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Aim Oxidized low-density lipoproteins (oxLDL) are formed as a result of lipid peroxidation and are highly immunogenic and proatherogenic. In this study, saliva antibodies binding to oxLDL, Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) were characterized and their cross-reactivity was evaluated. Materials and Methods Resting and stimulated saliva samples were collected from 36 healthy adults (mean age 26 years). Saliva IgA, IgG and IgM autoantibody levels to copper oxidized LDL (CuOx-LDL) and malondialdehyde acetaldehyde-modified LDL (MAA-LDL) were determined with chemiluminescence immunoassay. Results Saliva IgA and IgG antibodies binding to MAA-LDL and CuOx-LDL were detected in all samples and they were associated with the saliva levels of IgA and IgG to P. gingivalis and A. actinomycetemcomitans. Competitive immunoassay showed that saliva antibodies to MAA-LDL cross-reacted specifically with P. gingivalis. The autoantibody levels to oxLDL in saliva were not associated with the autoantibody levels to oxLDL in plasma or with saliva apolipoprotein B 100 levels. Conclusions Saliva contains IgA and IgG binding to oxLDL, which showed cross-reactive properties with the periodontal pathogens Porphyromonas gingivalis (P.g). The data suggest that secretory IgA to P.g may participate in immune reactions involved in LDL oxidation through molecular mimicry.

In the last decade, technological advances, combined with an improved appreciation of the ability of saliva to inform caregivers about both oral health and systemic disease, have led to the emergence of salivary diagnostic platforms. However, the majority of these assays have targeted diseases that more commonly affect the adult population, largely neglecting infants and children who arguably could benefit the most from non-invasive assessment tools for health monitoring. Gaining access into development, infection, and disease through comprehensive “omic” analyses of saliva could significantly improve care and enhance health access. In this review, we will highlight novel applications of salivary diagnostics in pediatrics across the “omic” spectrum, including at the genomic, transcriptomic, proteomic, microbiomic, and metabolomic level. The challenges to implementing salivary platforms into care, including the effects of age, diet, and developmental stage on salivary components, will be reviewed. Ultimately, large-scale, multicenter trials must be performed to establish normative biomarker values across the age spectrum to accurately discriminate between health and disease. Only then can salivary diagnostics truly translate into pediatric care.

Saliva contains biological information as blood and is recognized as a valuable diagnostic medium for their noninvasiveness. Although “-omics” researches have tried to investigate saliva, the origin and significance of its contents are not clear, and its usage is largely confined to oral disease in the diagnostic and prognostic field. In an attempt to broaden the applicability of saliva and to find systemic disease-derived RNA in saliva, we made mouse models that had human melanoma and isolated extracellular vesicles (EVs) from their saliva by an aqueous 2-phase system (ATPS), then identified and evaluated their expression of human melan-A RNA, which is associated with melanoma on skin. With ATPS, EVs were isolated efficiently and stably while taking less time compared to isolation by ultracentrifugation. When ATPS was used to isolate EVs from saliva, the mean ± SD percentage of EVs recovered from initial EVs was 38.22% ± 18.55% by the number of particles, and the mean ± SD percentage of RNA recovered from the initial amount was 60.33% ± 5.34%. RNAs within isolated EVs were analyzed subsequently by reverse transcription quantitative polymerase chain reaction and polymerase chain reaction from saliva and plasma. In melanoma mice, amplification of human melan-A was identified from saliva and plasma, even though a relative amount of normalized melan-A was lower than that of plasma. These results present a possibility that RNAs derived from systemic disease are transferred into saliva from blood in EVs. Also, they suggest that saliva could be exploited in obtaining information about systemic disease, not only about oral disease, by examining RNAs in EVs from saliva instead of blood.

Salivary diagnostics has great potential to be used in the early detection and prevention of many cancerous diseases. If implemented with rigour and efficiency, it can result in improving patient survival times and achieving earlier diagnosis of disease. Recently, extraordinary efforts have been taken to develop non-invasive technologies that can be applied without complicated and expensive procedures. Saliva is a biofluid that has demonstrated excellent properties and can be used as a diagnostic fluid, since many of the biomarkers suggested for cancers can also be found in whole saliva, apart from blood or other body fluids. The currently accepted gold standard methods for biomarker development include chromatography, mass spectometry, gel electrophoresis, microarrays and polymerase chain reaction-based quantification. However, salivary diagnostics is a flourishing field with the rapid development of novel technologies associated with point-of-care diagnostics, RNA sequencing, electrochemical detection and liquid biopsy. Those technologies will help introduce population-based screening programs, thus enabling early detection, prognosis assessment and disease monitoring. The purpose of this review is to give a comprehensive update on the emerging diagnostic technologies and tools for the early detection of cancerous diseases based on saliva. Sin financiación 4.302 JCR (2017) Q1, 25/133 Medicine, Research and Experimental; Q2, 67/190 Cell Biology UEM

Purpose The aim of this study was to compare microRNA (miRNA) gene expression in saliva using miRNA polymerase chain reaction (PCR) arrays in healthy and aggressive periodontitis (AP) patients. Methods PCR arrays of 84 miRNAs related to the human inflammatory response and autoimmunity from the saliva samples of 4 patients with AP and 4 healthy controls were performed. The functions and diseases related to the miRNAs were obtained using TAM 2.0. Experimentally validated targets of differentially expressed miRNAs were obtained from mirTarBase. Gene ontology terms and pathways were analyzed using ConsensusPathDB. Results Four downregulated miRNAs (hsa-let-7a-5p, hsa-let-7f-5p, hsa-miR-181b-5p, and hsa-miR-23b-3p) were identified in patients with AP. These miRNAs are associated with cell death and innate immunity, and they target genes associated with osteoclast development and function. Conclusions This study is the first analysis of miRNAs in the saliva of patients with AP. Identifying discriminatory human salivary miRNA biomarkers reflective of periodontal disease in a non-invasive screening assay is crucial for the development of salivary diagnostics. These data provide a first step towards the discovery of key salivary miRNA biomarkers for AP., Graphical Abstract

The field of salivary diagnostics to allow risk determination for periodontal diseases is advancing. New technologies in proteomics, genomics and nanotechnologies have continued the discovery of discriminatory periodontal disease biomarkers. This review briefly overviews biomarker studies that have been completed in saliva for the detection of periodontal disease since 2010. Disease specific biomarkers could be used in risk determination, treatment planning and disease progression. Currently, diagnostic tests are commercially available, and the development of point-of-care tests is expanding. Even though challenges remain, salivary diagnostics for periodontal disease is promising and could facilitate the diagnostics and treatment in a clinical practice by dental practitioners.

The ability to monitor the health and disease status of the patient through saliva is a highly desirable goal for the health professionals. Considering the microconcentration of salivary constituents, saliva is explored to be diagnostic tool as it also meets the demands for an inexpensive, noninvasive and easy to use screening method. The incorporation of salivary diagnostics into clinical practice is gaining reality and will be of diagnostic value in the prospective future. The investigative use of saliva is not being applied only in dental health but also in various other systemic disorders. The advent of molecular techniques is gaining attention and this has triggered its application as a specific and sensitive biomarker in proteomics, genomics, and transcriptomics. This review discusses the basics of salivary diagnostics, expectoration techniques, and its application in various local and systemic disorders.

Early detection of disease plays a crucial role for treatment planning and prognosis. Saliva has great potential as a diagnostic fluid and offers advantage over serum and other biological fluids by an economic and noninvasive collection method for monitoring of systemic health and disease progression. The plethora of components in this fluid can act as biomarkers for diagnosis of various systemic and local diseases. In this review paper, we have emphasized the role of salivary biomarkers as diagnostic tools.

Saliva, a biologic fluid, recently has been a boon in the field of diagnostic science, as the presence of viable biomarkers in saliva elaborate its applications in multiplexed assays, disease diagnosis, clinical monitoring and for clinical decisions of patient care. Application of saliva in diagnosis is undeniable as ease in sample collection and conventional procedure. This article provides emerging evidence to provide an overview of salivary diagnostics in various fields of diagnosis and affirm its application as diagnostic Bio-Fluid.

BACKGROUND The use of salivary diagnostics is increasing because of its noninvasiveness, ease of sampling, and the relatively low risk of contracting infectious organisms. Saliva has been used as a biological fluid to identify and validate RNA targets in head and neck cancer patients. The goal of this study was to develop a robust, easy, and cost-effective method for isolating high yields of total RNA from saliva for downstream expression studies. METHODS Oral whole saliva (200 μL) was collected from healthy controls (n = 6) and from patients with head and neck cancer (n = 8). The method developed in-house used QIAzol lysis reagent (Qiagen) to extract RNA from saliva (both cell-free supernatants and cell pellets), followed by isopropyl alcohol precipitation, cDNA synthesis, and real-time PCR analyses for the genes encoding β-actin (“housekeeping” gene) and histatin (a salivary gland–specific gene). RESULTS The in-house QIAzol lysis reagent produced a high yield of total RNA (0.89–7.1 μg) from saliva (cell-free saliva and cell pellet) after DNase treatment. The ratio of the absorbance measured at 260 nm to that at 280 nm ranged from 1.6 to 1.9. The commercial kit produced a 10-fold lower RNA yield. Using our method with the QIAzol lysis reagent, we were also able to isolate RNA from archived saliva samples that had been stored without RNase inhibitors at −80 °C for >2 years. CONCLUSIONS Our in-house QIAzol method is robust, is simple, provides RNA at high yields, and can be implemented to allow saliva transcriptomic studies to be translated into a clinical setting.

Background: The prevalence of chronic renal failure is increasing because of increase in chronic debilitating diseases and progressing age of population. These patients experience accumulation of metabolic byproducts and electrolyte imbalance, which has harmful effects on their health. Timely hemodialysis at regular intervals is a life-saving procedure for these patients. Salivary diagnostics is increasingly used as an alternative to the traditional methods. Thus, the aim of the present study was to determine the diagnostic effi cacy of saliva in chronic renal failure patients. Materials and Methods: This case–control study included 82 individuals, of which 41 were chronic renal failure patients and 41 were age- and sex-matched controls. Blood and saliva were collected and centrifuged. Serum and supernatant saliva were used for biochemical analysis. Serum and salivary urea, creatinine, sodium, potassium, calcium, and phosphorus were evaluated and correlated in chronic renal failure patients using unpaired t-test, Pearson’s correlation coeffi cient, diagnostic validity tests, and receiver operative curve. Results: When compared to serum; salivary urea, creatinine, sodium, and potassium showed diagnostic accuracy of 93%, 91%, 73%, and 89%, respectively, based on the fi ndings of study. Conclusion: It can be concluded that salivary investigation is a dependable, noninvasive, noninfectious, simple, and quick method for screening the mineral and metabolite values of high-risk patients and monitoring the renal failure patients. Key Words: Creatinine, dialysis,Renal Insufficiency,saliva, serum, urea

Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells and detected in saliva. Pathophysiological roles for salivary exosomes are beginning to be recognized in diseases including cancer, highlighting potential biomarkers and biological functions. Since early detection of cancer is vital for successful treatment, salivary exosomes would be advantageous in achieving a better survival rate due to their ready availability and noninvasiveness. The use of salivary exosomes may therefore be promising in the accurate detection of premalignant lesions and early-stage cancers, also for better our understanding of the molecular basis of tumorigenesis. In this chapter, we review our current knowledge of salivaomics, focusing on nucleic acids and proteins in saliva as potential cancer biomarkers. Since salivaomics is a rapidly evolving field, we hope to expand frameworks toward salivary exosomes, integrate new and existing information, and bridge salivaomics with other biomedical researches. Furthermore, we would like to coin the term "saliva-exosomics" as the next-generation salivaomics. Our goal in this chapter is to provide the most updated information on cancer-derived exosomes in the saliva as natural carriers of biomarkers and signaling molecules. Major advances include definitive structure analysis and molecular characterization of salivary exosomes. We also highlight the exosome biogenesis and cargo trafficking mechanisms in which recent animal studies have expanded our understanding of exosome-mediated transfer of cancer-derived products from distal tumor to salivary gland. The potential roles of the salivary exosomes in cancer progression and immune surveillance are also addressed.

The present study evaluated diurnal variations and day-to-day fluctuations of salivary oxidative stress (OS) markers in healthy adult individuals. Whole unstimulated saliva was collected at 2 time intervals over 3 consecutive days. Glutathione peroxidase (GPX), superoxide dismutase (SOD), total antioxidant capacity (TAC), and uric acid (UA) were analyzed using spectrophotometric methods, while 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA) were determined using immunoassays. No significant differences for salivary OS markers between men and women were observed. For all examined OS markers, no significant day-to-day variations were demonstrated. Significant diurnal variations were found in salivary GPX, TAC and MDA levels. For SOD, TAC, GPX, and UA, good-to-moderate intraindividual coefficients of variations (CVs) were observed in more than 75% of the subjects. For MDA and 8-OHdG, intraindividual CVs > 35% were observed in 60% and 40% of the subjects, respectively. Between-subject variance was wide for all examined OS markers (CV% 30.08%–85.70%). Due to high intraindividual variability in the salivary concentrations of MDA and 8-OHdG, those markers cannot be reliably verified based on single measurements and multiple measurements over several days would provide more reliable information. Salivary SOD, TAC, GPX, and UA proved stable across three days of measurement. Trial Registration. ClinicalTrials.gov NCT03029494. Registered on 2017-01-19.

The microbiome of the human body represents a symbiosis of microbial networks spanning multiple organ systems. Bacteria predominantly represent the diversity of human microbiota, but not to be forgotten are fungi, viruses, and protists. Mounting evidence points to the fact that the “microbial signature” is host-specific and relatively stable over time. As our understanding of the human microbiome and its relationship to the health of the host increases, it is becoming clear that many and perhaps most chronic conditions have a microbial involvement. The oral and gastrointestinal tract microbiome constitutes the bulk of the overall human microbial load, and thus presents unique opportunities for advancing human health prognosis, diagnosis, and therapy development. This review is an attempt to catalog a broad diversity of recent evidence and focus it toward opportunities for prevention and treatment of debilitating illnesses.

Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells and detected in saliva. Pathophysiological roles for salivary exosomes are beginning to be recognized in diseases including cancer, highlighting potential biomarkers and biological functions. Since early detection of cancer is vital for successful treatment, salivary exosomes would be advantageous in achieving a better survival rate due to their ready availability and noninvasiveness. The use of salivary exosomes may therefore be promising in the accurate detection of premalignant lesions and early-stage cancers, also for better our understanding of the molecular basis of tumorigenesis. In this chapter, we review our current knowledge of salivaomics, focusing on nucleic acids and proteins in saliva as potential cancer biomarkers. Since salivaomics is a rapidly evolving field, we hope to expand frameworks toward salivary exosomes, integrate new and existing information, and bridge salivaomics with other biomedical researches. Furthermore, we would like to coin the term “saliva-exosomics” as the next-generation salivaomics. Our goal in this chapter is to provide the most updated information on cancer-derived exosomes in the saliva as natural carriers of biomarkers and signaling molecules. Major advances include definitive structure analysis and molecular characterization of salivary exosomes. We also highlight the exosome biogenesis and cargo trafficking mechanisms in which recent animal studies have expanded our understanding of exosome-mediated transfer of cancer-derived products from distal tumor to salivary gland. The potential roles of the salivary exosomes in cancer progression and immune surveillance are also addressed.

Background Oral squamous cell carcinoma (OSCC) is the most common malignant tumour of the oral cavity. Detection of OSCC is currently based on clinical oral examination combined with histopathological evaluation of a biopsy sample. Direct contact between saliva and the oral cancer makes measurement of salivary metalloproteinase- 9 (MMP-9) an attractive alternative. Material and Methods In total, 30 OSCC patients and 30 healthy controls were included in this prospective study. Saliva samples from both groups were collected, centrifuged and supernatant fluid was subjected to ELISA for assessment of MMP-9. The median salivary MMP-9 values with interquartile range (IQR) of OSCC patients and the control group were statistically analysed using the Mann-Whitney U-test. The receiver operating characteristic (ROC) curve was constructed and the area under curve (AUC) was computed. Results The median absorbance MMP-9 value of the OSCC group was 0.186 (IQR=0.158) and that of control group was 0.156 (IQR=0.102). MMP-9 was significantly increased in the OSCC patients than in the controls by +19.2% (p=0.008). Median values in patients with recurrence and in patients with primary event were 0.233 (IQR=0.299) and 0.186 (IQR=0.134) respectively. MMP-9 was significantly increased in patients with primary event (p=0.017) compared to controls by +19.2%. No significant increase of MMP-9 level was detected when comparing patients with recurrence and healthy controls (+49.4%; p=0.074). The sensitivity value of MMP-9 was 100% whereas the specificity value was 26.7% with AUC of 0.698. Conclusions The present data indicates that the elevation of salivary levels of MMP-9 may be a useful adjunctive diagnostic tool for detection of OSCC. However, further studies are necessary to provide scientific and clinical validation. Key words:Oral squamous cell carcinoma, oral cancer, saliva, salivary diagnostics, cancer detection, MMP-9, metalloproteinases.

In this review, we provide an update on the current and future applications of saliva for diagnostic purposes. There are many advantages of using saliva as a biofluid. Its collection is fast, easy, inexpensive, and non-invasive. In addition, saliva, as a "mirror of the body," can reflect the physiological and pathological state of the body. Therefore, it serves as a diagnostic and monitoring tool in many fields of science such as medicine, dentistry, and pharmacotherapy. Introduced in 2008, the term "Salivaomics" aimed to highlight the rapid development of knowledge about various "omics" constituents of saliva, including: proteome, transcriptome, micro-RNA, metabolome, and microbiome. In the last few years, researchers have developed new technologies and validated a wide range of salivary biomarkers that will soon make the use of saliva a clinical reality. However, a great need still exists for convenient and accurate point-of-care devices that can serve as a non-invasive diagnostic tool. In addition, there is an urgent need to decipher the scientific rationale and mechanisms that convey systemic diseases to saliva. Another promising technology called liquid biopsy enables detection of circulating tumor cells (CTCs) and fragments of tumor DNA in saliva, thus enabling non-invasive early detection of various cancers. The newly developed technology-electric field-induced release and measurement (EFIRM) provides near perfect detection of actionable mutations in lung cancer patients. These recent advances widened the salivary diagnostic approach from the oral cavity to the whole physiological system, and thus point towards a promising future of salivary diagnostics for personalized individual medicine applications including clinical decisions and post-treatment outcome predictions. Impact statement The purpose of this mini-review is to make an update about the present and future applications of saliva as a diagnostic biofluid in many fields of science such as dentistry, medicine and pharmacotherapy. Using saliva as a fluid for diagnostic purposes would be a huge breakthrough for both patients and healthcare providers since saliva collection is easy, non-invasive and inexpensive. We will go through the current main diagnostic applications of saliva, and provide a highlight on the emerging, newly developing technologies and tools for cancer screening, detection and monitoring. Sin financiación 2.413 JCR (2017) Q3, 76/133 Medicine, Research and Experimental UEM

Saliva is an easily accessible fluid that has led to increasing interest in the development of salivary diagnostics. This chapter describes some of the newer tools and procedures for collection, stabilization, and storage of oral fluid matrices that aid in the successful use of saliva as a test specimen. This chapter focuses particularly on nucleic acid components for downstream molecular diagnostic (MDx) testing, since this is probably the area where saliva is likely to have the greatest impact in improving healthcare for the general population.

Salivary diagnostics is a rapidly advancing field that offers clinicians and patients the potential of rapid, noninvasive diagnostics with excellent accuracy. In order for the complete realization of the potential of saliva, however, extensive profiling of constituents must be conducted and diagnostic biomarkers must be thoroughly validated. This article briefly overviews the process of conducting a study of salivary biomarkers in a patient cohort and highlights the studies that have been conducted on different classes of molecules in the saliva. Emerging frontiers in salivary diagnostics research that may significantly advance the field will also be highlighted.

Saliva is an oral fluid, composed mainly of water, produced and secreted in large part from the acinar and ductal cells of the major and minor salivary glands. Whole saliva derives additional constituents from serum, gingival crevicular fluid and oral mucosal transudate, making it appealing as a potential diagnostic fluid reflective of circulating levels in blood.1 Several studies report systemic biomarkers appearing in saliva including electrolytes,2, 3 blood products,4 enzymes and tissue destruction molecules,5–9 inflammatory markers,10–16 as well as proteins putatively associated with deadly diseases.17–21 However, the clinical utility of salivary diagnostics for the assessment of systemic disease remains elusive.20, 22–27 Cardiovascular disease (CVD) is a major cause of mortality in the U.S. and worldwide. More than 17 million people died from CVD in 2008, with the majority due to atherosclerotic events including myocardial infarction.28, 29 With the extend and importance of this disease, easy, noninvasive and low cost screening methods for the detection of myocardial infarction continue to be sought. One such potential screening fluid is saliva.30 Support for the use of saliva for the detection of CVD and myocardial infarction is built upon the framework that many serum biomarkers associated with inflammation, atherosclerosis, plaque stability and myocardial damage are known and appear in oral fluids.4, 9, 31–35 However, only a limited number of studies have demonstrated correlations between serum and salivary biomarkers of CVD.14, 33, 36 Thus, additional research is required if we are to gain a greater understanding of the clinical utility of saliva compared to serum with respect to CVD and myocardial infarction assessment. This is particularly important for finding new diagnostics for situations where acquisition, processing or analysis of blood is problematic. In this research investigation, we hypothesized that select salivary biomarkers would correlate with serum biomarkers of CVD in patients with pre-existing heart disease and could yield important diagnostic information regarding the evolution and progression of myocardial necrosis. To test this hypothesis we employed a unique model of myocardial infarction in patients with a pre-existing CVD (i.e., hypertrophic cardiomyopathy [HCM]) who were scheduled to undergo alcohol septal ablation (ASA). This procedure results in a controlled myocardial infarction and subsequent remodeling processes that permit improved hemodynamics and improved patient outcomes.37, 38 Together, the pre-existing CVD and subsequent kinetic course of myocardial necrosis allowed us to predict that a spectrum of myocardial damage, inflammation and tissue remodeling biomarkers would appear in serum and saliva at baseline, and significant spikes would occur in these biomarker levels during the post-operative phase consistent with myocardial infarction.39–43 Here this unique study design permitted us to report the novel findings that salivary levels of troponin (Tn)I and C-reactive protein (CRP) reflected changes in serum consistent with myocardial damage, and select salivary biomarkers correlate with serum biomarkers of CVD.

Diagnostics that involve the use of oral fluids have become increasingly available commercially in recent years and are of particular interest because of their relative ease of use, low cost and noninvasive collection of oral fluid for testing.The authors discuss the use of salivary diagnostics for virus detection with an emphasis on rapid detection of infection by using point-of-care devices. In particular, they review salivary diagnostics for human immunodeficiency virus, hepatitis C virus and human papillomavirus. Oral mucosal transudate contains secretory immunoglobulin (Ig) A, as well as IgM and IgG, which makes it a good source for immunodiagnostic-based devices.Because patients often visit a dentist more regularly than they do a physician, there is increased discussion in the dental community regarding the need for practitioners to be aware of salivary diagnostics and to be willing and able to administer these tests to their patients.

Sjögren's syndrome (SS) is an autoimmune inflammatory disorder of exocrine glands. SS particularly affects the lacrimal and salivary glands. Dry mouth and dry eyes are frequently proffered as presenting symptoms, but nonspecific symptoms such as malaise and fatigue, and extraglandular manifestations like purpura, polyneuropathy and arthritis are also often present. Moreover, lymphomas develop in about 7.5% of SS patients, mostly marginal zone B-cell lymhomas. Futhermore, SS has a very substantial impact on the patients' quality of life and their daily activities. Recently, many breakthroughs have been seen in salivary diagnostics, which not only can be used for diagnosis but also for monitoring of disease activity and disease progression as well as for objectively scoring the effect of intervention treatment with biologicals. In addition, salivary proteomics, genomics and system biology have been shown to be very promising tools in unravelling the pathophysiology of SS, thus providing in depth insight in its underlying mechanisms which could give clues for intervention therapies with biologicals. The latter is of particular interest as B cell depletion therapy has been shown a very promising therapy for a subgroup of SS patients. When applying salivary diagnostics in combination with instruments to rate disease activity and progression in SS, one might be able to select those SS patients who respond to a particular type of biological. These topics are addressed in this review and promises for the near future are described.

Lung cancer is the leading cause of cancer death for both men and women worldwide. Since most of the symptoms found for lung cancer are nonspecific, diagnosis is mostly done at late and progressed stage with the consecutive poor therapy outcome. Effective early detection techniques are sorely needed. The emerging field of salivary diagnostics could provide scientifically credible, easy-to-use, non-invasive and cost-effective detection methods. Recent advances have allowed us to develop discriminatory salivary biomarkers for a variety of diseases from oral to systematic diseases. In this study, salivary transcriptomes of lung cancer patients were profiled and led to the discovery and pre-validation of seven highly discriminatory transcriptomic salivary biomarkers (BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and LZTS1). The logistic regression model combining five of the mRNA biomarkers (CCNI, EGFR, FGF19, FRS2, and GREB1) could differentiate lung cancer patients from normal control subjects, yielding AUC value of 0.925 with 93.75 % sensitivity and 82.81 % specificity in the pre-validation sample set. These salivary mRNA biomarkers possess the discriminatory power for the detection of lung cancer. This report provides the proof of concept of salivary biomarkers for the non-invasive detection of the systematic disease. These results poised the salivary biomarkers for the initiation of a multi-center validation in a definitive clinical context.

Identifying discriminatory human salivary RNA biomarkers reflective of disease in a low-cost non-invasive screening assay is crucial to salivary diagnostics. Recent studies have reported both mRNA and microRNA (miRNA) in saliva, but little information has been documented on the quality and yield of RNA collected. Therefore, the aim of the present study was to develop an improved RNA isolation method from saliva and to identify major miRNA species in human whole saliva.RNA samples were isolated from normal human saliva using a combined protocol based on the Oragene RNA collection kit and the mirVana miRNA isolation kit in tandem. RNA samples were analysed for quality and subjected to miRNA array analysis.RNA samples isolated from twenty healthy donors ranged from 2.59 to 29.4 μg/ml saliva and with 1.92-2.16OD(260/280 nm) ratios. RNA yield and concentration of saliva samples were observed to be stable over 48 h at room temperature. Analysis of total salivary RNA isolated from these twenty donors showed no statistical significance between sexes; however, the presence of high-, medium-, and low-yield salivary RNA producers was detected. MiRNA array analysis of salivary RNA detected five abundantly expressed miRNAs, miR-223, miR-191, miR-16, miR-203, and miR-24, that were similarly described in other published reports. Additionally, many previously undetected miRNAs were also identified.High quality miRNAs can be isolated from saliva using available commercial kits, and in future studies, the availability of this isolation protocol may allow specific changes in their levels to be measured accurately in various relevant diseases.

Periodontal disease results from interaction between oral bacteria and the host inflammatory response. This interaction triggers a cascade of inflammatory events, which in turn promote connective tissue destruction and alveolar bone remodeling.1-7 These unique biological events contain signatures of the microbial ecology, as well as downstream events involving inflammation, attachment loss and bone destruction. It is likely that identification of the dominant signatures for each of these biological phases could provide insight into biomarkers of periodontal disease in oral fluids. To this end, several investigators have identified salivary biomarkers of biological events associated with aspects of periodontal disease8-12 and reviews on this topic are available.13-15 However, lacking to date is a focused panel of biomarkers that encompass the early and later biological phases that would yield, at least theoretically, the specificity for the development of a real-time, accurate, biologically-based, periodontal disease diagnostic device. Progress continues to be made towards the achievement of a salivary diagnostic device based on the knowledge that saliva is a rich pool of proteins and molecules that reflects aspects of oral health. In one recent example, we found that biomarkers of inflammation, connective tissue destruction and bone remodeling were elevated in concentrations in the saliva of patients with periodontal disease.11 In that study, we observed that salivary levels of IL-1β and MMP-8 were significantly associated with clinical parameters of periodontal disease, and levels of IL-1β and MMP-8 elevated above a threshold (i.e., 2 standard deviations above the mean of healthy controls), together demonstrated an odds ratio of 45 for periodontal disease. While these results demonstrated proof of principle that biomarkers from two distinct biological phases could aid in distinguishing periodontal disease from health, the identification of a biomarker associated with aspects of bone remodeling – a late biological event that could improve the accuracy of salivary diagnostics - remains elusive. Bone resorption is mediated by osteoclasts that exhibit specific abilities to degrade organic and inorganic components of bone. Different mediators such as interleukin-1β, prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein (MIP)-1α, interleukin-6 (IL-6), interleukin-11 (IL-11), and interleukin-17 (IL-17) act upstream as activators of osteoclastogenesis.16-22 Within the resorption lacunae, receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) are important cytokines belonging to the TNF family that regulate differentiation of osteoclast progenitor cells into active osteoclasts or inhibiting the differentiation process, respectively.23, 24 As a result, type 1 collagen is degraded during bone destruction by proteolytic enzymes such as matrix metalloproteinases (MMPs) and cathepsin K which lead to release of cross-linked telopeptides into the circulation (serum, saliva, and urine) as stable fragments such as pyridinoline cross-linked carboxyterminal telopeptide of type 1 collagen (ICTP)25-27 and C-terminal type 1 collagen telopeptide (β-CTX).28-31 We and others have investigated salivary levels of several of these important molecules associated with cytokine signaling and alveolar bone resorption32-39 however no conclusive information has been yet reported on the best biomarker associated with alveolar bone remodeling in adults. Also, there is a lack of knowledge whether upstream pathways, midstream osteoclastogenic factors, or downstream degradation products are better salivary biomarkers of periodontal disease. Therefore, the purpose of this study was to test the hypothesis that a specific salivary biomarker associated with bone remodeling could be identified that would distinguish healthy and periodontal disease subjects. Proteins associated with the upstream, midstream and downstream processes of osteoclastogenesis (i.e., MIP-1α, OPG, β-CTX, and ICTP) were selected for evaluation.

Saliva, a biofluid historically well-studied biochemically and physiologically, has entered the post-genomic ‘omics’ era, where its proteomic, genomic, and microbiome constituents have been comprehensively deciphered. The translational path of these salivary constituents has begun toward a variety of personalized individual medical applications, including early detection of cancer. Salivary diagnostics is a late-comer, but it is catching up where dedicated resources, like the Salivaomics Knowledge Base (SKB), now have taken center stage in the dissemination of the diagnostic potentials of salivary biomarkers and other translational and clinical utilities.

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.

The purpose of this study was to determine the role of saliva-derived biomarkers and periodontal pathogens during periodontal disease progression (PDP). One hundred human participants were recruited into a 12-month investigation. They were seen bi-monthly for saliva and clinical measures and bi-annually for subtraction radiography, serum and plaque biofilm assessments. Saliva and serum were analyzed with protein arrays for 14 pro-inflammatory and bone turnover markers, while qPCR was used for detection of biofilm. A hierarchical clustering algorithm was used to group study participants based on clinical, microbiological, salivary/serum biomarkers, and PDP. Eighty-three individuals completed the six-month monitoring phase, with 39 [corrected] exhibiting PDP, while 44 [corrected] demonstrated stability. Participants assembled into three clusters based on periodontal pathogens, serum and salivary biomarkers. Cluster 1 members displayed high salivary biomarkers and biofilm; 71% [corrected] of these individuals were undergoing PDP. Cluster 2 members displayed low biofilm and biomarker levels; 76% [corrected] of these individuals were stable. Cluster 3 members were not discriminated by PDP status; however, cluster stratification followed groups 1 and 2 based on thresholds of salivary biomarkers and biofilm pathogens. The association of cluster membership to PDP was highly significant (p < 0.0007). [corrected] The use of salivary and biofilm biomarkers offers potential for the identification of PDP or stability (ClinicalTrials.gov number, CT00277745).

Salivary diagnostics is a dynamic and emerging field utilizing nanotechnology and molecular diagnostics to aid in the diagnosis of oral and systemic diseases. In this article the author critically reviews the latest advances using oral biomarkers for disease detection. The use of oral fluids is broadening perspectives in clinical diagnosis, disease monitoring, and decision making for patient care. Important elements determining the future possibilities and challenges in this field are also discussed.

This study investigated salivary cholesterol of children from 6 to 16 years of age in response to dietary intervention. One thousand sixty-two infants started in the prospective, randomized project. At 3 years of age, every fifth child was invited into the study (n = 178). Of these, 148 enrolled, and 86 completed the oral sub-study at 16 years of age. The intervention aimed at restricting the child’s saturated fat and cholesterol intake. Control children received no special recommendations. Every third year, paraffin-stimulated saliva samples (10.0 mL) were collected for cholesterol assays. Nutrient intakes and serum total cholesterol concentrations were regularly followed up by means of 4-day food records and blood samples. Intake of saturated fatty acids (SAFA) was lower in the intervention than in the control group (p < 0.001). Salivary cholesterol concentration increased from 1.9 (± 1.1) µmol/L at 6 years of age to 16.0 (± 9.0) µmol/L at 16 years of age. The increase was smaller in the intervention than in the control group (p < 0.001). The ratios of salivary to serum cholesterol concentrations tended to be higher in boys than in girls (p = 0.07). Thus, dietary intervention was reflected in children’s salivary cholesterol values more sensitively than in serum cholesterol values. (clinicaltrials.gov NCT00223600).

For the past two decades, salivary diagnostic approaches have been developed to monitor oral diseases such as periodontal diseases and to assess caries risk. Recently, the combination of emerging biotechnologies and salivary diagnostics has extended the range of saliva-based diagnostics from the oral cavity to the whole physiologic system as most compounds found in blood are also present in saliva. Accordingly, saliva can reflect the physiologic state of the body, including emotional, endocrinal, nutritional and metabolic variations and acts as a source for the monitoring of oral and also systemic health. This review presents an update on the status of saliva diagnostics and delves into their applications to the discovery of biomarkers for cancer detection and therapeutic applications. Translating scientific findings of nucleic acids, proteins and metabolites in body fluids to clinical applications is a cumbersome and challenging journey. Our research group is pursuing the biology of salivary analytes and the development of technologies for detection of distinct biomarkers with high sensitivity and specificity. The avenue of saliva diagnostics incorporating transcriptomic, proteomic and metabolomic findings will enable us to connect salivary molecular analytes to monitor therapies, therapeutic outcomes, and finally disease progression in cancer.

The protein compositions, or the proteomes, found in human salivary and plasma fluids are compared. From recent experimental work by many laboratories, a catalogue of 2290 proteins found in whole saliva has been compiled. This list of salivary proteins is compared with the 2698 proteins found in plasma. Approximately 27% of the whole-saliva proteins are found in plasma. However, despite this apparent low degree of overlap, the distribution found across Gene Ontological categories, such as molecular function, biological processes, and cellular components, shows significant similarities. Moreover, nearly 40% of the proteins that have been suggested to be candidate markers for diseases such as cancer, cardiovascular disease, and stroke can be found in whole saliva. These comparisons and correlations should encourage researchers to consider the use of saliva to discover new protein markers of disease and as a diagnostic non-proximal fluid to detect early signs of disease throughout the body.

Saliva is a noninvasive and accessible biofluid that permits early detection of oral and systemic diseases. Changes in saliva reflect the alterations in the blood and thus making saliva a suitable diagnostic tool. The use of saliva has many advantages, including simple and non-invasive sampling and easy, low-cost storage. Today salivary diagnostics is a promising tool for diagnostic processes and clinical monitoring. Saliva is used to detect illicit drugs, alcohol, to measure hormone levels, and in the diagnosis of wide range systemic diseases such as cardiovascular, infectious, renal, endocrine diseases, some types of cancer as well as nonsystemic oral diseases. The current review presents a critical overview of saliva as a promising tool for the development of valuable salivary biomarkers, their relevance to the prognosis, diagnosis and management of systemic and oral diseases. Development of point-of-care testing based on saliva samples as a screening tool will also support the diagnostic process in near future.

Purpose: We have previously shown that a transcriptome is found in saliva and subpanels of these mRNAs can be used as oral cancer biomarkers. In this study, we measured the presence of microRNAs (miRNA) in saliva and determined their potential as an additional set of oral cancer biomarkers. Experimental Design: A total of 314 miRNAs were measured using reverse transcriptase-preamplification-quantitative PCR in 12 healthy controls. Degradation pattern of endogenous and exogenous saliva miRNAs were measured at room temperature over time. Selected miRNAs were validated in saliva of 50 oral squamous cell carcinoma patients and 50 healthy matched control subjects. Results: We detected ∼50 miRNAs in both the whole and supernatant saliva. Endogenous saliva miRNA degraded much slower compared with exogenous miRNA. Two miRNAs, miR-125a and miR-200a, were present in significantly lower levels (P < 0.05) in the saliva of oral squamous cell carcinoma patients than in control subjects. Conclusions: Both whole and supernatant saliva of healthy controls contained dozens of miRNAs, and similar to saliva mRNAs, these miRNAs are stable. Saliva miRNAs can be used for oral cancer detection. (Clin Cancer Res 2009;15(17):5473–7)

Purpose: This study aims to explore the presence of informative protein biomarkers in the human saliva proteome and to evaluate their potential for detection of oral squamous cell carcinoma (OSCC). Experimental Design: Whole saliva samples were collected from patients (n = 64) with OSCC and matched healthy subjects (n = 64). The proteins in pooled whole saliva samples of patients with OSCC (n = 16) and matched healthy subjects (n = 16) were profiled using shotgun proteomics based on C4 reversed-phase liquid chromatography for prefractionation, capillary reversed-phase liquid chromatography with quadruple time-of-flight mass spectrometry, and Mascot sequence database searching. Immunoassays were used for validation of the candidate biomarkers on a new group of OSCC (n = 48) and matched healthy subjects (n = 48). Receiver operating characteristic analysis was exploited to evaluate the diagnostic value of discovered candidate biomarkers for OSCC. Results: Subtractive proteomics revealed several salivary proteins at differential levels between the OSCC patients and matched control subjects. Five candidate biomarkers were successfully validated using immunoassays on an independent set of OSCC patients and matched healthy subjects. The combination of these candidate biomarkers yielded a receiver operating characteristic value of 93%, sensitivity of 90%, and specificity of 83% in detecting OSCC. Conclusion: Patient-based saliva proteomics is a promising approach to searching for OSCC biomarkers. The discovery of these new targets may lead to a simple clinical tool for the noninvasive diagnosis of oral cancer. Long-term longitudinal studies with large populations of individuals with oral cancer and those who are at high risk of developing oral cancer are needed to validate these potential biomarkers.

There is a high demand for a non-invasive, rapid, and highly accurate tool for disease diagnostics. Recently, saliva based diagnostics for the detection of specific biomarkers has drawn significant attention since the sample extraction is simple, cost-effective, and precise. Compared to blood, saliva contains a similar variety of DNA, RNA, proteins, metabolites, and microbiota that can be compiled into a multiplex of cancer detection markers. The salivary diagnostic method holds great potential for early-stage cancer diagnostics without any complicated and expensive procedures. Here, we review various cancer biomarkers in saliva and compare the biomarkers efficacy with traditional diagnostics and state-of-the-art bioelectronics. We summarize biomarkers in four major groups: genomics, transcriptomics, proteomics, and metabolomics/microbiota. Representative bioelectronic systems for each group are summarized based on various stages of a cancer. Systematic study of oxidative stress establishes the relationship between macromolecules and cancer biomarkers in saliva. We also introduce the most recent examples of salivary diagnostic electronics based on nanotechnologies that can offer rapid, yet highly accurate detection of biomarkers. A concluding section highlights areas of opportunity in the further development and applications of these technologies.