Table 2. Salivary Cytokines levels in control and DS groups.
Figure 2. Salivary C. albicans prevelance according to DS severity.
Table 3. Organisms detected with DNA-DNA checkerboard of biofilm from denture surfaces. Numbers reflect counts × 106 CFU.
4.3. Serum CRP
There was no significant difference in the serum CRP level comparing health to DS individuals (p = 0.74). We also examined levels of clinically elevated CRP values using CRP < 3 mg/ml as a threshold, and no statistical differences between the groups could be detected (Table 4).
We investigated levels of the selected markers: IL-1α, IL-1b, IL-6, IL-8, and TNFα in saliva and found a statistically significant two fold increase in median salivary levels of IL-8 (p = 0.04) and a 1.8-fold increase in IL-1β. These increases are in accordance with other studies that looked at these pro-inflammatory cytokines in oral epithelial cells infected with C. albicans [17-19]. Increased levels of GM-CSF, IFN-γ, TNF-α IL-8, IL-18 and IL-1α, IL-1β, IL-6, have been identified in the oral mucosa epithelial cell infection models and saliva of subjects with oral candidiasis and in experimental models of oral candidiasis [11,18,19]. Recognition of C. albicans by oral epithelial cells through specific patternrecognition receptors (PRRs) leads to activation of intracellular signaling pathways that modulate inflammatory responses including release of cytokines such as IL-1α, IL-6, IL-8, IL-15, IL-18, TNFα as well as upregulation of synthesis of specific antimicrobials such as defensins and nitrous oxide (NO) . These mediators could also exhibit a regulatory effect on the secretion of each other, for example TNF-α, which showed a, increase, albeit no significant, in the DS group is known to activate secretion of other cytokines, such as IFN-γ, IL-1, IL-6, and IL-12 which further amplifies the protective immune response to C. albicans infection [19,20]. Furthermore, IL-1β which was significantly increased in the DS group,
Table 4. CRP levels in control and DS groups.
is known to play a crucial role in activating production of IL-1inducible cytokines, which attract and activate cells of the immune and inflammatory systems, e.g., IL-6, IL-8, granulocyte-macrophage (GM)-CSF, and G-CSF . We found a significant association between the salivary counts of C. albicans and the levels of IL-8 and IL-1α (Figure 2). We have recently found regarding C. albicans colonization in denture stomatitis patients, that the denture appears to be the primary source of salivary C. albicans rather than the inflamed mucosa. C. albicans was detected in saliva of 80% (12/15) of the DS subjects, but only 41.2% (7/17) of the Healthy subjects, (p = 0.03). Thus, C. albicans was twice as likely to be present in saliva if mild or severe DS was present. Therefore, the high levels in saliva likely represent the organisms released from denture surfaces with minor contribution from the mucosal surfaces. IL8 levels in saliva ranged from 447 - 1970 ng/ml in the DS group, this cytokine acts as a potent chemokine for neutrophils at nanomolar concentrations, thus this increase in IL-8 levels would lead to neutrophil recruitment within the inflamed mucosa and passage into the saliva via transepithelial migration. Such an increase in neutrophils within the saliva has previously been reported in mucositis and in DS subjects [11,18,22].
It is well understood that adequate attachment of candidal organisms to denture surface is a crucial step in the pathogenesis of denture stomatitis. The formation of biofilms on the surface of the denture protects these organisms from detachment and removal by salivary flow and physical forces [4,14]. It’s also worth noticing that quorum sensing (QS) occurs both in single species bacterial communities and in complex mixed bacterialyeast communities [23,24]. However, the role played by saliva or serum pellicles during the colonization process and subsequent multilayer biofilm formation is poorly understood [12,24-26].
Measuring the levels of 16 dental biofilm organisms by DNA-DNA checkerboard in the mucosa and dentures, only samples collected from dentures were within the detectable range of 1 × 104 counts, however, organisms could be detected in the biofilm samples collected from dentures in three control cases, 1-DS Type 2, and 1-DS Type 3 patients and there were no significant differences in the counts comparing Health to Diseased samples. Those findings suggest that a more sensitive method, like 16 s RNA, should be utilized to better assess changes in the bacterial mucosal bacterial flora when comparing DS and control samples [27-33].
Sachdeo et al.  have used a similar methodology to the one performed in our study to characterize the microbiota of edentulous oral cavities using checkerboard DNA-DNA hybridization, analyzing samples from mucosal and denture surfaces, and saliva from edentulous patients for 41 bacterial species where they found that A. actinomycetemcomitans and P. gingivalis, which were thought to be eliminated with the extraction of all natural teeth, were seen in significant numbers in the edentulous subjects. These pathogens were detected in our study as well. Also, when those authors compared the mean total DNA probe counts among the dorsal surfaces of the tongue, denture palates, and the subjects’ palates, the highest mean counts were found on the tongue dorsum, followed by the polished (exterior) surface of the denture palate, and were lowest on the hard palate. This finding is also consistent with our study where no organisms were detected from the mucosal samples. However, no samples were taken from the polished surfaces of the denture in our samples, as we collected the tissue-bearing surface.
Other studies have shown that the predominant cultivable flora of denture plaque was Gram-positive cocci (Streptococci) and rod-shaped bacteria [35-37]. Results from such studies suggest similarity between denture plaque bacterial composition and the periodontal niche in dentate patients. Therefore, consistent with the association of periodontal disease and specific periodontal pathogens on conditions such as cardiovascular disease and poor glycemic control among diabetics it has been suggested that denture cleaning is important to oral and systemic health and that the denture-wearing population requires care and follow-up similar to their non-denturewearing counterparts .
Analyzing levels of CRP in those patients, we found no significant difference in the levels of CRP between the two groups of patients. The findings are consistent with those of Kostial el. al who reported that CRP values in patients with acute fungal stomatitis in non-immunocompromised patients were within normal ranges . In contrast, another study, Ajwani et al. reported elevated levels of CRP and microbial counts in edentulous individuals with signs of oral candidiasis or denture stomatatis . It has been reported that CRP levels are higher in patients with periodontitis [40-44], and our data showed there is similarity between the microbial content of denture plaque and that associated with periodontitis. Thus, it is plausible that fungal infections, which activate a different microbial pathogen recognition system than bacteria, may specifically trigger the release of hepatic acute phase reactants, other than CRP, such as alpha antitrypsin, transferrin or orosomucoid synthesis.
Elevated levels of salivary IL-8 and IL-1β are associated with DS, and IL-8 is positively associated with increased levels of C. albicans in saliva. Our findings suggest that the overgrowth and colonization of C. albicans on acrylic denture surfaces may act as an early colonizer in the succession of biofilm formation (replacing many streptococci) and may serve to facilitate the emergence of anaerobic microorganisms. These data suggest that an unclean denture can serve as the primary source of C. albicans that enhances mucosal inflammation, pointing to the importance of denture hygiene in reducing denture stomatitis.
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*This work was supported by NIH research grant UL-1-RR025746 and GSK Consumer Health Care.