Biochemistry Review - A New Approach for Diagnosis of Systemic and Oral Diseases Based on Salivary Biomolecules (PDF)

Hindawi Disease Markers Volume 2019, Article ID 8761860, 11 pages https://doi.org/10.1155/2019/8761860 Review Article A New Approach for the Diagnosis of Systemic and Oral Diseases Based on Salivary Biomolecules Alexandra Roi ,1 Laura C. Rusu ,1 Ciprian I. Roi,2 Ruxandra E. Luca,3 Simina...

Hindawi Disease Markers Volume 2019, Article ID 8761860, 11 pages https://doi.org/10.1155/2019/8761860 Review Article A New Approach for the Diagnosis of Systemic and Oral Diseases Based on Salivary Biomolecules Alexandra Roi ,1 Laura C. Rusu ,1 Ciprian I. Roi,2 Ruxandra E. Luca,3 Simina Boia,4 and Roxana I. Munteanu3 1 Department of Oral Pathology, “Victor Babes” University of Medicine and Pharmacy, Timisoara, 2 Eftimie Murgu Sq., 300041, Romania 2 Department of Anaesthesiology and Oral Surgery, “Victor Babeș” University of Medicine and Pharmacy, Timisoara, 2 Eftimie Murgu Sq., 300041, Romania 3 Department of Oral Rehabilitation and Dental Emergencies, “Victor Babeș” University of Medicine and Pharmacy, Timisoara, 2 Eftimie Murgu Sq., 300041, Romania 4 Department of Periodontology, “Victor Babeș” University of Medicine and Pharmacy, Timisoara, 2 Eftimie Murgu Sq., 300041, Romania Correspondence should be addressed to Laura C. Rusu; [email protected] Guest Editor: Napoleon Waszkiewicz Copyright © 2019 Alexandra Roi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Early diagnosis represents the target of contemporary medicine and has an important role in the prognosis and further treatment. Saliva is a biofluid that generated a high interest among researchers due to its multiple advantages over other body fluids. The multitude of components that can act as biomarkers influenced the existing technologies to develop protocols that could allow saliva to become the new noninvasive diagnostic method. Saliva as a diagnostic tool can bring substantial addition to the diagnostic armamentarium, providing important information about oral and general health. The diagnostic applications of saliva extended and had a rapid evolution due to the advancement in salivaomics. The present review summarizes the latest researches in saliva-related studies and explores the information and correlations that saliva can offer regarding the systemic and oral diseases, highlighting its great potential of diagnosis. It is expected that in the future specific guidelines and results regarding the salivary diagnostics are to be available, together with high-sensitivity and specificity tests for multiple systemic and oral diseases. 1. Introduction human saliva, holding the key to an early diagnosis, a better treatment, and an improved prognosis. The early detec- Body fluids provide a wide perspective regarding the biolog- tion of the diseases is often a difficult task and implies more ical processes and the health of different organs. The human clinical and laboratory investigations that can delay the treat- body is composed of a variety of fluids, such as blood, urine, ment and highly influence the prognosis. and saliva, with a high quantity of proteins that can be asso- Systemic diseases are very challenging to diagnose with- ciated with several systemic and oral diseases. These fluids out more invasive supplementary investigations. In order to proved to have found widespread clinical applications in overcome this condition, medical researchers worked into order to diagnose and monitor human health. The high finding molecular disease biomarkers that can be easily iden- global impact of a large number of diseases including cancer tified and where they can successfully implement a noninva- and cardiovascular, metabolic, and neurological diseases sive and fast diagnosis. During this path of research, three challenged the clinicians to provide and improve the diagno- main limitations have influenced until recent the late devel- sis procedures and clinical evaluation of these patients. One opment and research of specific biomarkers for early disease of the most appealing diagnostic tools is thought to be the detection: (1) the lack of definitive molecular biomarkers for 2 Disease Markers specific diseases, (2) the lack of an easy and inexpensive constituents, the mechanism of using saliva as a diagnostic sampling method with minimal discomfort, and (3) the lack tool, and the clinical applications that can influence an of an accurate and easy-to-use platform that can facilitate early diagnosis. the early detection. Until now, it can be considered that lim- itations 1 and 3 have found solutions with the help of sali- 2. Biomarkers Era: An Evolution vary biomarkers and an ongoing development of salivary diagnosis. The definition of a biomarker refers to a pharmacological or Salivary diagnosis is viewed as a promising modality that physiological measurement that can be used to predict a can provide an early and accurate diagnosis, an improved toxic event, in this specific case a molecule that contains par- prognosis, and a good monitoring post-therapy. The whole ticular material that can be used in order to diagnose a dis- saliva is composed of the secretions of the minor and major ease or measure the progression and treatment outcome. salivary glands as well as mucosal transudations, gingival The characteristics of biomarkers make them proper for an crevicular fluid, serum and some blood derivatives, desqua- alternative diagnostic tool, with or without the help of other mated epithelial cells, bacteria, viruses, fungi, and food methods. debris. Saliva is a complex fluid that also contains a high The development of mass spectrometric technologies led number of hormones, proteins, enzymes, antibodies, cyto- medicine to a new era in biomarker discovery that will have kines, and antimicrobial constituents that can facilitate their an important impact on future disease diagnosis and therapy. associations with a variety of systemic diseases. The assay More studies in salivary proteins showed the fact that saliva of saliva represents a wide area of research at this time and contains actually hundreds of minor proteins or peptides that has implications that target basic and clinical purposes. The although are present in variable concentrations can have a indications suggest that saliva can be used as an investigative significant role in the diagnosis of diseases; these proteins tool for disease processes and disorders, and after a careful can receive the role of biomarkers in relation to specific con- analysis, it can provide multiple information about the func- ditions. Although proteomes play an important role in the tioning of the organs within the human body. diagnosis, the salivary transcriptomic technology succeeded The past research within the last 10 years proves the to improve the diagnostic potential of saliva for multiple fact that saliva as a diagnostic tool has gained a lot of atten- medical applications. tion and has become a translational research method. Saliva Proteomic technology helped to discover the salivary bio- has the potential to become a first-line diagnostic tool with markers by outlining the importance of the proteome and the the help of the advancement made in early detection and analysis of the expressed proteomics. The existence of the the development of biomolecules that have clinical impor- proteomes in the body fluids represents a high potential of tance. Salivary diagnostics has received attention due disease markers. An accurate analysis of the human saliva to its connections to various high-impact systemic diseases proteome can be related to the general health status. Many and physiological conditions that were shown to have an functional alterations of proteins result from posttransla- influence in the composition of saliva. Serious investments tional modifications such as phosphorylation, glycosylation, were made, motivating scientists, governments, and indus- acetylation, and methylation. These kinds of alterations try to direct resources in the saliva diagnostics. A good and modified proteins can be specific in some diseases such method for salivary diagnostics should have general func- as autism spectrum disorder and cervical cancer. tionality, high sensitivity and specificity, low cost, and effi- The transcriptomic technology allowed researchers to cient clinical application. Regarding saliva, many of these discover the salivary transcriptomes (RNA molecules) that requirements have been accomplished with the implication include the molecules the cells use to transport information of several fields such as chemistry, physics, biology, and provided by the DNA for protein production. This opportu- engineering, in order to develop an accurate and efficient nity provides medical research with a second diagnostic tool test. that involves saliva and that can provide more opportunities Saliva has several advantages over serum and tissue frag- for salivary diagnostics. ments in its use as a diagnostic tool. One of the most appeal- ing characteristics is the noninvasive approach that, 3. Salivary Biomarkers: Generalities combined with the easy collection method and storage, makes it a valuable tool. New technologies have proven their The most important and revealing components of the saliva efficacy and unveiled a large number of salivary biomarkers are the proteins. Human saliva has a specific proteomic that are connected to several general and oral diseases. content that allows researchers to perform assays in order The aim of this review is to emphasize the role and to discover novel saliva biomolecules associated with general importance of saliva as a diagnostic tool for the diagnosis health status. Proteomic studies of saliva help with the iden- of systemic and oral diseases. The use of this method tification of new proteins and peptides that can help quantify brings to light an efficient and easy approach that can the biological activity in pathological states. improve considerably the diagnosis, prognosis, treatment, The Saliva Proteome Knowledge Base (http://www.skb. and post-therapy monitoring. Various components in this ucla.edu) is the first database that contains all the proteomic fluid can act as biomarkers for multiple diseases providing data being accessible to the public. The techniques used by valuable information regarding the health status. The focus researches and biochemists in order to perform the prote- is on providing information about the important salivary ome work from saliva are gel electrophoresis, capillary Disease Markers 3 electrophoresis, nuclear magnetic resonance, MS, immuno- viruses, and gastrointestinal reflux liquid) [15, 16]. To the assay, and LC. Due to the great development, researchers total composition, there is also a contribution from the have proposed the term salivaomics. This specific term crevicular fluid (a fluid that derivates from the epitheliul of gathers all the technologies used for analyzing potentially sal- the gingival crevice) that is produced at approximately ivary biomarkers: proteomics, genomics, transcriptomics, 2-3 μl/h per tooth and it can be considered as a plasma tran- microRNA (miRNA), and metabonomics. The value of sudate. The oral fluid also can contain food debris and salivary biomarkers has long been overcome until recent blood-derivated compounds such as plasmatic proteins, research on upgraded saliva from the position of being use- erythrocytes, and leucocytes in case there is inflammation less to the one of being a high-sensitivity diagnostic method. present. The composition of saliva based on its constitu- Research proved the high potential of the salivary biomarkers ents is inorganic, organic nonprotein, protein/polypeptide, and their diagnostic capability, promoting it with uncontest- hormone, and lipid molecules [17, 18] (Table 1). able advantages over other body fluids. The number of total protein increases in the salivary secretion through β-sympathetic activity in the salivary 4. Particularities of Saliva: Composition, glands, since saliva secretion is mainly evoked by the action Functions, and Production of adrenergic mediators. Saliva contains a large number of protein compounds, and their structure and function have Saliva is a unique fluid that contributed to the development been studied with biochemical techniques, including liquid of a new diagnostic tool in the past few years. The research chromatography, gel electrophoresis, capillary electrophore- has shown that a wide spectrum of hormones, nucleic acids, sis (CE), nuclear magnetic resonance, mass spectrometry, electrolytes, and proteins/peptides can be related to multiple immunoassays (RIA, IRMA, EIA, and ELISA) and lectin local and systemic diseases. It is said that saliva reflects the probe analysis [10, 20] (Table 2). Along with time, with the “body’s health” and well-being, but until recently its use as help of proteomic techniques, complete patterns of all the a diagnostic tool has been hindered because the examination salivary proteins were accomplished. of the biomolecules that exist in saliva and their relevance Researchers that focused on the study of human saliva and association with different etiologies has been not enough have characterized 4 major types of salivary proteins: explored. Used for the diagnosis of systemic diseases, PRPs, cystatins, statherins, and histatins. The important saliva is an important advantage, primarily because saliva role of this type of proteins is maintaining the integrity contains a small amount of plasma. Plasma-derived bio- of tooth structures in the oral cavity, especially involved markers in saliva facilitate the continuous monitoring of in the demineralization and remineralization process of the oral and general health status. the enamel. The salivary fluid is an exocrine secretion that consists In the oral fluid, hormones that are especially detected in of approximately 99% water, with a variety of electrolytes plasma can also be present. Although certain correlations (sodium, potassium, calcium, magnesium, and phosphate), have been made, further studies are necessary in order to proteins such as enzymes, immunoglobulins, antimicrobial prove the connection of salivary hormone level with the factors, albumin, polypeptides and oligopeptides, traces of plasma ones so it can be a trustful association with patholog- albumin, and mucosal glycoproteins of great importance ical and physiological states. At the present time, there is still in maintaining a balance of the oral health. Saliva also few information regarding the association of salivary hor- contains glucose, urea, and ammonia in various quantities mones and different pathologies, but until now steroid detec- that can interact and be responsible for several general tion is a promising application in salivary hormonal studies. diseases. The most commonly assayed salivary biomarkers are corti- The oral fluid originates preponderantly from three pairs sol, testosterone, progesterone, aldosterone, and hydroxypro- of major salivary glands (parotid, sublingual, and subman- gesterone. Salivary cortisol measurement is nowadays an dibular) and from numerous minor salivary glands. Parotid accepted alternative, proved by the fact that both serum and glands are serous glands, and their secretion lacks mucin; salivary levels are equivalent. There were also important the submandibular and sublingual glands are mixed ones, advancements made, proving the existence of growth hor- with ser-mucous secretion. Minor salivary glands that are mone, prolactine, and insulin-like growth factor I with simi- situated in the connective tissue below the circumvallate lar levels to those found in serum directing the research to papillae are Von Ebner glands, and the mucous ones are exploiting new fields of interest. Blandin-Nühm glands. The salivary composition varies and depends on the type 5. Saliva as a Diagnostic Tool: Introduction into of the gland, mucous or serous ones. Its composition a New Perspective differs by the contribution of each gland in order to obtain the total of unstimulated saliva secretion, and the variations The use of saliva as a diagnostic fluid has gained attention in are from 65%, 23%, and 8% to 4% for the submandibular, the past few years, and researches have proved the high sen- parotid, Von Ebner, and sublingual glands. Components sitivity of this type of diagnosis regarding the detection and of saliva can have also a nonglandular origin; basically, the prediction of diseases. As a diagnostic fluid, saliva offers sev- oral fluid is considered to be a mixture of the production of eral advantages over serum: being a cost-effective approach, salivary glands and other fluids that originate from the oro- having real-time diagnostic values, having multiple samples pharingeal mucosa (oral mucosal transudate, fungi, bacteria, which can be obtained easily, requiring less manipulation 4 Disease Markers Table 1: Comparison of inorganic compounds between saliva and to identify diagnostic markers that can be successfully used plasma. in a clinic. Inorganic Whole Whole compounds unstimulated stimulated Plasma 6. Potentially Salivary Biomarkers for Oral and (mmol/l) saliva saliva Systemic Diseases Na+ 5 20- 80 145 For many years now, researchers investigated the importance K+ 22 20 4 of the changes that occurred in the saliva, changes that affect Cl− 15 30–100 120 the flow rate and composition. The changes in the fluid are Ca2+ 1–4 1–4 2.2 valuable regarding the diagnosis of oral and systemic diseases HCO3 − 5 15–80 25. At first, the examination of saliva was used in order to Mg2+ 0.2 0.2 1.2 identify the local gland diseases, such as inflammatory and NH3 6 3 0.05 autoimmune diseases , but later on the researchers expanded their work, highlighting the potential for diagnos- ing multiple general diseases. during the diagnostic procedure, and having a noninvasive 6.1. Periodontal Disease. Regarding the periodontal patho- collection method with a minimal risk of infections and genic processes, periodontitis can be classified based on addressing to all categories of patients, especially those to the three phases of evolution: inflammation, connective tis- whom blood sampling could be a challenge (children, anx- sue degradation, and bone turnover. There are, associated ious or uncooperative patients). In this review, we would with each phase of the periodontal disease, different salivary like to outline the diagnostic potential of saliva and its impli- biomarkers that can stage the evolution and the status of the cation in the detection of several diseases taking into consid- patient. At the beginning of the inflammatory phase, prosta- eration the high-quality DNA that this fluid possesses. glandin E2, interleukin-1, interleukin-6, and tumor necrosis Saliva is an important fluid, and interest in it has devel- factor-alpha are found in a high number, released from a oped due to the wide spectrum of proteins/peptides, electro- variety of cells. As the stages progress and the disease lytes, hormones, and nucleic acids that are in its composition becomes more advanced with severe bone loss, the levels and can provide important information about the body’s of tumor necrosis factor, interleukin-1, and RANKL are ele- health. The delay in the use of saliva as a diagnostic method vated and directly related to the degree of bone destruction was mainly because until recent there has been a lack of. The specific biomarkers for the bone, such as pyridi- understanding of the biomolecules that were found in the noline cross-linked carboxyterminal telopeptide of type I saliva. As a diagnostic tool, several disadvantages have been collagen, are being transported in the crevicular fluid into reported: the variations due to the diurnal/circadian rhythm, the periodontal pocket and finally become a component of the method of collection that can influence the salivary com- saliva [26, 27]. position, and the necessity of sensitive detection systems. An important cytokine with a proinflammatory role However, saliva is considered to have an enormous potential involved in the inflammation process associated with peri- of biomarkers that range from changes in biochemical, odontitis is interleukin-1. IL-1 can be the product of several DNA, RNA, and proteins to the oral environment. As a cells, as epithelial cells, monocytes, polymorphonuclear diagnostic tool, saliva can provide a new and noninvasive neutrophils, fibroblasts, endothelial cells, and osteoblasts perspective in order to obtain a diagnosis, and it can be [28, 29]. Interleukin-1 influences the production of prosta- expected in the future to become a substitute for serum glandin E2 and is involved in the regulation of metallopro- and urine tests. A part of the constituents enter the teinases and their inhibitors, and it induces the osteoclastic saliva through blood by passive/active transport or extracel- activity that sustains bone loss associated with periodontitis lular ultrafiltration. [28, 30, 31]. The entire activity of IL-1 is based on Clinical research has developed various protocols in interleukin-1alpha and interleukin-1beta (was proved to be order to assay saliva. At the time, saliva is most frequently elevated in association with periodontitis) [31–33]. Also, used as a diagnostic tool for systemic diseases and the future studies found increased salivary levels of IL-6 in patients relies in combinations of different biomarker panels that can diagnosed with periodontitis [34–36] and proved the fact be used for screening in order to improve the early diagnosis that it influences osteoclast differentiation and bone resorp- and the general outcome. The first choices in the analysis tion, being directly involved in tissue destruction [37, 38]. are the proteomic constituents, but genomic targets can be a Another key biomarker involved in periodontitis is valuable source of biomarkers also. Salivary diagnostics with mainly produced by macrophages and is represented by the help of biotechnologies made it possible for several bio- tumor necrosis factor-alpha. It is an important immune markers to be associated with multiple diseases such as can- mediator, and in relationship with this disease, it influences cer, autoimmune diseases, viral diseases, bacterial diseases, bone collagen synthesis and induces collagenases, similar to cardiovascular diseases, and HIV. The clinical need of a sim- IL-1 [28, 39]. Also involved in the periodontal disease, ple and easy diagnostic tool is sadly lacking although we are matrix metalloproteinase-9 is part of the process of peri- surrounded by multiple health risks and diseases. Saliva used odontal disease, especially immune response and tissue as a diagnostic is an important challenge based on the need degradation [40–42]. The elevated salivary levels of matrix Disease Markers 5 Table 2: Salivary proteins. Origin Functions Concentrations 0 47 ± 0 19 mg/ml 0 9 ± 0 2 mg/ml Total proteins 4 3 – 710 0 mg/dl 2 67 ± 0 54 mg/ml 3257 ± 1682 U/ml α-Amylase Starch digestion 1080 0 ± 135 6 IU/I 476 ± 191 μg/ml 0 2 ± 0 1 mg/ml Albumin Plasma Mainly from plasma leakage 0 8 – 192 0 mg/dl 14.3 kDa form 58 ± 25 μg/ml Cystatin group SM>SL Antimicrobial (cistein-proteinase inhibitor) 14.2 kDa form 91 ± 46 μg/ml Hystatin P Antifungal 1190 ± 313 μg/ml Secretory-IgA B lymphocytes Antimicrobial 124 3 – 335 3 μg/ml Lactoferrin Mucous>serous Antimicrobial 3 7 ± 2 5 μg/ml 3.5–92.0 μg/ml Lysozyme SL>SM, P Antimicrobial 21 8 ± 2 5 mg/dl 59.7–1062.3 μg/ml Acidic PRP: 456 ± 139 μg/ml PRPs P Binding to bacteria and with dietary tannins Basic PRP: 165 ± 69 μg/ml 4 93 ± 0 61 μmol/I Statherin Ca++ binding 36 ± 18 μg/ml Transferrin Plasma 0 58 ± 0 20 mg/dl metalloproteinase-9 prove that the characteristics of a bio- that can effectively estimate the number of neutrophils marker are being accomplished and associated with disease found in the saliva in order to certify the existence of peri- conditions, as low salivary levels are associated with a clin- odontal disease. ically normal condition [40, 43] (Table 3). A recent study outlined the existence of certain correla- 6.2. Sjögren’s Syndrome. Sjögren’s syndrome (SS) is an tions between salivary superoxide dismutase levels and the autoimmune chronic systemic disease that has important gingival index, pocket depth, and clinical attachment loss symptoms: xerostomia and keratoconjunctivitis. Patients found in patients that were diagnosed with chronic peri- diagnosed with SS have a decreased salivary flow rate and a odontitis. Saliva’s potential of diagnosis is seen as a nonin- modified composition of the saliva. It was shown the fact that vasive and easy way to diagnose patients with premalignant this syndrome is accompanied with significant changes in the conditions. Also, salivary macrophage inflammatory proteome and transcriptome, having also important alter- protein-1α, matrix metalloproteinase-8, interleukin- (IL-) ations in the levels of IL-4, IL-5, and cytokine clusters. 1β, IL-6, prostaglandin E2, and tumor necrosis factor- Another important research identified 19 genes (EPSTI1, (TNF-) α levels seem to be associated with gingivitis and IFI44, IFI44L, IFIT1, IFIT2, IFIT3, MX1, OAS1, SAMD9L, periodontitis, having a high potential to be used in their PSMB9, STAT1, HERC5, EV12B, CD53, SELL, HLA-DQA1, diagnosis. Based on another study, the salivary levels PTPRC, B2M, and TAP2) that were correlated with this syn- of uric acid, transaminase, procalcitonin, IL-8, and Toll-like drome and were responsible for the induction of interferons receptor-4 were higher in patients diagnosed with peri- and antigen presentation. The study of Hu et al. identi- odontitis than in the healthy control group, proving positive fied a panel of biomarkers that had high levels in patients correlations between the gingival index, pocket depth mea- with SS, including a number of three mRNA biomarkers surements, and clinical attachment loss (Table 4) [46, 47]. (guanylate-binding protein 2, myeloid cell nuclear differenti- More recently, a new oral rinse system has been developed ation antigen, and low-affinity IIIb receptor for the Fc 6 Disease Markers Table 3: Salivary biomarkers in periodontitis. Salivary biomarker Function References IL-1 Strong relation with periodontal disease; high inflammatory potential [25, 28–33, 45] Stimulates osteoclastic differentiation; increased levels in periodontal disease; IL-6 [34–38, 45] regulated the immune responses Tumor necrosis factor Influences the bone collagen synthesis [28, 39, 45] Matrix metalloproteinase-9 Mediator of the immune response and tissue destruction in periodontal disease [40–43] Table 4: Salivary biomarkers in oral cancer. The existence of gene mutations can often be associated Salivary biomarker Variation References and used as biomarkers in order to diagnose oral cancer. In saliva, the tumor-specific DNA was positive in 100% of the IL-8 High levels [53–56] patients diagnosed with oral cancer, and 47-70% of the Endothelin receptor type-B patients with tumors in other places of the body also carry High levels hypermethylation specific tumor DNA markers in the saliva. microRNAs (miR-200a, miR-125a, The p53 protein is responsible for tumor suppression, High levels [53, 54] and miR-31) and it is produced in cells as a response to multiple DNA S100 calcium-binding protein P High levels damages. The inactivation of p53 during a mutation is one IL-1beta High levels of the main causes of the development of malignancy. Studies Tissue polypeptide antigen (TPA) High levels have shown the fact that p53 antibodies were detected in the Cancer antigen CA125 High levels [57, 59] saliva of patients diagnosed with oral squamous cell carci- p53 antibodies High levels noma. CA 125 is a tumor-associated antigen that was found in high levels in the saliva of the patients with oral, H3 histone family 3A High levels breast, and ovarian cancer. Also, an important aspect Cyfra 21-1 High levels is the fact that salivary cortisol levels were found to be signif- Ornithine decarboxylase antizyme 1 High levels icantly high in the saliva of patients diagnosed with OSCC. This association suggests that this hormone can be used as a marker for clinical staging. fragment of IgG). These types of biomarkers from the It can be affirmed the fact that all the results prove that transcriptome and proteome can provide in the future a sim- saliva has an important charge of biomarkers that can be ple diagnostic tool for SS. used successfully in providing a screening and diagnosis of oral cancer. 6.3. Oral Cancer. Early diagnosis and treatment is the key to a good prognosis in almost all types of cancer. Saliva has 6.4. Cardiovascular Disease. Cardiovascular disease (CVD) been used in studies as a diagnostic tool for oral squamous includes atherosclerosis, coronary heart disease, and myocar- cell carcinoma (OSCC) based on the help of salivary analytes dial infarction. The studies performed by Kosaka et al. (proteins, mRNA, and DNA). Oral cancer is the sixth show that the salivary levels of IL-1β, IL-6, TNF-α, and most common cancer type worldwide, and 90% is repre- prostaglandin E2 are increased in atherosclerosis, suggesting sented by OSCC. The average 5-year survival rate is approx- that these cytokines could be potential biomarkers in the imately 60% , and usually the high mortality rate is diagnosis of atherosclerosis. Other studies concluded the associated with a late diagnosis. The solution for the future fact that other salivary markers can be C-reactive protein is to develop strategies to obtain an early diagnosis for (CRP), myoglobin (MYO), creatine kinase myocardial band OSCC. Until now, several biomarkers have been reported (CKMD), cardiac troponins (cTn), and myeloperoxidase. in association with OSCC, including IL-8, endothelin recep- Acute myocardial infarction was predicted by a correlation tor type B hypermethylation , and microRNAs (such as of an ECG with the CRP levels, proving 80% sensitivity miR-200a, miR-125a, and miR-31) [53–55]. Other previous and 100% specificity. In saliva, there were also salivary transcriptomic studies have discovered seven oral CK-MB and troponins identified, but their diagnostic squamous cell carcinoma-associated salivary RNAs (S100 potential was very low. Also, the levels of α-2-HS-gly- calcium-binding protein P, dual-specificity phosphatase 1, coprotein in saliva seem to decrease in patients diagnosed interleukin-8, interleukin-1beta, H3 histone family 3A, orni- with cardiovascular diseases, suggesting the fact that the thine decarboxylase antizyme 1, and spermine N1-acetyl- peptidome can contribute to the early diagnosis of these transferase) that showed a prediction accuracy of 81% as patients. biomarkers for OSCC. More research studies proved the importance of three tumor markers (Cyfra 21-1, tissue 6.5. Alzheimer and Other Neurodegenerative Disorders. Alz- polypeptide antigen (TPA), and cancer antigen CA125) that heimer’s disease (AD) is one of the most common neuro- were found to have a high level in the saliva of patients diag- vegetative disorders that occur to the aging population. It nosed with OSCC. is supposed that the process of Alzheimer’s is initiated Disease Markers 7 years before it becomes clinically manifest. Until now, RNA, antigens, and antibodies. Currently, several salivary the specific biomarkers for this disease could be found in tests are available based on the proteomic analysis of the the cerebrospinal fluid through the amyloid b levels saliva and the existing antibodies for hepatitis A, B, C or using structural and functional magnetic resonance viruses, HIV-1, rubella virus, mumps virus, and others imaging , procedures that proved to be invasive and. A new salivary test is used by the san Raffaele Scientific time-consuming. Further researches show that the exis- Institute in Milan that is named OraQuick hepatitis C virus tence of Ab and tau [68, 69] or a-Syn and DJ-1 in and represents a fast antibody test in order to detect easily human saliva can be considered proteins that are related the presence of the virus. Nefzi et al. conducted a to Alzheimer’s disease and Parkinson’s disease, suggesting study that showed the fact that human cytomegalovirus actually the implication of saliva and its potential in the (HHV-6) appears to be more easily identified in saliva than diagnosis of neurodegenerative diseases. Risk factors in in serum. the development of Alzheimer’s disease are systemic infec- The HIV (human immunodeficiency virus) is possible tions , brain infections due to bacteria or virus involve- through antibody-based screening assays. The diagnostic test ment , but the association of various antimicrobial is an antibody assay that can be a Western blot test via blood peptides in this disease is still not completely clear. or saliva or a polymerase chain reaction via blood. These The study performed by Carro et al. investigates the specific tests have the aim at identifying the p24 antigens and potential of lactoferrin as a salivary biomarker for Alzhei- antibodies against HIV-1 and HIV-2. However, the detection mer’s, based on the fact that lactoferrin is an antimicrobial of viral RNA is difficult to be performed during salivary anal- peptide that targets bacteria, fungi, protozoa, viruses, and ysis due to the decreased viral load. yeasts [73–75]. The results of their study show that lacto- ferrin can be used as a biomarker for Alzheimer’s disease, 6.7. Lung Cancer. Early diagnosis is an important aspect after the outcome was compared to a standard test per- regarding this type of cancer knowing the fact it is the most formed for the certain diagnosis of AD, proving a very high common cause of death in men and women. Until now, con- correlation with validated cerebrospinal fluid biomarkers. ventional diagnosis methods are not suited for screening, Although more studies are needed, lactoferrin has proved with a high false-negative rate [81, 82]. CT is being used for its correlations and has the potential of being a solid bio- routine screening for early lung cancer, with the disadvantage marker that can help the screening process of “apparently of a high false-positive rate. Salivary biomarkers have the healthy” individuals that can suffer from a preclinical stage potential to help the early diagnosis without using CT. of the disease. After studies were performed, 16 potentially biomarkers have Ahmadi-Motamayel et al. conducted a recent study been discovered that can efficiently contribute to the salivary with the aim at evaluating acetylcholinesterase (AChE) and diagnosis , three of them (haptoglobin, calprotectin, and pseudocholinesterase (PChE) in whole saliva in patients zinc-a-2-glycoprotein) with a high sensitivity and excellent with Alzheimer’s disease and in healthy subjects. Until specificity. The transcriptomic biomarker profile—B-Raf now, many studies have been performed focusing on the gene, cyclin I, the EGF receptor, FGF-19, fibroblast growth salivary biomarkers in Alzheimer’s disease and only a few factor receptor substrate 2, growth regulation by estrogen in regarding the salivary cholinesterase enzyme. The result of breast cancer 1, and leucine zipper putative tumor suppressor this study after the comparison of the salivary samples of 1—has been identified, and a panel of five of these bio- the healthy subjects and those diagnosed with Alzheimer’s markers accomplished to achieve a sensitivity of 93.75% disease concluded the fact that AChE and PChE levels were and a specificity of 82.81% regarding the diagnosis of lung increased in saliva samples of patients with Alzheimer’s dis- cancer. ease. Parkinson’s disease is characterized pathologically by 6.8. Orofacial Pain Salivary Biomarkers. The tissues that are progressive degeneration of dopaminergic (DA) neurons in found in the orofacial region are heterogeneous, a fact that the substantia nigra pars compacta. The formation of a-synu- makes the treatment of pain conditions a challenge for the clein- and ubiquitin-containing fibrillar inclusions (Lewy clinician. The main problem for an adequate treatment bodies and Lewy neurites) occurs in this cell population as option consists in the diversity of conditions for which well as variable changes in other neurotransmitter systems orofacial pain is a major symptom that makes it hard to. The aim of the research initiated by Song et al. differentiate many of these disorders clinically. Several was to evaluate the levels and implications of salivary HO-1 population-based cross-sectional studies revealed a 1-month in patients with idiopathic Parkinson’s disease. The results prevalence rate of self-reported orofacial pain that varies showed that salivary HO-1 concentrations are significantly from 19% to 26% [88, 89]. The current research must focus elevated in patients with idiopathic PD versus nonneurolo- on methods that combine different biomarkers for a condi- gical controls matched for sex. Importantly, the test most tion. Biomarkers evaluation combines physiological parame- effectively differentiated controls from Parkinson’s disease ters, psychological and behavioral aspects, genomics, and patients at the earliest motor stages of the disease and was molecular and protein characteristics. not influenced by age, sex, and various comorbidities. Orofacial pain is a sensory experience within a specific anatomical region and can be related to some common 6.6. Viral Infections. The existing tests for viral infections are chronic orofacial entities: TMJ myalgia and arthralgia, based on salivary biomarkers, basically on viral DNA and atypical odontalgia, persistent dentoalveolar pain disorder, 8 Disease Markers burning mouth syndrome, persistent idiopathic facial pain, Conflicts of Interest neuralgia of the head and neck, and primary headache syn- dromes. The authors declare that there is no conflict of interest Collecting saliva in order to identify biomarkers associ- regarding the publication of this paper. ated with orofacial pain is a painless method and is easy to collect and store. Recently, saliva and synovial fluids have piqued the interest of numerous researchers and clinicians Authors’ Contributions as possible alternatives to serum. All authors contributed equally to this work. Further researches conclude that saliva-based bio- markers are not only preferred but also are accurate in discerning healthy subjects from those afflicted with peri- References odontal disease or burning mouth syndrome [90–94]. Saliva has also been used as an indicator of stress and N. Malathi, S. Mythili, and H. R. Vasanthi, “Salivary diagnos- chronic pain. Several studies report substance P, a neuro- tics: a brief review,” ISRN Dentistry, vol. 2014, Article ID peptide associated with inflammation status and pain, as 158786, 8 pages, 2014. well as the stress hormone cortisol, and markers of oxida- Y. H. Lee and D. T. Wong, “Saliva: an emerging biofluid for tive stress can be repeatedly detected within salivary secre- early detection of diseases,” American Journal of Dentistry, tions [95, 96]. vol. 22, no. 4, pp. 241–248, 2009. The study performed by Jasim et al. focused on salivary S. Chiappin, G. Antonelli, R. Gatti, and E. F. De Palo, “Saliva biomarkers related to chronic pain by comparing blood sam- specimen: a new laboratory tool for diagnostic and basic inves- ples and saliva samples of the same subjects and revealed the tigation,” Clinica Chimica Acta, vol. 383, no. 1-2, pp. 30–40, fact that five specific biomarkers related to pain were tar- 2007. geted. The results showed that they were first to find several R. M. Nagler, O. Hershkovich, S. Lischinsky, E. Diamond, and isoforms of NGF, CGRP, and BDNF in saliva. The expression A. Z. Reznick, “Saliva analysis in the clinical setting: revisiting showed great variations between different saliva collection an underused diagnostic tool,” Journal of Investigative Medi- cine, vol. 50, no. 3, pp. 214–225, 2002. methods. Glutamate was mostly expressed in whole stimulated saliva, and in contrast, the concentration was T. Pfaffe, J. Cooper-White, P. Beyerlein, K. Kostner, and C. Punyadeera, “Diagnostic potential of saliva: current state moderately correlated between saliva types as well as in and future applications,” Clinical Chemistry, vol. 57, no. 5, plasma. The concentration of glutamate has also been shown pp. 675–687, 2011. to be elevated in different pain conditions [98, 99]. These B. M. Brinkman and D. T. W. Wong, “Disease mechanism and results suggest that glutamate may be an essential pain medi- biomarkers of oral squamous cell carcinoma,” Current Opin- ator in peripheral tissue and may therefore act as a potential ion in Oncology, vol. 18, no. 3, pp. 228–233, 2006. pain biomarker among others. M. Castagnola, I. Messana, R. Inzitari et al., “Hypo-phosphor- With the help of a standardized collection procedure and ylation of salivary peptidome as a clue to the molecular patho- protocol, the use of salivary biomarkers for different orofacial genesis of autism spectrum disorders,” Journal of Proteome pain disorders is a promising diagnostic method that will Research, vol. 7, no. 12, pp. 5327–5332, 2008. allow for a noninvasive approach. H. B. Cho, S. W. Hong, Y. J. Oh et al., “Clinical significance of osteopontin expression in cervical cancer,” Journal of Cancer 7. Conclusions Research and Clinical Oncology, vol. 134, no. 8, pp. 909–917, 2008. Saliva is an important biological fluid with a wide area of A. Marini and E. Cabassi, “La saliva: approccio complementare research and applications, having a high potential to become nella diagnostica clinica e nella ricerca biologica,” Annali della the future in early diagnosis. The effective contribution of Facoltà di Medicina Veterinaria, Università di Parma, vol. 22, genomic and proteomic technologies made possible for saliva pp. 295–311, 2002. to become an attractive solution to other invasive diagnostic S. Hu, Y. Xie, P. Ramachandran et al., “Large-scale identifi- methods. Saliva as a diagnostic tool for oral and systemic dis- cation of proteins in human salivary proteome by liquid eases has multiple advantages over other body fluids and chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry,” Proteomics, vol. 5, based on specific biomarkers can provide an accurate diagno- no. 6, pp. 1714–1728, 2005. sis. However, until saliva becomes a certified diagnostic test E. J. Helmerhorst, C. Dawes, and F. G. Oppenheim, “The com- that can replace the conventional ones, all the research values plexity of oral physiology and its impact on salivary diagnos- must be compared with the existing accepted methods. The tics,” Oral Diseases, vol. 24, no. 3, pp. 363–371, 2018. main problem consists in the fact that a standardized and V. de Almeida Pdel, A. M. Grégio, M. A. Machado, A. A. de accurate method of saliva collection needs to be associated Lima, and L. R. Azevedo, “Saliva composition and functions: with each type of diagnostic test, in order to avoid errors. a comprehensive review,” The Journal of Contemporary Dental This review has discussed several oral and systemic diseases Practice, vol. 9, no. 3, pp. 72–80, 2008. that could be diagnosed based on different salivary bio- M. Carranza, M. E. Ferraris, and M. Galizzi, “Structural and markers, but research needs to be extended in order for saliva morphometrical study in glandular parenchyma from alco- to become an effective and sure diagnostic tool that can be holic sialosis,” Journal of Oral Pathology and Medicine, used for screening and uncontestable diagnosis. vol. 34, no. 6, pp. 374–379, 2005.  Archives of Oral Biology 99 (2019) 31–42 Contents lists available at ScienceDirect Archives of Oral Biology journal homepage: www.elsevier.com/locate/archoralbio Review Human salivary proteins and their peptidomimetics: Values of function, T early diagnosis, and therapeutic potential in combating dental caries ⁎ Kun Wang, Xuedong Zhou, Wei Li, Linglin Zhang State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Dept. of Cariology and Endodontics West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, China A R T I C LE I N FO A B S T R A C T Keywords: Saliva contains a large number of proteins that play various crucial roles to maintain the oral health and tooth Salivary proteins integrity. This oral fluid is proposed to be one of the most important host factors, serving as a special medium for Dental caries monitoring aspects of microorganisms, diet and host susceptibility involved in the caries process. Extensive Antimicrobial peptides salivary proteomic and peptidomic studies have resulted in considerable advances in the field of biomarkers Remineralization discovery for dental caries. These salivary biomarkers may be exploited for the prediction, diagnosis, prognosis Biomarkers and treatment of dental caries, many of which could also provide the potential templates for bioactive peptides used for the biomimetic management of dental caries, rather than repairing caries lesions with artificial mate- rials. A comprehensive understanding of the biological function of salivary proteins as well as their derived biomimetic peptides with promising potential against dental caries has been long awaited. This review over- viewed a collection of current literature and addressed the majority of different functions of salivary proteins and peptides with their potential as functional biomarkers for caries risk assessment and clinical prospects for the anti-caries application. 1. Introduction are synthesized and secreted into the oral cavity by the acinar cells of the salivary glands, which can be divided into a few families: proline- Human saliva contains a large array of proteins, many of which rich proteins (PRPs), salivary mucins, salivary α-amylase, salivary cy- possess a distinct biological function to maintain the homeostasis of the statins, histatins (small cationic histidine-rich peptides), and statherin oral cavity system. Alternative splicing and post-translation modifica- and PeB peptide (Amado, Lobo, Domingues, Duarte, & Vitorino, 2010; tions occurring in the course of gene expression lead to the formation of Cabras et al., 2014; Gonzalez-Begne et al., 2009; Zhang, Sun, Wang, & salivary proteins with various structures. Since Krasnow and Oblatt Wang, 2013). The above findings have promoted the gaining of (Krasnow & Oblatt, 1933) firstly investigated the variation in salivary knowledge regarding the association between salivary protein compo- protein concentration in different individuals, there has been increasing sition and human health, and also provided a promising result in uti- interest in the application of salivary analyses to monitor general lizing saliva to explore biomarkers for diagnosis purposes. The non- health. The National Institute of Dental and Craniofacial Research invasive and simple nature of saliva collection made it interesting to be (NIDCR) started to fund three research groups comprising the Saliva used for the early diagnosis and risk assessment of a variety of oral Proteome Consortium in 2004, and they made an effort to identify and diseases, such as Sjögren’s syndrome, oral squamous cell carcinoma, catalogue the human saliva proteome including saliva proteins as well periodontitis, and dental caries (Gallo et al., 2016; Hall et al., 2017; as their structurally modified forms (Katsiougiannis & Wong, 2016). Nomura et al., 2012; Wang, Wang, Wang et al., 2018). With an ex- With the rapid development of proteomic technology and application of pectedly increasing number of salivary protein species to be identified high resolution MS, in-depth proteomic analysis for the salivary protein in the near future, now is the time to devote more attention to the polymorphisms has recently been achievable (Si, Ao, Wang, Chen, & comprehension of their function and to the application of their clinical Zheng, 2015; Sun et al., 2016; Wang, Wang, Wang et al., 2018). These practice. studies revealed the salivary proteome as a sizeable collection of up to Dental caries is one of the most common chronic diseases afflicting a 1166 proteins, including 914 in parotid and 917 in submandibular/ large proportion of the world’s population, involved in interactions sublingual saliva (Denny et al., 2008). The majority of these proteins between the tooth structure, the microbial biofilm formed on the tooth ⁎ Corresponding author at: No.14, Section 3 of Renmin South Road, Chengdu, China. E-mail address: [email protected] (L. Zhang). https://doi.org/10.1016/j.archoralbio.2018.12.009 Received 7 October 2018; Received in revised form 21 November 2018; Accepted 22 December 2018 0003-9969/ © 2018 Elsevier Ltd. All rights reserved. K. Wang et al. Archives of Oral Biology 99 (2019) 31–42 surface, as well as genetic influences and salivary function (Petersen, Maeda, Kim, Azen, & Smithies, 1985; Scherer et al., 2006). They are 2003; Pitts et al., 2017). Saliva contains a variety of proteins partici- mainly classified into three groups: acidic PRPs (aPRPs), basic PRPs pating in maintaining the tooth integrity and preventing caries through (bPRPs), and glycosylated PRPs (gPRPs) (Bennick, 1987; Schenkels, several mechanisms: I) formation of acquired enamel pellicle to con- Veerman, & Amerongen, 1995). Accounting for 25–30% of all salivary tinuously protect against tooth wear (mucins and proline-rich glyco- proteins, the aPRPs possess a 30-amino acid N-terminal domain rich in protein); II) inhibition of demineralization of exposed tooth surfaces aspartate and glutamate with a few serine phosphate residues, which (mucins); III) promotion of enamel remineralization by attracting cal- are involved in the formation of the acquired enamel pellicle and act as cium ions (proline-rich proteins and statherin); IV) antimicrobial ac- salivary receptors for several plaque-forming bacteria (Amano et al., tivities including prevention of cariogenic species adherence onto en- 1994). aPRPs binding to hydroxyapatite involves the acidic N-terminal amel surface (histatins and cystatins), microorganism aggregation and domain and exposes the proline-rich C-terminal domain to oral bacteria clearance from the oral cavity (agglutinin and immunoglobulins), and binding (Manconi et al., 2016). Also, aPRPs (PRP-3 and its derived the secretion of antimicrobial peptides (AMPs) (Gao, Jiang, Koh, & Hsu, peptides) play a key role in the protection of tooth enamel through 2016; Guo & Shi, 2013; Van Nieuw Amerongen, Bolscher, & Veerman, inhibiting calcium phosphate precipitation and thus promoting calcium 2004). Currently, the biological functions of salivary proteins have been homeostasis in oral cavity (Hay, Carlson, Schluckebier, Moreno, & extensively studied for their possible relevance to caries risk assess- Schlesinger, 1987; Vitorino et al., 2007). The family of bPRPs and ment. For example, low levels of statherin and truncated cystatin S in gPRPs, encoded by the polymorphic PRB1-PRB4 genes, includes 11 saliva are found to be associated with caries susceptibility (Rudney, parent peptides/proteins and more than 6 parent glycosylated proteins, Staikov, & Johnson, 2009). This enhances knowledge of the structure- but a higher number of proteoforms with similar structures derived function relationship of salivary proteins, making it possible to identify from polymorphisms and post-translational modifications (Padiglia potential biomarkers for dental caries, and further to design small, et al., 2018). More recently, 55 new components of the family were biologically active peptides as instruments to fight against caries, as extensively characterized by top-down liquid chromatography-mass well as restore functionality in patients in whom the natural protection spectrometry, bringing the total number of proteoforms to 109 is compromised. (Padiglia et al., 2018). The significant structural differences presenting This review aims to summarize the following: (1) the protective in the class of salivary PRPs suggest their crucial role in the oral pro- function of major salivary proteins in cariology; (2) the identification of tection. Some bPRP fragments are involved in enamel pellicle formation salivary proteins utilized as biomarkers for caries risk assessment; (3) (PRB-1, IB-1, IB-8a, and IB-9), and others exhibit the ability to modulate bioactive peptides derived from salivary proteins for the therapeutic use the oral flora (Ruhl, Sandberg, & Cisar, 2004; Vitorino et al., 2007). against dental caries. Some other bPRPs (IB-7) could attach to a major adhesion antigen on the surface of Streptococcus mutans and other oral streptococci, whose 2. Protective functions of salivary proteins and their potentials as arginine and lysine residues neutralize acids from carbohydrate meta- biomarkers for caries risk assessment bolism within the biofilm (Levine, 2011; Nobbs, Jenkinson, & Jakubovics, 2011). In addition, the gPRPs is able to interact particularly Saliva contains a large number of secreted proteins, including major with several types of microorganisms, such as Fusobacterium nucleatum, salivary glycoproteins (proline-rich proteins, mucins and im- and is involved in the plaque formation (Kolenbrander & London, 1993; munoglobulins), minor salivary proteins (cystatins, lysozyme, lacto- Schenkels et al., 1995). ferrin, agglutinin, and amylase), AMPs (histatins, cathelicidin peptide In terms of the association between caries prevalence and PRPs, LL-37, alpha-defensins, and beta-defensins), statherin and P-B peptide, contradictory results were reported in previous studies. A cross-sec- which protect the tooth integrity through either against losing calcium tional study showed that subjects with high DMFT indices presented and phosphate ions from the enamel surface or playing antimicrobial significant reduction of salivary aPRP (PRP-1), mucin 5B, and mucin 7 role directly or indirectly. The most common salivary proteins and their (Banderas-Tarabay, Zacarias-D’Oleire, Garduño-Estrada, Aceves-Luna, protective properties against dental caries are listed in Table 1. These & González-Begné, 2002). Similarly, Vitorino et al. evaluated differ- molecules as well as their post-translational modifications with specific ential protein expression patterns in the whole saliva of caries-free and function may be better indicators of oral diseases, and could provide a caries-susceptible individuals using 2-DE combined with matrix-as- promising repertoire in utilizing saliva to explore more sensitive and sisted laser desorption/ionization time-of-flight mass spectrometry specific biomarkers for dental caries (Al-Tarawneh, Border, Dibble, & (MALDI-TOF MS), and found significantly higher quantities of aPRPs in Bencharit, 2011). Initiation of salivary biomarker discovery and ex- caries-free samples (Vitorino et al., 2006). Based on iTRAQ and MRM panding the dataset of carious patients will help to establish a concise analysis, our recent study found significant lower amounts of bPRPs model for the early diagnosis and measurement of dental caries. (PRB2) in saliva samples from caries-susceptible children (Wang, Wang, Wang et al., 2018). On the contrary, Ribeiro et al. identified that the 2.1. Major salivary glycoproteins presence of proline-rich peptides IB-4 could significantly increase the risk of caries (Ribeiro et al., 2013). Another report by Bhalla et al. also Major salivary glycoproteins account for approximately 50% of total observed a higher number of PRP bands in saliva of subjects with caries salivary proteins, including proline-rich proteins (15–20%), mucous (Bhalla, Tandon, & Satyamoorthy, 2010). However, no obvious differ- glycoproteins (20–30%), and immunoglobulins (5–15%). ence in the salivary levels of aPRPs was reported in other studies be- tween the caries-free and caries-resistant subjects (Dodds, Johnson, 2.1.1. Proline-rich proteins Mobley, & Hattaway, 1997; Mandel & Bennick, 1983). The human salivary proline-rich proteins (PRPs) are a hetero- geneous group of proteins produced from the parotid, submandibular, 2.1.2. Mucous glycoproteins and sublingual salivary glands, which are abundant in saliva and con- Human saliva contains two structurally and functionally distinct stitute about 15–20% (w/w) of total proteins in whole human saliva populations of mucous glycoproteins (mucins), the high-molecular- and more than 50–60% (w/w) of proteins in parotid saliva (Kim et al., weight mucins (MG1, molecular mass > 1000 kDa), and low-molecular- 1993; Manconi et al., 2016). PRPs are coded by a multi-family of six weight mucins (MG2, molecular mass 150–200 kDa) (Amerongen & different genes (PRH1, PRH2, PRB1, PRB2, PRB3, and PRB4), strictly Veerman, 2002). Although secreted mucins are components of the non- associated in a segment of ˜4.0 Kb in length on chromosome 12 at band immune oral host defense system, they are considered the first line of 13.2, and a large number of PRPs have been discovered by differential defense for the oral health (Tabak, 1995). Mucins are identified as acid- RNA splicing and proteolytic cleavages after secretion (Kim et al., 1993; resistant proteins since it is still present in the acquired pellicle after 32 K. Wang et al. Archives of Oral Biology 99 (2019) 31–42 Table 1 Main properties of salivary proteins against dental caries. Salivary proteins Sources Main functions Reference Proline-rich proteins Parotid, submandibular, and Remineralization of enamel, formation of AEP, Amano et al. (1994), Hay et al. (1987), Kim et al. sublingual glands neutralization of acids within the biofilm (1993), Levine (2011), Manconi et al. (2016), Nobbs et al. (2011), Vitorino et al. (2007) Mucins Submandibular, sublingual, and Inhibition of demineralization, agglutination of Buzalaf et al. (2012), Delecrode et al. (2015), labial glands microorganisms, barrier protection, resistance of acid, Frenkel & Ribbeck (2015), Jordão et al. (2017), decrease attachment of S. mutans Levine et al. (1978) Immunoglobulins Major and minor salivary glands Adaptive immune defense against bacterial, inhibition of Law et al. (2007), Russell et al. (1999), Van Nieuw bacterial adherence and colonization, microorganism Amerongen et al. (2004) aggregation and clearance, enhance the activity of lactoferrins and lysozymes Cystatins Parotid, submandibular gland, and Protease inhibitor, formation of AEP, remineralization of Baron et al. (1999), Dickinson (2002a), Dickinson gingival crevicular enamel, bactericidal property (2002b), Lamkin et al. (1991), Vitorino et al. (2008) Lysozyme Major and minor salivary glands, Antibacterial, cell wall lysis, inhibition of biofilm formation Hatti et al. (2007), Kho et al. (2005) Krzysciak neutrophil granulocytes, and and S. mutans adhesion on HA et al. (2015), Moslemi et al. (2015) gingival crevicular fluid Lactoferrin Major and minor salivary glands, Antimicrobial activity, immunomodulatory activity, cell Baveye et al. (1999), Chierici (2001), Gorr & neutrophil granulocytes, and growth modulation, iron homeostasis regulation, bacterial Abdolhosseini (2011), Lenander-Lumikari & mucosal epithelial cells binding, agglutination of S. mutans Loimaranta (2000), Moslemi et al. (2015), Velusamy et al. (2016) Agglutinin Parotid, submandibular and Microorganism aggregation and clearance Bikker et al. (2002), Ligtenberg et al. (2010) sublingual glands Amylase Major and minor salivary glands Formation of AEP, modulation of bacterial colonization, Scannapieco et al. (1993), Singh et al. (2015) adhesion, and dental plaque formation Histatins Parotid and submandibular Formation of AEP, antibacterial and antifungal activities, Fernández-Presas et al. (2018), Gorr (2009), salivary duct cells regulation of oral hemostasis, adsorption on enamel Oppenheim et al. (1986), Oudhoff et al. (2009), surfaces, reduce S. mutans adhesion onto HA Richardson et al. (1993), Shimotoyodome et al. (2006) LL-37 Salivary glands, epithelial cells, Antibacterial and antifungal activity Isogai, Isogai et al. (2003, 2003b), Lo´ pez-Garcı´a neutrophil leukocytes and gingival et al. (2005), Zanetti et al. (2002) crevicular fluid Defensins Neutrophil granulocytes, epithelial Microbicidal activity and antiviral activity Ganz et al. (1985), Lehrer & Ganz (1999) cells, gingival crevicular fluid C-C motif chemokine Salivary glands, epithelial cells Antimicrobial activities, homing of certain types of Allen et al. (2007), Denny et al. (2008), Hieshima 28 lymphocytes, chemoattractant of IgA et al. (2003), Jiang et al. (2012) Azurocidin Azurophil granules of neutrophils Antibacterial activity, strong affinity for LPS Dhaifalah et al. (2014) Statherin Major and minor salivary glands Formation of AEP, remineralization of enamel, inhibition of Shimotoyodome et al. (2006), Trindade & Amado the spontaneous precipitation of calcium and phosphate (2015), Xiao et al. (2015) salts, inhibition of S. mutans adhesion onto HA, antimicrobial activity exposure to citric acid (Delecrode et al., 2015). The protective effect of associated with elevated S. mutans titers in saliva (Baughan, Robertello, mucins may be attributed to its high viscosity or chemical composition. Sarrett, Denny, & Denny, 2000). In addition, salivary mucins expression Jordão et al. reported that artificial saliva with mucins could protect in relation to dental caries was also found to be different between sound enamel against demineralization similarly to human saliva in situ preschool and school children (Angwaravong, Pitiphat, Bolscher, & (Jordão et al., 2017). MG1 is the primary mucin component of the Chaiyarit, 2015). Both mucin 5B and mucin 7 showed up-regulated dental pellicle coating the soft and hard tissues in the oral cavity, which levels in caries-susceptible children in response to dental caries (Wang, provides an attachment site for bacteria and affects the adhesion of Wang, Wang et al., 2018). For adolescents, the salivary mucin 1 and specific bacteria onto the tooth surface (Buzalaf, Hannas, & Kato, mucin 5B levels were significant higher in subjects with high intensity 2012). In spite that mucin 5B does not alter S. mutans growth, it could of dental caries (DMF > 11) compared to those with low DMF decrease surface attachment and biofilm formation by maintaining S. (Gabryel-Porowska et al., 2014). mutans in the planktonic form (Frenkel & Ribbeck, 2015). As an im- portant antimicrobial salivary agent, MG2 can agglutinate S. mutans 2.1.3. Immunoglobulins and S. sanguis via promoting the clearance of them from the oral cavity The salivary immunoglobulins constitute 5–15% of whole salivary (Levine et al., 1978). However, mucins have also been shown to provide proteins, belonging primarily to the IgA subclass and to a lesser extent nutrients for a variety of bacterial species that inhabit mucosal surfaces to the IgG and IgM subclass (Van Nieuw Amerongen et al., 2004). The (Derrien et al., 2010). Some previous studies found that S. mutans could secretory IgA immune response represents the first line of adaptive scavenge nutrients from mucins to enhance its growth and survival immune defense against mutans streptococci. SIgA composes 60% of (Mothey, Buttaro, & Piggot, 2014; Renye, Piggot, Daneo-Moore, & the immunoglobulin in saliva, which plays antimicrobial roles through Buttaro, 2004). neutralizing bacterial toxins and enzymes, inhibiting bacterial ad- The importance of mucin 5B and mucin 7 has been the focus of herence and colonization, reducing the hydrophobicity of bacteria and much research in the past two decades (Humphrey & Williamson, aggregating the bacteria together (Russell, Hajishengallis, Childers, & 2001). The presence or absence of mucin 5B and mucin 7 in the oral Michalek, 1999; Van Nieuw Amerongen et al., 2004). Salivary IgA has cavity could alter individuals’ susceptibility to dental cavity formation, also been shown to enhance the activity of several enzymes, such as which could then be an easily accessible and highly predictable clinical lactoferrins and lysozymes (Law, Seow, & Townsend, 2007). diagnostic biomarker of dental caries. The importance of mucin 7 in Previous investigations have reported contradictory results in the caries prevention has been demonstrated in elderly populations, in field regarding the association of salivary levels of sIgA and dental whom the diminished levels of mucin 7 were found to be significantly caries. A systematic review and meta-analysis presented evidence that 33 K. Wang et al. Archives of Oral Biology 99 (2019) 31–42 supported the presence of increased sIgA levels in caries-active subjects acids might be potential risk indicators for caries development (Rudney based on 314 abstracts (Fidalgo et al., 2014). Likewise, a recent study et al., 2009). collected saliva samples from 70 children and 43 elderly adults with and without dental caries, and demonstrated a significantly higher level 2.2.2. Lysozyme of sIgA in both children with severe early childhood caries (ECC) and In the oral cavity, lysozyme is an antibacterial enzyme, especially the elderly subjects with root caries (RC) using qPCR and ELISA (Yang against gram-positive bacteria, which is secreted from major and minor et al., 2015). On the contrary, Colombo et al. in a more recent study salivary glands, gingival crevicular fluid, and salivary leukocytes explored positive correlations between salivary IgA antibody response (Moslemi et al., 2015). It also has the ability to bind to gram-negative against S. mutans GbpB and S. mutans counts, and found children with bacteria lipopolysaccharides (LPS), thus contributing to the reduced severe ECC had significantly lower IgA antibody levels to S. mutans risk of development of an inflammatory response when high amounts of GbpB (Colombo, Pereira et al., 2016). However, no significant corre- exotoxin occur in the organism (Krzysciak et al., 2015). Due to the lation between IgA concentration and caries activity was observed in biological activity of lysozyme, the ability of cariogenic microorganisms other reports (Koga-Ito, Martins, Balducci, & Jorge, 2004; Shifa, Muthu, toward the formation of biofilm is inhibited, and also the adhesion of S. Amarlal, & Rathna Prabhu, 2008). Salivary IgG functions in the pro- mutans to HA is reduced (Hatti, Ravindra, Satpathy, Kulkarni, & tection of oral cavity by inhibiting the growth, adherence and acid Parande, 2007; Kho, Vacca Smith, Koo, Scott-Anne, & Bowen, 2005). production of S. mutans (Gregory, Kindle, Hobbs, Filler, & Malmstrom, Apart from the antibacterial effect, lysozyme also exhibits antiviral and 1990). Controversy still remains in research regarding IgG and IgM as antifungal properties (Krzysciak et al., 2015). biomarkers for caries. A cross-sectional study exhibited a higher sali- Various studies in the past have attempted to relate salivary lyso- vary IgG level among children with ECC (Bagherian, Jafarzadeh, zyme and caries activity. Mass et al. found an association between low Rezaeian, Ahmadi, & Rezaity, 2008). Another study supported the levels of lysozyme and decreased amounts of S. mutans and lactobacilli correlation between higher levels of total salivary IgA and IgG and (Mass, Gadoth, Harell, & Wolff, 2002). A recent study demonstrated children with ECC, but no significant difference in total IgM level was significantly increased lysozyme level in caries-free children compared found between children with and without ECC (de Farias & Bezerra, with ECC children, and suggested reduced amounts of lysozyme may be 2003). Based on the evidence currently available, the diagnostic value risk factor for childhood caries (Moslemi et al., 2015). Supporting this of salivary immunoglobulins as biomarker for caries risk has not yet claim, lower levels of salivary lysozyme were correlated with low caries been ascertained. increment over 4 years of 28 adults by cluster analysis (Jentsch, Beetke, & Gocke, 2004). However, equivocal conclusions of the resemblance of 2.2. Minor salivary proteins salivary lysozyme between caries-active samples and caries-free con- trols were reported in some other studies. Stuchell and Mandel found no Besides the major proteins, saliva is abundant in lots of proteins significant difference in lysozyme concentrations between caries-free with relatively lower concentrations, many of which are enzymes ex- and caries-active groups (Stuchell & Mandel, 1983). Also, a two-year erting significant biological activity even in low levels. cohort study based on longitudinal analysis indicated that single anti- microbial agents including lysozyme had not sufficiently strong power 2.2.1. Cystatins to have diagnostic significance in vivo with respect to future caries Cystatins are a superfamily of cysteine-peptidase inhibitors with (Kirstilä, Häkkinen, Jentsch, Vilja, & Tenovuo, 1998). various proteoforms, including type 1 cystatins (cystatins A and B), type 2 cystatins (C, D, E, F, S, SN, SA), type 3 cystatins, or kininogens 2.2.3. Lactoferrin (Manconi et al., 2017). The major “S-type” cystatins belong to type 2 As a ubiquitous biodiverse molecule, lactoferrin is typically found in comprising cystatin S, SN and SA with a molecular mass ranging from saliva at concentrations of 1 to 7ug/ml (Scannapieco, 1994). Lacto- 13.5 to 14.5 kDa, and they participate in multi-functions in the oral ferrin has been shown a diverse range of physiological functions, such cavity (Dickinson, 2002b). Cystatin S may be either monopho- as antimicrobial/antiviral activities, immunomodulatory activity, cell sphorylated on Ser3 (cystatin S1, accounting for 60–70% of total cy- growth modulation, and iron homeostasis regulation (Baveye, Elass, statin S forms) or diphosphorylated on Ser1 and Ser3 (cystatin S2, ac- Mazurier, Spik, & Legrand, 1999; Chierici, 2001). Its unique capability counting for 20–30%) (Messana et al., 2008; Vitorino et al., 2004). of binding iron leads to its characterization as a “metal iron chelator” Besides of modified forms identified in cystatin S, cystatin B in saliva is (Gorr & Abdolhosseini, 2011). The iron-free state known as apo-lacto- major present as S-modified derivatives, namely S-glutathionylated, S- ferrin, is capable of directly binding to bacteria via interactions through cysteinylated, and S-S 2-mer (Cabras et al., 2012). “S-type” cystatins are the tail end of the N-terminal region consisting of 47 amino acids, and major components of the acquired enamel pellicle and their phos- agglutinating S. mutans, thus helping to remove the agglutinated bac- phorylated forms have capacity of binding to hydroxyapatite (HA), teria from the oral cavity through mechanical action of saliva (Moslemi which is reduced but not eliminated by dephophorylation (Dickinson, et al., 2015). In an in vivo study, the lactoferrin-knockout infected mice 2002a; Lamkin, Jensen, Setayesh, Troxler, & Oppenheim, 1991; had significantly higher colonization with S. mutans and more carious Vitorino, Calheiros-Lobo, Duarte, Domingues, & Amado, 2008). They lesions, indicating that endogenous lactoferrin could exert a protective also inhibit calcium phosphate precipitation and control the process of effect against caries development (Velusamy, Markowitz, Fine, & enamel remineralization (Dickinson, 2002a). Besides, type-2 cystatins Velliyagounder, 2016). Lactoferrin does not stand alone in its multi- also exhibit strong bactericidal and virucidal properties (Baron, Gansky, functionality, which can also bind to salivary agglutinin acting together Ryder, & Featherstone, 1999; Dickinson, 2002a). Cystatin S, a putative to bind microbes (Lenander-Lumikari & Loimaranta, 2000). Besides, it indicator of caries, was present at a higher level in elderly with RC in a has also been proposed that lactoferrin’s effect can be correlated with previous study (Preza, Thiede, Olsen, & Grinde, 2009). In contrast, a lysozyme, lactoperoxidase, and IgA (Rudney, Hickey, & Ji, 1999). Due strong correlation between cystatin S, cystatin SN2, cystatin SAIII and to its antimicrobial activity, salivary lactoferrin has been thought to the lowest values of DMFT was reported in another work (Vitorino play an important role in the prediction of caries risk. Moslemi et al. et al., 2006). Consistent with this result, our previous study also found collected unstimulated saliva from 42 children, and reported a higher down-regulated levels of cystatin S and cystatin SN in high caries-sus- concentration of lactoferrin in caries-free individuals than that in ECC ceptible children using MRM validation, indicating their importance in individuals (Moslemi et al., 2015). This study suggested reduced the maintenance of teeth integrity (Wang, Wang, Wang et al., 2018). amounts of lactoferrin as a risk factor for childhood dental caries Using a modified proteomic approach, Rudney et al. suggested that (Moslemi et al., 2015). Similarly in adults, a 4-year study among 28 levels of a truncated cystatin S missing the first eight N-terminal amino young adults also linked low caries increment with low level of 34 K. Wang et al. Archives of Oral Biology 99 (2019) 31–42 lactoferrin in unstimulated saliva (Jentsch et al., 2004). However, the strains, as well as strong antifungal activity (Edgerton & Koshlukova, relationship between salivary lactoferrin and caries susceptibility has 2000). Histatin 1 and histatin 3 are derived from the available genes not yet shown consistent validity. HTN1 and HTN3 present in humans (Helmerhorst, Van’t Hof, Veerman, Simoons-Smit, & Nieuw Amerongen, 1997). Histatin 5, a peptide 2.2.4. Agglutinin composed of 24 amino acids derived from histatin 3, has potent in- Salivary agglutinin (encoded by the deleted in malignant brain tu- hibitory effect against Candida species (Candida albicans, Candida glab- mors 1 (DMBT1) gene) is an innate immune receptor glycoprotein rata, Candida krusei, and Cryptococcus neoformans) growth and bacterial comprising ˜10% of total salivary protein in children and 5% in adults co-aggregation (Gorr, 2009; Oppenheim et al., 1986). Recently, Fer- (Sonesson, Ericson, Kinnby, & Wickström, 2011), which was originated nández-Presas et al. found histatin 5 could induce ultrastructural da- as an S. mutans-agglutinating glycoprotein (Bikker et al., 2002). Ag- mage in S. mutans through an apoptosis-like death mechanism glutinin is known to bind a variety of oral bacteria and viruses, and (Fernández-Presas et al., 2018). Besides that, histatins are also involved mediate the attachment of S. mutans to HA on the surface of the tooth in the regulation of oral hemostasis and bonding of metal ions in saliva (Ligtenberg, Karlsson, & Veerman, 2010). Some studies revealed a (Oudhoff et al., 2009). Histatins, especially histatin 1, also possess high correlation between high levels of salivary agglutinin and increased affinity for enamel surfaces and participate in the formation of acquired amounts of S. mutans in dental plaque and caries susceptibility (Carlén, enamel pellicle (Richardson, Johnsson, Raj, Levine, & Nancollas, 1993). Olsson, & Ramberg, 1996). In contrast, a previous study found a two- Shimotoyodome et al. elucidated that the N-terminal domain of histatin fold enhancement of salivary agglutinin in caries-resistant group com- 1 could competitively reduce the adhesion of S. mutans onto HA sur- pared with caries-susceptible individuals (Rosan, Appelbaum, Golub, faces by inhibiting the adsorption of salivary high-molecular weight Malamud, & Mandel, 1982). More recently, protein variants of agglu- glycoproteins (Shimotoyodome, Kobayashi, Tokimitsu, Matsukubo, & tinin have been suggested to affect caries susceptibility (Jonasson et al., Takaesu, 2006). 2007). A previous study verified a strong correlation between large amounts of histatin 1 and the absence of dental caries using HPLC-MS 2.2.5. Amylase (Maeda et al., 1985). In a more recent work, a salivary peptidome Salivary α-amylase is the most plentiful and physiologically active profiling analysis was conducted using magnetic bead-based MALDI- enzyme in the oral cavity, and is also found in abundance in the ac- TOF MS separately at the different time point of before, 1 and 4 weeks quired enamel pellicle, which has ability to modulate bacterial colo- after dental treatment. Histatin 1 showed higher levels in children with nization and to provide additional glucose for the biofilm formation severe ECC after 4 weeks treatment compared with before treatment (Scannapieco, Torres, & Levine, 1993; Singh et al., 2015). In addition, it (Sun et al., 2016). In agreement with their results, our recent study also could bind to the membrane of S. mutans and Lactobacillus, pro- found significant lower amounts of histatin 1 in saliva samples from moting their removal from the oral cavity and lowering the risk of high caries-susceptible children based on iTRAQ and MRM analysis dental caries (Scannapieco et al., 1993). On the other hand, all these (Wang, Wang, Wang et al., 2018). More recently, histatin-1 was also activities depend on an intact enzyme configuration. Amylase binds to validated to exhibit decreased salivary abundance in both caries-sus- bacteria in plaque while free amylase in saliva may facilitate dietary ceptible adults and elderly subjects using a discovery-through-ver- starch hydrolysis to provide additional low-molecular weight carbo- ification pipeline (Wang, Wang, Zheng et al., 2018). On the other hand, hydrates for metabolism by plaque microorganisms. Then the resulting another study revealed a significant increase in the concentration of acid production may be added to the pool of acid in plaque to accelerate histatin 5 in children with severe ECC compared to children without tooth demineralization and further progression of dental caries (Arya & caries lesions and correlated with the progression of dental caries Taneja, 2015; Scannapieco et al., 1993). Until now, there is, however, (Jurczak, Kościelniak, Papież, Vyhouskaya, & Krzyściak, 2015). Gor- no sufficient evidence of a relationship between α-amylase and dental nowicz et al. found that adolescents with high severity of caries caries susceptibility. Rec