Walter L. Siqueira

Salivary proteome and its genetic polymorphisms.

Abstract:  Salivary diagnostics for oral as well as systemic diseases is dependent on the identification of biomolecules reflecting a characteristic change in presence, absence, composition, or structure of saliva components found under healthy conditions. Most of the biomarkers suitable for diagnostics comprise proteins and peptides. The usefulness of salivary proteins for diagnostics requires the recognition of typical features, which make saliva as a body fluid unique. Salivary secretions reflect a degree of redundancy displayed by extensive polymorphisms forming families for each of the major salivary proteins. The structural differences among these polymorphic isoforms range from distinct to subtle, which may in some cases not even affect the mass of different family members. To facilitate the use of modern state‐of‐the‐art proteomics and the development of nanotechnology‐based analytical approaches in the field of diagnostics, the salient features of the major salivary protein families are reviewed at the molecular level. Knowledge of the structure and function of salivary gland–derived proteins/peptides has a critical impact on the rapid and correct identification of biomarkers, whether they originate from exocrine or non‐exocrine sources.

New Insights into the Composition and Functions of the Acquired Enamel Pellicle

The acquired enamel pellicle (AEP) is a thin acellular film that forms on tooth surfaces upon exposure to the oral environment. It consists predominantly of salivary proteins, but also includes non-salivary-derived proteins, carbohydrates, and lipids. Since it is the interface between teeth and the oral environment, the AEP plays a key role in the maintenance of oral health by regulating processes including lubrication, demineralization, and remineralization and shaping the composition of early microbial flora adhering to tooth surfaces. Knowledge of the 3D structure of the AEP and how that correlates with its protective functions may provide insight into several oral pathological states, including caries, erosion, and periodontal disease. This review intends to update readers about the latest discoveries related to the formation, ultrastructure, composition, and functions of the AEP, as well as the future of pellicle research, with particular emphasis on the emerging role of proteomic and microscopy techniques in oral diagnosis and therapeutics.

Fiber-Optic Microsphere-Based Antibody Array for the Analysis of Inflammatory Cytokines in Saliva

Antibody microarrays have emerged as useful tools for high-throughput protein analysis and candidate biomarker screening. We describe here the development of a multiplexed microsphere-based antibody array capable of simultaneously measuring 10 inflammatory protein mediators. Cytokine-capture microspheres were fabricated by covalently coupling monoclonal antibodies specific for cytokines of interest to fluorescently encoded 3.1 microm polymer microspheres. An optical fiber bundle containing approximately 50,000 individual 3.1 microm diameter fibers was chemically etched to create microwells in which cytokine-capture microspheres could be deposited. Microspheres were randomly distributed in the wells to produce an antibody array for performing a multiplexed sandwich immunoassay. The array responded specifically to recombinant cytokine solutions in a concentration-dependent fashion. The array was also used to examine endogenous mediator patterns in saliva supernatants from patients with pulmonary inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). This array technology may prove useful as a laboratory-based platform for inflammatory disease research and diagnostics, and its small footprint could also enable integration into a microfluidic cassette for use in point-of-care testing.

Use of Colorimetric Test Strips for Monitoring the Effect of Hemodialysis on Salivary Nitrite and Uric Acid in Patients with End-Stage Renal Disease: A Proof of Principle

BACKGROUND Initial screening of potential biomarkers for monitoring dialysis was performed with saliva samples collected from patients with end-stage renal disease (ESRD). A more thorough analysis of the most promising markers identified in the initial screening was conducted with saliva samples acquired at hourly intervals throughout dialysis to monitor analyte concentrations as dialysis progressed. We observed that salivary nitrite (NO(2)(-)) and uric acid (UA) concentrations consistently decreased as dialysis proceeded. METHODS Solution-based colorimetric-detection chemistries for NO(2)(-) and UA were converted to a test strip format to produce a simple method for semiquantitatively measuring NO(2)(-) and UA concentrations in the clinic or at the patients home. We assessed the test strips with saliva samples collected from both ESRD patients undergoing dialysis and healthy control volunteers to qualitatively monitor the effect of dialysis on salivary NO(2)(-) and UA. We used computer software to analyze digital images of the resulting test strip color intensities. RESULTS Test strip measurements showed that mean salivary concentrations of NO(2)(-) and UA were decreased in ESRD patients by 86% and 39%, respectively, compared with 15% and 9% for time-matched controls. Comparison of test strip results with calibrated solution-based assays suggests that the test strips can semiquantitatively measure salivary concentrations of NO(2)(-) and UA. CONCLUSIONS The colorimetric test strips monitored changes in salivary NO(2)(-) and UA concentrations that occurred in ESRD patients during dialysis. The test strips may prove useful for noninvasively evaluating dialysis progress and may also be useful for monitoring renal disease status.

Proteome of Human Minor Salivary Gland Secretion

Recent research efforts in oral biology have resulted in elucidation of the proteomes of major human salivary secretions and whole saliva. One might hypothesize that the proteome of minor gland secretions may show significantly different characteristics when compared with the proteomes of parotid or submandibular/sublingual secretions. To test this hypothesis, we conducted the first exploration into the proteome of minor salivary gland secretion. Minor gland secretion was obtained from healthy volunteers, and its components were subjected to liquid-chromatography-electrospray-ionization-tandem-mass-spectrometry. This led to the identification of 56 proteins, 12 of which had never been identified in any salivary secretion. The unique characteristics of the minor salivary gland secretion proteome are related to the types as well as the numbers of components present. The differences between salivary proteomes may be important with respect to specific oral functions.

Small Molecular Weight Proteins/Peptides Present in the in vivo formed Human Acquired Enamel Pellicle

OBJECTIVE The aim of this study was to investigate the type and the nature of peptides present in the in vivo formed human acquired enamel pellicle. DESIGN Pellicle material was collected from 10 volunteers and subjected to sample preparations consisting of centrifugal filtration using a 10 kDa molecular weight cut-off membrane and high-resolution gel filtration chromatography. The fractions containing peptides <10 kDa obtained by both methods were analyzed by LC-ESI-MS/MS. RESULTS 78 natural pellicle peptides with molecular weights ranging from 766.9 Da to 3981.4 Da were identified originating from 29 different proteins. CONCLUSIONS The number of peptides present in acquired enamel pellicle appears to be large and this is likely to enhance the functional spectrum of this protein film. The presence of small peptides in pellicle may be functionally important since structure/function studies of many salivary proteins have shown that specific domains within these native proteins retain or even exhibit enhanced biological activities. The data present the basis for determining the precise function of these pellicle peptides and for gaining insights into the role pellicle plays in the oral cavity.

Whole-saliva Proteolysis and Its Impact on Salivary Diagnostics

There is growing interest in the use of human whole saliva for diagnostics and disease monitoring as an alternative to blood samples. In contrast to blood, whole saliva is a non-sterile body fluid. Proper hand-ling and storage are required to preserve the integrity of potential biomarkers. We investigated salivary autoproteolytic degradation using a variety of approaches. We determined inhibition of protease activities by monitoring the endogenous proteome. In addition, the stability of highly protease-susceptible proteins—histatin 5, statherin, and PRP1—was assessed. Experimental variables included (a) protease inhibitors, (b) salivary pH, (c) incubation temperatures, and (d) sample heating. A cocktail containing AEBSF, aprotinin, pancreatic trypsin inhibitor, leupeptin, antipain, and EDTA could not prevent histatin 5, statherin, or PRP1 degradation in whole saliva. Among the other treatments evaluated, short-term storage of freshly collected samples on ice was effective without interfering with the chemistry of the proteome. In conclusion, whole saliva contains a unique mixture of enzymes as evidenced from their resilience to protease inhibition. Analytical evidence on protein stability is needed to ensure the validity of salivary biomarker study outcomes. Analysis of the data presented will provide help and guidance for the use of saliva samples for diagnostic purposes.

Evidence of Intact Histatins in the in vivo Acquired Enamel Pellicle

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.

Meta‐Analysis of Salivary Flow Rates in Young and Older Adults

To determine whether salivary flow decreases as a function of aging.

In vitro performance of methods of approximal caries detection in primary molars

OBJECTIVE The aim was to compare the performance of different methods in detecting approximal caries lesions primary molars ex vivo. STUDY DESIGN One hundred thirty-one approximal surfaces were examined by 2 observers with visual inspection (VI) using the International Caries Detection and Assessment System, radiographic interpretation, and clinically using the Diagnodent pen (LFpen). To achieve a reference standard, surfaces were directly examined for the presence of white spots or cavitations, and lesion depth was determined after sectioning. The area under the receiver operating characteristic curve (A(z)), sensitivity, specificity, and accuracy were calculated, as well as the interexaminer reproducibility. RESULTS Using the cavitation threshold, all methods presented similar sensitivities. Higher A(z) values were achieved with VI at white spot threshold, and VI and LFpen had higher A(z) values at cavitation threshold. VI presented higher accuracy and A(z) than radiographic and LFpen at both enamel and dentin depth thresholds. Higher reliability values were achieved with VI. CONCLUSIONS VI performs better, but both radiographic and LFpen methods also show good performance in detecting more advanced approximal caries lesions.