Combined serum anti-SSA/Ro and salivary TRIM29 reveals promising high diagnostic accuracy in patients with primary Sjögren's syndrome.

OBJECTIVES: To determine the diagnostic potential of simultaneous presence of serum anti-SSA/Ro and upregulated salivary protein biomarkers in patients with primary Sjögren's syndrome (pSS). METHODS: Previous proteomics data on the intensity of neutrophil elastase, calreticulin, tripartite motif containing protein 29 (TRIM29), clusterin and vitronectin provided basis for performing extended analysis. Protein data was obtained by liquid chromatography tandem mass spectrometry technique in whole saliva from 24 patients with pSS and 16 patients having symptoms of pSS, but not fulfilling the American College of Rheumatology/European League against Rheumatism classification criteria (non-pSS). Serum anti-SSA/Ro antibody was measured using enzyme-linked immunosorbent assays. Receiver operating characteristic curve (ROC) value was calculated for combined biomarkers. RESULTS: Simultaneous presence of serum anti-SSA/Ro and upregulated salivary TRIM29 provided the most optimal combination with an area under curve (AUC) of 0.995 (95% CI 0.98-1.00, p = 2.0E-7 and standard error 0.007) and combinations of sensitivity and specificity within the interval of 91-100%. ROC analysis showed that salivary levels of TRIM29 alone enabled differentiation between pSS and non-pSS with an area under curve (AUC) of 0.88 (95%CI 0.77-1.00). All patients with pSS and 3 non-pSS patients were serum anti-SSA/Ro positive. CONCLUSIONS: Simultaneous presence of serum anti-SSA/Ro and upregulated salivary TRIM29 provided a high diagnostic accuracy exceeding that of currently available tools used in pSS diagnostics. This biomarker combination represents a promising less invasive diagnostic tool for pSS. The clinical applicability of TRIM29 needs further testing in independent cohorts using relevant analytical techniques.

Proteomics of saliva, plasma, and salivary gland tissue in Sjögren's syndrome and non-Sjögren patients identify novel biomarker candidates.

OBJECTIVE: The aim of this study was to characterize and compare the proteome in whole saliva, plasma, and salivary gland tissue in patients with primary Sjögren's syndrome (pSS) and patients having symptoms of pSS, but not fulfilling the classification criteria, and to search for diagnostic biomarker candidates for pSS. METHODS: Liquid chromatography tandem mass spectrometry was conducted on whole saliva, plasma, and labial salivary gland tissue samples from 24 patients with pSS and 16 non-Sjögren control subjects (non-pSS). Gene Ontology (GO)-terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-pathways were applied for functional annotation. RESULTS: 1013 proteins were identified in whole saliva, 219 in plasma, and 3166 in salivary gland tissue. In saliva, 40 proteins differed significantly between the two groups. In pSS, proteins involved in immunoinflammatory processes were upregulated, whereas proteins related to salivary secretion were downregulated. The combination of neutrophil elastase, calreticulin, and tripartite motif-containing protein 29 yielded a receiver-operating characteristic (ROC) value of 0.97 (CI 0.93-1.00). Protein expression in plasma and salivary gland tissue did not differ between the patient groups. CONCLUSION: The salivary proteome of patients with pSS differed from that of non-pSS patients, indicating that saliva proteomics represents a promising non-invasive diagnostic tool for pSS. SIGNIFICANCE: Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease, which clinically may present with a wide variety of symptoms and signs. Symptoms of dry eyes and dry mouth due to lacrimal and salivary gland dysfunction are prominent, but not pathognomonic, and an extensive diagnostic work-up including blood tests and labial salivary gland biopsy is often required to distinguish pSS from non-pSS. In this study, we used high throughput proteomics and identified a non-invasive biomarker candidate comprising a combination of three different upregulated salivary proteins, which enables differentiation between patients with pSS and non-pSS patients with an accuracy of 97%. In the future, this could contribute to earlier, more accurate and less costly diagnosis of pSS.

Ex vivo correlation of the permeability of metoprolol across human and porcine buccal mucosa.

The pH partition theory proposes a correlation between fraction of unionized drug substance and permeability. The aim of this study was to compare the permeability of metoprolol and mannitol in ex vivo human and porcine buccal mucosa models at varying pH to validate whether the porcine permeability model is predictive for human buccal absorption. Human (n = 9-10) and porcine (n = 6-7) buccal mucosa were mounted in a modified Ussing chamber, and the kinetics of metoprolol and mannitol transport was assessed for a period of 5.5 h with the pH values of donor medium set at 7.4, 8.5, and 9.0. In addition, hematoxylin-eosin and Alcian blue-van Gieson were used as tissue stains to evaluate the histology and the presence of acidic polysaccharides (e.g., mucins), respectively. The permeability of metoprolol was decreased in human buccal mucosa by almost twofold when compared with porcine buccal mucosa with a positive correlation (r(2) = 0.96) between the permeability assessed in porcine and human buccal mucosa. There was no change in the degree of either epithelial swelling or desquamation when treating with the pH 9.0 donor medium for 5.5 h. These data suggest that buccal mucosa from pigs can be used to predict human buccal absorption.

Development of an ex vivo retention model simulating bioadhesion in the oral cavity using human saliva and physiologically relevant irrigation media.

In recent years, there has been a particular interest in bioadhesive formulations for oromucosal drug delivery as this may promote prolonged local therapy and enhanced systemic effect. Saliva plays a vital role in oromucosal drug absorption by dissolving the drug and presenting it to the mucosal surface. However, the rheological, chemical, and interfacial properties of this complex biological fluid may strongly affect the adhesion of bioadhesive formulations. There is a need for well characterized in vitro models to assess the bioadhesive properties of oral dosage forms for administration in the oral cavity. Thus we aimed at developing an advanced ex vivo buccal retention model, with focus on choosing a physiologically relevant irrigation media closely resembling human saliva. Spray dried chitosan microparticles containing metformin hydrochloride as an example of a small hydrophilic drug, were employed as bioadhesive formulations. Chewing-stimulated human whole saliva was collected and characterized for use in retention studies in comparison with four artificial irrigation media; phosphate buffer, Saliva Orthana(®), porcine gastric mucin base media (PGM3), and xanthan gum based media (XG2). Retention of metformin, applied as spray dried microparticles on porcine buccal mucosa, greatly depended on the characteristics of the irrigation media. When rheology of the irrigation media was examined, changes in retention profiles could be interpreted, as irrigation media containing mucin and xanthan gum possessed a higher viscosity than phosphate buffer, which led to longer retention of the drug due to better hydration of the mucosa and the spray dried microparticles. Metformin retention profiles were comparable when human saliva, Saliva Orthana(®), or PGM3 were used as irrigation media. Moreover, PGM3 displayed physico-chemical properties closest to those of human saliva with regard to pH, protein content and surface tension. Saliva Orthana(®) and PGM3 are therefore considered as suitable irrigation media for further retention studies.