In vitro effects of curcumin on proinflammatory cytokines and expression of their genes in minor salivary gland tissue of patients with Sjogren's syndrome.

Curcumin reduces disease severity and ameliorates lupus-like/Sjögren's Syndrome-like disease in mice model. The immunological basis of these effects is largely unknown. This study examined the effects of curcumin on pro-inflammatory cytokines secreted by minor salivary glands in patients with primary Sjögren's syndrome (pSS). Minor salivary gland (MSG) tissue samples were collected from patients undergoing biopsy for suspected pSS. The tissues were treated with phytohemagglutinin (PHA) alone as well as PHA with curcumin (30 µM) and cultured in RPMI 1640 medium for 48 h at 37 °C in CO2 incubator. After the incubation period, culture supernatant and tissues were stored in the freezer (-80 °C). IL-6 levels were measured in supernatant by ELISA kit. Gene expressions of pro-inflammatory cytokines, namely IL-6, IL-8, TNF-α, IL-1ß, IL-4, IL-10, IL-17, IL-21, and IFN-γ, were measured by qPCR. IL-6 secretion levels and gene expressions were compared statistically between groups by Student's t test. Forty-seven patients were screened. Eight patients satisfied ACR/EULAR criteria for pSS. Seven patients with absent glandular inflammation and negative serology constituted sicca controls. These 15 subjects were included in final analysis. In pSS group, but not in controls, median IL-6 levels in supernatant were less in curcumin-treated as compared to PHA-alone subset [5.5 (0.7-13.34) vs 18.3 (12-32) ng/ml; p = 0.0156]. mRNA expression levels of IL-6 were also lower in curcumin-treated samples as compared to PHA alone, when cases and controls were analyzed together as well as in cases alone (p = 0.0009 and p = 0.0078, respectively); however, mRNA expression of IL-1ß was lower in curcumin-treated samples as compared to PHA alone, only when cases and controls were analyzed together (p = 0.0215). There was no difference in other cytokine gene expression levels between the subsets under the in-vitro experimental conditions. In conclusion, curcumin reduced mRNA expression as well as secretion of IL-6 levels by salivary gland tissue of patients with pSS. Curcumin also suppressed PHA-induced mRNA expression levels of IL-6 and IL-1ß in MSG tissue of patients with pSS and controls when analyzed together as a combined group.

Inflammatory Stratification in Primary Sjögren's Syndrome Reveals Novel Immune Cell Alterations in Patients' Minor Salivary Glands.

There is a critical need to deconvolute the heterogeneity displayed by the minor salivary glands of primary Sjögren's syndrome (pSS) patients. This is challenging primarily because the disease etiology remains unknown. The hypothesis includes that initial events in the disease pathogenesis target the salivary glands, thereby triggering the development of focal infiltrates (≥50 mononuclear cells) and finally germinal center-like structures. However, the proportion of key mononuclear immune cells residing at these sites, in combination with the overall ratio of morphometric tissue atrophy and adipose infiltration within the minor salivary glands (MSG) parenchyma at distinct phases of inflammatory disease establishment and progression have not been quantified in detail. In this cross-sectional study, we intended to address this problem by stratifying 85 patients into mild (S1), moderate (S2), and severe (S3) stages using the Inflammatory severity index. We found that mild (<3%) and marked (≥3%) levels of atrophy were accompanied by the respective levels of adipose infiltration in the non-SS sicca controls (p <0.01), but not in pSS patients. The percentage of adipose infiltration significantly correlated with the age of patients (r = 0.458, p <0.0001) and controls (r = 0.515, p <0.0001). The CD4+ T helper cell incidence was reduced in the focal infiltrates of the MSG of S2 patients compared to S1 (p <0.01), and in S2 compared to S1 and S3 combined (p <0.05). CD20+ B cells increased from S1 to S3 (p <0.01) and S2 to S3 (p <0.01), meanwhile CD138+ plasma cells diminished in S3 patients compared to both S1 and S2 groups combined (p <0.01). The proportion of patients with anti-Ro/SSA+, anti-La/SSB+, and RF+ increased over the course of inflammatory disease progression and they were significantly more common in the S3 group relative to S1 (p <0.05). On the other hand, S2 patients measured a higher mean salivary flow relative to S1 and S3 patients combined (p <0.05). Our results demonstrate how the proposed Inflammatory severity index stratification revealed pathological cell and tissue-associated aberrations in the salivary component over the course of inflammatory progression, and their correlations to clinical outcomes. This could be directly transferred to the optimization of available diagnostic strategies applied for pSS patients.

A biomarker for lymphoma development in Sjogren's syndrome: Salivary gland focus score.

The aim of this study is to explore the role of labial minor salivary gland (LMSG) focus score (FS) in stratifying Sjögren's Syndrome (SS) patients, lymphoma development prediction and to facilitate early lymphoma diagnosis. Ιn an integrated cohort of 1997 patients, 618 patients with FS ≥ 1 and at least one-year elapsing time interval from SS diagnosis to lymphoma diagnosis or last follow up were identified. Clinical, laboratory and serological features were recorded. A data driven logistic regression model was applied to identify independent lymphoma associated risk factors. Furthermore, a FS threshold maximizing the difference of time interval from SS until lymphoma diagnosis between high and low FS lymphoma subgroups was investigated, to develop a follow up strategy for early lymphoma diagnosis. Of the 618 patients, 560 were non-lymphoma SS patients while the other 58 had SS and lymphoma. FS, cryoglobulinemia and salivary gland enlargement (SGE) were proven to be independent lymphoma associated risk factors. Lymphoma patients with FS ≥ 4 had a statistically significant shorter time interval from SS to lymphoma diagnosis, compared to those with FS < 4 (4 vs 9 years, respectively, p = 0,008). SS patients with FS ≥ 4 had more frequently B cell originated manifestations and lymphoma, while in patients with FS < 4, autoimmune thyroiditis was more prevalent. In the latter group SGE was the only lymphoma independent risk factor. A second LMSG biopsy is patients with a FS ≥ 4, 4 years after SS diagnosis and in those with FS < 4 and a history of SGE, at 9-years, may contribute to an early lymphoma diagnosis. Based on our results we conclude that LMSG FS, evaluated at the time of SS diagnosis, is an independent lymphoma associated risk factor and may serve as a predictive biomarker for the early diagnosis of SS-associated lymphomas.

A case with overlapping features of IgG4-related autoimmune pancreatitis, Sjögren's syndrome and anti-aminoacyl-tRNA synthetase syndrome.

A 55-year-old man who had been diagnosed with autoimmune pancreatitis five years earlier was referred to our department because of finger swelling, finger stiffness and the presence of interstitial lung disease (ILD). The patient was diagnosed with Sjögren's syndrome according to the pathological findings of minor salivary glands and positive anti-SS-A antibodies. Later, at age 58, he was hospitalised due to the exacerbation of the ILD. Serum IgG4 level was checked and was found to be elevated (417 mg/dL). After the introduction of cyclosporine in addition to the prednisolone, at age 60, the ILD disease activity stabilised. However, at age 62, fever, myalgia and mechanic's hands appeared. His serum creatine kinase level was high, and magnetic resonance imaging showed inflammatory findings of muscle. In-house ELISA clarified that his serum carried anti-PL-7 antibody among anti-aminoacyl-tRNA synthetase antibodies. This is a unique case who had overlapping features of IgG4-related autoimmune pancreatitis, Sjögren's syndrome and anti-synthetase syndrome. Although the aetiology of the complications in this patient is obscure, autoimmunity might have played a significant role in the disease conditions and prognosis of the present case with IgG4-related disease.

LAMP3 induces apoptosis and autoantigen release in Sjögren's syndrome patients.

Primary Sjögren's syndrome (pSS) is a complex autoimmune disease characterized by dysfunction of secretory epithelia with only palliative therapy. Patients present with a constellation of symptoms, and the diversity of symptomatic presentation has made it difficult to understand the underlying disease mechanisms. In this study, aggregation of unbiased transcriptome profiling data sets of minor salivary gland biopsies from controls and Sjögren's syndrome patients identified increased expression of lysosome-associated membrane protein 3 (LAMP3/CD208/DC-LAMP) in a subset of Sjögren's syndrome cases. Stratification of patients based on their clinical characteristics suggested an association between increased LAMP3 expression and the presence of serum autoantibodies including anti-Ro/SSA, anti-La/SSB, anti-nuclear antibodies. In vitro studies demonstrated that LAMP3 expression induces epithelial cell dysfunction leading to apoptosis. Interestingly, LAMP3 expression resulted in the accumulation and release of intracellular TRIM21 (one component of SSA), La (SSB), and α-fodrin protein, common autoantigens in Sjögren's syndrome, via extracellular vesicles in an apoptosis-independent mechanism. This study defines a clear role for LAMP3 in the initiation of apoptosis and an independent pathway for the extracellular release of known autoantigens leading to the formation of autoantibodies associated with this disease.ClinicalTrials.gov Identifier: NCT00001196, NCT00001390, NCT02327884.

Immunohistochemical detection of IL-17 and IL-23 improves the identification of patients with a possible diagnosis of Sjogren's syndrome.

OBJECTIVES: The diagnosis of primary Sjogren's syndrome (pSS) continues to be difficult and several patients keep symptomatic for years with different diagnoses before confirmation of pSS. Since the IL-23-IL-17 axis is involved in the etiopathogenesis of pSS we evaluated by immunohistochemistry and morphometric methods the presence of IL-17 as well as IL-23 within minor salivary glands (MSG) obtained from patients with uncertain diagnosis of pSS. MATERIALS AND METHODS: 42 patients, with symptoms attributable to pSS, and 8 patients used as a control, were enrolled for the study. Autoantibody detection, histological analysis for the presence of Germinal Centers (GC+), immunohistochemistry to detect IL-23 and IL-17 were performed. RESULTS: The detection of GC + anti-SSA and anti-SSB antibody in parallel with the detection of IL-17 and IL-23, displays only a diagnostic reinforcement value. Instead, the detection of a positive reaction for both IL-17 and IL-23 without GC + or autoantibody within minor salivary glands, as detected in 36 % of patients with uncertain diagnosis, may be hold as a sensitive and specific marker to identify those patients who are likely to evolve into pSS. CONCLUSION: we suggest to use the IL-17/ IL-23 immunohistochemical detection to improve the identification of patients with a possible diagnosis in all cases which do not fully meet the American-European criteria for pSS, in particular when the GC + are not present at histopathological analysis and anti-SSA and anti-SSB antibody are undetectable in the serum.

Age-Associated Role of Lingual and Pharyngeal Glands in Local Immunity in Oral Cavity.

The macroscopic and histological methods were employed to examine the autopsy specimens of salivary lingual glands obtained from 299 patients of both sexes and various age ranging from newborn to longevity. The age-associated alterations of minor lingual and pharyngeal glands were revealed, and the topographical relations between the glands and lymphoid cells were described. The characteristic sparsity of the glands in infancy is caused by nutritional uniformity at this period, when diminished production of secretory IgA results in frequent inflammatory processes in oral and pharyngeal cavities. With age, the glandular orifices widen, and their number increases thereby augmenting local immunity in the oral cavity and in oral aspect of the pharynx. Starting from elderly and senile age, the involutive alterations were observed, which were accompanied by diminished production of secretory immunoglobulin A and related degradation of local and humoral immunity.

Long Term Delta-9-tetrahydrocannabinol Administration Inhibits Proinflammatory Responses in Minor Salivary Glands of Chronically Simian Immunodeficieny Virus Infected Rhesus Macaques.

HIV/SIV-associated oral mucosal disease/dysfunction (HAOMD) (gingivitis/periodontitis/salivary adenitis) represents a major comorbidity affecting HIV patients on anti-retroviral therapy. Using a systems biology approach, we investigated molecular changes (mRNA/microRNA) underlying HAOMD and its modulation by phytocannabinoids (delta-9-tetrahydrocannabinol (∆9-THC)) in uninfected (n = 5) and SIV-infected rhesus macaques untreated (VEH-untreated/SIV; n = 7) or treated with vehicle (VEH/SIV; n = 3) or ∆9-THC (THC/SIV; n = 3). Relative to controls, fewer mRNAs were upregulated in THC/SIV compared to VEH-untreated/SIV macaques. Gene enrichment analysis showed differential enrichment of biological functions involved in anti-viral defense, Type-I interferon, Toll-like receptor, RIG-1 and IL1R signaling in VEH-untreated/SIV macaques. We focused on the anti-ER-stress anterior gradient-2 (AGR2), epithelial barrier protecting and anti-dysbiotic WAP Four-Disulfide Core Domain-2 (WFDC2) and glucocorticoid-induced anti-inflammatory TSC22D3 (TSC22-domain family member-3) that were significantly downregulated in oropharyngeal mucosa (OPM) of VEH-untreated/SIV macaques. All three proteins localized to minor salivary gland acini and secretory ducts and showed enhanced and reduced expression in OPM of THC/SIV and VEH/SIV macaques, respectively. Additionally, inflammation associated miR-21, miR-142-3p and miR-29b showed significantly higher expression in OPM of VEH-untreated/SIV macaques. TSC22D3 was validated as a target of miR-29b. These preliminary translational findings suggest that phytocannabinoids may safely and effectively reduce oral inflammatory responses in HIV/SIV and other (autoimmune) diseases.

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 (r2 = 0.54, p < .02), while ductal epithelial expression showed a negative such correlation (r2 = 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.

Transcriptomic and Network Analysis of Minor Salivary Glands of Patients With Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized primarily by immune-mediated destruction of exocrine tissues, such as those of the salivary and lacrimal glands, resulting in the loss of saliva and tear production, respectively. This disease predominantly affects middle-aged women, often in an insidious manner with the accumulation of subtle changes in glandular function occurring over many years. Patients commonly suffer from pSS symptoms for years before receiving a diagnosis. Currently, there is no effective cure for pSS and treatment options and targeted therapy approaches are limited due to a lack of our overall understanding of the disease etiology and its underlying pathology. To better elucidate the underlying molecular nature of this disease, we have performed RNA-sequencing to generate a comprehensive global gene expression profile of minor salivary glands from an ethnically diverse cohort of patients with pSS. Gene expression analysis has identified a number of pathways and networks that are relevant in pSS pathogenesis. Moreover, our detailed integrative analysis has revealed a primary Sjögren's syndrome molecular signature that may represent important players acting as potential drivers of this disease. Finally, we have established that the global transcriptomic changes in pSS are likely to be attributed not only to various immune cell types within the salivary gland but also epithelial cells which are likely playing a contributing role. Overall, our comprehensive studies provide a database-enriched framework and resource for the identification and examination of key pathways, mediators, and new biomarkers important in the pathogenesis of this disease with the long-term goals of facilitating earlier diagnosis of pSS and to mitigate or abrogate the progression of this debilitating disease.

Distinct clinical characteristics of anti-Ro/SSA-negative primary Sjögren's syndrome: data from a nationwide cohort for Sjögren's syndrome in Korea.

OBJECTIVES: To investigate clinical characteristics of patients with primary Sjögren's syndrome (SS) who were negative for anti-Ro/SSA antibody but positive for minor salivary gland biopsy (MSGB) compared to patients who presented positivity for anti-Ro/SSA antibody. METHODS: The data of 355 patients from the Korean Initiative of primary Sjögren's Syndrome (KISS), a nationwide prospective cohort for primary SS in Korea, were analysed. All patients fulfilled the 2016 American College of Rheumatology/European League Against Rheumatism (EULAR) classification criteria. Of these patients, 326 were positive for anti-Ro/SSA antibody and 29 were antibody-negative, although they had positive findings in MSGB. Various clinical features including all kinds of tests for evaluating secretory function, disease-related clinical indices and serological values available in the cohort were compared between the two groups. RESULTS: The anti-Ro/SSA-negative group showed less rheumatoid factor positivity (p<0.001), leucopenia (p=0.003), hyper-gammaglobulinaemia (p<0.001), lower serum ß2-microglobulin level (p=0.034), more anti-centromere antibody positivity (p<0.001), higher score in dryness domain of EULAR SS patient-reported index (p=0.048) and more positivity for peripheral nervous system domain in EULAR SS disease activity index and loss of teeth in SS disease damage index (p=0.021 and 0.041, respectively) than patients who were positive for anti-Ro/ SSA antibody. CONCLUSIONS: Primary SS patients who are negative for anti-Ro/SSA antibody have different clinical characteristics compared to patients who are positive for such antibody in Korea. Therefore, clinicians should consider MSGB in patients with suspicious symptoms who are anti-Ro/SSA-negative.

Presence of intraepithelial B-lymphocytes is associated with the formation of lymphoepithelial lesions in salivary glands of primary Sjögren's syndrome patients.

OBJECTIVES: Lymphoepithelial lesions (LELs) in salivary glands are associated with primary Sjögren's syndrome (pSS). LELs are composed of hyperplastic epithelium infiltrated with lymphocytes. The objective of this study was obtaining insight in the relative roles of intraepithelial B- and T-lymphocytes in the formation of LELs in salivary glands of pSS patients. METHODS: Parotid and labial salivary gland biopsies of pSS patients (n=15), non-SS sicca patients (n=5) and non-sicca controls (n=5) were analysed. Serial sections were stained with H & E and for cytokeratin, CD20 and CD3. Striated ducts with lymphocytes, but without hyperplasia, and striated ducts with LELs were identified in H & E and cytokeratin stained sections. LELs were classified in successive stages of severity based on the amount of hyperplasia (stage1-3). Numbers of B- and T-lymphocytes within striated ducts and LELs were counted in CD20 and CD3 stained sections. RESULTS: Lymphocyte-containing striated ducts of both salivary glands of all pSS and control patients harboured T-lymphocytes, scattered throughout the ductal epithelium. In contrast, B-lymphocytes were exclusively found in a small fraction (21%) of striated ducts without hyperplasia and in nearly all striated ducts with LELs of pSS patients, but not in controls. In striated ducts with LELs B-lymphocytes were mostly located in the areas of proliferating epithelium. Numbers of B-lymphocytes and B/T-ratios increased significantly with higher severity of LELs. This was even more pronounced in the parotid than in the labial gland. CONCLUSIONS: We conclude there is an association between presence of intraepithelial B-lymphocytes and the formation of LELs in salivary glands of pSS patients.

Tissue-Resident Memory CD8+ T Cells Acting as Mediators of Salivary Gland Damage in a Murine Model of Sjögren's Syndrome.

OBJECTIVE: Although a role for CD4+ T cells in the pathogenesis of Sjögren's syndrome (SS) has been documented, the pathogenic significance of CD8+ T cells is unclear. The aim of this study was to investigate the role of CD8+ T cells in the development of SS. METHODS: Flow cytometry and immunofluorescence analyses were utilized to detect T cell infiltration within the labial salivary glands of patients with primary SS. In parallel, p40-/- CD25-/- mice were used as a murine model of SS. In addition, mice with genetic knockout of CD4, CD8a, or interferon-γ (IFNγ) were crossed with p40-/- CD25-/- mice to study the pathogenic significance of specific lineage subpopulations, including functional salivary gland tests as well as histopathologic and serologic data. A CD8+ T cell-specific depletion antibody was used in this murine SS model to evaluate its potential as a therapeutic strategy. RESULTS: CD8+ T cells with a tissue-resident memory phenotype outnumbered CD4+ T cells in the labial salivary glands of patients with SS, and were primarily colocalized with salivary duct epithelial cells and acinar cells. Furthermore, infiltrating CD8+ T cells with a CD69+CD103+/- tissue-resident phenotype and with a significant elevation of IFNγ production were dominant in the submandibular glands of mice in this murine SS model. CD8a knockout abrogated the development of SS in these mice. Knockout of IFNγ decreased CD8+ T cell infiltration and gland destruction. More importantly, depletion of CD8+ T cells fully protected mice against the pathologic manifestations of SS, even after the onset of disease. CONCLUSION: These data reveal the pathogenic significance of CD8+ T cells in the development and progression of SS in the salivary glands. Treatment directed against CD8+ T cells may be a rational therapy for the management of SS in human subjects.

IFI16 filament formation in salivary epithelial cells shapes the anti-IFI16 immune response in Sjögren's syndrome.

IFN-inducible protein 16 (IFI16) is an innate immune sensor that forms filamentous oligomers when activated by double-stranded DNA (dsDNA). Anti-IFI16 autoantibodies occur in patients with Sjögren's syndrome (SS) and associate with severe phenotypic features. We undertook this study to determine whether the structural and functional properties of IFI16 play a role in its status as an SS autoantigen. IFI16 immunostaining in labial salivary glands (LSGs) yielded striking evidence of filamentous IFI16 structures in the cytoplasm of ductal epithelial cells, representing the first microscopic description of IFI16 oligomerization in human tissues, to our knowledge. Transfection of cultured epithelial cells with dsDNA triggered the formation of cytoplasmic IFI16 filaments with similar morphology to those observed in LSGs. We found that a majority of SS anti-IFI16 autoantibodies immunoprecipitate IFI16 more effectively in the oligomeric dsDNA-bound state. Epitopes in the C-terminus of IFI16 are accessible to antibodies in the DNA-bound oligomer and are preferentially targeted by SS sera. Furthermore, cytotoxic lymphocyte granule pathways (highly enriched in the SS gland) induce striking release of IFI16•dsDNA complexes from cultured cells. Our studies reveal that IFI16 is present in a filamentous state in the target tissue of SS and suggest that this property of DNA-induced filament formation contributes to its status as an autoantigen in SS. These studies highlight the role that tissue-specific modifications and immune effector pathways might play in the selection of autoantigens in rheumatic diseases.

Morphological alterations in minor salivary glands of HTLV1+ patients: A pilot study.

BACKGROUND: Among the complex of HTLV-associated diseases, Sjögren's syndrome (SS) is one of the most controversial. This work aims to detect morphological and inflammatory alterations, including clues of the presence of HTLV-1, in minor salivary glands of patients with dryness symptoms. METHODS: We have assessed HTLV-1-seropositive patients (HTLV-1 group) and patients with SS (SS group). We used formalin-fixed, paraffin-embedded minor salivary gland tissue to evaluate the morphological aspects and, by means of immunohistochemistry, the presence of Tax protein, CD4, CD8 and CD20 cells. Additionally, viral particles and proviral load were analysed by PCR. RESULTS: The HTLV-1 group had the highest prevalence of non-specific chronic sialadenitis (85.71%; P = 0.017) and greater amount of T CD8+ cells. In the SS group, focal lymphocytic sialadenitis (80%; P = 0.017) prevailed, with a greater amount of B CD20+ . Both immunohistochemistry and PCR identified the Tax protein and its gene in the salivary glands of both groups and in similar proportions. CONCLUSION: The results indicate that HTLV-1-seropositive patients have different patterns of morphological/inflammatory alterations, suggesting a likely difference in the process of immune activation.

A clinical and histopathological analysis of the anti-centromere antibody positive subset of primary Sjögren's syndrome.

OBJECTIVES: ACA-positive/primary Sjögren's syndrome (pSS) represents a distinct overlapping entity with intermediate features in between limited systemic sclerosis (lSSc) and pSS. Few data are available on their general risk for lymphoproliferative complications, specifically regarding adverse predictors at the level of minor salivary gland (MSG) histology. The objectives of this work are: a) to characterise, through a detailed immunohistochemistry study, the organisation of the lymphomonocitic infiltrates in ACA-positive/pSS patient vs. ACA-negative/pSS patients focusing on the presence of GC-like structures in minor salivary gland biopsies; b) to compare the frequency of traditional clinical and serological risk factors for lymphoma between the two subgroups. METHODS: We analysed 28 MSG samples from ACA-positive/pSS patients and 43 consecutive MSGs from ACA-negative/pSS, using sequential IHC staining for CD3, CD20 and CD21 in order to define the T/B cell segregation within the periductal infiltrates and presence of ectopic GC-like on the detection of GC-like structures. Clinical and serological data of all the patients were retrieved and analysed. RESULTS: Ectopic lymphoid structures (ELS) with GC-like structures were observed in 7 out of 28 ACA-positive/pSS patients (25%) and in 13 out of 43 ACA-negative/pSS patients (30.2%). Similarly, no statistical significant difference was found between the two groups as far as the classical pSS risk factors for lymphoproliferative complications was concerned (i.e. salivary gland enlargement, purpura, low C4, leukocytopenia, clonal gammopathy). Finally, the 3 cases of non-Hodgkin's lymphoma observed were equally distributed between the two subsets. CONCLUSIONS: Overall, this study indicates that ACA-positive/and ACA-negative pSS patients apparently present a similar risk for lymphoproliferative complications as suggested indirectly by the analogies between the two groups observed at the histopathology level.

Association of high 5-hydroxymethylcytosine levels with Ten Eleven Translocation 2 overexpression and inflammation in Sjögren's syndrome patients.

Here, we determined the 5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5mC), Ten Eleven Translocation (TETs), and DNA methyltransferases (DNMTs) levels in epithelial and inflammatory cells of labial salivary glands (LSG) from Sjögren's syndrome (SS)-patients and the effect of cytokines on HSG cells. LSG from SS-patients, controls and HSG cells incubated with cytokines were analysed. Levels of 5mC, 5hmC, DNMTs, TET2 and MeCP2 were assessed by immunofluorescence. In epithelial cells from SS-patients, an increase in TET2, 5hmC and a decrease in 5mC and MeCP2 were observed, additionally, high levels of 5mC and DNMTs and low levels of 5hmC were detected in inflammatory cells. Cytokines increased TET2 and 5hmC and decreased 5mC levels. Considering that the TET2 gene.promoter contains response elements for transcription factors activated by cytokines, together to in vitro results suggest that changes in DNA hydroxymethylation, resulting from altered levels of TET2 are likely to be relevant in the Sjögren's syndrome etiopathogenesis.

Cytokine markers of B lymphocytes in minor salivary gland infiltrates in Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic autoimmune disorder characterised by the clinical presence of sicca syndrome. SS compromises the dysfunction of exocrine glands due to the presence of focal, mononuclear cell infiltrates that surround the ducts and replace the secretory units. Abnormal expression of different cytokines and chemokines such as B-cell activating factor, CXC Motif Chemokine Ligand 13, interleukin 6 (IL-6), IL-22, and FMS-like tyrosine kinase 3 ligand as well as that of their corresponding receptors has been implicated in the inflammatory process. The severity of glandular infiltration has been suggested to be associated with the presence of extra-glandular systemic manifestations, contributing to a clinical spectrum of the most severe disease. This review describes several cytokines and chemokines associated with B lymphocytes expressed in the minor salivary gland, their chemical structures, and their roles in SS as possible early predictors of lymphoma development and disease progression.

Lymphocytic focus score is positively related to airway and interstitial lung diseases in primary Sjögren's syndrome.

OBJECTIVE: Although high-resolution computed tomography (HRCT) is useful for the characterization of minute morphological changes in the lungs, no study has investigated risk factors for lung involvement detected by HRCT in patients with Sjögren's syndrome with or without respiratory symptoms. The aim of the current study was to investigate risk factors for lung involvement in patients with primary Sjögren's syndrome detected by HRCT, with a particular focus on airway and interstitial lung diseases. METHODS: We performed a retrospective cohort study of patients with primary Sjögren's syndrome and investigated risk factors for lung involvement detected by HRCT. A total of 101 patients with primary Sjögren's syndrome with initial HRCT examinations were enrolled. RESULTS: Higher age, dry mouth, and higher labial gland biopsy focus scores (≥4) were risk factors for airway diseases (odds ratio [OR] 1.064 confidence interval [CI] 1.026-1.102, OR 8.795 CI 2.317-33.378 and OR 3.261 CI 1.100-9.675, respectively) in the multivariable analysis. Higher age, male sex, and higher labial gland biopsy focus scores (≥4) were risk factors for interstitial lung diseases (OR 1.078 CI 1.032-1.127, OR 12.178 CI 1.121-132.307 and OR 3.954 CI 1.423-10.987, respectively) in the multivariable analysis. The presence of anti-T-lymphotropic virus type 1 antibodies was significantly more common in patients with airway diseases. CONCLUSIONS: This study showed significant associations of labial gland biopsy focus scores and dry mouth with pulmonary manifestations in patients with primary Sjögren's syndrome. Focus scores as well as dry mouth may reflect lymphoproliferative activity in the lungs in patients with primary Sjögren's syndrome.

The Ratio of Blood T Follicular Regulatory Cells to T Follicular Helper Cells Marks Ectopic Lymphoid Structure Formation While Activated Follicular Helper T Cells Indicate Disease Activity in Primary Sjögren's Syndrome.

OBJECTIVE: To investigate whether the balance of blood follicular helper T (Tfh) cells and T follicular regulatory (Tfr) cells can provide information about ectopic lymphoid neogenesis and disease activity in primary Sjögren's syndrome (SS). METHODS: We prospectively recruited 56 patients clinically suspected of having SS. Sixteen of these patients subsequently fulfilled the American-European Consensus Group criteria for SS and were compared to 16 patients with non-SS sicca syndrome. Paired blood and minor salivary gland (MSG) biopsy samples were analyzed to study Tfr cells and subsets of Tfh cells in both compartments. RESULTS: Patients with primary SS had normal Tfh cell counts in peripheral blood; however, activated programmed death 1-positive (PD-1+) inducible costimulator-positive (ICOS+) Tfh cells in peripheral blood were strongly associated with disease activity assessed by the European League Against Rheumatism Sjögren's Syndrome Disease Activity Index (r = 0.8547, P = 0.0008). Conversely, the blood Tfr cell:Tfh cell ratio indicated ectopic lymphoid structure formation in MSGs, being strongly associated with B cell, CD4+ T cell, and PD-1+ICOS+ T cell infiltration in MSGs, and was especially increased in patients with focal sialadenitis. Further analysis showed that the blood Tfr cell:Tfh cell ratio allowed discrimination between SS patients and healthy donors with excellent accuracy and was a strong predictor of SS diagnosis (odds ratio [OR] 12.96, P = 0.028) and the presence of focal sialadenitis (OR 10, P = 0.022) in patients investigated for sicca symptoms, thus highlighting the potential clinical value of this marker. CONCLUSION: The blood Tfr cell:Tfh cell ratio and PD-1+ICOS+ Tfh cells constitute potential novel biomarkers for different features of primary SS. While the blood Tfr cell:Tfh cell ratio is associated with ectopic lymphoid neogenesis, activated Tfh cells indicate disease activity.