BackgroundThere 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.MethodsLiquid 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.ResultsUpregulation 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.ConclusionsLC-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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-017-1228-x) contains supplementary material, which is available to authorized users.

Extracellular vesicles (EVs) are a heterogeneous population of biological particles released by cells. They represent an attractive source of potential biomarkers for early detection of diseases such as cancer. However, it is critical that sufficient amounts of EVs can be isolated and purified in a robust and reproducible manner. Several isolation methods that seem to produce distinct populations of vesicles exist, making data comparability difficult. While some methods induce cellular stress that may affect both the quantity and function of the EVs produced, others involve expensive reagents or equipment unavailable for many laboratories. Thus, there is a need for a standardized, feasible and cost-effective method for isolation of EVs from cell culture supernatants. Here we present the most common obstacles in the production and isolation of small EVs, and we suggest a combination of relatively simple strategies to avoid these. Three distinct cell lines were used (human oral squamous cell carcinoma (PE/CA-PJ49/E10)), pancreatic adenocarcinoma (BxPC3), and a human melanoma brain metastasis (H3). The addition of 1% exosome-depleted FBS to Advanced culture media enabled for reduced presence of contaminating bovine EVs while still ensuring an acceptable cell proliferation and low cellular stress. Cells were gradually adapted to these new media. Furthermore, using the Integra CELLine AD1000 culture flask we increased the number of cells and thereby EVs in 3D-culture. A combination of ultrafiltration with different molecular weight cut-offs and size-exclusion chromatography was further used for the isolation of a heterogeneous population of small EVs with low protein contamination. The EVs were characterized by nanoparticle tracking analysis, immunoaffinity capture, flow cytometry, Western blot and transmission electron microscopy. We successfully isolated a significant amount of small EVs compatible with exosomes from three distinct cell lines in order to demonstrate reproducibility with cell lines of different origin. The EVs were characterized as CD9 positive with a size between 60–140 nm. We conclude that this new combination of methods is a robust and improved strategy for the isolation of EVs, and in particular small EVs compatible with exosomes, from cell culture media without the use of specialized equipment such as an ultracentrifuge.

The COVID-19 (caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) epidemic started in Wuhan (Hubei Province, China) in mid-December 2019 and quickly spread across the world as a pandemic. As a key to tracing the disease and to implement strategies aimed at breaking the chain of disease transmission, extensive testing for SARS-CoV-2 was suggested. Although nasopharyngeal/oropharyngeal swabs are the most commonly used biological samples for SARS-CoV-2 diagnosis, they have a number of limitations related to sample collection and healthcare personnel safety. In this context, saliva is emerging as a promising alternative to nasopharyngeal/oropharyngeal swabs for COVID-19 diagnosis and monitoring. Saliva collection, being a non-invasive approach with possibility for self-collection, circumvents to a great extent the limitations associated with the use of nasopharyngeal/oropharyngeal swabs. In addition, various salivary biomarkers including the salivary metabolomics offer a high promise to be useful for better understanding of COVID-19 and possibly in the identification of patients with various degrees of severity, including asymptomatic carriers. This review summarises the clinical and scientific basis for the potential use of saliva for COVID-19 diagnosis and disease monitoring. Additionally, we discuss saliva-based biomarkers and their potential clinical and research applications related to COVID-19.

Objectives Primary Sjögren’s syndrome (pSS) is an autoimmune disease characterized by reduced lacrimal and salivary secretion. Sicca symptoms together with fatigue and musculoskeletal pain can significantly reduce the patients’ quality of life. Furthermore, low salivary secretion may disrupt the oral microbial homeostasis. The aim of this study was to compare the salivary microbiota from pSS patients with patients with sicca symptoms not fulfilling the classification criteria for pSS (non-SS), and with healthy controls without sicca complaints. Methods Pellets from centrifuged chewing-stimulated whole saliva from pSS patients (n = 15), non-SS sicca patients (n = 15) and healthy controls (n = 15) were prepared. DNA was extracted and analyzed by 16S rRNA gene sequencing. The acquired sequencing data were performed using the human oral microbiome database (HOMD). Results We detected 42, 45, and 34 bacterial genera in saliva samples from pSS patients, non-SS sicca patients, and healthy controls, respectively. The most abundant genera in all samples were Prevotella , Veillonella , Streptococcus , and Haemophilus . At species level Streptococcus intermedius , Prevotella intermedia , Fusobacterium nucleatum subsp . vincentii , Porphyromonas endodontalis , Prevotella nancensis , Tannerella spp ., and Treponema spp . were detected in the samples from pSS and non-SS only, while Porphyromonas pasteri was mostly found among the healthy controls. Conclusion Our study indicated dysbiosis in the salivary microbiota from pSS and non-SS patients compared to healthy controls. Additionally, the results showed that the salivary microbiome in the pSS group differed significantly from the non-SS group.

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs.