Salivary proteins and microbiota as biomarkers for early childhood caries risk assessment.

Early childhood caries (ECC) is a term used to describe dental caries in children aged 6 years or younger. Oral streptococci, such as Streptococcus mutans and Streptococcus sorbrinus, are considered to be the main etiological agents of tooth decay in children. Other bacteria, such as Prevotella spp. and Lactobacillus spp., and fungus, that is, Candida albicans, are related to the development and progression of ECC. Biomolecules in saliva, mainly proteins, affect the survival of oral microorganisms by multiple innate defensive mechanisms, thus modulating the oral microflora. Therefore, the protein composition of saliva can be a sensitive indicator for dental health. Resistance or susceptibility to caries may be significantly correlated with alterations in salivary protein components. Some oral microorganisms and saliva proteins may serve as useful biomarkers in predicting the risk and prognosis of caries. Current research has generated abundant information that contributes to a better understanding of the roles of microorganisms and salivary proteins in ECC occurrence and prevention. This review summarizes the microorganisms that cause caries and tooth-protective salivary proteins with their potential as functional biomarkers for ECC risk assessment. The identification of biomarkers for children at high risk of ECC is not only critical for early diagnosis but also important for preventing and treating the disease.

Assessment of using recombinant Ixodes ricinus AV422 saliva protein for confirmation of tick bites in hunting dogs as naturally infested hosts.

Due to the recorded spreading of ticks in past years, a higher incidence of tick-borne diseases (TBDs) can be expected in the future in endemic areas, but can also pose an emerging public health concern in areas where they have not yet been recognized. Assessment of the exposure of vulnerable hosts to ticks would be a very helpful tool for TBD epidemiological studies, as well as for their proper managing. To confirm previous tick bites, the method of choice is detection of antibodies in host serum as markers developed against injected tick saliva proteins during feeding. We recently showed that the recombinant form of Ixodes ricinus AV422 saliva protein (rIrAV422) can serve for detection of markers in experimentally infested rats. Here we examine whether it can be used in the same manner in naturally exposed hosts. We chose hunting dogs as good sentinel animals. The study group consisted of 15 dogs that varied in breed, age, sex, previous tick infestation history and repellent treatment. Western blot analysis with rIrAV422 as an antigen confirmed the presence of tick bite markers in all analysed dogs. For some of the dogs, their previous tick infestation history was unclear, which emphasizes the usefulness of rIrAV422 for revealing it. Since hunting dogs are naturally infested with different ticks, the potential of rIrAV422 in assessment of general exposure to ticks is highlighted. Use of rIrAV422 can also be helpful in veterinary practice and research as a tool for validation of the efficiency of tick repellent products.

Salivary proteomics in biomedical research.

Proteins that are important indicators of physiological or pathological states, can provide information for the identification of early and differential markers for disease. Saliva, contains an abundance of proteins, offers an easy, inexpensive, safe, and non-invasive approach for disease detection, and possesses a high potential to revolutionize the diagnostics. Discovery of salivary biomarkers could be used to scrutinize health and disease surveillance. The impact of human saliva proteome analysis in the search for clinically relevant disease biomarkers will be realized through advances made using proteomic technologies. The advancements of emerging proteomic techniques have benefited biomarker research to the point where saliva is now recognized as an excellent diagnostic medium for the detection of disease. This review presents an overview of the value of saliva as a credible diagnostic tool and we aim to summarize the proteomic technologies currently used for global analysis of saliva proteins and to elaborate on the application of saliva proteomics to the discovery of disease biomarkers, and discuss some of the critical challenges and perspectives in this field.

Quality assessment of saliva bank samples.

INTRODUCTION: Biospecimens, such as urine, blood, saliva, tissue, cells, DNA, RNA, and protein, are biological material to be stored in a biorepository. They constitute critical resources of molecular data for basic and translational research integrated with diagnostic, therapeutics, and prevention of human diseases. The reliability of the molecular data is dependent on the quality and the consistency of the biospecimen being analyzed. The potential of human saliva as a valuable diagnostic fluid for oral and systemic conditions is being increasingly recognized. The aim of this study is to determine the molecular quality of unstimulated whole saliva (UWS) samples stored over a period of 1 to 5 years. MATERIALS AND METHODS: UWS samples collected between 2006 and 2010 (20/year) and stored at -80°C were assessed for molecular integrity. The study was approved by the institutional review board of the Indiana University Purdue University at Indianapolis. Qualitative and quantitative measurements of salivary proteins were determined by gel electrophoresis and spectrophotometry. The salivary nucleic acid content was determined by the Nanodrop method and genetic analysis. The nature of the cellular sediment in the UWS was determined by amplification of specific gene. RESULTS: No significant differences were observed in the amount of proteins, nucleic acid, or in the number of viable cells in the UWS samples stored for 1 to 5 years. CONCLUSION: Archived UWS samples could function as excellent biospecimen resources for measurement of protein, DNA, and RNA analytes, and act as an efficient source for human epithelial cells.

Putting the teeth into the UK Biobank.

The author of this article has been involved in the development of the UK Biobank, and was instrumental in ensuring that dentistry has been included in the project. He describes what the UK Biobank is, what the project involves and aims to achieve, and how by July 2010 some 500,000 UK citizens aged from 40-69 years had been recruited. He then details the events that led to the inclusion of dentistry in the project, the key role that stored saliva samples will have, and how the project will link to data stored by the Dental Practice Board and now the National Health Service Business Services Authority. The article ends with a brief look into the future of the project.

Effect of protease inhibitors on the quantitative and qualitative assessment of oral microorganisms.

Protease inhibitor cocktails are routinely added to clinical samples used for proteomic studies to inactivate proteases. As these same samples are often used for microbial studies, we determined whether the addition of protease inhibitors could affect the quantitative or qualitative assessment of microbial profiles. Twenty-two saliva samples were collected and processed immediately with or without the addition of a protease inhibitor cocktail. Conventional cultivation methods were used to evaluate total bacterial growth. Total genomic DNA was isolated and a specific 16S rRNA gene-targeted region was PCR-amplified and separated by denaturing gradient gel electrophoresis. A combination of 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis and LC-MS/MS methods was used to determine the effect of the protease inhibitors on the integrity of salivary proteins and peptides. Interestingly, no significant differences were observed in either the bacterial growth and composition or the integrity of salivary proteins between the two groups. Correlation coefficients between the paired samples for total cultivable microbiota (r(2) =0.847), total mutans streptococci (r(2) =0.898), total oral lactobacilli (r(2) =0.933), and total Streptococcus mutans (r(2) =0.870) also exceeded expected values. The results suggest that the addition of a protease inhibitor cocktail in saliva samples does not impact the growth of oral microbiota or compromise the ability to characterize its composition.

Availability of saliva for the assessment of alterations in the autonomic nervous system caused by physical exercise training.

OBJECTIVE: The purpose of the study was to assess the alterations of the autonomic nervous system activities caused by physical exercise training under food restriction using a running wheel in mice based on an analysis of saliva. METHODS: Male ICR mice, weighing 39-50 g, at 32 weeks of age were divided into three groups; an exercise with food restriction (EXP), an exercise without food restriction (EXA) and a control (CTL). The EXP group was fed the same amount of diet as the CTL group (pair-feeding). The EXP and EXA groups used a "voluntary running wheel" for exercise. The pilocarpine stimulated whole saliva was collected from the oral cavity by micropipette over 15 min 4, 8 and 12 weeks after the beginning of the experiment. The salivary flow rate, protein concentration and amylase and kallikrein activities were determined, since amylase and kallikrein release have been shown to be evoked by beta- and alpha-adrenergic receptor stimulation, respectively. RESULTS: There was no significant difference in the sera corticosterone levels among the three experimental groups. The flow rate of saliva per total salivary gland weight in the EXP was significantly lower than that in the CTL and the EXA groups. The total protein secretion and kallikrein activity decreased by 20-30% in the EXP mice between 4 and 12 weeks after the starting of experiments in comparison to the CTL mice, thus suggesting that signal transduction in the alpha-sympathetic nervous system was downregulated. There was no significant difference in the amylase activity between the EXP and the CTL groups. CONCLUSION: Physical exercise under diet control induced alterations in saliva secretion, while exercise training alone did not affect the content of saliva. The results demonstrate the availability of saliva and suggest that the present experimental situation is a suitable experimental model for sports activity performed under diet control.

Quantitative assessment of IgA levels in the unstimulated whole saliva of caries-free and caries-active children.

Saliva is commonly referred to as the blood stream of the oral cavity. It has many functions, one of the major functions being protection of teeth against dental caries. There are many components in saliva, each one having a specific role in the prevention of dental caries. The composition of saliva varies from individual to individual and in the same individual it varies between the glands. The composition of whole saliva, especially when unstimulated, has gained much interest, because it is this which constantly bathes the teeth. The aim of this study was to determine the IgA levels in the unstimulated whole saliva of caries-free and caries-active children aged 3-6 years and to correlate its role in protection of the tooth against dental caries.

Assessment of SS-A and SS-B in parotid saliva of patients with Sjögren's syndrome.

BACKGROUND: The purpose of this study was to compare the sensitivity of parotid saliva to that of serum in detecting anti-SSA/Ro and anti-SSB/La autoantibodies in patients with Sjögren's syndrome. METHODS: Forty patients and 20 controls participated in the study; all patients met the 1993 European Community criteria for the diagnosis of Sjögren's syndrome. Healthy controls were age- and sex-matched individuals with no signs or symptoms of Sjögren's syndrome. Serum and saliva samples were evaluated using AffiniTech SSA/Ro and SSB/La antibodies kits (AffiniTech, Ltd. Bentonville, AR, USA). The results were also compared with serological status of SS-A and SS-B as reported by an independent clinical laboratory. RESULTS: Serum was significantly more sensitive than saliva in detecting SSA/Ro and SSB/La antibodies (P = 0.001). There was high agreement between the results with the AffiniTech kits and the independent laboratory (kappa = 0.80; P < 0.001). However, there was poor agreement between saliva and serum results (kappa = 0.174; P = 0.168). CONCLUSIONS: The overall results appear to support that serum analysis is effective method for evaluating the presence of SS-A and SS-B autoantibodies.

Feeding aggregation of the tick Rhipicephalus appendiculatus (Ixodidae): benefits and costs in the contest with host responses.

Gregariousness can be advantageous in interspecific competition while intraspecific competition may favour solitude. We examined feeding behaviour of the ixodid tick, Rhipicephalus appendiculatus, in the context of interspecific (tick-host) and intraspecific (tick-tick) competition. Such competition is mediated through host rejection responses to tick infestation to which ticks respond by secreting immunodulatory saliva. We observed that group feeding adults increased their blood-feeding rate, reducing the time to mating and repletion, compared with individual feeding of paired adults. The benefits of feeding aggregation indicate direct reciprocity between ticks, most likely resulting from the shared activities of their bioactive saliva. However, fast-feeding ticks appeared to impair blood-feeding success of slow-feeding females during group feeding. This may be explained by the faster feeders exacerbating host responses on detachment that are then directed against the slower feeders. As female fecundity is generally proportional to the size of the bloodmeal, there will be a selection pressure to feed gregariously. Greater understanding of the benefits and costs of feeding aggregation may help to improve tick control strategies.

Reliability assessment of soluble c-erbB-2 concentrations in the saliva of healthy women and men.

BACKGROUND: The protein c-erb B-2, also known as Her2/neu, is a prognostic breast cancer marker assayed in tissue biopsy specimens from women diagnosed with malignant tumors. Current studies suggest that soluble fragments of the c-erb B-2 oncogene may be released from the cell surface and become detectable in patients with a carcinoma of the breast. Consequently, the purpose of this study is to assay soluble c-erb B-2 protein in the saliva of healthy men and women to determine the reliability of the assay. METHODS: To determine the diagnostic utility of this oncogene, we assayed the soluble form of the c-erb B-2 protein in the saliva with an enzyme-linked immunosorbent assay. The study population consisted of 10 healthy women and 9 healthy men who were serially sampled for saliva 3 times a day for a 5-day period. Saliva was collected from each subject at 9 AM, 4 PM, and 9 PM during the 5-day period. RESULTS: We found the presence of c-erb B-2 protein in the saliva of both groups of subjects. The salivary levels of c-erb B-2 were not significantly different when compared for gender differences. Likewise, the results suggest that sampling during various times of the day for salivary c-erb B-2 levels has no effect on marker concentration. Reliability analyses showed that supervised salivary collections were more reliable than unsupervised collections. CONCLUSIONS: The results of this pilot study suggest that the assay for salivary c-erb B-2 protein is reliable and might have potential use in the initial detection and follow-up screening for the recurrence of breast cancer in both men and women.

Social class, sex, and age differences in mucosal immunity in a large community sample.

There have been very few reports addressing levels and distribution of commonly used PNI measures in large community samples. In this study, we report such data for secretion rates of secretory immunoglobulin A (sIgA), as determined from saliva samples taken from 1971 subjects interviewed as part of the West of Scotland Twenty-07 survey of health in West Central Scotland. Univariate analyses of demographic variables found lower sIgA and salivary flow to be significantly related to poorer social class, increased age, and being female. Smokers also had lower sIgA but not lower salivary flow. Multivariate analysis showed that demographic variables were significant predictors of sIgA independently of each other and assay variation. Adding smoking status to the equation confirmed it as an independent predictor and also indicated that social class differences in sIgA are partly explicable in terms of smoking status. In view of reported associations between sIgA levels and stress, its role as a first line of mucosal defense, and its relevance to health, these first results from a large survey are of interest. Further work is now needed to explore which factors, including psychosocial ones, may be contributing to subgroup differences.

[Devices for saliva collection from the major salivary glands. Results in normal subjects]. / Dispositivos para colección de saliva de las glándulas salivales mayores. Resultados en sujetos normales.

BACKGROUND: Collection of saliva produced by the major salivary glands may be accomplished either by cannulation of the glandular ducts or by the application of specific collecting devices to the emergence area of the glandular ducts. Those procedures are complex, slow, invasive and require skilled personnel. AIM: To report the design and application of a device to collect parotid saliva (snail collector) and another device to collect saliva from the submandibular/sublingual complex. MATERIAL AND METHODS: The saliva collection devices were tested in 40 healthy volunteers (20 male) aged 18 to 22 years old. Saliva was collected using conventional conditions, during 5 to 15 min. RESULTS: An average of 1 to 1.5 ml of saliva was collected in the 10-15 min period from both parotid and submandibular/sublingual glands. Flow rates from parotid glands were 80 microliters/min and 180 microliters/min from submandibular/sublingual glands. Parotid saliva had a protein and organic material concentration twice as high than saliva from submandibular/sublingual glands. The presence of human alpha-amylase duplet (Mr 55 kD and 58 kD) predominated in parotid saliva, whereas saliva from submandibular/sublingual glands had other molecular markers such as the lysozyme duplet (Mr 18.5 kD and 17 kD). CONCLUSIONS: The tested devices were easily applicable, comfortable and allowed the collection of both parotid saliva and submandibular/sublingual saliva from various subjects at once, under the supervision of a single professional.

Dispositivos para colección de saliva de las glándulas salivales mayores: resultados en sujetos normales / Assessment of devices for saliva collection from major salivary glands in groups of individuals

Background: Collection of saliva produced by the major salivary glands may be accomplished either by cannulation of the glandular ducts or by the application of specific collecting devices to the emergence area of the glandular ducts. Those procedures are complex, slow, invasive and require skilled personnel. Aim: To report the design and application of a device to collect parotid saliva (snail collector) and another device to collect saliva from the submandibular/sublingual complex. Material and methods: The saliva collection devices were tested in 40 healthy volunteers (20 male) aged 18 to 22 years old. Saliva was collected using conventional conditions, during 5 to 15 min. Results: An average of 1 to 1.5 ml of saliva was collected in the 10-15 min period from both parotid and submandibular/sublingual glands. Flow rates from parotid glands were 80 µl/min and 180 µl/min from submandibular/sublingual glands. Parotid saliva had a protein and organic material concentration twice as high than saliva from submandibular/sublingual glands. The presence of human a-amylase duplet (Mr 55 kD and 58 kD) predominated in parotid saliva, whereas saliva from submandibular/sublingual glands had other molecular markers such as the lysozyme duplet (Mr 18.5 kD and 17 kD). Conclusions: The tested devices were easily applicable, comfortable and allowed the collection of both parotid saliva and submandibular/sublingual saliva from various subjects at once, under the supervision of a single professional

Assessment of bromhexine as a treatment regimen in Sjögren's syndrome-like disease in the NOD (non-obese diabetic) mouse.

OBJECTIVE: Bromhexine has been reported to alleviate the xerostomia and xerophthalmia associated with secondary Sjögren's syndrome. The aim of this study was to determine if it might prove useful in the treatment of Sjögren's syndrome-like disease of the NOD mouse model for autoimmune sialoadenitis. METHODS: Groups of mice were divided into sets receiving 60 mg/kg bromhexine in drinking water and control pair-fed animals. The efficacy of drug treatment was assessed by weekly measurement of stimulated saliva volumes, protein concentration, and amylase activity. At termination (20 weeks) submandibular and lacrimal glands were removed to assess the levels of lymphocytic infiltration by histological evaluation under light microscopy. RESULTS: Control and bromhexine-treated groups of mice showed no difference in the loss or rate of reduction in stimulated saliva flow over the 12 weeks of treatment. No differences were noted in the protein concentration and amylase loss with increasing age of the animals. Similar temporal changes in total protein profiles and aberrant expression of the 20 kDa parotid secretory protein isoform were observed by SDS-polyacrylamide gel profiles and Western bolt analysis. Histological evaluation of exocrine gland sections failed to detect any reduction in focal lymphocyte infiltration. CONCLUSION: Bromhexine therapy did not alter the development or severity of Sjögren's syndrome-like disease in the NOD mouse model for autoimmune sialoadenitis.