Non-Coding RNA in Salivary Extracellular Vesicles: A New Frontier in Sjögren's Syndrome Diagnostics?

Sjögren's syndrome is an autoimmune rheumatic disease characterized by inflammation of the salivary and lacrimal glands, often manifesting as dry mouth and dry eyes. To simplify diagnostics of primary Sjögren's syndrome (pSS), a non-invasive marker is needed. The aim of the study was to compare the RNA content of salivary extracellular vesicles (EVs) between patients with pSS and healthy controls using microarray technology. Stimulated whole saliva was collected from 11 pSS patients and 11 age-matched controls. EV-RNA was isolated from the saliva samples using a Qiagen exoRNeasy Midi Kit and analyzed using Affymetrix Clariom D™ microarrays. A one-way ANOVA test was used to compare the mean signal values of each transcript between the two groups. A total of 9307 transcripts, coding and non-coding RNA, were detected in all samples. Of these transcripts, 1475 showed statistically significant differential abundance between the pSS and the control groups, generating two distinct EV-RNA patterns. In particular, tRNAs were downregulated in pSS patients, with the transcript tRNA-Ile-AAT-2-1 showing a 2-fold difference, and a promise as a potential biomarker candidate. This study therein demonstrates the potential for using salivary EV-RNA in pSS diagnostics.

Proteomic Profiling of Saliva and Tears in Radiated Head and Neck Cancer Patients as Compared to Primary Sjögren's Syndrome Patients.

Patients with head and neck cancer (HNC) and patients with primary Sjögren's syndrome (pSS) may exhibit similar symptoms of dry mouth and dry eyes, as a result of radiotherapy (RT) or a consequence of disease progression. To identify the proteins that may serve as promising disease biomarkers, we analysed saliva and tears from 29 radiated HNC patients and 21 healthy controls, and saliva from 14 pSS patients by mass spectrometry-based proteomics. The study revealed several upregulated, and in some instances overlapping, proteins in the two patient groups. Histone H1.4 and neutrophil collagenase were upregulated in whole saliva of both patient groups, while caspase-14, histone H4, and protein S100-A9 were upregulated in HNC saliva only. In HCN tear fluid, the most highly upregulated protein was mucin-like protein 1. These overexpressed proteins in saliva and tears play central roles in inflammation, host cell injury, activation of reactive oxygen species, and tissue repair. In conclusion, the similarities and differences in overexpressed proteins detected in saliva from HNC and pSS patients may contribute to the overall understanding of the different pathophysiological mechanisms inducing dry mouth. Thus, the recurring proteins identified could possibly serve as future promising biomarkers.

Characterization of Lipids in Saliva, Tears and Minor Salivary Glands of Sjögren's Syndrome Patients Using an HPLC/MS-Based Approach.

The diagnostic work-up of primary Sjögren's syndrome (pSS) includes quantifying saliva and tear production, evaluation of autoantibodies in serum and histopathological analysis of minor salivary glands. Thus, the potential for further utilizing these fluids and tissues in the quest to find better diagnostic and therapeutic tools should be fully explored. Ten samples of saliva and tears from female patients diagnosed with pSS and ten samples of saliva and tears from healthy females were included for lipidomic analysis of tears and whole saliva using high-performance liquid chromatography coupled to time-of-flight mass spectrometry. In addition, lipidomic analysis was performed on minor salivary gland biopsies from three pSS and three non-SS females. We found significant differences in the lipidomic profiles of saliva and tears in pSS patients compared to healthy controls. Moreover, there were differences in individual lipid species in stimulated saliva that were comparable to those of glandular biopsies, representing an intriguing avenue for further research. We believe a comprehensive elucidation of the changes in lipid composition in saliva, tears and minor salivary glands in pSS patients may be the key to detecting pSS-related dry mouth and dry eyes at an early stage. The identified differences may illuminate the path towards future innovative diagnostic methodologies and treatment modalities for alleviating pSS-related sicca symptoms.

Dental erosion in mice with impaired salivary gland function.

Objective: Salivary flow rate exerts an essential impact on the development and progression of dental erosion. In this work, the experimental dental erosion in non-obese diabetic (NOD) mice with reduced salivary flow rate was induced, and the erosive effect of acidic drinks on their dentition was studied.Material and methods: Three acidic drinks (sports drink, cola light drink and sugar containing cola drink) were given to adult NOD mice (groups: N = 11) as the only drink for 6 weeks. Two control groups were included; wild type and NOD control (groups: N = 9). Experimental and control (water) teeth were dissected out and observed by scanning electron microscopy (SEM). Mandibular first molars were subsequently embedded in Epon, ground transversely, observed again by SEM, and the enamel thickness and tooth height were measured.Results: Mandibular molars were considerably more eroded than maxillary molars. The erosive process started at the top of the cusps and subsequently extended in the cervical, mesio-distal, and pulpal direction. Erosive lesions were evident in increased succession from sports drink, cola light to cola drink exposed mandibular molars, with the lingual tooth height being approximately 23%, 26%, and 37% lower, respectively, compared to the control. The lingual enamel was approximately 48% thinner in sports drink molars and 62% thinner in cola light molars. In cola drink molars, the lingual enamel was totally eroded, and significant erosion of dentine was evident.Conclusion: Reduced salivary flow, together with a high consumption of acidic drinks, results in severe erosion of NOD mice molars.

Calcium signaling and cell volume regulation are altered in Sjögren's Syndrome.

OBJECTIVE: Sjögren's Syndrome (SS) is a chronic autoimmune disease, leading to deficient secretion from salivary and lacrimal glands. Saliva production is normally increased by cholinergic innervation, giving rise to intracellular calcium signaling and water transport through water channels (aquaporins, AQPs). The aim of this study was to investigate possible pathophysiological changes in cell volume regulation, AQP expression and localization, and intracellular calcium signaling in glandular cells from SS patients compared to controls. MATERIALS AND METHODS: A total of 35 SS patients and 41 non-SS controls were included. Real time qPCR was combined with immunohistochemistry to analyze the mRNA expression and cellular distribution of AQP1, 3 and 5. Cell volume regulation and intracellular calcium signaling were examined in fresh acinar cells. RESULTS: We show for the first time a reduced mRNA expression of AQP1 and 5 in SS compared to controls, accompanied by a decrease in staining intensity of AQP1, 3 and 5 in areas adjacent to local lymphocytic infiltration. Furthermore, we observed that the SS cells' capacity for volume regulation was abnormal. Similarly, the calcium response after parasympathetic agonist (carbachol) stimulation was markedly decreased in SS cells. CONCLUSIONS: It is concluded that mRNA expression of AQP1 and 5, protein distribution of AQP1, 3 and 5, glandular cell volume regulation and intracellular calcium signaling are all altered in SS, pointing to possible pathophysiological mechanisms in SS.

Proton- compared to X-irradiation leads to more acinar atrophy and greater hyposalivation accompanied by a differential cytokine response.

Proton therapy gives less dose to healthy tissue compared to conventional X-ray therapy, but systematic comparisons of normal tissue responses are lacking. The aim of this study was to investigate late tissue responses in the salivary glands following proton- or X-irradiation of the head and neck in mice. Moreover, we aimed at investigating molecular responses by monitoring the cytokine levels in serum and saliva. Female C57BL/6J mice underwent local fractionated irradiation with protons or X-rays to the maximally tolerated acute level. Saliva and serum were collected before and at different time points after irradiation to assess salivary gland function and cytokine expression. To study late responses in the major salivary glands, histological analyses were performed on tissues collected at day 105 after onset of irradiation. Saliva volume after proton and X-irradiation was significantly lower than for controls and remained reduced at all time points after irradiation. Protons caused reduced saliva production and fewer acinar cells in the submandibular glands compared to X-rays at day 105. X-rays induced a stronger inflammatory cytokine response in saliva compared to protons. This work supports previous preclinical findings and indicate that the relative biological effectiveness of protons in normal tissue might be higher than the commonly used value of 1.1.

A preclinical model to investigate normal tissue damage following fractionated radiotherapy to the head and neck.

Radiotherapy (RT) of head and neck (H&N) cancer is known to cause both early- and late-occurring toxicities. To better appraise normal tissue responses and their dependence on treatment parameters such as radiation field and type, as well as dose and fractionation scheme, a preclinical model with relevant endpoints is required. 12-week old female C57BL/6 J mice were irradiated with 100 or 180 kV X-rays to total doses ranging from 30 to 85 Gy, given in 10 fractions over 5 days. The radiation field covered the oral cavity, swallowing structures and salivary glands. Monte Carlo simulations were employed to estimate tissue dose distribution. The follow-up period was 35 days, in order to study the early radiation-induced effects. Baseline and post irradiation investigations included macroscopic and microscopic examinations of the skin, lips, salivary glands and oral mucosa. Saliva sampling was performed to assess the salivary gland function following radiation exposure. A dose dependent radiation dermatitis in the skin was observed for doses above 30 Gy. Oral mucositis in the tongue appeared as ulcerations on the ventral surface of the tongue for doses of 75-85 Gy. The irradiated mice showed significantly reduced saliva production compared to controls. In summary, a preclinical model to investigate a broad panel of normal tissue responses following fractionated irradiation of the H&N region was established. The optimal dose to study early radiation-induced effects was found to be around 75 Gy, as this was the highest tolerated dose that gave acute effects similar to that observed in cancer patients.

Saliva Metabolomics in Dry Mouth Patients with Head and Neck Cancer or Sjögren's Syndrome.

The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren's syndrome (pSS) share many of the same symptoms despite different causes. With the aim of better understanding the pathophysiology and biochemical processes behind dry mouth with different etiologies, we investigated the metabolic profile of 10 HNC patients, 9 pSS patients and 10 healthy controls using high-performance liquid chromatography-high resolution mass spectrometry (HPLC-MS) metabolomics. Principal component analysis (PCA) revealed different metabolic profiles when comparing all subjects included in the study. Both patient groups showed higher ratios of several pyrimidine nucleotides and nucleosides when compared to controls. This finding may indicate that purinergic signaling plays a role in dry mouth conditions. Moreover, significantly increased levels of DL-3-aminoisobutyric acid were found in HNC patients when compared to controls, and a similar tendency was observed in the pSS patients. Furthermore, a dysregulation in amino acid metabolism was observed in both patient groups. In conclusion, metabolomics analysis showed separate metabolic profiles for HNC and pSS patients as compared to controls that could be useful in diagnostics and for elucidating the different pathophysiologies. The demonstrated dysregulation of pyrimidine nucleotides and levels of metabolites derived from amino acids in the patient groups should be studied further.

Expression of NGAL-specific cells and mRNA levels correlate with inflammation in the salivary gland, and its overexpression in the saliva, of patients with primary Sjögren's syndrome.

Salivary gland involvement is a characteristic feature of primary Sjögren's syndrome (pSS), where tissue destruction is mediated by infiltrating immune cells, and may be accompanied by the presence of adipose tissue. Optimally diagnosing this multifactorial disease requires the incorporation of additional routines. Screening for disease-specific biomarkers in biological fluid could be a promising approach to increase diagnostic accuracy. We have previously investigated disease biomarkers in saliva and tear fluid of pSS patients, identifying Neutrophil gelatinase-associated lipocalin (NGAL) as the most upregulated protein in pSS. In the current study, we aimed to explore for the first time NGAL expression at the site of inflammation in the pSS disease target organ. Immunohistochemical staining was conducted on minor salivary gland biopsies from 11 pSS patients and 11 non-SS sicca subjects, targeting NGAL-specific cells. Additional NGAL/PNAd double staining was performed to study NGAL expression in high endothelial venules, known as specialised vascular structures. Moreover, NGAL mRNA expression was measured utilising quantitative real-time polymerase chain reaction (qRT-PCR) on minor salivary gland biopsies from 15 pSS patients and 7 non-SS sicca individuals that served as tissue controls. Our results demonstrated NGAL expression in acinar and ductal epithelium within the salivary gland of pSS patients, where significantly greater levels of acinar NGAL were observed in pSS patients (p < .0018) when compared to non-SS subjects. Also, acinar expression positively correlated with focus score values (r2 = 0.54, p < .02), while ductal epithelial expression showed a negative such correlation (r2 = 0.74, p < .003). Some PNAD+ endothelial venules also expressed NGAL. An increase in NGAL staining with increased fatty replacement was also observed in pSS patients. Concurringly, a 27% increase in NGAL mRNA levels were also detected in the minor salivary glands of pSS patients when compared to non-SS tissue control subjects. In conclusion, there is a positive association between increase in NGAL expression and inflammation in the pSS disease target organ, which also coincides with its previously demonstrated upregulation in the saliva of pSS patients. Additional functional analyses are needed to better understand the immunological implications of this potential biomarker.

Cytokines Explored in Saliva and Tears from Radiated Cancer Patients Correlate with Clinical Manifestations, Influencing Important Immunoregulatory Cellular Pathways.

Although radiotherapy is a common form of treatment for head and neck cancer, it may lead to tissue damage in the salivary and lacrimal glands, possibly affecting cytokine expression in the gland fluid of treated individuals. Cytokine profiles in saliva and tear fluid of 29 radiated head and neck cancer patients and 20 controls were screened using a multiplex assay. Correlations between cytokine expression and clinical oral and ocular manifestations were examined, and cellular pathways influenced by these cytokines were assessed using the Functional Enrichment Analysis Tool. Significantly elevated cytokines identified in patient saliva were CCL21, IL-4, CX3CL1, CCL2, CXCL1 and CCL15. Many of these cytokines correlated positively with objective signs of oral dryness, and reduced saliva production in the patients. Although CCL21 and IL-4 levels were significantly lower in patient tear fluid, they correlated with subjective ocular symptoms. These increased salivary cytokines affected pro-inflammatory and apoptotic cellular pathways, including T cell signalling, several interleukin signalling pathways, TNF and TGF-ß receptor signalling, and the apoptotic p53 pathway. In conclusion, the upregulated salivary cytokines identified suggest an interplay between innate and adaptive immunity, affecting immunoregulatory cellular pathways. Whether this is due to late effects of radiotherapy or tissue repair remains to be investigated.

Proteomic and histopathological characterisation of sicca subjects and primary Sjögren's syndrome patients reveals promising tear, saliva and extracellular vesicle disease biomarkers.

BACKGROUND: Mononuclear cell infiltration of exocrine glands, production of Ro/SSA and La/SSB autoantibodies, along with oral and ocular dryness, are characteristic features of primary Sjögren's syndrome (pSS). Non-SS sicca subjects, an underexplored group in relation to pSS, display similar sicca symptoms, with possible mild signs of inflammation in their salivary glands, yet with no serological detection of autoantibody production. In this study, we investigated inflammatory manifestations in the salivary gland tissue, tear fluid and saliva of non-SS subjects, as compared to pSS patients and healthy individuals. METHODS: Fifteen non-SS, 10 pSS and 10 healthy subjects were included in the analyses. Histological evaluation of salivary gland biopsies was performed. Liquid chromatography-mass spectrometry (LC-MS) was conducted on tear fluid and stimulated whole saliva, and proteomic biomarker profiles were generated. Extracellular vesicle (EVs) isolation and characterisation from both fluids were also combined with LC-MS. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold. Database for Annotation, Visualization and Integrated Discovery (DAVID) and Functional Enrichment Analysis Tool (FunRich) were applied for functional analyses. RESULTS: Histopathological evaluation of salivary gland biopsies showed implications of milder inflammation in non-SS subjects through mononuclear cell infiltration, fibrosis and fatty replacement, as compared to pSS patients. Although unaffected in the non-SS group, upregulation of proinflammatory pathways and proteins involved in ubiquitination (LMO7 and HUWE1) and B cell differentiation (TPD52) were detected in tear fluid of pSS patients. Moreover, overexpression of proteins STOM, ANXA4 and ANXA1, regulating cellular innate and adaptive immunological pathways, were further identified in EVs from tear fluid of pSS patients. Finally, whole saliva and EVs isolated from whole saliva of pSS patients expressed proteins vital for innate MHC class I cellular regulation (NGAL) and T cell activation (CD44). CONCLUSIONS: Non-SS sicca subjects may show implications of mild inflammation in their glandular tissue, while their protein profile was strikingly more similar to healthy controls than to pSS patients. Hence, the tear and salivary biomarkers identified could be implemented as potential non-invasive diagnostic tools that may aid in increasing diagnostic accuracy when evaluating non-SS subjects and pSS patients and monitoring disease progression.

Elevated cytokine levels in tears and saliva of patients with primary Sjögren's syndrome correlate with clinical ocular and oral manifestations.

Investigating cytokines in tear fluid and saliva may offer valuable information for understanding the pathogenesis of primary Sjögren's syndrome (pSS). Cytokine profiles in both tear fluid and saliva of pSS patients, non-Sjögren's syndrome (non-SS) subjects with sicca symptoms, and healthy controls without sicca complaints were analysed. Furthermore, relationships associating the severity of clinical ocular and oral manifestations with the upregulated cytokines were assessed. In tear fluid, pSS patients showed elevated levels of IL-1ra, IL-2, IL-4, IL-8, IL-12p70, IL-17A, IFN-γ, IP-10, MIP-1b, and Rantes compared to non-SS subjects and healthy controls. The increased cytokine levels (except IP-10) correlated significantly with reduced tear production, less stable tear film, and greater ocular surface damage. In saliva, pSS patients had a higher IP-10 level, which correlated with higher candida score; and an elevated MIP-1a level, which correlated significantly with lower unstimulated and stimulated whole saliva secretion rates. The upregulated cytokines identified in tear fluid and saliva of pSS patients show a clear interplay between innate and adaptive immune responses that may contribute to disease pathogenesis. The increase of IP-10 and MIP in both tears and saliva further emphasises the essential role of macrophages and innate immunity in pSS.

New animal model of extrinsic dental erosion-Erosive effect on the mouse molar teeth.

OBJECTIVE: Consumption of acidic food and drinks is considered as important risk factor for development of dental erosion. There are several in vitro and in situ studies focusing on the risk indicators and preventive treatment, however, the need for a standardized animal model has been emphasised for many years. The aim was to establish an animal model of extrinsic dental erosion, which may serve as a standard for future studies to improve our understanding of the erosion. DESIGN: Two acidic drinks, sports drink and cola drink, were given to young mice for six weeks. Experimental and control (water) molars and incisors were dissected out and observed by scanning electron microscopy (SEM). Mandibular first molars were subsequently ground transversely and observed again by SEM. The tooth height and enamel thickness were measured on the SEM images. RESULTS: The lingual surface of the mandibular molars was most eroded after consumption of acidic drinks. The cola drink exhibited higher erosive effect on mandibular molars compared to sports drink. The lingual tooth height, compared to control, was about 34% and 18% lower in the cola drink and sports drink molars, respectively. Compared to the control molars, the lingual enamel was about 23% thinner in the sports drink molars and totally eroded on the certain lingual areas of the cola drink molars. CONCLUSIONS: This new animal model of extrinsic dental erosion and the presented method with ground molars observed in SEM are suitable for further studies, which will gain deeper insights into the erosive disease.

Identification of potential saliva and tear biomarkers in primary Sjögren's syndrome, utilising the extraction of extracellular vesicles and proteomics analysis.

BACKGROUND: There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren's syndrome (pSS) patients. Incorporation of additional diagnostics involving screening for disease-specific biomarkers in biological fluid is a promising concept that requires further investigation. In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients. METHODS: Liquid chromatography-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated. LC-MS was also combined with size exclusion chromatography to isolate extracellular vesicles (EVs) from both fluids. Nanoparticle tracking analysis was conducted on joint fractions from the saliva and tears to determine size distribution and concentration of EVs. Further EV characterisation was performed by immunoaffinity capture of CD9-positive EVs using magnetic beads, detected by flow cytometry. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold, and the proteins were further analysed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontology overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network analysis. RESULTS: Upregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS. Saliva EVs also displayed biomarkers critical for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP). Tear analysis indicated overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3). Moreover, neutrophil gelatinase-associated lipocalin was upregulated in saliva and tears in pSS. Consistently, DAVID analysis demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metabolism and protein folding in tears in pSS patients. CONCLUSIONS: LC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatography allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs. This approach could provide additional diagnostic accuracy in pSS, and could possibly also be applied for staging and monitoring the disease.