Original article

Use of Candida-specific chicken egg yolk antibodies to inhibit the adhering of Candida to denture base materials: prevention of denture stomatitis Yoshiaki Kamikawa*1, Junichi Fujisaki*2, Tomohiro Nagayama1, Kiyotsugu Kawasaki2, Daisuke Hirabayashi2, Tomofumi Hamada2, Ryoich Sakamoto2, Hiroshi Mukai1,2 and Kazumasa Sugihara1,2 1

Department of Oral Surgery, Kagoshima University Medical and Dental Hospital, Kagoshima, Japan; 2Department of Maxillofacial Diagnostic and Surgical Sciences, Field of Oral and Maxillofacial Rehabilitation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan; *They contributed for this study at the same levels,.

Gerodontology 2014; doi:10.1111/ger.12163 Use of Candida-specific chicken egg yolk antibodies to inhibit the adhering of Candida to denture base materials: prevention of denture stomatitis Objectives: Polyclonal anti-Candida chicken egg yolk antibodies (anti-IgY) were used to investigate the prevention of adherence of Candida species to denture base material in vitro. Background: Candida is a potential virulence factor that can cause systemic infection and even death in immunocompromised individuals. Because long-term antifungal treatment may lead to the emergence of drug-resistant strains, it is necessary to develop novel preventive measures and treatments for candidiasis. Materials and methods: Three types of chicken egg yolk antibodies were used in this study: non-specific antibody (control IgY), Candida albicans-specific antibody (anti-C.a.IgY) and Candida glabrata-specific antibody (anti-C.g.IgY). A mixture of different dilutions of each antibody with a suspension of Candida species and denture base material was incubated for 3 h, and then the colony-forming units of Candida on the denture base material were counted. Results: Compared with control IgY, anti-C.a.IgY and anti-C.g.IgY significantly inhibited the adherence of C. albicans, but anti-C.a.IgY tended to be more potent than anti-C.g.IgY. The adherence of C. glabrata was also inhibited significantly by anti-C.a.IgY and anti-C.g.IgY with almost equivalent potency, indicating that their actions against C. glabrata were comparable. Conclusions: This study revealed the inhibitory effects of anti-C.a.IgY and anti-C.g.IgY against the adherence of C. albicans and C. glabrata to denture base material. This finding indicates the possibility of a beneficial effect of IgYs for the prevention of denture stomatitis and candidiasis in clinical settings. Keywords: egg yolk globulin, Candida, oral candidiasis, prophylaxis. Accepted 25 September 2014

Introduction Denture stomatitis is an erythematous lesion of the oral mucosa in contact with dentures due to dental plaque1. Compared with other microorganisms in dental plaque, Candida adheres more strongly to denture base material and produces biofilms, a process that forms the foundation of dental plaque2–4. The pathogenic properties of

Candida, such as adherence capability and proteolytic potential4,5, are believed to play an important role in denture stomatitis. Candida is a micro-organism that resides in the oral cavity, skin mucosa and gastrointestinal tract6–8 of healthy individuals and is the most prevalent micro-organism found on dentures9. Candida rarely causes infection in healthy individuals; however, in immunocompromised individuals,

© 2014 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd

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the micro-organism often causes skin, mucosal and systemic candidiasis. Candida may also cause systemic candidiasis and fatal complications in patients undergoing cancer chemotherapy10–12, immunosuppressive therapy13 or long-term antibiotic treatment14. Given the potential virulence of Candida, antifungal drugs have been used prophylactically and even indiscriminately. However, to prevent the emergence of antibiotic-resistant strains10,15,16, it is necessary to develop novel preventive measures and treatments for candidiasis. Passive immunization with antibodies has been used as immunotherapy17–22. Of the antibodies used for passive immunization, polyclonal chicken egg yolk antibodies (IgYs) exhibit inhibitory effects against a wide range of pathogens such as bacteria, viruses and parasites. IgYs are fast acting and safe and have attracted attention as a cost-effective substitute for antibiotics. In addition, IgYs are used in the production of antibodies against influenza virus, Helicobacter pylori and periodontal diseasecausing bacteria, and their effectiveness has been confirmed23–33. However, no effective antibody therapy against Candida-induced diseases has been established. Therefore, we focused our attention on IgYs in the prevention of such diseases because they would be safe, cost effective and readily massproduced for use in a clinical setting. Although a C. albicans-specific antibody (anti-C.a.IgY) isolated from chicken egg yolk blocked the adherence of C. albicans to host cells in a previous study31, no report has demonstrated such an inhibitory action using denture base material. In this study, we investigated the efficacy of IgYs to prevent the adherence of C. albicans and C. glabrata, which are highly prevalent Candida species, to denture base material2–4,33.

Materials and methods Yeast strains C. albicans (ATCC19880) and C. glabrata (ATCC90030) were stored at 80°C in yeast-peptone-dextrose broth (1% yeast extract, 2% polypeptone and 2% dextrose) (Sigma-Aldrich, Inc., St. Louis, MO, USA) containing 10% glycerol until use. Fungal cells were harvested by centrifugation for 10 min at 3000 g at 4°C and washed twice with sterile phosphate-buffered saline (PBS; pH 7.2) (TAKARA, Otsu, Japan). For use in the experiments, all organisms were grown aerobically on CHROMagar CANDIDA (B&D Japan, Fukushima, Japan) at 37°C.

Preparation of IgY The anti-C.a.IgY and C. glabrata-specific antibody (anti-C.g.IgY) antibodies were provided by the GHEN Corporation (Gifu, Japan). These antibodies were produced according to the method described by Yokoyama et al.26,31. Briefly, the vaccine was prepared by mixing 0.5 mg C. albicans antigen with 0.5 ml emulsion oil containing 5% Arlacel 80 (Maine Biological Laboratories, Waterville, ME, USA) and 5-month-old white leghorn hens (strain Hyline W36; GHEN Corporation) were immunized by injecting 0.5 ml of this mixture into the breast muscle. Six weeks after the initial immunization, a booster was given in the same manner. Eggs from the immunized hens were harvested daily from the week 2 to week 6 after the booster and stored at 4°C. Egg yolk was carefully separated from the albumen and yolk membrane. The yolk was then pooled, homogenised and filtered through a Teflon filter cloth. Partially purified specific IgY powder (anti-C.a.IgY) was prepared from the egg yolk by ammonium sulphate precipitation. Then, the precipitated IgY was suspended in PBS, dialysed and freeze-dried in a Labconco LL-12 freeze-drying machine (Labconco Corp., Kansas City, MO, USA) and a solution containing 10 mg/ml was prepared in PBS. Anti-C.g.IgY was prepared using the same method with the C. glabrata antigen. Control IgY powder was prepared from the eggs of nonimmunized hens by the same method. In vitro adhesion inhibition activity of anti-C.a.IgY and anti-C.g.IgY The ability of anti-C.a.IgY and anti-C.g.IgY to inhibit the adhesion of C. albicans and C. glabrata to denture base material was examined by an adhesion inhibition assay. Ten samples of denture base material (10 9 10 mm, 1 mm thick) (ACRON, GC, Tokyo, Japan) for each concentration of anti-C.a.IgY, anti-C.g.IgY and control IgY were weighed and inserted into the wells of a 12well plate. The anti-C.a.IgY mixture was prepared as follows: the concentration of the C. albicans or C. glabrata cell suspension was adjusted to 5.0 9 105 colony-forming units (CFUs)/ml in a TC10 automated cell counter (Bio-Rad, Hercules, CA, USA). One millilitre of the anti-C.a.IgY solution (0.0, 2.5, 5.0 and 10.0 mg/ml) was added to the C. albicans and C. glabrata suspensions. The mixtures were incubated aerobically for 3 h at 37°C. Thereafter, the denture base material was washed three times with PBS to remove non-

© 2014 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd

Anti-Candida IgY prevent denture stomatitis

adherent fungal cells and vigorously vortexed for 30 min with 1 ml of 0.1% Triton X-100 (CALBIOCHEM, San Diego, CA, USA). Then, the 0.1% Triton X-100 solution was centrifuged at 3000 g for 5 min, the supernatant was removed by aspiration, and pellets containing the adherent fungal cells were resuspended in 1 ml PBS. To count the adherent fungal cells, 10 ll of each PBS solution were inoculated on CHROMagar CANDIDA, the plates were incubated overnight at 37°C, and C. albicans and C. glabrata CFUs were counted. Control IgY-treated wells were used as controls. Statistical analysis The log10 CFU value of the adhered C. albicans and C. glabrata colonies was analysed using Student’s t-test. All statistical analyses were performed using SPSS software version 19.0 for Windows (SPSS, Chicago, IL, USA). A p value

Use of Candida-specific chicken egg yolk antibodies to inhibit the adhering of Candida to denture base materials: prevention of denture stomatitis.

Polyclonal anti-Candida chicken egg yolk antibodies (anti-IgY) were used to investigate the prevention of adherence of Candida species to denture base...
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