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Background: Salivary diagnostics promise an easily accessible, non-invasive approach for analyzing and monitoring various oral diseases including cancer. Objectives: This study was undertaken to establish the baseline salivary lactate dehydrogenase (LDH) levels in healthy controls and to discern the effect of smokeless tobacco (SLT) on the diagnostic efficacy of salivary LDH. Materials and Methods: Thirty-seven systemically and periodontally healthy individuals in the age group of 19-27 years without any deleterious habit and oral lesion participated in this study. After explaining the procedure to the participants, unstimulated whole saliva was collected by spit method under sterile conditions. Baseline LDH activity was measured spectrophotometrically. After evaluating baseline activity, 1 ml of saliva was mixed with 1 g of SLT and incubated at 37°C. LDH activity was measured at different time intervals. Results: The median value of baseline LDH levels at rest was 338 IU/L. The gender-based variation in LDH activity was found to be non-significant. Exposure of whole saliva to SLT resulted in an initial increase followed by reduction in the LDH activity. Conclusion: This study provides additional rationale for the use of salivary LDH as a biomarker to investigate the aggressiveness and biological nature of oral pathological conditions. Interpretation of changes in salivary LDH activity should be done with caution in individuals habituated to tobacco use.

Importance of the field: Periodontal disease is one of the most prevalent of all diseases. The chronic inflammatory process causes tooth loss and is associated with negative systemic effects. There are still major challenges when it comes to precise diagnosis, prognosis and appropriate disease therapy. The rapidly emerging field of salivary diagnostics could help to overcome these problems. Areas covered in this review: The article covers the last 20 years of periodontal disease biomarker research and gives an overview of the current status of salivary diagnostic techniques. What the reader will gain: The reader will gain insight into the periodontopathic process, associated biomarkers and biomarker detection techniques in the field of saliva-based molecular diagnostics. Take home message: Salivary diagnostics has great potential for the detection of oral and systemic diseases. Periodontal disease diagnostics, therapy and prognosis could benefit from this emerging diagnostic field.

OBJECTIVE: Regulators of peri-implant bone loss in patients with diabetes appear to involve multiple risk factors that have not been clearly elucidated. This study was conducted to explore putative local etiologic factors on implant bone loss in relation to type 2 diabetes mellitus, including clinical, microbial, salivary biomarker, and psychosocial factors. MATERIALS AND METHODS: Thirty-two subjects (divided into type 2 diabetes mellitus and non-diabetic controls), having at least one functional implant and six teeth, were enrolled in a 1-year longitudinal investigation. Analyses of clinical measurements and standardized intra-oral radiographs, saliva and serum biomarkers (via protein arrays for 20 selected markers), and plaque biofilm (via qPCR for eight periodontal pathogens) were performed at baseline and 1 year. In addition, the subjects were asked to respond to questionnaires to assess behavioral and psychosocial variables. RESULTS: There was a significant increase from baseline to 1 year in the probing depth of implants in the diabetes group (1.95 mm to 2.35 mm, P = 0.015). The average radiographic bone loss during the study period marginally increased at dental implants compared to natural teeth over the study period (0.08 mm vs. 0.05 mm; P = 0.043). The control group harbored higher levels of Treponema denticola at their teeth at baseline (P = 0.046), and the levels of the pathogen increased significantly over time around the implants of the same group (P = 0.003). Salivary osteoprotegerin (OPG) levels were higher in the diabetes group than the control group at baseline only; in addition, the salivary levels of IL-4, IL-10, and OPG associated with host defense were significantly reduced in the diabetes group (P = 0.010, P = 0.019, and P = 0.024), while controls showed an increase in the salivary OPG levels (P = 0.005). For psychosocial factors, there were not many significant changes over the observation period, except for some findings related to coping behaviors at baseline. CONCLUSIONS: The study suggests that the clinical, microbiological, salivary biomarker, and psychosocial profiles of dental implant patients with type 2 diabetes who are under good metabolic control and regular maintenance care are very similar to those of non-diabetic individuals. Future studies are warranted to validate the findings in longer-term and larger clinical trials (ClinicalTrials.gov # NCT00933491).

BACKGROUND AND OBJECTIVES We investigated if a combination of plasma or salivary interleukin-2 (IL-2), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), and troponin can improve estimation of the pretest probability of the left ventricular systolic dysfunction (LVSD). SUBJECTS AND METHODS Eighty patients with newly-diagnosed myocardial infarction (MI) were echocardiographically examined for LVSD (ejection fraction ≤40%). Measurements included traditional MI risk factors, plasma and salivary concentrations of troponin, IL-2, IL-6, TNF-α, and TGF-β. With the LVSD as the outcome variable, we developed logistic regression models, starting with a basic model incorporating traditional risk factors and consecutively adding salivary and plasma biomarkers. Models were compared using several criteria, including (but not limited to) C statistic (discrimination) and net reclassification improvement index (NRI). RESULTS APART FROM TROPONIN, PLASMA, AND SALIVARY VALUES OF THE BIOMARKERS WERE CORRELATED: spearman's ρ was 0.19 (p=0.088) for troponin, 0.36 (p=0.001) for IL-2, 0.74 (p

Saliva as a biological fluid is gaining wider acceptance for diagnosing diseases. The growing interest in saliva as a biological fluid is due to its noninvasiveness, ease of use, cost-effectiveness, and multiple sample collection possibilities as well as minimal risk to health care professionals of contracting infectious organisms such as HIV and Hep B. However, the clinical translation of saliva is hampered by our lack of understanding of the biomolecular transportation from blood into saliva, the diurnal variations of biomolecules present in saliva, and relatively low levels of analytes (100th to a 1000th fold less than in blood). We provide information on the current status of salivary research, salivary diagnostics empowered by nanotechnology, and future prospects in this emerging field of saliva diagnostics.

The importance of the genetic influence on disease susceptibility has been well-established. The genetic regulation of the immune response is a subject of considerable ongoing research in terms of periodontal inflammation and its squeal. The development of commercially available DNA-PCR salivary testing represents an important translational development in our understanding and management of periodontitis. This paper will discuss the importance of interleukin polymorphism, especially IL-6 and its role in the determination of disease susceptibility in the periodontal patient. The clinical significance of salivary diagnostics is emphasized as an important part of the decision-making process. This type of testing and the rapidly developing innovations can literally change the way we manage patients. 1.1. Clinical significance: By providing us with knowledge of the patient's predisposition to a hyper inflammatory reaction we can intervene at an earlier stage of disease development with considerably better long-term outcome.

Saliva is a non-invasive and accessible biofluid that provides accurate readouts for the detection of oral cancers. Recent technological advances have uncovered oral cancer specific salivary biomarkers. The availability of highly sensitive and high-throughput assays such as microarray, mass spectrometry, quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and nano-scale sensors can measure proteins and nucleic acids despite the low concentrations in saliva. This chapter will discuss these proteomic and transcriptomic salivary biomarkers development for oral cancer, and the translational applications of these markers for the detection of the disease.

Saliva is responsible for the formation of the acquired enamel pellicle (AEP), a protein integument formed as a result of selective adsorption of salivary proteins to the enamel surface. The AEP demonstrates an important role for modulating dental erosion as a result of its physical properties, along with its salivary and exogenous protein composition (Chapter 2). In addition, individual proteins that comprise the AEP have important physiological functions. Histatin 5 (H5) has potent antifungal effect against C. albicans, the yeast responsible for the initiation of oral candidiasis. We designed an in vitro model and found, for the first time, that H5 adhered in the form of pellicle retains its antifungal activity on C. albicans (Chapter 3). As a pellicle precursor protein, H5 demonstrates high affinity for hydroxyapatite, the primary mineral component of enamel. We used atomic force microscopy (AFM) to determine adhesion forces between H5 and the hydroxyapatite surface to be stronger compared to our protein control, albumin. This knowledge can be applied in the design of therapeutic proteins, and the methodology that we developed can be used for measuring adhesion forces between various other proteins and substrates of interest (Chapter 4). Finally, with the development of proteomics instruments, researchers have identified some protein biomarkers, hidden within salivary fluids. These can be used for diagnostic dentistry, in a clinical setting to identify patients’ susceptibility of developing oral diseases. In addition, the delivery proteins with antimicrobial properties via toothpastes or oral rinses can have tremendous therapeutic potential for a multitude of oral diseases (Chapter 5).

The use of saliva as a diagnostic sample provides a non-invasive, cost-efficient method of sample collection for disease screening without the need for highly trained professionals. Saliva collection is far more practical and safe compared with invasive methods of sample collection, because of the infection risk from contaminated needles during, for example, blood sampling. Furthermore, the use of saliva could increase the availability of accurate diagnostics for remote and impoverished regions. However, the development of salivary diagnostics has required technical innovation to allow stabilization and detection of analytes in the complex molecular mixture that is saliva. The recent development of cost-effective room temperature analyte stabilization methods, nucleic acid pre-amplification techniques and direct saliva transcriptomic analysis have allowed accurate detection and quantification of transcripts found in saliva. Novel protein stabilization methods have also facilitated improved proteomic analyses. Although candidate biomarkers have been discovered using epigenetic, transcriptomic, proteomic and metabolomic approaches, transcriptomic analyses have so far achieved the most progress in terms of sensitivity and specificity, and progress towards clinical implementation. Here, we review recent developments in salivary diagnostics that have been accomplished using genomic, transcriptomic, proteomic and metabolomic approaches.

Background and Overview The use of salivary diagnostics continues to develop and advance the field of risk determination for periodontal diseases. Researchers are investigating genetic, microbial and protein biomarkers with the objective of translating findings to such aspects of clinical care as broad patient screening, monitoring and treatment planning. Methods In this review, the author briefly explores currently available salivary diagnostics used to identify bacteria prevalent in periodontal disease, and focuses on the future development and use of a variety of rapid disease detection platforms, such as lab-on-a-chip, as a point-of-care device for identification of patients' risk. Conclusions and Clinical Implications Several diagnostic tests are commercially available, and point-of-care tests are under development. However, challenges remain regarding the introduction of these technologies to clinical practice and adoption by dental practitioners for promotion of personalized oral health care. The author explores currently available salivary diagnostics for periodontal disease and focuses on future developments in the field.

Metabolomics is the systematic study of the unique chemical fingerprints of low molecular weight endogenous metabolites or metabolite profiles in a biological sample. Metabolites that are important indicators of physiological or pathological states can provide information for the identification of early and differential markers for disease and help to understand its occurrence and progression. Analysis of these key biomarkers has become an important role to monitor the state of biological organisms and is a widely used diagnostic tool for disease. Metabolomic analyses are propelling the field of medical diagnostics forward at unprecedented rates because of its ability to reliably identify metabolites that are at the metabolic level in concentration. These advancements have benefited biomarker research to the point where saliva is now recognized as an excellent diagnostic medium for the detection of disease. Saliva contains a large array of metabolites, many of which can be informative for the detection of diseases. Salivary diagnostics offer an easy, inexpensive, safe, and noninvasive approach for disease detection. Discovery of salivary biomarkers that could be used to scrutinize health and disease surveillance has addressed its diagnostic value for clinical applications. Availability of emerging metabolomic techniques gives optimism that saliva can eventually be placed as a biomedium for clinical diagnostics. Comprehensive salivary metabolome will be an important resource for researchers who are studying metabolite chemistry, especially in the fields of salivary diagnostics, and will be helpful for analyzing and hence identifying corresponding disease-related salivary biomarkers. This review presents an overview of the value of saliva as a credible diagnostic tool, the discovery of salivary biomarkers, and the development of salivary diagnostics now and in the future. In particular, proof of principle has been demonstrated for salivary biomarker research.

Saliva is considered a mirror of body health and is composed of variety of analytes from systemic sources that reach the oral cavity through various pathways. Because water is a major constituent, saliva plays a key role in the lubrication and repair of oral mucosa, formation and swallowing of food bolus, digestion of starch, facilitation of food tasting and control of oropharyngeal microbial population (Lawrence, 2002).

Jill L. Maron, MD, MPH The integration of a noninvasive platform for disease surveillance into clinical care has been a long-sought-after goal of modern medicine. Saliva and its diverse constituents are known for both their healing and diagnostic potential. Combined with its ease of collection, saliva, in theory, is an ideal biofluid to noninvasively assess a patient’s health status, particularly when the feasibility of blood draws is limited or in remote locations where infections are numerous, resources are scarce, and timely diagnosis is essential for improved outcomes. However, although the concept of analyzing saliva for infection or disease may seem straightforward, the reality of integrating it into clinical practice has proven difficult. Not only must informative salivary biomarkers be identified and validated across large patient populations and demographics, but also technological advances must permit their rapid identification and quantification. Furthermore, patients, caregivers, and insurers must be assured that salivary diagnostic tests are better than current standard of care and may ultimately improve patient outcomes while reducing health care costs. Only once each of these goals is achieved can salivary diagnostics become a mainstay of our health care system. In October 2014, the first annual North American Salivary Symposium was held at the Tufts School of Dental Medicine in Boston, Massachusetts. For 3 days, multidisciplinary expert investigators, leaders in industry, and executive officers of insurance companies gathered to share ideas, form collaborations, and identify ongoing limitations to salivary diagnostics that could be targeted to expedite their integration into patient care. The meeting not only highlighted emerging salivary research and technological advances but also aimed to engage relevant stakeholders who will ultimately play a large role in its applicability and translatability to the bedside. Such comprehensive conferences are an essential and much needed aspect of ensuring the successful translation of this rapidly emerging field. In this issue of the Clinical Therapeutics, we highlight the research findings presented at the North American Salivary Symposium. Salivary research is moving well beyond detection of oral diseases and microbial infections and is delving into assessment of systemic disease. Research presented at the meeting included the use of saliva to better understand disrupted developmental patterns and infectious risks in the vulnerable preterm newborn and the development of real-time platforms to assess stress reactivity and regulation in psychobiological research. Exploration of cell-to-cell communication through the analysis of salivary exosomes is providing investigators with a noninvasive window to further our understanding of complex biological mechanisms. Finally, the emergence of point-of-service devices that are capable of rapid identification of protein, microbial, and/or gene targets is laying the foundation to finally bring the reality of salivary diagnostics into the 21st century.

Human saliva is a complex fluid, rich in immunological components, which reflects real time systemic concentrations. Advances in biotechnology have enabled us to measure minute concentrations of immunological components such as cytokines in saliva samples with precision and accuracy 1. There are few data evaluating how the collection method may influence the results in addition to other methodological challenges, resulting in lack of consensus on a universally accepted saliva collection technique2, especially in children. Specifically, the impact of saliva collection methods on detection and analysis of salivary concentrations of allergic inflammatory cytokines such as the interleukin (IL)-4, IL-5, IL-13, eotaxin 3 (Eo3) and thymic stromal lymphopoietin (TSLP), that mediate allergic inflammation3 has not been reported. These cytokines play an important role in the development of allergic inflammatory states in increasingly prevalent conditions such as allergic rhinitis (AR), food allergy (FA), asthma (AS), atopic dermatitis (AD), and eosinophilic esophagitis (EoE)4. The overall goal of this study was to compare two methods of saliva collection in the detection and analysis of salivary concentrations of IL-4, IL-5, IL-13, Eo3 and TSLP in children. Our study was approved by the Baylor Institutional Review Board and informed consent was obtained from the parent and assent, where appropriate, from the child. We studied 20 children; median age: 14 years (9–17, IQR), female: 14 (64%), Caucasian: 13 (59%). Three (15%) children did not report any allergies and the remaining 17 (85%) children reported one or more of following commonly prevalent allergic conditions: AD (n=1), oral allergy syndrome (n=1), AS (n=2), FA (n=2), AR (n=6) and EoE (n=9). No attempt was made to distinguish salivary cytokine profiles between children with and without allergies in this study. Participants were nil per os for at least 4 hours. They rinsed their mouth with water, and following a 10 min. period, passive drool (PD) was collected followed by saliva collection via an oral swab (OS), in order to minimize the effect of saliva on the flow rate and concentration of cytokines in PD. PD samples were obtained with the head tilted forward while drooling down a 5.5 cm polypropylene Salimetrics Collection Aid® (Salimetrics LLC, State College, PA). Next, the participant placed a Salimetrics Oral Swab® (Salimetrics LLC, State College, PA) in their mouth and gently rolled it side-to-side for approximately 60–90 sec. to saturate the swab, which was then placed in a storage tube that allows saliva to be centrifuged. All samples were collected between 7:30 am and 12:30 pm. Two (9%) participants aged 11 and 14 years were unable to provide PD because they were not able to direct their drool into the collection device. All participants were able to use the OS. After providing paired saliva samples, all children responded to a short questionnaire assessing their knowledge of salivary diagnostics, asking them to rate their experience in providing each saliva sample in terms of perceived ease of use, acceptability and safety. Only 23% were aware of saliva testing in the surveillance of health or disease. A significantly higher proportion of children preferred the OS over PD sampling (82 % vs. 18 %; P < 0.05). Ease (68%) and speed (14%) of providing samples were the most common reasons cited by those who preferred the OS. Saliva samples were maintained at 4°C and transport ed to the laboratory within 2 hr of collection and were centrifuged at 3,000 rpm for 15 minutes. Supernatants were stored in aliquots and frozen at −80°C. On the day of anal ysis, samples were brought to room temperature, vortexed, and centrifuged for 15 minutes at 3,000 rpm. A magnetic, high sensitivity human multi-analytes profiling bead-based assay kit was used (EMD Millipore, Billerica, MA; lower limit of detection (LLD) for IL-4, IL-5, and IL-13: 0.13 pg/mL, and coefficient of variation (CV) < 10%

Oral fluid-based (salivary) tests have the potential to create practical, point-of-care clinical instruments that are convenient, practical, and comfortable to use in dentistry and medicine. Currently, there are no simple, accurate, and inexpensive sampling, screening, or detection methods to support definitive diagnostic platforms across dental and medical disciplines. Though the benefits from advancing screening and detection technologies seem eminent, analytical, chemical, molecular, genetic, and protein markers are still under development. Clinical applications in patient care must be validated independently to ensure that they are clinically accurate, reliable, precise, and uniformly consistent for screening and detecting specific diseases or conditions. As technology designed to improve patient care through risk assessment, prevention, and disease management is transferred into clinical practice, dentistry may need to reassess its role in general health care.

BACKGROUND Over the past 10 years, the use of saliva as a diagnostic fluid has gained attention and has become a translational research success story. Some of the current nanotechnologies have been demonstrated to have the analytical sensitivity required for the use of saliva as a diagnostic medium to detect and predict disease progression. However, these technologies have not yet been integrated into current clinical practice and work flow. CONTENT As a diagnostic fluid, saliva offers advantages over serum because it can be collected noninvasively by individuals with modest training, and it offers a cost-effective approach for the screening of large populations. Gland-specific saliva can also be used for diagnosis of pathology specific to one of the major salivary glands. There is minimal risk of contracting infections during saliva collection, and saliva can be used in clinically challenging situations, such as obtaining samples from children or handicapped or anxious patients, in whom blood sampling could be a difficult act to perform. In this review we highlight the production of and secretion of saliva, the salivary proteome, transportation of biomolecules from blood capillaries to salivary glands, and the diagnostic potential of saliva for use in detection of cardiovascular disease and oral and breast cancers. We also highlight the barriers to application of saliva testing and its advancement in clinical settings. SUMMARY Saliva has the potential to become a first-line diagnostic sample of choice owing to the advancements in detection technologies coupled with combinations of biomolecules with clinical relevance.

In all, 350,000 new cases of oral cancer are reported annually worldwide, 35,000 of these occur in the United States. For decades, the 5-year survival rate has remained low at only 60%, which is mainly due to cancer diagnosis at late and progressed stage. Using saliva as a diagnostic medium could be the key for early detection and thus improved survival rates. Among all salivary constituents, the transcriptome has turned out to be a highly promising biomarker source. So far, seven mRNA and two microRNA markers were found to be discriminatory in saliva of oral cancer patients. This review will give an overview on the field of salivary transcriptome research with focus on oral cancer detection as well as the translation of salivary diagnostics into clinical reality.

Background: A sensitive assay to identify biomarkers using non-invasively collected clinical specimens is ideal for breast cancer detection. While there are other studies showing disease biomarkers in saliva for breast cancer, our study tests the hypothesis that there are breast cancer discriminatory biomarkers in saliva using de novo discovery and validation approaches. This is the first study of this kind and no other study has engaged a de novo biomarker discovery approach in saliva for breast cancer detection. In this study, a case-control discovery and independent preclinical validations were conducted to evaluate the performance and translational utilities of salivary transcriptomic and proteomic biomarkers for breast cancer detection. Methodology/Principal Findings: Salivary transcriptomes and proteomes of 10 breast cancer patients and 10 matched controls were profiled using Affymetrix HG-U133-Plus-2.0 Array and two-dimensional difference gel electrophoresis (2DDIGE), respectively. Preclinical validations were performed to evaluate the discovered biomarkers in an independent sample cohort of 30 breast cancer patients and 63 controls using RT-qPCR (transcriptomic biomarkers) and quantitative protein immunoblot (proteomic biomarkers). Transcriptomic and proteomic profiling revealed significant variations in salivary molecular biomarkers between breast cancer patients and matched controls. Eight mRNA biomarkers and one protein biomarker, which were not affected by the confounding factors, were pre-validated, yielding an accuracy of 92% (83% sensitive, 97% specific) on the preclinical validation sample set. Conclusions: Our findings support that transcriptomic and proteomic signatures in saliva can serve as biomarkers for the non-invasive detection of breast cancer. The salivary biomarkers possess discriminatory power for the detection of breast cancer, with high specificity and sensitivity, which paves the way for prediction model validation study followed by pivotal clinical validation.

Recent developments in biosensor technology allow point-of-use reporting of salivary alpha amylase (sAA) levels while approaching the precision and accuracy of conventional laboratory-based testing. We deployed a portable prototype sAA biosensor in 54 healthy, male dental students during a low stress baseline and during final exams. At baseline, participants completed the Brief Symptom Inventory (BSI). At baseline and the exam week, participants provided saliva samples at 10 AM, 1 PM, and 5 PM, and rated concurrent subjective distress. Although subjective distress was higher during exams compared to baseline, sAA levels did not differ between baseline and exams. Higher sAA levels were related to higher concurrent subjective distress, and higher depressive and social isolation symptoms on the BSI were related to lower sAA during exams. Results from this study, in combination with previous validation data, suggest that the sAA biosensor is a promising tool for point-of-use measures of exposure to stress.

Salivary diagnostics is an emerging field that has progressed through several important developments in the past decade, including the publication of the human salivary proteome and the infusion of federal funds to integrate nanotechnologies and microfluidic engineering concepts into developing compact point-of-care devices for rapid analysis of this secretion. In this article, we discuss some of these developments and their relevance to the prognosis, diagnosis and management of periodontitis, as an oral target, and cardiovascular disease, as a systemic example for the potential of these biodiagnostics. Our findings suggest that several biomarkers are associated with distinct biological stages of these diseases and demonstrate promise as practical biomarkers in identifying and managing periodontal disease, and acute myocardial infarction. The majority of these studies have progressed through biomarker discovery, with the identified molecules requiring more robust clinical studies to enable substantive validation for disease diagnosis. It is predicted that with continued advances in this field the use of a combination of biomarkers in multiplex panels is likely to yield accurate screening tools for these diagnoses in the near future.

Measurement of steroid levels in saliva has been proposed as a new laboratory tool for characterizing steroid metabolism, but it is not known whether the salivary levels of bile acids can be measured with accuracy and if so, whether such measurements provide information that is of clinical value. We developed and validated a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the quantification of chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA), representative primary non-amidated and glycine-conjugated bile acids, in whole saliva. We also examined whether the salivary bile acid concentrations were dependent on the saliva flow rate, because this is a very important aspect in a discussion of the utility of salivary diagnostics. Saliva was deproteinized with ethanol and purified using a Strata-X cartridge. Bile acids were converted to their hydrazide derivatives using 2-hydrazinopyridine, and subjected to LC-MS/MS. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated CDCA and GCDCA were used as internal standards. This method allowed the reproducible and accurate quantification of the salivary bile acids using a 200-microl sample and the limits of quantification for CDCA and GCDCA were 25 and 50pg/ml, respectively. Using this method, the effect of increased saliva flow rate by gum-chewing on the salivary concentrations of CDCA and GCDCA was determined. The salivary level of GCDCA was significantly decreased by gum-chewing, whereas the concentration of CDCA remained constant. These results indicate that there is a good possibility that saliva may be a clinical tool for non-amidated bile acid testing.

BACKGROUND & AIMS: Lack of detection technology for early pancreatic cancer invariably leads to a typical clinical presentation of incurable disease at initial diagnosis. New strategies and biomarkers for early detection are sorely needed. In this study, we have conducted a prospective sample collection and retrospective blinded validation to evaluate the performance and translational utilities of salivary transcriptomic biomarkers for the noninvasive detection of resectable pancreatic cancer. METHODS: The Affymetrix HG U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA) was used to profile transcriptomes and discover altered gene expression in saliva supernatant. Biomarkers discovered from the microarray study were subjected to clinical validation using an independent sample set of 30 pancreatic cancer patients, 30 chronic pancreatitis patients, and 30 healthy controls. RESULTS: Twelve messenger RNA biomarkers were discovered and validated. The logistic regression model with the combination of 4 messenger RNA biomarkers (KRAS, MBD3L2, ACRV1, and DPM1) could differentiate pancreatic cancer patients from noncancer subjects (chronic pancreatitis and healthy control), yielding a receiver operating characteristic plot, area under the curve value of 0.971 with 90.0% sensitivity and 95.0% specificity. CONCLUSIONS: The salivary biomarkers possess discriminatory power for the detection of resectable pancreatic cancer, with high specificity and sensitivity. This report provides the proof of concept of salivary biomarkers for the noninvasive detection of a systemic cancer and paves the way for prediction model validation study followed by pivotal clinical validation.

Objective: The objective of this study is to determine if LC/MS/MS based isotopic tagging technique is useful to detect putative breast cancer markers in saliva. Methods: Six pooled (n=10 subjects per pool) stimulated whole saliva specimens from women were analyzed. One pooled specimen was from healthy women, another pooled specimen from women diagnosed with a benign breast tumor and the other four-pooled specimens were from women diagnosed with carcinoma of the breast. The four cancer specimens were staged 0, I, IIa and IIb (lymph node involvement). Isotopically tagging proteins in the tumor groups and comparing them to the healthy control group measured differential expression of proteins. The iTRAQ labels are isobaric and chemically identical, thus labeled peptides from the different pools have identical elution times and masses. The relative concentration of identified proteins in each of the mixtures is determined from the intensities of each of the reporter ions. Results: The results of the salivary analyses yielded approximately 209 proteins in the saliva specimens. These proteins were able to distinguish between the healthy control, the benign and the cancer patients. Additionally, there were proteins able to distinguish between node positive and node negative patients.

The integration of semiconductor nanoparticle quantum dots (QDs) into a modular, microfluidic biosensor for the multiplexed quantitation of three important cancer markers, carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), and Her-2/Neu (C-erbB-2) was achieved. The functionality of the integrated sample processing, analyte capture and detection modalities was demonstrated using both serum and whole saliva specimens. Here, nano-bio-chips that employed a fluorescence transduction signal with QD-labeled detecting antibody were used in combination with antigen capture by a microporous agarose bead array supported within a microfluidics ensemble so as to complete the sandwich-type immunoassay. The utilization of QD probes in this miniaturized biosensor format resulted in signal amplification 30 times relative to that of standard molecular fluorophores as well as affording a reduction in observed limits of detection by nearly 2 orders of magnitude (0.02 ng/mL CEA; 0.11 pM CEA) relative to enzyme-linked immunosorbent assay (ELISA). Assay validation studies indicate that measurements by the nano-bio-chip system correlate to standard methods at R(2)=0.94 and R(2)=0.95 for saliva and serum, respectively. This integrated nano-bio-chip assay system, in tandem with next-generation fluorophores, promises to be a sensitive, multiplexed tool for important diagnostic and prognostic applications.

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

The use of saliva as a diagnostic fluid for various human ailments is gaining popularity as it offers distinct advantages over serum. These include the non-invasive nature of saliva collection compared with phlebotomy, simplicity of collection even for individuals with a modest training and the cost-effective applicability for screening large populations. Whole saliva is most frequently used for diagnosis of systemic diseases since it is readily collected and contains serum constituents while gland-specific saliva is useful for investigating pathology of major salivary glands. Broadly, saliva analysis is currently used for the diagnosis of infectious and malignant diseases, hereditary disorders, autoimmune diseases, and endocrine disorders, as well as for the assessment of therapeutic drug levels, particularly in monitoring drug abuse. This review addresses the current status of salivary diagnostics and their future potential.

Salivary secretions contain a variety of molecules that reflect important pathophysiological activities. Quantitative changes of specific salivary biomarkers could have significance in the diagnosis and management of both oral and systemic diseases. Modern point-of-care technologies with enhanced detection capabilities are needed to implement a significant advancement in salivary diagnostics. One such promising technology is the recently described lab-on-a-chip (LOC) assay system, in which assays are performed on chemically sensitized beads populated into etched silicon wafers with embedded fluid handling and optical detection capabilities. Using this LOC system, complex assays can be performed with small sample volumes, short analysis times, and markedly reduced reagent costs. This report describes the use of LOC methodologies to assess the levels of interleukin-1beta (IL-1beta), C-reactive protein (CRP), and matrix metalloproteinase-8 (MMP-8) in whole saliva, and the potential use of these biomarkers for diagnosing and categorizing the severity and extent of periodontitis. This study demonstrates that the results achieved by the LOC approach are in agreement with those acquired with standard enzyme-linked immunosorbent assay (ELISA), with significant IL-1beta and MMP-8 elevations in whole saliva of periodontitis patients. Furthermore, because of the superior detection capacities associated with the LOC approach, unlike those with ELISA, significant differences in CRP levels between periodontitis patients and normal subjects are observed. Finally, principal component analysis (PCA) is performed to yield an efficient method to discriminate between periodontally healthy and unhealthy patients, thus increasing the diagnostic value of these biomarkers for periodontitis when examined with the integrated LOC sensor system.

There have been increasing numbers of applications using oral fluids, saliva in particular, as the target substrate for performing clinical diagnostic tests. These have focused primarily on point-of-care (POC) testing. These POC testing approaches range from, for example, currently available, highly specialized screening tests for the presence of antibodies recognizing HIV to the potential development of "lab-on-a-chip" platforms. Broad claims have been made that the latter will revolutionize clinical laboratory testing. From the perspective of large centralized clinical laboratories, multiple issues must be considered before implementing individual tests using saliva as the target fluid in a POC format or using saliva as a universal test fluid for measuring multiple analytes in a centralized laboratory format. The current scope of laboratory testing is large and comprehensive, involving both POC and centralized testing. Current academic laboratory programs have the ability to qualitatively identify and/or quantitatively measure several thousand analytes in various target matrices including blood, plasma, serum, urine, joint fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, and tissue. These tests fall into multiple clinical pathology disciplines, including clinical chemistry, hematology, coagulation, transfusion medicine, microbiology, cytogenetics, molecular diagnosis, and immunology. In addition, before implementing a given test, multiple issues need to be evaluated to ensure the validity of the reported result; these include considerations involving the three major phases of testing: preanalytical (e.g., patient identification and specimen collection, stability, and transport), analytical (e.g., sensitivity, specificity, accuracy, and precision), and postanalytical (e.g., reporting results, quality improvement, and turn-around-time).

High-impact diseases, including cancer, cardiovascular disease, and neurological disease, are challenging to diagnose without supplementing clinical evaluation with laboratory testing. Even with laboratory tools, definitive diagnosis often remains elusive. The lack of three crucial elements presents a road block to achieving the potential of clinical diagnostic tests: (1) definitive disease-associated protein and genetic markers, (2) easy and inexpensive sampling methods with minimal discomfort for the subject, and (3) an accurate and quantitative diagnostic platform. Our aim is to develop and validate a solution for requirement (3) and also to develop a portable system. Requirements (1) and (2) will be addressed through the utilization of novel and highly specific oral cancer saliva proteomic and genomic biomarkers and the use of saliva as the biofluid of choice, respectively. The Oral Fluid NanoSensor Test (OFNASET) technology platform combines cutting-edge technologies, such as self-assembled monolayers (SAM), bionanotechnology, cyclic enzymatic amplification, and microfluidics, with several well-established techniques including microinjection molding, hybridization-based detection, and molecular purification. The intended use of the OFNASET is for the point of care multiplex detection of salivary biomarkers for oral cancer. We have demonstrated that the combination of two salivary proteomic biomarkers (thioredoxin and IL-8) and four salivary mRNA biomarkers (SAT, ODZ, IL-8, and IL-1b) can detect oral cancer with high specificity and sensitivity. Our preliminary studies have shown compelling results. We sequentially delivered a serial dilution of IL-8 antigen, probe solution, wash, enzyme solution, wash, and mediator solution to sensor reaction chambers housed in a prototype cartridge and demonstrated strong signal separation at 50 pg/mL above a negative control.

Saliva is an underused fluid with considerable promise for biomedical testing. Its potential is particularly great for monitoring small-molecule analytes since these are often present in saliva at concentrations that correlate well with their free levels in blood. We describe the development of a prototype diagnostic device for the rapid detection of the antiepileptic drug (AED) phenytoin in saliva. The multicomponent system includes a hand-portable surface plasmon resonance (SPR) imaging instrument and a disposable microfluidic assay card.

A multiplexed fiber optic microsphere-based immunoassay array capable of simultaneously measuring five inflammatory cytokines has been developed. Five groups of amine-functionalized 3.1 micron microspheres were internally encoded with five distinct concentrations of a europium dye and converted to cytokine probes by covalently coupling monoclonal capture antibodies specific for human VEGF, IFN-gamma, RANTES, IP-10, and Eotaxin-3 to the microspheres via glutaraldehyde chemistry. The microspheres were pooled and loaded into a 1 mm diameter fiber optic bundle containing ~50,000 individual etched microwells, producing the multiplexed cytokine immunoassay array. Multiple arrays can be created from a single microsphere pool for high throughput sample analysis. Sandwich fluoroimmunoassays were performed by incubating the probe array in a sample, followed by incubation in a mixture of biotin-labeled detection antibodies that are complementary to the five cytokines. Finally, universal detection of each protein was performed using a fluorescence imaging system after briefly immersing the array in a solution of fluorophore-labeled streptavidin. The multiplexed cytokine array has been shown to respond selectively to VEGF, IFNgamma, RANTES, IP-10, and Eotaxin-3, permitting multiplexed quantitative analysis. Ultimately, the multiplexed cytokine array will be utilized to evaluate the potential of using saliva as a noninvasive diagnostic fluid for pulmonary inflammatory diseases such as asthma.

Background The ability to monitor health status, disease onset and progression, and treatment outcome through noninvasive means is a highly desirable goal in health care promotion and delivery. Oral fluid is a perfect medium to be explored for health and disease surveillance. Methods Two prerequisites exist before the goal of salivary diagnostics can be achieved: identification of specific biomarkers associated with a health or disease state and the development of technologies that can discriminate between the biomarkers. A recent initiative of the National Institute of Dental and Craniofacial Research has created a roadmap to achieve these goals through the use of oral fluids as the diagnostic medium to scrutinize the health and/or disease status of patients. This is an ideal opportunity to optimize state-of-the-art saliva-based biosensors for salivary biomarkers that discriminate between diseases. Results Seven technology groups are developing point-of-care salivary diagnostic technologies. Three groups are working together toward deciphering the salivary proteome. Conclusion These collective efforts and the convergence of salivary diagnostic technologies and the salivary proteome will present unparalleled opportunities to explore the diagnostic potential of saliva for oral and systemic diseases.

Purpose: Oral fluid (saliva) meets the demand for noninvasive, accessible, and highly efficient diagnostic medium. Recent discovery that a large panel of human RNA can be reliably detected in saliva gives rise to a novel clinical approach, salivary transcriptome diagnostics. The purpose of this study is to evaluate the diagnostic value of this new approach by using oral squamous cell carcinoma (OSCC) as the proof-of-principle disease. Experimental Design: Unstimulated saliva was collected from patients (n = 32) with primary T1/T2 OSCC and normal subjects (n = 32) with matched age, gender, and smoking history. RNA isolation was done from the saliva supernatant, followed by two-round linear amplification with T7 RNA polymerase. Human Genome U133A microarrays were applied for profiling human salivary transcriptome. The different gene expression patterns were analyzed by combining a t test comparison and a fold-change analysis on 10 matched cancer patients and controls. Quantitative polymerase chain reaction (qPCR) was used to validate the selected genes that showed significant difference (P < 0.01) by microarray. The predictive power of these salivary mRNA biomarkers was analyzed by receiver operating characteristic curve and classification models. Results: Microarray analysis showed there are 1,679 genes exhibited significantly different expression level in saliva between cancer patients and controls (P < 0.05). Seven cancer-related mRNA biomarkers that exhibited at least a 3.5-fold elevation in OSCC saliva (P < 0.01) were consistently validated by qPCR on saliva samples from OSCC patients (n = 32) and controls (n = 32). These potential salivary RNA biomarkers are transcripts of IL8, IL1B, DUSP1, HA3, OAZ1, S100P, and SAT. The combinations of these biomarkers yielded sensitivity (91%) and specificity (91%) in distinguishing OSCC from the controls. Conclusions: The utility of salivary transcriptome diagnostics is successfully demonstrated in this study for oral cancer detection. This novel clinical approach could be exploited to a robust, high-throughput, and reproducible tool for early cancer detection. Salivary transcriptome profiling can be applied to evaluate its usefulness for other major disease applications as well as for normal health surveillance.

Since morbidity and mortality rates due to oral cavity and oropharyngeal squamous cell carcinoma (OSCC) have improved little in the past 30 years, early detection or prevention of this disease is likely to be most effective. Using laser-capture microdissection, we have identified the expression of 2 cellular genes that are uniquely associated with OSCC: interleukin (IL) 6 and IL-8. These cytokines may contribute to the pathogenesis of this disease, and have been linked with increased tumor growth and metastasis.To investigate whether IL-6 and/or IL-8 could serve as informative biomarkers for OSCC in saliva and/or serum and to determine if there is a role for saliva as a diagnostic medium for OSCC.Patients with newly diagnosed T1 or T2 oral cavity or oropharyngeal histologically confirmed squamous cell carcinoma were recruited for the study. Age and sex-matched disease-free subjects were used as controls. Using quantitative real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay, we respectively assessed the expression of IL-6 and IL-8 in serum (controls, n = 32; patients with OSCC, n = 19) and saliva (controls, n = 32; patients with OSCC, n = 32) at the messenger RNA (mRNA) and protein levels.Specificity and sensitivity of these biomarkers for OSCC and their predictive value.Interleukin 8 was detected at higher concentrations in saliva (P.01) and IL-6 was detected at higher concentrations in serum of patients with OSCC (P.01). We confirmed these results at both the mRNA and the protein levels, and the results were concordant. The concentration of IL-8 in saliva and IL-6 in serum did not appear to be associated with sex, age, or alcohol or tobacco use (P.75). Using statistical analysis, we were able to determine the threshold value, sensitivity, and specificity of each biomarker, as well as a combination of biomarkers, for detecting OSCC.Our findings indicate that IL-8 in saliva and IL-6 in serum hold promise as biomarkers for OSCC. A saliva-based test could be a cost-effective adjunctive tool in the diagnosis and follow-up of patients with OSCC.

Saliva, like other bodily fluids, has been used to monitor human health and disease. This study tests the hypothesis that informative human mRNA exists in cell-free saliva. If present, salivary mRNA may provide potential biomarkers to identify populations and patients at high risk for oral and systemic diseases. Unstimulated saliva was collected from ten normal subjects. RNA was isolated from the cell-free saliva supernatant and linearly amplified. High-density oligonucleotide microarrays were used to profile salivary mRNA. The results demonstrated that there are thousands of human mRNAs in cell-free saliva. Quantitative PCR (Q-PCR) analysis confirmed the present of mRNA identified by our microarray study. A reference database was generated based on the mRNA profiles in normal saliva. Our finding proposes a novel clinical approach to salivary diagnostics, Salivary Transcriptome Diagnostics (STD), for potential applications in disease diagnostics as well as normal health surveillance.

Human saliva mirrors body’s health and well-being and many of the biomolecules present in blood or urine can also be found in salivary secretions. However, biomolecular concentrations in saliva are usually one tenth to one thousandth of the levels in blood (Pfaffe et al., 2011). Sensitive detection technology platforms are therefore required to detect biomolecules in saliva. Another road block to the advancement of salivary diagnostics is the lack of information related to healthy state saliva vs. a diseased saliva, baseline levels and reference ranges and diurnal variations. Saliva has numerous advantages over blood or urine as a diagnostic fluid: (a) the non-invasive nature of sample collection and the simple, safe, painless and cost-effective methods to collect it; (b) unskilled personnel can collect saliva samples at multiple time points; and (c) the total protein concentration is approximately a quarter of that is present in plasma, which makes it easier to investigate low abundance proteins (Pfaffe et al., 2011). Currently, saliva assays are routinely used to determine, diseases such as HIV, drugs and substances of abuse to provide information on exposure and give qualitative information on the type of illicit drug used (Kintz et al., 2009), cortisol levels for diagnosing Cushing’s syndrome (Doi et al., 2008), and use for biomonitoring of exposure to chemicals (Caporossi et al., 2010) to measure hormones (Groschl, 2009)....