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Objective The aim was to study clinical, histopathological and immunological changes in the vagina and cervix of women with primary SS, which might explain vaginal dryness. Methods We included 10 pre-menopausal female primary SS patients with vaginal dryness and 10 pre-menopausal controls undergoing a laparoscopic procedure. The vaginal health index was recorded. Multiplex immunoassays and flow cytometry were performed on endocervical swab and cervicovaginal lavage samples to evaluate cellular and soluble immune markers. Mid-vaginal and endocervical biopsies were taken and stained for various leucocyte markers, caldesmon (smooth muscle cells), avian V-ets erythroblastosis virus E26 oncogene homologue (ERG; endothelial cells) and anti-podoplanin (lymphatic endothelium). The number of positive pixels per square micrometre was calculated. Results One patient was excluded because of Clamydia trachomatis, and two controls were excluded because of endometriosis observed during their laparoscopy. Vaginal health was impaired in primary SS. CD45+ cells were increased in vaginal biopsies of women with primary SS compared with controls. Infiltrates were predominantly located in the peri-epithelial region, and mostly consisted of CD3+ lymphocytes. In the endocervix, CD45+ infiltrates were present in patients and in controls, but a higher number of B lymphocytes was seen in primary SS. Vascular smooth muscle cells were decreased in the vagina of primary SS patients. No differences were found in leucocyte subsets in the vaginal and endocervical lumen. CXCL10 was increased in endocervical swab samples of primary SS patients. Conclusion Women with primary SS show impaired vaginal health and increased lymphocytic infiltration in the vagina compared with controls. Vaginal dryness in primary SS might be caused by vascular dysfunction, possibly induced by IFN-mediated pathways.

Objective The involvement of salivary glands in primary SS (pSS) can be assessed in different ways: histopathology, salivary flow and ultrasonography. To understand the relative value of these different approaches, it is crucial to understand the relationship between them. As we routinely perform these three modalities in the parotid gland for disease evaluation, our aim was to investigate the construct validity between these modalities in one and the same gland. Methods Consecutive sicca patients underwent a multidisciplinary diagnostic workup including parotid gland biopsy, collection of parotid gland–specific saliva and parotid gland ultrasonography. Patients who were classified as pSS according to the ACR-EULAR criteria were included. Construct validity was assessed using Spearman’s correlation coefficients. Results The 41 included pSS patients completed a full workup within a mean time interval of 2.6 months. Correlations between histopathological features and stimulated parotid salivary flow were fair (ρ = −0.123 for focus score and ρ = −0.259 for percentage of CD45+ infiltrate). Likewise, poor correlations were observed between stimulated parotid salivary flow and parotid ultrasonography (ρ = −0.196). Moderate to good associations were found between the histopathological items focus score and the percentage of CD45+ infiltrate, with parotid US scores (total US score: ρ = 0.510 and ρ = 0.560; highest for homogeneity: ρ = 0.574 and ρ = 0.633). Conclusion Although pSS-associated ultrasonographic findings did correlate with histopathological features, the three modalities that evaluate salivary gland involvement assess different (or at best partly related) constructs. Therefore histopathology, salivary flow and ultrasonography are complementary measurements and cannot directly replace each other in the workup of pSS.

BackgroundWhile all salivary glands (SGs) can be involved in primary Sjögren’s syndrome (pSS), their respective role in pathogenesis remains unclear. Our objective was to assess immunopathway activation in paired parotid and labial gland tissue from biopsy-positive and biopsy-negative pSS and non-SS sicca patients.MethodsParaffin-embedded, paired parotid and labial salivary gland tissue and peripheral blood mononuclear cells were obtained from 39 pSS and 20 non-SS sicca patients. RNA was extracted, complementary DNA libraries were prepared and sequenced. For analysis of differentially expressed genes (DEGs), patients were subdivided based on fulfillment of ACR-EULAR criteria and histopathology.ResultsWith principal component analysis, only biopsy-positive pSS could be separated from non-SS sicca patients based on SG gene expression. When comparing the transcriptome of biopsy-positive pSS and biopsy-negative non-SS sicca patients, 1235 and 624 DEGs (FDR1) were identified for parotid and labial glands, respectively. The number of DEGs between biopsy-negative pSS and non-SS sicca patients was scarce. Overall, transcript expression levels correlated strongly between parotid and labial glands (R2 = 0.86, p-valueConclusionTranscriptomes of paired major and minor SGs in pSS were overall comparable, although significant inter-individual heterogeneity in immunopathway activation existed. The SG transcriptome of biopsy-negative pSS was indistinguishable from non-SS sicca patients. Different patterns of SG immunopathway activation in pSS argue for personalized treatment approaches.

OBJECTIVE: Alterations in the microbiota composition of the gastro-intestinal tract are suspected to be involved in the etiopathogenesis of two closely related systemic inflammatory autoimmune diseases: primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE). Our objective was to assess whether alterations in gut and oral microbiota compositions are specific for pSS and SLE.METHODS: 16S ribosomal RNA gene sequencing was performed on fecal samples from 39 pSS patients, 30 SLE patients and 965 individuals from the general population, as well as on buccal swab and oral washing samples from the same pSS and SLE patients. Alpha-diversity, beta-diversity and relative abundance of individual bacteria were used as outcome measures. Multivariate analyses were performed to test associations between individual bacteria and disease phenotype, taking age, sex, body-mass index, proton-pump inhibitor use and sequencing-depth into account as possible confounding factors.RESULTS: Fecal microbiota composition from pSS and SLE patients differed significantly from population controls, but not between pSS and SLE. pSS and SLE patients were characterized by lower bacterial richness, lower Firmicutes/Bacteroidetes ratio and higher relative abundance of Bacteroides species in fecal samples compared with population controls. Oral microbiota composition differed significantly between pSS patients and SLE patients, which could partially be explained by oral dryness in pSS patients.CONCLUSIONS: pSS and SLE patients share similar alterations in gut microbiota composition, distinguishing patients from individuals in the general population, while oral microbiota composition shows disease-specific differences between pSS and SLE patients.

Dryness of epithelial surfaces is characteristic for patients with primary Sjogren’s syndrome (pSS). Vaginal dryness is frequently reported by pSS-women and is associated with sexual dysfunction.1 2 Recently, we showed that dysbiosis of the oral microbiome is largely similar between oral dryness patients with and without pSS when compared with healthy controls.3 4 The objective of our current study was to assess whether the vaginal microbiome of women with pSS-associated vaginal dryness differs from controls. In a case-control design, we compared the vaginal microbiome of 10 premenopausal pSS-women with that of 10 age-matched premenopausal women without pSS, who underwent general anaesthesia for a laparoscopic procedure. Exclusion criteria were genital inflammatory or infectious comorbidity, endometriosis and use of disease modifying antirheumatic drugs, corticosteroids, vaginal oestrogens or an intrauterine contraceptive device. All patients with pSS fulfilled the 2016 ACR/EULAR classification criteria. All participants completed a questionnaire on vaginal symptoms. Patient-reported vaginal dryness was scored using a Numeric Rating Scale (NRS, range 0–10). Vaginal health was assessed with the Vaginal Health Index (VHI).5 From all participants, a cervicovaginal lavage (CVL) and endocervical swab (ES) were collected. DNA from all samples was …

The oral microbiome of patients with primary Sjogren’s syndrome (pSS) differs from that of healthy individuals.1–3 Whether these changes are the consequence of reduced salivary secretion or specific for pSS is yet unknown. Therefore, the aim of our study was to assess whether patients with pSS have a specific oral microbiome compared with symptom controls (patients without SS, indicated as non-SS patients) and healthy controls (HCs). To capture the overall bacterial composition in the mouth, we collected oral washings instead of buccal swabs from 121 consecutive patients referred for a diagnostic pSS workup and 14 volunteers without oral or ocular dryness (HCs). Patients fulfilling the 2016 American College of Rheumatology/European League Against Rheumatism classification criteria were classified as pSS (n=36) and patients not fulfilling the criteria as non-SS (n=85) (table 1).4 View this table: Table 1 Patient characteristics* Bacterial composition was assessed with 16S rRNA gene sequencing of the V4 region. Reads were processed and diversity was analysed with QIIME V.1.9.1.5 All samples were equated to 8000 reads/sample. Comparative statistics were performed in R V.3.4.2.6 For details see online Supplementary text. ### Supplementary file 1 [SP1.docx] Alpha diversity, that is, the number of bacterial taxa and proportion in which each taxon is represented per sample was measured by the number of observed operational taxonomic units and Shannon index. Alpha diversity was similar between pSS, non-SS and HCs, indicating that oral washings of …

Background Patients with primary Sjogren’s syndrome (pSS) present with heterogeneous clinical symptoms and immune dysregulation. How immune dysregulation in SS arises is poorly understood, which hampers development of effective therapies. Identification of key immune pathways contributing to pSS pathogenesis is essential for successful drug development. Systemic treatment of patients early in the disease process will limit irreversible damage to salivary and lacrimal glands, as well as to other affected tissues. Objectives To identify early systemic biomarkers and dysregulated immune pathways in newly diagnosed pSS patients by multidimensional immuno-profiling. Methods We included 40 newly diagnosed pSS patients (39 female; mean age 51±14) and 20 age- and sex-matched non-SS sicca patients (19 female; mean age 50±13). All pSS patients fulfilled ACR-EULAR criteria. Serum and peripheral blood mononuclear cells (PBMC) were collected and cryopreserved. PBMCs were thawed for immunophenotyping by flow cytometry and RNA isolation. RNA sequencing was performed using TruSeq Stranded Total RNA Library Prep Gold (Illumina), following manufacturer’s recommendations, and RNAseq libraries were sequenced on a HiSeq2500 system. Additionally, serum proteomics and immunoassays for pro-inflammatory cytokines in serum were performed. Results Interferon (IFN) type I signaling pathways were at the top of enriched pathways in PBMCs from pSS patients, compared with non-SS sicca controls (adjusted p Conclusion Newly diagnosed patients with pSS show co-activation of the IFN type I and B cell activation pathways, compared to non-SS sicca controls. At the same time, CD4+ T cell subsets critical for B cell function are activated, which can enhance B cell activation and plasma cell formation. Personalized treatment based on the activity of each pathway in individual patients potentially increases the efficacy of such treatments. Acknowledgement Part of this work was funded by NIH grant RFA-DE-06-004 Disclosure of Interests Gwenny M. Verstappen: None declared, Lu Gao Employee of: Bristol-Myers Squibb, Sarah A. Pringle: None declared, Janneke H. Terpstra: None declared, Silvia C. Liefers: None declared, Jolien F. van Nimwegen Speakers bureau: Bristol-Myers Squibb, Arjan Vissink: None declared, Vishal Patel Employee of: Bristol-Myers Squibb, Aiqing He Employee of: Bristol-Myers Squibb, Amy Truong Employee of: Bristol-Myers Squibb, Sarah Hu Employee of: Bristol-Myers Squibb, Ashok Dongre Employee of: Bristol-Myers Squibb, Arathi Krishnakumar Employee of: Bristol-Myers Squibb, Stefan Kirov Employee of: Bristol-Myers Squibb, Julie Carman Employee of: Bristol-Myers Squibb, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant for: Roche, Bristol-Myers Squibb, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Bristol-Myers Squibb, Novartis, Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant for: Bristol-Myers Squibb, Speakers bureau: Bristol-Myers Squibb, Roche, Janssen-Cilag

Background:In patients with primary Sjögren’s syndrome (pSS), both minor and major salivary glands are targets of the disease. Infiltration of salivary glands by immune cells is characteristic for pSS. However, significant inter- and intra-individual variation exists in the size and composition of the infiltrates. Potential differences between minor and major salivary glands in immune cell presence and inflammatory pathway activation are unclear. This knowledge is essential for clinical trial design and precision therapy.Objectives:To compare the transcriptome of paired labial and parotid salivary gland tissue of patients with pSS and non-SS sicca controls.Methods:Thirty-nine pSS patients and 20 age- and sex-matched non-SS sicca controls, who participated in a prospective diagnostic cohort[1], were included. All pSS patients fulfilled 2016 ACR-EULAR criteria. RNA was isolated from formalin-fixed, paraffin-embedded labial and parotid gland tissue sections from the same individuals. Complementary DNA libraries were prepared and sequenced. Biopsies with evidence of sclerosing chronic sialoadenitis or mucosa-associated lymphoid tissue lymphoma were excluded in the current analysis. For differential gene expression analysis, patients were subdivided in four categories: I) non-SS sicca without lymphocytic infiltration, II) non-SS sicca with aspecific infiltration, III) pSS with negative biopsy (focus scoreResults:Principal component analysis (PCA) showed that only pSS patients with a positive biopsy (group IV) could be separated from the non-SS sicca patients based on gene expression. When comparing the labial and parotid gland transcriptome, resp. 798 and 1461 DEGs (FDR-adjusted p-value1) were identified between groups I and IV. The top differentially regulated genes were mostly related to T and B cells.CXCL13, CCR6, MS4A1(CD20), FCRL4andDAZLwere among the genes with the highest positive fold change in both glands of biopsy-positive pSS patients. Overall, there was a moderate to strong correlation between fold changes in labial and parotid glands (R2=0.58, p-valueConclusion:The transcriptome of labial and parotid gland tissue from pSS patients with a positive biopsy is overall comparable, while salivary gland tissue from biopsy-negative pSS patients shows a comparable gene expression profile to non-SS sicca controls. These results indicate that different treatment strategies may be necessary for biopsy-negative and biopsy-positive pSS patients.References:[1]van Nimwegen JF,et al.Validation of the ACR-EULAR criteria for primary Sjögren’s syndrome in a Dutch prospective diagnostic cohort.Rheumatology (Oxford) 2018;57:818–25.Disclosure of Interests:Gwenny M. Verstappen: None declared, Lu Gao Employee of: BMS, Sarah A. Pringle: None declared, Silvia C. Liefers: None declared, Bert van der Vegt Consultant of: Advisory board member for Philips and Visiopharm., Vishal Patel Employee of: BMS, Sarah Hu Shareholder of: Bristol-Myers Squibb, Employee of: Bristol-Myers Squibb, Sumanta Mukherjee Employee of: BMS, Arjan Vissink: None declared, Hendrika Bootsma Grant/research support from: Unrestricted grants from Bristol-Myers Squibb and Roche, Consultant of: Consultant for Bristol-Myers Squibb, Roche, Novartis, Medimmune, Union Chimique Belge, Speakers bureau: Speaker for Bristol-Myers Squibb and Novartis., Frans G.M. Kroese Grant/research support from: Unrestricted grant from Bristol-Myers Squibb, Consultant of: Consultant for Bristol-Myers Squibb, Speakers bureau: Speaker for Bristol-Myers Squibb, Roche and Janssen-Cilag

Objectives. Environmental factors in the aetiology of primary Sjogren's syndrome (pSS) are largely unknown. Host-microbiome interaction at mucosal surfaces is presumed to be involved in the aetiopathogenesis of pSS. Here, we assessed whether the microbiome of the buccal mucosa is specific for pSS compared with symptom-controls.Methods. The bacterial composition of buccal swab samples from 37 pSS patients, 86 non-SS sicca patients (with similar dryness symptoms to pSS patients, but not fulfilling the classification criteria) and 24 healthy controls (HCs) was determined with 16S rRNA sequencing. Multivariate Association with Linear Models was used to find associations between individual taxa and pSS, taking into account smoking and dental status. Associations were replicated in a general population cohort (n = 103).Results. The buccal mucosa microbiome of pSS and non-SS sicca patients both differed from HCs. A higher Firmicutes/Proteobacteria ratio was characteristic for both pSS and non-SS sicca patients. Disease status (pSS, nonSS sicca, HCs) and salivary secretion rate contributed almost equally to the variation in bacterial composition between individuals (3.8 and 4.3%, respectively). Two taxa were associated with pSS compared with non-SS sicca patients and 19 compared with HCs. When salivary secretion rate was taken into account, no taxon was associated with pSS compared with non-SS sicca. Twelve of the 19 pSS-associated taxa were correlated with salivary secretion.Conclusion. Dysbiosis of the buccal mucosa microbiome in pSS patients resembles that of symptom-controls. The buccal mucosa microbiome in pSS patients is determined by a combination of reduced salivary secretion and disease-specific factors.

The autoimmune disease primary Sjögren’s syndrome (pSS) is characterized by hypofunction of the salivary glands (SGs), the cause of which is not correlated to lymphocytic SG infiltration, as prevailing dogma often states. We knocked out the NF-κB proinflammatory pathway inhibitor A20 in keratin14+ epithelial cells, to investigate if immune activated epithelial cells are capable of initiating pSS SG hallmarks. We show that immune activated epithelial cells can cause T cell dominated leukocytic infiltration and immune foci development of the SGs, reflecting the early clinical picture. Infiltrating leukocytes invaded striated ducts, similar to early stage lymphoepithelial lesions observed clinically. Expression of proinflammatory cyto-/chemokines IFN, TNFα, IL-6, CXCL10 and CXCL13 increased in A20-/- SGs, and functionally both volume and mucin 10 content of whole stimulated saliva from A20-/-mice was significantly reduced. Epithelial cells may therefore represent the initial trigger for pSS SG pathologies, as opposed to simple reactionaries to pre-existing stimuli.

Background Hyposalivation and lymphocytic infiltration in salivary glands are common manifestations of primary Sjogren’s syndrome[1]. NF-kappa B (NFκB) signalling is one of the most important proinflammatory pathways, and is inhibited by A20 (also known as TNFAIP3)[2]. Although mounting studies are pointing to the central role of epithelial cells in pSS[3–5], whether pSS-like features can be initiated by immune activation of epitheliums remains to be explored. Objectives We sought to investigate the hypothesis that epithelial cells are capable of initiating the major pathological salivary gland hallmarks of primary Sjogren’s syndrome. In order to achieve this we employ a keratin 14 (KRT14) promoter-driven knockout of the A20 NFκB signalling inhibitor. Methods A20 gene expression was knocked out in KRT14+ cells, namely ductal and myoepithelial cells. Whole pilocarpine-stimulated saliva was collected from A20-/- mice and wildtype (WT) littermate controls at 10, 20 and 30 weeks of age. Submandibular SGs were harvested at all time points for histological examination and qPCR. Results In submandibular SGs of A20-/- mice at 30 weeks of age, 10% of all cells were CD45+ leukocytes and 3% were CD3+ T cells, both significantly more than controls. B cell proportion increased over time in A20-/- mice, but was not significantly different to controls. CD45+ cells formed immune foci (>50 CD45+ cells together) localised to striated ducts, present at significanty greater frequencies than control mice. CD45+ cells, T cells and occasional B cells in A20-/- mice also invaded striated ducts. Expression of the pro-inflammatory cyto/chemo-kines IFNγ, TNFα, IL-6, CXCL10 and CXCL13 was also significantly greater in A20-/- mice. Functionally, both volume and mucin 10 content of whole stimulated saliva from A20-/- mice was significantly reduced compared to controls. Conclusions We present a model for epithelial cell involvement in pSS SG pathology development. We confirm that saliva production defects, foci formation and striated duct invasion can be triggered solely by immune activated epithelial cells. References [1] Brito-Zeron P, et al. Early diagnosis of primary Sjogren’s syndrome: EULAR-SS task force clinical recommendations. Expert Rev. Clin. Immunol. 2016;12:137–156. [2] Shembade N, Ma A, Harhaj EW. Inhibition of NF-kappaB signaling by A20 through disruption of ubiquitin enzyme complexes. Science2010;327:1135–9. [3] Spachidou MP, et al. Expression of functional Toll-like receptors by salivary gland epithelial cells: increased mRNA expression in cells derived from patients with primary Sjogren’s syndrome. doi:10.1111/j.1365-2249.2006.03311.x [4] Ittah M, et al. B cell-activating factor of the tumor necrosis factor family (BAFF) is expressed under stimulation by interferon in salivary gland epithelial cells in primary Sjogren’s syndrome. Arthritis Res. Ther. 2006;8:R51. [5] Fox RI, Kang HI, Ando D, Abrams J, Pisa E. Cytokine mRNA expression in salivary gland biopsies of Sjogren’s syndrome. J. Immunol1994;152:5532–9. Disclosure of Interest None declared

International audience; Leptin is a hormone produced by adipocytes, and its expression is regulated by body fatness and energy balance. This study describes the association of four leptin gene polymorphisms in dairy cows (R4C, A59V, RFLP1, and BM1500) with circulating leptin concentrations during the periparturient period. A59V is located at a between-species conserved region of leptin, and R4C might have effect on the tertiary structure of the leptin protein because of the presence of an extra cystein. RFLP1 is an intronic SNP and BM1500 is a microsatellite located 3.6 kb downstream of the leptin locus. The four polymorphisms were genotyped in 323 HF heifers with known pedigree. Leptin concentrations were determined biweekly from 30 days before until 80 days after parturition. The effect of genotype on leptin concentrations was modeled by fitting a spline in ASREML describing leptin concentrations as a function of days relative to parturition for each genotype/allele. Surprisingly, associations were found during pregnancy, but not during lactation. This indicates that the polymorphism could be more effective during pregnancy. If further studies demonstrate that more leptin-binding protein (Ob-Re) is present in this stage, it is hypothesized that a structural difference in the leptin protein could cause a sub-optimal binding stringency to Ob-Re. Free leptin could be cleared faster than bound leptin, and this could result in lower leptin concentrations during pregnancy for the polymorphism. The effects found might be ascribed to R4C. However, more study on the Ob-Re receptor, like binding stringencies between R4C and wild-type leptin and glycosylation during pregnancy, would provide more insight in the results found.