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Sirtuins are conserved NAD+ -dependent deacylases. SIRT1 is a nuclear and cytoplasmic sirtuin involved in the control of histones a transcription factors function. SIRT3 is a mitochondrial protein, which regulates mitochondrial function. Although, both SIRT1 and SIRT3 have been implicated in resistance to cellular stress, the link between these two sirtuins has not been studied so far. Here we aimed to unravel: i) the role of SIRT1-SIRT3 axis for cellular response to oxidative stress and DNA damage; ii) how mammalian cells modulate such SIRT1-SIRT3 axis and which mechanisms are involved. Therefore, we analyzed the response to different stress stimuli in WT or SIRT1-silenced cell lines. Our results demonstrate that SIRT1-silenced cells are more resistant to H2 O2 and etoposide treatment showing decreased ROS accumulation, γ-H2AX phosphorylation, caspase-3 activation and PARP cleavage. Interestingly, we observed that SIRT1-silenced cells show an increased SIRT3 expression. To explore such a connection, we carried out luciferase assays on SIRT3 promoter demonstrating that SIRT1-silencing increases SIRT3 promoter activity and that such an effect depends on the presence of SP1 and ZF5 recognition sequences on SIRT3 promoter. Afterwards, we performed co-immunoprecipitation assays demonstrating that SIRT1 binds and deacetylates the transcription inhibitor ZF5 and that there is a decreased interaction between SP1 and ZF5 in SIRT1-silenced cells. Therefore, we speculate that acetylated ZF5 cannot bind and sequester SP1 that is free, then, to increase SIRT3 transcription. In conclusion, we demonstrate that cells with low SIRT1 levels can maintain their resistance and survival by increasing SIRT3 expression. J. Cell. Physiol. 232: 1835-1844, 2017. © 2016 Wiley Periodicals, Inc.

Pregnancy problems are common in patients with rheumatic disease; indeed, autoimmune disorders and autoantibodies can affect pregnancy progress and lead to maternal complications. Recent studies have highlighted a close association between HMGB1, chronic inflammation, and autoimmune diseases. Thus, in this investigation, we analyzed serum levels of HMGB1, an alarmin which plays a pivotal role in inducing and enhancing immune cell function. Sera from 30 patients with antiphospholipid syndrome (11 primary and 19 secondary APS), 35 subjects with pregnancy morbidity, and 30 healthy women were analysed for HMGB1 and its putative receptor RAGE (sRAGE) by Western blot and for TNF-α by ELISA. Results revealed that APS patients showed significantly increased serum levels of HMGB1, sRAGE, and the proinflammatory cytokine TNF-α, as compared to healthy women. However, also, the pregnancy morbidity subjects showed significantly increased levels of HMGB1 and sRAGE as well as TNF-α compared to healthy women. Our findings suggest that in subjects with pregnancy morbidity, including obstetric APS, elevated levels of HMGB1/sRAGE may represent an alarm signal, indicating an increase of proinflammatory triggers. Further studies are needed to evaluate the role of HMGB1/sRAGE as a possible tool to evaluate the risk stratification of adverse pregnancy outcomes.

Objectives Autophagy may represent a functional processing event that creates a substrate for autoreactivity. In particular, autophagy may play a role in the pathogenesis of RA, since autophagy is a key cellular event involved in the generation of citrullinated peptides, with consequent breakage of tolerance. Thus, in RA, autophagy may be the common feature in several situations (including smoking, joint injury and infection) that may drive the adaptive responses to citrullinated self-proteins. The aim of this study was the analysis, in vitro, of the role of autophagy in the generation of citrullinated peptides and, in vivo, of the relationship between autophagy and the production of anti-CCP antibodies (Abs). Methods For autophagy induction, fibroblast-like synoviocytes, primary fibroblasts and monocytes were stimulated with tunicamycin or rapamycin. Peptidyl arginine deiminase activity was tested by enzyme-linked immunosorbent assay, and protein citrullination was evaluated by western blotting. The main citrullinated RA candidate antigens, vimentin, α-enolase and filaggrin, were demonstrated by immunoprecipitation. The relationship between autophagy and anti-CCP Abs was analysed in 30 early-active RA patients. Results Our results demonstrated in vitro a role for autophagy in the citrullination process. Cells treated with tunicamycin or rapamycin showed peptidyl arginine deiminase 4 activation, with consequent protein citrullination. Immunoblotting and immunoprecipitation experiments, using specific Abs, identified the main citrullinated proteins: vimentin, α-enolase and filaggrin. In vivo, a significant association between levels of autophagy and anti-CCP Abs was observed in treatment-naive early-active RA patients. Conclusion These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA.

Background Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterised by the presence of at least one clinical event among vascular thrombosis and/or pregnancy morbidity, in the presence of circulating antiphospholipid antibodies (aPL). High-mobility group box-1 (HMGB1) is a non-histonic protein belonging to the family of alarmins. It is associated with chromatin and has a dual function depending on the cell state: in basal conditions it is located in the nucleus and promotes the interaction of some transcription factors with DNA, in inflammatory conditions it is secreted in the extracellular space and exerts the functions of a pro-inflammatory cytokine. One of the main receptor system responsible for the HMGB1 activity is the ”receptor for advanced glycation end products” (RAGE). Increased serum HMGB1 levels have been reported in patients with Systemic Lupus Erythematosus and pre-eclampsia, as other alarmins are increased in patients with early abortions. Objectives To evaluate the serum levels of HMGB1 and soluble RAGE (sRAGE) in patients with obstetric and thrombotic APS. Methods 43 consecutive patients with APS, diagnosed according to the Sapporo criteria, were enrolled. The study cohort included both primary APS (=15) and APS associated with SLE (=28). In addition, 30 healthy subjects (HC) matched for age and sex were studied as controls. Serum levels of HMGB1 and sRAGE were analysed by Western blot. Results The clinical features of the enrolled patients (40 females and 3 males, mean age 40.98±13.48 years,) are reported in table 1. HMGB1 and sRAGE serum levels were significantly increased in APS patients in comparison with controls (p Conclusions In this study, we investigated for the first time the serum levels of HMGB1 and sRAGE in patients with APS, showing increased levels in both primary and secondary APS compared to controls. Larger studies are needed to assess whether monitoring serum HMGB1/sRAGE levels could be a useful tool for risk stratification in patients with APS. Disclosure of Interest None declared

Sirtuins are conserved NAD

Aim: Type 2 diabetes increases the risk for cardiovascular disease, and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) reduce cardiovascular events in these patients. The benefits of statin therapy cannot be explained only by the lipid-lowering effect. The aim of this study was to test the effect of atorvastatin therapy on CD36 scavenger receptor expression, nuclear factor-kappaB (NFκB) levels and markers of inflammation (C-reactive protein, CRP, Tumor Necrosis Factor-α, TNF-α) in circulating monocytes from diabetic patients.Methods: Twenty-two type 2 diabetic patients were treated for 8 weeks with atorvastatin (20 mg/day). At baseline and after treatment a blood sample was collected for measurement of glucose, lipid profile (total cholesterol, HDL, LDL cholesterol, triglycerides), glycated hemoglobin (HbA1c), CRP and for isolation of monocytes.Results: Atorvastatin decreased total (p

The molecular mechanism by which the lipido-sterolic extract of Serenoa repens (LSESr, Permixon®) affects prostate cells remains to be fully elucidated. In androgen-independent PC3 prostate cancer cells, the LSESr-induced effects on proliferation and apoptosis were evaluated by counting cells and using a FACScan cytofluorimeter. PC3 cells were stained with JC-1 dye to detect mitochondrial membrane potential. Cell membrane lipid composition was evaluated by thin layer chromatography and gas chromatographic analysis. Akt phosphorylation was analyzed by Western blotting and cellular ultrastructure through electron microscopy. LSESr (12.5 and 25 µg/ml) administration exerted a biphasic action by both inhibiting proliferation and stimulating apoptosis. After 1 h, it caused a marked reduction in the mitochondrial potential, decreased cholesterol content and modified phospholipid composition. A decrease in phosphatidylinositol-4,5-bisphosphate (PIP2) level was coupled with reduced Akt phosphorylation. After 24 h, all of these effects were restored to pre-treatment conditions; however, the saturated (SFA)/unsaturated fatty acid (UFA) ratio increased, mainly due to a significant decrease in ω6 content. The reduction in cholesterol content could be responsible for both membrane raft disruption and redistribution of signaling complexes, allowing for a decrease of PIP2 levels, reduction of Akt phosphorylation and apoptosis induction. The decrease in ω6 content appears to be responsible for the prolonged and more consistent increase in the apoptosis rate and inhibition of proliferation observed after 2–3 days of LSESr treatment. In conclusion, LSESr administration results in complex changes in cell membrane organization and fluidity of prostate cancer cells that have progressed to hormone-independent status. J. Cell. Physiol. 219: 69–76, 2009. © 2008 Wiley-Liss, Inc.

We induced apoptosis in primary cultures of cerebellar granule neurons by switching the growing medium into a medium containing lower concentrations of K(+) (5 or 10 mM instead of 25 mM) or, alternatively, by addition of staurosporine. The apoptotic phenotype was always preceded by an early increase in the intracellular levels of the disialoganglioside GD3, which peaked at 2-6 h and returned back to normal at 12 h. GD3 synthase, the enzyme that forms GD3 from the monosialoganglioside GM3, was also induced at early times after the induction of apoptosis in granule cells. Immunofluorescent staining showed that GD3 increased in neuronal cell bodies and neurites, but was never localized in cell nuclei. In cultures switched into a low K(+)-containing medium, exogenously applied GD3, but not the disialoganglioside GD1a, accelerated the development of neuronal apoptosis. In contrast, the antisense-induced knock-down of GD3 synthase was protective against granule cell death induced by lowering extracellular K(+) from 25 to 10 - but not 5 - mM. These results demonstrate that an early and transient increase in GD3 synthesis is one of the factors that contribute to the induction of neuronal apoptosis in culture.

Background Autophagy is a fundamental self-degradative process in eukaryotic cells via the lysosomal pathway, involved both in basal turnover of cellular components and in the response to nutrient starvation (1). The immune system utilizes autophagy to restrict intracellular pathogens and regulate adaptive immunity, thus autophagy, as well as apoptosis, could be relevant in induction or loss of self tolerance to intracellular molecules and might act as a source of autoantigens in autoimmune diseases (2). Objectives Aim of our study was to study the monocyte autophagy in patients with active early RA naive to steroids and DMARDs before and after 3 months of therapy. Methods 17 early RA patients (mean age 49 yrs, mean disease duration 21 weeks), fulfilling both 1987 and 2010 ACR criteria, naive to treatment, were recruited after written informed consent. Before (T0) and after 3 months of treatment (T1), DAS28 was calculated, CRP was assessed. Autophagy markers (LC3, Ambra, beclin) were evaluated by flow cytometry on isolated monocytes. As a control group, 10 healthy donors were also evaluated. Results As expected, CRP, GH and DAS 28 score were significantly reduced following treatment with steroids and DMARDs (Table 1). Autophagy markers (LC3, Ambra, beclin) showed a similar behaviour, reducing their expression in monocytes after treatment (Tab.1). Interestingly, the expression of autophagy markers was significantly related to DAS28 (p=0.04 for LC3, p=0.03 for Ambra), to GH (p=0.01 for Ambra), and to CRP (p=0.01 for Ambra, p=0.006 for beclin). Conclusions These data indicate an increased expression of autophagy markers in isolated monocytes from patients with active early RA, suggesting a possible role for autophagy in the pathogenesis of this disease. The relationship between autophagy and acute inflammation is supported by the significant reduction of autophagy markers after therapy, together with their relationship with DAS28, GH, and CRP. Our results may contribute to clarify whether endogenous or pharmacological regulation of autophagy might modify the autoimmune response and the progression of the disease. References Lleo A et Al,J.Autoimmun 2007;29:61-68. Deretic V,Curr Opin Immunol 2009;21:53-62. Disclosure of Interest None Declared

Introduction High mobility group box 1 (HMGB1) is a key mediator of inflammation that is actively secreted by macrophages and/or passively released from damaged cells. The proinflammatory role of HMGB1 has been demonstrated in both animal models and humans, since the severity of inflammatory response is strictly related to serum HMGB1 levels in patients suffering from traumatic insult, including operative trauma. This study was undertaken to investigate HMGB1 production kinetics in patients undergoing major elective surgery and to address how circulating mononuclear cells are implicated in this setting. Moreover, we explored the possible relationship between HMGB1 and the proinflammatory cytokine interleukin-6 (IL-6). Methods Forty-seven subjects, American Society of Anesthesiologists physical status I and II, scheduled for major abdominal procedures, were enrolled. After intravenous medication with midazolam (0.025 mg/Kg), all patients received a standard general anesthesia protocol, by thiopentone sodium (5 mg/Kg) and fentanyl (1.4 μg/Kg), plus injected Vecuronium (0.08 mg/Kg). Venous peripheral blood was drawn from patients at three different times, t0: before surgery, t1: immediately after surgical procedure; t2: at 24 hours following intervention. Monocytes were purified by incubation with anti-CD14-coated microbeads, followed by sorting with a magnetic device. Cellular localization of HMGB1 was investigated by flow cytometry assay; HMGB1 release in the serum by Western blot. Serum samples were tested for IL-6 levels by ELISA. A one-way repeated-measures analysis ANOVA was performed to assess differences in HMGB1 concentration over time, in monocytes and serum. Results We show that: a) cellular expression of HMGB1 in monocytes at t1 was significantly higher as compared to t0; b) at t2, a significant increase of HMGB1 levels was found in the sera of patients. Such an increase was concomitant to a significant down-regulation of cellular HMGB1, suggesting that the release of HMGB1 might partially derive from mononuclear cells; c) treatment of monocytes with HMGB1 induced in vitro the release of IL-6; d) at t2, high amounts of circulating IL-6 were detected as compared to t0. Conclusions This study demonstrates for the first time that surgical/anesthesia trauma is able to induce an early intracellular upregulation of HMGB1 in monocytes of surgical patients, suggesting that HMGB1 derives, at least partially, from monocytes.