The effect of Candida albicans on the permeability ...

Journal of Medical and Veterinary Mycology (1987) 25, 19-28

The effect of Candida albicans on the permeability of rat palatal epithelium: an ultrastructural and biochemical study

SMALLEY2

Departments of Dental Surgery and 2Dental Sciences, The Dental School, University o f Liverpool, Pembroke Place, Liverpool L69 3BX, England, U.K. (Accepted 17 October 1986)

The effect of a palatal acrylic appliance and/or infection with Candida albicans on the permeability of rat palatal epithelium has been investigated. Although normal rats, or rats inoculated with Candida albicans but without a prosthesis, had a barrier to perfused lanthanum, some low-molecular-weight proteins were able to pass through the epithelium. When infection was established by inoculation of Candida albicans under an acrylic plate, the epithelium became permeable to perfused lanthanum. Polyacrylamide gel electrophoresis of perfusates showed that a selective permeability to proteins was retained in such animals. Removal of acrylic plates from infected animals resulted in healing and restoration of the barrier to lanthanum.

Oral candidosis and in particular chronic atrophic candidosis is a c o m m o n condition in denture wearers [6]. It usually affects the palatal epithelium and is caused by the fungus Candida albicans [7, 8]. The histopathological features of chronic atrophic candidosis have been described [6] and are largely non-specific. The palatal tissue is chronically inflamed and hyperplastic, with occasional fungal hyphae penetrating as far as the inferior border of the stratum granulosum. With the exception of some clinical and histopathological observations there is little information available on the effect of C. albicans on palatal tissues. Healthy palatal epithelium has barrier properties which prevent the ingress of water and water-soluble compounds, and toxins or other deleterious substances [14]. The permeability barrier can be demonstrated ultrastructurally by the use of electron-dense materials such as lanthanum nitrate [23]. Hyperplasia, induced by turpentine or mild trauma has been shown to affect the permeability of oral epithelium [23]. No reports have been traced concerning the effects of infection on barrier function. The aim of this investigation was to study the effect o f C. albicans on the barrier properties of palatal epithelium. A rat model for the induction of palatal candidosis Correspondence address: Dr M. V. Martin, Department of Dental Surgery, The Dental School, University of Liverpool, Pembroke Place, Liverpool L69 3BX, U.K. 19

Downloaded from http://jmvm.oxfordjournals.org/ at University of Liverpool on March 23, 2015

M. V. MARTIN ~, J. APPLETON, JANETTE C H E S T E R S ~ AND J. W.

20

MARTINET AL.

[ 18] was used for this purpose. In addition, it was decided to analyse qualitatively oral epithelial transudates to determine their nature and resemblance to serum.

METHODS Source and maintenance of yeast C. albicans NCPF 3091 (serotype A) was used throughout this study. It was maintained by monthly subculture on Sabouraud's agar (Lab M, London). Inocula for rat infection were prepared by scraping growth from the surface of a plate that had been incubated for 48 h at 37°C.

Perfusion The animals were anaesthetized and if an acrylic plate was present this was removed. The right jugular vein was exposed by blunt dissection and ligatured near the superior end of this exposure. A cannula (outer diameter 0.63 mm, Portex, Kent) was inserted into the inferior part of the exposed vein and tied in position. The cannula was connected to a peristaltic pump (Gilson, France) set at a flow rate of 10 ml min -1. The animal was then perfused for 5 min with 0.15 M sodium chloride, a small cut being made in the jugular vein superior to the ligature to allow fluid to escape. After 5 min the animals were perfused with a mixture of 1% (w/v) lanthanum chloride, 2.5% (w/v) glutaraldehyde and 3% (w/v) dextran in 0.1 M cacodylate buffer pH 7.4 for a further 15 min. After perfusion was complete the palatal mucosa was removed from the intermolar area, as defined by Kutuzov Sicher [10], cut into 1 mm 3 pieces and fixed in 2.5% (w/v) glutaraldehyde, 0.1 ra cacodylate buffer pH 7.4 for 2 h. The treatment of the animals is summarized in Table 1; all animals were perfused at the end of the experimental period shown. The experiments were designed to investigate whether the permeability barrier was affected by infection. In addition, the effect of palatal healing on the permeability was also determined.

Electron microscopy The tissue removed was postfixed in 1% (w/v) osmium tetroxide in 0.1 M cacodylate buffer pH 7.4 before dehydration in an ascending series of concentrations of ethanol.


Induction of candidosis Randomly inbred male Wistar rats were used throughout this investigation. The rats were provided with food (Spratt's Expanded Rodent Diet, K & K Greef Chemicals, Suffolk) and water ad libitum. Animals were anaesthetized with intramuscular Immobilon (C Vet, Bury St Edmunds, Suffolk) at a dose of 9 mg Kg-~ body weight. Palatal candidosis was induced by fitting the animals with an acrylic appliance that covered the palate but did not occlude the posterior teeth [ 18]. The appliance was maintained in position by notching the distopalatal aspect of the upper incisor teeth and cementing the appliance to these teeth with cold-cure resin (WHW Plastics, Hull). Immediately before cementation of the appliances the animals were inoculated by smearing their palatal tissues and the appliance with approximately 30 mg wet weight of C. albicans 3091. Previous work [ 18] has shown that this technique results in histological and mycological evidence of candidosis 2 weeks after inoculation. If the acrylic plates are removed after induction of candidosis, then 2 weeks later the palatal tissues are histologically indistinguishable from normal [19].

21

C. ALBICANS AND ORAL EPITHELIUM

TABLE 1. A summary of the permeability experiments

Treatment

Total time of experiment (weeks)

t 1 1

2 2 2

5

2

2

2

3

4

3

6

2

10

Acrylic plate only Inoculated with C. albicans only Untreated Acrylic plated inoculated with C. albicans

Acrylic plate inoculated with C. albicans for 2 weeks Acrylic plate inoculated with C. albicans for 2 weeks. Plate removed for 2 weeks. Acrylic plate inoculated with C. albicans for 2 weeks. Plate removed for 4 weeks. Acrylic plate inoculated with C. albicans for 2 weeks. Plate removed for 8 weeks.

The dehydrated tissue was then embedded in Spurr resin [20] and sections 1/tm thick were stained with toluidine blue to ensure correct orientation. Ultrathin sections (60-90 mm) of the tissue were then cut with a diamond knife on a Reichert OMU2 microtome (Reichert-Jung, Slough), stained with uranyl acetate and examined with a JEOL 100CX electron microscope (JEOL, Tokyo, Japan).

Transudation

Proteins which transuded the oral epithelium were qualitatively investigated by sodium dodecyl sulphate polyacrylamide gel electrophoresis; 15 animals were used. The number of animals and the procedures are shown in Table 2. One additional animal was anaesthetized and minor palatal salivary gland excretions collected as described below, after stimulation with pilocarpine (5 m g k g l; Sigma, Poole, England). Serum was also collected from this rat via the tail vein.

TABLE 2. A summary of the transudation experiments

Treatment Appliance C. albicans inoculation only

Untreated C. atbicans acrylic plate C. albicans acrylic plate for

2 weeks, removed for 2 weeks Acrylic plate for 2 weeks, removed for 2 weeks Pilocarpine stimulation

Total time of experiment Number (weeks) of before animals perfusion 2 2 2 2

2 2 4 2

5

4

2 1

4 0


Number of animals

22

MARTINE T AL.

Transudates were collected from anaesthetized animals in which the mouth was propped open with a small acrylic wedge placed between the posterior teeth. I f an appliance was present this was removed. The palatal tissues were irrigated to remove debris with sterile water and the mucosa gently dried with a sterile cotton wool swab. A small strip of cellulose acetate filter (Millipore H A 0-45 #m, France) was cut to the shape of the intermolar area. The cellulose acetate filter was then applied to the intermolar area and held in position with clean tweezers for 15 rain then replaced with another filter paper for further 15 min. The filters containing the transudates were kept at - 2 0 ° C in airtight containers until required.


FIG. 1. Normal rat palatal epithelium showing an electron-dense lanthanum deposit between cells of the stratum granulosum (sg, arrowed), ×4640.


23

FIG. 2. Rat palatal epithelium inoculated with C. albicans and fitted with an acrylic plate. Note the presence of lanthanum throughout the stratum corneum, × 3876.

RESULTS

Ultrastructural A deposit of electron-dense lanthanum was localized as a distinct layer between cells


Electrophoresis of the transudates The proteins present in the transudates were electrophoresed in discontinuous SDS polyacrylamide gels (SDS gels) according to the method of Laemmli [11 ]. The cellulose acetate strips were cut into 1 × 4 mm pieces and the absorbed material extracted with 1 ml of a 1 in 50 ml dilution of sample buffer [11] for 6 h at room temperature on an orbital shaker set at 100 rev. min ~. The standard, undiluted sample buffer contained, in addition, 2 M urea, 1 mM phenylmethylsulphonyl fluoride, 0.3 mM sodium azide and 50 mM dithiothreitol. The acetate strips were re-extracted in a further 1 ml of diluted sample buffer and this was combined with the first extracts before freeze-drying. Samples, including a control extract of cellulose acetate alone, were reconstituted to the volume of the original undiluted sample buffer by the addition of 40/tl of distilled water and electrophoresed on a 7% (w/v) acrylamide slab gel with a 3% (w/v) stacking gel. Samples of rat serum were diluted 1:20 with Laemmli [11] sample buffer to provide appropriate concentrations for electrophoresis. Myosin, fl-galactosidase, phosphorylase B, bovine serum albumin, ovalbumin and carbonic anhydrase were used as molecular weight standards. Saliva samples were diluted 50-fold with Laemmli [11] sample buffer and electrophoresed as described above. Gels were stained with 0.1% Coomassie blue in methanol:acetic acid:water (50:7:43 v/v) for 18 h and destained in an aqueous 5% methanol/7% acetic acid (v/v). The gels were scanned densitometrically at 575 nm in a Beckman DU-8 spectrophotometer (Beckman, U.S.A.) to obtain molecular weights and integrated peak areas of the migrated bands.

24

M A R T I N E T AL.

within the stratum granulosum (Fig. 1). Apparent discontinuities in the deposit were found when the cell junctions left the plane of the section. A similar appearance was noted in animals fitted with an acrylic plate alone or only inoculated with C. albicans. Animals fitted with an acrylic plate and inoculated with C. albicans had lanthanum deposited throughout the tissue, including the stratum corneum (Fig. 2); no discrete barrier to lanthanum was seen. Two weeks after removal of the acrylic plate a distinct barrier had reformed between cells at the interface of the strata spinosum and granulosum (Fig. 3). Four weeks after removal of the plate from infected animals the lanthanum permeability was located between cells of the stratum granulosum (sg, arrowed; Fig. 4). A similar appearance was seen 8 weeks after removal of the acrylic plate from infected animals (Fig. 5). Downloaded from http://jmvm.oxfordjournals.org/ at University of Liverpool on March 23, 2015

FIG. 3. Two weeks after removal of the acrylic plate in animals inoculated with C. albicans. A lanthanum barrier (arrowed) is present between the stratum spinosum (ss) and granulosum (sg), × 5640.


25


FIG. 4. Four weeks after removal of the acrylic plate in animals inoculated with C. albicans. The lanthanum barrier is present in the lower part of the stratum granulosum, × 3850.

Electrophoresis Densitometric scans of palatal transudates and serum are shown in Fig. 6. Normal palatal epithelium allowed the transudation of small amounts of protein of low molecular weight (39 000-80 000), despite the presence of a permeability barrier to lanthanum (Fig. 6A). The presence of a palatal acrylic plate enhanced this effect further, but proteins of molecular weight 52 000 and 59 000 were particularly prominent (Fig. 6c). Some other high-molecular weight proteins were also present in infected areas (Fig. 6c). The protein profile of serum (Fig. 6D) was markedly different to that of

26

MARTIN E T AL.

the transudates, but one protein of molecular weight 59 000 was common to both. Rats that had been inoculated with C. albicans only or which had their acrylic plates removed 2 weeks earlier had transudate protein profiles identical to Fig. 6A (results not shown). Proteins found in pilocarpine-stimulated salvia did not resemble those of palatal transudates or serum (results not shown). DISCUSSION This investigation has used a rat model to study the effects of C. albicans on the oral epithelium. Previous work [ 13, 15, 19, 20,] has shown that this model is reproducible and reliable. Pilot studies (results not shown) have demonstrated that the model can be used to study epithelial permeability but that care is necessary with the perfusion techniques. The perfusion methods used in this investigation are time-consuming but are necessary if reproducible results are to be achieved. The presence of a permeability barrier to the passage of lanthanum could be demonstrated in healthy rats; a finding in agreement with that of Squier and colleagues [21-23]. However, the maintenance of an epithelial barrier in the presence of an oral prosthesis has not been demonstrated previously. Although it has been suggested that the presence of an oral prosthesis traumatizes the palatal epithelium [2-5, 16], no histological evidence for this contention could be found in previous reports citing this model [18]. The presence of an ill-fitting prosthesis was found not to affect the ultrastructural appearance of the permeability barrier adversely. It did, however, cause the transudation of protein to alter qualitatively with low molecular weight molecules predominating. The presence of a fungal infection radically changed the distribution of perfused lanthanum within the epithelium. This electron dense material was distributed throughout the tissue the permeability barrier having apparently being destroyed.


FIG. 5. Eight weeks after removal of the acrylic plate in animals inoculated with C. albicans. Lanthanum is present between cells of the stratum granulosum (arrowed, sg), × 4624.

C. ALBICANS AND ORAL EPITHELIUM 59

27

47 39

A

59

FiG. 6. Densitometric scans of SDS gels. The origin is to the left: molecular weights (× ]03) of major proteins arc shown: A is transudatc from untreated palates; B is transudate from a rat fitted with an acrylic plate; C is transudat¢ from a rat fitted with an acrylic plate and inoculated with C. albicans; D is rat serum. Analysis o f the protein transudates, however, showed that the barrier was still selectively operational, with the low-molecular-weight proteins predominant. Thus even when no effective barrier could be demonstrated ultrastructurally, oral epithelium still exerted some selective action on the transudation o f serum proteins. Removal of the prosthesis resulted in a healing of the oral epithelium, comparable to the healing o f human mucosa that occurs when a palatal prosthesis is removed [1, 4,

12]. This investigation has demonstrated that the permeability barrier completely reforms, but deeper in the oral epithelium than originally, i.e. in the stratum spinosum. Restitution of impermeability appeared to be associated with membrane-coated granules (results not shown) as described by previous workers [23]. As the epithelial cells matured and migrated towards the keratin layer the barrier moved to the level of the stratum granulosum: the reason for location remains unclear. The combination of ultrastructural and biochemical methods used in this study has shown that oral epithelium exerts a selective action on the transudation of molecules. Some of the transudated proteins co-migrate with those from serum and thus it is likely that blood is the source. However, this remains to be confirmed by purification and identification of the transudates. A number of studies have shown that there is an increased transepithelial water transudation when the permeability barrier is lost [9, 17]. It would logically be assumed that if destruction of the epithelial barrier is complete then serum proteins would also escape. This study has shown that although the barrier is permeable to


17o ~4o123~o3s7

28

MARTINE T AL.

tracer molecules some selective action o n the m a c r o m o l e c u l e s occurs. T h u s the molecules reaching the k e r a t i n layer are n o t a complete reflection of s e r u m proteins. This selectivity could be a f u n c t i o n o f m o l e c u l a r charge, shape or o f the properties o f the surface o f the epithelial cells that they are passing. W h a t is clear is t h a t i n f e c t i o n of the e p i t h e l i u m associated with the wearing o f a prosthesis does affect the p e r m e a b i l i t y b a r r i e r b u t this c a n be reversed if the prosthesis is removed.

ACKNOWLEDGEMENTS The authors would like to acknowledge the expert technical assistance of Pauline McGowan, Heather Dunn and Andrew Birss.

1. ANDERUP, B., ANDERSSON, B. & HEDEGARD, B. 1977. Proteshygien III. Racker rengoring or helprotesen fur stomatitulakning. Tandlakartidringen, 69, 394-398. 2. BASTIAAN,R. J. 1976. Denture sore mouth. Aetiological aspects and treatment. Australian Dental Journal, 21, 375-387. 3. BERGMAN, B., CARLSSON,G. E. & ERICSSON, S. 1971. Effect of differences in habitual use of complete dentures on underlying tissues. Scandinavian Journal of Dental Research, 79, 449-460. 4. BUDTZ-JORGENSEN.E. & LOE, H. 1972. Chlorhexidine as a denture disinfectant in the treatment of denture stomatitis. Scandinavian Journal of Dental Research, 80, 457-464. 5. BUDTZ-JORGENSEN,E. 1974. The significance of Candida albicans in denture stomatitis. Scandinavian Journal of Dental Research, 82, 151-190. 6. BUDTZ-JORGENSEN, E. 1981. Oral mucosal lesions associated with the wearing of removable dentures. Journal of Oral Pathology, 10, 65-80. 7. CAWSON, R. A. 1965. Denture sore mouth and angular cheilitis. British Dental Journal, 115, 441-449. 8. CAWSON,R. A. 1976. Infections of the oral mucous membrane. In: B. COHEN& 1. R. H. KRAMER (eds) Scientific Foundations of Dentistry, pp. 484-487. William Hieneman, London. 9. KAABER,S. 1971. Studies on the permeability of oral mucosa. Acta Odontologica Scandinavica, 29, 653-662. 10. KUTUZOV,H. & SICHER,H. 1952. Anatomy and function of the palate in the white rat. Anatomical Record, 114, 67-84. 11. LAEMMLI, V. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature, 227, 680-685. 12. LUNDQUIST, L., ANDERUP, B. 86 HEDEGARD, B. 1975. Proteshygien 11. Klinisk vardering ar elt hygienprogram fur patienter med protesstomatit. Tandlakartidringen, 67, 872-879. 13. LAMB, D. J. 86 MARTIN, M. V. 1983. An in vitro and in vivo study of the treatment of palatal candidosis by incorporation of chlorhexidine into autopolymerising resin plates. Biomaterials, 4, 205-210. 14. MIMS,C. A. 1982. The Pathogenesis oflnfectious Disease. Academic Press, London. I5. NORRIS, i . i . , LAMB,D. J., CRAIG,G. T. ,9. MARTIN, M. V. 1985. The effect of miconazole on palatal candidosis in the Wistar rat. Journal of Dentistry, I3, 288-297. 16. NYQUIST, G. 1952. A study of denture sore mouth. An investigation of traumatic, allergic and toxic lesions of the oral mucosa arising from the use of full dentures. Acta Odontologica Seandinavica, 10, Suppl. 9, 11-54. 17. REBER, E. 8¢ KAABER,S. 1978. Barrier properties of inflamed denture loaded mucosa to water. Scandinavian Journal of Dental Research, 86, 386-391. 18. SHAK1R,B. S., MARTIN,M. V. & SMITH, C. J. 1981. Induced palatal candidosis in the Wistar rat. Archives of Oral Biology, 26, 787-793. 19. SHAKIR,B. S., MARTIN,M. V. & SMITH, C. J. Rat palatal candidosis: effect of alternate removal and insertion. Archives of Oral Biology, in press. 20. SPURR, A. R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research, 26, 31-43. 21. SQUIER, C. A. 1973. The permeability of keratinised and non-keratinised oral epithelia to horseradish peroxidase. Journal of Ultrastructural Research, 43, 160-177. 22. SQUIER, C. A. & ROONEY, S. L. 1976. Permeability of keratinised and non-keratinised oral epithelia to lanthanum in vivo. Journal of Ultrastructural Research, 54, 286-295. 23. SQUIER, C. A. & HALL, B. K. 1985. The permeability of hyperplastic oral epithelium. Journal of Oral Pathology, 14, 357-362.


REFERENCES