Quantitative light-induced fluorescence to measure enamel remineralization in vitro.

UNLABELLED: The aim of this study was to compare the ability of quantitative light-induced fluorescence (QLF) and surface microhardness (SMH) to measure the remineralization of enamel subsurface lesions, using a pH-cycling model including treatment with 0-ppm, 550-ppm or 1,100-ppm sodium fluoride (NaF) dentifrices. METHODS: Subsurface lesions were created in human enamel specimens (n = 36) and exposed to a remineralization pH-cycling model for 14 days. The pH-cycling model was performed in an automated system where specimens were subjected to a demineralizing solution for 20 min and treatment for 1 min and were then remineralized for 7 h 39 min, 3 times daily. The treatments consisted of 3 NaF, silica-containing dentifrices (0 ppm F; 550 ppm F; 1,100 ppm F). The outcome variables were: change from baseline in surface hardness and percentage change from baseline in fluorescence. An ANCOVA explored differences between different treatment groups (at the p < 0.05 level). Associations between QLF and SMH were evaluated using Spearman's correlation coefficient. RESULTS: The percentage SMH changes were 14.9 ± 2.1%, 56.6 ± 9.6% and 103.9 ± 14.6% for the 0-, 550- and 1,100-ppm F dentifrices, respectively. The percentage fluorescence changes were 15.6 ± 7.1%, 59.8 ± 11.9% and 85 ± 13.2%, respectively. The differences between all pairwise comparisons were statistically significant for both methods (p = 0.001). QLF correlated with SMH (r = 0.67). CONCLUSIONS: Both the SMH and QLF methods demonstrated a significant F dose response for toothpaste in this in vitro remineralization model, and both methods were able to distinguish treatments with different F levels.

Salivary proteolysis of histidine-rich polypeptides and the antifungal activity of peptide degradation products.

Incubation of purified synthetic histidine-rich polypeptides, HRP-2, -3, -4, -5, -6 (histatins), with diluted human parotid saliva yielded a series of peptide degradation products whose structures could be determined by gas-phase sequencing of cationic polyacrylamide gel electroblots. Sequencing indicated that two and sometimes three peptides were present in the same Coomassie blue-stained band. By comparing different individuals' salivas it was observed that structural variation occurs, perhaps due to differences in the concentrations or specific activities of salivary proteases. Based on the structural data, four proteolytic enzyme activities are proposed. A trypsin-like and chymotrypsin-like enzymatic activity(s) appear to represent the most active salivary protease; however, both an alanine-lysine endopeptidase and a histidine peptidase activity are also present in parotid saliva. In comparison to HRP-4 or HRP-6, degraded products were less active as antifungal agents against Candida albicans both in blastospore and germ-tube assays.

Salivary anti-candidal assays.

Inhibition of Candida albicans blastospore viability by parotid, submandibular-sublingual and whole salivas could not be determined by direct assay of yeast cells in each respective saliva. Determination of antifungal activity could, however, be carried out if saliva was first preincubated with Candida cells and this was immediately followed by removal of saliva and resuspension of yeast cells in nonenriched buffers of pH 5-7 for appropriate incubation periods. To attain accurate reproducible quantitative data, parotid, submandibular-sublingual and whole salivas each required different preincubation times with C. albicans as well as prior acidification and boiling. Acidification was also necessary for optimizing the germ tube assay although, in contrast to blastospore viability, inhibition of blastospore-germ tube conversion could be determined directly in saliva. Salivary antifungal effects on blastospore division were negligible at yeast cell concentrations greater than 10(6) colony-forming units per ml and were found to be independent of pH, whereas salivary inhibition of germ tube formation was significant only at pH 5 in the assay systems employed. The requirement for acidification and an observed enhancement of antifungal activity on aqueous dilution of the saliva suggested that only a fraction of the salivary antifungal components present in saliva were available in the free form to exert their biological activity. These results open up the possibility of investigating salivary antifungal activity in human health and disease.

One-step purification of histidine-rich polypeptides form human parotid saliva and determination of anti-candidal activity.

Immunoadsorption affinity chromatography was used to selectively purify the family of the histidine-rich polypeptides (HRPs) from human parotid saliva. The immunoadsorbent was prepared by coupling an enriched preparation of horse anti-(HRPs 2, 3, 4, 5 and 6) IgG to protein G. Both freshly collected stimulated untreated and acidified boiled salivas (5 ml) were applied to the affinity column. When native saliva was used it appeared that all of the components of saliva, with the exception of the HRPs, were present in the fraction nonadsorbed to the affinity column; however, recovery of the HRPs with 0.2 M sodium acetate-HCl, pH 1.8, was poor. Yields of HRPs desorbed from the column with the pH 1.8 treatment were significantly improved if salivary HRP proteolysis was delayed immediately after collection by acidifying the saliva to pH 4.5 followed by a short boiling time period, which neither affected HRP quantification nor biological activity. Affinity chromatography results were checked both by cationic polyacrylamide gel and by capillary electrophoresis. Antifungal activity was found to reside only in the low pH HRP fraction of the immunoadsorbent column, suggesting that it is the histidine-rich family of polypeptides that is responsible for salivary antifungal action.

Antifungal activities of salivary histidine-rich polypeptides against Candida albicans and other oral yeast isolates.

Twenty-six oral yeast isolates from 26 donors were tested for their susceptibility to salivary histidine-rich polypeptide-4 (HRP-4) in blastospore viability assays. HRP-4 was observed to inhibit blastospore division in all of the yeast isolates, although inhibition was variable depending upon both species and strain tested. Nine species of Candida and 2 strains of Trichosporon pullulans were included in the study. No significant differences in susceptibility to HRP-4 could be seen, irrespective of where in the oral cavity the yeast isolate was obtained.

Pilot study comparing the salivary cationic protein concentrations in healthy adults and AIDS patients: correlation with antifungal activity.

This investigation compared the salivary cationic protein concentrations of 12 healthy adult controls with those of 12 hospitalized patients with AIDS. Salivas were quantified by capillary electrophoresis using purified cationic protein standards. In parotid saliva, histidine-rich polypeptides (HRPs) 1-6, histatin 6, and lysozyme concentrations were determined. In addition to these eight cationic proteins, submandibular-sublingual saliva was also quantified for histatin 2 and the histatin 2 degradation product. When comparisons were made on the basis of individual proteins, the HRP-histatin concentrations in the AIDS patients showed either statistically significant decreases or a decreasing trend compared with healthy adult controls. When HRP-histatin concentrations were summed for each patient, there were statistically significant differences between the healthy adult controls and the individuals with AIDS in both parotid and submandibular-sublingual salivas. Closer examination revealed that some individuals with AIDS had HRP-histatin concentrations that fell within the normal range of the healthy adult controls. For these individuals, lower than expected salivary antifungal values were obtained. Either decreasing histidine-rich protein concentrations and/or an inability of these proteins in saliva to interact with Candida albicans may contribute to the defective salivary antifungal activity seen in AIDS patients.

Determination of salivary anticandidal activities in healthy adults and patients with AIDS: a pilot study.

This investigation compared the salivary anticandidal activities of 12 healthy adults with 12 hospitalized patients with AIDS. Stimulated parotid, submandibular-sublingual, and whole salivas were collected during a period of 10 min, immediately acidified, boiled, and then centrifuged to isolate salivary supernatants. Supernatants were then tested for antifungal activity against Candida albicans in blastospore viability inhibition and germ tube formation assays. A unit of blastospore or germ tube antifungal activity was established as that activity yielding 90% or greater inhibition during a defined time period in each salivary assay. Each of the patients with AIDS were found to be defective in one or more of their salivary antifungal activities, and in comparison with healthy adults the differences in antifungal units per milliliter of saliva and total antifungal units were statistically significant for each saliva and each antifungal assay. Defective salivary antifungal activity may contribute to the oral candidiasis seen in patients with AIDS.

Assessment of antimicrobial treatment of denture stomatitis using an in vivo replica model system: therapeutic efficacy of an oral rinse.

Five denture stomatitis patients demonstrating Candida albicans on both maxillary dentures and palates volunteered to test the effects of Peridex oral rinse in treating their oral disease. They used Peridex rinse both as a mouthrinse and as a denture soak for a period of 24 days. Agar replicas of the tissue-fitting surfaces of the maxillary dentures revealed elimination of C. albicans. Significant decreases in palatal inflammation were also noted, although some inflammation was still evident. Several weeks after the termination of Peridex oral rinses, inflammation increased as concentrations of C. albicans on the denture surface returned to pretreatment levels. A marked similarity in the site-specific localization of this yeast species on the denture was noted before and after Peridex rinse treatment.

In vivo antifungal efficacy of salivary histidine-rich polypeptides: preliminary findings in a denture stomatitis model system.

Six denture stomatitis patients, all found to have Candida albicans on their maxillary denture and palatal tissue surfaces, volunteered in this preliminary study to test the in vivo efficacy of human salivary antifungal histidine-rich polypeptides (HRPs) in treating their oral disease. The patients were equally divided among the Newton types classification and, as expected, the severity of the inflammation was greatest in the Newton type III patients and least in the Newton type I patients. Patients received sterile solutions of either HRP-3 or HRP-4, which they used both as a mouthrinse and as a denture soak for a period of 1 week. Agar replicas of the tissue-fitting surface of the maxillary dentures revealed HRP reduction and/or elimination of C. albicans from the denture; in one Newton type II individual, this finding directly correlated with a site-specific reduction in palatal inflammation. In the Newton type II and type III individuals alike, there was a significant generalized decrease in inflammation suggesting the therapeutic efficacy of the HRPs. Killing of this yeast species by the HRPs, as determined by scanning electron microscopy (SEM), was probably responsible for the observed clinical benefits noted in this investigation. In the SEM, HRP-treated blastospores appeared severely deflated, as if they had been emptied of significant quantities of intracellular material.

Sensitivity of the replica method in the detection of candidal infection among denture wearers with clinically healthy oral mucosa.

To ascertain the role of Candida in denture stomatitis, the practitioner must conduct a mycologic examination of the acrylic resin denture surface, because it acts as a reservoir for continuous reinfection of the palate. Twenty-two patients were examined to compare the sensitivity of the standard technique of swabbing the denture to that of a newly developed cast agar replica technique for detecting Candida albicans. The dentures were swabbed and cast replicas of the tissue-fitting surface of the dentures were made of both study populations. The majority of cultures obtained by swabbing failed to detect the presence of Candida albicans, while all cast agar replicas grew Candida albicans. The replica method for the detection of Candida albicans in edentulous patients seemed to be a more sensitive method than currently available mycologic methods.

Role of pH in salivary histidine-rich polypeptide antifungal germ tube inhibitory activity.

Purified synthetic salivary histidine-rich polypeptides (HRPs) 2, 3, 4, 5 and 6 were found to inhibit Candida albicans conversion of blastospores to germ tubes. HRP-4 was the best inhibitor within the pH 5 to 7 range tested and all of the HRPs were observed to lose potency as the pH was raised from 5 to 7. The pH pattern obtained with a synthetic homologous histidine peptide suggested that the protonated form of the histidine imidazole residues of the HRPs was important to the germ tube antifungal activity. Similar pH inhibition profiles of germ tube formation by parotid saliva and the HRPs were also observed.

Parameters affecting the inhibition of Candida albicans GDH 2023 and GRI 2773 blastospore viability by purified synthetic salivary histidine-rich polypeptides.

Purified synthetic salivary histidine-rich polypeptides, HRPs 2, 3, 4, 5, and 6, were observed to inhibit Candida albicans blastospore viability at yeast cell concentrations ranging from 10(2) to greater than 10(6) colony forming units per ml. Among the HRPs, HRP-4 was the best inhibitor with significant killing activity noted at a peptide concentration of 0.5 microgram per ml. Antifungal potency under growth conditions was observed to be dependent upon pH. In contrast, killing did not vary throughout the pH range tested under non-growth conditions. Electron microscopy results demonstrated HRP damage at pH 5 which appeared to be initiated at the membrane. At pH 7.4, micrographs revealed clear evidence of intracellular destruction suggesting more extensive damage at neutral as compared to acidic pH. These results suggest that within the changing realm of the oral cavity, the HRPs would be expected to be potent killers of C. albicans.

An in vivo replica method for the site-specific detection of Candida albicans on the denture surface in denture stomatitis patients: correlation with clinical disease.

A site-specific agar replica technique for detecting Candida albicans on the acrylic resin denture surface of denture stomatitis patients has been developed. The method is selective for C. albicans during a finite incubation period with a specific synthetic growth medium. C. albicans colonies can be geographically observed on the replica and their presence can be correlated with inflammatory lesions visible on the mucosa of the maxillary and mandibular residual ridges. In 12 denture stomatitis patients studied, a close clinical correlation of Newton type III patients was noted but this clinical correlation could not be observed in Newton type I and II patients. In general, the number of C. albicans colonies increased with the severity of the inflammation. The findings are discussed in light of lack of knowledge of the etiology of the stomatitis. The importance of the replica method is also discussed.

In vitro study on the inhibiting effect of different agents on the growth of Candida albicans on acrylic resin surfaces.

This study evaluated at the in vitro level the antifungal effectiveness of nystatin, chlorhexidine, and a homologous histidine polypeptide on the surface of acrylic resin disks. The agents were used in a way that simulated storage of a denture by a denture wearer. Results indicated that pretreatment with poly-L-histidine was not protective against C albicans adherence and growth regardless of whether disks were stored in water or in the open air for the 8-hour period following yeast contamination. Chlorhexidine was totally effective in preventing C albicans attachment to, and growth on, the acrylic resin, even after a period of 8 days of turbidimetric monitoring. Pretreatment with Nystatin, followed by drying, was protective, yielding results similar to those obtained with chlorhexidine.

Model system for the in vitro testing of a synthetic histidine peptide against Candida species grown directly on the denture surface of patients with denture stomatitis.

The denture surface provides a nidus for the growth of microbial species that act to initiate, aggravate, and maintain clinical disease. The present investigation describes the development of a model system for the testing of the effectiveness of agents against these microbial species inhabiting the denture surface. It was observed through in vitro growth patterns that the model permitted the testing of representative samples of the microbial flora. Poly-L-histidine was observed to inhibit both Candida albicans and C. glabrata from growing from the denture surface into nutrient broth. Scanning electron microscopy of control and treated denture disks revealed that poly-L-histidine had either eliminated most microbial flora from the denture surface or had effected a noticeable distortion of those Candida blastospores still present on the surface. From microbiologic studies, it appeared that poly-L-histidine had inflicted direct but not lethal damage to the still-attached distorted blastospores because the latter were still able to promote growth in agent-free broth. The antifungal effects of poly-L-histidine were observed to be dependent on the concentration of the polypeptide. The data obtained were consistent for all of the patients regardless of their denture stomatitis classification.

A comparison of the inhibition of blastospore viability and germ-tube development in Candida albicans by histidine peptides and ketoconazole.

Synthetic homologous peptides of L-histidine, ranging in length from 3 to 64 amino-acid residues, suppressed blastospore viability. Killing activity was dependent upon the peptide molecular size and concentration, and the time of cell exposure to the agent, but was independent of cell concentration in the range 10(5)-10(7) colony-forming units (c.f.u.) per ml. A 25 amino-acid residue polypeptide, similar to the human parotid salivary histidine-rich peptide (HRP-5), also affected yeast viability. Its killing effect was dependent upon the number of c.f.u. in the assay, as well as contact time with the blastospores and the final peptide concentration. HRP-5 inhibition increased with rising pH in the range 5-7.4, in contrast to poly-L-histidine and ketoconazole, which had optimal candidacidal activity at about pH 6. Poly-L-histidine, HRP-5, and ketoconazole each prevented conversion of blastospores to germ tubes, but their rank order of effectiveness varied with the assay selected. In N-acetylglucosamine-supplemented fetal calf serum, poly-L-histidine and HRP-5 were more effective inhibitors than ketoconazole, but the reverse was true in amino-acid-supplemented glucose beef-extract medium. Reduction of both germ-tube numbers and germ-tube size by HRP-5 was concentration-dependent.