Expression of Secreted Aspartyl Proteinases in an Experimental Model of Candida albicans-Associated Denture Stomatitis ´ Priscila Lie Tobouti, DDS,1 Ana Regina Casaroto, DDS, PhD,1 Ricardo Sergio Couto de Almeida, PhD,2 3 4 Solange de Paula Ramos, PhD, Thiago Jose´ Dion´ısio, PhD, Vinicius Carvalho Porto, PhD,5 Carlos Ferreira Santos, PhD,4 & Vanessa Soares Lara, PhD1 1

˜ Paulo, Bauru, Brazil Department of Stomatology (Oral Pathology), Bauru School of Dentistry, University of Sao Department of Microbiology, State University of Londrina, Londrina, Brazil 3 Department of Histology, State University of Londrina, Londrina, Brazil 4 ˜ Paulo, Bauru, Brazil Department of Biological Sciences, Bauru School of Dentistry, University of Sao 5 ˜ Paulo, Bauru, Brazil Department of Prosthodontics, Bauru School of Dentistry, University of Sao 2

Keywords Pathogenesis; secreted aspartyl proteinase; virulence; clinical aspect; microscopic aspect. Correspondence Vanessa Soares Lara, ˜ Bauru School of Dentistry, University of Sao Paulo, Department of Stomatology (Oral ´ Pathology), Alameda Octavio Pinheiro Brisola, ´ 9–75, Vila Universitaria, Bauru, SP, 17012–901 Brazil. E-mail: [email protected] ˜ Paulo Research Funded by The Sao Foundation (FAPESP 2008/03539–0) and The Coordination for Enhancement of Higher ˜ Paulo, Education Personnel (CAPES), Sao Brazil. The authors deny any conflicts of interest. Accepted October 1, 2014 doi: 10.1111/jopr.12285

Abstract Purpose: Candida albicans is known to produce secreted aspartyl proteinases (SAPs) to aid adhesion, invasion, and host tissue destruction. SAPs may contribute to denture stomatitis (DS) pathogenesis. The aim of this study was to develop an in vivo experimental model for Candida-associated DS that allows the analysis of SAP2, SAP5, and SAP9 expression by C. albicans from biofilm induced on the denture surface. Materials and Methods: Thirty-five male Wistar rats were divided into three groups: control, denture, and denture/Candida group. The last two groups remained with dentures for 2, 4, and 6 days, with or without induced biofilm. SAP expression was concomitant with leukocyte counts as well as clinical and histological changes shown by animal palate. Results: The signs observed at 4 days in the denture/Candida group were clinically closer to the Candida-associated DS, showing a significant increase of neutrophils and decrease of lymphocytes in peripheral blood, presence of inflammation signs on the palate similar to DS Newton type I, and fungal invasion in the epithelial layer. Accordingly, the denture/Candida group at 4 days presented the highest relative expression of all SAPs studied. Conclusion: The results showed a coincidence between SAP expression and clinical, microscopic, and blood data. Finally, the molecular findings were consistent with the virulence capacities of C. albicans from biofilm formed on the denture resin, which possibly allowed epithelial invasion by the fungus.

Denture stomatitis (DS) is an inflammatory reaction of the alveolar mucosa underlying removable dental prostheses1 and is the most common form of oral candidiasis.2,3 This disease has a multifactorial etiology, and poor hygiene and continuous denture wearing have been the most frequently associated factors.1 The human pathogenic fungus Candida albicans is commonly found in the biofilm formed on the palatal surface of the acrylic denture, supporting the theory that the maxillary denture acts as a reservoir enabling this fungus to reinfect the mucosal surface continually, thus contributing to DS pathogenesis.4 Moreover, the fungi present in these biofilms are able to withstand host defenses and survive antifungal therapy.5 It has been shown that adhesion of C. albicans to acrylic surfaces and development of biofilm could be influenced by various factors, such as the

presence of other microorganisms and diets rich in sucrose.6 Additionally, other factors are important to adhesion of this fungus, such as hydrophobic interactions and surface roughness, which provide a greater surface area and protected sites for colonization.7 C. albicans is a commensal fungus constituent of the normal mucosal microbiota in humans.8-11 However, under suitably predisposing conditions, it is able to cause several mucosal diseases such as DS.12,13 The morphological switch from yeasts to filamentous hyphae is crucial for C. albicans pathogenicity in addition to the virulence attributes associated with the transition.14-16 Environmental changes such as range of oxygen and carbon dioxide levels, pH, temperature, and nutrient availability contribute to the fungal morphologic transition.14 These

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changes are also observed in biofilms of continuous denture wearing patients with poor hygiene conditions.2 Furthermore, other factors have been suggested to be virulence attributes of C. albicans that assist in its ability to colonize host tissues, cause infections, and overcome host defenses.17 Among these virulence factors, the secreted aspartyl proteinases (SAPs) have been the most widely studied5 besides the hyphal formation.17,18 SAPs are extracellular hydrolytic enzymes considered key virulence determinants of C. albicans. They are encoded by a family of 10 genes (SAP1 to SAP10) that can be expressed under a variety of in vitro and in vivo environmental conditions. SAP2, SAP5, and SAP9 play crucial roles during the different types/stages of fungal infections.5 SAP2 is highly expressed in acidic pH and has the capacity to cleave many human proteins such as extracellular matrix, collagen, laminin, fibronectin, mucin, and immunoglobulins,19 and is the most expressed SAP in an in vitro model.20 SAP5 is expressed more under neutral pH and seems to degrade E-cadherin molecules, facilitating mucosal colonization, penetration, and infection by C. albicans.20,21 On the other hand, SAP9 expression seems to be pH-independent, occurring in both pathogenic and commensal stages. It is found on the cell surface and contributes to cell wall integrity.22,23 Moreover, SAP5 and SAP9 are highly expressed by Candida in patients with oral and vaginal candidosis.20 Studies involving the biofilm process on dentures have been primarily limited to in vitro models,24-28 which consequently deprive the environment of important factors, such as salivary immune components that may influence SAP expression and biofilm growth.29,30 Therefore, although the expression of different types of SAPs by C. albicans has been widely investigated, the majority of these studies involve in vitro models or immunocompromised patients.31-34 Thus, in this study, we developed an in vivo experimental model for Candida-associated DS to analyze SAP2, SAP5, and SAP9 expression by C. albicans from biofilm induced on the denture surface. Finally, we compared the results with leukocyte counts as well as clinical and histological changes on the rat palate.

Materials and methods

chloramphenicol (Sigma Chemical Co., St. Louis, MO) at 37°C. After 24 hours, 0.1 ml broth samples were plated onto CHROMagar Candida plates to quantify possible colony-forming units (CFU). None of the samples of the animals included in the study showed C. albicans or nonalbicans CFU before the infection. Rats were divided into animals without a denture base made of acrylic resin (control group, n = 5), animals with a denture base without C. albicans (denture group, n = 15), and animals with denture base with C. albicans adhered on its internal surface (denture/Candida group, n = 15). The last two groups remained with dentures for 2, 4, and 6 days (n = 5 animals for each experimental time). Dentures stayed in contact with the oral mucosa area throughout the study. This experimental model of Candida-associated DS was based on an adaptation of the method by Nett et al29 obtaining a more faithful model to the reality of the dentures of edentulous patients. As a parameter of SAP production by the biofilm before contact with the oral environment (90 minutes), two denture bases with C. albicans grown on the internal surfaces were not placed in the animals, and were subjected to analysis of SAP expression immediately after the fungal adhesion, being considered and referred to as 0 day. Denture fabrication

Initially, one preliminary dental impression tray was fabricated for each rat. The animals had the palate impressed by polyether impression material (Impregum Soft; 3M ESPE, Sumar´e, Brazil) under anesthesia with equal parts ketamine hydrochloride (100 mg/ml, Dopalen; Vetbrands, Jacare´ı, Brazil) and xylazine hydrochloride (20 mg/ml, Anasedan; Vetbrands) at 1 ml/kg. Then, the individual denture base for each rat was made from the replication molds of type IV dental stone. To hold the denture to the palate, stainless steel wires (5 cm in length, 0.2 mm in diameter) were fixed in the incisive region by heat-cured resin (Lucitone 550; Dentsply International Inc., York, PA). Additionally, two holes were made in the first and second molar region of the plaque. The dentures remained in distilled water for 2 days at 37°C to release the monomer35 and were dried and sterilized with ethylene oxide.

Animals

Thirty-five male nonimmunosuppressed Wistar rats (Rattus novergicus albinus), weighing from 300 to 350 g, and 3 months old, were purchased from the State University of Londrina (UEL, Londrina, Brazil) and were housed under specific pathogen-free conditions in routinely cleaned cages. Animals were maintained in accordance with the UEL Care Criteria in an environmentally controlled room, after approval by the Institutional Animal Care Committee (150/2010). In addition to water ad libitum, the animals were fed by a liquid diet, supplemented with vitamins and amino acids (Glicopan Pet; Vetnil, Louveira, Brazil). As inclusion criteria, the animals had to be C. albicansnegative before the denture placement. Therefore, rats had their salivary samples collected by sterile swabs incubated in Sabouraud dextrose broth (Difco; Becton, Dickinson, Le Pont de Claix, France) supplemented with 0.1 mg/ml

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Adhesion of C. albicans to the denture base

C. albicans strain SC5314 was used for this study. Prior to adhesion of the fungus on the internal surface of the denture base, fungal cells were grown at 30°C in trypticase soy broth medium with orbital shaking at 180 rpm overnight to obtain stationary phase cells.20,29 The yeast cells were washed with phosphatebuffered saline (PBS), counted with a hemocytometer and reR , New York, NY) at suspended in RPMI1640 medium (Gibco 7 1 × 10 cells/ml. To allow C. albicans adhesion on the denture internal surface, the denture bases of the denture/Candida group and 0 day were added in six-well tissue culture plates with each well containing 1 × 107 yeast cells/ml suspension in RPMI medium as described above. After incubation at 37°C for 90 minutes with shaking at 75 rpm,32 the dentures were washed with PBS to remove the cells that did not adhere to the acrylic.

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Table 1 Primer sequences used in reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for SAP2, SAP5, and SAP9 expression by C. albicans of biofilm formed on the dentures Target SAP2 SAP5 SAP9 ACT1

FP TCCTGATGTTAATGTTGATTGTCAAG CATTGTGCAAAGTAACTGCAACAG ATTTACTCCACAGTTTATATCACTGAAGGT GACAATTTCTCTTTCAGCACTAGTAGTGA

RP TGGATCATATGTCCCCTTTTGTT CAGAATTTCCCGTCGATGAGA CCACCAGAACCACCCTCAGTT GCTGGTAGAGACTTGACCAACCA

FP, forward primer; RP, reverse primer; ACT1, reference gene.

Denture base placement

Rats were anesthetized, and two stainless steel wires (5 cm in length, 0.25 mm in diameter) were threaded between the first and second molars of both sides of the upper maxilla, and through the holes in the posterior region of the denture. The wire ends were tied together, the excess was cut off, and the tips were covered with self-cured resin to protect the tongue of the animal. The wires fixed in the incisive region of the denture were threaded between the incisors of the animal and tied together with self-cured resin. Blood analysis, clinical and histopathological study

After the experimental times of 2, 4, and 6 days, denture bases with C. albicans were removed and immediately frozen in liquid nitrogen for later analysis of SAP expression. Dentures from the group without C. albicans were swabbed and seeded in CHROMagar Candida to verify the absence of contamination as described before. Moreover, all animals had their tongue and palate clinically evaluated for inflammation signs. The DS was assessed according to Newton’s classification as Newton Type I, petechiae dispersed throughout all or any part of the palatal mucosa in contact with the denture; Newton Type II, macular erythema without hyperplasia; or Newton Type III, diffuse or generalized erythema with papillary hyperplasia.36 Under anesthesia, a blood sample obtained by cardiac puncture of each animal was taken in a vacuum blood collection tube with EDTA (K3EDTA; VAcuette, Americana, Brazil), and sent to the Veterinary Hospital of the State University of Londrina for leukocyte counting. After euthanasia, the palate of each animal was dissected for microscopic analysis. All specimens were fixed in 10% buffered formalin, embedded in paraffin and stained with hematoxylin and eosin, and periodic acid-Schiff (PAS) stain. The palate was cut in two halves in the median plane to the microscopic view, and not from the tissue close to the stainless steel wires, thus discarding tissue alterations related to trauma caused by the wires. Microscopic data, including epithelial tissue and inflammatory infiltrate features as well the presence, distribution, and morphology of fungal cells, were qualitatively evaluated by two calibrated observers. Real-time PCR analysis of SAP2, SAP5, and SAP9 expressions

Nitrogen-frozen biofilm cells were dislodged from denture bases by scraping, and fungal total RNA from denture/Candida group and 0 day denture was isolated using the RiboPure-Yeast

kit (Ambion, Inc., Austin, TX) following the manufacturer’s instructions. A pool of total RNA from each group as well as 0 day denture was quantified in a spectrophotometer (NanoDrop ND 1000; UV-Vis, Wilmington, DE). RNA was treated for 30 to 45 minutes with DNA-free DNase (Ambion, Inc.). Purified RNA from each sample was confirmed DNA-free by the absence of an amplified product after real-time PCR using C. albicans primers (Table 1). Complementary DNA (cDNA) was generated from total RNA through reverse transcription (RT) following the manufacturer’s instructions (QuantiTect Reverse Transcription kit; Qiagen, Hilden, Germany). The mRNAs of SAP2, SAP5, and SAP9 were quantitatively assessed by quantitative real-time polymerase chain reaction (qPCR) using the fluorophore labelR R ing cDNA by SYBR Green I (SYBR Green Master Mix ; Applied Biosystems, Grand Island, NY). Primer specificities were confirmed at NCBI-GenBank databases (National Center for Biotechnology Information; Table 1). Each reaction mixture consisted of fluorophore mix, 0.6 μM of forward and reverse primers, pure water, and 2 μl of cDNA in a final volume of 12 μl. Following RT and initial denaturation at 95°C for 10 minutes, thermoycling conditions were 95°C for 15 seconds followed by 60°C for 1 minute for 45 cycles. Negative (water) and positive (C. albicans SC5314 DNA) controls were included in each run. SAP2, SAP5, and SAP9 were normalized to the housekeeping gene ACT1, and analyzed using the comparative Ct method (Ct). Relative expression of SAPs by biofilm at 0 day and different times of denture use was determined. In addition, SAP relative expression was compared with 0 day and SAP2 in all experimental times studied. Statistical analysis

The blood data were analyzed via two-way ANOVA and Tukey’s test at a 5% significance level (STATISTIC v.7; IBM Software, Armonk, NY).

Results Denture stomatitis changes neutrophil and lymphocyte percentages in blood

Blood leukocyte counts such as neutrophils and lymphocytes demonstrated significant differences on the 4th day (Table 2). An increase in neutrophil percentage was observed in the denture/Candida group when compared with the control (p = 0.03) and denture groups (p = 0.04). On the other hand, lymphocyte percentage decreased in the denture/Candida group when

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Table 2 Leukocyte counts presented by animals after the use of dentures with biofilm of C. albicans in different experimental times (average ± standard deviation) Blood cells Leukocytes (cells/mm3 )

Neutrophils (%)

Lymphocytes (%)

Control group

Days

8275.0 ± 2947.74

2 4 6 2 4 6 2 4 6

25.0 ± 6.48

73.0 ± 7.79

Denture group 5762.5 7283.33 5766.67 30.75 28.0 39.17 68.5 72.0 60.17

± ± ± ± ± ± ± ± ±

1025.81 1575.07 1925.53 1.50 7.21 17.22 1.92 7.21 17.92

Denture/Candida group 6037.5 8780.0 6270.0 26.75 54.4 46.0 73.0 44.4 53.75

± ± ± ± ± ± ± ± ±

1881.21 2651.79 1257.48 7.54° 25.79∗ # 12.73 8.04 ° 25.24∗ # 12.97

°

°

Two-way ANOVA, Tukey’s test; symbols represent p ࣘ 0.05. *Experimental groups compared with respective control groups. # Denture/Candida groups compared with respective denture groups, at the same period. °Comparison between the different experimental times for each variable of blood cells, separately.

signs in any animal involved in this study. In addition, all rats showed healthy hair and no hair loss with the exception of the denture/Candida group on the 6th day after infection (data not shown). Microscopic examination of the palatal mucosa surface of the denture/Candida group revealed hyperplastic stratified squamous epithelium with hyperorthokeratinization and exophytic papillary projections, mainly at 4 and 6 days. Only on the 4th day of infection were Munro’s microabscess (neutrophils among epithelial cells) and tissue invasion by C. albicans hyphae on the superficial epithelial layer PAS-positive (Fig 2) observed. Connective tissue presented an increase of the mononuclear inflammatory infiltrate over time. The palatal mucosa surface of the control and denture groups demonstrated epithelial and connective tissue without inflammatory features or fungus presence (negative PAS; data not shown). Quantitative analysis of SAP2, SAP5, and SAP9 expression of denture/Candida and 0 day groups Figure 1 Clinical images of the denture/Candida group at 4 days of use of the denture with biofilm of C. albicans. Inflammation signs on the hard palate similar to denture stomatitis (black arrows).

compared with the other groups (p = 0.03). As far as the experimental periods, neutrophil percentage was higher at 4 days than at 2 days (p = 0.04), whereas the opposite was observed for lymphocytes (p = 0.03; Table 2). There were no statistically significant differences between the absolute values of leukocytes. Analysis of histopathological changes caused by C. albicans

Clinically, inflammation signs on the palate similar to DS Newton Type I were observed only in the denture/Candida group on the 4th day (Fig 1). There were no tongue disease

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To evaluate proteinase expression, during DS in vivo, cDNA was generated from isolated total RNA from denture biofilms through RT reaction. Gene expression was quantified by qPCR. SAP9 relative expression from Candida biofilm of 0 day was higher than the expression of the other SAPs. After 2 days of denture use, the fungal biofilm in the denture/Candida group presented changes in SAP expression. SAP5 followed by SAP9 expression had a relative increase when compared with SAP2. On the 4th day, mRNA relative expression was similar for all SAPs analyzed, whereas on the 6th day, results were similar to 0 day (Fig 3). The highest relative expression of SAP2, SAP5, and SAP9 occurred on the 4th day when compared with 0 day. Although the results are relative expressions, SAP2 expression was the lowest when compared with the other SAPs, regardless of the period the rats used the acrylic dentures (Fig 4). The efficiency of the PCR with the primers used in this study (ACT1, SAP2, SAP5, and SAP9) was 98%.

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Figure 2 Histopathological features of the palatal mucosa surface of the denture/Candida group at 4 days of use of denture with biofilm of C. albicans. (A), (B) hyperplastic stratified squamous epithelium with Munro’s microabscess (black arrows) and tissue invasion by Candida hyphae on the superficial epithelial layer (red arrow; PAS stain).

Discussion

Figure 3 SAP5 and SAP9 relative expression by C. albicans of biofilm formed on the denture when compared with SAP2. (A) 0 day, (B) Denture/Candida at 2 days, (C) Denture/Candida at 4 days, and (D) Denture/Candida at 6 days.

The rat denture model developed in this study mimicked human Candida-associated DS29,37 in terms of Candida biofilm formation, host factors, and anatomic location. Therefore, it allowed the investigation of SAP virulence factors produced by the fungus in the form of biofilm attached on the acrylic resin that stayed in the oral microenvironment for different periods. Proteinases studied in this work are known to be highly expressed in vitro (SAP2)5,28 and in patients (SAP538 and SAP920 ). The first in vivo study detecting C. albicans mRNA from whole saliva of patients with oral C. albicans infection indicated that the pathogenesis of Candida-associated DS may be associated with the differential expression of individual SAP genes.38 Furthermore, the significant differences in SAP activity between C. albicans strain isolates from diseased and healthy patients suggested a correlation between in vitro biofilm-associated proteinase activity and severity of disease.28 However, given the clinical spectrum of disease for patients with DS, the salivary flow or presence of biofilm alone does not explain the clinical presentation, since it is clear that host immune factors are likely to play a role in the pathogenesis.28,29 Similarly, an alternative model also used rats with molded acrylic plates29 but with limitations. This model was not designed to make an individual denture base for each rat that resulted in variability in the placement of the devices, damaging the contact between the denture and palate. In addition, prior to placement of denture appliances, the rats were immunosuppressed.29 Since there is no scientific evidence that DS is associated with invasive infection in humans, unlike in the previous model cited above,29 our model used nonimmunosuppressed animals, ensuring the participation of the host immune response during rat mucosal surface colonization with Candida. We highlight that in this model, thermal-cured rather than self-cured resin was used for denture fabrication, since the latter leads to an inflammation of the palate by toxic monomer release.39 Moreover, the denture was fabricated from materials currently used in clinical dentistry and was custom-fitted to each animal to facilitate optimal contact between the denture and palate.37 Therefore, through this model we could relate SAPs analyzed with leukocyte counts as well as clinical and histopathological features of the palate. The results showed a concordance of the molecular findings with clinical, microscopic, and blood aspects. After 4 days of use of the contaminated denture, the palate of animals and their

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Figure 4 SAP2, SAP5, and SAP9 relative expre– ssion by C. albicans of biofilm formed on the denture of denture/Candida at 2, 4, and 6 days when compared with 0 day.

blood cell counts demonstrated signals of disease, including fungal invasion in epithelial layer. Overall, following this period, the denture/Candida group had a significant increase in neutrophils and a decrease in lymphocytes in the peripheral blood compared with control. The presence of inflammation signs on the palate such as small erythematous spots similar to DS Newton type I in humans, and presence of Candida in the epithelial superficial layers as well as fungus invasion of the epithelial tissue were also observed. These findings are highly associated with Candida colonization rather than a response to acrylic and/or ligatures, since this was not observed in the denture group. Probably, this period of use was the one that most likely mimicked the Candida-associated DS. In accordance, the denture/Candida group at day 4 presented the highest relative expression of all studied SAPs (SAP2, SAP5, and SAP9) when compared with 0 day (i.e., acrylic dentures with C. albicans on the surfaces but without interaction with the oral microenvironment). These results reinforce the view that C. albicans produces higher levels of SAP2, SAP5, and SAP9 when in the oral cavity than on acrylic surfaces not placed in the animals, thus suggesting a virulence capacity of the fungi present in the biofilm on the internal surfaces of the complete denture. This aspect highlights the need for proper hygiene or disinfection of this complete denture. Moreover, in this study, the molecular results regarding SAP’s relative expression were consistent with the different virulence capacities of C. albicans, which formed a biofilm on the denture resin and possibly invaded the adjacent epithelial tissue. SAP9 was the most expressed proteinase by C. albicans at 0 day, whereas SAP5 was the least expressed enzyme. These results are coherent with the tendency of the fungus to adhere to dentures, since SAP9 contributes to the process of fungal adhesion to acrylic resin.22,23,40 On the other hand, SAP5 contributes to degrade E-cadherin in the oral mucosa in order to invade the lining epithelium.21,41 Therefore, the increase of SAP5 expression in the denture/Candida group at 0 day and 2 days when compared with SAP2 suggests the fungal adherence in the oral environment and the change of virulence factor synthesis by C. albicans.22,23 As SAP5 is a strongly hyphal-associated gene, the 132

high levels of SAP5 expression observed may indicate hyphal formation and thus higher adhesion and invasion potential. As already mentioned, on the 4th day of use of a contaminated denture, a significant increase in neutrophil percentage was observed in the serum of the animals when compared with the other groups. These results corroborate the activation of the innate immune response to Candida,42 since fungal factors and chemical mediators locally released can stimulate neutrophil recruitment. Furthermore, the serum findings observed here can be directly related to the presence of neutrophils among epithelial cells and microabscess of Munro, detected in palatal samples from rats of the denture/Candida group after 4 days, thus representing an acute inflammation. Also, in another study, the presence of neutrophils and microabscess of Munro in animal samples of DS was verified.29 Those cells are the first to migrate to the site of infection, and their function includes phagocytosis and activation of other cells involved in the elimination of C. albicans, therefore collaborating for infection elimination and epithelium homeostasis.43 Abscess of Munro is very common during an oral Candida infection, such as DS. Neutrophils are the central weapon of the innate immune system for rapid clearance of infecting microorganisms from the blood and have been shown to efficiently repress the yeast-to-hyphal morphological transition, thus counteracting the transcriptional program associated with C. albicans morphogenesis and pathogenesis.14 Also, its dysfunction or numeric alteration can lead to a predisposition to an infection. A previous report suggested that a deficiency in neutrophil function may play a role in DS, because a decreased phagocytosis against Candida was observed in systemic neutrophils from elderly denture wearers. Additionally, a lower number of neutrophils were found in the saliva of Candidarelated DS patients in comparison with healthy subjects.44 In this work, after 6 days of acrylic denture use, microabscesses of Munro and presence of fungus in the epithelium were no longer observed in any of the groups. A desquamative superficial epithelium layer was noticed in the denture/Candida group besides a hyperplasic and hyperkeratinized epithelium. Desquamation of the superficial epithelial cells may help in the

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elimination of the fungus adhering to the surface, thus representing an important mechanism against Candida infection that prevents fungus invasion of the connective tissue.45 Moreover, SAP expression on the 6th day of use of contaminated dentures was similar to 0 day, suggesting that the animal immune system was able to change the production of these important virulence factors, leading to regression of the inflammatory process, in addition to epithelial desquamation. With regard to our SAP analysis by RT-qPCR, RNA extraction from C. albicans was difficult mainly on the 6th day (data not shown). In this period, we believe that the epithelial response acted together with the immune system response against the fungus, justifying the small amount of fungal RNA extracted and corroborating the clinical, microscopic, and leukocyte counts results. This model was an appropriate representation for acute cases, since analysis on the 6th day showed neutrophils among epithelial cells and microabscess of Munro in the denture/Candida group. Investigation including a prolonged time course experiment may be necessary to represent a chronic inflammation; however, longer experimental periods were tested in previous experiments (data not shown), but the animals had become debilitated with tongue inflammatory signs in these periods. All rats showed healthy hair, no signs of tongue disease, and continued a normal food and water intake until the 6th day, justifying the experimental period analyzed. Furthermore, despite representing only an acute inflammation, the model mimicked human Candida-associated DS in terms of Candida biofilm formation, host factors, and anatomic location. The various microscopic alterations observed after the different periods of contact between epithelial surface and Candida in this experimental model suggest a defensive epithelial response against the fungus, since such alterations were not seen in the denture or control group. In fact, epithelium is the first physical barrier and has an immune response against pathogens. Therefore, epithelial alterations observed in this DS model may represent important aspects of innate immunity in response to Candida. Protection seems to depend on the epithelial hyperplasia, keratinization, and desquamation,18,32 in addition to superficial recruitment of leukocytes such as polymorphonuclears.39

Conclusions Within the limitations of this study, the following conclusions were drawn: 1. Taking together the clinical, microscopic, and serum leukocyte results, there was an increase in the palatal inflammatory response over time of use of denture bases contaminated with C. albicans. 2. SAP expression findings were consistent with the different virulence capacities of C. albicans to form a biofilm on the denture resin and possibly invade the epithelial tissue. 3. The concordance of the molecular findings with clinical, microscopic, and blood aspects observed at 4 days demonstrated that this experimental animal model can be used for further experiments such as drug resistance, biofilm analysis, immune cells, and proteomic findings.

SAP Expression in an In Vivo Model of DS

Acknowledgments The authors would like to thank Dr. Emerson Jos´e Venˆancio and Dr. Luciana Rezende Pinto for the support and advice. We thank Rafaela Alves da Silva Alavarce, Marcelo Milanda, F´atima Aparecida Silveira, and Marcia Graeff for excellent technical assistance.

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C 2015 by the American College of Prosthodontists Journal of Prosthodontics 25 (2016) 127–134 

Expression of Secreted Aspartyl Proteinases in an Experimental Model of Candida albicans-Associated Denture Stomatitis.

Candida albicans is known to produce secreted aspartyl proteinases (SAPs) to aid adhesion, invasion, and host tissue destruction. SAPs may contribute ...
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