Introduction
Down syndrome (DS) is an autosomal chromosomal anomaly associated with trisomy of chromosome 21 [1] and displays many functional and physical characteristics including intellectual impairment, congenital heart disease, leukaemia, dementia, chylothorax, and increased susceptibility to infection (especially respiratory infections and periodontal disease) [2-4]. DS is also characterized by abnormalities in the immune response including cell-mediated responses [5], humoral immunity [6], phagocytosis [7,8], and interferon production [9].
Many microorganisms exist as normal commensals in the oral cavity, but become pathogenic when the host's immune system is compromised. Oral candidiasis is an opportunistic infection that usually occurs when the host is immune-compromised. In the oral cavity, the prevalence of Candida has been noted to be 45 - 65% in normal children [10] and 30 - 45% in normal adults [11]. Especially, Candida albicans is a potent endogenous fungal pathogen, and oral carriage of Candida albicans has been reported to be 26 - 44% in normal individuals not demonstrating any clinical signs or symptoms of mucosal disease [12].
Saliva is crucial for the oral defence against infections since saliva contains a multitude of antimicrobial proteins and peptides, and pH and buffering capacity in recovering drug abusers [13]. They play critical roles in the innate immune response and the non-immune host defence system to avoid microbial colonization and infection in the oral cavity. Histatin 5 is a major component of a family of small proteins produced by the parotid and submandibular glands [14-17]. It exhibits strong fungicidal activity against pathogenic fungi, including Candida albicans [15,18] and other medically meaningful Candida species [19,20].
DS individuals display increased susceptibility to fungal infections and clinical studies has been conducted on oral fungal colonization [21]. However, no in vivo studies have been conducted to investigate levels of histatin 5 in DS patients. Therefore, the present investigation was undertaken to study the relationship between salivary levels of histatin 5 and the prevalence of oral carriage of Candida species in children and adults with DS and in age- and sex-matched systemically healthy controls with non-DS.
Subjects and Methods
Study population
The participants in the study had DS with karyotypes of 47 XX, 21+ (female) and 47 XY, 21+ (male) and were systemically healthy individuals. The exclusion criteria were treatment with antibiotics and steroids for the previous 2 months; use of non-steroidal anti-inflammatory medications and antifungals within the previous 3 months, use of corticosteroids in case of asthma; use of oral topical corticosteroids within the previous 3 months; use of a partial or complete dental prosthesis; a history of chemotherapy or radiation treatment; and systemic diseases such as diabetes mellitus, hepatitis B and hepatitis C infections, infection with human immunodeficiency virus (HIV), and acquired immunodeficiency syndrome. In accordance with the Helsinki Declaration (Fortaleza, 2013), approval for the study was obtained from the Institutional Ethics Committee of Kanagawa Dental University Graduate School (No. 169). Moreover, the participants or their legally acceptable representatives were informed about the study and informed consent was obtained from all volunteer participants or their legally acceptable representatives.
A total of 51 individuals with DS (34 male subjects and 17 female subjects; age range, 5 - 62 years) and a control group of 48 age-matched systemically healthy individuals (20 male subjects and 28 female subjects; age range, 8 - 57 years) were included in the study.
The DS and C groups were subdivided according to age: group 1 consisted of DS (DS-1) and control (C-1) subjects under 20 years of age and group 2 consisted of DS (DS-2) and control (C-2) subjects over 40 years of age. The sex compositions and mean ages of the study groups are shown in Table 1.
An oral evaluation, sample collection for fungal culture, and un-stimulated saliva collection were performed by an oral medical professional.
Saliva collection and measurement of the salivary flow rate
Un-stimulated whole saliva was collected by using the absorbent method [22]. Salivary samples were collected between 10:00 AM and 2:00 PM and at least 1 h after food intake and before clinical measurements because of difficulty in collecting stimulated saliva from individuals with intellectual disability. One neutral, non-covered absorbent cotton roll (Salivette degrees, Sarstedt, Numbrecht, Germany) was placed into the mouth under the tongue for exactly 5 min. To determine the salivary flow rate, the cotton rolls were weighed before and after saliva collection using an electronic scale, which was sensitive to 0.01 g. The gain in weight during the 5-min interval was then converted into ml of saliva min-1 [23]. Immediately after weighing, saliva samples were clarified by centrifugation at 10,000 multiplication g for 15 min at 4 degree centigrade [24]. The supernatants were stored at -40degree centigrade for up to 1 month prior to analysis.
Measurement of salivary histatins
Histatin 5 levels in saliva samples were determined using an Enzyme-Linked Immunosorbent Assay (ELISA) kit (Wuhan Eiaab Science Co., LTD; Hubei, China). The microtiter plates had been pre-coated with an antibody specific to natural human histatin 5 (Wuhan Eiaab Science Co., LTD). No significant cross-reactivity or interference was observed. Standards and samples (100 μl each) were added to the microtiter plate wells in duplicate and incubated for 2 h at 37degree centigrade. After incubation, 100 μl of a biotin-conjugated polyclonal antibody solution preparation specific for histatin 5 was added to each well and incubated for 1 hour at 37degree centigrade. After washing away any unbound substances, 100 μl of avidin conjugated to horseradish peroxidase solution was added to each well for 1 h at 37degree centigrade . Then, 90 μl of substrate solution was added to each well and reacted for 30 min at 37degree centigrade. The enzyme-substrate reaction was terminated by the addition of 50 μl of sulphuric acid solution and the colour change was measured spectrophotometrically at a wavelength of 450 nm. Only wells that contain histatin 5, biotin-conjugated antibody, and enzyme-conjugated avidin show a change in colour. The concentrations of histatin 5 in the samples were calculated from a standard curve.
The total salivary protein concentration was determined by the Bicinchoninic Acid (BCA) method [25] using a Micro BCA Protein Assay Kit (#23235, Thermo Fisher Scientific K.K, Kanagawa, Japan) according to the manufacturer's instructions.
Isolation and identification of Candida species
Oral candidal samples were collected from behind the centre of the dorsal surface of the tongue by swabbing; the swab was rotated thoroughly in one direction with moving back and forth. The cotton swabs were placed in 1 mL of transport medium composed of 5.0 mg NaCl, 1.1 mg Na2HPO4, 0.09 mg CaCl2, 1.5 mg thioglycolic acid sodium salt, and 5.6 mg agar (pH 7.2; 0.4% thioglycollate / 0.15% phosphate-buffered saline) in sterile bottles. (Seedswab No. 1, Eiken Chemical Co., Ltd., Tokyo, Japan) [26]. These were suspended in 1 ml of physiological saline to facilitate dissolution of the swabs and to improve recovery of the oral microflora. After agitating a cotton swab in 1 ml of physiological saline, 100 μl of the sample was aerobically inoculated onto CHROMagar Candida (Becton Dickinson, Sparks, MD, USA) at 37degree centigrade for 48 h. Three Candida species (C. albicans, C. tropicalis, and C. krusei) were identified on CHROMagar based on colony morphology and pigmentation by following the manufacturer's instructions and the description by Tan and Peterson (2005). C. albicans, C. tropicalis, and C. krusei produced green, blue, and pink and fuzzy colonies, respectively. Colonies of non C. albicans species were certified by the API 20C AUX system for yeasts (bio-Merieux VITEK, Tokyo, Japan).
Statistical analyses
Descriptive data including means and standard deviations were determined for each parameter in each group and were used for analysis. A one-way analysis of variance (ANOVA) was used for multiple group comparisons followed by Scheffe's test for pairwise comparisons. Correlations between concentrations of total salivary protein and salivary histatin 5 were analysed by Pearson's correlation test. For all tests, a P value of 0.05 or less was considered statistically significant. The statistical analysis was performed using SPSS software version 23 (IBM SPSS Statistics, Tokyo, Japan).
Results
The ratio of Candida carriers among the participants is shown in (Figure 1). Candida carriers were detected in 7 (29.2%) of 24 in the C-1 group, 2 (8.3%) of 24 in the C-2 group, 13 (65.0%) of 20 in the DS-1 group, and 23 (74.2%) of 31 in the DS-2 group with mean colony counts of 0.71 +/- 1.71 colony forming units (CFU) (range, 0 - 8 CFU), 0.29 +/- 1.23 CFU (range, 0-6 CFU), 13.0 +/- 33.6 CFU (range, 0 - 152 CFU), and 72.8 +/- 204.3 CFU (range, 0 - 641 CFU), respectively. The prevalence of Candida species colonization and total bacteria on the centre of the dorsal surface of the tongue is shown as follows. C. albicans was most frequently isolated; C. albicans carriers was found in 7 subjects (29.2%) in the C-1 group, 1 subject (4.2%) in the C-2 group, 13 subjects (65.0%) in the DS-1 group, and 23 subjects (74.2%) in the DS-2 group (Figure 1) with mean colony counts of 2.04 +/- 1.70 CFU (range, 0 - 2 CFU), 0.04 +/- 0.20 CFU (range, 0 - 1 CFU), 12.98 +/- 33.58 CFU (range, 0 - 152 CFU), and 61.13 +/- 159.88 CFU (range, 0 - 659 CFU), respectively. In contrast, C. tropicalis carriers was only found in 1 subject (4.2%) in the C-1 group, 2 subjects (10.0%) in the DS-1 group, and 6 subjects (19.4%) in the DS-2 group with mean colony counts of 0.24 +/- 0.20 CFU (range, 0 - 1 CFU), 0.15 +/- 0.49 CFU (range, 0-2 CFU), and 2.35 +/- 4.83 CFU (range, 0 - 21 CFU), respectively. C. krusei carriers was found in 1 subject (4.2%) in the C-2 group, 1 subject (5.0%) in the DS-1 group, and 6 subjects (19.4%) in the DS-2 group with mean colony counts of 0.25 +/- 1.22 CFU (range, 0 - 6 CFU), 0.05 +/- 0.22 CFU (range, 0 - 1 CFU), and 12.17 +/- 58.16 CFU (range, 0 - 324 CFU), respectively (Figure 1). C. krusei was not recovered from the C-1 group (Figure 1). C. tropicalis was not recovered from the C-2 group (Figure 1). Fungal cultures demonstrated differences in both of the colonization of Candida species and the prevalence rate of Candida carriers between the C-2 and DS-2 groups.
(Table 2) shows a comparison of salivary flow rates between the DS and C groups. The mean un-stimulated salivary flow rates were 0.43 +/- 0.08 ml min−1 in the C-1 group, 0.33 +/- 0.17 ml min−1 in the C-2 group, 0.38 +/- 0.23 ml min−1 in the DS-1 group, and 0.16 +/- 0.10 ml min−1 in the DS-2 group. Statistical analyses revealed a significantly higher mean un-stimulated salivary flow rate in the C-1 group than in the C-2 groups, in the C-2 group compared with the DS-2 group (P < 0.01; Table 2), and also in the DS-1 group compared with the DS-2 group (P < 0.01; Table 2). There was no difference between the C-1 and DS-1 groups (Table 2).
The mean total salivary protein concentrations were 649 +/- 178 μg ml−1 in the C-1 group, 884 +/- 252 μg ml−1 in the C-2 group, 1019 +/- 250 μg ml−1 in the D-1 group, and 1194 +/- 438 μg ml−1 in the D-2 group, as shown in Table 3. Statistical analyses revealed a significantly lower mean total salivary protein concentration in the C-1 group compared with the C-2 group (P < 0.01). Statistical analyses also revealed a significantly lower mean total salivary protein concentration in the C-1 group compared with the DS-1 group (P < 0.01; Table 3). Furthermore, statistical analyses revealed a significantly lower mean total salivary protein concentration in the C-2 group compared with the DS-2 group (P < 0.01; Table 3). On the other hand, there was no difference between the DS-1 and DS-2 groups (Table 3) [27].
The mean salivary concentration of histatin 5 was 2.09 +/- 1.40 μg ml−1 (range, 1.27 - 6.14 μg ml−1) in the C-1 group, 4.47 +/- 1.33 μg ml−1 (range, 2.18 - 7.01 μg ml−1) in the C-2 group, 2.12 +/- 0.97 μg ml−1 (range, 0.75 - 4.56 μg ml−1) in the DS-1 group, and 3.12 +/- 1.24 μg ml−1 (range, 2.12 - 7.65 μg ml−1) in the DS-2 group (Table 4). Statistical analyses revealed a significantly lower mean salivary concentration of histatin 5 in the C-1 group compared to the C-2 group (P < 0.01; Table 4), and in the DS-1 group compared to the DS-2 group (P < 0.05; Table 4). The mean salivary concentration of histatin 5 was significantly higher in the C-2 group compared to the DS-2 group (P < 0.01; Table 4). However, there was no difference between the C-1 and DS-1 groups (Table 4).
The salivary concentration of histatin 5 was strongly correlated with the total salivary protein concentration in only the C-2 group (Figure 2 and Table 5); the other groups showed no correlation between them (Table 5). No correlation was observed in any group between histatin 5 levels and age, sex, or un-stimulated salivary flow rate (data not shown).
Discussion
The oral cavity is an environment heavily colonised by microorganisms, which are commonly in harmony with the mammalian host. The relationship between microorganisms and the mammalian host appears to be related to the immune status of the individual. The rate of Candida carriers in the healthy children and adolescents (C-1 group) was higher than the adults (C-2 group). Since younger children are under the state of developing immunity, Candida species has the potential to increase in number during low immune states [28].
On the contrary to the results of C groups, the rates of Candida carriers were high status in both the children and adolescents and the adults with DS. 65.0%of the children and adolescents with DS were colonised by one or more Candida species, with C. albicans being the most frequent species followed by C. tropicalis, C. kruse, (Figure 1). These results concerning children and adolescents with DS are very similar to those from previous reports. In a study performed by [29], it was recognised from samples taken from the dorsum of tongue that 74% of DS patients were colonised with Candida species in the age range from 3 - 22 years and that strains of C. albicans were the most prevalent species at 84% of the culture-positive samples, followed by C. tropicalis. Another study reported that 69% of children and adolescents with DS were colonised with C. albicans from samples taken from their oral cavities [21]. The other probable explanation is that the number of fungi is greatest in patients with geographic and fissured tongues [30], which occur in 38% of patients with DS [29].
The salivary protein concentrations among DS subjects were higher than those among healthy control subjects of the same age (Table 3); there was no difference between the DS-1 and DS-2 groups (Table 3). In essence, the sources of proteolytic enzymes in whole saliva are mostly derived from oral microbiota associated with periodontopathic bacteria [31]. The children with DS could have higher number of periodontopathic bacteria as well as the adults with DS [32]. The results of the high concentration of proteins in DS may be thought of as damaging salivary proteins by proteolysis, since oral fluid proteolysis could serve to free smaller peptides from larger, less active or inactive, precursor proteins [33].
The concentrations of histatin 5 were comparable between DS and healthy subjects (Table 4). In agree with our study, the salivary proteomics has shown that the antimicrobial peptide histatin 5 showed similar salivary levels in the 10-17 year old DS patient and healthy control groups [34]. It may be consistently to replenish with intact hisitatin 5 against to the proteolytic breakdown of histain 5 since the salivary flow rate is no different between DS-1 and C-1 (Table 2).
The children with DS may have different compositions of biochemical components in their saliva due to alterations in the metabolism of the ducts and/or acinar cells of the salivary glands [35]. The abnormalities of biochemical components other than histatin may also effect on the increase of Candida prevalence in DS children.
In contrast to results obtained with the younger groups, in the adult groups, ELISA results demonstrated a significant (P < 0.01; Table 4) reduction in histatin 5 concentration in the DS-2 group compared with the C-2 group. At the same time, our fungal culture results demonstrated a significantly higher prevalence of Candida colonization in the DS-2 group compared with the C-2 group. The histatin 5 in whole saliva of DS-2 were reduced as compared with C-2 with the reduction of the salivary flow rate (Tables 2 and 5). Prevalence rates of periodontitis were characterized by significant bacterial and inflammatory burden increase with age in people with DS.
It may be presumed that whole saliva proteolysis from oral bacteria-derived proteases may rapidly degrade histatin 5 in the oral cavity of the DS-2 with low salivary flow rate, since the presence of a microbial imbalance in the oral cavity could be the result of some major local factors causing increased Candida carrier in individuals with DS (Figure 1).
These results are in accordance with previous studies on immune-compromised patients by [36,37], which showed lower histatin 5 levels in HIV-positive individuals and an increased prevalence of C.albicans among these patients. Among those immune-compromised individuals, innate immune system involved in secretion of histatin 5 may be depressed with decrease of saliva flow and increase of oral microbiota or not work to response as the adequate reaction. It is tempting to speculate therefore that the lower salivary histatin levels are related with the higher oral Candida levels in adults with immune-compromised or DS individuals.
Furthermore, our fungal culture results demonstrated increase of prevalence of candidal colonization with slightly increase of rate of Candida carriers in the DS-2 group compared with the DS-1 group. The histatin 5 concentrations in the DS-2 group were also increased (Table 4). However, as compare the change of value in histatin 5 concentrations between C-1 and C-2, that of DS was smaller. The increase of the rate of Candida carrier in adults DS may in part be a result of a lack of sufficient increase of histatin 5, along with salivary immunoglobulin deficiency [38,39]. It was already shown that one or more salivary glands in the areas of the bilateral parotid and submandibular glands were absent in some children with DS [35,40]. Also reported parotid salivary hypofunction in subjects with DS. It could account for the decreased salivary histatin 5 concentration (Table 4) with accelerating the diminution of salivary output, since histatin 5 is secreted mainly by the parotid glands. Such a finding suggests that the expression of salivary histatin 5 in DS would be affected by salivary gland dysfunction. It has been well documented that in the oral cavity, the adsorption of proteins to oral structures may cause, in part, the decreased concentration of histatin 5 in expectorated samples [41,42] reported that calcium levels were significantly higher in DS children with caries compared with control groups with and without caries. The calcium ion might be involved in the reducing the concentration of histatin 5 by binding histatin and inhibiting the antifungal effect of histatin 5 [43].
Moreover, there was a correlation between the concentration of histatin 5 and total protein in only the C-2 group (Figure 2); no association was found between histatin 5 concentration and total protein in the other groups (Table 6). The innate oral defence from salivary histatin 5 in healthy individuals plays a role in enhanced susceptibility to oral candidiasis. Consequently, it may be associated with the increased incidence of Candida colonization related to salivary histatin 5 defence mechanisms, at least in healthy adult individuals. The adaptive immunity of the adults with DS is impaired in the oral cavity. It is possible that the subtle abnormalities in both humoral and cellular immune response, involved the concentration of histatin 5, related with gene coded on chromosome 21 may effect on the large number of carrier in DS. The healthy young children are more vulnerable to infections, though by then better armed with the maturing innate and adaptive immune systems. However, for DS, it does not have a fully developed effective immune response even though mature of age. There are different mechanisms in DS and C individuals; the immune defence of DS individuals could be decreased from childhood and their defences may not increase, as they become adults.
Conclusion
In conclusion, Candida colonization was significantly higher in the DS groups than in the control groups. Histatin 5 concentrations were not different between the DS group and groups in healthy children and adolescents, but were significantly lower in the DS-2 group compared with the C-2 group in the adults. The progression of candidal colonization in the DS population will contribute to the enhanced native propensity in vulnerable populations.
Acknowledgements
This research was supported by a Grant-in-Aid for Scientific Research (no. 23593049 to T.K. & M.L.; no. 26463125 to T.K. & M.L.; no. 16K11901 to T.K. & M.L.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and NIH/NIAID grant AI101067 to EJH.
Conflict of interest
The authors report no conflict of interest with respect to this work.
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