The in vitro and in vivo reproducibility of a video-based digital imaging system for tooth colour measurement.

OBJECTIVES: To assess the robustness of a new custom built video-based digital imaging system (VDIS) for measuring tooth colour and whiteness under in vitro and in vivo conditions. METHODS: The VDIS imaging system was developed for tooth colour measurement and evaluated in vitro and in vivo. The in vitro validation used extracted human teeth (HT, n=14) stored in water and VITA Classical shade guide tabs (SG, n=16). These were measured by the VDIS at baseline, 5min, 2h, 1 week and 2 weeks to evaluate the system repeatability. For in vivo validation, adult volunteers (male/female, n=34) with two natural, unrestored central incisors had their teeth imaged using the VDIS at baseline, 5min and 2h (3 images each) by two different operators to evaluate time and operator effects. Between taking individual images, subjects moved from the imaging-frame to assess the effect of re-positioning on reproducibility. From the in vitro and in vivo images, the average tooth RGB values were obtained, and the CIELAB values and a tooth whiteness index WIO value were calculated. Repeatability and reproducibility of VDIS imaging system was assessed using appropriate repeated measurement analysis techniques and ANOVA. RESULTS: The measurement variations in vitro were between 1 and 2 units of ΔWIO and the average colour differences were less than 1 ΔE*ab unit. For the in vivo study, analysis of the CIELAB parameters and WIO showed that subject variability accounted for between 82 and 99% of the observed variability in the measurement process. The operator variability was less than 0.5% and the overall measurement error was found to be only 0.3% for WIO. Across assessment times the variability was less than 0.5%. CONCLUSIONS: The dental imaging system V-DIS was shown to be a highly reproducible means for tooth colour and whiteness measurement. CLINICAL SIGNIFICANCE: Digital imaging based techniques gives a highly reproducible approach to measuring tooth colour.

Tooth whitening evaluation of blue covarine containing toothpastes.

OBJECTIVES: To measure the tooth whitening effects delivered immediately after brushing with silica-based toothpastes containing blue covarine in vitro and in vivo. METHODS: Salivary pellicle coated human extracted teeth were brushed with either a slurry of a toothpaste containing blue covarine (BC), a formulation containing an increased level of blue covarine (BC+) or a negative control toothpaste containing no blue covarine. The colour of the specimens were measured in vitro using either a Minolta chromameter or a VITA Easyshade spectrophotometer, before and after brushing and changes in CIELAB values and tooth Whiteness Index (WIO) values calculated. In a double-blind cross-over clinical study, subjects brushed with either BC or BC+ toothpaste and tooth colour changes were measured with a digital image analysis system. RESULTS: The in vitro studies demonstrated that toothpastes containing blue covarine gave a significantly (p<0.05) greater change in b* and WIO values than the negative control toothpaste; the BC+ toothpaste gave a significantly greater increase in b* and WIO values than the BC toothpaste, and BC+ gave a significant increase in shade change versus the negative control. Clinical results showed that BC and BC+ gave a significant reduction in b* (p<0.0001) and increase in WIO (p<0.0001) from baseline indicating significant tooth whitening had occurred. The parameter changes were significantly greater when brushing with the BC+ toothpaste than with the BC toothpaste (WIO p=0.006; b* p=0.013). CONCLUSIONS: Toothpastes containing blue covarine gave a statistically significant reduction in tooth yellowness and improvement in tooth whiteness immediately after brushing in both in vitro and clinical studies. In addition, the higher concentration blue covarine toothpaste gave statistically significant greater tooth whitening benefits than the lower concentration blue covarine toothpaste. CLINICAL SIGNIFICANCE: The silica-based toothpastes containing blue covarine evaluated in the current study gave tooth whitening benefits immediately after one brush.

In vitro and clinical evaluation of optical tooth whitening toothpastes.

OBJECTIVES: To measure tooth whitening effects delivered immediately after brushing with silica-based toothpastes containing either blue covarine or a combination of blue covarine and FD&C Blue No. 1 in vitro and in vivo. METHODS: Salivary pellicle coated human extracted teeth were brushed with either a slurry of a toothpaste containing blue covarine (BC) or a formulation containing a matched level of blue covarine and FD&C Blue No.1 (BC+D). The colour of the specimens were measured in vitro using a colorimeter, before and after brushing and changes in CIELAB and tooth Whiteness Index (WIO) values calculated. In a double-blind cross-over clinical study, subjects brushed with BC toothpaste, a toothpaste containing increased levels of blue covarine (BC+) or BC+D toothpaste and tooth colour changes were measured with a digital image analysis system. RESULTS: The in vitro study demonstrated that BC+D gave a significantly (p=0.002) greater change in WIO value than BC. Clinical results showed that BC, BC+ and BC+D gave a significant increase in WIO (p<0.0001) from baseline. The WIO change was significantly greater when brushing with BC+D toothpaste than with either toothpaste BC (p<0.0001) or BC+ (p<0.05). CONCLUSIONS: Toothpastes containing blue covarine or a combination of blue covarine and FD&C Blue No. 1 gave a statistically significant improvement in tooth whiteness immediately after brushing in both in vitro and clinical studies. In addition, the toothpaste containing both blue covarine and FD&C Blue No. 1 gave statistically significant greater tooth whitening from baseline than the blue covarine containing toothpastes. CLINICAL SIGNIFICANCE: The silica-based toothpastes containing blue covarine or a combination of blue covarine and FD&C Blue No. 1 evaluated in the current study gave significant tooth whitening benefits immediately after one brush.

Mode of action studies on the formation of enamel minerals from a novel toothpaste containing calcium silicate and sodium phosphate salts.

OBJECTIVES: To investigate in vitro and in situ the deposition and formation of hydroxyapatite (HAP) on enamel surfaces following brushing with a novel toothpaste containing calcium silicate (CaSi), sodium phosphate salts and fluoride. METHODS: Polished enamel blocks were brushed in vitro with a slurry of the CaSi toothpaste. After one brush and four weeks simulated brushing the enamel surfaces were analysed. In an in situ protocol, enamel blocks were attached to first or second molar teeth of healthy subjects, exposed to 4 weeks twice per day brushing with the CaSi toothpaste and then analysed. The surface deposits were analysed using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). In addition, the CaSi toothpaste was slurried in simulated oral fluid (SOF) over a 3 hour period and the solids were isolated and analysed by Fourier transform infrared spectroscopy (FTIR). RESULTS: The FTIR study demonstrated that calcium phosphate phases had formed and these became increasingly crystalline over 3 hours. CaSi was deposited onto enamel surfaces following one brushing with the toothpaste in vitro.The deposited particles showed evidence of HAP crystalline phases associated with the CaSi. Following 4 weeks brushing in vitro, the deposition increased and analyses showed that the deposited material was HAP. These results were confirmed by the in situ study. CONCLUSIONS: Calcium silicate can be deposited onto enamel surfaces from a novel toothpaste formulation where it can form the enamel mineral HAP. CLINICAL SIGNIFICANCE: A novel toothpaste formulation containing CaSi can form HAP on enamel surfaces. The potential of this technology is for a novel approach to the repair of demineralised enamel and the protection of enamel during acid exposure.

Host cell invasion and virulence mediated by Candida albicans Ssa1.

Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease.

Salivary histatin 5 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans.

Salivary histatin 5 (Hst 5) is a cationic salivary protein with high fungicidal activity against Candida albicans. Binding to the cell wall followed by intracellular translocation is required for killing; however, specific binding components and critical toxic events are not understood. In this study, laminarin (beta-1,3-glucan) but not sialic acid, mannan or pustulan mediated Hst 5 binding to C. albicans, and was disassociated by 100 mM NaCl. Time-lapse confocal microscopy revealed a dose-dependent rate of cytosolic uptake of Hst 5 that invariably preceded propidium iodide (PI) entry, demonstrating that translocation itself does not disrupt membrane integrity. Cell toxicity was manifest by vacuolar expansion followed by PI entrance; however, loss of endocytotic vacuolar trafficking of Hst 5 did not reduce killing. Extracellular NaCl (100 mM), but not sorbitol, prevented vacuolar expansion and PI entry in cells already containing cytosolic Hst 5, thus showing a critical role for ionic balance in Hst 5 toxicity. Hst 5 uptake, but not cell wall binding, was blocked by pretreatment with azide or carbonyl cyanide m-chlorophenylhydrazone; however, 10% of de-energized cells had membrane disruption. Thus, Hst 5 is capable of heterogeneous intracellular entry routes, but only direct cytosolic translocation causes cell death as a result of ionic efflux.

Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis.

The commensal fungus Candida albicans causes oropharyngeal candidiasis (OPC; thrush) in settings of immunodeficiency. Although disseminated, vaginal, and oral candidiasis are all caused by C. albicans species, host defense against C. albicans varies by anatomical location. T helper 1 (Th1) cells have long been implicated in defense against candidiasis, whereas the role of Th17 cells remains controversial. IL-17 mediates inflammatory pathology in a gastric model of mucosal candidiasis, but is host protective in disseminated disease. Here, we directly compared Th1 and Th17 function in a model of OPC. Th17-deficient (IL-23p19(-/-)) and IL-17R-deficient (IL-17RA(-/-)) mice experienced severe OPC, whereas Th1-deficient (IL-12p35(-/-)) mice showed low fungal burdens and no overt disease. Neutrophil recruitment was impaired in IL-23p19(-/-) and IL-17RA(-/-), but not IL-12(-/-), mice, and TCR-alphabeta cells were more important than TCR-gammadelta cells. Surprisingly, mice deficient in the Th17 cytokine IL-22 were only mildly susceptible to OPC, indicating that IL-17 rather than IL-22 is vital in defense against oral candidiasis. Gene profiling of oral mucosal tissue showed strong induction of Th17 signature genes, including CXC chemokines and beta defensin-3. Saliva from Th17-deficient, but not Th1-deficient, mice exhibited reduced candidacidal activity. Thus, the Th17 lineage, acting largely through IL-17, confers the dominant response to oral candidiasis through neutrophils and antimicrobial factors.

Uptake of the antifungal cationic peptide Histatin 5 by Candida albicans Ssa2p requires binding to non-conventional sites within the ATPase domain.

Candida albicans Hsp70 Ssa1/2 proteins have been identified as cell wall binding partners for the antifungal cationic peptide Histatin 5 (Hst 5) in vivo. C. albicans Ssa2p plays a major role in binding and translocation of Hst 5 into fungal cells, as demonstrated by defective peptide uptake and killing in C. albicans SSA2 null mutants. Candidal Hsp70 proteins are classical chaperone proteins with two discrete functional domains consisting of peptide binding and ATP binding regions. Pull-down assays with full-length and truncated Ssa2 proteins found that the ATPase domain was required for Hst 5 binding. Further mapping of Ssa2p by limited digestion and peptide array analyses identified two discrete Hst 5-binding epitopes within the ATPase region. Expression of Ssa2p in C. albicans cells carrying mutations in the first epitope identified by thermolysin digestion (Ssa2128-132A3) significantly reduced intracellular transport and fungicidal activity of Hst 5, confirming its importance as a binding site for Hst 5 function in vivo. Since this Hst 5 binding site lies within the Ssa2p ATPase domain near the ATP-binding cleft, it is possible that ATP modulates Hst 5 binding to Ssa2p. Indeed, gel filtration assays demonstrated that although nucleotides are not required for Hst 5 binding, their presence improved binding affinity by 10-fold. Thus, C. albicans Ssa2p binds Hst 5 at a surface-localized epitope in a subunit of the ATPase domain; and this region is required for intracellular translocation and killing functions of Hst 5.

The P-113 fragment of histatin 5 requires a specific peptide sequence for intracellular translocation in Candida albicans, which is independent of cell wall binding.

The activity of histatin 5 (Hst 5) against Candida albicans is initiated through cell wall binding, followed by translocation and intracellular targeting. The C. albicans cell wall protein Ssa2 is involved in the transport of Hst 5 into cells as part of cell killing. P-113 (a 12-amino-acid candidacidal active fragment of Hst 5) and P-113Q2.10 (which is inactivated by a glutamine substitution of the Lys residues at positions 2 and 10) were compared for their levels of cell wall binding and intracellular translocation in Candida wild-type (wt) and ssa2Delta strains. Both P-113 and P-113Q2.10 bound to the walls of C. albicans wt and ssa2Delta cells, although the quantity of P-113Q2.10 in cell wall extracts was higher than that of P-113 in both strains. Increasing the extracellular NaCl concentration to 100 mM completely inhibited the cell wall association of both peptides, suggesting that these interactions are primarily ionic. The accumulation of P-113 in the cytosol of wt cells reached maximal levels within 15 min (0.26 microg/10(7) cells), while ssa2Delta mutant cells had maximal cytosolic levels of less than 0.2 microg/10(7) cells even after 30 min of incubation. Furthermore, P-113 but not P-113Q2.10 showed specific binding with a peptide array of C. albicans Ssa2p. P-113Q2.10 was not transported into the cytosol of either C. albicans wt or ssa2Delta cells, despite the high levels of cell wall binding, showing that the two cationic lysine residues at positions 2 and 10 in the P-113 peptide are important for transport into the cytosol and that binding and transport are independent functional events.

The role of released ATP in killing Candida albicans and other extracellular microbial pathogens by cationic peptides.

A unifying theme common to the action of many cationic peptides that display lethal activities against microbial pathogens is their specific action at microbial membranes that results in selective loss of ions and small nucleotides-chiefly ATP. One model cationic peptide that induces non-lytic release of ATP from the fungal pathogen Candida albicans is salivary histatin 5 (Hst 5). The major characteristic of Hst 5-induced ATP release is that it occurs rapidly while cells are still metabolically active and have polarized membranes, thus precluding cell lysis as the means of release of ATP. Other cationic peptides that induce selective release of ATP from target microbes are lactoferricin, human neutrophil defensins, bactenecin, and cathelicidin peptides. The role of released extracellular ATP induced by cationic peptides is not known, but localized increases in extracellular ATP concentration may serve to potentiate cell killing, facilitate further peptide uptake, or function as an additional signal to activate the host innate immune system at the site of infection.

Candida albicans cell wall ssa proteins bind and facilitate import of salivary histatin 5 required for toxicity.

Fungicidal activity of Hst 5 is initiated by binding to cell surface proteins on Candida albicans, followed by intracellular transport to cytoplasmic effectors leading to cell death. As we identified heat shock 70 proteins (Ssa1p and/or Ssa2p) from C. albicans lysates that bind Hst 5, direct interactions between purified recombinant Ssa proteins and Hst 5 were tested by pull-down and yeast two-hybrid assays. Pulldown of both native complexes and those stabilized by cross-linking demonstrated higher affinity of Hst 5 for Ssa2p than for Ssa1p, in agreement with higher levels of interactions between Ssa2p and Hst 5 measured by yeast two-hybrid analyses. C. albicans ssa1Delta and ssa2Delta mutants were constructed to examine Hst 5 binding, translocation, and candidacidal activities. Both ssa1Delta and ssa2Delta mutants were indistinguishable from wild-type cells in growth and hyphal formation. However, C. albicans ssa2Delta mutants were highly resistant to the candidacidal activity of Hst 5, although the ssa1Delta mutant did not have any significant reduction in killing by Hst 5. Total cellular binding of 125I-Hst 5 in the ssa2Delta mutant was reduced to one-third that of wild-type cells, in contrast to the ssa1Delta mutant whose total cellular binding of Hst 5 was similar to the wild-type strain. Intracellular transport of Hst 5 was significantly impaired in the ssa2Delta mutant strain, but only mildly so in the ssa1Delta mutant. Thus, C. albicans Ssa2p facilitates fungicidal activity of Hst 5 in binding and intracellular translocation, whereas Ssa1p appears to have a lesser functional role in Hst 5 toxicity.

The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5.

The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K(+), which can be blocked by the anion channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organism's principal K(+) transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K(+) ((86)Rb(+)) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5-7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.