons Supernatants for epithelial cell enriched populations from HIV+ subjects with and without OPC cocultured with C. albicans for 48 or 96 h were assayed for proinammatory (TNF-a, IL-1a, IL-6), Th1 (IFN-c, IL-2), and Th2 (IL-10) cytokines, and the results of the specic cytokine production is illustrated in Figures 1 and 2. Candida cultured alone as a negative control showed no production of any cytokine (data not shown). Of the proinammatory cytokines, TNF-a (Figure 1b) and IL-6 (Figure 1c), but not IL-1a (Figure 1a) were induced by Candida in higher amounts by epithelial cells from OPC+ persons at 96 h post-culture compared to cells from OPC) persons (P = 0.018 and 0.014, respectively). Moreover, TNF-a was induced by Candida in greater amounts from cells of OPC+ persons at 96 compared to 48 h post-culture (P = 0.008). Th cytokines, IFN-c (Figure 2a), IL-2 (Figure 2b), and IL-10 (Figure 2b), induced by Candida were similar for epithelial cells from OPC) or OPC+ persons over the 96 h period. Anti-Candida activity by cytokine-stimulated oral epithelial cells IL-1a, TNF-a, IFN-c, and IL-10 were each added to the standard growth inhibition assay to determine the inuence of select cytokines on oral epithelial cell anti-Candida activity in vitro. Results in Figure 3 show no eect of 1, 10, or 100 pg/ml of TNF-a (frame A) and IFN-c (frame B). Similar results were observed for IL-10 and IL-1a (data not shown).

Discussion Epithelial cells and cytokines are considered important host defenses against mucosal pathogens and are not mutually exclusive. Indeed, epithelial cells from the oral and vaginal mucosa of HIV) persons have anti-Candida activity [6, 22, 23] and produce cytokines in response to microorganisms including Candida [1921]. Cytokines are also found in the saliva and tissue of HIV) and HIV+ persons and are elevated or distinct in those with OPC [4, 5, 11]. Thus, we were interested in whether epithelial cells from HIV+ persons with and without OPC could contribute positively or negatively to the oral cytokine milieu. For this, epithelial cells from HIV+ persons with and without OPC were evaluated for Candida-induced proinammatory and Th cytokines. Furthermore, we were also interested in determining what inuence, if any, cytokines had on oral epithelial cell anti-Candida activity in healthy HIV) persons. Considering the prevalence of OPC in HIV+ persons and the role of the host response in controlling Candida infection, changes in cytokine responses and an inuence of cytokines on anti-Candida activity were expected along with some potential for use in future therapeutic strategies. Results overall showed low Candida-induced epithelial cell cytokine production from HIV+ persons. This was consistent for some cytokines (Th cytokines and IL-6) evaluated in similar cultures from HIV) persons, but was quite dierent compared to the relatively high concentrations of TNF-a and IL-1a in the same cultures [21]. Additionally, baseline levels of both proinammatory and Th cytokines were greater in those with HIV (the present study) compared to previous studies in HIV) individuals [21] (e.g., TNF-a $15 versus $6.3 pg/ml; IL-10 $50 versus $1.4 pg/ ml) possibly due to the presence of Candida in the samples. Irrespective though, since the HIV) and HIV+ cohorts were not tested or analyzed in

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Figure 1. Candida-induced proinammatory cytokine production by oral epithelial cells of HIV+ persons. Epithelial cells collected from saliva of HIV+ persons with (n = 5) and without (n = 6) OPC were cultured with C. albicans at an epithelial cell/Candida ratio of 1:100 for up to 96 h. Supernatants of the Candida-epithelial cell coculture were collected at the specied timepoints, and cytokines were quantied by ELISA. The gure shows the mean concentrations of Candida-specic (minus concentrations of medium alone) production of IL-1a (a), TNF-a (b), and IL-6 (c). Signicance (P < 0.05) was determined using the Students t test. Lines between specic bars represent signicant dierences. SEM=Standard error of the mean.

parallel, it is dicult to interpret such dierences. The relatively low IL-1a production in the cell cultures from HIV+ persons together with the reduced tissue-associated IL-1a mRNA expression in those with HIV [11] suggests some regulation of cytokine production/expression by HIV. The time of culture with Candida had little eect since cytokine production was relatively similar at 48 and 96 h. We recognize, however, that cytokine production in HIV+ persons may be delayed, and higher concentrations could have been detected past 96 h. Future studies could address this. It also should be noted that the fully dierentiated state of the epithelial cells compromises the metabolic activity that will aect cytokine responses.

In comparing Candida-induced cytokine production by cells from OPC) and OPC+ persons, few dierences were detected. The exceptions were increased TNF-a and IL-6 in OPC+ individuals at 96 h of culture. These results suggest that epithelial cells can contribute to the local inammatory cytokine milieu in those with OPC [4]. Interestingly, IL-6 mRNA was shown to be increased in lesions of those with OPC. On the other hand, TNF-a mRNA was shown to be decreased in OPC lesions [11]. While we recognize that protein may not always correspond to mRNA, and these comparisons are between in vitro and in vivo conditions, it remains possible that the cytokines present in the oral cavity during infection or

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Figure 3. Eects of cytokines on epithelial cell anti-Candida activity. Oral epithelial cells collected from saliva of healthy HIV) subjects (n = 9) were cocultured with C. albicans (5:1 ratio) alone or in the presence of 1, 10, or 100 pg/ml of TNF-a (a) or IFN-c (b). [3H]-glucose incorporation after 9 h was used as a measure of Candida growth and results were expressed as the mean percent inhibition of growth. SEM, Standard Error of the Mean.

48 h

96 h

Figure 2. Candida-induced Th cytokine production by oral epithelial cells of HIV+ persons. Epithelial cells collected from saliva of HIV+ persons with (n = 5) and without (n = 6) OPC were cultured with C. albicans at an epithelial cell/Candida ratio of 1:100 for up to 96 h. Supernatants of the Candida-epithelial cell coculture were collected at the specied timepoints, and cytokines were quantied by ELISA. The gure shows the mean concentrations of Candida-specic (minus concentrations of medium alone) production of IFN-c (a), IL-2 (b), and IL-10 (c). SEM = Standard error of the mean.

asymptomatic colonization can, in part, be a result of the epithelial cell-Candida interaction. Although real-time PCR studies of the same cohort would be useful in conrming these data, such studies are not possible due to the low integrity of RNA obtained from the fully dierentiated oral epithelial cells. Considering that previous studies have shown reduced anti-Candida activity in those with HIV [6] and that locally present cytokines bathe the tissue and inuence immune reactivity [4, 11], it was

important to determine if the presence of specic cytokines could inuence oral epithelial cell antiCandida activity. The proinammatory cytokines, TNF-a and IL-1a, were chosen because of previous observations in which both were released at the highest levels by epithelial cells from HIV) persons in response to Candida compared to other cytokines [21]. In addition, we chose two other cytokines, IFN-c and IL-10, that were increased in the tissue of those with OPC [11]. We also chose an HIV) cohort since it would rst be important to evaluate inuences in a population of cells not aected by HIV. Results showed that anti-Candida activity of this HIV) cohort, while reduced compared to a previous study ($65% versus 8090%) [6], was not aected positively or negatively by any cytokine. This suggests that the fungistatic inhibitory eect of the epithelial cells, requiring cell

31 contact, may not be easily inuenced by immunomodulators, and activity may not be enhanced by cytokines in HIV+ individuals who have reduced activity. If so, this would be unfortunate, as the epithelial cell antifungal activity is postulated to be more critical when CD4+ T cells are reduced [24], and the ability to enhance the activity under such conditions would be extremely benecial. It remains possible, however, that other cytokines may inuence the activity or that epithelial cells not terminally dierentiated and thus less metabolically compromised may be more strongly inuenced. It still remains possible, too, that cytokines may inuence the activity of cells from HIV+ persons that were not evaluated in this study. In summary, our data suggest that oral epithelial cells can contribute to the local cytokine milieu in response to Candida in those with OPC, but that proinammatory and Th cytokines present in saliva or tissue of both HIV) and HIV+ persons do not appear able to inuence oral epithelial cell anti-Candida activity.
6. Steele C, Leigh JE, Swoboda RK, Fidel PL Jr. Growth inhibition of Candida by human oral epithelial cells. J Infect Dis 2000; 182: 14791485. 7. Steele C, Leigh JE, Swoboda RK, Ozenci H, Fidel PL Jr. Potential role for a carbohydrate moiety in anti-Candida activity of human oral epithelial cells. Infect Immun 2001; 69: 70917099. 8. Nomanbhoy F, Steele C, Yano J, Fidel PL Jr. Vaginal and oral epithelial cell anti-Candida activity. Infect Immun 2002; 70: 70817088. 9. Yano J, Lilly E, Steele C, Fortenberry D, Fidel PL Jr. Oral and vaginal epithelial cell anti-Candida activity is acidlabile and does not require live epithelial cells. Oral Microbiol Immunol 2005; 20: 199205. 10. Romani L, Puccetti P, Bistoni F. Biological role of Th cell subsets in candidiasis. In: Romagnani S), ed. Th1 and Th2 Cells in Health and Disease, Karger: Farmington, CT, 1996: 114137. 11. Lilly E, Hart DJ, Leigh JE, Hager S, McNulty KM, Mercante DE et al. Tissue-associated cytokine expression in HIV-positive persons with oropharyngeal candidiasis. J Infect Dis 2004; 190: 605612. 12. Gonella PA, Neutra MR. Membrane-bound and uidphase macromolecules enter separate prelysosomal compartments in absorptive cells of suckling rat ileum. J Cell Biol 1984; 99: 909917. 13. Gonella PA, Wilmore DW. Co-localization of class II antigen and endogenous antigen in the rat enterocyte. J Cell Sci 1993; 106: 937940. 14. Wira CR, Rossoll RM. Antigen-presenting cells in the female reproductive tract: Inuence of the estrous cycle on antigen presentation by uterine epithelial and stromal cells. Endocrinology 1995; 136: 45264534. 15. Lehrer R, Ganz T, Szklarek D, Seisted M. Modulation of the in vitro candidiacidal activity of human neutrophil defensins by target cell metabolism and divalent cations. J Clin Invest 1988; 81: 18291835. 16. Weinberg A, Krisanaprakornkit S, Dale BA. Epithelial antimicrobial peptides: Review and signicance for oral applications. Crit Rev Oral Bio Med 1998; 9: 399414. 17. Xu T, Levitz SM, Diamond RD, Oppenheim FG. Anticandidal activity of major human salivary histatins. Infect Immun 1991; 59: 25492554. 18. Brandtzaeg P, Gabrielsen TO, Dale I, Muller F, Steinbakk M, Fagerhol MK. The leucocyte protein L1 (calprotectin): A putative nonspecic defence factor at epithelial surfaces. Adv Exp Med Bio 1995; 371: 201206. 19. Hedges SR, Agace WW, Svanborg C. Epithelial cytokine responses and mucosal cytokine networks. Trend Microbiol 1995; 3: 266270. 20. Hedges SR, Agace WW, Svensson M, Sjogren A, Ceska M, Svanborg C. Uroepithelial cells are part of a mucosal cytokine network. Infect Immun 1994; 62: 23152321. 21. Steele C, Fidel PL Jr. Cytokine and chemokine production by human oral and vaginal epithelial cells in response to Candida albicans. Infect Immun 2002; 70: 577583. 22. Barousse MM, Steele C, Dunlap K, Espinosa T, Boikov D, Sobel JD et al. Growth Inhibition of Candida albicans

Acknowledgements This work was supported by Public Health Service grant DE 12178 from the National Institute of Dental and Craniofacial Research (NIDCR) at the NIH.

Reference
1. 2. Fidel PL Jr, Sobel JD. The role of cell-mediated immunity in candidiasis. Trends Micro 1994; 2: 202206. Salmi M, Jalkanen S. How do lymphocytes know where to go: Current concepts and enigmas of lymphocyte homing. Adv Immunol 1997; 64: 139218. Fidel PL Jr. Distinct protective host defenses against oral and vaginal candidiasis. Med Mycol 2002; 40: 359375. Leigh JE, Steele C, Wormley FL Jr, Luo W, Clark RA, Gallaher WR et al. Th1/Th2 cytokine expression in saliva of HIV-positive and HIV-negative individuals: A pilot study in HIV-positive individuals with oropharyngeal candidiasis. J Acquir Immune Dec Syndr Hum Retrovirol 1998; 19: 373380. Slavinsky III J, Myers T, Swoboda RK, Leigh JE, Hager S, Fidel PL Jr. Th1/Th2 cytokine proles in saliva of HIVpositive smokers with oropharyngeal candidiasis. Oral Microbiol Immunol 2002; 17: 3843.

3. 4.

5.

32
by human vaginal epithelial cells. J Infect Dis 2001; 184: 14891493. 23. Steele C, Ozenci H, Luo W, Scott M, Fidel PL Jr. Growth inhibition of Candida albicans by vaginal cells from naive mice. Med. Mycol 1999; 37: 251260. 24. Leigh JE, Shetty K., Fidel PL Jr. Oral opportunistic infections in HIV-positive individuals: Review and role of mucosal immunity. AIDS Patient Care STDs 2004; 18: 443456. Address for correspondence: P.L. Fidel Jr., Center of Excellence in Oral and Craniofacial Biology, Louisiana State University Health Sciences Center and School of Dentistry, 100 Florida Ave, New Orleans, LA, 70119, USA Phone: +504-670-2734; Fax: +504-670-2736 E-mail: pdel@lsuhsc.edu

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