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Background: Mass indoor gatherings were banned in early 2020 to prevent the spread of SARS-CoV-2. We aimed to assess, under controlled conditions, whether infection rates among attendees at a large, indoor gathering event would be similar to those in non-attendees, given implementation of a comprehensive prevention strategy including antigen-screening within 3 days, medical mask wearing, and optimised ventilation., Methods: The non-inferiority, prospective, open-label, randomised, controlled SPRING trial was done on attendees at a live indoor concert held in the Accor Arena on May 29, 2021 in Paris, France. Participants, aged 18-45 years, recruited via a dedicated website, had no comorbidities, COVID-19 symptoms, or recent case contact, and had had a negative rapid antigen diagnostic test within 3 days before the concert. Participants were randomly allocated in a 2:1 ratio to the experimental group (attendees) or to the control group (non-attendees). The allocation sequence was computer-generated by means of permuted blocks of sizes three, six, or nine, with no stratification. The primary outcome measure was the number of patients who were SARS-CoV-2-positive by RT-PCR test on self-collected saliva 7 days post-gathering in the per-protocol population (non-inferiority margin <0·35%). This trial is registered with ClinicalTrials.gov, NCT04872075., Findings: Between May 11 and 25, 2021, 18 845 individuals registered on the dedicated website, and 10 953 were randomly selected for a pre-enrolment on-site visit. Among 6968 who kept the appointment and were screened, 6678 participants were randomly assigned (4451 were assigned to be attendees and 2227 to be non-attendees; median age 28 years; 59% women); 88% (3917) of attendees and 87% (1947) of non-attendees complied with follow-up requirements. The day 7 RT-PCR was positive for eight of the 3917 attendees (observed incidence, 0·20%; 95% CI 0·09-0·40) and three of the 1947 non-attendees (0·15%; 0·03-0·45; absolute difference, 95% CI -0·26% to 0·28%), findings that met the non-inferiority criterion for the primary endpoint., Interpretation: Participation in a large, indoor, live gathering without physical distancing was not associated with increased SARS-CoV-2-transmission risk, provided a comprehensive preventive intervention was implemented., Funding: French Ministry of Health., Translation: For the French translation of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests All authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ovarian cancer has the highest mortality rate in the world. Therefore, it is urgent but still challenging to develop an efficient circulating tumor cell (CTC) detection method to sensitively detect ovarian cancer. To address such issues, herein, for the first time, we present a novel CTC detection method for ovarian cancer cells by designing sensitive and rapid graphene-based biosensors. This graphene-based sensor, consisting of a cell pool and two electrodes, can be prepared by a conventional chip fabrication process. It demonstrates high-sensitivity detection even for several ovarian cancer cells by comparing the electrical signal before and after adding cell solution. Moreover, the graphene-based biosensors can perform rapid detection with good repeatability. This suggests that this novel method is possible to use for the early detection of ovarian cancer with very low CTC cell concentration. This work provides a novel and quick strategy to detect ovarian cancer and further judge or predict the risk of the transfer of ovarian cancer.

Introduction: Asthma is a common chronic inflammatory air way disease which poses a high disease burden worldwide. Asthma is a heterogenous disease with various phenotypes and endotypes. Refractory asthma requires new and personalized approaches to manage it effectively. Periostin is a promising biomarker that may help in predicting severity, prognosis and could be a therapeutic target. We aimed in this study to investigate periostin levels in the sputum of asthmatic Egyptian patients of inflammatory phenotypes and its relation to asthma severity., Subjects and Methods: This study included 96 adult asthmatic patients; 48 patients with mild-to-moderate bronchial asthma, 48 patients with severe bronchial asthma according to ATS criteria and 10 healthy controls. All participants were subjected to full history taking and clinical examination; pulmonary function tests; skin prick test; induced-sputum analysis for inflammatory cells and periostin., Results: Sputum periostin concentrations were significantly higher in patients with asthma than in controls. Sputum periostin is strongly correlated with age and sputum TLC and inversely correlated with FEV1. It is correlated with sputum neutrophil count and sputum eosinophil percentage. Best cut off value for sputum periostin is >528.25 ng/ml to differentiate between mild-to-moderate and severe asthma., Conclusion: Sputum periostin levels provide a satisfying diagnostic accuracy in severe asthmatic with persistent airflow limitation than mild-to-moderate asthmatic adults. There is a modest positive correlation between sputum periostin and sputum eosinophilia.

Objective: Parasympathetic network damage results in facial nerve damage, sublingual ganglion degeneration, sublingual gland dysfunction, and dry mouth. In this study, subarachnoid hemorrhage (SAH) was considered to be the cause of dry mouth., Methods: We assessed 23 hybrid rabbits, including 5 control (group 1, Control). One milliliter of serum saline was injected into the cisterna magna of 5 animals (group 2). SAH was induced by injecting 1 mL of autologous blood into the cisterna magna of 13 animals (group 3). The animals were killed after 3 weeks of induction. The animals' sublingual ganglion and sublingual gland were excised for histopathological examination. The number of degenerated cells in the sublingual ganglion, secretory vesicles, and secretory granules in the sublingual gland that contain salivary components were estimated using Sequential Window Acquisition of All Theoretical Mass Spectra data analysis. The values were compared by the Mann-Whitney U-test., Results: The numbers of secretory vesicles in the sublingual gland were 5.3 ± 1.1 × 10 3 (group 1), 4.23 ± 0.45 × 10 3 (group 2), and 1.56 ± 0.22 × 10 3 (group 3); the numbers of secretory vesicles containing saliva in the sublingual gland were 324 ± 12.18 (group 1), 263 ± 36.23 (group 2), and 114 ± 23.14 (group 3); and the numbers of degenerated cells in the sublingual ganglion were 11 ± 3/mm 3 (group 1), 98.43 ± 15.54/mm 3 (group 2), and 346 ± 12.28/mm 3 (group 3) (P < 0.05)., Conclusions: Clinical findings in infection and diseases such as Sjögren syndrome, aseptic meningitis, and SAH are similar. However, until now, SAH has not been demonstrated experimentally to cause dry mouth. Discovering that SAH might cause dry mouth might prevent unnecessary use of antibiotics and decrease morbidity due to the wrong or late diagnosis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Peripheral blood leucocytes (PBL) have been traditionally used to investigate DNA damage by the comet assay in population studies, but validating alternative non-invasive samples would expand the application of this assay in human biomonitoring. The objectives of this study were (i) to test the validity of salivary leucocytes as a proper biomatrix for the comet assay, (ii) to evaluate the ability of this approach to detect different types of primary and oxidative DNA damage, and (iii) to determine whether frozen salivary leucocytes are still suitable for displaying those types of DNA damage. Fresh and frozen leucocytes isolated from saliva samples (six healthy non-smoking volunteers), were exposed to four genotoxic agents inducing different types of DNA damage, both primary (methyl methanesulfonate, actinomycin-D, ultraviolet radiation) and oxidative (potassium bromate), and standard or enzyme-modified comet assay was conducted. Results were compared with those obtained from PBL. Cells exposed to the four genotoxic agents showed dose-dependent increases of primary and oxidative DNA damage, demonstrating the suitability of all these samples to detect genetic damage from different origin. When comparing baseline levels of DNA damage, just a slight significant increase in primary DNA damage was observed in frozen salivary leucocytes regarding the other biomatrices, but similar results were obtained regarding sensitivity to DNA damage induction by all agents tested. This study demonstrates that salivary leucocytes can be employed in comet assay as an alternative or complement to blood samples. Frozen salivary leucocytes were proved to be a very convenient sample in large biomonitoring studies.

Cerebral stroke is a serious worldwide health problem, as can be seen by the global epidemic of the disease. In this disorder, when the blood flow is compromised by ruptures or blocked arteries, sudden death of neurons is observed as a result of a lack of oxygen and nutrients. Numerous severe problems and frequent complications also exist in stroke patients; therefore, there is an urgent need to develop new therapeutic, diagnostic, and prognostic methods for the disease. At present, the diagnosis of stroke is based on a neurological examination, medical history, and neuroimaging, due to the fact that rapid and noninvasive diagnostic tests are unavailable. Nevertheless, oxidative stress and inflammation are considered key factors in stroke pathogenesis. Oxygen free radicals are responsible for oxidation of lipids, proteins, and DNA/RNA, which in turn contributes to oxidative damage of the brain. Toxic products of the oxidation reactions act cytostatically on the cell by damaging cell membranes and leading to neuronal death by apoptosis or necrosis. Thus, it seems that redox/inflammatory biomarkers might be used in the diagnosis of the disease. Nowadays, saliva is of increasing interest in clinical laboratory medicine. Redox biomarkers could be obtained easily, noninvasively, cheaply, and stress-free from saliva. This minireview is aimed at presenting the current knowledge concerning the use of salivary biomarkers of oxidative stress and inflammation in the diagnosis and prognosis of stroke., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Mateusz Maciejczyk et al.)

Mosaic loss of chromosome Y (mLOY) in leukocytes has attracted much attention as an emerging biomarker of aging and aging-related diseases. We evaluated the usefulness of saliva for mLOY analysis and showed that saliva-derived mLOY is significantly associated with aging and increased physical activity, but not with smoking. While these data support the robust association between saliva-derived mLOY and aging, caution is required when comparing data from saliva-derived and blood-derived mLOY.

The composition of the oral milieu reflects oral health. Saliva provides an environment for multiple microorganisms, and contains soluble factors and immune cells. Neutrophils, which rapidly react on the changes in the microenvironment, are a major immune cell population in saliva and thus may serve as a biomarker for oral pathologies. This review focuses on salivary neutrophils in the oral cavity, their phenotype changes in physiological and pathological conditions, as well as on factors regulating oral neutrophil amount, activation and functionality, with special emphasis on oral cancer and its risk factors., (Copyright © 2020 Domnich, Riedesel, Pylaeva, Kürten, Buer, Lang and Jablonska.)

Human microbiome contains billions of microorganisms that play important roles in the biological system and different diseases. Due to its complexity, conventional culture-independent technology may underestimate the value of low-abundance bacteria, which calls for a highly efficient method for its enrichment and comprehensive analysis. In this study, we developed a recycling free-flow isoelectric focusing (RFFIEF) method-based electrophoresis method to separate salivary microbiome. First, we used Escherichia coli (DH5α) as a model for RFFIEF method development, which was focused in a narrow pH range (0.38 pH unit). The recovery rate was 80.81% with 5.85% relative standard deviation ( n = 5). The optimized method was then adopted to separate the human salivary microbiome into 32 fractions, followed by 16 S rRNA gene sequencing and metaproteomics analysis. After RFFIEF fractionation, we identified 508 bacterial genera, which increased by 225% on average ( n = 3) when compared to the results before fractionation. We further compared the compositional change of microbiome in the saliva of lung cancer group ( n = 22) and control group ( n = 21) through RFFIEF. Quantitative results demonstrated that six bacterial genera were upregulated dramatically in the lung cancer group, while two genera were downregulated. Through qPCR verification in an independent sample set ( n = 48), we confirmed that genus Granulicatella was significantly upregulated in the lung cancer group, whereas Pseudomonas was remarkably downregulated ( p < 0.001). RFFIEF is an efficient and reproducible technology to fractionate the microbiome for its comprehensive analysis, which can be further applied to the in-depth study of the complex microbiomes and contribute to the discovery of disease-associated bacteria.

Salivary exosomes are extracellular vesicles (EVs) with abundant CD63 immunoreactivity on their surface. Based on their size and protein composition, these exosomes can be categorized into two classes of exosomes I (mean diameter of 83.5 nm) and II (mean diameter of 40.5 nm). We have attempted to review the features of these exosomes, including origin, composition, separation methods, and their application in medicine. Not only the composition of salivary exosomes is invaluable in term of diagnosis, but can also afford an understanding in roles of the contents and components of these exosomes in the fundamental pathophysiologic processes of different diseases. since these EVs can cross the epithelial barriers they may be essential for transporting of multifarious components from the blood into saliva. Thus, in comparison to other bodily fluids, salivary exosomes are probably a better and accessible tool to examine the function of exosomes in the diagnosis and treatment of disease.

Purpose: The aim of this study is to investigate the effect of an edible saliva substitute, oral moisturizing jelly (OMJ), and a topical saliva gel (GC) on dry mouth, swallowing ability, and nutritional status in post-radiotherapy head and neck cancer patients., Methods: Sixty-two post-radiation head and neck cancer patients with xerostomia completed a blinded randomized controlled trial. They were advised to swallow OMJ (n = 31) or apply GC orally (n = 31) for 2 months. Outcome measures were assessed at baseline, 1, and 2 months, including subjective and objective dry mouth (Challcombe) scores, subjective swallowing problem scores (EAT-10), water swallowing time, clinical nutritional status (PG-SGA), body weight, and dietary intake., Results: After 1 and 2 months of interventions, subjective and objective dry mouth scores, subjective swallowing problem scores, swallowing times, and clinical nutritional status in both groups were significantly improved (p < 0.0001). Compared to GC, OMJ group had higher percent improvement in all outcome measures (p < 0.001) except swallowing time and clinical nutritional status. Interestingly, subjective dry mouth scores were significantly correlated with subjective swallowing problem scores (r = 0.5321, p < 0.0001)., Conclusions: Continuous uses of saliva substitutes (OMJ or GC) for at least a month improved signs and symptoms of dry mouth and enhanced swallowing ability. An edible saliva substitute was superior to a topical saliva gel for alleviating dry mouth and swallow problems. These lead to improved clinical nutritional status. Thus, palliation of dry mouth may be critical to support nutrition of post-radiotherapy head and neck cancer patients., Clinical Trial Registry: Clinicaltrials.gov NCT03035825.

Background: Enhancing angiogenesis is critical for accelerating wound healing. Application of different types of exosomes (Exos) to promote angiogenesis represents a novel strategy for enhanced wound repair. Saliva is known to accelerate wound healing, but the underlying mechanisms remain unclear., Results: Our results have demonstrated that saliva-derived exosomes (saliva-Exos) induce human umbilical vein endothelial cells (HUVEC) proliferation, migration, and angiogenesis in vitro, and promote cutaneous wound healing in vivo. Further experiments documented that Ubiquitin-conjugating enzyme E2O (UBE2O) is one of the main mRNAs of saliva-Exos, and activation of UBE2O has effects similar to those of saliva-Exos, both in vitro and in vivo. Mechanistically, UBE2O decreases the level of SMAD family member 6 (SMAD6), thereby activating bone morphogenetic protein 2 (BMP2), which, in turn, induces angiogenesis., Conclusions: The present work suggests that administration of saliva-Exos and UBE2O represents a promising strategy for enhancing wound healing through promotion of angiogenesis.

This pilot study aims to investigate whether salivary small extracellular vesicle (sEV)-associated microRNAs could act as potential biomarkers for periodontal disease status. Twenty-nine participants (10 who were healthy, nine with gingivitis, 10 with stage III/IV periodontitis) were recruited and unstimulated whole saliva samples were collected. Salivary sEVs were isolated using the size-exclusion chromatography (SEC) method and characterised by morphology, EV-protein and size distribution using transmission electron microscopy (TEM), Western Blot and Nanoparticle Tracking Analysis (NTA), respectively. Ten mature microRNAs (miRNAs) in salivary sEVs and saliva were evaluated using RT-qPCR. The discriminatory power of miRNAs as biomarkers in gingivitis and periodontitis versus healthy controls was evaluated by Receiver Operating Characteristics (ROC) curves. Salivary sEVs were comparable to sEVs morphology, mode, size distribution and particle concentration in healthy, gingivitis and periodontitis patients. Compared to miRNAs in whole saliva, three significantly increased miRNAs (hsa-miR-140-5p, hsa-miR-146a-5p and hsa-miR-628-5p) were only detected in sEVs in periodontitis when compared to that of healthy controls, with a good discriminatory power (area under the curve (AUC) = 0.96) for periodontitis diagnosis. Our study demonstrated that salivary sEVs are a non-invasive source of miRNAs for periodontitis diagnosis. Three miRNAs that are selectively enriched in sEVs, but not whole saliva, could be potential biomarkers for periodontal disease status.

Gastrointestinal (GI) symptoms are common in anorexia nervosa (AN), can predate illness onset, complicate renourishment, and persist after recovery. We explore how, through processes of aversive visceral conditioning, early GI pain and discomfort may increase vulnerability to AN in some individuals. Processes include enhanced preoccupation with the gut resulting from aversive visceral memories and disruptions in the typical acquisition of self-attunement when children learn to map and interpret interoceptive sensations and develop adaptive actions. We question whether a fear of weight gain, in some cases, may be an epiphenomenon of the recapitulation of actual or perceived GI symptoms that is especially relevant during puberty, especially in girls. This conceptualization has immediate clinical implications and offers ideas for future research. We propose that GI discomfort associated with renourishment may reignite prior aversive visceral experiences. We encourage development of a formulation that organizes the individual's current experience of the body with respect to these prior aversive experiences. Our conceptualization underscores the importance of assessment of GI experiences in individuals with AN; the examination of dietary strategies that minimize GI symptoms and enhance renourishment efficacy; and strategies that attempt to alter this aversive visceral conditioning by mapping sensations to meanings and adaptive actions., (© 2020 Wiley Periodicals, Inc.)

Objective: Cigarette tobacco smoking has been shown to cause cancer through different mechanisms that include epigenetic modulation of tumor-suppressor genes. In the present study, the association between global and MLH1 gene promoter methylation and waterpipe tobacco smoking was investigated. Materials and Methods: Blood lymphocytes and oral epithelium were sampled from 150 pure waterpipe smokers and 150 never-smokers from Jordan. Methylation assessment was performed using the methylation-specific PCR technique for MLH1 gene and ELISA for global DNA methylation. Results: Significant increases were shown in global DNA methylation as measured in blood lymphocytes ( p  < 0.01). In addition, increases in MLH1 gene promoter methylation among waterpipe smokers compared to nonsmokers ( p  < 0.001) in both oral epithelium and blood lymphocytes was also observed. In addition, strong correlation was found between LWDS-10J dependence score and magnitude of promoter specific methylation of MLH1 (r2 = 0.74-0.78, p  < 0.001). Moreover, the percentage of methylated MLH1 promoter was not affected by age or gender ( p  > 0.05). Discussion and Conclusion: Collectively, the results indicate that waterpipe tobacco use is associated with epigenetic changes that might predispose users to lung and blood cancers. The results highlight the need for actions to discourage waterpipe smoking and can be used in cessation interventions that target this type of smoking.

Oxidative stress biomarkers can be found at detectable concentrations in saliva. These salivary biomarkers reflect specific oxidation pathways associated with caries and periodontitis. Our study evaluated the influence of dental caries severity (assessed using the ICCMS™ criteria) on the levels of oxidative stress biomarkers in saliva from children. Unstimulated saliva samples were collected from patients (from one to three years old) in a day care center in Birigui, SP, Brazil, two hours after fasting. Children were divided into four groups ( n = 30/group), according to caries severity: caries free (group A), early carious lesions (group B), moderate carious lesions (group C), and advanced carious lesions (group D). The following salivary biomarkers were determined: total proteins (TP), measured by the Lowry method; oxidative damage, measured by the TBARS method; total antioxidant capacity (TAC); superoxide dismutase (SOD) enzymatic antioxidant activity; and uric acid (UA) non-enzymatic antioxidant activity. Data were analyzed by ANOVA, followed by the Student-Newman-Keuls test, Pearson and Spearman correlation coefficients, and multivariable linear regression ( p < 0.05). TP, TAC, SOD enzymatic antioxidant activity, and UA non-enzymatic antioxidant activity increased with caries severity, consequently reducing salivary oxidative damage. It was concluded that higher caries severity increases salivary antioxidant system activity, with consequent reduction in salivary oxidative damage., Competing Interests: All authors declare that they have no conflicts of interest., (Copyright © 2020 Heitor Ceolin Araujo et al.)

Alzheimer's disease (AD) is a progressive and multifactorial disease. Many scientific advances have advanced our understanding of the pathogenesis of AD. However, the clinical diagnosis of AD remains difficult, with only post-mortem assays confirming its definitive diagnosis. There is a crucial need for an early and accurate detection of AD related symptoms. To date, current diagnosis techniques are costly or invasive. Finding a peripheral biomarker that could provide a sensitive, reproducible, and accurate detection prior to the onset of the AD clinical symptoms will allow identification of "at risk" individuals, thereby facilitating early initiation of treatments that may prove more effective. Salivary glands contain stem cells, which are affected by aging, suggesting that tissue samples from these glands may reveal a stem cell biomarker of AD, but also stem cells may be harvested from these glands, with proper timing and isolation technique, for cell-based regenerative medicine. Alternatively, instead of the salivary glands, saliva may represent an attractive source for biomarkers due to minimal discomfort to the patient, non-invasive collection, and the possibility of cost-effective screening large populations, encouraging greater compliance in clinical trials and frequent testing. In addition, salivary glands contain stem cells, which are likely also present in the saliva, making these cells as potentially sensitive cellular biomarker of and a therapeutic agent for AD. The aim of this review is to critically analyze the use of saliva for the identification of circulating biological markers to help the diagnosis of early cognitive impairment associated with AD and to generate insights into the potential application of stem cells derived from salivary glands or saliva as therapeutics (i.e., stem cell transplantation) for the disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study tested the feasibility of collecting saliva samples from Pacific Islanders (PIs) via a community-based participatory research approach. Collection of saliva samples were conducted by trained and trusted PI community leaders at various partner sites. A total of 214 saliva samples were donated by PIs living in Southern California, more than half of whom were females between the ages of 18 and 35 years. Donors indicated that they donated because they wanted to help science and their community. A majority of donors reported a very positive experience with the donation process and were willing to donate saliva and hair samples in the future. The positive findings of this article highlight the importance of community input and participation.

Despite the proven role of oxidative stress in numerous systemic diseases and in the process of aging, little is still known about the salivary redox balance of healthy children, adults, and the elderly. Our study was the first to assess the antioxidant barrier, redox status, and oxidative damage in nonstimulated (NWS) and stimulated (SWS) saliva as well as blood samples of healthy individuals at different ages. We divided 90 generally healthy people into three equally numbered groups based on age: 2-14 (children and adolescents), 25-45 (adults), and 65-85 (elderly people). Antioxidant enzymes (salivary peroxidase (Px), glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase-1 (SOD)), nonenzymatic antioxidants (reduced glutathione (GSH) and uric acid (UA)), redox status (total antioxidant capacity (TAC), total oxidant status (TOS), and oxidative stress index (OSI)), and oxidative damage products (advanced glycation end products (AGE), advanced oxidation protein products (AOPP), and malondialdehyde (MDA)) were evaluated in NWS and SWS as well as in erythrocyte/plasma samples. We demonstrated that salivary and blood antioxidant defense is most effective in people aged 25-45. In the elderly, we observed a progressive decrease in the efficiency of central antioxidant systems (↓GPx, ↓SOD, ↓GSH, and ↓TAC in erythrocytes and plasma vs. adults) as well as in NWS (↓Px, ↓UA, and ↓TAC vs. adults) and SWS (↓TAC vs. adults). Both local and systemic antioxidant systems were less efficient in children and adolescents than in the group of middle-aged people, which indicates age-related immaturity of antioxidant mechanisms. Oxidative damage to proteins (↑AGE, ↑AOPP) and lipids (↑MDA) was significantly higher in saliva and plasma of elderly people in comparison with adults and children/adolescents. Of all the evaluated biomarkers, only salivary oxidative damage products generally reflected their content in blood plasma. The level of salivary redox biomarkers did not vary based on gender., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 Mateusz Maciejczyk et al.)

Infants undergo extensive developments during their first year of life. Although the biological mechanisms involved are not yet fully understood, changes in the DNA methylation in mammals are believed to play a key role. This study was designed to investigate changes in infant DNA methylation that occurs between 6 and 52 weeks. A total of 214 infant saliva samples from 6 or 52 weeks were assessed using principal component analyses and t-distributed stochastic neighbor-embedding algorithms. Between the two time points, there were clear differences in DNA methylation. To further investigate these findings, paired two-sided student's t-tests were performed. Differently methylated regions were defined as at least two consecutive probes that showed significant differences, with a q-value < 0.01 and a mean difference > 0.2. After correcting for false discovery rates, changes in the DNA methylation levels were found in 42 genes. Of these, 36 genes showed increased and six decreased DNA methylation. The overall DNA methylation changes indicated decreased gene expression. This was surprising because infants undergo such profound developments during their first year of life. The results were evaluated by taking into consideration the extensive development that occurs during pregnancy. During the first year of life, infants have an overall three-fold increase in weight, while the fetus develops from a single cell into a viable infant in 9 months, with an 875-million-fold increase in weight. It is possible that the findings represent a biological slowing mechanism in response to extensive fetal development. In conclusion, our study provides evidence of DNA methylation changes during the first year of life, representing a possible biological slowing mechanism. We encourage future studies of DNA methylation changes in infants to replicate the findings by using a repeated measures model and less stringent criteria to see if the same genes can be found, as well as investigating whether other genes are involved in development during this period.

Background: Hazardous alcohol consumption has significant adverse medical consequences. These effects may be mediated, in part, by alterations in DNA methylation. Thus, DNA methylation signatures in peripheral cells may provide biomarkers of the medical impact of alcohol use and the risk for future alcohol consumption., Method: Using a high-density methylation array, we characterized epigenome-wide DNA methylation in saliva cells with respect to alcohol consumption in a large cohort of male European American veterans. In this study, DNA methylation of over 870,000 CpG DNA sites was profiled in 1,135 European American men. Alcohol consumption was assessed using the Alcohol Use Disorder Identification Test-Consumption (AUDIT-C). Linear regression was applied in an epigenome-wide association study (EWAS), adjusted for confounders. Gene set enrichment analysis was performed in the KEGG database with a correction for gene length., Results: We found that a total of 70 CpG sites reached EWAS-corrected significance (p < 6E-08) with small effects on alcohol consumption for individual CpG sites, including 64 new CpG sites and 6 CpG sites that were previously reported as associated with alcohol use disorder, liver function, body mass index, and lipid metabolism. The most significant CpG site was located in SLC7A11 (t = -11.34, p = 2.66E-28), a gene involved specifically in cysteine and glutamate transportation. The 70 significant CpG sites were located on 44 genes, including genes involved in amino acid transport and metabolism systems. We identified 68 pathways with a false discovery rate < 0.05., Conclusions: We identified novel DNA methylation sites associated with alcohol consumption. Results may shed light on peripheral mechanisms of alcohol consumption on adverse health outcomes among heavy drinkers., (© 2019 by the Research Society on Alcoholism.)

Objective: To examine associations between maternal experiences of discrimination and child biomarkers of toxic stress in a multiethnic, urban sample of mothers and children (4-9 years)., Methods: Data were drawn from a cross-sectional study of maternal-child dyads (N = 54) living in low-income neighborhoods in New Haven, Connecticut, USA. Mothers reported experiences of discrimination. Noninvasive biomarkers of toxic stress were collected to assess neuroendocrine (hair cortisol), immune (salivary cytokines, c-reactive protein), and cardiovascular (blood pressure) functioning in children., Results: Maternal experiences of discrimination were associated with increased log-transformed salivary interleukin-6 (IL-6) levels in children (β = 0.15, p = 0.02)., Conclusions: Vicarious racism, or indirect exposure to discrimination experienced by caregivers, is associated with poor health outcomes for children. Immune pathways may be a biological mechanism through which racial discrimination "gets under the skin," but additional research is needed to fully understand these relationships. Uncovering the physiological mechanisms linking vicarious racism with child health is an important step towards understanding possible early roots of racial and ethnic health inequities.

Severe oral problems, including oral mucositis (OM) and xerostomia, often occur after conditioning therapy for hematopoietic stem cell transplantation (HSCT). Saliva plays a major role in protecting the oral mucosa and teeth. Alterations in salivary flow rate or salivary components resulting in decreased salivary defence mechanisms may affect oral/mucosal health and may influence the severity of OM. A systematic review was conducted to assess the current scientific knowledge on changes in salivary function and composition before and after HSCT. All English or Dutch articles examining salivary flow rate or salivary components before and after HSCT were included after title/abstract selection by 2 independent reviewers (weighted κ = .91). After quality assessment and exclusion of all research groups with both children age <14 years and adults, 33 articles were included for data analysis. Overall, the salivary flow rate was decreased at several days and months after HSCT. Although several salivary components were studied, most components were examined in only 1 or 2 studies with different patient populations or at different time points after HSCT. At 7 days after HSCT, albumin and proinflammatory cytokines were increased, whereas secretory IgA and components of the salivary antioxidant system were decreased. Secretory IgA levels were still reduced at 1 month after HSCT but returned to pre-HSCT values at 6 months after HSCT. Lactoferrin, secretory leukocyte protease inhibitor, and β 2 -microglobulin levels were increased at 6 months after HSCT. Our findings show that changes in saliva reflect an inflammatory response occurring immediately after HSCT, followed by evidence of increased salivary antimicrobial defense mechanisms by 6 months after HSCT., (Copyright © 2019 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Evaluating the genetic diversity of natural populations of endangered species is important for conservation. Although the genetic analysis of wildlife usually requires collecting DNA non-invasively, the variety of non-invasive DNA sampling methods is limited for each species. We present a method to obtain DNA of an endangered species, the bonobo (Pan paniscus), in which the pith of the terrestrial herbaceous vegetation (THV) that they consumed was newly utilized. We investigated the (1) frequency of encountering remnant saliva on three types of THV pith; (2) concentrations of DNA in the saliva samples by the real-time quantitative PCR; and (3) rates of positive PCR, accurate genotyping, and allelic drop out by analyzing two autosomal microsatellite loci (D7s817 and D9s910). The number of remnant saliva samples was recorded by following the bonobo groups on a daily basis. The frequency of encountering DNA samples was higher in saliva samples than in fecal samples. More than half of the saliva samples remaining on two types of THV pith provided sufficient concentrations of bonobo DNA (> 200 pg/μl). Rates of positive PCR and accurate genotyping were high, and allelic drop out rate was low when the amount of template DNA was above 200 pg per reaction. Our results suggest that the remnants of bonobo saliva on the pith of THV are a potential resource for obtaining DNA, and better than other kinds of samples from the perspective of the abundant sampling opportunities.

Cortisol, a steroid hormone, is a main biomarker of psychological stress. Early diagnosis and proper treatment of such stress is crucial to prevent the excessive secretion of cortisol. However, cortisol has a low molecular weight and cannot provide sufficient recognition sites for sandwich immunoreaction; it has previously been measured using a competitive immunoassay instead of a general sandwich immunoassay. The disadvantage of this approach is that quantitative measurements are limited because of the narrow measurable range that is key for biosensors. To overcome this limitation, we propose a new detection platform that enables small molecules such as cortisol to be quantified with high detection sensitivity. A trap lateral flow immunoassay (trapLFI) sensor has deletion and detection zones instead of the test and control zones in general lateral flow immunoassay (LFI) sensors. The conjugates used to minimize possible detection targets at low concentration are gold nanoparticles that include an antibody against cortisol and an enzyme for signal generation. Target-bound conjugates are captured in the detection zone, whereas conjugates not binding with targets are trapped in the deletion zone. Using this platform, enzyme-catalyzed color signals increase in the detection zone and decrease in the deletion zone with the concentration of cortisol. The ratio of signal from deletion zone and detection zone supplied a wide analytical range (0.01-100 ng mL-1) with high detection sensitivity (9.9 pg mL-1). Analysis of 15 human saliva samples showed a good correlation with conventional ELISA results (R2 = 0.9432).

Aim: An outstanding challenge in epigenome studies is the estimation of cell-type proportions in complex epithelial tissues., Materials & Methods: Here, we construct and validate a DNA methylation reference and algorithm for complex tissues that contain epithelial, immune and nonimmune stromal cells., Results: Using this reference, we show that easily accessible tissues such as saliva, buccal and cervix exhibit substantial variation in immune cell (IC) contamination. We further validate our reference in the context of oral cancer, where it correctly predicts an increased IC infiltration in cancer but suppressed in patients with highest smoking exposure. Finally, our method can improve the specificity of differentially methylated CpG calls in epithelial cancer., Conclusion: The degree and variation of IC contamination in complex epithelial tissues is substantial. We provide a valuable resource and tool for assessing the epithelial purity and IC contamination of samples and for identifying differential methylation in such complex tissues.

Essentials Human salivary extracellular vesicles (EVs) expose coagulant tissue factor (TF). Salivary EVs expose CD24, a ligand of P-selectin. CD24 and coagulant TF co-localize on salivary EVs. TF + /CD24 + salivary EVs bind to activated platelets and trigger coagulation., Summary: Background Extracellular vesicles (EVs) from human saliva expose coagulant tissue factor (TF). Whether such TF-exposing EVs contribute to hemostasis, however, is unknown. Recently, in a mice model, tumor cell-derived EVs were shown to deliver coagulant TF to activated platelets at a site of vascular injury via interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin. Objectives We hypothesized that salivary EVs may deliver coagulant TF to activated platelets via interaction with P-selectin. Methods We investigated the presence of two ligands of P-selectin on salivary EVs, PSGL-1 and CD24. Results Salivary EVs expose CD24 but PSGL-1 was not detected. Immune depletion of CD24-exposing EVs completely abolished the TF-dependent coagulant activity of cell-free saliva, showing that coagulant TF and CD24 co-localize on salivary EVs. In a whole blood perfusion model, salivary EVs accumulated at the surface of activated platelets and promoted fibrin generation, which was abolished by an inhibitory antibody against human CD24. Conclusions A subset of EVs in human saliva expose coagulant TF and CD24, a ligand of P-selectin, suggesting that such EVs may facilitate hemostasis at a site of skin injury where the wound is licked in a reflex action., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Xerostomia is a state of oral dryness associated with salivary gland dysfunction and is induced by stress, radiation and chemical therapy, various systemic and autoimmune diseases, and specific medications. Fluid secretion is interrupted by the stimulation of neurotransmitter-induced increase in cytosolic calcium ([Ca 2+ ] i ) in salivary gland acinar cells, prompting the mobilization of ion channels and their transporters. Salivary fluid and protein secretion are principally dependent on parasympathetic and sympathetic nerves. Various inflammatory cytokines allied with lymphocytic infiltration cause glandular damage and Sjogren's syndrome, an autoimmune exocrinopathy associated with hyposalivation. A defect in IP 3 Rs, a major calcium release channel, prompts inadequate agonist-induced [Ca 2+ ] i in acinar cells and deters salivary flow. The store-operated calcium entry-mediated Ca 2+ movement into the acini activates K + and Cl - channels, which further opens a water channel protein, aquaporin-5, and triggers the release of fluid secretion from the salivary glands. The cellular mechanism of salivary gland dysfunction and hyposalivation has not yet been elucidated. In this review, we focused mainly on the proteins responsible for deficient saliva, the correlation between inflammation and salivation, autoimmune disorders and other ailments or complications associated with hyposalivation., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

The ability to diagnose oral lichen planus (OLP) based on saliva analysis using THz time-domain spectroscopy and chemometrics is discussed. The study involved 30 patients (2 male and 28 female) with OLP. This group consisted of two subgroups with the erosive form of OLP (n  =  15) and with the reticular and papular forms of OLP (n  =  15). The control group consisted of six healthy volunteers (one male and five females) without inflammation in the mucous membrane in the oral cavity and without periodontitis. Principal component analysis was used to reveal informative features in the experimental data. The one-versus-one multiclass classifier using support vector machine binary classifiers was used. The two-stage classification approach using several absorption spectra scans for an individual saliva sample provided 100% accuracy of differential classification between OLP subgroups and control group., ((2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

Objectives: Saliva contains biomarkers for systemic as well as oral diseases. This study was undertaken to assess the variability in the sources of such biomarkers (plasma, cells) and attempted to identify saliva deterioration markers in order to improve saliva diagnostic outcomes., Materials and Methods: Inter- and intrasubject variations in salivary gingival crevicular fluid levels were determined by measuring salivary albumin and transferrin levels. The purity of collected glandular secretions was determined by bacterial culture, and the variability in epithelial cell numbers by cell counting and optical density measurement. Saliva sample deterioration markers were identified by RP-HPLC and LC-ESI-MS/MS., Results: Tenfold variations were observed in plasma-derived albumin and transferrin levels, emphasizing the need for biomarker normalization with respect to plasma contributions to saliva. Epithelial cell levels varied 50-fold in samples collected before and after a meal. Salivary fungal levels varied within subjects and among subjects from 0 to >1,000 colony-forming units per milliliter. In saliva samples incubated for various time intervals at 37°C, five peptides were identified that steadily increased in intensity over time and which could be explored as "deterioration markers.", Conclusion: Taking saliva characteristics appropriately into account will help realize the promise that this body fluid is suitable to be exploited for reliable healthcare monitoring and surveillance., (Published 2017. This article is a U.S. Government work and is in the public domain in the USA.)

Healthy human skin sustains an effective immune defense mechanism, formed by a complex physical and chemical epidermal barrier that coordinates with different cellular components of the skin immune system. However, the mechanism by which skin cells regulate local immune homeostasis in health and disease contexts is not well known. To investigate whether exosomes exist in sweat, sweat samples from healthy individuals were collected after aerobic exercise. Sweat exosome was isolated via differential ultracentrifugation, observed under transmission electron microscopy, measured by dynamic light scattering, and confirmed by immunoblot. Further, shotgun liquid chromatography (LC)-mass spectrometry (MS)/MS analysis was conducted to investigate the proteomic profiling of sweat exosome. Secreted exosome was detected in human sweat. A total of 1,062 proteins were identified in sweat exosome, including 997 different proteins compared with sweat proteomics and 896 unique proteins compared with urine, saliva, and plasma exosomes. Diverse antimicrobial peptides and immunological factors were found in sweat exosome, suggesting the involvement of exosome in skin immunity. This study provides direct evidence that secreted exosomes exist in human sweat. The proteomic profiling of sweat exosome provides insight into sweat features and the potential physiological significance of exosomes in immune homeostasis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In Spain, two smoke-free laws have been passed (Law 28/2005 and Law 42/2010).This study evaluates the association between Spanish smoking legislations and the second-hand smoke (SHS) exposure in an adult non-smoking population cohort in Barcelona (Spain)., Methods: This is a longitudinal study, before and after the implementation of two national smoking bans, in a representative sample of adults (≥16years old) from Barcelona (Spain) surveyed in 2004-2005 and followed up in 2013-2014 (n=736). We only analyzed non-smokers (n=397). We obtained 9ml of saliva sample for analysis of cotinine, a biomarker of recent tobacco exposure. We calculated geometric means of salivary cotinine concentration and their geometric standard deviation. We used linear mixed effect models, with individuals as random effects, to model the percentage change in salivary cotinine concentration and their 95% confidence intervals., Results: The percentage of participants with saliva samples with measurable concentrations of cotinine fell from 92.4% to 64.2% after both Spanish smoking legislations. The geometric mean of salivary cotinine concentration significantly decreased 88% (from 0.98ng/mL to 0.12ng/mL, p<0.001) after the implementation of the two Spanish smoke-free legislations. The decrease of the GM salivary cotinine concentration was statistically significant independently of the sociodemographic variables., Conclusion: There was a large reduction in the salivary cotinine concentration among adult non-smokers and higher cotinine concentrations among those declaring exposure to SHS at home after both legislations. Moreover, after both Spanish smoke-free laws salivary cotinine concentration was homogenized according to sociodemographic variables., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Aims: Insulin resistance (IR) detection is challenging and no test is currently used in clinical practice. We developed salivary biomarkers that could be used for IR detection., Methods: We collected saliva from 186 healthy and 276 pre-diabetic participants, divided them into high and low IR groups based on a HOMA cutoff of 2.5. We profiled extracellular transcriptome by microarray in saliva supernatant from 23 high IR and 15 low IR participants, and pre-validated the top ten extracellular mRNA (exRNA) markers in a new cohort of 40 high and 40 low IR participants. A prediction panel was then built and validated in an independent cohort of 149 high and 195 low IR participants., Results: Transcriptomic analyses identified 42 exRNA candidates differentially present in saliva of high and low IR participants. From the top ten candidates, six were individually validated (PRKCB, S100A12, IL1R2, CAMP, VPS4B, CAP1) (p<0.01) and yielded AUC values ranging from 0.66 to 0.76. Body mass index (BMI) was significant higher in high compared to low IR group with AUC of 0.66, and showed no correlation with any of candidate biomarkers. The combination of four exRNA markers (IL1R2, VPS4B, CAP1, LUZP6) with BMI achieved excellent results in the prediction panel building dataset (AUC=0.79, sensitivity=79%, specificity=64%). The prediction model was validated in an independent cohort (AUC=0.82, sensitivity=63%, specificity=92%)., Conclusions: A panel of four salivary exRNA biomarkers (IL1R2, VPS4B, CAP1, LUZP6) and BMI was validated that can distinguish high and low IR participants, overall and in subgroups of healthy and pre-diabetic participants., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Several articles suggest that DNA methylation levels in blood relate to Parkinson's disease (PD) but there is a need for a large-scale study that involves suitable population based controls. The purposes of the study were: (1) to study whether PD status is associated with DNA methylation levels in blood/saliva; (2) to study whether observed associations relate to blood cell types; and (3) to characterize genome-wide significant markers ("CpGs") and clusters of CpGs (co-methylation modules) in terms of biological pathways., Methods: In a population-based case control study of PD, we studied blood samples from 335 PD cases and 237 controls and saliva samples from another 128 cases and 131 controls. DNA methylation data were generated from over 486,000 CpGs using the Illumina Infinium array. We identified modules of CpGs (clusters) using weighted correlation network analysis (WGCNA)., Results: Our cross-sectional analysis of blood identified 82 genome-wide significant CpGs (including cg02489202 in LARS2 p = 8.3 × 10 -11 and cg04772575 in ABCB9 p = 4.3 × 10 -10 ). Three out of six PD related co-methylation modules in blood were significantly enriched with immune system related genes. Our analysis of saliva identified five significant CpGs. PD-related CpGs are located near genes that relate to mitochondrial function, neuronal projection, cytoskeleton organization, systemic immune response, and iron handling., Conclusions: This study demonstrates that: (1) PD status has a profound association with DNA methylation levels in blood and saliva; and (2) the most significant PD-related changes reflect changes in blood cell composition. Overall, this study highlights the role of the immune system in PD etiology but future research will need to address the causal structure of these relationships.

Objectives: We aimed to assess the cross-sectional relation between levels of cortisol and specific symptom clusters, symptom severity and duration of symptoms in patients with medically unexplained physical symptoms (MUPS)., Methods: Baseline data of a cohort of MUPS patients were used. We chose the Cortisol Awakening Response (CAR) as a cortisol parameter, using saliva samples. We used confirmatory factor analysis for the identification of 4 specific symptom clusters: (1) gastro-intestinal symptoms; (2) pain; (3) cardio-pulmonary symptoms; and (4) fatigue. For this factor analysis we used the Physical Symptom Questionnaire (PSQ), which assesses the occurrence and frequency of 51 physical symptoms. Symptom severity was measured with the Patient Health Questionnaire-15 (PHQ-15). Duration of symptoms was based on self-reported duration of top 3 symptoms. We performed multiple linear regression to assess relations between CAR and individual factor scores on symptom clusters, symptom severity and duration of symptoms., Results: Data from 296 patients (76% female) were included in the analyses. The majority of patients suffered from symptoms in multiple organ systems. Factor analysis confirmed that the model with 4 symptom clusters fitted our data. For the total study population, we found no significant relation between CAR and participants' factor scores on any of the symptom clusters. We also found no significant relations between CAR and severity or duration of symptoms., Conclusion: Our results suggest that within a heterogeneous MUPS population there is no relation between CAR and symptom severity and duration. However, more studies are needed to confirm our findings., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Latest genotyping technologies allow to achieve a reliable genetic profile for the offender identification even from extremely minute biological evidence. The ultimate challenge occurs when genetic profiles need to be retrieved from a mixture, which is composed of biological material from two or more individuals. In this case, DNA profiling will often result in a complex genetic profile, which is then subject matter for statistical analysis. In principle, when more individuals contribute to a mixture with different biological fluids, their single genetic profiles can be obtained by separating the distinct cell types (e.g. epithelial cells, blood cells, sperm), prior to genotyping. Different approaches have been investigated for this purpose, such as fluorescent-activated cell sorting (FACS) or laser capture microdissection (LCM), but currently none of these methods can guarantee the complete separation of different type of cells present in a mixture. In other fields of application, such as oncology, DEPArray™ technology, an image-based, microfluidic digital sorter, has been widely proven to enable the separation of pure cells, with single-cell precision. This study investigates the applicability of DEPArray™ technology to forensic samples analysis, focusing on the resolution of the forensic mixture problem. For the first time, we report here the development of an application-specific DEPArray™ workflow enabling the detection and recovery of pure homogeneous cell pools from simulated blood/saliva and semen/saliva mixtures, providing full genetic match with genetic profiles of corresponding donors. In addition, we assess the performance of standard forensic methods for DNA quantitation and genotyping on low-count, DEPArray™-isolated cells, showing that pure, almost complete profiles can be obtained from as few as ten haploid cells. Finally, we explore the applicability in real casework samples, demonstrating that the described approach provides complete separation of cells with outstanding precision. In all examined cases, DEPArray™ technology proves to be a groundbreaking technology for the resolution of forensic biological mixtures, through the precise isolation of pure cells for an incontrovertible attribution of the obtained genetic profiles., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Chen, Y, Hill, HZ, Lange, G, and Falvo, MJ. Salivary mitochondrial DNA copy number is associated with exercise ventilatory efficiency. J Strength Cond Res 31(7): 2000-2004, 2017-Mitochondrial DNA copy number (mtDNAcn) is an index of mitochondrial content and is responsive to changes in exercise training volume. Therefore, assessment of mtDNAcn may help to optimize exercise prescription and aid in athlete monitoring. Although previous work has assessed mtDNAcn derived from skeletal muscle and blood using invasive approaches, no study has examined salivary mtDNAcn and its relationship with sport performance. Fifteen adults (32.2 ± 7.1 years) volunteered to participate in this study. Each participant provided a saliva sample for the analysis of mtDNAcn via real-time polymerase reaction. In addition, participants completed an exercise challenge test to assess oxygen consumption relative to body weight (V[Combining Dot Above]O2·kg) and ventilatory efficiency (VE/V[Combining Dot Above]CO2). Using multiple linear regression, we examined the association of V[Combining Dot Above]O2·kg and VE/V[Combining Dot Above]CO2 with salivary mtDNAcn, adjusting for self-reported physical activity (min·wk). Greater mtDNAcn was associated with lower VE/V[Combining Dot Above]CO2 (p < 0.01) and higher V[Combining Dot Above]O2·kg (p < 0.05). In our model adjusted for physical activity, greater mtDNAcn remained associated with lower VE/V[Combining Dot Above]CO2 (β = -0.186; 95% confidence interval [CI], -0.348 to -0.025; p < 0.05), but not with V[Combining Dot Above]O2·kg (β = -0.022; 95% CI, -0.113 to 0.063). Our findings suggest that salivary mtDNAcn is associated with ventilatory efficiency, which may reflect enhanced exercise efficiency as a consequence of greater total mitochondrial content. As saliva collection is noninvasive, stable at room temperature, and less costly in comparison to skeletal muscle and blood, future studies may consider using saliva for the evaluation of mitochondrial content for the purposes of monitoring exercise training as well as optimizing exercise prescription.

Fc receptor-like protein 4 (FcRL4) is normally expressed on a small subset of mucosa-associated B-cells, as well as on mucosa-associated lymphoid tissue (MALT) lymphoma B-cells. Primary Sjögren's syndrome (pSS) patients have an increased risk of developing MALT lymphomas, preferentially in the parotid glands. For this reason we studied here by immunohistochemistry and mRNA analysis whether FcRL4 expressing B-cells are present in salivary gland tissue (labial and parotid) of pSS patients (n = 54) and non-pSS sicca patients (n = 16) and whether parotid gland MALT lymphomas in pSS patients (n = 49) also express this receptor. We also studied the effect of treatment (rituximab and abatacept) on the presence of FcRL4 + B-cells, and whether numbers in labial gland biopsies at time of diagnosis of pSS can predict whether patients are at risk for MALT lymphoma development. We demonstrate that FcRL4 + cells are present in salivary gland tissue of pSS patients where they are closely associated with ductal epithelial cells forming lymphoepithelial lesions. The glandular FcRL4 + cells are highly proliferative, genuine PAX5 + B-cells. These FcRL4 + B-cells are far more frequent in parotid gland than in labial gland tissue (p = 0.003). We further show that expression of FcRL4 is present in pSS-related parotid MALT lymphomas. The FcRL4 mRNA expression level in parotid MALT lymphoma is increased compared to parotid gland tissue of pSS patients without lymphoma (p = 0.017). However, numbers of FcRL4 + B-cells in labial gland biopsies taken at the time of pSS diagnosis, are not predictive for later development of MALT lymphoma. Reduction of parotid gland FcRL4 + B-cells by rituximab, but not abatacept is accompanied by restoration of the glandular epithelium, illustrating the crosstalk between these B-cells with the ductal cells. In conclusion, intraepithelial FcRL4 + B-cells are present in the salivary glands of pSS patients. These cells are likely involved in the epithelial changes seen in pSS. Their enrichment in parotid glands may explain why MALT lymphomas in pSS patients preferentially develop at this specific location., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background.: It is standard practice for laboratories to assess the cellular quality of expectorated sputum specimens to check that they originated from the lower respiratory tract. The presence of low numbers of squamous epithelial cells (SECs) and high numbers of polymorphonuclear (PMN) cells are regarded as indicative of a lower respiratory tract specimen. However, these quality ratings have never been evaluated for induced sputum specimens from children with suspected pneumonia., Methods.: We evaluated induced sputum Gram stain smears and cultures from hospitalized children aged 1-59 months enrolled in a large study of community-acquired pneumonia. We hypothesized that a specimen representative of the lower respiratory tract will contain smaller quantities of oropharyngeal flora and be more likely to have a predominance of potential pathogens compared to a specimen containing mainly saliva. The prevalence of potential pathogens cultured from induced sputum specimens and quantity of oropharyngeal flora were compared for different quantities of SECs and PMNs., Results.: Of 3772 induced sputum specimens, 2608 (69%) had <10 SECs per low-power field (LPF) and 2350 (62%) had >25 PMNs per LPF, measures traditionally associated with specimens from the lower respiratory tract in adults. Using isolation of low quantities of oropharyngeal flora and higher prevalence of potential pathogens as markers of higher quality, <10 SECs per LPF (but not >25 PMNs per LPF) was the microscopic variable most associated with high quality of induced sputum., Conclusions.: Quantity of SECs may be a useful quality measure of induced sputum from young children with pneumonia., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Carlson, LA, Lawrence, MA, LeCavalier, K, and Koch, AJ. Salivary lymphocyte responses following acute anaerobic exercise in a cool environment. J Strength Cond Res 31(5): 1236-1240, 2017-The purpose of this study was to examine the impact of anaerobic training on salivary lymphocytes (s-LYMPH), and further determine whether these responses differ between cool vs. thermoneutral environments. Nine lightly clothed (∼0.3 clo) volunteers (7/2 women/men: age, 21 ± 1 years; height, 168.7 ± 7.3 cm; weight, 66.4 ± 8.4 kg; body fat, 20.6 ± 7.6%) completed speed, agility, and quickness (SAQ) sessions in both warm (18.9° C; Biddeford) and cool (10.4° C; Thorsmörk) temperatures. The SAQ sessions consisted of 3 trials of 20-m sprints, 40-m sprints, t-tests, and box drills, and two 300-yd shuttle runs in both conditions. Saliva samples via passive drool were collected at baseline, immediately postexercise, and after 2 hours of recovery. The s-LYMPH increased (p < 0.001) immediately postexercise, followed by a decrease (p < 0.001) below baseline values after 2 hours of recovery in both environments. The s-LYMPH counts were lower (p < 0.001) for the cool environment than for the thermoneutral environment. The s-LYMPH counts increased postexercise, followed by a decrease after 2 hours of recovery regardless of environment. Acute anaerobic exercise induced transient changes in s-LYMPH counts similar to that observed in peripheral blood. Compared with baseline measures, changes in s-LYMPH were of a smaller magnitude after exercise in the cool environment compared with thermoneutral environment. In summary, there is no indication that exercise in the cool environment presented a greater challenge to the subjects' immunity. Rather, these data indicate exercise in a cool environment produces smaller fluctuations in salivary immune cells compared with resting levels.

Introduction: Intracellular lactoferrin (Lac) and lysozyme (Lys) content play an important role in regulating inflammation and promoting host protection. While exercise has demonstrated an increase in Lac and Lys concentration in exocrine solutions, little is known regarding intracellular concentration changes in response to exercise., Purpose: To quantify intracellular Lac and Lys concentration before and after exercise in salivary CD45 + CD15 + cells., Methods: 11 males (20.3 ± 0.8 years, 57.2 ± 7.6 mL/kg/min V̇O 2pk , 11.1 ± 3.9% body fat) ran for 45 min at 75% of VO 2pk . 12 mL of stimulated saliva were collected pre and immediately post exercise. Saliva was filtered through a 30-µm filter before analysis of leukocytes (CD45 + ) and granulocytes (CD45 + CD15 + ) using flow cytometry., Results: Median fluorescent intensity (MFI) of Lac increased from pre (64,268 ± 46,036 MFI) to post (117,134 ± 88,115 MFI) exercise (p <0.05). Lys MFI decreased with exercise (pre: 16,933 ± 8249; post: 11,616 ± 6875) (p <0.05)., Conclusion: Acute running resulted in an increased Lac concentration which could lead to a decrease in inflammation, adding further evidence of the anti-inflammatory effects of exercise. Conversely, the exercise-associated decrease of intracellular Lys content could be the cause of increased Lys in exocrine solutions.

Academic stress (AS) is one of the most important health problems experienced by students, but no biomarker of the potential psychological or physical problems associated with AS has yet been identified. As several cross-sectional studies have shown that psychiatric conditions accelerate aging and shorten telomere length (TL), we explored whether AS affected TL.Between June 2014 and July 2014, we recruited 200 junior high school students with imminent final examinations for participation in this study. The students were divided into three subgroups (mild, moderate, and severe anxiety) using the Sarason Test Anxiety Scale (TAS). Saliva samples were collected for TL measurement via quantitative polymerase chain reaction (qPCR).Students from both a specialized and a general school suffered from anxiety (p > 0.05). A total 35% had severe anxiety (score: 26.09±3.87), 33% had moderate anxiety (16.98±2.64), and 32% had mild anxiety (7.89±1.92). The TAS values differed significantly (p < 0.05) among the three subgroups, but the TLs of saliva cells differed only slightly (p > 0.05): 1.14±0.46 for those with severe anxiety, 1.02±0.40 for those with moderate anxiety, and 1.12±0.45 for those with mild anxiety.Previous reports have found that AS is very common in Asian adolescents. We found no immediate telomere shortening in adolescents with AS. Longitudinal observations are required to determine if TL is affected by AS.

Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells and detected in saliva. Pathophysiological roles for salivary exosomes are beginning to be recognized in diseases including cancer, highlighting potential biomarkers and biological functions. Since early detection of cancer is vital for successful treatment, salivary exosomes would be advantageous in achieving a better survival rate due to their ready availability and noninvasiveness. The use of salivary exosomes may therefore be promising in the accurate detection of premalignant lesions and early-stage cancers, also for better our understanding of the molecular basis of tumorigenesis. In this chapter, we review our current knowledge of salivaomics, focusing on nucleic acids and proteins in saliva as potential cancer biomarkers. Since salivaomics is a rapidly evolving field, we hope to expand frameworks toward salivary exosomes, integrate new and existing information, and bridge salivaomics with other biomedical researches. Furthermore, we would like to coin the term "saliva-exosomics" as the next-generation salivaomics. Our goal in this chapter is to provide the most updated information on cancer-derived exosomes in the saliva as natural carriers of biomarkers and signaling molecules. Major advances include definitive structure analysis and molecular characterization of salivary exosomes. We also highlight the exosome biogenesis and cargo trafficking mechanisms in which recent animal studies have expanded our understanding of exosome-mediated transfer of cancer-derived products from distal tumor to salivary gland. The potential roles of the salivary exosomes in cancer progression and immune surveillance are also addressed., (© 2017 Elsevier Inc. All rights reserved.)

Exosomes are derived from various sources, including primary and cultured cell lines and body fluids. It is now evident that they are important for communication between cells. They have, therefore, been proposed as potential carriers to deliver drugs to specific sites. In this study, we examined stability of exosomes derived from human saliva. Exosomes were stored at 4°C for up to 20 months and their membrane integrity assessed. Several exosomal markers, such as dipeptidyl peptidase IV (DPP IV; membrane marker) and programmed cell death 6-interacting protein (Alix, lumen marker), were retained intact after 20 months storage at 4°C. Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release of Alix into the supernatant. In contrast, sodium dodecyl sulfate treatment caused a complete disruption of the membrane. In addition, membrane stability was maintained after freezing and thawing. These results indicated that human saliva-derived exosomes are stable, maintaining their membrane integrity over a long storage period.

A composite transposon is a mobile genetic element consisting of two insertion sequences (ISs) flanking a segment of cargo DNA often containing antibiotic resistance (AR) genes. Composite transposons can move as a discreet unit. There have been recently several reports on a novel mechanism of movement of an IS26-based composite transposon through the formation of a translocatable unit (TU), carrying the internal DNA segment of a composite transposon and one copy of a flanking IS. In this study, we determined the presence of composite transposons and TUs in human oral metagenomic DNA using PCR primers from common IS elements. Analysis of resulting amplicons showed four different IS1216 composite transposons and one IS257 composite transposon in our metagenomic sample. As our PCR strategy would also detect TUs, PCR was carried out to detect circular TUs predicted to originate from these composite transposons. We confirmed the presence of two novel TUs, one containing an experimentally proven antiseptic resistance gene and another containing a putative universal stress response protein (UspA) encoding gene. This is the first report of a PCR strategy to amplify the DNA segment on composite transposons and TUs in metagenomic DNA. This can be used to identify AR genes associated with a variety of mobile genetic elements from metagenomes., (© FEMS 2016.)

Background: Cadmium and arsenic are ubiquitous metals commonly found in the environment which can harm human health. A growing body of research shows telomere length as a potential biomarker of future disease risk. Few studies have examined the effects of metals on telomere length and none have focused on adolescents., Objectives: In this study, the impact of cadmium and arsenic on salivary telomere length was studied in adolescents in Terai, Nepal., Methods: Adolescents aged 12-16 years old (n=351)were recruited where questionnaire interviews and both saliva and urine collection took place. Telomere length was determined by quantitative polymerase chain reaction using DNA extracted from saliva. Urinary cadmium and arsenic concentration were measured by inductively coupled plasma mass spectrometry. Multivariable linear regression was used to examine associations between urinary metals and salivary telomere length., Results: The geometric means and standard deviations of cadmium and arsenic were 0.33±0.33μg/g creatinine and 196.0±301.1μg/g creatinine, respectively. Urinary cadmium concentration was negatively associated with salivary telomere length after adjustment for confounders (β=-0.24, 95% CI -0.42,-0.07). Arsenic showed positive associations with telomere length but did not reach statistical significance., Conclusions: This is the first study to demonstrate that cadmium may shorten adolescent telomeres, even at exposure levels that may be considered low. These results agree with prior experimental and adult epidemiological studies, and also help identify the mechanism of DNA damage by cadmium. This study expanded current evidence on the harmful effects of cadmium exposure on telomere length even to adolescents., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Reference genes are needed as internal controls to determine relative expression for clinical application of gene expression panels. Candidate constitutively expressed genes must be validated as suitable reference genes in each body fluid and disease entity. Prior studies have predominantly validated oral squamous cell carcinoma associated messenger RNAs (mRNAs) based on quantitative polymerase chain reaction (qPCR) quantification cycle (Cq) values without adjustment for housekeeping genes., Methods: One hundred sixty eight patients had saliva collected before clinically driven biopsy of oral lesions suspicious for cancer. Seven potential housekeeping mRNAs and six pre-specified oral cancer associated mRNAs were measured with qPCR by personnel blinded to tissue diagnosis. Housekeeping gene stability was determined with the NormFinder program in a training set of 12 randomly selected cancer and 24 control patients. Genes with stability indices <0.02 were then tested in the validation set consisting of the remaining cancer and control patients and were further validated by the geNorm program. Cancer gene delta Cqs were compared in case and control patients after subtracting the geometric mean of the reference gene raw Cqs., Results: B2M and UBC had stability indices >0.02 in the training set and were not further tested. MT-ATP6, RPL30, RPL37A, RPLP0 and RPS17 all had stability indices <0.02 in the training set and in the verification set. The geNorm M values were all ≤1.10. All six pre-specified cancer genes (IL8, IL1, SAT, OAZ1, DUSP1 and S100P) were up-regulated in cancer versus control patients with from nearly twofold to over threefold higher levels (p<0.01 for all based on delta Cq values)., Conclusions: Five reference genes are validated for use in oral cancer salivary gene expression panels. Six pre-specified oral carcinoma associated genes are demonstrated to be highly significantly up-regulated in cancer patients based on delta Cq values. These cancer and reference genes are suitable for inclusion in gene expression panels for research and clinical applications., Trial Registration: ClinicalTrials.gov NCT01587573.

Objective: The protective mechanisms that maintain periodontal homeostasis in gingivitis and prevent periodontal tissue destruction are poorly understood. The aim of this study was to identify changes in the salivary proteome during experimental gingivitis., Study Design: We used oral neutrophil quantification and whole saliva (WS) proteomics to assess changes that occur in the inflammatory and resolution phases of gingivitis in healthy individuals. Oral neutrophils and WS samples were collected and clinical parameters measured on days 0, 7, 14, 21, 28, and 35., Results: Increased oral neutrophil recruitment and salivary cytoprotective proteins increased progressively during inflammation and decreased in resolution. Oral neutrophil numbers in gingival inflammation and resolution correlated moderately with salivary β-globin, thioredoxin, and albumin and strongly with collagen alpha-1 and G-protein coupled receptor 98., Conclusions: Our results indicate that changes in salivary cytoprotective proteins in gingivitis are associated with a similar trend in oral neutrophil recruitment and clinical parameters., Clinical Relevance: We found moderate to strong correlations between oral neutrophil numbers and levels of several salivary cytoprotective proteins both in the development of the inflammation and in the resolution of gingivitis. Our proteomics approach identified and relatively quantified specific cytoprotective proteins in this pilot study of experimental gingivitis; however, future and more comprehensive studies are needed to clearly identify and validate those protein biomarkers when gingivitis is active.

Background: Inorganic nitrate from exogenous and endogenous sources is accumulated in saliva, reduced to nitrite by oral bacteria and further converted to nitric oxide (NO) and other bioactive nitrogen oxides in the acidic gastric lumen. To further explore the role of oral microbiota in this process we examined the gastric mucus layer in germ free (GF) and conventional mice given different doses of nitrate and nitrite., Methods: Mice were given either nitrate (100mg/kg/d) or nitrite (0.55-11 mg/kg/d) in the drinking water for 7 days, with the lowest nitrite dose resembling the levels provided by swallowing of fasting saliva. The gastric mucus layer was measured in vivo., Results: GF animals were almost devoid of the firmly adherent mucus layer compared to conventional mice. Dietary nitrate increased the mucus thickness in conventional animals but had no effect in GF mice. In contrast, nitrite at all doses, restored the mucus thickness in GF mice to the same levels as in conventional animals. The nitrite-mediated increase in gastric mucus thickness was not inhibited by the soluble guanylyl cyclase inhibitor ODQ. Mice treated with antibiotics had significantly thinner mucus than controls. Additional studies on mucin gene expression demonstrated down regulation of Muc5ac and Muc6 in germ free mice after nitrite treatment., Conclusion: Oral bacteria remotely modulate gastric mucus generation via bioactivation of salivary nitrate. In the absence of a dietary nitrate intake, salivary nitrate originates mainly from NO synthase. Thus, oxidized NO from the endothelium and elsewhere is recycled to regulate gastric mucus homeostasis., (Copyright © 2015 Elsevier Inc. All rights reserved.)