Salivary Gland Samples Research Articles

Molecular characterization of the salivary adenoid cystic carcinoma immune landscape by anatomic subsites

Adenoid cystic carcinoma (AdCC) is a slow-growing salivary gland malignancy that relapses frequently. AdCCs of the submandibular gland exhibit unique differences in prognosis and treatment response to adjuvant radiotherapy compared to other sites, yet the role of tumor anatomic subsite on gene expression and tumor immune microenvironment (TIME) composition remains unclear. We used 87 samples, including 48 samples (27 AdCC and 21 normal salivary gland tissue samples) from 4 publicly available AdCC RNA sequencing datasets, a validation set of 33 minor gland AdCCs, and 39 samples from an in-house cohort (30 AdCC and 9 normal salivary gland samples). RNA sequencing data were used for single sample gene set enrichment analysis and TIME deconvolution. Quantitative PCR and multiplex immunofluorescence were performed on the in-house cohort. Wilcoxon rank-sum, nonparametric equality-of-medians tests and linear regression models were used to evaluate tumor subsite differences. AdCCs of different anatomic subsites including parotid, submandibular, sublingual, and minor salivary glands differed with respect to expression of several key tumorigenic pathways. Among the three major salivary glands, the reactive oxygen species (ROS)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway signature was significantly underexpressed in AdCC of submandibular compared to parotid and sublingual glands while this association was not observed among normal glands. Additionally, the NRF2 pathway, whose expression was associated with favorable overall survival, was overexpressed in AdCCs of parotid gland compared to minor and submandibular glands. The TIME deconvolution identified differences in CD4+ T cell populations between AdCC of major and minor glands and natural killer (NK) cells among AdCC of minor, submandibular, and parotid glands while plasma cells were enriched in normal submandibular glands compared to other normal gland controls. Our data reveal key molecular differences in AdCC of different anatomic subsites. The ROS and NRF2 pathways are underexpressed in submandibular and minor AdCCs compared to parotid gland AdCCs, and NRF2 pathway expression is associated with favorable overall survival. The CD4+ T, NK, and plasma cell populations also vary by tumor subsites, suggesting that the observed submandibular AdCC tumor-intrinsic pathway differences may be responsible for influencing the TIME composition and survival differences.

The role of M1/M2 macrophage polarization in primary Sjogren’s syndrome

BackgroundThe purpose of this study was to investigate the role of macrophage polarization in the pathogenesis of primary Sjogren’s syndrome (pSS).MethodsPeripheral venous blood samples were collected from 30 patients with pSS and 30 healthy controls. Minor salivary gland samples were abtainted from 10 of these patients and 10 non-pSS controls whose minor salivary gland didn’t fulfill the classification criteria for pSS. Enzyme-linked immuno sorbent assay was used to examine the serum concentration of M1/M2 macrophage related cytokines (TNF-a, IL-6, IL-23, IL-4, IL-10 and TGF-β). Flow cytometry was used to examine the numbers of CD86+ M1 macrophages and CD206+ M2 macrophages in peripheral blood mononuclear cells (PBMCs). Immunofluorescence was used to test the infiltration of macrophages in minor salivary glands.ResultsThis study observed a significant increase in pSS patients both in the numbers of M1 macrophages in peripheral blood and serum levels of M1-related pro-inflammatory cytokines (IL-6, IL-23 and TNF-α). Conversely, M2 macrophages were downregulated in the peripheral blood of pSS patients. Similarly, in the minor salivary glands of pSS patients, the expression of M1 macrophages was increased, and that of M2 macrophages was decreased. Furthermore, a significantly positive correlation was found between the proportions of M1 macrophages in PBMCs and serum levels of IgG and RF.ConclusionsThis study reveals the presence of an significant imbalance in M1/M2 macrophages in pSS patients. The M1 polarization of macrophages may play an central role in the pathogenesis of pSS.

Alpha-Gal, epitope responsible for allergy to red meat, in the Mediterranean tick Hyalomma lusitanicum.

Alpha-Gal/α-Gal is an oligosaccharide produced by non-primate mammals. Humans have developed an immune response mediated by anti-α-Gal antibodies that can trigger an allergic reaction and cause anaphylaxis. In recent years, cases of patients with delayed allergic reaction to mammalian meat have been reported worldwide. In Spain, these cases have been related to the species Ixodes ricinus L. (Ixodida: Ixodidae), whose distribution is located in the north of the country. In this work, the presence of α-Gal in water-soluble extracts from samples of salivary glands and digestive tracts of Hyalomma lusitanicum Koch (Ixodida: Ixodidae) both engorged and collected from vegetation were studied. The presence of that epitope was confirmed by the presence of reactive proteins of >250 kDa in both samples. The highest concentrations of α-Gal were detected in salivary glands. Neither sex nor diet influenced the concentration of α-Gal, which seems to indicate its endogenous production and its possible inoculation to the host during tick feeding.

Abstract 1076: Single nucleus RNA-sequencing using normal human salivary glands revealed a potential etiologic relationship between each salivary gland cell and salivary gland malignancies

Abstract Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including Adenoid cystic carcinoma (ACC), Acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In this study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, total 13,643 salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2, and myoepithelial cells/stromal cells, with differentially expressed genes of each group. The localization of each cell group was anatomically validated by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Gene ontology analysis for this group included “epithelial cell differentiation” confirming the intercalated duct. Furthermore, to elucidate the potential etiologic relationship between normal salivary glands cell type and salivary gland carcinoma histology, we compared the expression of each cluster marker to SGC public RNA-seq data. In comparison between AciCC and ACC, we used normalized RNA-seq data of 54 ACC and 21 AciCC. ACC had significantly less expression of acinar cells markers (STATH, CST2, and LPO) compared to other markers expression(***P = 4 × 10−51), unlike AciCC (P = 0.54), suggesting a possible contribution of acinar cells to the carcinogenesis of AciCC. In another cohort, we compared 16 SDC, and 38 MECA with publicly available RNA-seq data. Remarkably, HES1 and ELF3, each of two different ductal cell markers, were significantly upregulated in SDC consistent with origin of SDC from ductal cells (P=0.001, P=0.0007, respectively). Consistent with bulk RNA-seq results, expression of HES1 in IHC using clinical human SDC and MECA samples was higher in SDC than MECA, suggesting that the expression of the derived cells is involved in salivary gland carcinogenesis. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship. On the other hand, there was no significant difference in myoepithelial marker ACTA2 expression between SDC and MECA, suggesting that the markers of origin are not always highly expressed, depending on cancer subtypes. Although publicly available SGC RNA-seq data used in this study are limited, the results remarkably reflect the diverse modes of carcinogenesis of SGC. These findings might provide a better understanding of the molecular basis of SGC and treatment strategies for SGC. Citation Format: Takuya Nakagawa, Chanond Nasamran, Jingjing Hu, Prakriti Sen, Theresa Guo, Kathleen Fisch, Joseph Califano. Single nucleus RNA-sequencing using normal human salivary glands revealed a potential etiologic relationship between each salivary gland cell and salivary gland malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1076.

Abstract 2999: Evaluation of microRNA expression in metastatic and non-metastatic salivary gland mucoepidermoid carcinoma

Abstract Salivary gland tumors are a heterogeneous group of lesions, being mucoepidermoid carcinoma (MEC) the malignant neoplasm with the highest incidence, occurring mainly in the parotid gland. Characterization of its histological grade (high, intermediate, and low) is important for determining patient prognosis. Metastasis is more common high-grade tumors and can occur at regional lymph nodes or distant sites. Although the mechanisms underlying the metastatic process are largely unknown, large-scale microRNA expression profiling studies of human cancers have demonstrated that dysregulation of miRNA is frequently associated with many cancer types. The aim of this study is to validate the use of microRNA expression for the discrimination of the metastatic potential of MEC samples. Using Real Time RT-PCR (qPCR) we have analyzed the expression of 384 miRNAs and controls in 4 non-metastatic MECs, 3 MECs with lymph node metastasis, 3 MECs with distant metastasis, and 2 non-neoplastic human salivary gland samples. Comparing the expression among these groups we identified 45 differentially expressed miRNAs that may be related with the metastatic potential of these tumors. To validate the microRNA expression associated with the metastatic potential, we selected 7 out of the 45 differentially expressed microRNAs and 7 MEC samples from the different groups of samples. Considering the 7 microRNAs evaluated, we validated that the expression of miR-191 was increased in non-neoplastic samples when compared to MECs with lymph node metastasis and the expression of miR-494 was increased in non-neoplastic samples when compared to MECs with distant metastasis, suggesting that loss of these microRNAs might be involved in the development of metastasis in mucoepidermoid carcinoma. The determination of a miRNA expression profile could provide a better understanding of the molecular basis of MEC and facilitate the determination of diagnosis and prognosis of patients. Citation Format: Maria E. Trevizani, Katia K. Oliveira, Fabio A. Marchi, Daniela Bizinelli, Fernanda V. Mariano, Cibele P. Nagano, Felipe A. Costa, Clovis A. Pinto, Luiz P. Kowalski, Silvia V. Lourenço, Cláudia M. Coutinho-Camillo. Evaluation of microRNA expression in metastatic and non-metastatic salivary gland mucoepidermoid carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2999.

TGFβ signaling pathway in salivary gland tumors

ObjectivePleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFβ signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFβ signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFβ cascade. DesignThirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR. ResultsCases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFβ signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC. ConclusionsOur study demonstrated the differential expression of TGFβ cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression.

Investigation of pyroptosis-related hub genes and the immune microenvironment in primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is an inflammatory systemic autoimmune disease, while the role and mechanisms of pyroptosis in pSS remain largely undefined. Pyroptosis-related genes and gene expression data were obtained from the Molecular Signatures Database and NCBI GEO databases. Differentially expressed genes (DEGs) and pyroptosis-related hub genes were identified by R software. Functional enrichment analyses were conducted using the "ClusterProfiler" R package and WebGestalt7. CIBERSORTx was used to calculate the correlations between immune cells and pyroptosis. Subsequently, histological staining was performed on salivary gland samples from non-pSS and pSS patients to identify the expression of pyroptosis-related genes. Immunofluorescence double staining was conducted to validate the correlation between immune cells and pyroptosis. A total of 1494 DEGs were identified between eight pSS samples and 10 healthy volunteer samples. Five pyroptosis-related hub genes (AIM2, CASP1, CASP3, IL6, TNF) were recognised. DEGs were mostly enriched in immunity-related terms and several immune cells were associated with the hub genes in pSS. Among them, delta gamma T cell was significantly positively correlated with CASP3. Finally, the protein levels of these hub genes were validated to be elevated in the labial minor salivary gland biopsies of pSS patients compared to those of healthy volunteers using immunohistochemical staining. Immunofluorescence double staining further showed that IL-6, AIM2, CASP1and CASP3 were related to delta gamma T cells, and TNF was related to dendritic cells. This study uncovered a significant interaction between pyroptosis and the immune microenvironment in pSS patients. Besides, we identified five pyroptosis-related hub genes that might play a role in the pathogenesis of pSS. These findings could offer valuable insights for the development of novel treatment strategies for pSS.

Defining the relationship of salivary gland malignancies to novel cell subpopulations in human salivary glands using single nucleus RNA-sequencing.

Salivary glands have essential roles in maintaining oral health, mastication, taste and speech, by secreting saliva. Salivary glands are composed of several types of cells, and each cell type is predicted to be involved in the carcinogenesis of different types of cancers including adenoid cystic carcinoma (ACC), acinic cell carcinoma (AciCC), salivary duct carcinoma (SDC), myoepithelial carcinoma (MECA) and other histology. In our study, we performed single nucleus RNA-seq on three human salivary gland samples to clarify the gene expression profile of each complex cellular component of the salivary glands and related these expression patterns to expression found in salivary gland cancers (SGC) to infer cell of origin. By single nucleus RNA-seq, salivary gland cells were stratified into four clusters: acinar cells, ductal cells 1, ductal cells 2 and myoepithelial cells/stromal cells. The localization of each cell group was verified by IHC of each cluster marker gene, and one group of ductal cells was found to represent intercalated ductal cells labeled with HES1. Furthermore, in comparison with SGC RNA-seq data, acinar cell markers were upregulated in AciCC, but downregulated in ACC and ductal cell markers were upregulated in SDC but downregulated in MECA, suggesting that markers of origin are highly expressed in some SGC. Cell type expressions in specific SGC histology are similar to those found in normal salivary gland populations, indicating a potential etiologic relationship.

A Prospective Study Comparing Diagnosis of Neck Swellings by Fine Needle Aspiration Cytology with Histopathology

Aim: To to correlate the fine needle aspiration cytology findings with histopathology findings of specimens from neck swellings. Study Design; Cross-Sectional Observational Setting: Pathology Department, Chandka Medical College, Larkana Period: June 2021 to June 2023 Methods: This study consisted of neck swelling samples received in the Department of Pathology by FNAC or excision. Both Cytology and Histopathology were evaluated by an experienced pathologist. Diagnoses of cytology were compared with the histopathological examination and the data was recorded into groups that comprised of lymph nodes, thyroid gland, and salivary glands. Lesions that did not fit into these groups were classified into as “others”. Data was entered and analyzed using SPSS Version-22. Results: There were a total of 261 cases of neck swelling samples received in our department. Most of the cases were due to lymph node (n=104) pathologies notably due to reactive hyperplasia and tuberculosis. The thyroid gland (n=95) was the second most common region for FNAC and most samples showed the diagnosis of colloid goiter followed by follicular adenomas. There were n=30 samples of salivary glands whereas n=32 were classified into others. Overall sensitivity and specificity were variable among the regions of FNAC with the highest being (100%) in the salivary glands and lesions classified into others category. Practical Implication: Comparing neck swelling diagnosis via Fine Needle Aspiration Cytology (FNAC) with Histopathology is vital to validate FNAC's accuracy before definitive treatment. It ensures informed decision-making, avoiding unnecessary surgeries and minimizing risks. Conclusion: FNAC of the neck swellings is a highly accurate technique and provides the first line of diagnosis. However, FNAC does have its own limitations and this technique should always be complemented by a confirmatory histopathology. Keywords: Fine needle Aspiration Cytology, Neck Swellings, Lymph node, Salivary Glands, Thyroid Glands

Mitochondria-related genes and metabolic profiles of innate and adaptive immune cells in primary Sjögren's syndrome.

Primary Sjogren's syndrome (pSS) is a prototypical systemic autoimmune disease characterised by lymphocyte infiltration and immune-complex deposition in multiple organs. The specific distribution of immune cell populations and their relationship with mitochondria remain unknown. Histological analysis was performed to assess the specific distribution of innate and adaptive immune cell populations in labial salivary gland (LSG) samples from 30 patients with pSS and 13 patients with non-pSS. The ultrastructural morphometric features of mitochondria within immune cells were observed under the transmission electron microscope (TEM). RNA sequencing was performed on LSG samples from 40 patients with pSS and 7 non-pSS patients. The Single-sample Gene Set Enrichment Analysis (ssGSEA), ESTIMATE, and CIBERSORT algorithms and Pearson correlation coefficients were used to examine the relationship between mitochondria-related genes and immune infiltration. Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify the mitochondria-specific genes and the related pathways based on the immune cell types. HE staining revealed a massive infiltration of plasma cells with abundant immunoglobulin protein distributed around phenotypically normal-appearing acinar and ductal tissues of patients with pSS. Immunohistochemical analyses revealed that innate immune cells (macrophages, eosinophils and NK cells) were distributed throughout the glandular tissue. Dominant adaptive immune cell infiltration composed of B cells, CD4+T cells and CD8+ T cells or ectopic lymphoid follicle-like structures were observed in the LSGs of patients with pSS. TEM validated the swelling of mitochondria with disorganised cristae in some lymphocytes that had invaded the glandular tissue. Subsequently, bioinformatic analysis revealed that innate and adaptive immune cells were associated with different mitochondrial metabolism pathways. Mitochondrial electron transport and respiratory chain complexes in the glandular microenvironment were positively correlated with innate immune cells, whereas amino acid and nucleic acid metabolism were negatively correlated with adaptive immune cells. In addition, mitochondrial biogenesis and mitochondrial apoptosis in the glandular microenvironment were closely associated with adaptive immune cells. Innate and adaptive immune cells have distinct distribution profiles in the salivary gland tissues of patients with pSS and are associated with different mitochondrial metabolic pathways, which may contribute to disease progression.

Diagnostic role of minor salivary glands biopsy in Sjögren's syndrome: correlations between histology and autoimmunity in a large, monocentric cohort.

Based on ACR/EULAR classification criteria, minor salivary glands biopsy (MSGB) is a useful diagnostic tool for the diagnosis of primary Sjögren's syndrome (SS). The main objective of our study was to evaluate the diagnostic role of MSGB, as well as to highlight correlations between histological findings and autoimmune profiles. We retrospectively evaluated histological and autoimmunity data from patients who underwent MSGB in our department in cases of suspected SS, from March 2011 to December 2018. Salivary gland samples were evaluated using Chisholm and Mason (CM) grading and the focus score (FS). A total of 1,264 patients (108 males, 1,156 females) were included. The median age was 55.22 ±13.51 years (range: 15-87). In univariate binary logistic regression, CM ≥ 3 and FS ≥ 1 were significantly predicted by antinuclear antibodies (ANA), anti-extractable nuclear antigens (ENA) and anti-Ro/SSA titer as well as anti-La/SSB, anti-Ro/SSA, rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) positivity. In multivariate analysis, CM ≥ 3 and MSGB positivity were significantly associated with ANA titer; FS ≥ 1 was not associated with laboratory findings. A positive biopsy was associated with laboratory findings, as ANA and ENA titers, anti-Ro/SSA, anti-La/SSB, RF and ACPA positivity may discriminate patients with SS-related histological findings. Minor salivary glands biopsy is a useful tool to diagnose SS in cases of highly suggestive clinical symptoms but in the absence of a specific autoimmunity.

Abstract 3763: microRNAs associated with metastatic potential in salivary gland mucoepidermoid carcinoma

Abstract Mucoepidermoid carcinoma (MEC) is the most common malignant tumor of the salivary glands. Metastatic spread occurs in up to 80% of high-grade tumors and it is a strong predictor of poor outcome, however, the mechanisms underlying this process are largely unknown. Large-scale microRNA expression profiling studies of human cancers have demonstrated that dysregulation of miRNA is frequently associated with many cancer types. The aim of this study was to investigate the microRNA profile in metastatic versus non-metastatic MECs. Using Real Time RT-PCR (qPCR) we have analyzed the expression of 384 miRNAs and controls in 4 non-metastatic MECs, 3 MECs with lymph node metastasis, 3 MECs with distant metastasis, and 2 non-neoplastic human salivary gland samples. The microRNA profile was able to discriminate between non-neoplastic and tumor samples and between metastatic and non-metastatic tumors. Considering non-neoplastic samples and non-metastatic MECs, 12 microRNAs were differentially expressed. The non-neoplastic versus lymph node metastasis MEC analysis demonstrated that 10 microRNAs were differentially expressed. Considering non-neoplastic versus distant metastasis MECs, 3 microRNAs were differentially expressed: 1 down-regulated and 2 up-regulated. Comparing non-metastatic MECs versus lymph node metastasis MECs we observed 17 up-regulated microRNAs. Considering non-metastatic MECs versus distant metastasis MECs, 2 microRNAs were up-regulated. One microRNA is differentially expressed between lymph node metastasis MECs and distant metastasis MECs. Our results suggest that microRNA profiles could discriminate the metastatic potential of salivary MECs and might be helpful in diagnostic and prognostic evaluation of patients. Citation Format: Maria Eduarda S. Trevizani, Katia K. Oliveira, Fabio A. Marchi, Daniela Bizinelli, Fernanda V. Mariano, Cibele P. Nagano, Felipe A. Costa, Clóvis A. Pinto, Luiz P. Kowalski, Silvia V. Lourenço, Cláudia M. Coutinho-Camillo. microRNAs associated with metastatic potential in salivary gland mucoepidermoid carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3763.

Phosphorylated signal transducer and activator of transcription proteins 1 in salivary glandular tissue: an important histological marker for diagnosis of primary Sjögren’s syndrome

ObjectivesThe pathological diagnostic criteria for primary Sjögren’s syndrome (SjS) have certain limitations. We first explored the key pathogenic pathways of SjS through a bioinformatics approach, and then evaluated the diagnostic...

Correction of LAMP3-associated salivary gland hypofunction by aquaporin gene therapy

Sjögren’s disease (SjD) is a chronic autoimmune sialadenitis resulting in salivary gland hypofunction with dry mouth symptom. Previous studies showed that lysosome-associated membrane protein 3 (LAMP3) overexpression is involved in the development of salivary gland hypofunction associated with SjD. However, the molecular mechanisms are still unclear, and no effective treatment exists to reverse gland function in SjD. Analysis on salivary gland samples from SjD patients showed that salivary gland hypofunction was associated with decreased expression of sodium–potassium-chloride cotransporter-1 (NKCC1) and aquaporin 5 (AQP5), which are membrane proteins involved in salivation. Further studies revealed that LAMP3 overexpression decreased their expression levels by promoting endolysosomal degradation. Additionally, we found that LAMP3 overexpression enhanced gene transfer by increasing internalization of adeno-associated virus serotype 2 (AAV2) via the promoted endolysosomal pathway. Retrograde cannulation of AAV2 vectors encoding AQP1 gene (AAV2-AQP1) into salivary glands induced glandular AQP1 expression sufficient to restore salivary flow in LAMP3-overexpressing mice. LAMP3 could play a critical role in the development of salivary gland hypofunction in SjD by promoting endolysosomal degradation of NKCC1 and AQP5. But it also could enhance AAV2-mediated gene transfer to restore fluid movement through induction of AQP1 expression. These findings suggested that AAV2-AQP1 gene therapy is useful in reversing salivary gland function in SjD patients.

CROSS-TALK OF PROTEINS RELATED TO INVADOPODIA AND THE INVASION PROCESS IN SALIVARY GLAND MUCOEPIDERMOID CARCINOMA

<h3>Objectives</h3> To evaluate the immunoexpression of Tks4, Tks5, cortactin, and MT1-MMP proteins in salivary gland mucoepidermoid carcinoma (MEC). <h3>Study Design</h3> Immunohistochemistry was performed to compare the expression of the 4 proteins in samples of MEC (<i>n</i> = 19) and healthy salivary glands (SG) (<i>n</i> = 10). The immunostaining assessment was performed by measuring the area (in µm) and the fraction (in %) of immunostaining of antibodies using the software ImageJ. The data were statistically analyzed using the Kruskal-Wallis test with Dunn's post hoc test for different proteins. Spearman correlation test was also performed. <h3>Results</h3> All MEC samples expressed Tks4, Tks5, cortactin, and MT1-MMP. There was a high immunoexpression of the 4 proteins in the MEC samples compared with the SG samples (*<i>P</i> < .05). In neoplastic MEC cells, expression of Tks4 and Tks5 proteins was correlated (*<i>P</i> < .05). <h3>Conclusions</h3> From the data obtained and in view of the important role of these proteins in the formation of invadopodia and in the cell invasion process, the expression of these proteins together in the MEC, as well as the correlation of Tks4 and Tks5, may suggest a possible relationship between these proteins and the invasive behavior of this neoplasm.

RABIES IN ARCTIC FOX (VULPES LAGOPUS) AND REINDEER (RANGIFER TARANDUS PLATYRHYNCHUS) DURING AN OUTBREAK ON SVALBARD, NORWAY, 2011–12

Rabies is an important zoonotic disease with high fatality rates in animals and humans. In the Arctic, the Arctic fox (Vulpes lagopus) is regarded as the principal reservoir, but there is considerable debate about how the disease persists at the low population densities that are typical for this species. We describe an outbreak of rabies among Arctic foxes and Svalbard reindeer (Rangifer tarandus platyrhynchus) during 2011-12 on the remote Arctic archipelago of Svalbard, an area with a very low and relatively stable Arctic fox density. The aim of the research was to increase knowledge of Arctic rabies in this ecosystem and in the presumed spillover host, the Svalbard reindeer. Phylogenetic analysis of rabies virus (RABV) RNA isolates from Arctic fox and reindeer was performed, and clinical observations and histologic and immunohistochemical findings in reindeer were described. An ongoing capture-mark-recapture project allowed collection of serum samples from clinically healthy reindeer from the affected population for detection of rabies virus-neutralizing antibodies. The outbreak was caused by at least two different variants belonging to the RABV Arctic-2 and Arctic-3 clades, which suggests that rabies was introduced to Svalbard on at least two different occasions. The RABV variants found in Arctic fox and reindeer were similar within locations, suggesting that Arctic foxes and reindeer acquired the infection from the same source(s). The histopathologic and immunohistochemical findings in 10 reindeer were consistent with descriptions in other species infected with RABV of non-Arctic lineages. Evidence of RABV was detected in both brain and salivary gland samples. None of 158 examined serum samples from clinically healthy reindeer had virus-neutralizing antibodies against RABV.

A Link Between Mitochondrial Dysfunction and the Immune Microenvironment of Salivary Glands in Primary Sjogren's Syndrome.

BackgroundPrimary Sjogren’s syndrome (pSS) is a slowly progressive, inflammatory autoimmune disease characterized by lymphocytic infiltration into salivary and lacrimal glands. It becomes more recognized that morphology alterations of epithelial mitochondria are involved in altered cellular bioenergetics in pSS patients. The integrated analysis of the mitochondrial role in the pathogenesis and aberrant immune microenvironment in pSS remains unknown.MethodsThe mitochondria-related genes and gene expression data were downloaded from the MitoMiner, MitoCarta, and NCBI GEO databases. We performed novel transcriptomic analysis and constructed a network between the mitochondrial function and immune microenvironment in pSS-salivary glands by computer-aided algorithms. Subsequently, real-time PCR was performed in clinical samples in order to validate the bioinformatics results. Histological staining and transmission electron microscopy (TEM) were further studied on labial salivary gland samples of non-pSS and pSS patients characterized for mitochondria-related phenotypic observation in the different stages of the disease.ResultsThe bioinformatic analysis revealed that the expression of several mitochondria-related genes was altered in pSS. Quantitative real-time PCR showed that four hub genes, CD38, CMPK2, TBC1D9, and PYCR1, were differentially expressed in the pSS clinical samples. These hub genes were associated with the degree of immune cell infiltration in salivary glands, the mitochondrial respiratory chain complexes, mitochondrial metabolic pathway in gluconeogenesis, TCA cycle, and pyruvate/ketone/lipid/amino acid metabolism in pSS. Clinical data revealed that the gene expression of fission (Fis1, DRP1, and MFF) and fusion (MFN1, MFN2, and OPA1) was downregulated in pSS samples, consistent with the results from the public validation database. As the disease progressed, cytochrome c and Bcl-2 proteins were regionally distributed in salivary glands from pSS patients. TEM revealed cytoplasmic lipid droplets and progressively swollen mitochondria in salivary epithelial cells.ConclusionOur study revealed cross talk between mitochondrial dysfunction and the immune microenvironment in salivary glands of pSS patients, which may provide important insights into SS clinical management based on modulation of mitochondrial function.

Association between Increased Bcl-2, Fas and FasL Levels and Inflammation Extent in Labial Salivary Glands During Primary Sjögren's Syndrome.

Primary Sjögren Syndrome (pSS) is a chronic autoimmune disease characterized by epithelial atrophy, mononuclear infiltration in exocrine glands resulting in the defective function of these glands. In pSS, atrophy of the epithelium is caused by an increased amount of apoptosis. The main aim of this study is to investigate the role of the apoptosis-related factors by studying Bcl-2, Fas and FasL expression in relation to the extent of inflammation as well as the effect of therapy on the expression of these mediators. In pSS patients (n=62) documented for their serological and clinical features, Fas, FasL and Bcl-2 plasma levels were assessed using enzyme-linked immunosorbent assays. In the same context, we investigated their expression by immunohistochemistry analysis in the labial salivary glands samples in association with the extent of inflammation. Interestingly, our results indicated that in pSS patients, the plasmatic Bcl-2, Fas and FasL levels, which appeared to be associated with the severity of inflammation and were significantly elevated in comparison to the healthy controls. Moreover, a significant decrease in all these factors was observed in patients after combined corticosteroids-hydroxychloroquine therapy. Importantly, we report a strong positive correlation between Bcl-2 and NO levels. The immunohistochemical staining reveals a strong Bcl-2 expression in infiltrating mononuclear cells and a total absence in the acinar cells. The Bcl-2 level varies according to the severity of pathology. However, the expression of Fas and FasL was less important and predominantly localized in infiltrating mononuclear cells. Our current study highlights the involvement of Bcl-2, Fas and FasL in pSS glands injury. These factors may act as useful predictor markers of a clinical course in pSS, suggesting a novel approach in the pSS patients monitoring.

First isolation and molecular characterization of pseudorabies virus detected in Turkey.

Pigs are the main host species for the pseudorabies virus. It causes fatal encephalitis in many species, including humans. This article aims to report the first clinical case of pseudorabies as well as isolation and molecular characterization of the virus from a hunting dog in Bursa province, Turkey. The dog shows clinical signs including pruritus and neurological signs such as stumbling and inability to stand up compatible with pseudorabies. The virus isolates were obtained from the supernatant of fresh tissue samples from the cerebellum, cornu ammonis, spleen, salivary gland, conjunctival swab, serum, and PBMC samples. The glycoprotein C region is targeted for viral DNA amplification. Pseudorabies virus genome detected both in fresh tissues and supernatants of third passage on Vero cells. The number of PCR positive samples was dramatically increased after cell culture inoculations. Genome sequencing of strain Bursa-10303, which was isolated from a non-endemic area, identified it to belong to clade A. This study confirms the possible presence of pseudorabies infection in the wildlife reservoirs in Turkey. Future studies may clarify the importance of the infection in Turkey region, where there is no prevalent pig production.

Exosomes Derived from Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSC) Protect Submandibular Glands in Diabetic Rats

Our study assess whether exosomes derived from bone marrow mesenchymal stem cells (BM-MSC) ameliorates diabetic salivary gland complications. 10 SD rats were assigned into diabetes group I and exosome treatment group II. Diabetic rats were induced by streptozotocin (STZ) and injected with DMSO or exosomes through tail vein followed by collection of submandibular salivary gland samples for histological analysis and TGFβ, Smad2 and Smad3 level by PCR, saliva IgA and serum amylase level. Compared with control mice, exosome treatment mice showed less fibrosis of the submandibular salivary glands and duct components with a more complete structure. Exosome treatment inhibited TGFβ, Smad2 and Smad3 level to reduce diabetic salivary gland complications, effectively decreased blood sugar level, improved salivary glands function with significantly reduced serum amylase and salivary IgA levels. In conclusion, BM-MSC-derived exosomes may be a new therapeutic strategy for treating diabetic salivary gland complications.