Histopathological comparison of Sjögren-related features between paired labial and parotid salivary gland biopsies of sicca patients.

OBJECTIVES: To compare focus score and other histopathological features between paired labial and parotid salivary gland biopsies in a diagnostic cohort of suspected Sjögren's disease (SjD) patients. METHODS: Labial and parotid salivary gland biopsies were simultaneously obtained from patients with sicca complaints, suspected of having SjD. Biopsies were formalin fixed and paraffin embedded. Sections were stained with haematoxylin & eosin, and for CD3, CD20, CD45, cytokeratin, CD21, Bcl6, activation-induced deaminase (AID) and IgA/IgG. Focus score and other histopathological features characteristic for SjD were analysed. RESULTS: Based on the expert opinion of three experienced rheumatologists, 36 patients were diagnosed as SjD and 63 as non-SjD sicca patients. When taking all patients together, absolute agreement of various histopathological features between labial and parotid biopsies was high and varied between 80% (focus score) and 93% [(pre-)lymphoepithelial lesions (LELs)]. More labial gland biopsies had a focus score ≥1 compared with their parotid counterpart. Accordingly, the area of infiltrate was larger in labial gland biopsies. When considering only SjD patients, labial glands contained significantly fewer B-lymphocytes and germinal centres/mm2, and less severe LELs compared with parotid glands. CONCLUSION: Labial and parotid glands from SjD patients contain similar histopathological key features, and thus both glands can be used for diagnosis and classification of SjD. However, parotid salivary glands reveal more evident B-lymphocyte-related features, while labial glands exhibit more inflammation, which may be partially unrelated to SjD.

Increased Interferon Signaling in Vaginal Tissue of Patients With Primary Sjögren Syndrome.

OBJECTIVE: Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS. METHODS: Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs]). RESULTS: The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway (P < 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher (P < 0.01 for both pathways). Conversely, transforming growth factor-ß signaling and angiogenesis pathway scores were lower in pSS (P = 0.02 and P = 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity. CONCLUSION: Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness).

Histopathology, salivary flow and ultrasonography of the parotid gland: three complementary measurements in primary Sjögren's syndrome.

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.

Vaginal dryness in primary Sjögren's syndrome: a histopathological case-control study.

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.

Shared gut, but distinct oral microbiota composition in primary Sjögren's syndrome and systemic lupus erythematosus.

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.

Dysbiosis of the buccal mucosa microbiome in primary Sjögren's syndrome patients.

Objectives: Environmental factors in the aetiology of primary Sjögren'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, non-SS 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.

Increased Diagnostic Accuracy of the Labial Gland Biopsy in Primary Sjögren Syndrome When Multiple Histopathological Features Are Included.

OBJECTIVE: The aim of this study was to evaluate the diagnostic accuracy of the labial salivary gland biopsy based on multiple histopathological features in patients with suspected primary Sjögren syndrome (pSS). METHODS: Patients from a diagnostic sicca cohort with clinically suspected pSS who underwent a labial gland biopsy were included. Patients were categorized as having pSS or non-Sjögren syndrome sicca (non-SS sicca) based on vignettes scored by an expert panel. Labial gland biopsies were analyzed for the presence of four histopathological features: focus score (FS) ≥1, prelymphoepithelial and lymphoepithelial lesions, immunoglobulin G plasma cell shift, and germinal centers. Sensitivity and specificity of histologic features were calculated, and the optimal cutoff value for the number of histopathological features needed to diagnose pSS was determined with receiver operating curve analysis. RESULTS: A total of 38 patients were categorized as having pSS and 65 as having non-SS sicca. In labial gland biopsies of patients with pSS, the prevalence of FS ≥1 was 82%, followed by 68% for pre-lymphoepithelial and lymphoepithelial lesions, 63% for plasma cell shift, and 24% for germinal centers. Although FS ≥1 showed the highest sensitivity for patients with pSS (82%), specificity was higher for the other three features (98%-100%). The presence of two or more (of four) histopathological features had almost comparable sensitivity to FS alone, but specificity increased with 12% to 100%. For fulfillment of American College of Rheumatology/EULAR criteria, specificity increased from 84% to 95% when an abnormal biopsy was defined by the presence of two or more histopathological features instead of FS ≥1 only. CONCLUSION: The diagnostic accuracy of the labial gland biopsy increases when other histopathological features besides FS are taken into account, by reducing the number of false-positive biopsies.

More severe parotid gland histopathology in paediatric-onset than in adult-onset Sjögren's disease.

OBJECTIVES: The aim of this study was to assess the histopathological features of the parotid glands in patients with paediatric-onset Sjögren's disease (pedSjD) in comparison to patients with adult-onset Sjögren's disease (adSjD). METHODS: This study was performed in Groningen, the Netherlands. Patients with pedSjD from a diagnostic paediatric cohort (n=19), patients with adSjD from a diagnostic adult cohort (n=32) and patients with adSjD who participated in a clinical trial (n=42) with a baseline parotid gland biopsy were included. Parotid gland biopsies were analysed after (immuno)histological staining for SjD-related histopathological markers and compared between groups. RESULTS: All characteristic histopathological features of adSjD were also observed in pedSjD. There were no significant differences in lymphoepithelial lesions or immunoglobulin A (IgA)/IgG plasma cell shift between the pedSjD and the adSjD cohorts. However, compared with the diagnostic adSjD cohort (with comparable total EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI) scores), pedSjD showed more severe lymphocytic infiltration as reflected by a higher focus score (p=0.003), a higher relative surface area of CD45+ infiltrate (p=0.041), higher numbers of B and T lymphocytes/mm2 (p=0.004 and p=0.029, respectively), a higher B/T lymphocyte ratio (p=0.013), higher numbers of CD21+ follicular dendritic cell networks/mm2 (p=0.029) and germinal centres (GC)/mm2 (p=0.002). Compared with the trial adSjD cohort, with significant higher total ESSDAI scores (p=0.001), only the B/T lymphocyte ratio and numbers of GC/mm2 were significantly higher in the pedSjD cohort (p=0.023 and p=0.018, respectively). CONCLUSION: Patients with pedSjD exhibit more pronounced histopathological features compared with patients with adSjD at diagnosis. Notably, the histopathology of patients with pedSjD aligns more closely with that observed in an adSjD clinical trial cohort, with even stronger B lymphocyte involvement.

The Transcriptome of Paired Major and Minor Salivary Gland Tissue in Patients With Primary Sjögren's Syndrome.

Background: While 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. Methods: Paraffin-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. Results: With 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 (FDR<0.05, log2FC<-1 or >1) 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-value<0.0001). Gene signatures present in both glands of biopsy-positive pSS patients included IFN-α signaling, IL-12/IL-18 signaling, CD3/CD28 T-cell activation, CD40 signaling in B-cells, DN2 B-cells, and FcRL4+ B-cells. Signature scores varied considerably amongst pSS patients. Conclusion: Transcriptomes 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.

Association of leptin gene polymorphisms with serum leptin concentration in dairy cows.

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.