hsa-miR-424-5p and hsa-miR-513c-3p dysregulation mediated by IFN-γ is associated with salivary gland dysfunction in Sjögren's syndrome patients.

Salivary secretory dysfunction in SS-patients is associated with altered proteostasis, upregulation of ATF6α and components of the ERAD complex, such as SEL1L, and downregulation of XBP-1s and GRP78. Hsa-miR-424-5p is downregulated and hsa-miR-513c-3p is overexpressed in salivary glands from SS-patients. These miRNAs emerged as candidates that could regulate ATF6/SEL1L and XBP-1s/GRP78 levels, respectively. This study aimed to evaluate the effect of IFN-γ on hsa-miR-424-5p and hsa-miR-513c-3p expression and how these miRNAs regulate their targets. In labial salivary glands (LSG) biopsies from 9 SS-patients and 7 control subjects and IFN-γ-stimulated 3D-acini were analyzed. hsa-miR-424-5p and hsa-miR-513c-3p levels were measured by TaqMan assays and their localization by ISH. mRNA, protein levels, and localization of ATF6, SEL1L, HERP, XBP-1s and GRP78 were determined by qPCR, Western blot, or immunofluorescence. Functional and interaction assays were also performed. In LSGs from SS-patients and IFN-γ-stimulated 3D-acini, hsa-miR-424-5p was downregulated and ATF6α and SEL1L were upregulated. ATF6α and SEL1L were decreased after hsa-miR-424-5p overexpression, while ATF6α, SEL1L and HERP increased after hsa-miR-424-5p silencing. Interaction assays revealed that hsa-miR-424-5p targets ATF6α directly. hsa-miR-513c-3p was upregulated and XBP-1s and GRP78 were downregulated. XBP-1s and GRP78 were decreased after hsa-miR-513c-3p overexpression, while increases in XBP-1s and GRP78 were observed after hsa-miR-513c-3p silencing. Furthermore, we determined that hsa-miR-513c-3p targets XBP-1s directly. Significant correlations were found between both miRNA levels and clinical parameters. In conclusion, IFN-γ-dependent hsa-miR-424-5p and hsa-miR-513c-3p levels affect the expression of important factors involved in cellular proteostasis that control secretory function in LSG from SS-patients.

Tofacitinib counteracts IL-6 overexpression induced by deficient autophagy: implications in Sjögren's syndrome.

OBJECTIVE: Altered homeostasis of salivary gland (SG) epithelial cells in Sjögren's syndrome (SS) could be the initiating factor that leads to inflammation, secretory dysfunction and autoimmunity. Autophagy is an important homeostatic mechanism, whose deficiency is associated with inflammation and accumulation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) components. We aimed to evaluate whether autophagy is altered in labial SG (LSG) epithelial cells from primary SS (pSS) patients and whether this contributes to inflammation through the JAK-STAT pathway. Furthermore, we investigated the anti-inflammatory effect of the JAK inhibitor tofacitinib in autophagy-deficient (ATG5 knockdown) three-dimensional (3D)-acini. METHODS: We analysed LSG biopsies from 12 pSS patients with low focus score and 10 controls. ATG5-deficient 3D-acini were generated and incubated with IL-6 in the presence or absence of tofacitinib. Autophagy markers, pro-inflammatory cytokine expression, and JAK-STAT pathway activation were evaluated by PCR or western blot, along with correlation analyses between the evaluated markers and clinical parameters. RESULTS: LSG from pSS patients showed increased p62 and decreased ATG5 expression, correlating negatively with increased activation of JAK-STAT pathway components (pSTAT1 and pSTAT3). Increased expression of STAT1 and IL-6 correlated with EULAR Sjögren's syndrome disease activity index and the presence of anti-Ro antibodies. ATG5-deficient 3D-acini reproduced the findings observed in LSG from pSS patients, showing increased expression of pro-inflammatory markers such as IL-6, which was reversed by tofacitinib. CONCLUSION: Decreased expression of ATG5 in LSG epithelial cells from pSS patients possibly contributes to increased inflammation associated with JAK-STAT pathway activation, as evidenced in ATG5-deficient 3D-acini. Interestingly, these results suggest that tofacitinib could be used as an anti-inflammatory agent in pSS patients.

Aberrant MUC1 accumulation in salivary glands of Sjögren's syndrome patients is reversed by TUDCA in vitro.

OBJECTIVES: Xerostomia in SS patients has been associated with low quality and quantity of salivary mucins, which are fundamental for the hydration and protection of the oral mucosa. The aim of this study was to evaluate if cytokines induce aberrant mucin expression and whether tauroursodeoxycholic acid (TUDCA) is able to counteract such an anomaly. METHODS: Labial salivary glands from 16 SS patients and 15 control subjects, as well as 3D acini or human submandibular gland cells stimulated with TNF-α or IFN-γ and co-incubated with TUDCA, were analysed. mRNA and protein levels of Mucin 1 (MUC1) and MUC7 were determined by RT-qPCR and western blot, respectively. Co-immunoprecipitation and immunofluorescence assays for mucins and GRP78 [an endoplasmic reticulum (ER)-resident protein] were also performed. mRNA levels of RelA/p65 (nuclear factor-κB subunit), TNF-α, IL-1ß, IL-6, SEL1L and EDEM1 were determined by RT-qPCR, and RelA/p65 localization was evaluated by immunofluorescence. RESULTS: MUC1 is overexpressed and accumulated in the ER of labial salivary gland from SS patients, while MUC7 accumulates throughout the cytoplasm of acinar cells; however, MUC1, but not MUC7, co-precipitated with GRP78. TUDCA diminished the overexpression and aberrant accumulation of MUC1 induced by TNF-α and IFN-γ, as well as the nuclear translocation of RelA/p65, together with the expression of inflammatory and ER stress markers in 3D acini. CONCLUSION: Chronic inflammation alters the secretory process of MUC1, inducing ER stress and affecting the quality of saliva in SS patients. TUDCA showed anti-inflammatory properties decreasing aberrant MUC1 accumulation. Further studies are necessary to evaluate the potential therapeutic effect of TUDCA in restoring glandular homeostasis in SS patients.

Synaptotagmin-1 overexpression under inflammatory conditions affects secretion in salivary glands from Sjögren's syndrome patients.

Sjögren's syndrome (SS) is an autoimmune exocrinopathy associated with severe secretory alterations by disruption of the glandular architecture integrity, which is fundamental for a correct function and localization of the secretory machinery. Syt-1, PI(4,5)P2 and Ca2+ are significant factors controlling exocytosis in different secretory cells, the Ca2+ role being the most studied. Salivary acinar cells from SS-patients show a defective agonist-regulated intracellular Ca2+ release together with a decreased IP3R expression level, and this condition may explain a reduced water release. However, there are not reports where Syt-1, PI(4,5)P2 and Ca2+ in acinar cells of SS patients had been studied. In the present study, we analyzed the expression and/or localization of Syt-1 and PI(4,5)P2 in acinar cells of labial salivary gland biopsies from SS-patients and control individuals. Also, we evaluated whether the overexpression of Syt-1 and the loss of cell polarity induced by TNF-α or loss of interaction between acinar cell and basal lamina, alters directionality of the exocytosis process, Ca2+ signaling and α-amylase secretion in a 3D-acini model stimulated with cholinergic or ß-adrenergic agonists. In addition, the correlation between Syt-1 protein levels and clinical parameters was evaluated. The results showed an increase of Syt-1 mRNA and protein levels, and a high number of co-localization points of Syt-1/STX4 and PI(4,5)P2/Ezrin in the acinar basolateral region of LSG from SS-patients. With regard to 3D-acini, Syt-1 overexpression increased exocytosis in the apical pole compared to control acini. TNF-α stimulation increased exocytic events in the basal pole, which was further enhanced by Syt-1 overexpression. Additionally, altered acinar cell polarity affected Ca2+ signaling and amylase secretion. Overexpression of Syt-1 was associated with salivary gland alterations revealing that the secretory dysfunction in SS-patients is linked to altered expression and/or localization of secretory machinery components together with impaired epithelial cell polarity. These findings provide a novel insight on the pathological mechanism implicated in ectopic secretory products to the extracellular matrix of LSG from SS-patients, which might initiate inflammation.

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.

Impaired IRE1α/XBP-1 pathway associated to DNA methylation might contribute to salivary gland dysfunction in Sjögren's syndrome patients.

Objectives: Labial salivary glands (LSGs) of SS patients show alterations related to endoplasmic reticulum stress. Glandular dysfunction could be partly the consequence of an altered inositol-requiring enzyme 1α (IRE1α)/X box-binding protein 1 (XBP-1) signalling pathway of the unfolded protein response, which then regulates genes involved in biogenesis of the secretory machinery. This study aimed to determine the expression, promoter methylation and localization of the IRE1α/XBP-1 pathway components in LSGs of SS patients and also their expression induced by IFN-γ in vitro. Methods: IRE1α, XBP-1 and glucose-regulated protein 78 (GRP78) mRNA and protein levels were measured by qPCR and western blot, respectively, in LSGs of SS patients (n = 47) and control subjects (n = 37). Methylation of promoters was evaluated by methylation-sensitive high resolution melting, localization was analysed by immunofluorescence and induction of the IRE1α/XBP-1 pathway components by IFN-γ was evaluated in 3D acini. Results: A significant decrease of IRE1α, XBP-1u, XBP-1s, total XBP-1 and GRP78 mRNAs was observed in LSGs of SS patients, which was correlated with increased methylation levels of their respective promoters, and consistently the protein levels for IRE1α, XBP-1s and GRP78 were observed to decrease. IFN-γ decreased the mRNA and protein levels of XBP-1s, IRE1α and GRP78, and increased methylation of their promoters. Significant correlations were also found between IRE1α/XBP-1 pathway components and clinical parameters. Conclusion: Decreased mRNA levels for IRE1α, XBP-1 and GRP78 can be partially explained by hypermethylation of their promoters and is consistent with chronic endoplasmic reticulum stress, which may explain the glandular dysfunction observed in LSGs of SS patients. Additionally, glandular stress signals, including IFN-γ, could modulate the expression of the IRE1α/XBP-1 pathway components.

Pro-inflammatory cytokines enhance ERAD and ATF6α pathway activity in salivary glands of Sjögren's syndrome patients.

Salivary gland (SG) acinar-cells are susceptible to endoplasmic reticulum (ER) stress related to their secretory activity and the complexity of synthesized secretory products. SGs of Sjögren's syndrome patients (SS)-patients show signs of inflammation and altered proteostasis, associated with low IRE1α/XBP-1 pathway activity without avert increases in apoptosis. Acinar-cells may avoid apoptosis by activation of the ATF6α pathway and ER-associated protein degradation (ERAD). The aim of this study was to evaluate the role of pro-inflammatory cytokines in ATF6α pathway/ERAD activation and cell viability in labial salivary glands (LSG) of SS-patients. In biopsies from SS-patients increased ATF6α signaling pathway activity, as evidenced by generation of the ATF6f cleavage fragment, and increased expression of ERAD machinery components, such as EDEM1, p97, SEL1L, gp78, UBE2J1, UBE2G2, HERP and DERLIN1, were observed compared to controls. Alternatively, for pro- (active-caspase-3) and anti-apoptotic (cIAP2) markers no significant difference between the two experimental groups was detected. Increased presence of ATF6f and ERAD molecules correlated significantly with increased expression of pro-inflammatory cytokines. These observations were corroborated in vitro in 3D-acini treated with TNF-α and/or IFN-γ, where an increase in the expression and activation of the ATF6α sensor and ERAD machinery components was detected under ER stress conditions, while changes in cell viability and caspase-3 activation were not observed. Cytokine stimulation protected cells from death when co-incubated with an ERAD machinery inhibitor. Alternatively, when cytokines were eliminated from the medium prior to ERAD inhibition, cell death increased, suggesting that the presence of pro-inflammatory cytokines in the medium is essential to maintain cell viability. In conclusion, the ATF6α pathway and the ERAD machinery are active in LSG of SS-patients. Both were also activated by TNF-α and IFN-γ in vitro in 3D-acini and aided in preventing apoptosis. IFN-γ levels were elevated in SS-patients and UPR responses triggered in vitro by this cytokine closely matched those observed in LSG from SS-patients, suggesting that cytokines may induce ER stress.

Salivary mucins induce a Toll-like receptor 4-mediated pro-inflammatory response in human submandibular salivary cells: are mucins involved in Sjögren's syndrome?

OBJECTIVES: A hallmark characteristic of SS patients is the ectopic presence of the mucins MUC5B and MUC7 in the extracellular matrix of salivary glands that have lost apical-basolateral acinar-cell polarity. This study aims to determine whether exogenous salivary mucins induce gene expression of pro-inflammatory cytokines, as well as to evaluate whether the Toll-like receptor-4 (TLR4) pathway is involved in this response. METHODS: Differentiated human submandibular gland (HSG) cells were stimulated with mucins or oligosaccharide residues at different concentrations and for different periods of time. The expression of pro-inflammatory cytokines and their receptors was determined by semi-quantitative real time PCR (sqPCR). TLR4-mediated responses induced by mucin were evaluated with the Toll-IL-1 receptor domain containing adaptor protein (TIRAP) inhibitory peptide or using anti-hTLR4 blocking antibody. TLR4-receptor expression was also determined in SS patients, controls and HSG cells. RESULTS: Mucins induced a significant increase in CXCL8, TNF-α, IFN-α, IFN-ß, IL-6 and IL-1ß, but not B cell activating factor (BAFF). Cytokine induction was mediated by TLR4, as shown using TIRAP or using anti-hTLR4 antibody. Sugar residues present in MUC5B, such as sulpho-Lewis (SO3-3Galß1-3GlcNAc), also induced cytokines. Unexpectedly, mucins induced MUC5B, but not MUC7 expression. CONCLUSION: Salivary mucins were recognized by TLR4 in epithelial cells initiating a pro-inflammatory response that could attract inflammatory cells to amplify and perpetuate inflammation and thereby contribute to the development of a chronic state characteristic of SS. The ectopic localization of MUC5B and MUC7 in the salivary gland extracellular matrix from SS patients and the current results reveal the importance of salivary epithelial cells in innate immunity, as well as in SS pathogenesis.

Giantin mediates Golgi localization of Gal3-O-sulfotransferases and affects salivary mucin sulfation in Sjögren's disease patients.

Sjögren's disease is a chronic autoimmune disease characterized by symptoms of oral and ocular dryness and extra-glandular manifestations. Mouth dryness is not only due to reduced saliva volume but also to alterations in the quality of salivary mucins in these patients. Mucins play a leading role in mucosa hydration and protection, where sulfated and sialylated oligosaccharides retain water molecules at the epithelial surface. The correct localization of glycosyltransferases and sulfotransferases within the Golgi apparatus determines adequate O-glycosylation and sulfation of mucins, which depends on specific golgins that tether enzyme-bearing vesicles. Here, we show that a golgin called Giantin is mislocalized in salivary glands from patients with Sjögren's disease and forms protein complexes with Gal3-O-sulfotransferases (Gal3STs), which change their localization in Giantin knockout and knockdown cells. Our results suggest that Giantin could tether Gal3ST-bearing vesicles and that its altered localization could affect Gal3ST activity, explaining the decreased sulfation of MUC5B observed in salivary glands from patients with Sjögren's disease.

The integrated stress response is activated in the salivary glands of Sjögren's syndrome patients.

Introduction: Primary Sjögren's syndrome (SS) is an autoimmune exocrinopathy that affects the structure and function of salivary and lachrymal glands. Labial salivary gland (LSG) acinar cells from SS patients lose cellular homeostasis and experience endoplasmic reticulum and oxidative stress. The integrated cellular stress response (ISR) is an adaptive pathway essential for restoring homeostasis against various stress-inducing factors, including pro-inflammatory cytokines, and endoplasmic reticulum and oxidative stress. ISR activation leads eIF2α phosphorylation, which transiently blocks protein synthesis while allowing the ATF4 expression, which induces a gene expression program that seeks to optimize cellular recovery. PKR, HRI, GCN2, and PERK are the four sentinel stress kinases that control eIF2α phosphorylation. Dysregulation and chronic activation of ISR signaling have pathologic consequences associated with inflammation. Methods: Here, we analyzed the activation of the ISR in LSGs of SS-patients and non-SS sicca controls, determining the mRNA, protein, and phosphorylated-protein levels of key ISR components, as well as the expression of some of ATF4 targets. Moreover, we performed a qualitative characterization of the distribution of ISR components in LSGs from both groups and evaluated if their levels correlate with clinical parameters. Results: We observed that the four ISR sensors are expressed in LSGs of both groups. However, only PKR and PERK showed increased expression and/or activation in LSGs from SS-patients. eIF2α and p-eIF2α protein levels significantly increased in SS-patients; meanwhile components of the PP1c complex responsible for eIF2α dephosphorylation decreased. ATF4 mRNA levels were decreased in LSGs from SS-patients along with hypermethylation of the ATF4 promoter. Despite low mRNA levels, SS-patients showed increased levels of ATF4 protein and ATF4-target genes involved in the antioxidant response. The acinar cells of SS-patients showed increased staining intensity for PKR, p-PKR, p-PERK, p-eIF2α, ATF4, xCT, CHOP, and NRF2. Autoantibodies, focus score, and ESSDAI were correlated with p-PERK/PERK ratio and ATF4 protein levels. Discussion: In summary, the results showed an increased ISR activation in LSGs of SS-patients. The increased protein levels of ATF4 and ATF4-target genes involved in the redox homeostasis could be part of a rescue response against the various stressful conditions to which the LSGs of SS-patients are subjected and promote cell survival.

Aberrant localization of fusion receptors involved in regulated exocytosis in salivary glands of Sjögren's syndrome patients is linked to ectopic mucin secretion.

Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease that mainly affects tear and salivary glands, whereby SS-patients frequently complain of eye and mouth dryness. Salivary acinar cells of SS-patients display alterations in their cell polarity; which may affect the correct localization and function of proteins involved in regulated exocytosis. Here we determined whether the expression and localization of SNARE proteins (membrane fusion receptors) involved in regulated secretion, such as VAMP8, syntaxin 3 (STX3), STX4 and SNAP-23 were altered in salivary glands (SG) from SS-patients. Additionally, we investigated SNARE proteins function, by evaluating their ability to form SNARE complexes under basal conditions. In SG from SS-patients and control subjects mRNA and proteins levels of SNARE complex components were determined by real-time PCR and Western blotting, respectively. SNARE protein distribution and mucin exocytosis were determined by indirect immunofluorescence. In SS-patients, the expression levels of mRNA and protein for VAMP8, STX4 and STX3 were altered. STX4, STX3, SNAP-23 and VAMP8 relocated from the apical to the basal region of acinar cells. Increased formation of SNARE complexes in a manner independent of external stimuli for secretion was detected. Mucins were detected in the extracellular matrix (ECM). Presence of mucins in the ECM, together with the observed alterations in SNARE protein localization is indicative of ectopic exocytosis. In the context of SS, such aberrantly localized mucins are likely to favor a pro-inflammatory response, which may represent an important initial step in the pathogenesis of this disease.

Decreased salivary sulphotransferase activity correlated with inflammation and autoimmunity parameters in Sjogren's syndrome patients.

OBJECTIVES: To determine the expression and enzymatic activities of sulphotransferases involved in mucin hyposulphation in labial salivary glands (LSGs) from SS patients and to correlate sulphotransferase activity with clinical parameters such as secretion, inflammation and serology. METHODS: LSG from 31 SS patients and 31 control subjects were studied. Relative mRNA and protein levels of Gal3-O-sulphotransferases (Gal3STs) and ß1,3-galactosyltransferase-5 (ß3GalT5) were determined by quantitative RT-PCR and western blotting, respectively. Enzymatic activities were quantified using radioactively labelled donor substrates and specific acceptor substrates. Products were purified by chromatography. Spearman's correlation analysis was used to compare data. RESULTS: The levels of Gal3ST activity were significantly decreased in SS patients, without changes in mRNA and protein levels, while the enzymatic activities of glycosyltransferases involved in mucin glycosylation were similar in both groups. An inverse correlation was observed between Gal3ST activity and glandular function measured by scintigraphy, but not with unstimulated salivary flow. Gal3ST activity was inversely correlated with focus score, TNF-α levels and presence of the autoantibodies Ro/SS-A and La/SS-B. CONCLUSION: The decrease in sulphotransferase activity provides an explanation for mucin hyposulphation observed in the LSGs from SS patients. The decrease in Gal3STs activity was not a consequence of reduced gene expression, but probably due to alterations in the enzyme activity regulation. Interestingly, the levels of sulphotransferase activity detected correlated well with secretory function, inflammation and serology. Finally, we postulate that pro-inflammatory cytokines induced by autoantibodies, such as Ro/SS-A and La/SS-B in SS patients, may modulate Gal3ST activity, thereby altering mucin quality and leading to mouth dryness.

Changes in Rab3D expression and distribution in the acini of Sjögren's syndrome patients are associated with loss of cell polarity and secretory dysfunction.

OBJECTIVE: Oral and ocular dryness are frequent and serious symptoms of Sjögren's syndrome (SS) that reflect problems in secretion due to glandular dysfunction. Exocytosis, an important process in the secretory pathway, requires the participation of Rab family GTPases. This study was undertaken to analyze the expression and localization of Rab3D and Rab8A and to examine their correlation with acinar cell polarity and glandular secretory function. METHODS: Nineteen patients with SS and 17 controls were evaluated. Levels of Rab3D and Rab8A messenger RNA (mRNA) and protein were determined by real-time polymerase chain reaction and Western blotting. Subcellular localization of proteins was determined by indirect immunofluorescence analysis. RESULTS: In patients with SS, total Rab3D protein levels decreased significantly, while mRNA levels remained unchanged. For Rab8A, no changes in either mRNA or protein levels were detected. In serous acini of labial salivary glands from patients with SS, the following 4 patterns of Rab3D staining were distinguishable: severely decreased, distribution throughout the cytoplasm, distribution throughout the cytoplasm combined with loss of nuclear polarity, and normal apical localization. Basal localization of Rab8A was not modified. Rab3D changes were accompanied by apicobasolateral redistribution of ezrin, loss of nuclear polarity, thicker Golgi stacks, and mucin 7 accumulation in the cytoplasm. Finally, low Rab3D protein levels correlated with alterations in scintigraphy measurements. CONCLUSION: Our findings indicate that Rab3D regulates the exocytosis of many components critical for the maintenance of oral physiology. Hence, the changes observed in Rab3D expression and distribution are likely to contribute to the decrease in or loss of saliva components (i.e., mucins), which may explain the variety of oral and ocular symptoms associated with SS.

Small RNA Expression Profiling Reveals hsa-miR-181d-5p Downregulation Associated With TNF-α Overexpression in Sjögren's Syndrome Patients.

MicroRNAs (miRNAs) are small non-coding RNAs (sRNA), that alter gene expression by binding to target messenger RNAs (mRNAs) and repressing translation. Dysregulated miRNA expression has been implicated in the pathogenesis of autoimmune diseases such as Sjögren's syndrome (SS). The aim of this study was to characterize the global profile of sRNAs in labial salivary glands (LSG) from SS-patients and to validate potential miRNA candidates implicated in glandular inflammation. LSG from 21 SS-patients and 9 sicca controls were analyzed. A global next generation sequencing (NGS)-based sRNA profiling approach was employed to identify direct targets whereby differentially expressed miRNAs were predicted using bioinformatics tools. miRNA levels were validated by TaqMan and target mRNA levels were determined by quantitative real-time PCR. We also performed in vitro assays using recombinant TNF-α. NGS shows that ~30% of sRNAs were miRNAs. In comparison with samples from sicca controls, four miRNAs were found differentially expressed in LSG from SS-patients with low focus score (LFS) and 18 from SS-patients with high focus score (HFS). The miRNA with the most significant changes identified by NGS was hsa-miR-181d-5p and downregulation was confirmed by TaqMan analysis. Levels of TNF-α mRNA, a direct target of hsa-miR-181d-5p, were significantly increased and negatively correlated with hsa-miR-181d-5p presence. Moreover, positive correlations between TNF-α transcript levels, focus score, ESSDAI, and autoantibody levels were also detected. Furthermore, TNF-α stimulation decreased hsa-miR-181d-5p levels in vitro. Downregulation of hsa-miR-181d-5p in LSG from SS-patients could contribute to the glandular pro-inflammatory environment by deregulation of its direct target TNF-α. Further dissection of the pathophysiological mechanisms underlying the hsa-miR-181d-5p-mediated action in inflammatory conditions could be useful to evaluate the benefits of increasing hsa-miR-181d-5p levels for restoration of salivary gland epithelial cell architecture and function.

What is Currently Known about Odontogenic Keratocysts?

PURPOSE: To present updated information on odontogenic keratocyst (OKC) classification, etiology, genetic and molecular alterations, epidemiology, clinical presentation, radiographic characteristics, histological and immune histochemical features, differential diagnosis, treatment, and controversies, as well as a literature review of case frequencies in different countries. MATERIALS AND METHODS: Studies were selected using the key words 'odontogenic keratocyst,' 'odontogenic cysts,' 'odontogenic keratocyst and clinical study'. Full-text papers were reviewed on the basis of the inclusion and exclusion criteria. The literature search aimed to find articles that would show the frequency of OKC, dentigerous cyst, radicular cyst, and other cysts. RESULTS: OKC presents local aggression and high recurrence; therefore, a better understanding of its clinical characteristics and the genetic and molecular factors involved in this peculiar and controversial lesion is required. It is always essential to discuss treatment alternatives. Although OKC is an entity with a high recurrence, aggressive treatment is not advisable in all cases because factors such as commitment to anatomical structures and possible complications should be considered. However, periodic radiographic controls are advised. CONCLUSION: To reduce the high number of present cases worldwide, it is important to improve knowledge on this pathology so that accurate diagnoses can be achieved and appropriate treatment can be provided. OKC presents local aggression and high recurrence; therefore, a better understanding is needed of the clinical characteristics and genetic and molecular factors involved in OKC. Furthermore, it is always essential to discuss treatment alternatives.

Primary Chemoradiotherapy Treatment (PCRT) for HER2+ and Triple Negative Breast Cancer Patients: A Feasible Combination.

Primary systemic treatment (PST) downsizes the tumor and improves pathological response. The aim of this study is to analyze the feasibility and tolerance of primary concurrent radio−chemotherapy (PCRT) in breast cancer patients. Patients with localized TN/HER2+ tumors were enrolled in this prospective study. Radiation was delivered concomitantly during the first 3 weeks of chemotherapy, and it was based on a 15 fractions scheme, 40.5 Gy/2.7 Gy per fraction to whole breast and nodal levels I-IV. Chemotherapy (CT) was based on Pertuzumab−Trastuzumab−Paclitaxel followed by anthracyclines in HER2+ and CBDCA-Paclitaxel followed by anthracyclines in TN breast cancers patients. A total of 58 patients were enrolled; 25 patients (43%) were TN and 33 patients HER2+ (57%). With a median follow-up of 24.2 months, 56 patients completed PCRT and surgery. A total of 35 patients (87.5%) achieved >90% loss of invasive carcinoma cells in the surgical specimen. The 70.8% and the 53.1% of patients with TN and HER-2+ subtype, respectively, achieved complete pathological response (pCR). This is the first study of concurrent neoadjuvant treatment in breast cancer in which three strategies were applied simultaneously: fractionation of RT (radiotherapy) in 15 sessions, adjustment of CT to tumor phenotype and local planning by PET. The pCR rates are encouraging.

Disruption of tight junction structure in salivary glands from Sjögren's syndrome patients is linked to proinflammatory cytokine exposure.

OBJECTIVE: Disorganization of acinar cell apical microvilli and the presence of stromal collagen in the acinar lumen suggest that the labial salivary gland (LSG) barrier function is impaired in patients with Sjögren's syndrome. Tight junctions define cell polarity and regulate the paracellular flow of ions and water, crucial functions of acinar cells. This study was undertaken to evaluate the expression and localization of tight junction proteins in LSGs from patients with SS and to determine in vitro the effects of tumor necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma) on tight junction integrity of isolated acini from control subjects. METHODS: Twenty-two patients and 15 controls were studied. The messenger RNA and protein levels of tight junction components (claudin-1, claudin-3, claudin-4, occludin, and ZO-1) were determined by semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting. Tight junction protein localization was determined by immunohistochemistry. Tight junction ultrastructure was examined by transmission electron microscopy. Isolated acini from control subjects were treated with TNFalpha and IFNgamma. RESULTS: Significant differences in tight junction protein levels were detected in patients with SS. ZO-1 and occludin were strongly down-regulated, while claudin-1 and claudin-4 were overexpressed. Tight junction proteins localized exclusively to apical domains in acini and ducts of LSGs from controls. In SS patients, the ZO-1 and occludin the apical domain presence of decreased, while claudin-3 and claudin-4 was redistributed to the basolateral plasma membrane. Exposure of isolated control acini to TNFalpha and IFNgamma reproduced these alterations in vitro. Ultrastructural analysis associated tight junction disorganization with the presence of endocytic vesicles containing electron-dense material that may represent tight junction components. CONCLUSION: Our findings indicate that local cytokine production in LSGs from SS patients may contribute to the secretory gland dysfunction observed in SS patients by altering tight junction integrity of epithelial cells, thereby decreasing the quality and quantity of saliva.

Type I Interferon Dependent hsa-miR-145-5p Downregulation Modulates MUC1 and TLR4 Overexpression in Salivary Glands From Sjögren's Syndrome Patients.

Sjögren's syndrome (SS) is an autoimmune disease that mainly affects salivary glands (SG) and is characterized by overactivation of the type I interferon (IFN) pathway. Type I IFNs can decrease the levels of hsa-miR-145-5p, a miRNA with anti-inflammatory roles that is downregulated in SG from SS-patients. Two relevant targets of hsa-miR-145-5p, mucin 1 (MUC1) and toll-like receptor 4 (TLR4) are overexpressed in SS-patients and contribute to SG inflammation and dysfunction. This study aimed to evaluate if hsa-miR-145-5p modulates MUC1 and TLR4 overexpression in SG from SS-patients in a type I IFN dependent manner. Labial SG (LSG) biopsies from 9 SS-patients and 6 controls were analyzed. We determined hsa-miR-145-5p levels by TaqMan assays and the mRNA levels of MUC1, TLR4, IFN-α, IFN-ß, and IFN-stimulated genes (MX1, IFIT1, IFI44, and IFI44L) by real time-PCR. We also performed in vitro assays using type I IFNs and chemically synthesized hsa-miR-145-5p mimics and inhibitors. We validated the decreased hsa-miR-145-5p levels in LSG from SS-patients, which inversely correlated with the type I IFN score, mRNA levels of IFN-ß, MUC1, TLR4, and clinical parameters of SS-patients (Ro/La autoantibodies and focus score). IFN-α or IFN-ß stimulation downregulated hsa-miR-145-5p and increased MUC1 and TLR4 mRNA levels. Hsa-miR-145-5p overexpression decreased MUC1 and TLR4 mRNA levels, while transfection with a hsa-miR-145-5p inhibitor increased mRNA levels. Our findings show that type I IFNs decrease hsa-miR-145-5p expression leading to upregulation of MUC1 and TLR4. Together, this suggests that type I interferon-dependent hsa-miR-145-5p downregulation contributes to the perpetuation of inflammation in LSG from SS-patients.

Dysfunctional mitochondria as critical players in the inflammation of autoimmune diseases: Potential role in Sjögren's syndrome.

Relevant reviews highlight the association between dysfunctional mitochondria and inflammation, but few studies address the contribution of mitochondria and mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) to cellular homeostasis and inflammatory signaling. The present review outlines the important role of mitochondria in cellular homeostasis and how dysfunctional mitochondrion can release and misplace mitochondrial components (cardiolipin, mitochondrial DNA (mtDNA), and mitochondrial formylated peptides) through multiple mechanisms. These components can act as damage-associated molecular patterns (DAMPs) and induce an inflammatory response via pattern recognition receptors (PRRs). Accumulation of damaged ROS-generating mitochondria, accompanied by the release of mitochondrial DAMPs, can activate PRRs such as the NLRP3 inflammasome, TLR9, cGAS/STING, and ZBP1. This process would explain the chronic inflammation that is observed in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type I diabetes (T1D), and Sjögren's syndrome. This review also provides a comprehensive overview of the importance of MERCs to mitochondrial function and morphology, cellular homeostasis, and the inflammatory response. MERCs play an important role in calcium homeostasis by mediating the transfer of calcium from the ER to the mitochondria and thereby facilitating the production of ATP. They also contribute to the synthesis and transfer of phospholipids, protein folding in the ER, mitochondrial fission, mitochondrial fusion, initiation of autophagosome formation, regulation of cell death/survival signaling, and regulation of immune responses. Therefore, alterations within MERCs could increase inflammatory signaling, modulate ER stress responses, cell homeostasis, and ultimately, the cell fate. This study shows severe ultrastructural alterations of mitochondria in salivary gland cells from Sjögren's syndrome patients for the first time, which could trigger alterations in cellular bioenergetics. This finding could explain symptoms such as fatigue and malfunction of the salivary glands in Sjögren's syndrome patients, which would contribute to the chronic inflammatory pathology of the disease. However, this is only a first step in solving this complex puzzle, and several other important factors such as changes in mitochondrial morphology, functionality, and their important contacts with other organelles require further in-depth study. Future work should focus on detecting the key milestones that are related to inflammation in patients with autoimmune diseases, such as Sjögren´s syndrome.

Definition and development of a digital system for the empowerment and activation of type 1 diabetic patient.

Health education is essential for type 1 diabetic patients to actively participate in the decision-making process about their disease. Under the framework of the INCAP project, a mobile application has been designed and developed with an easy-to-use interface for type 1 diabetic patients to improve their empowerment, activation and thus their self-control and improvement of their treatment adherence.