Labial Salivary Gland Biopsy Specimens Research Articles

Value of multilevel sectioning of labial salivary gland biopsies in the diagnosis of Sjögren's syndrome

The objective of this study was to examine the diagnostic value of cutting labial salivary gland (LSG) biopsies at 2 levels in the histological evaluation for Sjögren's syndrome (SS). This retrospective study included LSG biopsy specimens from 112 consecutive patients evaluated for SS from 2007 to 2019. Three observers, blinded with regard to patient data, independently scored the degree of focal lymphocytic infiltration (foci) and calculated the focus score in specimens cut at 2 levels 60 µm apart. Unblinded analysis revealed that the LSG specimens derived from 107 women and 5 men, aged 49.2 ± 22.3 years. Seventy-six patients had SS (70 primary SS and 6 secondary SS) according to the American-European Consensus Group and American College of Rheumatology/European League Against Rheumatism criteria. The average number of LSGs was 5.0 ± 1.4 and the focus scoring area was 16.1 ± 7.6 mm2. Compared to baseline, the average number of foci (4.4 vs 5.1, P < .001), focus score (1.7 vs 1.9, P = .01), and cases with focus score >1.0 (61 vs 74%; P=.03) were higher in the second level. Subsequently, an additional 11 cases of SS were confirmed (14%), and 8 non-SS cases were reclassified as SS (22%). Histological assessment of an additional section level improves the diagnostic accuracy of the labial salivary gland biopsy to detect histopathological changes consistent with the diagnosis of SS.

Leflunomide-hydroxychloroquine combination therapy in patients with primary Sjögren's syndrome (RepurpSS-I): a placebo-controlled, double-blinded, randomised clinical trial.

Primary Sjögren's syndrome is a systemic autoimmune disease characterised by secretory gland dysfunction, for which no effective therapy is available. Based on the complementary properties of leflunomide and hydroxychloroquine in inhibiting activation of key immune cells in primary Sjögren's syndrome, we aimed to evaluate the clinical efficacy and safety of leflunomide-hydroxychloroquine combination therapy in patients with primary Sjögren's syndrome. We did a placebo-controlled, double-blinded, phase 2A randomised clinical trial in patients with primary Sjögren's syndrome at the University Medical Center Utrecht (Utrecht, Netherlands). Eligible patients were aged 18-75 years, had a European League Against Rheumatism (EULAR) Sjögren's syndrome disease activity index (ESSDAI) score of 5 or higher, and a lymphocytic focus score of 1 or higher in labial salivary gland biopsy specimens. Patients were randomly assigned (2:1) with block randomisation (block size of six) to receive leflunomide 20 mg and hydroxychloroquine 400 mg daily or placebo for 24 weeks. The primary endpoint was the between-group difference in change in ESSDAI scores from 0 to 24 weeks, adjusted for baseline ESSDAI score. Patients were analysed according to the intention-to-treat principle. This study is registered with EudraCT, 2014-003140-12. Between March 7, 2016, and Nov 30, 2017, 37 patients were screened, of whom 29 patients (28 women and one man) were enrolled. 21 patients were assigned to receive leflunomide-hydroxychloroquine and eight patients were assigned to receive placebo. One patient in the placebo group required high-dose prednisone to treat polymyalgia rheumatica at week 13 and was excluded from the primary analysis. From 0 to 24 weeks, the mean difference in ESSDAI score, adjusted for baseline values, in the leflunomide-hydroxychloroquine group compared with the placebo group was -4·35 points (95% CI -7·45 to -1·25, p=0·0078). No serious adverse events occurred in the leflunomide-hydroxychloroquine group and two serious adverse events occurred in the placebo group (hospital admission for pancreatitis and hospital admission for nephrolithiasis). The most common adverse events in the leflunomide-hydroxychloroquine group were gastrointestinal discomfort (11 patients [52%] vs two [25%] in the placebo group), modest transient increases in alanine aminotransferase (ten [48%] vs one [13%]), and short episodes of general malaise and shivering (nine [43%] vs one [13%]). Leflunomide-hydroxychloroquine was safe and resulted in a clinical response in patients with primary Sjögren's syndrome. These results warrant further evaluation of leflunomide-hydroxychloroquine combination therapy in larger clinical trials. ZonMw.

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.

Cytometry by time-of-flight immunophenotyping identifies a blood Sjögren's signature correlating with disease activity and glandular inflammation

Mass cytometry has recently emerged as a promising tool for clinical research. However, few studies have demonstrated its benefit for patient stratification and biomarker identification. Primary Sjögren's syndrome (pSS) is a prototype of chronic autoimmune disease, the pathogenesis of which remains unclear and for which treatment does not exist. This observational case-control study was designed to discover new cellular biomarkers and therapeutic targets in patients with pSS. Forty-nine patients with pSS and 45 control subjects were enrolled for clinical evaluation and mass cytometry quantification of 34 protein markers in blood. For a third of these subjects, matched labial salivary gland biopsy specimens were also analyzed by mass cytometry and immunohistochemistry. In salivary gland biopsy specimens from patients with pSS, we identified a high number of activated CD8(+) Tcells, terminally differentiated plasma cells, and activated epithelial cells, pointing to new pathogenic mechanisms for future clinical intervention. In blood, we identified a 6-cell disease signature defined by decreased numbers of CD4 and memory B lymphocytes, decreased plasmacytoid dendritic cell numbers, and increased representation of activated CD4 and CD8 Tcells and plasmablasts. These blood cellular components correlated with clinical parameters and, when taken together, clustered patients into subsets with distinct disease activity and glandular inflammation. This first application of mass cytometry to a well-stratified clinical cohort and small biopsy tissues establishes the benefits of such an approach for the discovery of new biomarkers and therapeutic targets. Similar high-dimensional immunophenotyping strategies could be implemented in longitudinal and interventional clinical settings in this and other disease areas.

Associations between salivary gland histopathologic diagnoses and phenotypic features of Sjögren's syndrome among 1,726 registry participants

To examine associations between labial salivary gland (LSG) histopathology and other phenotypic features of Sjögren's syndrome (SS). The database of the Sjögren's International Collaborative Clinical Alliance (SICCA), a registry of patients with symptoms of possible SS as well as those with obvious disease, was used for the present study. LSG biopsy specimens from SICCA participants were subjected to protocol-directed histopathologic assessments. Among the 1,726 LSG specimens exhibiting any pattern of sialadenitis, we compared biopsy diagnoses against concurrent salivary, ocular, and serologic features. LSG specimens included 61% with focal lymphocytic sialadenitis (FLS; 69% of which had focus scores of ≥1 per 4 mm²) and 37% with nonspecific or sclerosing chronic sialadenitis (NS/SCS). Focus scores of ≥1 were strongly associated with serum anti-SSA/SSB positivity, rheumatoid factor, and the ocular component of SS, but not with symptoms of dry mouth or dry eyes. Those with positive anti-SSA/SSB were 9 times (95% confidence interval [95% CI] 7.4-11.9) more likely to have a focus score of ≥1 than were those without anti-SSA/SSB, and those with an unstimulated whole salivary flow rate of <0.1 ml/minute were 2 times (95% CI 1.7-2.8) more likely to have a focus score of ≥1 than were those with a higher flow rate, after controlling for other phenotypic features of SS. Distinguishing FLS from NS/SCS is essential in assessing LSG biopsies, before determining focus score. A diagnosis of FLS with a focus score of ≥1 per 4 mm², as compared to FLS with a focus score of <1 or NS/SCS, is strongly associated with the ocular and serologic components of SS and reflects SS autoimmunity.

Association between IFN-α and primary Sjogren's syndrome

To determine the level of IFN-alpha in labial salivary glands, plasma, and peripheral blood cells from patients with primary Sjogren's syndrome (pSS). Labial salivary gland biopsy specimens, plasma, and peripheral blood cells from patients with pSS were investigated. The IFN-alpha-positive cells, measurable IFN-alpha level, and IFN-alpha gene mRNA level were determined by using immunohistochemistry, ELISA, and real-time PCR, respectively. Statistical analysis was performed by Student t test or Fisher's exact test. About 60% of patients (22/37) with pSS had significantly higher scores of IFN-alpha-positive cells in labial gland biopsy and most IFN-alpha-positive cells were localized predominantly in the lymphocytes and ductal epithelial cells. But in 3 of the control samples (3/24), the IFN-alpha-positive cells existed only in the ductal epithelial cells with lower scores. Forty-three percents of the patients with pSS were found with detectable IFN-alpha concentration in plasma (> or = 12.5 pg/mL), and their concentration was higher than that of control group. Furthermore, the IFN-alpha mRNA levels in peripheral blood cells were up-regulated in the patients with pSS. No matter in labial salivary glands or hematoplasma, or peripheral blood cells, IFN-alpha expression levels are up-regulated in patients with primary Sjogren's syndrome.

Retrovirus in salivary glands from patients with Sjögren's syndrome.

AIMS: To investigate the possibility of an immune response to retroviral antigens or of detecting retrovirus in Sjögren's syndrome. METHODS: Retroviruses were sought in labial salivary glands and peripheral blood...

シェーグレン症候群患者唾液腺からのレトロウイルスの検出

Sjögren's syndrome (SS) is an autoimmune disease that is characterized by dryness of the mouth and eyes. The loss of salivary and lacrimal gland function is accompanied by lymphocytic infiltration. Because similar symptoms and glandular pathology are observed in certain persons infected with human immunodeficiency virus (HIV), a search was initiated for a possible retroviral etiology in this syndrome. To investigate the possibility of an immune response to retroviral antigens or that of detecting retrovirus in primary SS, I performed immunoblotting assay, immunohistochemical assay, polymerase chain reaction (PCR), and reverse transcriptase (RT) activity assay. Sera from 33% of 15 patients with SS, reacted against p24 group specific antigen (gag) of HIV. Labial salivary gland biopsy specimens from 47% of the 15 patients with SS contained an epithelial cytoplasmic protein reactive with a monoclonal antibody to p24 of HIV. I performed PCR to detect HIV and human T lymphotropic virus type I (HTLV-I)genes in salivary gland tissues and peripheral blood mononuclear cells (PBMC) from patients with SS. I also detected Mn2+ dependent, Mg2+ independent RT activity in the salivary gland tissues in 3 of 10 patients with SS. Target genes for HIV and HTLV-I were not found in any of the salivary gland tissues or PBMC from patients with SS. These data suggested the presence of an unknown retrovirus similar to HIV in the salivary gland that may contribute to chronic inflammation in SS.

Detection of Epstein-Barr virus DNA by in Situ hybridization and polymerase chain reaction in salivary gland biopsy specimens from patients with Sjögren's syndrome

PURPOSE: To determine whether Epstein-Barr virus (EBV) could be involved in the pathogenesis of Sjögren's syndrome (SS). PATIENTS AND METHODS: In situ hybridization using the BamH1-W fragment of EBV DNA was performed using labial salivary gland biopsy specimens from 14 patients with SS (eight with primary SS and six with secondary SS) and 39 control subjects. Furthermore, labial salivary gland biopsy specimens from 12 patients with SS (seven with primary SS and five with secondary SS) and 24 control subjects were submitted to the polymerase chain reaction to detect EBV DNA. RESULTS: In situ hybridization detected EBV DNA in epithelial cells of labial salivary gland biopsy specimens from four of eight (50%) patients with primary SS, zero of six patients with secondary SS, and three of 39 (8%) control subjects. The difference between patients with primary SS and control subjects was statistically significant (p <0.02). The polymerase chain reaction detected EBV DNA in six of seven (86%) patients with primary SS, three of five (60%) patients with secondary SS, and seven of 24 (29%) control subjects. The difference between patients with primary SS and control subjects was statistically significant (p <0.01). CONCLUSION: Both newly developed techniques showed that the presence of EBV DNA was significantly increased in patients with primary SS in comparison with control subjects. In all the positive SS patients who underwent in situ hybridization, epithelial cells of the labial salivary gland were the target of EBV infection. Our results suggests that this virus may play a role in the pathogenesis of SS. We cannot yet determine whether EBV is directly responsible for the destruction of the gland, or if its presence is a secondary event following gland injury. To determine whether Epstein-Barr virus (EBV) could be involved in the pathogenesis of Sjögren's syndrome (SS). In situ hybridization using the BamH1-W fragment of EBV DNA was performed using labial salivary gland biopsy specimens from 14 patients with SS (eight with primary SS and six with secondary SS) and 39 control subjects. Furthermore, labial salivary gland biopsy specimens from 12 patients with SS (seven with primary SS and five with secondary SS) and 24 control subjects were submitted to the polymerase chain reaction to detect EBV DNA. In situ hybridization detected EBV DNA in epithelial cells of labial salivary gland biopsy specimens from four of eight (50%) patients with primary SS, zero of six patients with secondary SS, and three of 39 (8%) control subjects. The difference between patients with primary SS and control subjects was statistically significant (p <0.02). The polymerase chain reaction detected EBV DNA in six of seven (86%) patients with primary SS, three of five (60%) patients with secondary SS, and seven of 24 (29%) control subjects. The difference between patients with primary SS and control subjects was statistically significant (p <0.01). Both newly developed techniques showed that the presence of EBV DNA was significantly increased in patients with primary SS in comparison with control subjects. In all the positive SS patients who underwent in situ hybridization, epithelial cells of the labial salivary gland were the target of EBV infection. Our results suggests that this virus may play a role in the pathogenesis of SS. We cannot yet determine whether EBV is directly responsible for the destruction of the gland, or if its presence is a secondary event following gland injury.

Detection of epstein-barr virus DNA by in situ hybridization and polymerase chain reaction in salivary gland biopsy specimens from patients with sjögren's syndrome

purpose: To determine whether Epstein-Barr virus (EBV) could be involved in the pathogenesis of Sjögren's syndrome (SS). patients and methods:, In situ hybridization using the BamH1-W fragment of EBV DNA was performed using labial salivary gland biopsy specimens from 14 patients with SS (eight with primary SS and six with secondary SS) and 39 control subjects. Furthermore, labial salivary gland biopsy specimens from 12 patients with SS (seven with primary SS and five with secondary SS) and 24 control subjects were submitted to the polymerase chain reaction to detect EBV DNA. results:, In situ hybridization detected EBV DNA in epithelial cells of labial salivary gland biopsy specimens from four of eight (50%) patients with primary SS, zero of six patients with secondary SS, and three of 39 (8%) control subjects. The difference between patients with primary SS and control subjects was statistically significant (p <0.02). The polymerase chain reaction detected EBV DNA in six of seven (86%) patients with primary SS, three of five (60%) patients with secondary SS, and seven of 24 (29%) control subjects. The difference between patients with primary SS and control subjects was statistically significant (p <0.01). conclusion: Both newly developed techniques showed that the presence of EBV DNA was significantly increased in patients with primary SS in comparison with control subjects. In all the positive SS patients who underwent in situ hybridization, epithelial cells of the labial salivary gland were the target of EBV infection. Our results suggest that this virus may play a role in the pathogenesis of SS. We cannot yet determine whether EBV is directly responsible for the destruction of the gland, or if its presence is a secondary event following gland injury.

The histopathology of Sjögren's syndrome in labial salivary gland biopsies

Labial salivary gland biopsy specimens from seventy-five patients evaluated for Sjögren's syndrome were examined histologically. Clinically severe cases of Sjögren's syndrome showed the most marked histologic change. Focus score was found to be the most useful histologic index of severity of the disease. Large numbers of plasma cells were found in small foci and mild cases. Plasma cells were decreased in proportion, compared to lymphocytes, in larger foci and severe cases. These observations are discussed in the light of possible mechanisms of tissue damage in this disease.