non-SS Subjects Research Articles

The Toll-like Receptor 7-Mediated Ro52 Antigen-Presenting Pathway in the Salivary Gland Epithelial Cells of Sjögren's Syndrome.

To investigate whether stimulation with toll-like receptor (TLR) 7 leads to pathways that proceed to tripartite motif-containing protein 21 (TRIM21) or Ro52/SS-A antigen presentation through major histocompatibility complex (MHC) class I in salivary gland epithelial cells (SGECs) from Sjögren's syndrome (SS) patients. Cultured SGECs from SS patients were stimulated with TLR7 agonist, loxoribine, and interferon-β. Cell lysates immunoprecipitated by anti-MHC class I antibody were analyzed by Western blotting. The immunofluorescence of salivary gland tissue from SS and non-SS subjects and cultured TLR7-stimulated SGECs was examined. Significantly increased MHC class I expression was observed in SS patients' ducts versus non-SS ducts; no significant difference was detected for ubiquitin. Upregulated MHC class I in the cell membrane and cytoplasm and augmented Ro52 expression were observed in SGECs stimulated with TLR7. The formation of peptide-loading complex (PLC), including tapasin, calreticulin, transporter associated with antigen processing 1, and endoplasmic reticulum-resident protein 57 in labial salivary glands (LSGs) from SS patients, was dominantly observed and colocalized with MHC class I, which was confirmed in TLR7-stimulated SGEC samples. These findings suggest that the TLR7 stimulation of SS patients' SGECs advances the process toward the antigen presentation of TRIM21/Ro52-SS-A via MHC class I.

Correlation Between Salivary Microbiome of Parotid Glands and Clinical Features in Primary Sjögren's Syndrome and Non-Sjögren's Sicca Subjects.

Recent studies have demonstrated that the oral microbiome in patients with Sjögren’s syndrome (SS) is significantly different from that in healthy individuals. However, the potential role of the oral microbiome in SS pathogenesis has not been determined. In this study, stimulated intraductal saliva samples were collected from the parotid glands (PGs) of 23 SS and nine non-SS subjects through PG lavage and subjected to 16S ribosomal RNA amplicon sequencing. The correlation between the oral microbiome and clinical features, such as biological markers, clinical manifestations, and functional and radiological characteristics was investigated. The salivary microbial composition was examined using bioinformatic analysis to identify potential diagnostic biomarkers for SS. Oral microbial composition was significantly different between the anti-SSA-positive and SSA-negative groups. The microbial diversity in SS subjects was lower than that in non-SS sicca subjects. Furthermore, SS subjects with sialectasis exhibited decreased microbial diversity and Firmicutes abundance. The abundance of Bacteroidetes was positively correlated with the salivary flow rate. Bioinformatics analysis revealed several potential microbial biomarkers for SS at the genus level, such as decreased Lactobacillus abundance or increased Streptococcus abundance. These results suggest that microbiota composition is correlated with the clinical features of SS, especially the ductal structures and salivary flow, and that the oral microbiome is a potential diagnostic biomarker for SS.

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 (r 2 = 0.54, p < .02), while ductal epithelial expression showed a negative such correlation (r 2 = 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.

Dysbiotic oral microbiota and infected salivary glands in Sjögren's syndrome.

Key events in the pathogenesis of Sjӧgren syndrome (SS) include the change of salivary gland epithelial cells into antigen-presenting cell-like phenotypes and focal lymphocytic sialadenitis (FLS). However, what triggers these features in SS is unknown. Dysbiosis of the gut and oral microbiomes is a potential environmental factor in SS, but its connection to the etiopathogenesis of SS remains unclear. This study aimed to characterize the oral microbiota in SS and to investigate its potential role in the pathogenesis of SS. Oral bacterial communities were collected by whole mouthwash from control subjects (14 without oral dryness and 11 with dryness) and primary SS patients (8 without oral dryness and 17 with dryness) and were analyzed by pyrosequencing. The SS oral microbiota was characterized by an increased bacterial load and Shannon diversity. Through comparisons of control and SS in combined samples and then separately in non-dry and dry conditions, SS-associated taxa independent of dryness were identified. Three SS-associated species and 2 control species were selected and used to challenge human submandibular gland tumor (HSG) cells. Among the selected SS-associated bacterial species, Prevotella melaninogenica uniquely upregulated the expression of MHC molecules, CD80, and IFNλ in HSG cells. Concomitantly, P. melaninogenica efficiently invaded HSG cells. Sections of labial salivary gland (LSG) biopsies from 8 non-SS subjects and 15 SS patients were subjected to in situ hybridization using universal and P. melaninogenica-specific probes. Ductal cells and the areas of infiltration were heavily infected with bacteria in the LSGs with FLS. Collectively, dysbiotic oral microbiota may initiate the deregulation of SGECs and the IFN signature through bacterial invasion into ductal cells. These findings may provide new insights into the etiopathogenesis of SS.

Proteomic and histopathological characterisation of sicca subjects and primary Sj\xf6gren\u2019s syndrome patients reveals promising tear, saliva and extracellular vesicle disease biomarkers

BackgroundMononuclear 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.MethodsFifteen 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.ResultsHistopathological 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).ConclusionsNon-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.

Initial human T-cell leukemia virus type 1 infection of the salivary gland epithelial cells requires a biofilm-like structure

The initial phase of the human T cell leukemia virus-1 (HTLV-1) infection of salivary gland epithelial cells (SGECs) was examined. SGECs of patients with Sjögren’s syndrome (SS) and non-SS subjects were co-cultured with the HTLV-1-infected cell line HCT-5 or MOLT-4, then immunofluorescence (IF), scanning and transmission electron microscopy (SEM/TEM) were employed.The extracellular matrix and linker proteins galectin-3, agrin, and tetherin were expressed on the surfaces of both HCT-5 and MOLT-4 cells. HTLV-1 Gag-positive spots were observed on adjacent SGECs after 1 h of co-culture with HCT-5. Both in subjects with and those without SS, agrin and tetherin were co-expressed with HTLV-1 Gag on SGECs after co-culture with HCT-5, although no polarization of HTLV-1 Gag and relevant molecules was observed. SEM showed HTLV-1 virions that were found on HCT-5 were observed in the interfaces between HCT-5 cells and SGECs. TEM imaging showed that HTLV-1 virions were transmitted to SGECs at the interface with thin film-like structure, while HTLV-1 virions were released from the surface of HCT-5 cells. No endogenous retroviruses were observed. These results showed that the initial phase of HTLV-1 infection toward SGECs of SS was mediated not by viral synapses, but by biofilm-like components.

Elevated cytokine levels in tears and saliva of patients with primary Sj\xf6gren\u2019s 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.

Connection Between Dysbiotic Oral Microbiota and Deregulation of Salivary Gland Epithelial Cells in Sjogren's Syndrome

Background: Dysbiosis of the gut and oral microbiomes is a potential environmental factor in Sjogren's syndrome (SS), but its connection to the etiopathogenesis of SS remains unclear. This study aimed to characterize the oral microbiota in SS and to investigate its potential role in the pathogenesis of SS. Methods: Oral bacterial communities collected by whole mouthwash from 15 healthy individuals, 10 drug/radiation-induced sicca patients, and 25 primary SS patients were analyzed by pyrosequencing. Four SS-associated species and 2 control species were selected and used to challenge human submandibular gland tumor (HSG) cells. The expression of MHC I, MHC II, CD80, CD86, and IFNλ in HSG cells and the bacterial invasion into HSG cells were examined. Sections of labial salivary gland (LSG) biopsies from 8 non-SS subjects and 15 SS patients were subjected to in situ hybridization using universal and Prevotella melaninogenica-specific probes. Findings: The SS oral microbiota was characterized by an increased bacterial load and Shannon diversity. Although a substantial portion of the changes observed in SS patients overlapped with those in drug/radiation-induced sicca patients, SS-specific changes were identified. Among the selected SS-associated bacterial species, P. melaninogenica uniquely upregulated the expression of MHC molecules, CD80, and IFN λ in HSG cells. Concomitantly, P. melaninogenica efficiently invaded HSG cells. Ductal cells and the areas of infiltration were heavily infected with bacteria in the LSGs with focal lymphocytic sialadenitis. Interpretation: Dysbiotic oral microbiota is associated with deregulation of salivary gland epithelial cells and acquisition of IFN signatures through bacterial invasion into ductal cells. Funding Statement: This study was supported by a grant of the Korean Health Technology R&D Project (HI13C0016) from Ministry of Health & Welfare, Republic of Korea. Labial salivary gland biopsy specimens used in this manuscript are from the Sjogren’s International Collaborative Clinical Alliance [SICCA], funded under contract N01 DE-32636 by the National Institute of Dental and Craniofacial Research, with funding support from the National Eye Institute and Office for Research in Women’s Health. The funders had no role in study design, data collection, analysis, and interpretation, and writing of the report. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All procedures involving human subjects and materials were performed in accordance with the Helsinki Declaration under approved protocols from the Institutional Review Boards at Seoul St. Mary’s Hospital (KC13ONMI0646) and at Seoul National University, School of Dentistry (S-D20140022, S-D20170004). Informed consent was obtained from all subjects.

Meibomian gland dropout in Sjögren's syndrome and non-Sjögren's dry eye patients.

The purpose of this study is to explore the differences in ocular symptoms and signs between Sjögren's syndrome (SS) and non-SS aqueous-deficient dry eye (ADDE) patients. Twenty-two eyes of 22 SS patients (Group 1) and 22 eyes of 22 non-SS ADDE patients (Group 2) were enrolled. The evaluated variables included the Standard Patient Evaluation of Eye Dryness (SPEED), the Ocular Surface Disease Index (OSDI), tear meniscus height, first and average non-invasive keratographic breakup time (fNIKBUT and avNIKBUT), Schirmer I test, lipid layer thickness (LLT), meibomian gland expressibility, Marx line, corneal staining, conjunctival congestion, incomplete blinking, and meibomian gland dropout using two novel, non-invasive instruments, the Keratograph and LipiView II. Ocular signs of the NIKBUT (fNIKBUT: 3.8 (2.7, 5.2)s and 6.3 (3.7, 8.9)s, P = 0.024; avNIKBUT: 5.4 (4.5, 8.9)s and 7.6 (5.8, 13.7)s, P = 0.041), meibomian gland dropout of the upper eyelid (35.5% (29.1%, 54.8%) and 21.9% (16.7%, 24.9%), P = 0.000), and corneal staining (P = 0.050) were more severe but were associated with less severe symptoms, i.e., a lower SPEED score (P = 0.001), in SS subjects than in non-SS subjects. SS patients exhibit more severe meibomian gland destruction of the upper eyelid than non-SS patients. Meibomian gland dysfunction is another key cause of SS-associated dry eye.

Sensitive Skin: Assessment of the Skin Barrier Using Confocal Raman Microspectroscopy

Background/Aims: Sensitive skin (SS), a frequently reported condition in the Western world, has been suggested to be underlined by an impaired skin barrier. The aim of this study was to investigate the skin barrier molecular composition in SS subjects using confocal Raman microspectroscopy (CRS), and to compare it with that of non-SS (NSS) individuals as well as atopic dermatitis (AD) and allergic rhinoconjunctivitis (AR) subjects, who frequently report SS. Methods: Subjects with SS (n = 29), NSS (n = 30), AD (n = 11), and AR (n = 27) were included. Stratum corneum (SC) thickness, water, ceramides/fatty acids, and natural moisturizing factor (NMF) were measured by CRS along with transepidermal water loss and capacitance on the ventral forearm, thenar, and cheek. Sebum levels were additionally measured on the forearm and cheek. Results: No differences between SS and NSS subjects were found regarding SC thickness, water, and NMF content, yet a trend towards lower ceramides/fatty acids was observed in the cheek. Compared to AD subjects, the SS group showed higher ceramides/fatty acid content in the forearm, whereas no differences emerged with AR. The correlation of macroscopic biophysical techniques and CRS was weak, yet CRS confirmed the well-known lower content of NMF and water, and thinner SC in subjects with filaggrin mutations. Conclusion: The skin barrier in SS is not impaired in terms of SC thickness, water, NMF, and ceramides/fatty acid content. The failure of biophysical techniques to follow alterations in the molecular composition of the skin barrier revealed by CRS emphasizes a strong need in sensitive and specific tools for in vivo skin barrier analysis.

The status of glucocorticoid-induced leucine zipper protein in the salivary glands in Sjögren's syndrome: predictive and prognostic potentials.

BackgroundWe recently showed that an imbalance between the pro-inflammatory cytokine, interleukin (IL)-17, and the developmental endothelial locus-1 (Del-1) likely contributes to inflammation and salivary gland abnormalities in Sjögren’s syndrome (SS). The glucocorticoid-induced leucine zipper (GILZ) protein is a pivotal player in mediating the anti-inflammatory effects of glucocorticoids. However, its status and role in salivary gland inflammation and dysfunction in SS are not established. Thus, we tested the hypothesis that SS is associated with reduced GILZ expression, thereby contributing to Del-1/Il-17 imbalance and inflammation in salivary glands.MethodsWe utilized the nonobese diabetic (NOD) mice, a model of SS-like disease as well as lower-lip biopsy samples of subjects without or with a diagnosis of SS in association with immunostaining studies. These studies were complemented with in vitro and flow-cytometry studies whereby interleukin (IL)-23-treated salivary gland cells were co-cultured with GILZ-expressing cells or control cells; IL-23 is known to increase generation of IL-17.ResultsSalivary glands of NOD mice displayed marked leukocyte infiltration and reduced GILZ expression in association with increased IL-17 but decreased Del-1 expression. A similar pattern was observed for lower-lip biopsy samples of SS than non-SS subjects. Further, IL-23-treated salivary gland cells displayed marked increase in IL-17 but reduced Del-1 positive cells which were reversed with co-culturing with GILZ-expressing cells but not control cells. Collectively, the results are suggestive of dysregulation of GILZ playing a role in inflammation and associated Del-1/Il-17 imbalance in SS.ConclusionsComplementing our demonstration of Del-1/IL-17 imbalance in salivary glands in SS, the present study has established the relevance and significance of GILZ as a novel predictive and prognostic molecular fingerprint for SS. Thus, assessment of minor salivary gland GILZ expression, in conjunction with Del-1/IL-17 imbalance, could potentially offer a more sensitive approach to help with diagnosis of SS, at early stage of the disease, than that based on leukocyte infiltration. Future studies should establish whether treatment with GILZ ameliorates signs and symptoms of salivary malfunction of SS for which effective treatment options remain elusive.

Reciprocal relation between GADD153 and Del-1 in regulation of salivary gland inflammation in Sjögren syndrome

Endoplasmic reticulum (ER) stress response is a pivotal regulator of inflammation and cell death. An integral component of ER stress-induced apoptosis is expression of growth arrest- and DNA damage-inducible protein 153 (GADD153). Further, ER stress response is implicated in leukocyte adhesion and recent studies have discovered endogenous inhibitors of leukocyte adhesion including the developmental endothelial locus-1 (Del-1). Accordingly, we tested the hypothesis that Sjögren's syndrome (SS) is associated with increased salivary gland expression of GADD153 and increased leukocyte infiltration in association with decreased Del-1 thereby contributing to inflammation and cell death. We utilized the non-obese diabetic (NOD) mice, a model of SS-like disease, in association with immunostaining and flow cytometry-based studies. Salivary glands of 14-week-old NOD mice displayed a) increased GADD153 expression, b) marked reduction in Del-1, c) inflammatory cell infiltrates including CD3+ T and CD19+ B lymphocytes as well as M1 and M2 macrophages and d) increased pro-inflammatory interleukin (IL)-17 but reduced anti-inflammatory cytokine, IL-10. These changes were accompanied with disruption of mitochondrial membrane potential and significant increase in apoptosis and necrosis of salivary gland cells of NOD than control mice. Our collective observations suggested that GADD153 directly and/or indirectly through downregulation of Del-1 contributes importantly to salivary gland inflammation and cell death. To establish the relevance of GADD153 and Del-1 for the human condition, lower lip biopsy samples of non-SS subjects and those with a diagnosis of SS were subjected to immunohistochemistry. The results show intense GADD153 immunostaining but marked reduction in Del-1 expression in biopsy samples of SS compared to non-SS subjects. Collectively, the results indicate that GADD153 regulates inflammation and cell death in salivary gland in SS. Further, Del-1 expression likely provides a mechanistic link between increased GADD153 and leukocyte infiltration and accompanying inflammation of salivary gland tissue in this condition.

Prevalence of Sjögren's syndrome with dementia in a memory clinic

IntroductionSjögren's syndrome (SS) is an autoimmune disorder involving the exocrine glands, which affects 1.9–3.0% of the elderly population. Approximately 20% of all patients with SS have CNS involvement, including dementia, as a result of angiitis. AimsThe aim of the study was to clarify the prevalence and impact of SS among patients in a memory clinic. MethodsThis study prospectively recruited patients with cognitive dysfunction in a memory clinic from 2007 to 2010. In addition to the examinations for dementia, the patients' levels of anti-SSA and SSB antibodies were measured. Schirmer's test and/or a lip biopsy were added if required. SS was diagnosed based on the American European consensus criteria. ResultsOut of 276 cases who completed the examinations, 265 (97/168 males/females, mean age: 77.9, median MMSE score: 23) did not demonstrated cognitive decline. Sixteen (6.3%) and seven (2.7%) patients were positive for anti-SS-A and SS-B antibodies, respectively. Twenty patients (7.5%) were diagnosed with primary SS (mean age: 77.2years old, median MMSE: 21). Seven of these patients had previously been diagnosed with MCI (VCIND: 5, aMCI: 2), and 13 had been diagnosed with dementia. All had asymmetrical focal hypoperfusion on SPECT, and eighteen had subcortical lesions on MRI. Twelve were treated for dementia (median time: 2.1years), and their MMSE significantly improved (median MMSE: 26, p=0.0019), while the non-SS subjects' MMSE declined (n=126, median: 22). ConclusionThe patients with SS accounted for 7.5% of those with a cognitive decline as determined at a memory clinic, and are characterized by subcortical white matter lesions and asymmetric hypoperfusion.

Identification of parotid salivary biomarkers in Sjögren's syndrome by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and two-dimensional difference gel electrophoresis

To identify the most significant salivary biomarkers in Sjögren's syndrome (SS) using proteomic methods. Parotid saliva from 20 non-SS subjects and 41 primary SS patients was analysed. Protein expression profiles for each sample were generated by surface-enhanced laser desorption/ionization time-of-flight-mass spectrometry (SELDI-TOF-MS). Mean peak intensities of SS patients and non-SS subjects were compared by univariate analyses. Samples pooled by diagnosis (SS and non-SS) and labelled with different Cy dyes were compared by two-dimensional difference gel electrophoresis (2D-DIGE). Two protein levels that were most significantly different by SELDI-TOF-MS and 2D-DIGE were validated by enzyme-linked immunosorbent assay in individual samples. SELDI-TOF-MS of 10-200 kDa peaks revealed eight peaks with >2-fold changes in the SS group that differed from non-SS at P < 0.005. Peaks of 11.8, 12.0, 14.3, 80.6 and 83.7 kDa were increased, while 17.3, 25.4, and 35.4 kDa peaks were decreased in SS samples. 2D-DIGE identified significant increases of beta-2-microglobulin, lactoferrin, immunoglobulin (Ig) kappa light chain, polymeric Ig receptor, lysozyme C and cystatin C in all stages of SS. Two presumed proline-rich proteins, amylase and carbonic anhydrase VI, were reduced in the patient group. Three of these ten biomarkers have not been associated previously with SS. The salivary proteomic profile of SS is a mixture of increased inflammatory proteins and decreased acinar proteins when compared with non-SS. Future studies will test the ability of these biomarker levels, alone and in combination, to diagnose the salivary component of SS.