Objectives
Saliva contains biomarkers for systemic as well as oral diseases. This study was undertaken to assess the variability in the sources of such biomarkers (plasma, cells) and attempted to ...identify saliva deterioration markers in order to improve saliva diagnostic outcomes.
Materials and Methods
Inter‐ and intrasubject variations in salivary gingival crevicular fluid levels were determined by measuring salivary albumin and transferrin levels. The purity of collected glandular secretions was determined by bacterial culture, and the variability in epithelial cell numbers by cell counting and optical density measurement. Saliva sample deterioration markers were identified by RP‐HPLC and LC‐ESI‐MS/MS.
Results
Tenfold variations were observed in plasma‐derived albumin and transferrin levels, emphasizing the need for biomarker normalization with respect to plasma contributions to saliva. Epithelial cell levels varied 50‐fold in samples collected before and after a meal. Salivary fungal levels varied within subjects and among subjects from 0 to >1,000 colony‐forming units per milliliter. In saliva samples incubated for various time intervals at 37°C, five peptides were identified that steadily increased in intensity over time and which could be explored as “deterioration markers.”
Conclusion
Taking saliva characteristics appropriately into account will help realize the promise that this body fluid is suitable to be exploited for reliable healthcare monitoring and surveillance.
Aims: To elucidate the first colonizers within in vivo dental biofilm and to establish potential population shifts that occur during the early phases of biofilm formation.
Methods and Results: A ...‘checkerboard’ DNA–DNA hybridization assay was employed to identify 40 different bacterial strains. Dental biofilm samples were collected from 15 healthy subjects, 0, 2, 4 and 6 h after tooth cleaning and the composition of these samples was compared with that of whole saliva collected from the same individuals. The bacterial distribution in biofilm samples was distinct from that in saliva, confirming the selectivity of the adhesion process. In the very early stages, the predominant tooth colonizers were found to be Actinomyces species. The relative proportion of streptococci, in particular Streptococcus mitis and S. oralis, increased at the expense of Actinomyces species between 2 and 6 h while the absolute level of Actinomyces remained unaltered. Periodontal pathogens such as Tannerella forsythensis(Bacteroides forsythus), Porphyromonas gingivalis and Treponema denticola as well as Actinobacillus actinomycetemcomitans were present in extremely low levels at all the examined time intervals in this healthy group of subjects.
Conclusion: The data provide a detailed insight into the bacterial population shifts occurring within the first few hours of biofilm formation and show that the early colonizers of the tooth surface predominantly consist of beneficial micro‐organisms.
Significance and Impact of the Study: The early colonizers of dental plaque are of great importance in the succession stages of biofilm formation and its overall effect on the oral health of the host.
Understanding the composition and function of the acquired enamel pellicle (AEP) has been a major goal in oral biology. The aim of this study was to test the hypothesis that intact histatins are part ...of the in vivo AEP and that histatins after adsorption to HA have effects on in vitro enamel demineralization. This is the first study demonstrating the presence of intact histatins in vivo in the AEP. The in vitro experiments show that all naturally occurring histatins in the AEP have the potential to provide some level of protection against acid injury.
Previous studies have shown that the human salivary antifungal peptide histatin 5 is taken up by Candida albicans cells and associates intracellularly with mitochondria. The purpose of the present ...study was to investigate the biological consequence of this specific subcellular targeting. Histatin 5 inhibited respiration of isolated C. albicans mitochondria as well as the respiration of intact blastoconidia in a dose and time-dependent manner. A nearly perfect correlation was observed between histatin-induced inhibition of respiration and cell killing with either logarithmic- or stationary-phase cells, but stationary-phase cells were less sensitive. Because nonrespiring yeast cells are insensitive to histatin 5, the potential mechanistic relationship between histatin 5 interference with the respiratory apparatus and cell killing was explored by using an oxygen radical sensitive probe (dihydroethidium). Fluorimetric measurements showed that histatin 5 induced the formation of reactive oxygen species (ROS) in C. albicans cells as well as in isolated mitochondria and that ROS levels were highly correlated with cell death. In the presence of an oxygen scavenger (L-cysteine), cell killing and ROS formation were prevented. In addition, the membrane-permeant superoxide dismutase mimetic 2,2,6,6-tetramethylpiperidine-N-oxyl, abolished histatin-induced ROS formation in isolated mitochondria. In contrast to histatin 5, the conventional inhibitors of the respiratory chain, sodium cyanide or sodium azide, neither induced ROS nor killed yeast cells. These data provide strong evidence for a comprehensive mechanistic model of histatin-5-provoked yeast cell death in which oxygen radical formation is the ultimate and essential step.
All organisms need protection against microorganisms, e. g. bacteria, viruses and fungi. For many years, attention has been focused on adaptive immunity as the main antimicrobial defense system. ...However, the adaptive immune system, with its network of humoral and cellular responses is only found in higher animals, while innate immunity is encountered in all living creatures. The turning point in the appreciation of the innate immunity was the discovery of antimicrobial peptides in the early eighties. In general these peptides act by disrupting the structural integrity of the microbial membranes. It has become clear that membraneactive peptides and proteins play a crucial role in both the innate and the adaptive immune system as antimicrobial agents. This review is focused on the functional and structural features of the naturally occurring antimicrobial peptides, and discusses their potential as therapeutics.
Periodontal disease is characterised by proteolytic processes involving enzymes that are released by host immune cells and periodontal bacteria. These enzymes, when detectable in whole saliva, may ...serve as valuable diagnostic markers for disease states and progression. Because the substrate specificities of salivary proteases in periodontal health and disease are poorly characterised, we probed these activities using several relevant substrates: (i) gelatin and collagen type IV; (ii) the Arg/Lys–rich human salivary substrate histatin‐5; and (iii) a histatin‐derived synthetic analog benzyloxycarbonyl‐Arg‐Gly‐Tyr‐Arg‐methyl cumaryl amide (Z‐RGYR‐MCA). Substrate degradation was assessed in gel (zymography) and in solution. Whole saliva supernatant enzyme activities directed at gelatin, quantified from the 42 kDa, 92 kDa and 130 kDa bands in the zymograms, were 1.3, 1.4 and 2.0‐fold higher, respectively, in the periodontal patient group (P < 0.01), consistent with enhanced activities observed towards collagen type IV. On the other hand, histatin 5 degraded equally fast in healthy and periodontal patients' whole saliva supernatant samples (P > 0.10). Likewise, the hydrolysis rates of the Z‐RGYR‐MCA substrate were the same in the healthy and periodontal patient groups (P > 0.10). In conclusion, gelatinolytic/collagenolytic activities but not trypsin‐like activities in human saliva differentiate health from periodontal disease and may thus provide an adjuvant to diagnosis for monitoring disease activity.
Histatin 5 is a human basic salivary peptide with strong fungicidal properties in vitro. To elucidate the mechanism of action, the effect of histatin 5 on the viability ofCandida albicans cells was ...studied in relation to its membrane perturbing properties. It was found that both the killing activity and the membrane perturbing activity, studied by the influx of a DNA-specific marker propidium iodide, were inhibited by high salt conditions and by metabolic inhibitors, like sodium azide. In addition, exposure to histatin 5 resulted in a loss of the mitochondrial transmembrane potential in situ, measured by the release of the potential-dependent distributional probe rhodamine 123. Localization studies using tetramethylrhodamine isothiocyanate-labeled histatin 5 or fluorescein isothiocyanate-labeled histatin 5 showed a granular intracellular distribution of the peptide, which co-localized with mitotracker orange, a permeant mitochondria-specific probe. Like the biological effects, uptake of labeled histatin 5 was inhibited by mitochondrial inhibitors and high salt conditions. Our data indicate that histatin 5 is internalized, and targets to the energized mitochondrion.
The hemolytic and fungicidal activity of a number of cationic antimicrobial peptides was investigated. Histatins and magainins were inactive against human erythrocytes and
Candida albicans cells in ...phosphate buffered saline, but displayed strong activity against both cell types when tested in 1 mM potassium phosphate buffer supplemented with 287 mM glucose. The HC
50/IC
50 ratio, indicative of the therapeutic index, was about 30 for all peptides tested. PGLa was most hemolytic (HC
50=0.6 μM) and had the lowest therapeutic index (HC
50/IC
50=0.5). Susceptibility to hemolysis was shown to increase with storage duration of the erythrocytes and also significant differences were found between blood collected from different individuals. In this report, a sensitive assay is proposed for the testing of the hemolytic activities of cationic peptides. This assay detects subtle differences between peptides and allows the comparison between the hemolytic and fungicidal potency of cationic peptides.
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