Galectin-9 potentiates salivary gland damage by inducing ferroptosis in Sjogren's disease.

Background:Sjögren’s disease (SjD) is a systemic autoimmune disorder that primarily affects the exocrine glands, particularly the salivary and lacrimal glands. Recent efforts have exploited serum and saliva metabolome analysis to...

Sjögren's syndrome (SS) is a chronic systemic autoimmune inflammatory disease. In addition to its destructive effects upon salivary and lacrimal glands, causing xerostomia and keratoconjunctivitis sicca, upward of 30% of patients develop systemic involvement, including a 5%-10% lifetime risk of non-Hodgkin lymphoma. Correct diagnosis of SS is crucial in order to optimally manage patients with respect to management of oral and ocular disease, detection and treatment of extra-glandular disease, and monitoring for lymphoma. Confirmation of primary SS (pSS) is contingent upon objective measures of dysfunction of salivary or lacrimal glands, in addition to evidence of autoimmunity, comprising either serum anti-Ro/SSA antibodies, or salivary gland histopathology demonstrating focal sialadenitis. The process to diagnose pSS mirrors published research classification criteria, recently revised following large collaborative efforts. This multistep process may be seen as arduous, and diagnostic accuracy can be compromised by the omission of objective tests for glandular dysfunction, resulting in misdiagnosis of undifferentiated connective tissue disease, systemic lupus erythematosus, rheumatoid arthritis, fibromyalgia, or menopausal symptoms. Conversely, patients with non-autoimmune sicca syndrome may receive a clinical diagnosis of pSS, and unnecessarily labelled for life as having an autoimmune disease. There is a need therefore for a readily available, accurate and reliable test to aid in the diagnosis of pSS, and to supplant the need for other tests such as salivary collection and salivary gland biopsy. With the advent of clinical trials of biologics and kinase inhibitors in pSS, a safe and reliable test for longitudinal evaluation of salivary gland function and architecture is also paramount. In 2002 the American-European Consensus Group (AECG) published the most widely used classification criteria for SS,1 refined and most likely to be superseded by the 2016 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria for pSS.2 Whereas the AECG criteria included abnormal sialography, salivary scintigraphy, or unstimulated whole saliva production as evidence of salivary gland dysfunction, sialography and scintigraphy are absent from the latest ACR/EULAR criteria. Although sialography is considered the most reliable imaging method, major limitations include its invasiveness, radiation exposure, potential for complications including sialadenitis, and contraindication in patients with infection, inflammation or allergy to iodine. Major salivary gland scintigraphy is sensitive for the diagnosis of pSS; however, specificity is poor, and it is not widely available. Magnetic resonance imaging offers good sensitivity and specificity to detect structural abnormalities in pSS, but access to this modality is limited and expensive. Major salivary gland ultrasound (SGUS) has emerged as a promising tool for the diagnosis, prognosis, and monitoring of response to treatment in patients with pSS. It is inexpensive, readily available, non-invasive, non-irradiating, rapidly performed, repeatable, and convenient for patients. Literature in this area is in its infancy, and studies conducted to date scoring parenchymal changes on B-mode images such as echogenicity, heterogeneity, border features, or vascularization have shown wide variability in sensitivity and specificity. A recent meta-analysis assessing the diagnostic properties of SGUS in pSS reported a pooled sensitivity of 69% and specificity 92%.3 This meta-analysis noted great clinical and methodological heterogeneity between studies, which hampered interpretation of pooled outcomes and influenced results reported within the various studies. This heterogeneity included the use of differing classification criteria, different resolutions of the US transducers used, absence of a standardized scanning method, lack of standardized definitions of US gland pathologies, use of different scoring systems for parenchymal changes, subjectivity in the assessment of US images, and inadequate demonstration of intra- and inter-observer reliability. The EULAR US-pSS Study Group assessed the validity of SGUS compared with parotid and labial gland biopsy outcome in 103 consecutive patients with clinically suspected pSS, and found high rates of agreement between SGUS, scored according to the Hocevar scoring system,4 and parotid (83%) and labial (79%) gland biopsies. Compared with AECG classification, SGUS demonstrated absolute agreement in 82%, with a sensitivity of 71% and specificity 92%. Compared with ACR/EULAR classification, absolute agreement was seen in 86%, with a sensitivity of 67% and specificity 94%. The combination of positive SGUS and the presence of anti-Ro/SSA antibodies highly predicted classification of pSS,5 raising the possibility of forgoing labial biopsy in this population. The SGUS Outcome Measures in Rheumatology task force group, composed of international clinicians and experienced sonographers, aims to develop a standardized scanning method using standardized definitions of gland pathology. A consensual Delphi process about definitions and scoring using B-mode SGUS showed good to excellent intra- and inter-operator reliabilities between 18 sonographers.6 A new US technique under investigation for the diagnosis of pSS is elastography, where ultrasound is used to investigate the elasticity of soft tissues. The principle underlying this method is that tissue compression produces strain (displacement) within the tissue, which is less pronounced in harder than in softer tissues. Real time 'sono' elastography (RTS) has been used in oncology to discriminate between hard and soft nodules, and more recently in rheumatology for the assessment of skin involvement in systemic sclerosis, rheumatoid nodules and tophi. RTS of the major SGs was prospectively investigated in 45 patients with pSS according to AECG criteria, 24 individuals with sicca complaints, and 11 healthy controls.7 In patients with an inconclusive B-mode SGUS, RTS provided a sensitive (66.7%) and specific (85.7%) classification of patients and sicca controls. Increased signal in SG of pSS patients may reflect increased tissue rigidity due to lymphocytic infiltration, hyperplasia of ductal epithelial cells, and fibrosis, although histopathologic confirmation is lacking. Cindil et al8 studied 58 patients with pSS by AECG criteria and 25 healthy controls by B-mode US and elastography using a semi-quantitative strain ratio method. They also found statistically significant differences between the groups in parotid glands (sensitivity 83% specificity 88%) and submandibular glands (sensitivity 83% and specificity 92%), with pSS SG again being stiffer. Notably, this technology is dependent on the operator applying repetitive similar pressure with the ultrasound probe, and reproducibility is poor. Soft tissue elasticity can also be measured using acoustic radiation force impulse (ARFI) US, where local tissue displacement by a brief acoustic radiation induces the emission and propagation of shear waves, which are then digitally recorded. Shear wave velocity (SWV) is measured in meters per second and increases with tissue stiffness. The advantage of the ARFI technique is that the strength of acoustic radiation does not require external compression, and acoustic radiation of short duration generates tissue displacement; however, results can vary with differing applied pressure of the US probe, thus accuracy and reproducibility are strongly operator-dependent. Strategies to avoid error, particularly in serial assessments, include the use of the same machine, transducer settings and frequency, and taking multiple measures with minimal pressure. Parotids are more diffusely fatty than submandibular glands, especially in older patients, highlighting the importance of scoring glands separately, and controlling for age in comparative studies. The deep parotid lobe is often more affected than the superficial lobe; thus tissue plane standardization is essential and should be documented, particularly for follow up and where variant anatomy has been encountered. Care is needed to avoid taking values from planes containing intra-glandular parotid lymph nodes and vessels. ARFI elastometry was performed in 10 patients with pSS according to ACR criteria and 15 healthy controls.9 In parotid glands the mean SWV was significantly higher in the pSS group, whereas mean SWG values for the submandibular glands were not significantly different between patients and controls. Zhang et al10 evaluated SG stiffness using quantitative ARFI imaging, including Virtual Touch tissue quantification (VTQ) and Virtual Touch tissue imaging quantification (VTIQ). VTQ was used to calculate the SWV, providing an objective numerical evaluation of the tissue stiffness, whereas VTIQ, a 2D shear wave imaging displaying a color-coded image, was used to enable the detection of SWV in multiple locations. Twenty-one patients with pSS according to the AECG criteria and 11 healthy controls were included. Parotid gland VTQ values were significantly higher in the pSS group than controls, and VTIQ values for both the parotid and submandibular glands were also significantly higher in pSS. Turnaoglu et al11 studied 25 patients with pSS by AECG criteria and 25 healthy controls by ARFI and reported that SWV values of both the parotid and submandibular glands were significantly higher in patients with pSS, even in early stages of disease. In this issue of the journal, Pia et al12 studied patients with SS classified by AECG criteria (pSS n = 30, secondary SS n = 13), patients with sicca symptoms not meeting SS criteria (n = 18), and healthy controls (n = 29), to assess the utility of ARFI VTQ in the diagnosis of SS. Their results were consistent with the two most recent reports cited above: in patients with SS, parotid and submandibular gland SWV were significantly higher than in control subjects, with average parotid gland sensitivity 67% and specificity 53%, and submandibular gland sensitivity 68% and specificity 50%. These results may underestimate the utility of this technique in pSS, given that patients with pSS and sSS were grouped together in the analysis, and previous studies have shown increased severity of US parameters in pSS. Indeed, within the text of this paper, the authors report that rigidity of the salivary glands was higher in pSS vs sSS; however, the data were not shown. It would also have been of interest to know the underlying diagnoses of patients with sSS in this study. With respect to the requirements for classification by AECG criteria, there was some confusion within the Materials and Methods section, with the statement that the patient must fulfill 4 histopathological or autoantibody criteria to reach SS classification. For classification as pSS, patients must: fulfill 4 of 6 criteria, including at least one of the histopathological or autoantibody criteria; or fulfill 3 of the 4 objective criteria; or be classified via use of a classification tree procedure, which is mainly intended for clinical-epidemiological surveys. For classification as sSS, patients need to fulfill a subjective criterion, and at least 2 objective criteria, which need not include histopathology; the autoantibody criterion does not apply. There is clearly an unmet need to simplify the diagnosis of SS, and to monitor response to therapy. Increased confidence in pSS diagnosis will lead to a deeper understanding of the scope of this disease, and will facilitate both observational research and clinical trials in the development of targeted therapies. Major salivary gland elastography, performed simultaneously with B-mode ultrasound using the same equipment, holds promise as an adjunct in the diagnosis of SS. Ongoing efforts to standardize and validate methodologies and reliability, will be central to this aim.

Salivary function provides host protection, assists in the initiation of food and fluid intake, and enables communication through speech. Without adequate salivary output, oral and pharyngeal health declines along with a person’s quality of life. The complaint of a dry mouth (xerostomia) and the objective finding of salivary dysfunction are common occurrences in older individuals, producing transient and permanent oral and systemic problems. Salivary dysfunction, however, is not a normal consequence of growing older, and is due to systemic diseases, medications, and head and neck radiotherapy. Diagnosis of salivary disorders begins with a careful medical history, head, and neck examination. While complaints of xerostomia may be indicative of a salivary gland disorder, salivary diseases can present without symptoms. Therefore, routine examination of salivary function must be part of any head, neck, and oral examination. Therapies are designed to prevent the development of oral and pharyngeal sequelae of salivary hypofunction. Current xerostomia-based treatments include replacement therapies and gustatory, masticatory, and pharmacological stimulants. Healthcare professionals can play a vital role in identifying patients at risk for developing salivary dysfunction, and should provide appropriate preventative and interventive techniques that will help preserve a person’s health, function, and quality of life.

AB0196 MESENCHYMAL STEM CELLS TUNE THE DIFFERENTIATION OF MYELOID-DERIVED SUPPRESSOR CELLS IN SJöGREN’S SYNDROME THROUGH INHIBITING IL-12

Sjögren's syndrome (SS) is a systemic autoimmune disease that is characterized by focal lymphocytic infiltration into exocrine organs such as salivary and lacrimal glands, resulting in dry mouth and eyes, and other systemic injuries. There is no curative clinical therapy for SS, and stem cell therapy has shown great potential in this area. The mesenchymal stem cells (MSCs) in the salivary glands of healthy individuals and in patients with SS have not been extensively studied. The aim of this study was to elucidate the characteristics of MSCs from the labial glands of healthy controls and of those from patients with SS to elucidate the related pathogenesis and to uncover potential avenues for novel clinical interventions. Labial glands from patients with SS and healthy subjects were obtained, and MSCs were isolated and cultured by using the tissue adherent method. The MSC characteristics of the cultured cells were confirmed by using morphology, proliferation, colony forming-unit (CFU) efficiency, and multipotentiality, including osteogenic, adipogenic, and salivary gland differentiation. The MSCs from the healthy controls and SS patients expressed characteristic MSC markers, including CD29, CD44, CD73, CD90, and CD105; they were negative for CD34, CD45, and CD106, and also negative for the salivary gland epithelium markers (CD49f and CD117). Labial gland MSCs from both groups were capable of osteogenic and adipogenic differentiation. The CFU efficiency and adipogenic differentiation potential of MSCs were significantly lower in the SS group compared with the healthy controls. Cells from both groups could also be induced into salivary gland-like cells. Real-time polymerase chain reaction and immunofluorescence staining showed that the gene and protein expression of AMY1, AQP5, and ZO-1 in cells from the SS group was lower than that in cells from the healthy group. Thus, MSCs from the labial glands in patients with SS could lack certain characteristics and functions, especially related to salivary secretion. These preliminary data provided insights that could lead to the development of novel therapeutic strategies for the treatment of SS.

Sjögren's syndrome (SS) is a systemic autoimmune disease that mainly affects exocrine salivary and lacrimal glands. Local inflammation in the glands is thought to trigger glandular dysfunction and symptoms of dryness. However, the mechanisms underlying these processes are incompletely understood. Our work suggests T cell exosome-derived miR-142-3p as a pathogenic driver of immunopathology in SS. We first document miR-142-3p expression in the salivary glands of patients with SS, both in epithelial gland cells and within T cells of the inflammatory infiltrate, but not in healthy volunteers. Next, we show that activated T cells secreted exosomes containing miR-142-3p, which transferred into glandular cells. Finally, we uncover a functional role of miR-142-3p-containing exosomes in glandular cell dysfunction. We find that miR-142-3p targets key elements of intracellular Ca2+ signaling and cAMP production - sarco(endo)plasmic reticulum Ca2+ ATPase 2b (SERCA2B), ryanodine receptor 2 (RyR2), and adenylate cyclase 9 (AC9) - leading to restricted cAMP production, altered calcium signaling, and decreased protein production from salivary gland cells. Our work provides evidence for a functional role of the miR-142-3p in SS pathogenesis and promotes the concept that T cell activation may directly impair epithelial cell function through secretion of miRNA-containing exosomes.

Developmental biology has long been ignored in the etiology and diverse manifestations of autoimmune diseases. Yet a role for development is suggested by intriguing overlaps in particular organs targeted in autoimmune diseases, in this case type 1 diabetes and Sjogren's syndrome. Patients with type 1 diabetes have high rates of co-occurring Sjogren's syndrome, and both conditions are associated with hearing loss and tongue abnormalities. All of these co-occurrences are found in organs tracing their lineage to the developmental transcription factor Hox11, which is expressed in embryonic cells destined for the pancreas, salivary glands, tongue, cranial nerves and cochlea. To determine whether development contributes to autoimmunity, we compared four target organs in NOD mice (an animal model for type 1 diabetes and Sjogren's syndrome) with NOD-SCID mice (which lack lymphocytes) and normal controls. We examined the structure and/or function of the cochlea, salivary glands, pancreas and tongue at early time points after birth. Before the usual time of the onset of type 1 diabetes or Sjogren's syndrome, we show that all four Hox11-derived organs are structurally abnormal in both NOD mice and NOD-SCID mice versus controls. The most striking functional defect is near complete hearing loss occurring before the normal time of the onset of autoimmunity. The hearing loss is associated with severe structural defects in the cochlea, suggesting that near-deafness occurs independent of autoimmune attack. The pancreas and salivary glands are also structurally abnormal in NOD and NOD-SCID mice, but they are functionally normal. This suggests that autoimmune attack of these two organs is required for functional failure. We conclude that a developmental lineage of cells contributes to autoimmunity and predicts which organs may be targeted, either structurally and/or functionally. Taken together, our findings challenge the orthodoxy that autoimmunity is solely caused by a defective immune system.

The underlying pathogenetic mechanisms of Sjögren syndrome (SjS) have not been elucidated yet [1]. Leptin is a glycosylated peptide structurally similar to some cytokines [2]. Leptin has proinflammatory effects via various mechanisms. It has been previously reported that serum leptin levels increase in some autoimmune diseases and that leptin levels are also associated with disease activity [3, 4]. In addition, leptin and its receptor in the salivary glands stimulate epithelial proliferation and modulate the local immune system with their autocrine effects [5]. In this study, we assessed the presence of leptin in minor salivary glands (MSG) of the patients with and without SjS. Additionally, we evaluated the association between leptin staining intensity and disease activity among patients with SjS. We included patients who underwent MSG biopsy with the suspicion of SjS between 2013 and 2014. Among these patients, those who met the 2012 American College of Rheumatology Sjögren's syndrome classification criteria (6) and had at least 50 mononuclear cell infiltrates in a 4 mm2 minor salivary gland section were classified as the SjS group. The rest of the patients who underwent MSG biopsy but did not meet the histopathological and/or clinical criteria for SjS were considered the control group. Leptin staining was assessed by immunohistochemistry (Bio-Rad AbDSerotec, Oxford, UK) in the stroma, acinar and ductal epithelium, and staining intensities were graded by a semiquantitative method; no staining (score 0), mild (score 1), moderate (score 2), and diffuse (score 3). Definitions of leptin staining intensities were described as mild; focal staining of few cells in one focus, moderate; mild staining in more than one focus, diffuse; diffuse staining in all assessed sections (Figure 1). The sum of leptin staining in all areas was defined as total leptin staining. The SjS group was divided into 3 groups according to the focus scores (FS) (FS1: FS = 1, FS2: FS = 2, FS3: FS ≥ 3). In addition, patients' disease activities were assessed by EULAR Sjogren's syndrome disease activity index (ESSDAI) [7]. Visual (histograms, probability plots) and analytical methods (Kolmogorov-Smirnov test) were used to analyze the variables' distribution and select the test method. Descriptive analyses were presented as mean or median according to their distribution patterns. A chi-square test was used for univariate analyses to identify variables associated with leptin staining and clinical features. Patients' disease activities were assessed by ESSDAI [9]. The correlations between ESSDAI scores and leptin staining intensities were analyzed using Spearman's test. A p value of 66%, in FS3 group: 100%) areas were higher in the patients with FS3. There was a positive correlation between ESSDAI and leptin staining scores in the acinar area (p < 0.05; r = 0.406) regardless of focus scores. However, the correlations between disease activity and leptin staining in ductal areas (p: 0.1; r: 0.334), in stromal areas (p: 0.1; r: 0.184), and total leptin (p: 0.2; r: 0.268) staining was not significant. We identified that majority of the patients in both groups had leptin staining in acinar (92% and 84%, respectively), ductal (94% and 84%, respectively), and stromal areas (96% and 100%, respectively) of their MSGs. Patients with higher FSs had significantly more intense leptin staining patterns. The possible explanations for the similar staining pattern in patients with an FS of ≤2 was the insufficient number of patients (β error) or absolute indifference. In contrast, a study by Erbasan F et al. argued that leptin has no role in primary SjS due to similar leptin staining patterns among patients with SjS and healthy controls. However, they did not compare patients according to the ESSDAI scores [8]. Thus, we still need more robust data to identify whether leptin has an inciting or prognostic role in pSS. Limitations of our study were the lack of some clinical characteristics and metabolic parameters which may affect leptin metabolism and lack of leptin receptor staining patterns in MSG biopsies, which together with leptin staining could better define the role of leptin. Lack of power analysis was another limitation. In conclusion, leptin staining properties were similar in both SjS and non-SjS groups. Additionally, the only clinical significance of leptin density in the MSG was in the acinar region. The authors have no financial or competing interests. All authors declare no conflict of interest We received funding support from Kartal Dr. Lutfi Kirdar City Hospital research assistance department. Authors contributed to writing this study and have approved the final version. Ethical approval: This article does not contain any studies with human participants performed by any of the authors. The study protocol was approved by the Local Ethics Committee of Dr Lutfi Kirdar Kartal City Hospital.

Downregulated GPX4 in salivary gland epithelial cells contributes to salivary secretion dysfunction in Sjogren's syndrome via lipid ROS/pSTAT4/AQP5 axis

Sjögren's syndrome (SS) is an autoimmune disease that targets the salivary and lacrimal glands. While all patients demonstrate inflammatory infiltration and abnormal secretory function in the target tissues, the disease features, pathology, and clinical course can vary. Activation of distinct inflammatory pathways may drive disease heterogeneity. The purpose of this study was to investigate whether activation of the interferon (IFN) pathway correlates with key phenotypic features. Clinical data and 1 labial salivary gland (stored frozen) were obtained from each of 82 participants (53 patients with primary SS and 29 control subjects) in the Sjögren's International Collaborative Clinical Alliance (SICCA) registry. Salivary gland lysates were immunoblotted with markers of type I or type II IFN, and patterns of IFN activity were determined by hierarchical clustering. Correlations between SS phenotypic features and IFN activity in the salivary gland were performed. A total of 58% of the SS participants had high IFN activity and differed significantly from those with low IFN activity (higher prevalence of abnormal findings on sialometry, leukopenia, hyperglobulinemia, high-titer antinuclear antibody, anti-SSA, and high focus score on labial salivary gland [LSG] biopsy). Three distinct patterns of IFN were evident: type I-predominant, type II-predominant, and type I/II mixed IFN. These groups were clinically indistinguishable except for the LSG focus score, which was highest in those with type II-predominant IFN. The SS phenotype includes distinct molecular subtypes, which are segregated by the magnitude and pattern of IFN responses. Associations between IFN pathways and disease activity suggest that IFNs are relevant therapeutic targets in SS. Patients with distinct patterns of high IFN activity are clinically similar, demonstrating that IFN-targeting therapies must be selected according to the specific pathway(s) that is active in vivo in the individual patient.

Interferon-inducible protein-16 (IFI16) is an intracellular DNA receptor involved in innate immunity. We evaluated the frequency, phenotypic characteristics, and clinical associations of anti-IFI16 antibodies in patients with primary Sjögren's syndrome (SS), and quantitated expression levels of IFI16 in SS and control salivary gland lysates. Anti-IFI16 antibodies were assayed by enzyme-linked immunosorbent assay using sera from patients with primary SS (n = 133) and from healthy controls (n = 47). Sera from systemic lupus erythematosus (SLE) patients (n = 132) were included as disease controls. Immunoprecipitation of in vitro transcription-translated IFI16 was used to determine which portion of IFI16 the antibodies recognized. Expression of IFI16 in salivary gland lysates was quantitated by immunoblotting. Anti-IFI16 antibodies were present in the sera of 38 of 133 SS patients (29%) compared to 1 of 47 healthy controls (2.1%) (SS versus controls; P < 0.0002) and in 31 of 132 SLE controls (24%). In SS, anti-IFI16 antibodies were associated with an abnormal Schirmer's test (P = 0.003), hyperglobulinemia (P = 0.02), antinuclear antibody ≥1:320 (P = 0.01), germinal center-like structures in labial salivary gland lymphoid infiltrates (P = 0.01), and higher focus scores (3.4 versus 2.4; P = 0.005). High-titer IFI16 antibodies were directed against an epitope outside the N-terminus in 9 of 13 SS patients (69%). IFI16 was expressed in 4 of 5 (80%) of SS and 1 of 6 (17%) of control labial salivary glands. Anti-IFI16 antibodies are a prominent specificity in primary SS and are associated with markers of severe disease. IFI16 is expressed at higher levels in SS salivary glands compared to controls. These high levels in disease target tissue may contribute to the ongoing anti-IFI16 immune response.

Sjögren’s disease (SjD) is a systemic auto-immune disease characterized by salivary gland inflammation and glandular dysfunction. Diagnosis is challenging due to its heterogeneity and currently relies on a variety of tests that are present in the ACR-EULAR classification criteria. These include invasive and resource-intensive tests, highlighting the unmet need for a single, accurate, non-invasive diagnostic modality. Multispectral optoacoustic tomography (MSOT), enabling functional imaging of hemoglobin-related parameters, may address this gap. This pilot study evaluates MSOT's potential for salivary gland imaging in patients suspected of SjD. This study included 20 patients clinically suspected of SjD. Which underwent MSOT imaging of the major salivary glands, alongside the full ACR-EULAR diagnostic workup, including serology, salivary gland biopsy, and sialometry, alongside salivary gland ultrasound. MSOT parameters were compared to standard of care diagnostic modalities and ultrasound scoring systems. A novel MSOT scoring system based on 800 nm hemoglobin signals was developed to evaluate diagnostic performance. Patients classified as SjD (n = 13) show significantly higher hemoglobin-related signals compared to non-SjD patients (n = 7). When ≥ 2 salivary glands, either submandibular or parotid, exceeded their predefined MSOT cut-off values of 371.6 a.u. and 374.2 a.u., respectively, MSOT achieved 92 % sensitivity and 100 % specificity for SjD classification, outperforming other diagnostic tests and established ultrasound scoring systems. MSOT shows promise as non-invasive imaging modality for SjD classification, and may offer higher sensitivity compared to established ultrasound scoring systems and other diagnostic tests. These explorative findings support further investigation of MSOT as non-invasive diagnostic tool in SjD.

To characterize the chronologic disease course and possible interrelationships between salivary gland inflammation, hyposalivation, and cytokine levels in NOD mice, a model for Sjögren's syndrome (SS). NOD mice of different ages were used to mimic different disease stages of SS. Histopathologic findings and rates of salivary secretion were compared between 8-week-old, 17-week-old, and 24-week-old female mice. In addition, 10 cytokines were analyzed in serum and saliva obtained from NOD and BALB/c mice. In NOD mice, the salivary flow rate did not change between 8 weeks and 17 weeks of age, while a significant decrease in the salivary flow rate occurred between 17 weeks and 24 weeks of age (P < 0.001). In contrast, significant histopathologic changes in the salivary glands occurred before 17 weeks of age. Chronic inflammatory cell infiltrates were characterized by T and B cell infiltration. Interestingly, in one-third of the mice, proliferating cells were observed in the focal infiltrates. Significant changes in the levels of interleukin-2 (IL-2), IL-5, and granulocyte-macrophage colony-stimulating factor in serum, and in the levels of IL-4 and tumor necrosis factor alpha (TNFalpha) in saliva occurred contemporarily with the decrease in salivary flow. Correlation analyses revealed a negative association between salivary secretion and the levels of IL-4, interferon-gamma, and TNFalpha in saliva obtained from NOD mice, while the correlation with inflammatory changes in the glands was consistently weak. Consistent with previous findings, our results indicate at least 2 phases of SS-like disease in NOD mice. Hyposalivation was preceded by inflammatory changes in the salivary glands, whereas abrupt changes in secretion occurred without significant progression of inflammation. Changes in cytokine levels are an indication of the mechanisms involved in the adaptive immune response in the transition from early to overt disease.

Salivary Gland Involvement in Chronic Graft-Versus-Host Disease: Prevalence, Clinical Significance, and Recommendations for Evaluation

Systemic and Local Interleukin-17 and Linked Cytokines Associated with Sjögren’s Syndrome Immunopathogenesis