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Decreased production of proinflammatory cytokines by monocytes from individuals presenting Candida-associated denture stomatitis
Cytokine 77 (2016) 145–151
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Decreased production of proinflammatory cytokines by monocytes from individuals presenting Candida-associated denture stomatitis Karen Henriette Pinke a, Patrícia Freitas a, Narciso Almeida Viera b, Heitor Marques Honório c, Vinicius Carvalho Porto d, Vanessa Soares Lara a,⇑ a
Department of Stomatology, Bauru School of Dentistry, University of São Paulo: Al. Dr. Octávio Pinheiro Brisola, 9-75, Vila Universitária, 17012-901 Bauru, SP, Brazil Clinical Analysis Laboratory, Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo, Bauru, SP, Brazil Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, University of São Paulo: Al. Dr. Octávio Pinheiro Brisola, 9-75, Vila Universitária, 17012901 Bauru, SP, Brazil d Department of Prosthodontics, Bauru School of Dentistry, University of São Paulo: Al. Dr. Octávio Pinheiro Brisola, 9-75, Vila Universitária, 17012-901 Bauru, SP, Brazil b c
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 29 October 2015 Accepted 30 October 2015 Available online 14 November 2015 Keywords: Candida-associated denture stomatitis Elderly Monocytes Cytokines
a b s t r a c t Candida-associated denture stomatitis (DS) is the most frequent lesion among denture wearers, especially the elderly. DS is strongly associated with Candida albicans, as well as local and systemic factors, such as impaired immune response. Monocytes are important in the protective immune response against the fungus by the production of cytokines that recruit and activate leukocytes. There are functional changes in these cells with age, and individual alterations involving monocyte response may predispose the host to developing infections by Candida spp. In this study, our aim was to evaluate the production of TNF-a, IL-6, CXCL8, IL-1b, MCP-1 and IL-10 by monocytes from elderly denture wearers with/without DS and elderly or young non-denture wearers. We detected that monocytes from elderly denture wearers with Candida-related denture stomatitis produced lower levels of CXCL-8, IL-6 and MCP-1. This imbalance in cytokine levels was observed in spontaneous or LPS-stimulated production. Therefore, our data suggested that inherent aspects of the host, such as changes in cytokine production by monocytes, might be associated with the development and the persistence of DS irrespective of aging. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction During aging, the human body undergoes many physiological and morphological changes that affect the quality of life and immunological functions, among them, the complete or partial loss of teeth [1–4]. Apart from problems with esthetics and chewing food, the use of dental prostheses may lead to oral infections, especially in the elderly [2]. Candida-associated denture stomatitis (DS) is the most frequent lesion among prosthesis wearers, especially complete maxillary dentures, and is characterized by inflammation of the denture-bearing mucosa. DS lesions are classified by their clinical characteristics, as proposed by Newton in 1962: Type I – localized inflammation or pinpoint hyperemia, being more common; Type II – diffuse erythema; and Type III – inflammation with papillary hyperplasia (nodular mucosa with hyperemic surface) [5–7]. Although multifactorial and associated with local and systemic factors, such as impaired immune response, the etiology of DS is ⇑ Corresponding author. E-mail address: [email protected] (V.S. Lara). http://dx.doi.org/10.1016/j.cyto.2015.10.017 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
strongly associated with the fungus Candida spp., mainly Candida albicans (C. albicans), present on denture surfaces and/or mucosa [6,8–15]. C. albicans is commonly found in the oral cavity of healthy individuals, whether they wear dentures, or not, however, denture wearing causes increased presence of the fungus in the palatal mucosa [16]. Its change from commensal to pathogen, causing candidiasis, is a complex process and relies on the formation of hyphae and fungal virulence factors, for example, adherence capacity and secretion of enzymes such as proteases and phospholipases [9,17,18]. Host systemic factors related to depressed immune response, such as age, vitamin deficiency, diabetes and use of immunosuppressors favor proliferation of the pathogenic fungus and onset of DS, in addition to decreased salivary flow, smoking, use of antibiotics or drugs with xerostomic side effects. Indeed, the etiology includes local factors associated with dentures, such as trauma, continual use, poor hygiene and improper stability [8,9,19–25]. Considering the cellular response in individuals with DS, some authors have stated the presence of ‘‘suppressor T cells” and defects in the response of phagocytes and lymphocytes favor the onset of DS [24,25]. Similarly, individuals with DS presented
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changes in phenotype and function of salivary and blood neutrophils, including the production of immunomodulatory cytokines [26–30]. Other researchers have argued that the suppression of the immune adaptive cellular response is unlikely to be the direct cause of DS, suggesting that chronic Candida infection can result in such suppression [31]. However, there are no reports about monocytes from patients with DS. Neutrophils and macrophages are the first line of defense against Candida infection and neutrophil and macrophage knockout mice develop several types of oral candidosis [32,33]. While neutrophils are crucial to eliminate the fungi via phagocytosis, macrophages/monocytes are key cells in the development of adaptive cellular response, mainly via cytokine production [32–39]. Although neutrophils are key players in the phagocytosis of Candida spp., Netea et al. showed that macrophages/monocytes are also able to kill yeasts and damage C. albicans hyphae in a similar manner among them, and more efficiently than dendritic cells [40]. Another study reported the ability of monocytes to kill hyphae, but less efficiently than phagocytized yeasts [41]. However, the candidacidal function of monocytes can be modulated by some cytokines [42,43]. Indeed, pro-inflammatory cytokines (IL-1a, IL-1b, CXCL8, IL-6, IL-17, TNF-a and GM-CSF) regulate the proliferation and migration of leukocytes and activate the antimicrobial mechanisms of immune cells. Anti-inflammatory cytokines, such as transforming growth factor-b (TGF-b) and IL10, inhibit pro-inflammatory cytokine secretion and impair the antifungal effector functions of phagocytes [34,38,44–46]. Few studies have evaluated the production of cytokines and DS, however, none of them have focused on their exclusive production by monocytes from elderly patients compared with young persons, as a contribution toward elucidating the phenomena related to Immunosenescence. Previous studies by our group showed alterations in salivary or blood neutrophils from volunteers with DS, such as increased production of IL-4 and IL-10, low GM-CSF levels by neutrophils challenged with C. albicans and impaired phagocytic activity [26,28]. Other alterations such as increased serum IL-4 levels, and reduced salivary IL-12 levels have also been detected in volunteers with DS [30]. Furthermore, it is important to assess whether monocytes from removable denture wearers with DS show functional alterations, which may represent particular systemic aspects associated with susceptibility to the development and persistence of DS in a specific population, irrespective of aging. Thus, we determined patterns of cytokine production (TNF-a, IL-6, IL-1b, CXCL8, MCP-1 and IL-10) by monocytes from elderly volunteers presenting DS and compared them the profile of cytokines produced by elderly or young volunteers without DS.
2. Material and methods 2.1. Study population Volunteers were recruited from the Bauru School of Dentistry, University of São Paulo (USP). The exclusion criteria were: volunteers with any oral lesions induced by microorganisms and with a medical history indicating systemic modifiers of DS and/or immune response, such as: systemic infectious diseases, either acute or chronic; with or without oral manifestation; malignancies; individuals undergoing chemotherapy or radiotherapy; endocrine disorders (diabetes, pregnancy, hypothyroidism, hyperthyroidism); individuals under treatment with antibiotics or antifungal agents; hematologic diseases; autoimmune diseases and other diseases involving the immune system; smokers; individuals being treated with antipsychotics, heavy metals, anticonvulsant and cardiotonic medications. All volunteers were previously informed about the blood sampling and intraoral
examination. Written informed consent was obtained from all volunteers and the research was conducted with the approval of the local Ethics Committee (No 100/2008). The patients with Candida-associated denture stomatitis were selected and classified with regard to the clinical aspect of the lesion, according to the classification proposed by Newton, by clinical evaluation in Undergraduate and Graduate Clinic of the Department of Prosthodontics (FOB/USP) [7]. After screening, the volunteers were divided into four groups, each consisting of 10–18 volunteers, according to the characteristics shown in Table 1.
2.2. Isolation and identification of Candida spp. To confirm the presence of Candida spp. in denture wearers, especially in those with the DS, biological samples were collected from both the palatal mucosa and denture surface using sterile cotton swabs. The samples collected were transferred to test tubes containing Sabouraud Dextrose broth (Difco) (plus chloramphenicol, 50 mg/L) and cultivated at 37 °C for 48–72 h. C. albicans/ dublinienses, C. tropicalis, C. krusei were presumptively differentiated by using CHROMagar Candida (Difco), a selective differential medium [47].
2.3. Monocyte isolation and lipopolysaccharide challenge Monocytes were obtained from the peripheral blood samples of volunteers, as described by Pinke et al. [4]. Briefly, after obtaining the peripheral blood mononuclear cells by cell separation using Histopaque 1083 gradients, 1 106 monocytes were counted by using neutral red (0.02%), and incubated in 24-well culture plates with round microscope coverslips, in 5% CO2, at 37 °C for 2 h. After this, non-adherent cells (lymphocytes) were removed by aspiration. Cell viability was assessed by morphological analysis of the cells using antibodies anti-CD14-FITC and DAPi. Over 95% of viable cells were adherent monocytes that had intensely expressed the CD14 receptor, and were morphologically viable. Monocytes were challenged with 100 ng/mL of lipopolysaccharide (LPS) of Escherichia coli O55B5 (Sigma–Aldrich, St. Louis, MO, USA), in 5% CO2, at 37 °C for 18 h. For spontaneous production, the monocytes were incubated only with medium. Culture supernatants were harvested and stored at 80 °C until assayed.
2.4. Enzyme-linked immunosorbent assay (ELISA) quantitative assay Cell-free supernatants obtained as mentioned were analyzed with BD OptEIATM kits (BD Biosciences) to quantify concentrations of TNF-a, IL-6, IL-1b, CXCL8, MCP-1 and IL-10. Evaluations were performed according to the manufacturer’s instructions. At the end, the results were read in the SynergyTM Mx Monochromatorbased Multi-Mode Microplate Reader (Bio-Tek Instruments) at 450 nm. The sensitivity of the ELISA kits was 5 pg/mL.
Table 1 Classification of volunteers enrolled in the study. Group
Characteristics of volunteers
ElPT/DS
60 years old and above (elderly) and denture wearers for at least two years, with DS 60 years old and above (elderly) and denture wearers for at least two years, without DS 60 years old and above (elderly) and non-denture wearers 25–45 years old (young) and non-denture wearers
ElPT El Yg
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2.5. Statistical methods Statistical tests were performed using the statistical program Statistica 11 (Statsoft Software Inc. Tulsa, Ok, USA), and p values <0.05 were considered statistically significant. To analyze the relationship between DS and the presence of Candida spp. or C. albicans in the palatal mucosa or on the inner surface of the denture, the Fisher exact test was applied. The cytokine concentrations were expressed as mean ± standard deviation of the values obtained for each group and analyzed by two-way ANOVA followed by the Tukey post-test. 3. Results 3.1. Characterization of volunteers: demographic data and microbiological evaluation The demographic characteristics of the age groups were summarized in Table 2. Among ElPT/DS volunteers, only one subject had a lesion classified as Newton type II (10%) and the remaining volunteers presented with Newton type I lesions (90%). The presence of Candida spp. in acrylic resin denture and palatal mucosa was also characterized. As expected, Candida spp. growth was found in all samples from Group ElPT/DS, at least in the palatal mucosa or on the denture surface. In contrast, approximately 24% of the samples of denture wearers without DS (ElPT) showed no presence of Candida spp. C. albicans/C. dubliniensis were the species more commonly found in both groups, totaling approximately 70% (ElPT/DS) and 59% (ElPT) in the palatal mucosa, and 80% (ElPT/DS) and 59% (ElPT) in the dentures evaluated (Table 3). Despite the difference in the percentage of volunteers who showed, or did not show growth of Candida spp., the Fischer Exact test showed no association between matched groups. 3.2. Volunteers with Candida-associated denture stomatitis produced fewer proinflammatory cytokines LPS stimulus induced higher levels of CXCL-8, TNF-a, IL-6 and IL-1b in all age groups; increased IL-10 production, except in ElPT/DS (Fig. 1); and showed no change only in the MCP-1 production by monocytes (Fig. 1E). In comparisons among age groups, we showed decreased spontaneous production of CXCL-8 and MCP-1 by monocytes from volunteers with DS (ElPT/DS). These differences were observed among ElPT/DS and all analyzed age groups, except for MCP-1, which was similar between Yg and ElPT/DS (Fig. 2). Likewise, LPS-stimulated monocytes from ElPT/DS volunteers showed lower values of CXCL8, MCP-1 and IL-6 than those from elderly without DS or young volunteers (Fig. 2). As noted in spontaneous MCP-1 production, the levels produced by monocytes from ElPT/DS or Yg volunteers were also similar. The age groups were statistically similar for production of IL-10 in all matched experimental conditions (Fig. 2F).
Table 2 Demographic data of volunteers in Groups ElPT/DS (elderly denture wearers with Candida-associated denture stomatitis, n = 10), ElPT (elderly denture wearers without Candida-associated denture stomatitis, n = 17), El (elderly non-wearers of denture prosthesis, n = 17) and Yg (young non-wearers of denture prosthesis, n = 18). Groups
Age in years (mean ± SD)
Minimum and maximum age (in years)
Gender
ElPT/DS ElPT El Yg
69.4 ± 5.0 68.9 ± 6.7 66.7 ± 7.1 31.6 ± 6.2
60–79 60–83 60–86 25–45
3 M/7F 1 M/16F 7 M/10F 8 M/10F
M = male and F = female.
Table 3 Percentage of elderly denture wearers with Candida-associated denture stomatitis (ElPT/DS, n = 10) and without disease (ElPT, n = 17), showing different species of Candida, alone or mixed, or showing no fungal growth on the palatal mucosa and inner surface of denture. Presumptive identification was performed by culture medium CHROMagar Candida. C. albicans and C. dubliniensis appeared as green colonies, C. tropicalis and C. krusei colonies as blue and pink colonies, respectively. Species of Candida
ElPT/DS
ElPT
ElPT/DS
ElPT
Palatal mucosa
Denture
albicans/dubliniensis tropicalis krusei albicans/dubliniensis + tropicalis albicans/dubliniensis + krusei tropicalis + krusei albicans/dubliniensis + tropicalis + krusei Without growth
40 10 10 10 10 0 10 10
58.8 5.9 5.9 0 0 5.9 0 23.5
50 10 0 0 10 0 20 10
35.3 11.8 5.9 17.6 5.9 0 0 23.5
Total
100 (%)
100 (%)
100 (%)
100 (%)
No differences were observed between the production of CXCL8, IL-6, MCP-1, TNF-a and IL-1b by monocytes from elderly and young volunteers. 4. Discussion In this study, monocytes from elderly denture wearers with Candida-associated denture stomatitis (DS) presented immunomodulatory aspects not observed among the elderly without the disease, although they were denture wearers. These findings led us to speculate whether inherent aspects not associated with age, could result in individual predisposition to DS, in the presence of local denture- related factors, including Candida spp. in biofilm covering the denture inner surface [10–12,22,48]. Indeed, aged populations undergo physiological changes such as decreased salivary flow and aspects related to immunosenescence that make them more susceptible to the development of DS [20,49]. However, the changes observed in this study were clearly associated with increasing susceptibility to the development of DS due to host factors. In this context, emergent researches have discussed the influence of host genetic polymorphisms in susceptibility to fungal infections, such as vulvovaginal candidiasis [50,51]. The immunomodulatory aspects observed in elderly with DS (ELPT/DS) involved the production of pro-inflammatory cytokines. Briefly, inherent alterations in proinflammatory cytokine production may have a negative effect on antimicrobial defense and contribute to DS development, since Candida spp. and other microorganisms are frequently present in the oral cavity of denture wearers; and the transition from commensal Candida spp. to pathogen is closely linked to the host immune conditions [24,25,31,52–54]. Spontaneous or stimulated CXCL-8 production by monocytes from ELPT/DS was strongly impaired in comparison with young and elderly volunteers without disease, even in denture wearers. CXCL-8, a chemokine produced by phagocytes, activates and recruits neutrophils – key cells during infection by Candida spp. – to the site of inflammation or infection [55–57]. Using CXCR2 / mice (defects in neutrophil trafficking) and neutrophil depletion in IL-17RA / or IL-23 / mice, Huppler et al. showed the importance of neutrophils in protection against oropharyngeal candidiasis, even in the absence of the Th17 response that is also important in antifungal defense and mucosa immunity [58,59]. Th17 response development is influenced by IL-23, IL-1b and IL-6, and only IL-23 was not explored in this study [59]. LPS stimulated IL-6 production by monocytes from ELPT/DS was impaired in comparison with the other groups. As mentioned above, this cytokine is also important in anti-Candida defense,
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Fig. 1. LPS stimulated the production of CXCL-8, TNF-a, IL-6 and IL-1b by monocytes from volunteers with DS, but not IL-10 production. In general, LPS induced increased levels of CXCL-8 (A – p<0.001), TNF-a (B – p<0.001), IL-6 (C – p = 0.03) and IL-1b (D – p < 0.001), while LPS-stimulated MCP-1 levels were similar to spontaneous production, in all age groups (E). After LPS stimulation, only monocytes from healthy volunteers (ElPT, El and Yg) produced increased levels of IL-10 (F – p < 0.001). Two-way Anova followed by Tukey test were applied with a level of significance of 95%. Statistically significant differences were represented with asterisks for comparisons among spontaneous and LPS-stimulated production values.
mainly due its action in developing Th17 response; maintaining the Th17/Th2 balance; and activating neutrophils [60–62]. We found no significant differences in IL-1b production between groups, although we observed a strong tendency toward diminished monocyte production from volunteers with DS. IL-1b stimulates the production of IL-6, and together with TNF-a, increases the production of CXCL-8 [56,63,64]. The production of another important chemokine – MCP-1 – was impaired in ELPT/DS. This molecule recruits monocytes to the site of local infection [41,65], and although it is mainly produced as a result of inflammatory stimuli, it is also a key factor in maintaining homeostatic levels of circulating monocytes by the recruiting these cells from bone marrow [64,66,67]. Thus, impaired production of CXCL-8, IL-1b, IL-6 and MCP-1 may result in poor mononuclear inflammatory infiltrate, reduced recruitment and activation of monocytes and imbalance in Th17 response. Further studies involving this complex network of cells and cytokines are needed
to elucidate these aspects. Indeed, the dysfunctions observed in monocytes from elderly volunteers with DS do not appear to be related to immunosenescence, since healthy elderly subjects responded in a manner similar to that of the young subjects. Lastly, the alterations observed after LPS stimulation were important not only because they showed differential responses inherent to volunteers with DS, but also because the biofilm covering the denture is formed by Candida spp. association with oral bacteria, that modulates the immune responses; microenvironment; and fungal virulence [52,68–71]. Furthermore, differential productions after LPS stimulation in ELPT/DS may also represent alterations in receptors such as TLR4, and other accessory molecules such as LBP and CD14 [72], as well as defects in signaling pathways. Therefore, we could verify the influence of each of the above-mentioned cytokines and the great impact that alterations in production of these mediators may cause on the defense against denture biofilms, and the onset and establishment of DS.
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Fig. 2. Monocytes from patients with Candida-associated denture stomatitis produced fewer proinflammatory cytokines. The spontaneous or LPS-stimulated production of CXCL-8 (A – p < 0.001) and MCP-1 (B – p = 0.006) by monocytes from ElPT/DS was lower than those cells from all healthy volunteers (ElPT, El and Yg) or from elderly, respectively. After LPS stimulation, monocytes from ElPT/DS also produced less IL-6 (C – p = 0.03) than other age groups. Unlike the production of IL-1b (D), TNF-a (E) and IL-10 (F) production was similar between the age groups in all experimental conditions. Two-way Anova followed by Tukey test were applied with a level of significance of 95%. Different letters represent statistically significant differences among age groups for each stimulus.
As regards the immunological aspects of DS, the majority of studies based on saliva or serum samples from DS patients in comparison with healthy volunteers, makes it difficult to perform a reasonable comparison with our study that involved isolated cells in an in vitro system. Discrepant results are observed among these studies. We previously demonstrated that DS patients showed increased levels of IL-6, IL-4, CCL-3 and TGF-b [27,30], and diminished presence of IL-12 in saliva [30]. On the other hand, Pesee & Arpormsuwan found no correlation with pro-inflammatory salivary cytokines in DS patients [73]. In serum, increased levels of IL-6, TNF-a, IL-4 and IL-10, and lower levels of IL-12 [26,30,74] have been shown. In vitro studies about DS are rare in the literature, however they involve cell types other than monocytes, such
as peripheral blood mononuclear cells; blood neutrophils; and oral epithelial cells, demonstrating higher levels of some cytokines [26,75,76]. These discrepancies may be result from differences in the selection and composition of experimental groups. For example, in general, our volunteers with DS (ELPT/DS group) presented Newton’s type I lesions, characterized by low-grade and localized inflammation, while the majority of the published studies were conducted with subjects with different types of lesions, including the most varied forms. Radovic et al. showed that salivary vascular endothelial growth factor (VEGF) levels changed according to the clinical type of lesion in patients with DS [77]. Briefly, our study is the first to report an impaired production of proinflammatory cytokines in vitro by monocytes from DS
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volunteers. These data are biologically relevant, since they demonstrate alterations in important immune cells that were not activated or influenced by locally produced immune factors in patients with DS. These results suggested impairment in the inherent capability of cells that would be recruited to the site of infection. 5. Conclusions In summary, we showed that elderly patients with DS presented altered immunomodulatory function of monocytes, with damaged spontaneous or stimulated production of cytokines. This imbalance in the production of pro- and anti-inflammatory cytokines may represent an individual predisposition to infectious diseases. With specific regard to DS, this predisposition takes on importance in establishing whether the patient presents associated factors other than the local type, since DS has a multifactorial etiology. Once DS has been acquired, this individual response profile contributes to progression and maintenance of the disease. Understanding of the pathogenic mechanisms of the disease is an important step in studies with the goal of improving the treatment of DS. However, further studies are needed for improved understanding of DS, preferably by means of investigations into genetic polymorphisms in both elderly and young populations. Our data could be strengthened if these immunomodulatory changes were also observed in young subjects with DS. However, it is rare to find young edentulous individuals and denture wearers. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements The authors thank the Professor Maria Sueli Parreira, Professor Maria Terezinha Serrão Peraçoli, Dr. James Venturini and Dr. Erika Nakaira Takahagi for their support during the beginning of this research. They also thank Msc. Márcia Sirlene Zardin Graeff, Marcelo Milanda Ribeiro Lopes and Rafaela Alves da Silva Alavarce for their technical support. This study was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil – FAPESP, (Grant Number 2008/03539-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (Process Number 135206/2006-5) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil. References [1] P.E. Petersen, The World Oral Health Report 2003: continuous improvement of oral health in the 21st century–the approach of the WHO Global Oral Health Programme, Commun. Dent. Oral Epidemiol. 31 (Suppl. 1) (2003) 3–23. [2] P.E. Petersen, T. Yamamoto, Improving the oral health of older people: the approach of the WHO Global Oral Health Programme, Commun. Dent. Oral Epidemiol. 33 (2005) 81–92. [3] M.P. do Nascimento, K.H. Pinke, M. Penitenti, M.R. Ikoma, V.S. Lara, Aging does not affect the ability of human monocyte-derived dendritic cells to phagocytose Candida albicans, Aging Clin. Exp. Res. (2015). [4] K.H. Pinke, B. Calzavara, P.F. Faria, M.P. do Nascimento, J. Venturini, V.S. Lara, Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence, Immun. & Age. I & A 10 (2013) 22. [5] F.R. Pires, E.B. Santos, P.R. Bonan, O.P. De Almeida, M.A. Lopes, Denture stomatitis and salivary Candida in Brazilian edentulous patients, J. Oral Rehabil. 29 (2002) 1115–1119. [6] M.H. Figueiral, A. Azul, E. Pinto, P.A. Fonseca, F.M. Branco, C. Scully, Denturerelated stomatitis: identification of aetiological and predisposing factors – a large cohort, J. Oral Rehabil. 34 (2007) 448–455. [7] A.V. Newton, Denture sore mouth, Brit Dent J1962. p. 4. [8] J.D. Shulman, F. Rivera-Hidalgo, M.M. Beach, Risk factors associated with denture stomatitis in the United States, J. Oral Pathol. Med.: Official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology 34 (2005) 340–346.
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Decreased production of proinflammatory cytokines by monocytes from individuals presenting Candida-associated denture stomatitis Karen Henriette Pinke a, Patrícia Freitas a, Narciso Almeida Viera b, Heitor Marques Honório c, Vinicius Carvalho Porto d, Vanessa Soares Lara a,⇑ a
Department of Stomatology, Bauru School of Dentistry, University of São Paulo: Al. Dr. Octávio Pinheiro Brisola, 9-75, Vila Universitária, 17012-901 Bauru, SP, Brazil Clinical Analysis Laboratory, Hospital for Rehabilitation of Craniofacial Anomalies, University of São Paulo, Bauru, SP, Brazil Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, University of São Paulo: Al. Dr. Octávio Pinheiro Brisola, 9-75, Vila Universitária, 17012901 Bauru, SP, Brazil d Department of Prosthodontics, Bauru School of Dentistry, University of São Paulo: Al. Dr. Octávio Pinheiro Brisola, 9-75, Vila Universitária, 17012-901 Bauru, SP, Brazil b c
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 29 October 2015 Accepted 30 October 2015 Available online 14 November 2015 Keywords: Candida-associated denture stomatitis Elderly Monocytes Cytokines
a b s t r a c t Candida-associated denture stomatitis (DS) is the most frequent lesion among denture wearers, especially the elderly. DS is strongly associated with Candida albicans, as well as local and systemic factors, such as impaired immune response. Monocytes are important in the protective immune response against the fungus by the production of cytokines that recruit and activate leukocytes. There are functional changes in these cells with age, and individual alterations involving monocyte response may predispose the host to developing infections by Candida spp. In this study, our aim was to evaluate the production of TNF-a, IL-6, CXCL8, IL-1b, MCP-1 and IL-10 by monocytes from elderly denture wearers with/without DS and elderly or young non-denture wearers. We detected that monocytes from elderly denture wearers with Candida-related denture stomatitis produced lower levels of CXCL-8, IL-6 and MCP-1. This imbalance in cytokine levels was observed in spontaneous or LPS-stimulated production. Therefore, our data suggested that inherent aspects of the host, such as changes in cytokine production by monocytes, might be associated with the development and the persistence of DS irrespective of aging. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction During aging, the human body undergoes many physiological and morphological changes that affect the quality of life and immunological functions, among them, the complete or partial loss of teeth [1–4]. Apart from problems with esthetics and chewing food, the use of dental prostheses may lead to oral infections, especially in the elderly [2]. Candida-associated denture stomatitis (DS) is the most frequent lesion among prosthesis wearers, especially complete maxillary dentures, and is characterized by inflammation of the denture-bearing mucosa. DS lesions are classified by their clinical characteristics, as proposed by Newton in 1962: Type I – localized inflammation or pinpoint hyperemia, being more common; Type II – diffuse erythema; and Type III – inflammation with papillary hyperplasia (nodular mucosa with hyperemic surface) [5–7]. Although multifactorial and associated with local and systemic factors, such as impaired immune response, the etiology of DS is ⇑ Corresponding author. E-mail address: [email protected] (V.S. Lara). http://dx.doi.org/10.1016/j.cyto.2015.10.017 1043-4666/Ó 2015 Elsevier Ltd. All rights reserved.
strongly associated with the fungus Candida spp., mainly Candida albicans (C. albicans), present on denture surfaces and/or mucosa [6,8–15]. C. albicans is commonly found in the oral cavity of healthy individuals, whether they wear dentures, or not, however, denture wearing causes increased presence of the fungus in the palatal mucosa [16]. Its change from commensal to pathogen, causing candidiasis, is a complex process and relies on the formation of hyphae and fungal virulence factors, for example, adherence capacity and secretion of enzymes such as proteases and phospholipases [9,17,18]. Host systemic factors related to depressed immune response, such as age, vitamin deficiency, diabetes and use of immunosuppressors favor proliferation of the pathogenic fungus and onset of DS, in addition to decreased salivary flow, smoking, use of antibiotics or drugs with xerostomic side effects. Indeed, the etiology includes local factors associated with dentures, such as trauma, continual use, poor hygiene and improper stability [8,9,19–25]. Considering the cellular response in individuals with DS, some authors have stated the presence of ‘‘suppressor T cells” and defects in the response of phagocytes and lymphocytes favor the onset of DS [24,25]. Similarly, individuals with DS presented
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changes in phenotype and function of salivary and blood neutrophils, including the production of immunomodulatory cytokines [26–30]. Other researchers have argued that the suppression of the immune adaptive cellular response is unlikely to be the direct cause of DS, suggesting that chronic Candida infection can result in such suppression [31]. However, there are no reports about monocytes from patients with DS. Neutrophils and macrophages are the first line of defense against Candida infection and neutrophil and macrophage knockout mice develop several types of oral candidosis [32,33]. While neutrophils are crucial to eliminate the fungi via phagocytosis, macrophages/monocytes are key cells in the development of adaptive cellular response, mainly via cytokine production [32–39]. Although neutrophils are key players in the phagocytosis of Candida spp., Netea et al. showed that macrophages/monocytes are also able to kill yeasts and damage C. albicans hyphae in a similar manner among them, and more efficiently than dendritic cells [40]. Another study reported the ability of monocytes to kill hyphae, but less efficiently than phagocytized yeasts [41]. However, the candidacidal function of monocytes can be modulated by some cytokines [42,43]. Indeed, pro-inflammatory cytokines (IL-1a, IL-1b, CXCL8, IL-6, IL-17, TNF-a and GM-CSF) regulate the proliferation and migration of leukocytes and activate the antimicrobial mechanisms of immune cells. Anti-inflammatory cytokines, such as transforming growth factor-b (TGF-b) and IL10, inhibit pro-inflammatory cytokine secretion and impair the antifungal effector functions of phagocytes [34,38,44–46]. Few studies have evaluated the production of cytokines and DS, however, none of them have focused on their exclusive production by monocytes from elderly patients compared with young persons, as a contribution toward elucidating the phenomena related to Immunosenescence. Previous studies by our group showed alterations in salivary or blood neutrophils from volunteers with DS, such as increased production of IL-4 and IL-10, low GM-CSF levels by neutrophils challenged with C. albicans and impaired phagocytic activity [26,28]. Other alterations such as increased serum IL-4 levels, and reduced salivary IL-12 levels have also been detected in volunteers with DS [30]. Furthermore, it is important to assess whether monocytes from removable denture wearers with DS show functional alterations, which may represent particular systemic aspects associated with susceptibility to the development and persistence of DS in a specific population, irrespective of aging. Thus, we determined patterns of cytokine production (TNF-a, IL-6, IL-1b, CXCL8, MCP-1 and IL-10) by monocytes from elderly volunteers presenting DS and compared them the profile of cytokines produced by elderly or young volunteers without DS.
2. Material and methods 2.1. Study population Volunteers were recruited from the Bauru School of Dentistry, University of São Paulo (USP). The exclusion criteria were: volunteers with any oral lesions induced by microorganisms and with a medical history indicating systemic modifiers of DS and/or immune response, such as: systemic infectious diseases, either acute or chronic; with or without oral manifestation; malignancies; individuals undergoing chemotherapy or radiotherapy; endocrine disorders (diabetes, pregnancy, hypothyroidism, hyperthyroidism); individuals under treatment with antibiotics or antifungal agents; hematologic diseases; autoimmune diseases and other diseases involving the immune system; smokers; individuals being treated with antipsychotics, heavy metals, anticonvulsant and cardiotonic medications. All volunteers were previously informed about the blood sampling and intraoral
examination. Written informed consent was obtained from all volunteers and the research was conducted with the approval of the local Ethics Committee (No 100/2008). The patients with Candida-associated denture stomatitis were selected and classified with regard to the clinical aspect of the lesion, according to the classification proposed by Newton, by clinical evaluation in Undergraduate and Graduate Clinic of the Department of Prosthodontics (FOB/USP) [7]. After screening, the volunteers were divided into four groups, each consisting of 10–18 volunteers, according to the characteristics shown in Table 1.
2.2. Isolation and identification of Candida spp. To confirm the presence of Candida spp. in denture wearers, especially in those with the DS, biological samples were collected from both the palatal mucosa and denture surface using sterile cotton swabs. The samples collected were transferred to test tubes containing Sabouraud Dextrose broth (Difco) (plus chloramphenicol, 50 mg/L) and cultivated at 37 °C for 48–72 h. C. albicans/ dublinienses, C. tropicalis, C. krusei were presumptively differentiated by using CHROMagar Candida (Difco), a selective differential medium [47].
2.3. Monocyte isolation and lipopolysaccharide challenge Monocytes were obtained from the peripheral blood samples of volunteers, as described by Pinke et al. [4]. Briefly, after obtaining the peripheral blood mononuclear cells by cell separation using Histopaque 1083 gradients, 1 106 monocytes were counted by using neutral red (0.02%), and incubated in 24-well culture plates with round microscope coverslips, in 5% CO2, at 37 °C for 2 h. After this, non-adherent cells (lymphocytes) were removed by aspiration. Cell viability was assessed by morphological analysis of the cells using antibodies anti-CD14-FITC and DAPi. Over 95% of viable cells were adherent monocytes that had intensely expressed the CD14 receptor, and were morphologically viable. Monocytes were challenged with 100 ng/mL of lipopolysaccharide (LPS) of Escherichia coli O55B5 (Sigma–Aldrich, St. Louis, MO, USA), in 5% CO2, at 37 °C for 18 h. For spontaneous production, the monocytes were incubated only with medium. Culture supernatants were harvested and stored at 80 °C until assayed.
2.4. Enzyme-linked immunosorbent assay (ELISA) quantitative assay Cell-free supernatants obtained as mentioned were analyzed with BD OptEIATM kits (BD Biosciences) to quantify concentrations of TNF-a, IL-6, IL-1b, CXCL8, MCP-1 and IL-10. Evaluations were performed according to the manufacturer’s instructions. At the end, the results were read in the SynergyTM Mx Monochromatorbased Multi-Mode Microplate Reader (Bio-Tek Instruments) at 450 nm. The sensitivity of the ELISA kits was 5 pg/mL.
Table 1 Classification of volunteers enrolled in the study. Group
Characteristics of volunteers
ElPT/DS
60 years old and above (elderly) and denture wearers for at least two years, with DS 60 years old and above (elderly) and denture wearers for at least two years, without DS 60 years old and above (elderly) and non-denture wearers 25–45 years old (young) and non-denture wearers
ElPT El Yg
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2.5. Statistical methods Statistical tests were performed using the statistical program Statistica 11 (Statsoft Software Inc. Tulsa, Ok, USA), and p values <0.05 were considered statistically significant. To analyze the relationship between DS and the presence of Candida spp. or C. albicans in the palatal mucosa or on the inner surface of the denture, the Fisher exact test was applied. The cytokine concentrations were expressed as mean ± standard deviation of the values obtained for each group and analyzed by two-way ANOVA followed by the Tukey post-test. 3. Results 3.1. Characterization of volunteers: demographic data and microbiological evaluation The demographic characteristics of the age groups were summarized in Table 2. Among ElPT/DS volunteers, only one subject had a lesion classified as Newton type II (10%) and the remaining volunteers presented with Newton type I lesions (90%). The presence of Candida spp. in acrylic resin denture and palatal mucosa was also characterized. As expected, Candida spp. growth was found in all samples from Group ElPT/DS, at least in the palatal mucosa or on the denture surface. In contrast, approximately 24% of the samples of denture wearers without DS (ElPT) showed no presence of Candida spp. C. albicans/C. dubliniensis were the species more commonly found in both groups, totaling approximately 70% (ElPT/DS) and 59% (ElPT) in the palatal mucosa, and 80% (ElPT/DS) and 59% (ElPT) in the dentures evaluated (Table 3). Despite the difference in the percentage of volunteers who showed, or did not show growth of Candida spp., the Fischer Exact test showed no association between matched groups. 3.2. Volunteers with Candida-associated denture stomatitis produced fewer proinflammatory cytokines LPS stimulus induced higher levels of CXCL-8, TNF-a, IL-6 and IL-1b in all age groups; increased IL-10 production, except in ElPT/DS (Fig. 1); and showed no change only in the MCP-1 production by monocytes (Fig. 1E). In comparisons among age groups, we showed decreased spontaneous production of CXCL-8 and MCP-1 by monocytes from volunteers with DS (ElPT/DS). These differences were observed among ElPT/DS and all analyzed age groups, except for MCP-1, which was similar between Yg and ElPT/DS (Fig. 2). Likewise, LPS-stimulated monocytes from ElPT/DS volunteers showed lower values of CXCL8, MCP-1 and IL-6 than those from elderly without DS or young volunteers (Fig. 2). As noted in spontaneous MCP-1 production, the levels produced by monocytes from ElPT/DS or Yg volunteers were also similar. The age groups were statistically similar for production of IL-10 in all matched experimental conditions (Fig. 2F).
Table 2 Demographic data of volunteers in Groups ElPT/DS (elderly denture wearers with Candida-associated denture stomatitis, n = 10), ElPT (elderly denture wearers without Candida-associated denture stomatitis, n = 17), El (elderly non-wearers of denture prosthesis, n = 17) and Yg (young non-wearers of denture prosthesis, n = 18). Groups
Age in years (mean ± SD)
Minimum and maximum age (in years)
Gender
ElPT/DS ElPT El Yg
69.4 ± 5.0 68.9 ± 6.7 66.7 ± 7.1 31.6 ± 6.2
60–79 60–83 60–86 25–45
3 M/7F 1 M/16F 7 M/10F 8 M/10F
M = male and F = female.
Table 3 Percentage of elderly denture wearers with Candida-associated denture stomatitis (ElPT/DS, n = 10) and without disease (ElPT, n = 17), showing different species of Candida, alone or mixed, or showing no fungal growth on the palatal mucosa and inner surface of denture. Presumptive identification was performed by culture medium CHROMagar Candida. C. albicans and C. dubliniensis appeared as green colonies, C. tropicalis and C. krusei colonies as blue and pink colonies, respectively. Species of Candida
ElPT/DS
ElPT
ElPT/DS
ElPT
Palatal mucosa
Denture
albicans/dubliniensis tropicalis krusei albicans/dubliniensis + tropicalis albicans/dubliniensis + krusei tropicalis + krusei albicans/dubliniensis + tropicalis + krusei Without growth
40 10 10 10 10 0 10 10
58.8 5.9 5.9 0 0 5.9 0 23.5
50 10 0 0 10 0 20 10
35.3 11.8 5.9 17.6 5.9 0 0 23.5
Total
100 (%)
100 (%)
100 (%)
100 (%)
No differences were observed between the production of CXCL8, IL-6, MCP-1, TNF-a and IL-1b by monocytes from elderly and young volunteers. 4. Discussion In this study, monocytes from elderly denture wearers with Candida-associated denture stomatitis (DS) presented immunomodulatory aspects not observed among the elderly without the disease, although they were denture wearers. These findings led us to speculate whether inherent aspects not associated with age, could result in individual predisposition to DS, in the presence of local denture- related factors, including Candida spp. in biofilm covering the denture inner surface [10–12,22,48]. Indeed, aged populations undergo physiological changes such as decreased salivary flow and aspects related to immunosenescence that make them more susceptible to the development of DS [20,49]. However, the changes observed in this study were clearly associated with increasing susceptibility to the development of DS due to host factors. In this context, emergent researches have discussed the influence of host genetic polymorphisms in susceptibility to fungal infections, such as vulvovaginal candidiasis [50,51]. The immunomodulatory aspects observed in elderly with DS (ELPT/DS) involved the production of pro-inflammatory cytokines. Briefly, inherent alterations in proinflammatory cytokine production may have a negative effect on antimicrobial defense and contribute to DS development, since Candida spp. and other microorganisms are frequently present in the oral cavity of denture wearers; and the transition from commensal Candida spp. to pathogen is closely linked to the host immune conditions [24,25,31,52–54]. Spontaneous or stimulated CXCL-8 production by monocytes from ELPT/DS was strongly impaired in comparison with young and elderly volunteers without disease, even in denture wearers. CXCL-8, a chemokine produced by phagocytes, activates and recruits neutrophils – key cells during infection by Candida spp. – to the site of inflammation or infection [55–57]. Using CXCR2 / mice (defects in neutrophil trafficking) and neutrophil depletion in IL-17RA / or IL-23 / mice, Huppler et al. showed the importance of neutrophils in protection against oropharyngeal candidiasis, even in the absence of the Th17 response that is also important in antifungal defense and mucosa immunity [58,59]. Th17 response development is influenced by IL-23, IL-1b and IL-6, and only IL-23 was not explored in this study [59]. LPS stimulated IL-6 production by monocytes from ELPT/DS was impaired in comparison with the other groups. As mentioned above, this cytokine is also important in anti-Candida defense,
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Fig. 1. LPS stimulated the production of CXCL-8, TNF-a, IL-6 and IL-1b by monocytes from volunteers with DS, but not IL-10 production. In general, LPS induced increased levels of CXCL-8 (A – p<0.001), TNF-a (B – p<0.001), IL-6 (C – p = 0.03) and IL-1b (D – p < 0.001), while LPS-stimulated MCP-1 levels were similar to spontaneous production, in all age groups (E). After LPS stimulation, only monocytes from healthy volunteers (ElPT, El and Yg) produced increased levels of IL-10 (F – p < 0.001). Two-way Anova followed by Tukey test were applied with a level of significance of 95%. Statistically significant differences were represented with asterisks for comparisons among spontaneous and LPS-stimulated production values.
mainly due its action in developing Th17 response; maintaining the Th17/Th2 balance; and activating neutrophils [60–62]. We found no significant differences in IL-1b production between groups, although we observed a strong tendency toward diminished monocyte production from volunteers with DS. IL-1b stimulates the production of IL-6, and together with TNF-a, increases the production of CXCL-8 [56,63,64]. The production of another important chemokine – MCP-1 – was impaired in ELPT/DS. This molecule recruits monocytes to the site of local infection [41,65], and although it is mainly produced as a result of inflammatory stimuli, it is also a key factor in maintaining homeostatic levels of circulating monocytes by the recruiting these cells from bone marrow [64,66,67]. Thus, impaired production of CXCL-8, IL-1b, IL-6 and MCP-1 may result in poor mononuclear inflammatory infiltrate, reduced recruitment and activation of monocytes and imbalance in Th17 response. Further studies involving this complex network of cells and cytokines are needed
to elucidate these aspects. Indeed, the dysfunctions observed in monocytes from elderly volunteers with DS do not appear to be related to immunosenescence, since healthy elderly subjects responded in a manner similar to that of the young subjects. Lastly, the alterations observed after LPS stimulation were important not only because they showed differential responses inherent to volunteers with DS, but also because the biofilm covering the denture is formed by Candida spp. association with oral bacteria, that modulates the immune responses; microenvironment; and fungal virulence [52,68–71]. Furthermore, differential productions after LPS stimulation in ELPT/DS may also represent alterations in receptors such as TLR4, and other accessory molecules such as LBP and CD14 [72], as well as defects in signaling pathways. Therefore, we could verify the influence of each of the above-mentioned cytokines and the great impact that alterations in production of these mediators may cause on the defense against denture biofilms, and the onset and establishment of DS.
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Fig. 2. Monocytes from patients with Candida-associated denture stomatitis produced fewer proinflammatory cytokines. The spontaneous or LPS-stimulated production of CXCL-8 (A – p < 0.001) and MCP-1 (B – p = 0.006) by monocytes from ElPT/DS was lower than those cells from all healthy volunteers (ElPT, El and Yg) or from elderly, respectively. After LPS stimulation, monocytes from ElPT/DS also produced less IL-6 (C – p = 0.03) than other age groups. Unlike the production of IL-1b (D), TNF-a (E) and IL-10 (F) production was similar between the age groups in all experimental conditions. Two-way Anova followed by Tukey test were applied with a level of significance of 95%. Different letters represent statistically significant differences among age groups for each stimulus.
As regards the immunological aspects of DS, the majority of studies based on saliva or serum samples from DS patients in comparison with healthy volunteers, makes it difficult to perform a reasonable comparison with our study that involved isolated cells in an in vitro system. Discrepant results are observed among these studies. We previously demonstrated that DS patients showed increased levels of IL-6, IL-4, CCL-3 and TGF-b [27,30], and diminished presence of IL-12 in saliva [30]. On the other hand, Pesee & Arpormsuwan found no correlation with pro-inflammatory salivary cytokines in DS patients [73]. In serum, increased levels of IL-6, TNF-a, IL-4 and IL-10, and lower levels of IL-12 [26,30,74] have been shown. In vitro studies about DS are rare in the literature, however they involve cell types other than monocytes, such
as peripheral blood mononuclear cells; blood neutrophils; and oral epithelial cells, demonstrating higher levels of some cytokines [26,75,76]. These discrepancies may be result from differences in the selection and composition of experimental groups. For example, in general, our volunteers with DS (ELPT/DS group) presented Newton’s type I lesions, characterized by low-grade and localized inflammation, while the majority of the published studies were conducted with subjects with different types of lesions, including the most varied forms. Radovic et al. showed that salivary vascular endothelial growth factor (VEGF) levels changed according to the clinical type of lesion in patients with DS [77]. Briefly, our study is the first to report an impaired production of proinflammatory cytokines in vitro by monocytes from DS
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volunteers. These data are biologically relevant, since they demonstrate alterations in important immune cells that were not activated or influenced by locally produced immune factors in patients with DS. These results suggested impairment in the inherent capability of cells that would be recruited to the site of infection. 5. Conclusions In summary, we showed that elderly patients with DS presented altered immunomodulatory function of monocytes, with damaged spontaneous or stimulated production of cytokines. This imbalance in the production of pro- and anti-inflammatory cytokines may represent an individual predisposition to infectious diseases. With specific regard to DS, this predisposition takes on importance in establishing whether the patient presents associated factors other than the local type, since DS has a multifactorial etiology. Once DS has been acquired, this individual response profile contributes to progression and maintenance of the disease. Understanding of the pathogenic mechanisms of the disease is an important step in studies with the goal of improving the treatment of DS. However, further studies are needed for improved understanding of DS, preferably by means of investigations into genetic polymorphisms in both elderly and young populations. Our data could be strengthened if these immunomodulatory changes were also observed in young subjects with DS. However, it is rare to find young edentulous individuals and denture wearers. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements The authors thank the Professor Maria Sueli Parreira, Professor Maria Terezinha Serrão Peraçoli, Dr. James Venturini and Dr. Erika Nakaira Takahagi for their support during the beginning of this research. They also thank Msc. Márcia Sirlene Zardin Graeff, Marcelo Milanda Ribeiro Lopes and Rafaela Alves da Silva Alavarce for their technical support. This study was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil – FAPESP, (Grant Number 2008/03539-0), Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (Process Number 135206/2006-5) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil. References [1] P.E. Petersen, The World Oral Health Report 2003: continuous improvement of oral health in the 21st century–the approach of the WHO Global Oral Health Programme, Commun. Dent. Oral Epidemiol. 31 (Suppl. 1) (2003) 3–23. [2] P.E. Petersen, T. Yamamoto, Improving the oral health of older people: the approach of the WHO Global Oral Health Programme, Commun. Dent. Oral Epidemiol. 33 (2005) 81–92. [3] M.P. do Nascimento, K.H. Pinke, M. Penitenti, M.R. Ikoma, V.S. Lara, Aging does not affect the ability of human monocyte-derived dendritic cells to phagocytose Candida albicans, Aging Clin. Exp. Res. (2015). [4] K.H. Pinke, B. Calzavara, P.F. Faria, M.P. do Nascimento, J. Venturini, V.S. Lara, Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence, Immun. & Age. I & A 10 (2013) 22. [5] F.R. Pires, E.B. Santos, P.R. Bonan, O.P. De Almeida, M.A. Lopes, Denture stomatitis and salivary Candida in Brazilian edentulous patients, J. Oral Rehabil. 29 (2002) 1115–1119. [6] M.H. Figueiral, A. Azul, E. Pinto, P.A. Fonseca, F.M. Branco, C. Scully, Denturerelated stomatitis: identification of aetiological and predisposing factors – a large cohort, J. Oral Rehabil. 34 (2007) 448–455. [7] A.V. Newton, Denture sore mouth, Brit Dent J1962. p. 4. [8] J.D. Shulman, F. Rivera-Hidalgo, M.M. Beach, Risk factors associated with denture stomatitis in the United States, J. Oral Pathol. Med.: Official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology 34 (2005) 340–346.
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