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J Dent Res 81(1): 28-32, 2002
© 2002 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Saliva Promotes Candida albicans Adherence to Human Epithelial Cells

A.R. Holmes, B.M.K. Bandara, and R.D. Cannon*,

Department of Oral Sciences and Orthodontics, University of Otago, PO Box 647, Dunedin, New Zealand;

*corresponding author, richard.cannon{at}stonebow.otago.ac.nz


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adhesion of Candida cells to oral surfaces is an initial event in pathogenesis. Since specific immobilized salivary components mediate the binding of Candida albicans to hydroxyapatite, we hypothesized that saliva may also promote adherence to oral epithelia via a similar mechanism. In an in vitro model, C. albicans ATCC 10261 yeast cells adhered in a saturable manner to monolayers of three cultured human epithelial cell lines (A549, HEp-2, and HET-1A). The addition of whole saliva to the assay promoted the binding of C. albicans to all cell lines in a dose-dependent manner, but pre-incubation of the epithelial cells with pooled whole saliva had no effect on subsequent adherence. Pre-incubation of the yeast cells with pooled whole saliva, however, significantly enhanced (by up to 120%, P < 0.05) binding to epithelial cell monolayers, and pooled saliva that had been pre-incubated with C. albicans yeast cells was defective in promoting yeast adherence. There was a negative correlation (r = 0.68, P < 0.005) between specific IgA titers against whole cells of C. albicans and adherence-promoting activities for individual saliva samples. The adhesion-inhibitory effect of specific anti- C. albicans IgA was reversed by depletion of IgA from saliva by affinity chromatography. Factors in whole saliva, therefore, bound to the yeast cells, counter the C. albicans-specific salivary IgA inhibitory effect on adhesion and promote the adherence of C. albicans yeast cells to cultured epithelial cells.

KEY WORDS: saliva • Candida albicans • epithelial cell lines • adherence • IgA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Candida albicans is a pathogenic yeast that causes superficial and systemic infections in immunocompromised individuals (Darouiche, 1998). Oral candidiasis affects many sectors of the population, including the very young, the elderly, and severely immunodeficient people. AIDS patients, for example, frequently suffer from esophageal candidiasis. Adhesion ofCandida cells to oral surfaces is an initial event in the development of candidiasis (reviewed by Cannonet al., 1995a; Cannon and Chaffin, 1999), andC. albicans has been shown to bind to various oral surfaces such as dental materials (Radfordet al., 1999), oral bacteria (Holmeset al., 1995), and epithelial cells (Tosh and Douglas, 1992; Hostetter, 1994; Williset al., 2000).

All oral surfaces are coated with an acquired pellicle containing salivary components; therefore, it is important to investigate the contributions of salivary components to Candida adherence. Previous studies from this laboratory have shown that C. albicans adheres specifically to immobilized salivary basic proline-rich proteins (bPRPs) (O'Sullivan et al., 1997), and that saliva promotes the binding of C. albicans cells to immobilized streptococcal cells (Holmes et al., 1995; O'Sullivan et al., 2000). There are conflicting reports, however, regarding the influence of soluble salivary components on C. albicans adherence to oral surfaces, particularly the role of salivary IgA. Anti-IgA antibodies abolished an inhibitory effect of saliva on the binding of C. albicans to human epithelial cells (Umazume et al., 1995). In contrast, whole saliva was shown to promote the attachment of Candida yeast cells to hard surfaces (Edgerton et al., 1993; San Millán et al., 2000), and non-immune mammary IgA was shown to enhance adherence of yeast cells to exfoliated epithelial cells (Vudhichamnong et al., 1982). None of these studies addressed the question of whether specific anti-Candida IgA antibodies in saliva were affecting adherence to oral surfaces. Salivary IgA antibodies specific to C. albicans yeast cell surface components are readily detectable in human saliva, and titers can be elevated in asymptomatic carriers (Bikandi et al., 2000) as well as in patients diagnosed with oropharyngeal candidiasis (Coogan and Challacombe, 2000; Millon et al., 2001). In this study, we investigated the effect of whole saliva on the interaction between C. albicans yeast cells and cultured human epithelial cells and related the effect to the presence of specific anti-C. albicans IgA antibodies in the saliva.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Strains and Culture Conditions
C. albicans strains used in this study were: ATCC 10261; five clinical isolates (hp11an, ko-2c, gaymc-c, RIH010, RIH030; Schmid et al., 1995) kindly provided by Dr. J. Schmid, Massey University, New Zealand; and two commensal isolates from the University of Otago School of Dentistry. Yeast cells were grown in glucose, salts, and biotin (GSB; Holmes and Shepherd, 1988) at 30°C with shaking (200 rpm).

Epithelial Cell Lines and Culture Conditions
Human epithelial cell lines HEp-2 and A549 were obtained from the European Collection of Cell Cultures, Centre for Applied Microbiological Research, Salisbury, UK. Cultures were passaged and maintained in Eagle's MEM medium (Gibco BRL, Life Technologies, Rockville, MD, USA) supplemented with 10% fetal calf serum (FCS) (Gibco, BRL) at 37°C in an atmosphere of 5% CO2. Confluent cells were prepared for subculture with trypsin-EDTA (Gibco, BRL). For adherence assays, trypsinized, washed cell suspensions (100 µL) were transferred to sterile 96-well flat-bottomed microtiter plates (Nalgene Nunc, Rochester, NY, USA) and cultured for 24 hrs (37°C, 5% CO2) until confluent. HET1-A cells were kindly provided by Dr. C.C. Harris, Laboratory of Human Carcinogenesis, NCI, NIH, USA, and grown and maintained in fibronectin-coated 20-mL tissue culture flasks with serum-free tissue culture medium (LHC 8, Biofluids, Rockville, MD, USA). For adherence assays, suspensions of HET1-A cells (100 µL) were transferred to sterile 96-well flat-bottomed microtiter plates, pre-coated with fibronectin (10 µg/mL), and cultured for 48 hrs (37°C, 5% CO2) until confluent.

Saliva Collection
Unstimulated saliva was collected on ice from six donors, and an equal volume from each donor was pooled. Saliva was collected after informed consent had been obtained and according to a protocol that had been reviewed and approved by the University of Otago Human Ethics Committee. The saliva was clarified by centrifugation, and protein inhibitors were added as described by O'Sullivan et al. (2000).

Adherence Assays
C. albicans yeast cells were radiolabeled with [35S]methionine as described by Cannon et al. (1995b). Briefly, cell cultures (50 mL) were grown to mid-exponential phase in GSB medium before the addition of [35S]methionine (2 µL, 20 µCi) and incubation at 30°C for 2 hrs with shaking. The cells were harvested by centrifugation (3000 x g, 5 min, 4°C) and washed twice in 10 mL of KCl buffer (2 mmol/L KH2PO4; 2 mmol/L K2HPO4; 5 mmol/L KCl, 1 mmol/L CaCl2, pH 6.5). Cells were then re-suspended for use in adherence assays, at a concentration of 1.1 x 106/mL (or at concentrations described in the text), in an ‘artificial saliva’ (AS) buffer constituted to mimic the ionic composition of saliva (1.75 mmol/L KH2PO4, 1.5 mmol/L CaCl2, 18.3 mmol/L KCl, 0.35 mmol/L NaSCN, 15 µmol/L NaF, 26 mmol/L NaHCO3, pH 6.5) and supplemented with glucose (1 g/L).

Radiolabeled C. albicans cells (50 µL) and saliva samples (50 µL, diluted in AS buffer to concentrations between 0 and 60%) were added to quadruplicate microtiter plate wells containing epithelial cell monolayers, and incubated at 37°C in an atmosphere of 5% CO2 for 1.5 hrs. The liquid in the wells was then aspirated and the monolayers washed three times with pre-warmed PBS. This wash procedure was shown in preliminary experiments to remove non-adherent yeast cells consistently, whereas further washes (up to three) did not remove bound yeast cells. Then the plates were air-dried at 37°C before the addition of 100 µL Optiphase scintillation fluid (Wallac Oy, Turku, Finland) and determination of radioactivity present in each well by means of a Wallac 1450 MicroBeta TriLux Scintillation counter (Wallac Oy).

In experiments to determine the effect of pre-treating tissue culture monolayers with saliva on subsequent adherence of radiolabeled C. albicans cells, we incubated quadruplicate microtiter plate wells of confluent tissue culture monolayers with pooled whole saliva (50 µL, diluted in AS buffer to concentrations between 0 and 60%) for 60 min before the aspiration of the saliva and the addition of radiolabeled C. albicans cells as above. Adherence was measured as described above.

In some experiments, C. albicans cells were pre-treated by incubation with saliva. Radiolabeled C. albicans cells at a concentration of 2.2 x 106/mL were incubated end-over-end with the saliva sample, diluted in AS buffer, at room temperature for 60 min. The cells were collected by centrifugation and washed twice with AS buffer before being re-suspended in AS buffer for use in adherence assays at the concentrations described in the text.

For experiments to examine the effect of pre-absorbing saliva with C. albicans yeast cells on its adherence-promoting activity, saliva samples were incubated end-over-end for 60 min at room temperature with non-radiolabeled C. albicans ATCC 10261 yeast cells (at concentrations described in the text), and then cells were removed by centrifugation.

Enzyme-linked Immunosorbent Assay (ELISA)
To determine the specific immune reactivity of salivary IgA with C. albicans, we used a modification of a whole-cell ELISA technique (Holmes et al., 1996). Briefly, yeast cells (50 µL, 1 x 107/mL) were immobilized onto microtiter plates' wells (Immunosorp, Nalgene Nunc) by low-speed centrifugation. Non-specific protein binding sites were blocked by incubation with Tris-buffered saline (100 µL, TBS: 1 mmol/L Tris-HCl, 0.15 mol/L NaCl, pH 8.0) containing 0.5% (w/v) gelatin at 4°C for 16 hrs. The blocking solution was aspirated, and doubling dilutions (1:4 to 1:256) of whole saliva (50 µL, in TBS containing 0.05% gelatin) were added to duplicate wells and incubated at 37°C for 1 hr. Wells were washed three times with TBS containing 0.05% (v/v) Tween 20 (Bio-Rad, Hercules, CA, USA), and IgA binding was detected by incubation with peroxidase-conjugated rabbit immunoglobulins raised against human IgA (alpha chains) (Dako Corp., Carpenteria, CA, USA), diluted 1:1000 in TBS gelatin (50 µL) at 37°C for 1 hr. Plates were developed with o-phenylenediamine as substrate, and after the reaction was stopped with 1 mol/L H2SO4 (50 µL), the A492 was measured. Antibody reactivities were expressed either as titers based on the highest saliva dilution giving greater than twice the background, blocked well, absorbance value, or as mean relative absorbance values for a 1:16 dilution of saliva (expressed, to eliminate inter-plate variability, as a percentage of the A492 value for a constant control positive sample). Non-specific, total IgA content of saliva samples was also determined by ELISA. Clarified whole saliva samples, diluted between 1:100 and 1:6400 in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), were adsorbed to wells of microtiter plates (Immunosorp, Nalgene Nunc) at 4°C for 16 hrs. Non-specific protein binding sites were blocked with TBS gelatin, and bound total IgA was detected as described above. Total IgA titers were expressed as the highest saliva dilution giving greater than twice the background absorbance value.

In some experiments, saliva samples were treated with immobilized jacalin (Pierce, Rockford, IL, USA) for the depletion of IgA content. Saliva samples (0.5 mL, whole saliva, clarified as described above) were added to 0.5 mL of washed jacalin-agarose slurry in binding buffer (PBS as per manufacturer's instructions) and mixed end-over-end at 4°C for 1 hr. The agarose beads were removed by centrifugation and the saliva samples assayed for adherence-promoting activity and specific anti-C. albicans IgA as described above.

Statistical Analysis
We used the one-tailed t test to assess the significance of differences between means in adherence assays. We used linear correlation and regression analysis to determine the relationship between specific anti-C. albicans IgA titers and adherence, applying a t test to determine the significance of the Pearson product-moment correlation coefficient, r.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Whole Saliva on the Adherence of C. albicans Strains to Cultured Epithelial Cells
The adherence of radiolabeled C. albicans cells to confluent monolayers of three epithelial cell lines was determined. A549 is a pneumocyte type II cell line originating from a human lung carcinoma, and the HEp-2 cell line originated from a human laryngeal carcinoma. Both cultures were maintained on a serum-based medium. The HET-1A cell line originated from SV40 T-antigen-immortalized human esophageal epithelial cells (Stoner et al., 1991) and was maintained on serum-free medium. Initial experiments, undertaken with the laboratory strain C. albicans ATCC 10261, showed that yeast cells bound to epithelial cell line monolayers, in the absence of added saliva (Fig. 1Go). Enumeration of the epithelial cells per microtiter plate well indicated that, on average, between 24 and 53 yeast cells adhered to each epithelial cell. Pooled whole human saliva added to the assay significantly (P < 0.05) promoted the binding of C. albicans ATCC 10261 yeast cells to epithelial cell monolayers of the three cell lines (Fig. 1Go). Yeast cells bound in highest numbers (58 ± 3.5% of the input yeast cells, in the presence of 40% pooled saliva) to the HET-1A cells. The higher binding to this cell line was not due to C. albicans cells adhering to the fibronectin coating of the microtiter wells, since the use of fibronectin-coated wells for growth of the other cell lines did not increase the binding of C. albicans. Increasing the saliva concentration in the assay beyond 60% did not increase further the number of yeast cells bound to any cell line.



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  		Figure 1. Adherence of C. albicans ATCC 10261 yeast cells to cultured monolayers of epithelial cell lines A549 ({blacksquare}), HEp-2 (), or HET-1A ({blacktriangleup}) in the presence of increasing concentrations of pooled human whole saliva added to the assay. Numbers of C. albicans cells bound are expressed as percentages of the number of input cells (5.5 x 104 per well). Data are expressed as the means ± SE of quadruplicate determinations from three separate experiments. The mean adherence in the presence of saliva was significantly higher (P < 0.05) than that in the absence of saliva for all data points, except for the adherence to A549 cells when 20% saliva was added to the assay.

 
The adherence of C. albicans ATCC 10261 to all cell lines, in the presence of 60% whole saliva, demonstrated saturable kinetics. The number of cells bound in the assay increased in proportion to the number of yeast cells added, up to the following maxima (means of quadruplicate determinations from representative experiments repeated twice, SEs did not exceed 15%): 6.5 x 105 cells bound per well (A549 cells); 5.5 x 105 cells bound per well (HEp-2 cells); and 8.0 x 105 cells bound per well (HET-1A cells). For all three cell lines, the number of yeast cells bound did not increase significantly for input cell concentrations above 4.0 x 107 mL-1. An input cell concentration of 1.1 x 106 mL-1 (within the linear portion of the saturation curves) was used in all subsequent experiments.

Significant (P < 0.05) saliva-mediated promotion of adherence was also observed when radiolabeled cells of 7 other C. albicans strains, including 5 clinical isolates, were tested in assays with each of the cell lines. The observed promotion of adherence ranged between 14.3% and 133% of adherence values obtained in the absence of saliva.

Pre-treatment of C. albicans Cells with Whole Saliva Promotes Adherence to Cultured Epithelial Cells
To determine whether the adherence-promoting effect of whole saliva was due to the binding of saliva components to receptors on the C. albicans cells or on the epithelial cells, we pre-incubated each with saliva before including them in separate adherence assays. C. albicans yeast cells that were pre-incubated with saliva and washed before use in the adherence assays showed greater adherence than yeast cells pre-incubated with buffer only (Fig. 2Go), which showed adherence levels similar to those of yeast cells assayed in the absence of saliva (Fig. 1Go). The increase in adherence was positively related to the concentration of saliva used to pre-treat the C. albicans yeast cells. In contrast, pre-treatment of each epithelial monolayer with various concentrations of saliva before use in adherence assays did not change C. albicans adherence by more than 10% relative to binding in the absence of saliva (results not shown).



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  		Figure 2. Adherence of C. albicans ATCC 10261 yeast cells to cultured monolayers of epithelial cell lines A549 ({blacksquare}), HEp-2 (), or HET-1A ({square}) after pre-treatment of the yeast cells with increasing concentrations of pooled whole saliva. Yeast cells (2.2 x 106 mL-1) were pre-treated with whole saliva diluted in assay buffer and washed before use in adherence assays, at an input concentration of 1.1 x 106 mL-1. Results are expressed as the percentage increase in binding relative to the binding of yeast cells pre-treated with buffer alone, and are the means ± SE of quadruplicate determinations from three separate experiments. Values significantly (P < 0.05) greater than adherence of untreated yeast cells are marked with an asterisk (*). Values for adherence of yeast cells pre-treated with buffer alone were 17.1 ± 1.9%, 19.2 ± 2.1%, and 36.9 ± 4.2% for A549, HEp-2, and HET-1A, respectively.

 
Depletion of the Adherence-promoting Activity of Saliva by Pre-incubation of Saliva with C. albicans Yeast Cells
Pooled saliva samples were incubated with various concentrations of yeast cells before the yeast cells were removed by centrifugation. The pre-absorbed saliva preparations were then tested in adherence assays with fresh C. albicans cells. Pre-treated saliva (60%) showed reduced adherence-promoting activity for all cell lines, and the depletion effect was positively related to the concentration of yeast cells used to pre-treat the saliva (Fig. 3Go).



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  		Figure 3. Depletion of the adherence-promoting effect of whole saliva by pre-absorbed saliva samples (60% in AS buffer) with C. albicans ATCC 10261 yeast cells. Pooled saliva samples were pre-absorbed by incubation with various concentrations of yeast cells before use in adherence assays to cultured cell monolayers of epithelial cell lines A549 ({blacksquare}), HEp-2 (), or HET-1A ({square}). The yeast cells used to adsorb the saliva were removed by centrifugation, and the adherence assays performed with the absorbed saliva and a fresh preparation of yeast cells at an input concentration of 1.1 x 106/mL. Results are expressed as the percentage increase in binding relative to the adherence of the yeast cells in the absence of saliva added to the assay, and are the means ± SE of quadruplicate determinations from three separate experiments. Values significantly (P < 0.05) less than adherence of untreated yeast cells are marked with an asterisk (*).

 
The Adherence-promoting Activity of Whole Saliva is Reduced in the Presence of Specific Salivary IgA Recognizing C. albicans
The saliva used in the assays described above was pooled from six individuals. Saliva samples from each individual and from five other subjects were tested by ELISA for C. albicans-specific IgA and were used in adherence assays with HEp-2 cells. There was a significant negative correlation between specific IgA values and adherence (r = 0.68, P = < 0.005) (Fig. 4Go). Saliva from individuals with low C. albicans-specific IgA promoted yeast cell adherence to epithelial cells more than did saliva samples from individuals with high C. albicans-specific IgA activity.



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  		Figure 4. Relationship between the amount of C. albicans-specific IgA in saliva samples and the adherence-promoting effect of saliva. The effect on C. albicans ATCC 10261 adherence to HEp-2 monolayers of 30 saliva samples taken from 11 individuals was determined. Values for yeast cell adherence were the means of quadruplicate determinations for each saliva sample, and expressed as percentages of the number of input cells (5.5 x 104 per well). Specific anti-C. albicans IgA in saliva samples was measured by ELISA titration, and results are expressed as the mean relative A492 values of triplicate determinations for 1:16 dilutions of saliva samples as described in MATERIALS & METHODS. Regression analysis of the data gave an r value of 0.68, P < 0.005.

 
To confirm that IgA was responsible for the reduced adherence-promotion by saliva samples containing high-titer anti-C. albicans antibodies, we treated selected saliva samples with immobilized jacalin, a lectin that specifically binds IgA. Saliva samples from two individuals, which possessed either high or low titers of salivary IgA to C. albicans (antibody titers were > 256 and 8, respectively), were treated with immobilized jacalin as described in MATERIALS & METHODS. A mean reduction in both specific and non-specific IgA reactivities (30 ± 9.8% [P < 0.05] and 14.8 ± 1.2% [P < 0.05], respectively), as determined by ELISA, was observed following jacalin treatment. The jacalin-treated saliva from the high-IgA subject showed an increased (26.6 ± 5.2 %) ability to promote binding of C. albicans to Hep-2 cells relative to replicate samples given a mock jacalin treatment (P < 0.05, data from quadruplicate determinations in duplicate experiments). In contrast, treatment of saliva from the low-IgA subject with immobilized jacalin had no effect on the saliva-mediated promotion of C. albicans adherence to HEp-2 cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Adherence of C. albicans yeast cells to oral surfaces is the initial step in colonization and hence in any subsequent development of candidiasis. Determination of the adherence mechanisms involved will facilitate development of new approaches to treatment by preventing colonization. We have shown previously that C. albicans adheres specifically to a family of salivary proteins, the basic proline-rich proteins (bPRPs), when the bPRPs are immobilized on material mimicking the tooth surface or on oral bacteria (O'Sullivan et al., 1997,2000). Although C. albicans can be found in dental plaque (Hodson and Craig, 1972), it is the mucosal surfaces of the oral cavity that are most frequently colonized by C. albicans (Arendorf and Walker, 1980), and so it is important to investigate Candida/epithelial cell interactions. C. albicans adheres to several host cell types, including exfoliated oral epithelial cells (Critchley and Douglas, 1987; Fukayama and Calderone, 1991; de Repentigny et al., 2000; Willis et al., 2000), reconstituted human epithelium (Schaller et al., 1999), and cultured epithelial cell lines (Umazume et al., 1995; Fu et al., 1998; Ueta et al., 2000). The use of cultured monolayers of human epithelial cells allows for the controlled investigation of the effects of other factors, such as saliva, which is precluded by the use of exfoliated cells. Previous studies using cell lines have mostly used cells (such as HeLa) that are not of oral origin. We selected three relevant epithelial cell lines to investigate the effect of whole saliva on C. albicans adherence. Protease inhibitors were included in the assay to exclude artefacts resulting from degradation of either salivary or yeast surface proteins by salivary proteases. C. albicans aspartyl proteases (SAPs) are not induced in the minimal medium used to produce our inoculum, and there would have been insufficient time during the course of the assay for SAP induction by saliva.

C. albicans adhered to monolayers of all three epithelial cell lines, in the absence of saliva. There was significantly higher adherence to the HET-1A cell line. Use of these cells presents conditions more approximating those in vivo, since added serum is not required for growth of the cells, and the cells retain normal expression of surface proteins such as cytokeratins (Stoner et al., 1991).

The addition of whole saliva pooled from six donors significantly enhanced the binding of C. albicans yeast cells to epithelial cell monolayers of all three cell lines. The promotion of C. albicans adherence to the HET-1A cell line was less marked than to the other two cell lines, due to higher baseline adhesion, but a significant (P < 0.05) increase was still observed for saliva concentrations of 40% or 60% added to the assay. Pre-treatment of yeast cells with saliva increased adherence potential to all cell lines, and the adherence-promoting activity of saliva was depleted by incubation with a yeast cell suspension. Thus, the adherence-promoting activity of whole saliva apparently resulted from the attachment of saliva components to the yeast cells. Conversely, pre-treating the tissue culture monolayers with saliva did not significantly alter adherence. The components of saliva that promote adherence have not been identified, but the presence of specific IgA against C. albicans antagonized the adherence-promoting effects of such components.

We conclude that factors in whole saliva other than C. albicans-specific IgA promote the adherence of C. albicans yeast cells to cultured epithelial cells, and that the saliva-mediated enhancement of adherence is reduced in the presence of specific salivary IgA against C. albicans. This observation may explain why individuals can remain colonized with C. albicans despite high levels of salivary IgA. The results also demonstrated that the saliva-promoted adherence of C. albicans yeast cells to epithelial cells resulted from the binding of saliva to receptors on the yeast cells rather than to human epithelial cell receptors.


   ACKNOWLEDGMENTS
 
This investigation was funded by a University of Otago Research Grant.

Received February 21, 2001; Last revision November 24, 2001; Accepted November 28, 2001


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IADR Journals Advances in Dental Research ®
Journal of Dental Research ® Critical Reviews (1990-2004)