Abstract Sjo¨gren’s syndrome (SS) is characterized by the features of systemic autoimmunity and exocrine gland dysfunction and inflammation. Deregulated cytokine production is known to contribute to the etiology of SS but the underlying molecular mechanism is still remains to be unclear. TNF-a-induced protein 3 or TNFAIP3 is involved in the negative feedback regulation of nuclear factor-jB (NF-jB) signaling in response to specific pro-inflammatory stimuli in different cell types. To define the contribution of TNFAIP3 to SS, the levels of TNFAIP3 expression in human salivary gland epithelial cells (SGEC) derived from active primary SS patients were analyzed. Histological analysis was performed on paraffin-embedded human Sjo¨gren’s samples and healthy tissues. In separate experiments, immunofluorescence staining, western blot analysis and quantitative real-time PCR for TNFAIP3 was conducted in SGEC from SS and healthy subjects. Our findings clearly demonstrate changes in levels of the protein and gene expression between healthy controls and SS patients, depicting a very weak positivity for TNFAIP3 in SS samples. TNFAIP3 was found down-regulated in SGECs derived from SS patients in comparison with controls, and the cells with down-regulated TNFAIP3 expression exhibited enhanced NF-jB activities. In addition, to investigate the role of TNFAIP3 in the activation of NFjB, we depleted TNFAIP3 expression by siRNA in healthy SGEC after treatment with or without TNF-a. Intriguingly, the silencing of TNFAIP3 by its siRNA in healthy SGEC increased NF-jB activation that could explain the deregulated cytokines production observed in SS.

Chemokines, small pro-inflammatory cytokines, are involved in migration of inflammatory cells in inflamed tissues and recent studies established their role in angiogenesis, hematopoiesis, cancer and autoimmune conditions. Growth related oncogene-alpha (GRO-α), a member of the CXC chemokine family, and its receptor CXCR2 are involved in the inflammatory processes. Since there is no previous report that supports a possible role of GRO-α/CXCR2 receptor complex during inflammation and neovascularization existing in the autoimmune disease Sjögren's syndrome (SS), in this study, we examined CXCR2 and its ligand GRO-α expression in SS tissues. Immunohistochemistry revealed that GRO-α and its receptor CXCR2 were expressed at high levels in diseased tissues compared to healthy controls. In addition, human salivary gland epithelial cells (SGEC) cultures were submitted to a pro-inflammatory microenvironment using cytokines IL-6 and TNF-α in order to demonstrate that CXCR2 may change its initial expression pattern to another under inflammatory condition. The data show an increased expression of CXCR2 depending on the inflammatory cytokine used in culture in a time-dependent manner. Furthermore, silencing of the pro-angiogenic chemokine GRO-α is proportionally correlated with decreased expression of CXCR2 in pro-inflammatory cytokine-stimulated SGEC indicating the GRO-α/CXCR2 complex as a novel therapeutic target for the chronic inflammatory disease Sjögren's syndrome.

A decreased saliva production occurs in primary Sjögren's syndrome (pSS), an autoimmune disease characterized by oral and ocular dryness due to dysfunction of the lacrimal and salivary glands (SGs). Since water movement is involved in saliva secretion, the expression, localization, and function of the water channels aquaporins (AQPs) have been extensively studied in SGs. To date, the presence of AQP4 remains controversial and ambiguous in human SGs. We investigated by immunohistochemistry, high-resolution confocal microscopy and quantitative image analysis, Western blot and real-time RT-PCR, the presence of the AQP4 gene, and the distribution of AQP4 protein in healthy controls and pSS SG biopsies. Through the immunohistochemical analysis, we demonstrated that AQP4 presence is confined to the basal region of acini, to the lateral and apical membrane of intercalated and striated ducts in both control and pSS glands. The most striking observation was the discovery of AQP4 localization in myoepithelial cells (MECs) that surround acini lobules and intercalated ducts, and the demonstration of AQP4-downregulated immunoreactivity in pSS MECs. Our studies suggest that the capacity for water flow across the membrane of MECs may be altered in pSS, identifying AQP4 as a promising new therapeutic agent to treat xerostomia.

Receptors for the Fc fragment of immunoglobulin G (FcgammaRs) are important molecules not only to mediate and control the effectors' functions of IgG antibodies, but they also control the autoimmunity-tolerance balance in the periphery. In humans, three different types of FcgammaRs, belonging to the Ig gene superfamily, have been identified; FcgammaRI (cluster of differentiation (CD64), FcgammaRII (CD32) and FcgammaRIII (CD16). A wide range of inflammatory and autoimmune diseases, such as vasculitis, glomerulonephritis, and autoimmune hemolytic anemia, seems to be mediated, in part, by FcgammaRs. Recent findings supposed that, under certain conditions, FcgammaRs are involved in the penetration of antibodies into cells and FcgammaRs constitute one of the main effector mechanisms through which autoantibodies exert their action. In this review, we concentrate on the role of human FcgammaRs in autoantibodies penetration and summarize the current knowledge on the structure, ligand binding capacity and their role in autoimmunity and pathogenic effect of autoantibodies.

Aims: To study the importance of IkBa in NF-kB signal transduction, we analysed the IkBa expression in monocytes from Sjo¨gren’s syndrome (SS) patients versus healthy controls. Methods: Monocytes were obtained from the peripheral blood of 30SSpatients and 23 healthy subjects. IkBaexpressionwas studied by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, immunoblotting, flow cytometry and enzyme linked immunosorbent assay (ELISA). Results: Analysis of the gene and protein expression profiles of SS monocytes revealed a down-regulation of IkBa, and in all the Sjo¨gren’s syndrome cases examined, serum IkBa levels were significantly decreased in comparison with controls. Conclusions: Our findings clearly demonstrate changes in the levels of IkBa in SS monocytes, suggesting that the attenuated expression of IkBa could contribute to the deregulation of NF-kB pathways in the SS pathogenesis. Decreased expression of IkBa may specifically amplify cytokines production and inflammatory response linked to Sjo¨gren’s syndrome.

The endoplasmic reticulum (ER) is a complex and multifunctional organelle. It is the intracellular compartment of protein folding, a complex task, both facilitated and monitored by ER folding enzymes and molecular chaperones. The ER is also a stress-sensing organelle. It senses stress caused by disequilibrium between ER load and folding capacity and responds by activating signal transduction pathways, known as unfolded protein response (UPR). Three major classes of transducer are known, inositol-requiring protein-1 (IRE1), activating transcription factor-6 (ATF6), and protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK), which sense with their endoluminal domain the state of protein folding, although the exact mechanism(s) involved is not entirely clear. Depending on whether the homeostatic response of the UPR is successful in restoring an equilibrium between ER load and protein folding or not, the two possible outcomes of the UPR so far considered have been life or death. Indeed, recent efforts have been devoted to understand the life/death switch mechanisms. However, recent data suggest that what appears to be a pure binary decision may in fact be more complex, and survival may be achieved at the expenses of luxury cell functions, such as expression of differentiation genes.

We have investigated whether increase in the oxidation rate of exogenous cytochrome c (cyto-c), induced by long chain ceramides, might be due to an increased rate of cytosolic NADH/cyto-c electron transport pathway. This process was identified in isolated liver mitochondria and has been studied in our laboratory for many years. Data from highly specific test of sulfite oxidase prove that exogenous cyto-c both in the absence and presence of ceramide cannot permeate through the mitochondrial outer membrane. However, the oxidation of added NADH, mediated by exogenous cyto-c and coupled to the generation of a membrane potential supporting the ATP synthesis, can also be stimulated by ceramide. The results obtained suggest that ceramide molecules, by increasing mitochondrial permeability, with the generation of either channels or raft-like platforms, may have a dual function. They can promote the release of endogenous cyto-c and activate, with an energy conserving process, the oxidation of cytosolic NADH either inducing the formation of new respiratory contact sites or increasing the frequency of the pre-existing porin contact sites. In agreement with the data in the literature, an increase of mitochondrial ceramide molecules level may represent an efficient strategy to activate and support the correct execution of apoptotic program.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune exocrine disease associated with variable lymphocytic infiltration of the affected organs (primarily salivary and lachrymal glands). To investigate the potential implication of nerve growth factor-β (NGF-β) and its high affinity receptor tyrosine kinase receptor A (TrkA) in the regulation of pSS inflammatory responses, we studied their expression in the human salivary gland epithelial cells (SGEC) cultures from pSS minor salivary glands (MSG) biopsies and their relationship with histopathological disease parameters. Here, we demonstrated an increased expression of the NGF-β/TrkA system in pSS SGEC, correlated with the MSG inflammation grade. The results demonstrate that the pro-inflammatory cytokines TNF-α and IL-6 enhance NGF-β production; on the contrary, NGF-β production was reduced in the presence of both Raf-1 kinase and MEK inhibitors. Furthermore, TNF-α/IL-6 treatment increased ERK1/2 phosphorylation. Inhibition of the EGF/EGFR system also decreased NGF-β release by pSS SGEC, indicating that the chronic inflammatory condition characteristic of pSS enhances NGF-β production via EGFR/Raf-1/MEK/ERK pathway activation. KEY MESSAGE: NGF-β and TrkA expression is elevated in salivary gland epithelial cells of primary Sjögren's syndrome (pSS). Overexpression of NGF-β/TrkA system in pSS occurs via EGFR/Raf-1/MEK/ERK pathway. In pSS, NGF-β overexpression was prevented by EGFR/Raf-1/MEK/ERK pathway inhibition.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder of the exocrine glands with associated lymphocytic infiltrates in the affected glands. Dryness of the mouth and eyes results from involvement of the salivary and lacrimal glands. The efficacy of Rituximab (RTX) in pSS is still open to debate. This study delineates the signaling pathway involved in RTX-mediated down-regulation of pro-inflammatory factors in a co-culture system of pSS salivary gland epithelial cells (SGEC) with syngeneic pSS B-lymphocytes. In addition, the effects of RTX on the activation of the Raf-1/ERK1/2 pathway in pSS SGEC co-cultured with syngeneic pSS T-lymphocytes were also investigated. This study demonstrated that RTX may interfere with the ERK1/2 pathway in a syngeneic co-culture of pSS SGEC with pSS B-lymphocytes, leading to decreased cytokine production by SGEC. These novel findings reveal that syngeneic co-culture of pSS SGEC with pSS B-lymphocytes leads to a down-regulation of Raf-1 in epithelial cells that adversely regulates the activity of the ERK1/2 pathway and determines a subsequent reduction of the release of pro-inflammatory factors.

Sjögren's syndrome (SS) is an autoimmune disease and the second most common chronic systemic rheumatic disorder. Prevalence of primary SS in the general population has been estimated to be approximately 1-3%, whereas secondary SS has been observed in 10-20% of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE) and scleroderma. Despite this, its exact aetiology and pathogenesis are largely unexplored. Nuclear factor-kappa B (NF-κB) signalling mechanisms provide central controls in SS, but how these pathways intersect the pathological features of this disease is unclear. The ubiquitin-editing enzyme A20 (tumour necrosis factor-α-induced protein 3, TNFAIP3) serves as a critical inhibitor on NF-κB signalling. In humans, polymorphisms in the A20 gene or a deregulated expression of A20 are often associated with several inflammatory disorders, including SS. Because A20 controls the ectodysplasin-A1 (EDA-A1)/ectodysplasin receptor (EDAR) signalling negatively, and the deletion of A20 results in excessive EDA1-induced NF-κB signalling, this work investigates the expression levels of EDA-A1 and EDAR in SS human salivary glands epithelial cells (SGEC) and evaluates the hypothesis that SS SGEC-specific deregulation of A20 results in excessive EDA1-induced NF-κB signalling in SS. Our approach, which combines the use of siRNA-mediated gene silencing and quantitative pathway analysis, was used to elucidate the role of the A20 target gene in intracellular EDA-A1/EDAR/NF-κB pathway in SS SGEC, holding significant promise for compound selection in drug discovery.

Sjögren's syndrome (SS) is an autoimmune disease characterized by an inflammatory mononuclear infiltration and the destruction of epithelial cells of the lachrymal and salivary glands. The aetiology is unknown. The expression "autoimmune epithelitis" has been proposed as an alternative to SS, in view of the emerging central role of the epithelial cells in the disease pathogenesis. At the biomolecular level, the epithelial cells play an important role in triggering the autoimmune condition via antigen presentation, apoptosis, and chemokine and cytokines release. Inflammation and angiogenesis are frequently coupled in the pathological conditions associated to autoimmune diseases, and an angiogenic imbalance contributes to the pathogenesis of a number of inflammatory disorders. This work reviews the current knowledge of the molecular and cellular mechanisms underlying the pathogenesis of the inflammatory reactions that characterize SS. The literature and our data on the role of angiogenesis in the pathophysiology of the disease are discussed.

The tumor-necrosis-factor-converting-enzyme (TACE)-TNF-alpha-Amphiregulin (AREG) axis plays an important pathogenic role in inflammatory and autoimmune disorders. However, the pathological roles of these proteins in the chronic autoimmune disease Sjogren's syndrome (SS) remain to be elucidated. It is known that the TACE-AREG axis is clearly part of a larger cascade of signals that starts with the activation of Furin, responsible for maturation of TACE that, in turn, determines the production of active TNF-alpha, directly involved in the up-regulation of AREG expression. This study showed that Furin, TACE, TNF-alpha, and AREG proteins, detected in acinar and ductal cells of human salivary glands from SS patients, increased remarkably in comparison with biopsies of labial salivary glands from healthy controls. The changes in Furin, TACE, TNF- alpha, and AREG proteins' level detected in salivary glands biopsies of SS patients could be responsible for pro-inflammatory cytokines overexpression characterizing Sjogren's syndrome.

The chemokine GRO-α and its receptor CXCR2 are associated with the chronic inflammation in Sjögren's syndrome (SS). To better understand the molecular mechanisms by which the GRO-α/CXCR2 system is involved in the SS inflammatory condition, our studies were designed to clarify the role of ADAM17 activation in the modulation of the GRO-α/CXCR2 chemokine system in epithelial cells (SGEC) from SS salivary glands. The CXCR2 overexpression observed in SS SGEC was dramatically decreased by ADAM17 inhibitor TAPI-1. In addition, comparing the expression levels of ADAM17 in healthy SGEC in presence or not of GRO-α treatment, we observed that GRO-α dose-dependently influences ADAM17 activation, an effect that was inhibited by blocking the interaction of GRO-α with its CXCR2 receptor. Our data show for the first time that ADAM17 has an important role in GRO-α/CXCR2 system activity regulation, suggesting that regulating CXCR2/ADAM17 interaction could be an attractive therapeutic target in SS

Activated microglia secrete an array of pro-inflammatory factors, such as prostaglandins, whose accumulation contributes to neuronal damages. Prostaglandin endoperoxide synthases or cyclooxygenases (COX-1 and COX-2), which play a critical role in the inflammation, are the pharmacological targets of non-steroidal anti-inflammatory drugs, used to treat pain and inflammation. Since it was reported that COX-1 is the major player in mediating the brain inflammatory response, the aim of this study was to evaluate the effects of highly selective COX-1 inhibitors, such as P6 and mofezolac, in neuroinflammation models. Lipopolysaccharide (LPS)-activated mouse BV-2 microglial cells and LPS intracerebroventricular-injected mice as in vitro and in vivo neuroinflammation models, respectively, were used to probe the antiinflammatory efficacy of P6 and mofezolac. Both P6 and mofezolac reduce COX-1 expression in LPS-activated BV-2 cells. This reduction was accompanied with PGE2 release reduction and NF-kB activation downregulation. Coextensively, in the in vivo model, both glial fibrillary acidic protein and ionized calcium-binding adapter molecule-1 expression, two markers of inflammation, were reduced by mofezolac to a rank depending on the encephalon area analyzed. The increase of COX-1 expression observed in all the brain sections of LPS-treated mice was selectively downregulated by the in vivo treatment with mofezolac as well as PGE2 release and Ikβα phosphorylation amount assayed in the brain areas tested. These results indicate the capability of P6 and mofezolac to modulate the NF-kB signaling pathway, emphasizing the neuroprotective effect and therapeutic potential of COX-1 inhibitors in the control of neuroinflammatory diseases.

The development of agents that can modulate microglial activation has been suggested as one potential strategy for the treatment or prevention of neurodegenerative diseases. Among these agents, resveratrol, with its anti-inflammatory action, has been described to have neuroprotective effects. In this paper we demonstrate that in LPS-stimulated microglia resveratrol pretreatment reduced, in a dose-dependent manner, pro-inflammatory cytokines IL-1β, TNF-α and IL-6 mRNA expression and increased the release of anti-inflammatory interleukin (IL)-10. Moreover, resveratrol pretreatment up-regulated the phosphorylated forms of JAK1 and STAT3, as well as suppressor of cytokine signaling (SOCS)3 protein expression in LPS activated cells, demonstrating that the JAK-STAT signaling pathway is involved in the anti-inflammatory effect exerted by resveratrol. By supplementing the cultures with an IL-10 neutralizing antibody (IL-10NA) we obtained the opposite effect. Taken together, these data allow us to conclude that the LPS-induced pro-inflammatory response in microglial cells can be markedly reduced by resveratrol, through IL-10 dependent up-regulation of SOCS3, requiring the JAK-STAT signaling pathway.

AIMS/HYPOTHESIS: Beta cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (decline of glucose-stimulated insulin secretion, downregulation of specific gene expression). Apoptosis and dysfunction are caused, at least in part, by lipoglucotoxicity. The mechanisms implicated are oxidative stress, increase in the hexosamine biosynthetic pathway (HBP) flux and endoplasmic reticulum (ER) stress. Oxidative stress plays a role in glucotoxicity-induced beta cell dedifferentiation, while glucotoxicity-induced ER stress has been mostly linked to beta cell apoptosis. We sought to clarify whether ER stress caused by increased HBP flux participates in a dedifferentiating response of beta cells, in the absence of relevant apoptosis. METHODS: We used INS-1E cells and murine islets. We analysed the unfolded protein response and the expression profile of beta cells by real-time RT-PCR and western blot. The signal transmission pathway elicited by ER stress was investigated by real-time RT-PCR and immunofluorescence. RESULTS: Glucosamine and high glucose induced ER stress, but did not decrease cell viability in INS-1E cells. ER stress caused dedifferentiation of beta cells, as shown by downregulation of beta cell markers and of the transcription factor, pancreatic and duodenal homeobox 1. Glucose-stimulated insulin secretion was inhibited. These effects were prevented by the chemical chaperone, 4-phenyl butyric acid. The extracellular signal-regulated kinase (ERK) signal transmission pathway was implicated, since its inhibition prevented the effects induced by glucosamine and high glucose. CONCLUSIONS/INTERPRETATION: Glucotoxic ER stress dedifferentiates beta cells, in the absence of apoptosis, through a transcriptional response. These effects are mediated by the activation of ERK1/2.

Parkinson's disease (PD) is a common neurodegenerative disease characterised by a slow and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Despite intensive research, the cause of neuronal loss in PD is poorly understood. Inflammatory mechanisms have been implicated in the pathophysiology of PD. In this study, conducted on an experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, we investigated the expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6 and their receptors (IL-1RI, TNF-αRI, IL-6Rα) at the SN and caudate-putamen (CP) levels. In MPTP-treated animals we observed a significant increase in IL-1β, TNF-α and IL-6 mRNA expression levels both in the SN and CP in comparison with untreated mice. In addition, both mRNA and protein levels of IL-1RI, TNF-αRI and IL-6Rα were significantly enhanced in the SN of MPTP-treated mice in comparison to controls, whereas no significant differences were observed in the CP between treated and untreated mice. Overall, these results indicate a role of both pro-inflammatory cytokines and their receptors in the pathogenesis of PD.

Angiogenesis is a common finding in chronic inflammatory diseases; however, its role in Sj€ogren’s syndrome (SS) remains to be elucidated. Previous SS studies have demonstrated an increase in VEGF-A/VEGFR-2 system expression in minor salivary gland (MSG) biopsies from patients with SS, but differences in the new blood vessel formation between the different grades of disease severity have not been reported. Therefore, experiments were performed to demonstrate angiogenesis during different phases of primary SS (pSS) and to define the relationship between the microvessel density (MVD), macrophage infiltration and histiocyte distribution in SS MSG inflammatory lesions. In this series of experiments, immunohistochemistry was used to examine angiogenesis in serial sections of pSS MSG. Patients with pSS were classified accordingly with the grade of inflammatory lesions as I = low-grade (low focus score of 1 or 2), II = intermediate-grade (focus score of 3–6) and III = extensive inflammation in the MSG (high focus score of 12). Histological examination demonstrated that the MVD increased with the severity of the inflammatory lesions, and in addition, we found an increased infiltration of inflammatory and pro-angiogenic cells.These findings reveal that angiogenesis is intimately involved in the progression of pSS, may be central to the propagation of the chronic immune response observed in pSS and could represent a novel potential biomarker of pSS disease activity.

Neuropilin-1 (NRP1) is a transmembrane co-receptor for members of the vascular endothelial growth factor family. Recent studies revealed an important role of NRP1 in angiogenesis and progression of many diseases. The role of NRP1 in the development of Sjögren's syndrome (SS), one of the most common rheumatic diseases, has not yet been investigated. Molecular studies and protein expression techniques were performed to elucidate the gene and protein expression profile of NRP1 in human salivary gland epithelial cells (SGEC) from primary SS. We used human microarrays and transient transfection with a mutant form of the negative inhibitory κBα proteins (IκBαDN) to investigate whether selective inhibition of nuclear Factor-κB (NF-κB) improves NRP1-mediated pro-angiogenic factors release from SS SGEC. The selective NRP1 function inhibition with an antibody to human NRP1, was employed to evaluate the therapeutic potential of targeting NRP1. We demonstrate that NRP1 is expressed in SGEC of both human healthy biopsies and in SS samples, and increased NRP1 expression in SS SGEC is significantly associated with pro-angiogenic factors release. Neutralizing anti-NRP1 antibody decreased pro-angiogenic factor production from SS SGEC and blocking NF-κB activation could be a way to inhibit NRP1-mediated angiogenesis in Sjögren's syndrome.

In the present study we used a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease (PD) mouse model to analyze resveratrol neuroprotective effects. The MPTP-induced PD model is characterized by chronic inflammation, oxidative stress and loss of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). We observed that resveratrol treatment significantly reduced glial activation, decreasing the levels of IL-1b, IL-6 and TNF-a, as well as their respective receptors in the SNpc of MPTP-treated mice, as demonstrated by Western blotting, RT-PCR and quantitative PCR analysis. This reduction is related to possible neuroprotection as we also observed that resveratrol administration limited the decline of tyrosine hydroxylase-immunoreactivity induced in the striatum and SNpc by MPTP injection. Consistent with these data, resveratrol treatment up-regulated the expression of the suppressor of cytokine signaling-1 (SOCS-1), supporting the hypothesis that resveratrol protects DA neurons of the SNpc against MPTP-induced cell loss by regulating inflammatory reactions, possibly through SOCS-1 induction

Prolonged inflammation can be detrimental because it may cause host toxicity and tissue damage. Indeed, excessive production of inflammatory cytokines is often associated with many autoimmune diseases. In this study we demonstrate that the anti-Ro/SSA autoantibodies (Abs) stimulate the production of pro-inflammatory cytokines IL-6 and IL-8 by human healthy salivary gland epithelial cells (healthy SGEC). The secretion of these cytokines is due to amphiregulin (AREG) that is overexpressed in healthy SGEC treated with anti-Ro/SSA Abs and in Sjögren's syndrome. We have discovered that the up-regulation of AREG occurs through TNF-alpha produced following anti-Ro/SSA Abs treatment. The gene silencing technique was used to study the AREG-TNF-alpha-IL-6/IL-8 secretion pathway, demonstrating that: (i) TNF-alpha gene silencing provokes a significant decrease of proinflammatory cytokines production and AREG expression in anti-Ro/SSA Abs-treated healthy SGEC; (ii) AREG gene silencing has a potent inhibitory effect on TNF-alpha-induced IL-6 and IL-8 secretion in healthy SGEC treated with anti-Ro/SSA Abs. These findings indicate that TACE-mediated AREG shedding plays a critical role in TNF-alpha-induced IL-6 and IL-8 secretion by the human healthy salivary gland epithelial cells, suggesting that this may be one of the possible intracellular mechanisms involved in the salivary glands inflammatory response in Sjögren's syndrome.

Tribulus terrestris L. (Zygophyllaceae) is an annual plant commonly known as Puncture vine. It is dramatically gaining interest as a rich source of saponins. T. terrestris is a promising ingredient for many industries and recent patents on dermatological applications support the use of this plant for cosmetics and hygiene. Nonetheless problems arise in the selection of the material to be used. The extracts of different origins may differ substantially. Natural speciation processes normally influence ‘variations’ in wild-crafted medicinal plants. The genus Tribulus is emblematic. Taxonomic status of T. terrestris is complicated by the wide geographical distribution leading to high levels of genetic polymorphism. Being aware of such variability we selected 3 commercial Tribulus extracts and compared their biological effect on Candida albicans with the effect produced by an extract from local plants (South of Apulia, Italy). One of the commercial extracts with the best anti-candida performance was used to substitute triclosan in a detergent formulation and it proved to improve the product performance in the control of potentially pathogenic skin flora such as C. albicans.

In this study, the effects of Radio Electric Asymmetric Conveyer (REAC), a non-invasive physical treatment, on neuroinflammatory responses in a mouse model of parkinsonism induced by intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), were investigated in vivo. We found that the REAC tissue optimization treatment specific for neuro-regenerative purposes (REAC TO-RGN-N) attenuated the inflammatory picture evoked by MPTP-induced nigro-striatal damage in mice, decreasing the levels of pro-inflammatory molecules and increasing anti-inflammatory mediators. Besides, there was a significant reduction of both astrocyte and microglial activation in MPTP-treated mice exposed to REAC TO-RGN-N. These results indicated that REAC TO-RGN-N treatment modulates the pro-inflammatory responses and reduces neuronal damage in MPTP-induced parkinsonism.

Apoptosis of the acinar and ductal epithelial cells of the salivary glands has been proposed as a mechanism possibly responsible for the impairment of the secretory function in Sjögren's syndrome, an organ-specific autoimmune disorder characterized by destruction of these glandular structures. The presence of serum autoantibodies (Abs) directed against the ribonucleoproteic antigens Ro and La is one of the classification criteria used to identify Sjögren patients, and there is increasing evidence of the direct involvement of Abs in tissue pathogenesis. Our recent report demonstrated that anti-Ro and anti-La Abs are able to trigger the extrinsic pathway of apoptosis in the human salivary gland cells. To better understand how the anti-Ro and anti-La Abs exert their apoptotic effect, human caspase-8 gene expression was examined in primary human salivary gland epithelial cell (SGEC) cultures established from biopsies of labial minor salivary glands. To measure mRNA expression changes of initiating caspase-8, the real-time polymerase chain reaction was employed. This was combined with western blot to study the activation of caspase-8 detecting the cleaved form of caspase-8 and the cleaved poly (ADP-ribose) polymerase, downstream consequences of caspases activation. Data obtained suggest that the anti-Ro and anti-La Abs determine a transcriptional up-regulation and activation of caspase-8. Study of the mRNA in SGEC experimental model may provide insight into the signal transduction pathway stimulated by anti-nuclear autoantibodies.

Primary Sjögren's syndrome (pSS) is an autoimmune disorder characterized by an epithelium injury surrounded by dense lymphocytic infiltrates. The conditions for the long-term maintenance of human salivary gland epithelial cells (SGEC) from pSS patients, and a coculture system with pSS lymphocytes were used to assess the effect of Rituximab (RTX) on the inflammatory condition and progression in pSS. Quantitative Real-Time PCR, genes and proteins Array analysis, Western blot, flow cytometry, siRNAs transfection and NF-kB DNA binding assays were used as methods. Supporting the RTX's benefits, this study demonstrates that RTX decreases NF-κB activity and interrupts NF-κB signalling pathway through the upregulation of the Raf-1 kinase inhibitor protein (RKIP). RKIP overexpression down-regulates interleukins, their receptors and the expression of genes encodes proteins that attracted lymphocytes. RKIP gene silencing leads to significantly increased expression and/or release of pro-inflammatory mediators supporting that RKIP expression could be involved in the suppression of NF-κB activation in pSS SGEC

Diagnosis and therapeutic strategies in Sjögren's syndrome (SS) might greatly benefit of the present multidisciplinary approach to studying the molecular pathogenesis of the disease. A deregulated inflammatory response has been described in the SS. The research in the last years sheds light on the importance of the NF-κB pathway regulating the pro-inflammatory cytokine production and leukocyte recruitment. These are important contributors to the inflammatory response during the development of SS. In this study we examine the expression of the NF-κB inhibitory protein termed IκBα in salivary glands epithelial cells (SGEC) comparing it with SGEC from healthy controls, to test the hypothesis that an altered expression of IκBα occurs in SGEC from SS biopsies. Real-Time PCR, western blot and immunohistochemistry demonstrated that the expression level of IκBα was significantly lower in SS with respect to healthy controls leading to an increased NF-κB activity. Our results suggest that the analysis of IκBα expression at salivary gland epithelial cell level could be a potential new hallmark of SS progression and sustain a rationale to more deeply investigate the therapeutic potential of specific NF-κB inhibitors in SS

Chronic exposure to solar UVB radiation damages skin, increasing the risk to develop cancer. Hence the identification of compounds with a photoprotective efficacy is essential. This study examined the role of saponins derived from Tribulus terrestris L. (TT) on the modulation of apoptosis in normal human keratinocytes (NHEK) exposed to physiological doses of UVB and to evaluate their antitumoral properties. In NHEK, TT saponins attenuate UVB-induced programmed cell death through inhibition of intrinsic apoptotic pathway. In squamous cell carcinomas (SCC) TT saponins do not make the malignant keratinocytes more resistant to UVB and determine an enhanced apoptotic response. The photoprotective effect of TT saponins is tightly correlated to the enhancement of NER genes expression and the block of UVB-mediated NF-κB activation. Collectively, our study shows experimental evidence that TT has a preventive efficacy against UVB-induced carcinogenesis and the molecular knowledge on the mechanisms through which TT saponins regulate cell death suggests great potential for TT to be developed into a new medicine for cancer patients.

Gastrointestinal damage (GD) is commonly associated with the inhibition of cyclooxygenase (COX)-1, one of the two known COXs, by traditional non-steroidal anti-inflammatory drugs. More recent evidences have proven that GD is caused by the simultaneous inhibition of the two COXs. This study was designed to evaluate the effect of the selective COX-1 inhibition on gastric integrity.

We explore the association of the inflammatory gene expression profile observed in the chronic inflammatory autoimmune disorder Sjögren's syndrome (SS) with changes in TNF-α converting enzyme (TACE), tumor necrosis factor (TNF)-α and nuclear factor (NF)-κB levels showing that pathways that include TNF-α signaling converge on NF-κB contributing to exacerbate the diseases. The treatment of human salivary gland epithelial cells (SGECs) with SS anti-Ro/SSA autoantibodies (Abs) result in a progressive increase in NF-κB-DNA binding, that includes a marked enhancement in NF-κB subunit p65 protein-DNA binding. A human cytokine multi-analyte array demonstrated that the NF-κB proinflammatory target genes, increased by anti-Ro/SSA Abs treatment, includes CXC chemokines (CXCL1, CXCL6 and CXCL9), CC chemokines (CCL2, CCL13 and CCL20), interleukins (IL-1α, IL-1β, IL-1F8, IL-6, IL-8, IL-9, IL-13, IL-17 and IL-22) and their receptors (IL-1RN, IL-10Rα, IL-13Rα, CCR1, CCR2, CCR3, CCR4 and CXCR1). Blockade of TACE through the use of the specific inhibitor TAPI-1 regulates proinflammatory cytokines production in SGEC treated with anti-Ro/SSA Abs inhibiting NF-κB nuclear translocation and activation. To further investigate the role of NF-κB on anti-Ro/SSA Abs-determined proinflammatory gene expression, we used the inhibitory protein IκB-α dominant negative super-repressor as inhibitor of NF-κB-DNA binding, demonstrating that transfection with dominant-negative IκB-α in anti-Ro/SSA-treated SGEC determined a marked reduction of proinflammatory cytokines gene expression. Although further studies are needed to clarify the mechanisms underlying SS, our results demonstrate that SS Abs exert their pathogenic effects via triggering the TACE/TNF-α/NF-κB axis

We explore the involvement of tumor necrosis factor α (TNF-α)-converting enzyme (TACE) in vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR2) (VEGF-A/VEGFR2)-mediated angiogenesis in Sjögren’s syndrome (SS), one of the most common autoimmune rheumatic diseases. To test the hypothesis that SS autoantibodies (Abs) regulate VEGF-A/VEGFR2 expression by a TACE-dependent nuclear factor-κB (NF-κB) activation pathway, their effects on the expression and activation of the VEGF-A/TACE/VEGFR2/NF-κB pathway were determined in human salivary gland epithelial cells (SGEC). An enhanced angiogenesis in SS salivary gland biopsies was observed, associated with an increased VEGF-A expression and activation of VEGF-A/VEGFR2 signaling. Human cytokine array analysis of the pro-inflammatory and pro-angiogenic protein response in SGEC treated with SS Abs revealed an overexpression of multiple pro-angiogenic factors. TACE RNA knockdown, the use of anti-VEGF-A monoclonal antibody and the inhibition of NF-κB activity significantly abrogated the release of pro-angiogenic factors, demonstrating that VEGF-A/TACE/VEGFR2/NF-κB axis dysfunction may be contributory to pathogenesis and exacerbation of this autoimmune condition.

Intrinsic and extrinsic apoptosis are both characterised by the presence of cytochrome c (cyto-c) in the cytosol. We present data on the extra-mitochondrial NADH oxidation catalysed by exogenous (cytosolic) cyto-c, as a possible answer to the paradox of apoptosis being an energy-dependent program but characterized by the impairment of the respiratory chain. The reduction of molecular oxygen induced by the cytosolic NADH/cyto-c pathway is coupled to the generation of an electrochemical proton gradient available for ATP synthesis. Original findings show that SH reagents inhibit the NADH/cyto-c system with a conformational change mechanism. The mitochondrial integrity-test of sulfite oxidase unequivocally demonstrates that this enzyme (120kDa) can be released outside but exogenous cyto-c (12.5kDa) does not permeate into mitochondria. Valinomycin at 2nM stimulates both the energy-dependent reversible mitochondrial swelling and the NADH/cyto-c oxidation pathway. The pro-apoptotic activity of valinomycin, as well as to the dissipation of membrane potential, can be also ascribed to the increased activity of the NADH/cyto-c oxidation pathway useful as an additional source of energy for apoptosis. It can be speculated that the activation of the NADH/cyto-c system coupled to valinomycin-induced mitochondrial osmotic swelling may represent a strategy to activate apoptosis in confined solid tumours.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder that particularly compromises the function of exocrine glands. The pathogenetic mechanisms of this autoimmune exocrinopathy have not been fully elucidated. Since increasing evidence actually suggests that the epidermal growth factor receptor (EGFR) pathway has a major impact on the inflammatory/immune reactions of the epithelial cells, in the apparent effort of enhancing innate immune defense while opposing overactivation of pro-inflammatory functions, the focus of the work presented here is clarify whether the EGFR-extracellular-signal-regulated kinase (ERK) pathway plays a role in the pro-inflammatory responses mounted by pSS salivary gland epithelial cells (SGEC). Investigations revealed that the EGFR-mediated activation of the downstream effectors ERK1/2 in pSS SGEC appeared to require ADAM17-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing Ab significantly reduced EGFR transactivation and ERK1/2 phosphorylation. In addition, pSS SGEC treated with the specific ADAM17 inhibitor TAPI-1 and with the EGFR inhibitor AG1478 exhibited deactivated AREG/EGFR/ERK signaling pathway and reduced pro-inflammatory cytokines released.

Novel biologic therapies targeted against specific components of the immune system, including blockade of TNF-α have revolutionized therapeutic approaches to inflammatory conditions and systemic inhibitors of TNF-α have been approved for the treatment of a wide variety of autoimmune diseases. No studies aimed to elucidate the effects of anti-TNF-α blockers on tumour necrosis factor-α convertase (TACE) expression and activation have yet been published. TACE is the principal protease involved in the activation of pro-TNF-α and is a target for anti-TNF-α therapy. Here we focused on regulation of TACE expression in human salivary gland epithelial cells (SGEC) treated by anti-Ro/SSA autoantibodies (autoAbs), characterizing primary Sjögren's syndrome and on the effect of anti-Ro/SSA autoAbs on TACE pro-domain shedding and activation. To test the hypothesis that anti-TNF-α blocker drugs affect TACE expression, we used Adalimumab and Etanercept to block TNF-α and evaluate the effects of these biological agents on post-translational regulation of TACE. Anti-Ro/SSA autoAbs determines TACE pro-domain shedding suggesting that TACE activity is necessary for the release of TNF-α observed in anti-Ro/SSA autoAbs-stimulated cells. The comparative efficacy analysis of the regulation of TACE activity by Adalimumab and Etanercept revealed that Adalimumab appear to be significantly more efficacious than Etanercept in preventing TACE activation caused by anti-Ro/SSA autoAbs. It is intriguing to consider that regulation of TACE may participate in the pathogenic role of autoantibodies and the modulation of TACE expression by TNF-α antagonists might contribute to the beneficial effect of these drugs in inflammatory and autoimmune diseases.

Background: Secretory proteins acquire their native three-dimensional conformation through repeated brief interactions with ER chaperones and oxidoreductases. Results: We have captured and defined previously-unidentified disulfide adducts of newly-synthesized thyroglobulin with ERp72 and CaBP1/P5. Conclusion: Multiple oxidoreductases simultaneously engage thyroglobulin during its early folding in the ER. Significance: Distinct chaperone/oxidoreductase partners coordinately engage this multi-domain secretory protein to promote its advancement to the native state.

Wound healing is the process by which a complex cascade of biochemical events is responsible of the repair the damage. In vivo, studies in humans and mice suggest that healing and post-healing heterogeneous behavior of the surgically wounded myometrium is both phenotype and genotype dependent. Uterine wound healing process involves many cells: endothelial cells, neutrophils, monocytes/macrophages, lymphocytes, fibroblasts, myometrial cells as well a stem cell population found in the myometrium, myoSP (side population of myometrial cells). Transforming growth factor beta (TGF-β) isoforms, connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha (TNF-β) are involved in the wound healing mechanisms. The increased TGF- β1/β3 ratio reduces scarring and fibrosis. The CTGF altered expression may be a factor involved in the abnormal scars formation of low uterine segment after cesarean section and of the formation of uterine dehiscence. The lack of bFGF is involved in the reduction of collagen deposition in the wound site and thicker scabs. The altered expression of TNF-β, VEGF, and PDGF in human myometrial smooth muscle cells in case of uterine dehiscence, it is implicated in the uterine healing process. The over-and under-expressions of growth factors genes involved in uterine scarring process could represent patient's specific features, increasing the risk of cesarean scar complications.

In valinomycin induced stimulation of mitochondrial energy dependent reversible swelling, supported by succinate oxidation, cytochrome c (cyto-c) and sulfite oxidase (Sox) [both present in the mitochondrial intermembrane space (MIS)] are released outside. This effect can be observed at a valinomycin concentration as low as 1 nM. The rate of cytosolic NADH/cyto-c electron transport pathway is also greatly stimulated. The test on the permeability of mitochondrial outer membrane to exogenous cyto-c rules out the possibility that the increased rate of exogenous NADH oxidation could be ascribed either to extensively damaged or broken mitochondria. Accumulation of potassium inside the mitochondria, mediated by the highly specific ionophore valinomycin, promotes an increase in the volume of matrix (evidenced by swelling) and the interaction points between the two mitochondrial membranes are expected to increase. The data reported and those previously published are consistent with the view that ‘‘respiratory contact sites’’ are involved in the transfer of reducing equivalents from cytosol to inside the mitochondria both in the absence and the presence of valinomycin. Magnesium ions prevent at least in part the valinomycin effects. Rather than to the dissipation of membrane potential, the pro-apoptotic property of valinomycin can be ascribed to both the release of cyto-c from mitochondria to cytosol and the increased rate of cytosolic NADH coupled with an increased availability of energy in the form of glycolytic ATP, useful for the correct execution of apoptotic program.

Microglia-mediated neuroinflammation has been described as a common hallmark of Parkinson's disease (PD) and is believed to further exacerbate the progressive degeneration of dopaminergic neurons. Current therapies are unable to prevent the disease progression. A significant association has been demonstrated between PD and low levels of vitamin D in patients serum, and vitamin D supplement appears to have a beneficial clinical effect. Herein, we investigated whether vitamin D administered orally in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced preclinical animal model of PD protects against glia-mediated inflammation and nigrostriatal neurodegeneration. Vitamin D significantly attenuated the MPTP-induced loss of tyrosine hydrlase (TH)-positive neuronal cells, microglial cell activation (Iba1-immunoreactive), inducible nitric oxide synthase (iNOS) and TLR-4 expression, typical hallmarks of the pro-inflammatory (M1) activation of microglia. Additionally, Vitamin D was able to decrease pro-inflammatory cytokines mRNA expression in distinct brain areas of the MPTP mouse. Importantly, we also assessed the anti-inflammatory property of vitamin D in the MPTP mouse, in which it upregulated the anti-inflammatory cytokines (IL-10, IL-4 and TGF-β) mRNA expression as well as increasing the expression of CD163, CD206 and CD204, typical hallmarks of alternative activation of microglia for anti-inflammatory signalling (M2). Collectively, these results demonstrate that vitamin D exhibits substantial neuroprotective effects in this PD animal model, by attenuating pro-inflammatory and up-regulating anti-inflammatory processes.