The effect of saliva or serum on bacterial and Candida albicans colonization on type I collagen.

Colonization of Candida albicans on oral surfaces can serve as a reservoir for disseminated infections, such as aspiration pneumonia and gastrointestinal infection, particularly in the immunocompromised host. Therefore, the aim of this study was to investigate the effects of salivary and serum pellicles on C. albicans, Streptococcus mutans, S. sanguis, Lactobacillus and Actinomyces colonization on type I collagen, a major organic component of periodontal ligaments. The colonization potential of two isolates each of C. albicans, S. mutans and S. sanguis, and a single isolate each of Lactobacillus and Actinomyces to uncoated (control), saliva-coated or serum-coated type I collagen plates (surface area 143 mm(2), Cell Disk; Sumitomo, Tokyo, Japan) was examined using a bioluminescent adenosine triphosphate assay based on firefly luciferase-luciferin system. The results revealed that with mutans streptococci, a saliva pellicle was significantly more effective in promoting bacterial colonization compared with the pellicle-free collagen disc, and the serum-coated sample significantly inhibited the colonization of streptococci (anova; P < 0b01). In contrast, in the case of C. albicans, Lactobacillus and Actinomyces isolates, a serum pellicle was significantly more effective in promoting the colonization, followed by saliva pellicle and uncoated specimen (anova; P < 0b01). These results suggested that crevicular fluid rich in seruminous components would promote the colonization of Candida, Lactobacillus and Actinomyces on type I collagen as opposed to streptococci which showed greater avidity to saliva-coated collagen.

An in vitro evaluation of the adhesion of Candida species to oral and lung tissue cells.

The analysis of the adherence capacity of fungi to surfaces of both oral tissue and different tissues would be of interest in the fungal dissemination as an oral and systemic pathogen. We developed an in vitro adenosine triphosphate (ATP)-based assay technique to extract the cellular and fungal ATP separately, which allowed the quantitative evaluation of the adhesion of the yeast to monolayers of human gingival epithelial cells (GEC), gingival fibroblasts (GF) and pulmonary fibroblasts (PF). Seven oral isolates of Candida species (three of Candida albicans, three of Candida tropicalis and one of Candida glabrata) were used in the study. The adherent level of the Candida species varied depending on both the isolates and the cell origins, although all the Candida isolates had a significantly higher level of adherence to GEC than to GF except the single isolate of C. tropicalis. Whereas the adherent level of the five isolates to GEC was significantly higher than that to PF, the adherent level of the remaining two isolates of C. tropicalis to GEC was significantly lower than that to PF. These results suggest that candidal adherence to host tissue cells should be regulated in an isolate-dependent and cell-origin-dependent manner, and that the phenomena may be involved in the colonisation and/or dissemination of the fungi.

Lactobacillus reuteri in bovine milk fermented decreases the oral carriage of mutans streptococci.

The effect of Lactobacillus reuteri against one of the major cariogenic organism, Streptococcus mutans, was studied. Yogurt products containing L.reuteri showed a significant growth inhibitory effect against S. mutans, whilst yoghurts with lactobaccilli other than L. reuteri did not show such inhibition. Further, double-blind, placebo-controlled trial demonstrated that consuming yogurt with L. reuteri significantly reduced the oral carriage of mutans streptococci, compared with the placebo yogurt.

Fungicidal effect of three new synthetic cationic peptides against Candida albicans.

OBJECTIVE: Peptide antibiotics are considered a new class of antifungal agents. Of these, an alpha-helical, cationic peptide termed Dhvar 4, a relative of salivary histatin has been shown to be an antifungal of relatively high potency. Similarly, lactoferricin B (LFB) and a derivative thereof, LFB(17-30), disrupts the fungal cell membrane and acts against Candida albicans. As Dhvar 4 and LFB(17-30), exhibit almost identical amino acid sequences at their C-terminal, we hypothesized that laboratory synthesis of peptides with an alpha-helical structure and having similar amphipathic properties could lead to products with candidacidal activity. Hence, three such peptides - JH8194, JH8195 and JH 8944, were synthesized and their antifungal properties compared with recognized antifungals LFB, LFB(17-30), human lactoferricin (LFH), Histatin-5 and Dhvar 4, against two isolates of C. albicans. MATERIALS AND METHODS: The antifungal agents were synthesized and their secondary structures evaluated according to a previously described protocol of Situ and Bobek (2000)Antimicrob Agents Chemother44: 1485-1493. The C. albicans strains were oral isolates from a human immunodeficiency virus-infected (isolate A2) and a healthy (A6) individual. A standard concentration of yeasts was exposed to a range of dilutions of the agents for a specific duration and the cell death (viability) in terms of the resultant colony forming units ml(-1) was quantified. RESULTS: Dhvar 4, showed the most alpha-helical propensity, and was the least fungicidal while LFB and LFB(17-30) showed the highest antifungal potential, and demonstrated total kill of A6, and A2 at 5 and 10 microM concentrations, respectively whilst LFH killed both isolates at a l0 microM concentration. Of the three new synthetic peptides, JH 8194 was the most potent (total kill of A6/A2 strains at 1.25/2.5 microM), followed by JH 8195 (total kill of A6/A2 strains at 5/10 microM while JH 8944 was the least potent as a 25 microM concentration was required to kill either strain of Candida. On further analyses of the relationship between pI value of the peptides and their anticandicidal activity, a significant positive correlation was noted. In order to rule out a cytotoxic effect of the new synthetic peptides we compared the fungicidal and hemolytic activities under similar incubation conditions using freshly isolated erythrocytes and all three peptides exhibited no detectable hemolysis upto an concentration of 100 microM in contrast to the polyene antifungal amphotericin B that elicited significant initiation of hemolysis at a concentration of 5.0 microM. CONCLUSION: Our data suggest that laboratory synthesis of agents with an alpha-helical structure and having amphipathic properties similar to known, natural antifungal agents may be a promising avenue to generate products with improved antifungal activity.

In vitro cariogenic potential of Candida albicans.

The adherence and dissociation of Candida albicans, C. tropicalis, Streptococcus mutans and S. sanguis to six substrates including hydroxylapatite (HAP) which exhibit various hydrophobicity, was examined by the use of a bioluminescent adenosine triphosphate (ATP) assay. Dissolution of HAP by C. albicans or S. mutans was determined spectrophotometrically by the use of o-cresolphthalein complexone. In the adherence of C. tropicalis, S. mutans and S. sanguis, the amount of adherent cells correlated with the hydrophobicity of the substrates. In contrast, the adherence of C. albicans to HAP was extraordinary high, although the adherence of the fungi also correlated with the hydrophobicity of the substrates, except for HAP. The yeasts attached to HAP was effectively removed by high concentration of either phosphate or calcium ions. The amount of calcium-release from HAP caused by C. albicans and S. mutans was 113 microg ml(-1) (final pH = 3.45), and 5.4 microg ml(-1) (final pH 4.81), respectively and the maximum growth of C. albicans and S. mutans was 10(7) cfu ml(-1) and 7.4 x 10(12) cfu ml(-1), respectively. The results, taken together, suggest that C. albicans adhere to HAP specifically through electrostatic interaction, and that, in a much smaller number (1.0/7.4 x 10(5)), C. albicans possesses the ability to dissolve HAP to a greater extent (approximately 20-fold) when compared with S. mutans.

A novel technique to evaluate the adhesion of Candida species to gingival epithelial cells.

We developed an in vitro ATP assay technique to extract cellular and fungal ATP separately, which allowed to evaluate quantitatively the adhesion of the yeasts to monolayers of human gingival epithelial cells. Thirteen isolates of Candida spp. representing three species (i.e. Candida albicans, C. tropicalis and C. glabrata) were used in the present study. When the adherent capacity of the Candida species was compared, C. albicans exhibited highest capacity of adherence to gingival epithelial cells, followed by C. tropicalis, and C. glabrata was the lowest [analysis of variance (ANOVA), P < 0.01]. The germ tubes of C. albicans exhibited significantly higher adherence capacity than their blastoconidia cells (ANOVA, P < 0.01), which was not observed with a C. albicans isolate, defect of germ tube formation. Our results suggested that the adherence of C. albicans is promoted by germ tube formation and may play an important role in the pathogenesis of the fungus.

Biofilm formation of Candida albicans on the surfaces of deteriorated soft denture lining materials caused by denture cleansers in vitro.

Candidal colonization and subsequent biofilm formation on denture materials are important in the development of pathogenesis, such as denture stomatitis. Routine use of denture cleansers is one of the most effective methods of denture plaque control, although the incompatibility of soft liners and denture cleansers cause damage to the materials. The present study, biofilm formation of Candida albicans on the surfaces of soft denture lining materials, immersed in denture cleansers for 180 days were studied. Seven commercially available soft denture lining materials, were artificially deteriorated by immersion into three commercially available denture cleansers for 180 days, and subsequent fungal growth and biofilm formation were studied by measuring pH of the media and by the use of adenosine triphosphate (ATP) analysis. Fungal biofilm formation on the deteriorated soft liners varied depending upon the combination of the soft liners and denture cleansers. Several combinations of soft liners with denture cleansers exhibited the significantly high colonization capacity as compared with each sample immersed in distilled water, used as individual controls. The relationship between the biofilm formation on the samples of each material and the surface roughness of the soft lining materials was analyzed. However, no significant correlation was observed. The results, taken together, suggested that fungal colonization could be predominantly regulated by the combination of lining material with denture cleansers. In clinical terms, our findings suggests that daily cleansing of soft lining materials with mismatched denture cleansers promoted the subsequent biofilm formation of fungi on the materials.

Changes in surface roughness and colour stability of soft denture lining materials caused by denture cleansers.

Soft denture lining materials were immersed into solutions of denture cleansers for 8 h at room temperature, and immersed into distilled water for the remainder of the 24-h period at 37 degrees C. Surface roughness of the soft denture lining materials was measured by contact type surface roughness instrument. For the colour stability test, soft denture lining materials were immersed in the denture cleansers as described above for 180 days. Finally, the colour changes of each material were quantitatively measured by a photometrical instrument to obtain the colour differences between newly processed specimen and immersed specimens (P < 0.01). An autopolymerizing silicone material, Evatouch, exhibited severe changes in surface roughness by all denture cleanser, and the generic material GC Denture Relining showed the minimal changes. Severe colour changes were also observed with some liner and cleanser combinations (P < 0.01). Except for Evatouth, the four silicone soft liners were more stable in surface roughness and in colour change than the two acrylic soft liners. One autopolymerizing silicone (GC denture relining) and one heat curing silicone (Molloplast B) demonstrated the best stability.

Bacterial and Candida adhesion to intact and denatured collagen in vitro.

Several recent reports imply the possibility of cariogenicity and periodontal disease linked to denture plaque containing Candida albicans. Adhesion of oral bacteria and Candida species to the extracellular matrix, such as type I collagen, fibronectin and denatured type I collagen, was examined by using adenosine triphosphate (ATP) analysis. The adhesion of C. albicans to intact and denatured type I collagen was significantly greater than those of oral bacteria and other species of Candida. This result suggests that C. albicans possesses the ability to adhere specifically to extracellular matrix, as compared with other Candida species or oral bacteria.

Differences in Candida albicans adhesion to intact and denatured type I collagen in vitro.

An inhibition assay of Candida albicans adhesion to gelatin-immobilized membranes was compared with that to intact type I collagen-immobilized membranes using an arginine-glycine-aspartic acid (RGD) containing peptide. As compared with a protein-free membrane, gelatin and collagen significantly enhanced the adherence of C. albicans. The adhesion of the yeast to gelatin was significantly inhibited by the RGD peptides, but not by arginine-glycine-glutamic acid (RGE) peptides. In contrast, attachment to collagen was not inhibited by RGD peptides. These results suggest that the RGD sequence of gelatin and the integrin-like proteins of yeasts may be involved in adherence.

Impact of components of denture acrylic resin on gingival cell growth and sensitivity to Candida albicans adhesion.

The effects of four liquid components of denture acrylic resin on host cell activity and fungal adhesion were investigated in this study. The low concentration (1 micromol l(-1)) of the liquid components caused no change in the activities and morphologies of the gingival fibroblast cells, compared with control and dimethylsulphoxide-exposed cells. However, when the cells were exposed to high concentrations (1 mmol l(-1)) of benzqyl peroxide, morphological change was observed, implying that the exposure of the cells to high concentrations of the liquid components of denture acrylic causes the loss of adhesion proteins from the cells. Thus the amount of Candida adhesion to human gingival cells was analysed, and the adherence of fungi to the cell was significantly reduced when the cells were pre-exposed to methyl methacrylate, hydroquinone and benzoyl peroxide at a concentration of 1 micromol l(-1) (P < 0.01), which did not affect either the cell viability or the cell morphology. These results, taken together, suggested that the renewal of dentures could be a possible therapeutic and/or preventive aid for oral candidosis in denture-wearing patients.

Alteration of the coadherence of Candida albicans with oral bacteria by dietary sugars.

Interactions between bacterial oral flora and Candida albicans are important in denture plaque formation. This study therefore first aimed to quantify the coadherence of C. albicans and bacteria by the use of a bioluminescent adenosine triphosphate (ATP) assay based on the firefly luciferase-luciferin system. The second aim was to examine the effect of i) dietary sugars (used for preculture) and ii) enzymatic digestion of fungi on the coadherence. When yeast was preincubated in yeast nitrogen base medium (YNB) supplemented with 250 mM glucose, the yeast coadhered with all isolates of Streptoccus mutans and Streptococcus sanguis, and no significant coadhesion was observed with the isolates of Streptococcus sobrinus, Streptococcus salivarius, Lactobacillus and Actinomyces. However, when the yeast was precultured in YNB supplemented with 500 mM galactose, the yeast coadhered with S. salivarius and Actinomyces, which was not observed when the yeast was grown in YNB with glucose. In addition, the coadherence of the yeast with the isolates of S. sanguis was significantly reduced. Enzymatic digestion of yeast and a reverse transcription polymerase chain reaction assay revealed that expression of at least two types of proteinaceous adhesins are involved in these phenomena.

Candida albicans growth on thermal cycled materials for maxillofacial prostheses in vitro.

In the present study, the growth of a single isolate of Candida albicans on saliva-, serum-coated or protein free (uncoated), thermocycled (4-70 degrees C for 1 min, respectively; 0, 1000 and 10 000 times) 15 commercial maxillofacial materials was investigated, by monitoring pH changes in growth media. The inhibitory effect of the tissue conditioners on fungal growth was analysed using three parameters viz: (i) delay in the onset of the rapid decline in pH (ii) reduction in the rate of pH change and (iii) the pH minima reached. In the case of control materials (non-thermocycled and uncoated), significant antifungal effect was observed with two products. However, the antifungal effect of the materials was significantly reduced both by thermal cycling (Analysis of covariance [ANOVA]; P < 0.01) and a layer of protein coating (saliva, P < 0.05; serum, P < 0.01). When the interrelation between three parameters of fungal growth and the surface hydrophobicity of the materials were analysed, minimum pH of fungal growth on 10 000-thermocycled materials correlated well with the contact angles of the materials (Student t-test, P < 0.01), suggesting that thermocycling process reduced the unpolymerized components of the materials which showed the antifungal effects, resulted in that the cell growth depends on the surface hydrophobicity of the specimens. These results, taken together, suggest that the ageing of the materials and the biological fluids of the host enhanced the fungal growth on maxillofacial materials.

Antifungal activity of histatin-5 against non-albicans Candida species.

Fungicidal effects of histatin-5 against 26 oral isolates belonging to 5 non-albicans Candida species were examined. Fifty microM of histatin-5 killed more than 95% of Candida tropicalis and Candida guilliermondii isolates and more than 90% of Candida parapsilosis and Candida krusei. However, Candida glabrata was less sensitive to the peptide (mean 62.9%). Our results, taken together, demonstrated that histatin-5 possessed the fungicidal activity against Candida species other than C. glabrata.

Induction of basic helix-loop-helix protein DEC1 (BHLHB2)/Stra13/Sharp2 in response to the cyclic adenosine monophosphate pathway.

DEC1 (BHLHB2)/Stra13/Sharp2, a basic helix-loop-helix (bHLH) transcription factor has been suggested to be involved in the control of proliferation and/or differentiation of several cells including nerve cells, fibroblasts and chondrocytes. In the present study, we examined the effect of parathyroid hormone (PTH), dibutyryl cAMP (Bt2cAMP) and forskolin on the expression of DEC1 in various cells. In rabbit chondrocyte cultures, PTH or Bt2cAMP increased the DEC1 mRNA level within 1 h. Thereafter, the DEC1 mRNA level rapidly decreased to the basal level at 3 h, and increased at 6-24 h. In cultures of a mouse embryo prechondrogenic cell line ATDC5, PTH or forskolin, an activator of adenylate cyclase, also increased the DEC1 mRNA level within 1 h. Furthermore, in all evaluated cell lines of human fibroblasts, canine epithelial cells, human carcinoma, human glioblastoma and human melanoma, Bt2cAMP increased the DEC1 mRNA level within 1-3 h. Studies with actinomycin D and cycloheximide indicated that the enhancement of DEC1 mRNA by cAMP was not due to mRNA stabilization and did not require new protein synthesis. These findings suggest that DEC1 is a novel direct target for cAMP in wide types of cells, and that the bHLH protein is involved in the control of gene expression in cAMP-activated cells.

Growth of Candida species on commercial denture adhesives in vitro.

PURPOSE: Although denture adhesives are widely used by the elderly, it is unknown whether denture adhesives support microbial growth. Therefore, we investigated the growth of Candida species on six commercial denture adhesives. MATERIALS AND METHODS: The growth of a single isolate of C albicans and C tropicalis on six commercial denture adhesives was investigated by monitoring pH changes in growth media. RESULTS: In the preliminary study, the effect of each product on the pH value of the medium was examined; a single product itself significantly reduced the pH of medium below 5.0. When the yeast was grown on the materials, the pH changes in the media varied depending on the adhesive materials and, to a greater or lesser extent, on whether they showed antifungal activity. Two products (Cushion Collect and Collect Soft A) significantly suppressed C albicans growth (P < 0.01), and one product (Collect Soft A) effectively reduced C tropicalis growth (P < 0.01). CONCLUSION: Our results suggest that denture adhesives possess antifungal activity to a greater or lesser degree. However, one product caused the reduction of pH below 5.0. Thus, in the daily use of denture adhesives, attention should be paid to both the materials and their microbiologic properties.

Effect of serum concentration on Candida biofilm formation on acrylic surfaces.

The biofilm formation of the oral fungal pathogen Candida on denture acrylic strips coated with saliva, serum and, saliva-serum pellicle were examined in vitro using Candida albicans (four isolates), Candida glabrata (three isolates) and Candida tropicalis (three isolates). The degree of biofilm activity varied depending upon both the isolate and the pellicle. Significantly increased biofilm activity on the pellicle (particularly serum)-coated strips was observed with three isolates of C. albicans and another of C. glabrata on protein-coated acrylics, with increasing concentration of serum in the pellicle. Similar trends were observed with one isolate of C. albicans and C. glabrata, although the effects of pellicles were not significant. In contrast, with all three isolates of C. tropicalis and a single isolate of C. glabrata, although the biofilm activity on the protein-free control strips was significantly higher than that of saliva-coated strips, the increase in activity of pellicle-admixed biofilm depended upon the serum concentration. Candidal biofilm formation on acrylic surfaces is essentially promoted with increasing concentration of serum in the pellicle. This suggests that inflammation in the oral environment would facilitate fungal colonization on denture acrylic.

Interactions between thermal cycled resilient denture lining materials, salivary and serum pellicles and Candida albicans in vitro. Part II. Effects on fungal colonization.

In the present study, the growth of a single isolate of Candida albicans on saliva-, serum-coated or protein free (uncoated), thermocycled (4-70 degrees C for 1 min, respectively; 0, 1000 and 10 000 times) seven commercial soft lining materials were investigated, by adenosine triphosphate (ATP) analysis. In the case of control resilient liners (not thermocycled and uncoated), the fungal colonization appeared to depend upon the type of commercial resilient liner used. Thus, the lowest colonization was observed with fluoric and heat-cured silicone materials, cold-cured silicone materials, except for one product, and heat-cured acrylic resin exhibited the highest colonization capacity, and cold-cured acrylic resilient liners exhibited the intermediate. However, the fungal colonization on the materials was significantly promoted both by thermal cycling (ANOVA, P<0.01) and a layer of protein coating (saliva, P<0.01; serum, P<0.01). These results, taken together, suggest that the ageing of the materials and the biological fluids of the host promote yeast colonization on resilient lining materials.

RGD-CAP ((beta)ig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin alpha(1)beta(1).

In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human (beta)ig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-beta. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or beta-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg(2+) or Mn(2+)), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin alpha(1) or beta(1) subunit, but not to the alpha(2), alpha(3), alpha(5) or beta(2) subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the alpha(5) subunit, but not the mAb to the alpha(1) subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin alpha(1)beta(1) that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.

Enhancement of cell adhesion and spreading by a cartilage-specific noncollagenous protein, cartilage matrix protein (CMP/Matrilin-1), via integrin alpha1beta1.

Cartilage matrix protein (CMP; also known as matrilin-1), one of the major noncollagenous proteins in most cartilages, binds to aggrecan and type II collagen. We examined the effect of CMP on the adhesion of chondrocytes and fibroblasts using CMP-coated dishes. The CMP coating at 10-20 micrograms/ml enhanced the adhesion and spreading of rabbit growth plate, resting and articular chondrocytes, and fibroblasts and human epiphyseal chondrocytes and MRC5 fibroblasts. The effect of CMP on the spreading of chondrocytes was synergistically increased by native, but not heated, type II collagen (gelatin). The monoclonal antibody to integrin alpha1 or beta1 abolished CMP-induced cell adhesion and spreading, whereas the antibody to integrin alpha2, alpha3, alpha5, beta2, alpha5beta1, or alphaVbeta5 had little effect on cell adhesion or spreading. The antibody to integrin alpha1, but not to other subunits, coprecipitated 125I-CMP that was added to MRC5 cell lysates, indicating the association of CMP with the integrin alpha1 subunit. Unlabeled CMP competed for the binding to integrin alpha1 with 125I-CMP. These findings suggest that CMP is a potent adhesion factor for chondrocytes, particularly in the presence of type II collagen, and that integrin alpha1beta1 is involved in CMP-mediated cell adhesion and spreading. Since CMP is expressed almost exclusively in cartilage, this adhesion factor, unlike fibronectin or laminin, may play a special role in the development and remodeling of cartilage.