P Sfriso

University of Padova, Padua, Veneto, Italy

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Publications (79)266.36 Total impact

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    P Galozzi · O Negm · P Sfriso · P Tighe · I Todd · L Punzi

    Preview · Article · Sep 2015 · Pediatric Rheumatology
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    ABSTRACT: Background Crystal-induced inflammation is characterized by a marked release of cytochines, chemokines and other factors in the synovial compartments. The resolution of this process is quite easy to achieve in patients through the use of NSAIDs and other more specific drugs such as colchicines and anti-IL1β biologics. In view of the potential long term adverse effects of these drugs, we aimed to investigate the role of a small polyphenolic compound, resveratrol (RES), best known as a constituent of grapes and wine. Objectives We used a monocytic cell line to assess the effect of RES in monosodium urate (MSU) and calcium pyrophosphate (CPP) crystal-induced inflammation. Methods THP-1 cells were stimulated with synthetic MSU (0.05mg/ml) and CPP (0.025mg/ml) crystals after a 3h priming with phorbol myristate acetate (PMA) (100ng/ml). Resveratrol was added to cultures at 10μM. Epigallocatechin-3-gallate (EGCG), an already known nutraceutic inhibitor of crystal-induced inflammation (1), was used as control at the same concentration. The cytokines IL-1β, IL-8 and TGFβ were determined in the culture supernatants by ELISA assays. To assess whether RES could interfere with crystal internalization, the phagocytosis index was calculated at different time points. These experiments were preceded by the evaluation of the role of phagocytosis on cytokine release by using cytochalasin D at 1μM. Results After the 3h of priming with PMA, THP-1 cells produced high basal levels of both IL-1β and IL-8 and these further increased after 24h treatment with MSU and CPP crystals. The addition of RES together with the stimulus lead to a marked decrease in IL-1β (3 fold and 5 fold in presence of CPP and MSU respectively) and IL-8 release (2 fold and 3 fold in presence of CPP and MSU respectively). The inhibition of the inflammatory response by RES was dose-dependent. As regards TGFβ, EGCG showed a greater inhibition with respect to RES which, in any case, showed a 1.7 and 1.5 fold inhibition when added to cultures simultaneously to CPP and MSU respectively. The kinetic study showed a rapid internalization of crystals. Within 24 hours 50% of crystals were actively phagocytized by the cells and RES did not show any inhibitory effect on crystal phagocytosis. Conclusions The results of this study highlighted the anti-inflammatory potential of resveratrol in crystal-induced inflammation. Although the possible mechanism by which resveratrol exerts its biological functions is under evaluation (using sirtuin inhibitors), the direct effect on the inflammatory outcome may have important implications in the prevention and treatment of crystal-related arthropathies. References Disclosure of Interest None declared
    No preview · Article · Jun 2015 · Annals of the Rheumatic Diseases
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    ABSTRACT: The objective was to study both ex vivo and in vitro secretion of pro-inflammatory cytokines in patients affected by Blau syndrome (BS) and carrying p.E383K mutation in the CARD15/NOD2 gene associated with the disease. For ex vivo studies, peripheral blood mononuclear cells (PBMCs), serum from three patients and healthy controls have been collected. PBMCs have been cultured in the presence or absence of inflammatory enhancers, such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP). The levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were assayed by either immunoassay or array-based system. For in vitro studies, different constructs were created cloning human wild-type and p.E383K-mutated NOD2 cDNA into the expression vector pCMV-Tag2c. HEK293 cell lines were stably transfected, cultured with or without MDP and IL-8 level was assayed in their surnatants. Statistical analysis in both studies was performed using non-parametric tests. Both ex vivo and in vitro studies have not identified a significant increase in secretion of the analyzed proinflammatory cytokines. p.E383K-mutated NOD2 transfected cells express low level of IL-8. The ex vivo basal level results from both serum and PBMCs surnatants present similar levels of IL-1β, IL-6, TNF-α and IFN-γ in patients and controls. The presence of the stimulant agents (LPS and MDP), either individual or paired, does not lead to significant increases in all cytokines concentrations in patients compared to controls. Taken together, the ex vivo and in vitro data suggest that there is not a primary mediation of IL-1β and other pro-inflammatory cytokines in BS patients carrying p.E383K.
    Preview · Article · Mar 2015 · Reumatismo
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    ABSTRACT: The objective was to study both ex vivo and in vitro secretion of pro-inflammatory cytokines in patients affected by Blau syndrome (BS) and carrying p.E383K mutation in the CARD15/NOD2 gene associated with the disease. For ex vivo studies, peripheral blood mononuclear cells (PBMCs), serum from three patients and healthy controls have been collected. PBMCs have been cultured in the presence or absence of inflammatory enhancers, such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP). The levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were assayed by either immunoassay or array-based system. For in vitro studies, different constructs were created cloning human wild-type and p.E383K-mutated NOD2 cDNA into the expression vector pCMV-Tag2c. HEK293 cell lines were stably transfected, cultured with or without MDP and IL-8 level was assayed in their surnatants. Statistical analysis in both studies was performed using non-parametric tests. Both ex vivo and in vitro studies have not identified a significant increase in secretion of the analyzed proinflammatory cytokines. p.E383K-mutated NOD2 transfected cells express low level of IL-8. The ex vivo basal level results from both serum and PBMCs surnatants present similar levels of IL-1β, IL-6, TNF-α and IFN-γ in patients and controls. The presence of the stimulant agents (LPS and MDP), either individual or paired, does not lead to significant increases in all cytokines concentrations in patients compared to controls. Taken together, the ex vivo and in vitro data suggest that there is not a primary mediation of IL-1β and other pro-inflammatory cytokines in BS patients carrying p.E383K.
    No preview · Article · Mar 2015 · Reumatismo
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    ABSTRACT: This study aimed to investigate, using an in vitro model, the mechanisms involved in the effects linked to a novel hexadecylamide derivative of hyaluronic acid (HA), HYADD®4 (HS), on some inflammatory aspects related to the osteoarthritis process. The human leukemic monocytic cell line THP-1 was stimulated with calcium pyrophosphate (CPP) crystals or lipopolysaccaride (LPS) and cultured in the presence of HS or two unmodified HAs (500-730 kDa and >1,500 kDa, respectively). The effects of the three HA derivatives were compared by examining the inhibition of IL-1ß and IL-8 release, the phagocytic capacity of THP-1, and HA's physical interference with the cytokines and their biological activity. Adding HS simultaneously with the stimuli led to a marked (nearly 100%) decrease in cytokine release and biological activity with respect to the two unmodified HAs. The effect was not altered when a CD44 function-blocking monoclonal antibody was used. Incubation of the three derivatives with IL-1ß and IL-8 led to a reduced bioavailability of the cytokines in the medium in the presence of HS but not of unmodified HA. This study examines a novel mechanism inhibiting cytokine bioactivity. The HA hexadecylamide derivative was found to suppress, in vitro, the inflammatory response induced by CPP crystals and LPS by reducing the bioavailability of the two cytokines that were analyzed. This article is protected by copyright. All rights reserved. Copyright © 2015 Wiley Periodicals, Inc., A Wiley Company.
    Full-text · Article · Feb 2015 · Journal of Biomedical Materials Research Part A
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    ABSTRACT: Background Epigallocatechin gallate (EGCG) is the most powerful phenolic compound of green tea. Although its antioxidant properties are the most widely known, EGCG is able to affect the activity and expression of many inflammatory substances through specific intracellular mechanisms. We recently demonstrated that EGCG inhibits the production of inflammatory cytokines and chemokines and cell migration in an in vitro model of calcium crystal-induced inflammation. Objectives The aim of this study was to investigate the effect of EGCG in an in vitro and in vivo model of monosodium urate (MSU) crystal-induced inflammation through the assessment of key inflammatory cytokines involved in gout. Methods The human leukemic monocytic cell line THP-1 was primed for 3 hours with phorbole myristate acetate (300 ng/ml), re-incubated overnight and treated with MSU 0.05 mg/ml for 24 hours in presence or absence of EGCG (range 10-100 μM). The levels of IL-1β, IL-8, IL-6 and CCL2 were determined in the culture supernatants by enzyme-linked immunosorbent assay (ELISA). As regard the study in vivo, the effect of EGCG was evaluated in the acute peritonitis model. Twenty-four mice was randomly subdivided into four groups and injected with 0.5 ml phosphate buffered saline containing: 1- MSU crystals 2mg/ml, 2- MSU crystals and EGCG 20mg/kg, 3- EGCG 20mg/kg, 4- the vehicle alone. The animals were sacrificed after 6 h. Peritoneal inflammation was assessed by cell recruitment (total number of leukocytes and neutrophils) and cytokine levels in the peritoneal lavage fluid. Cell populations were analyzed by flow cytometry while the production of IL-1β, IL-6, KC, CCL2 and TNF was determined by ELISA. Results EGCG inhibited IL-1β, IL-8, IL-6 and CCL2 release by stimulated THP-1 cells in a dose-dependent manner. Intraperitoneal injection of 1 mg of MSU crystals significantly increased leukocyte infiltrate (2.2 x 10e4 control mice vs 74.5 x 10e4 MSU-injected mice, p<0.05), the levels of IL -6 (7.2±11.1 pg/ml vs 87.5.5±93.4 pg/ml, p<0.05) and those of CCL2 (0 pg/ml vs 149.7±86.1 pg/ml, p<0.05). KC and TNF were also higher (3.5 and 10 times, respectively) with respect to controls, although not significantly. The concentrations of IL- 1β were low or undetectable. EGCG significantly decreased the inflammatory infiltrate induced by crystals (1.8x10e4, p<0.01), the number of neutrophils (5800/ml vs 64000/ml, p<0.05) and levels of all cytokines considered. Highly significant correlations were found between the total number of leukocytes and all the studied cytokines with the exception of TNF. Conclusions The results of this study show that the main catechin of green tea inhibits MSU crystal-induce peritonitis in mice. It is possible that EGCG acts by receptor-mediated mechanisms as shown in other models, or by activating anti- inflammatory signaling pathways. The identification of EGCG as a natural and potentially non toxic substance, capable of affording protection or modulating the inflammatory response to MSU crystals, may have important implications on therapy and prevention of gout and more generally, of all microcrystalline arthropathies. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.4306
    No preview · Article · Jun 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background BD is a systemic vasculitis with different subsets of clinical manifestations (1). The recurrent fever, detectable only in a subgroup of patients, along with the relapsing course of disease, the presence of signs and symptoms typical of autoinflammatory syndromes, the good response to anti-cytokines treatment and the increasing detection of mutations in genes encoding for molecules involved in innate-immunity regulation suggest to consider a subgroup of BD as auto-inflammatory disease (2). Objectives The aim of this study was to understand weather different disease subsets may correlate with the great variability observed in obstetric outcome and disease activity during pregnancy (3). Methods All patients affected with BD, according to diagnostic criteria (1), who experienced at least one pregnancy and seen in four Italian referral centers (Rheumatology Unit of Padua, Clinic Immunology Unit of Padua, Ophthalmology Unit of Padua, Rheumatology Unit of Siena) were included in the study. Data regarding fetal and maternal outcome was retrospective collected by researchers with clinical interviews based on a common questionnaire. Pregnancies were sub-divided into two groups: group 1 included patients with recurrent fever, while group 2 included patients without recurrent fever. Recurrent fever has been previously defined as three or more episodes of fever of unknown origin per year. Results 36 women affected with BD reported 71 pregnancies and 60 newborns. Group 1 included 13 patients and 24 pregnancies. 23 pregnancies occurred after the disease onset, while in 1 case BD started during pregnancy. Group 2 included 23 patients and 47 pregnancies. 24 pregnancies occurred after the disease onset, while 22 occurred before the disease onset. One case of BD started during pregnancy. Mean of disease duration was 16 years in group 1 and 11 years in group 2. Febrile BD started before pregnancy more often than not febrile: 83% vs 52%, p=0,008. Regarding disease activity during pregnancy, we observed (group 1 vs group2): stable disease activity 45% vs 20%, increased disease activity 40% vs 8%, improved disease activity 10% vs 8%, complete remission 5% vs 64%, p=0,003. As for pregnancy complications, we recorded (group 1 vs group 2): gestational hypertension 29,1% vs 0, p=0,001, preeclampsia 20,8% vs 0 p=0,001, miscarriage 20,8% vs 12,7% and preterm delivery 8,3% vs 6,4%. Conclusions Febrile BD activity remains unaffected or it worsens during pregnancy, while not febrile BD usually improves, even to a complete remission. Patients with recurrent fever have an higher risk of gestational complications than patients without recurrent fever, particularly gestational hypertension and preeclampsia. References Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4502
    No preview · Article · Jun 2014 · Annals of the Rheumatic Diseases
  • P. Galozzi · E. Greco · A. Gava · D. Basso · P. Sfriso · P. Tighe · I. Todd · L. Punzi
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    ABSTRACT: Background The Blau syndrome (BS) is a rare autosomal dominant autoinflammatory disease clinically characterized by symmetrical arthritis, granulomatous dermatitis and recurrent uveitis. The disease is caused by single mutations in CARD15/NOD2, encoding the NOD2 protein that is known to regulate the defense against pathogens by activating the NF-κB signalling pathway (1). Objectives In vitro and ex vivo functional analysis aimed at characterizing p.E383K mutation found in an Italian family affected by Blau syndrome. Methods For ex vivo studies, peripheral blood mononuclear cells (PBMC) are collected from patients carrying p.E383K mutation, then lysed and analyzed by a protein microarray technique (RPPA) to determine the NF-κB activity. IL1β, IL6, IL8, TNFα, IFNγ, IL12, IL17, IL22, IL23 releases are quantified by ELISA and antibody microarray techniques in PBMC cultured for 7 hours in presence or absence of lipopolysaccharide (LPS) and muramyldipeptide (MDP). In vitro analysis started from the creation and stably transfection of constructs containing human NOD2 cDNA wild-type and mutated p.E383K into HEK293 cells. The cells were cultured for 7 and 24 hours with or without MDP. NF-κB activity was determined by Western blot, evaluating the expression of IKBα and its phosphorylated form in cellular lysates, and also by RPPA. IL8 was assayed in the supernatants of the reported cell cultures through antibody microarray technique. The significance of all the data was tested using non-parametric analysis. Results Confocal microscopy revealed that p.E383K NOD2 resides in cytoplasm of transfected HEK293 cells, as well as wild-type. The expression of p.E383K NOD2, assessed by Western blot, did not present any variations after MDP stimulation. Regarding the NF-κB activity, both in vitro RPPA and Western blot studies showed an inactivation of this pathway, presenting lower expression of phosphorylated IKBα than IKBα in presence or absence of MDP stimulation (p<0.05). However, all the pathway components (NF-κB, IKBα and IKKα/β and their phosphorylated forms) were found upregulated ex vivo compared with controls (p<0.05). analysis Concerning the cytokines, both in vitro and ex vivo studies have not identified a significant increase secretion of most of the proinflammatory cytokines analyzed. IL8 showed both in vitro and ex vivo a significant decrease (p<0.05) compared to controls. IL17, IL22 and IL23 release ex vivo was higher in patients than in controls (p<0.05 p<0.001 and p<0.005 respectively), while IL12 is reduced in patients (p<0.05). After stimulation (LPS, MDP or both), IL1β, IL6, IL8, TNFα and IFNγ production was not augmented in patients compared to controls. Conclusions The contrasting results presented by our in vitro and ex vivo data on NF-κB pathway may indicate that the activation do not depend only on NOD2 in carriers of p.E383K mutation. Further experiments may clarify these results, using also different in vitro models, such as THP1 cells. Regarding the cytokines profile, it seems that there is not a primary mediation of IL1β and other pro-inflammatory cytokines in patients presenting p.E383K mutation, as reported in literature for p.R334W/Q carriers (2), with the exception of IL17/22/23 whose role has to be deeply investigated. References Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2906
    No preview · Article · Jun 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background Adult onset Still's disease (AOSD) is a rare rheumatic disease with an estimated prevalence of less than 1 case per 100,000 population. The diagnosis is difficult and is often delayed due to the lack of specific diagnostic tests and the need to rule out other pathological entities. Objectives To describe the clinical characteristics of a multicenter Italian case series of patients with AOSD. Methods 14 Italian University Hospital centers participated in the study. A standardized medical record containing clinical data, laboratory investigations, disease patterns and the different therapies has been sent to all participating centers. Each center collected data retrospectively. Results In the first wave of data collection, from March to May 2013, 233 patients were included with AOSD according to Yamaguchi criteria (125 males, 53.6%, mean age at onset: 40.2±16.2 years, time between onset and diagnosis: 242±650 days). The most frequent manifestations at onset were: arthralgia (93.0%), fever (92.6%), leukocytosis (89.0%), typical rash (67.7%), sore throat (61.8%), lymphadenopathy/splenomegaly (60.4%), increased liver enzymes (53.5%). In 56.4% of patients ferritin was increased more than 5 times the normal threshold. Fever intensity was recorded up to 41 ° C (mean 39.1±0.7), with a single daily peak in 27.3% of cases and 2 peaks in 51.9% of cases. The average duration of febrile episodes was 10.3 days with variable interval between episodes (average 3.2 days). Based on clinical evolution the disease was classified polycyclic systemic in 40.8%, chronic articular polycyclicin 30.7%, systemic monocyclic in 23.9% and chronic articular monocyclic in 4.6%. In 21.9% of cases, corticosteroids and traditional DMARDs have not been able to control the disease while biological drugs have been shown to be effective. 56 cycles of biological drugs were used in 51 patients: anakinra in 22 cases, etanercept in 15 cases, infliximab in 8 cases, adalimumab in 7 cases, rituximab in 3 cases, tocilizumab in 2 cases, abatacept and golimumab in 1 case each (anti- TNFalpha 52.5%, 37.3% anakinra, other 10.2%). Conclusions This study presents the largest Italian multicentre series of AOSD patients. The main clinical and laboratory characteristics are in line with those reported in the literature. In more than 20% of patients biologics are the only drugs that allow to control the disease. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5300
    No preview · Article · Jun 2014 · Annals of the Rheumatic Diseases

  • No preview · Article · Apr 2014 · Osteoarthritis and Cartilage
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    ABSTRACT: Background Direct cell activation by crystals in vitro provokes the release of a number of cytokines and chemokines but not that of IL-1ß whose production and secretion needs the activation of NALP3 inflammasome and that of procaspase 1. It is now clear that a second stimulus in cell culture experiments is needed to prime cells to generate IL-1ß. Such stimulus is mostly represented by the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) which influences cell differentiation and stimulate monocyte functions including phagocytosis. Objectives The purpose of this study was to investigate the mechanism though which crystals induce IL-1ß production after PMA stimulation. Methods Human THP-1 were incubated for 3 hours with different concentrations of PMA (range 0-300ng/ml) or a mixture of IL-1ß/TNFa (0.5/1 ng/ml) and then re-incubated with fresh medium. The day after cells were stimulated with calcium pyrophosphate (CPP) or monosodium urate (MSU) crystals for 24hours. The rate of intact, necrotic and apoptotic cells induced by PMA before crystal stimulation were quantified by surface annexin V-FITC and propidium iodide staining. The levels of IL-1ß were determined in the culture supernatants by enzyme-linked immunosorbent assay while IL-1 expression was quantify by real time PCR. Results PMA is a potent inducer of pro-IL-1ß mRNA expression in THP1 cells. After 24h from the 3h treatment with PMA, IL-1ß mRNA is about 10 fold and 35 fold higher using PMA at 100 and 300 ng/ml respectively. At the same time high levels of extracellular IL-1ß are observed. PMA also induces a dose-dependent apoptosis in THP-1 cells after 3h treatment. This effect is much more stronger 24 h after the stimulation and using PMA at 300 ng/ml. The pre-treatment of cells with IL-1ß/TNFa did not affect the rate of intact cells and their viability. IL-1ß/TNFa did not stimulate the IL-1ß mRNA expression nor the release of the protein in the supernatants. MSU and CPP crystals used after a cell washout of 24h, induce high IL-1ß release in culture supernatants only after PMA treatment but slightly affect pro-IL-1ß gene transcription. CPP induced a mild increased expression of IL-1ß mRNA in cells pre-treated with IL-1ß/TNFa. The involvement of inflammasome was confirm by using KCl which returned extracellular IL-1ß to control levels. Conclusions The results of our study show that high concentrations of PMA used in in vitro experiments to reproduce some aspects of crystal-induced inflammation have a strong effect on cell apoptosis and consequently in their viability. The fact that MSU and CPP crystals have a strong effect on IL-1ß protein levels but not on IL-1ß mRNA, and that this effect is evident only in cells pre-treated with PMA and not with IL-1ß/TNF, confirms that crystals may act on inflammasome assembly or directly on caspase-1 activation once pro-IL-1ß transcription has been already induced. Furthermore, the results of our study prompt us to hypothesize that apoptosis may be considered as one of the signals which lead to IL-1ß activation and release by pathogenetic crystals. Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
  • A. Scanu · F. Oliviero · D. Mavilio · P. Sfriso · L. Punzi
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    ABSTRACT: Background Natural killer (NK) cells represent one of the major components of innate immune system and are very important in both early and chronic phases of inflammatory responses. NK cells might contribute to the disease onset through the release of great amount of pro-inflammatory cytokines and through a direct cytolitic activity. However, the role of NK cells in joint inflammation remains unclear. Objectives To determine whether NK cells are present in synovial fluid (SF) and whether they might play a role in amplifying the inflammatory process. Methods Mononuclear cells were isolated by density gradient centrifugation from SF collected by arthrocentesis from the knees of 4 untreated patients with inflammatory arthritis (3 seronegative spondiloarthritis and 1 psoriatic arthritis). NK cells were isolated from mononuclear cells by negative immunomagnetic selection. Human fibroblast-like synoviocytes (FLS) were obtained by synovial tissue explants from patients with OA and cocultured for 24 h at 10×103 cells/well in 96-well plates with increasing ratios of NK cells (1:1; 1:2; 1:5; 1:10; 1:20). FLS or NK cells were cultured alone as control. Culture supernatants were tested by ELISA for the production of IL-1β, IL-8, CCL2 and TGFβ1. Results NK cells were isolated from all 4 SF collected. The percentage of NK cells in mononuclear cells was 0.14 in SF1, 0.16 in SF2, 1.06 in SF3 and 0.56 in SF4. IL-1β and TGFβ1 were not detectable in all culture supernatants. When FLS were cocultured with NK cells the production of IL-8 and CCL2 increased in a NK ratio-dependent manner. FLS or NK alone did not release significant levels of IL-8 and CCL2. Conclusions This study confirms that NK cells are also present in synovial fluid and they might potentially contribute to amplification of the inflammatory process within the joint. The increased cytokine production might be due to the capacity of NK cells to produce fibroblast-activating factors or to direct interactions with synovial cells. Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
  • P. Galozzi · E. Greco · A. Gava · P. Sfriso · D. Basso · M. Plebani · L. Punzi
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    ABSTRACT: Background Blau syndrome (BS) is a rare, autosomal dominant autoinflammatory disease, characterized by granulomatous dermatitis, symmetrical arthritis and recurrent uveitis. The caspase recruitment domain gene CARD15/NOD2 has been identified as responsible for BS (1). To date, 11 BS-associated mutations have been identified. Few functional data on p.E383K mutation are available in literature, whereas p.R334W/Q is the most frequent and studied mutations. Several in vitro observations have reported an elevated basal NF-κB activity and a “gain of function” hypothesis has been proposed (2), suggesting a spontaneous release of inflammatory cytokines from BS patients. Objectives We aimed at studying the functional profile of CARD15/NOD2 in patients carrying p.E383K mutation compared to healthy controls; in particular we focused on the cytokine levels since there is no evidence in literature. Methods IL-1β, IL-6, IL-8 and TNF-α releases were measured by ELISA assays in peripheral blood mononuclear cells (PBMC) obtained from 3 patients with p.E383K mutation (3). All patients were members of the only one Italian affected family. Cells were cultured in vitro either with or without stimulation of muramyldipeptide (MDP, NOD2 agonist), lipopolysaccaride (LPS, a TLR-4 agonist) or a combination of MDP and LPS. Statistical comparisons were made between the means of patients and controls data, using multiple comparison with Fisher post hoc analysis. Results In absence of stimulation, no differences in cytokine levels were detected in PBMC of both controls and patients. Interestingly, stimulation with LPS or MDP did not induce augmented production of each cytokine in PBMC from patients compared with those from controls; besides, the cytokine levels in patients appeared to be attenuated compared with the control group. Both in patients and controls, there were no statistically significant differences in each cytokine release comparing LPS or MDP stimuli and no stimulation, except for IL-8 (p=0.011 and p=0.045, after LPS stimulation in patients and in controls respectively). Notably, the synergistic stimulatory effect of the combination of MDP and LPS is observed for each cytokine only in control group (IL-1β, p=0.027; IL-6, p=0.001 and IL-8, p=0.002 referred to MDP; TNF-α, p=0.000 and 0.04 referred to MDP and LPS respectively). Conclusions Our findings for p.E383K mutation correlate with those reported in literature for patients carrying p.R334W/Q mutations (4), showing a similar cytokine profile of BS patients for whichever mutations. Moreover, according to our data, it would seem not to exist a primarily mediation of IL-1β in Blau syndrome. More repeated measures should be made in order to justify our results for all cytokines. Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Objectives To analyse a panel of synovial fluid (SF) and synovial tissue (ST) biological markers expression at single joint level to find candidate biomarkers in resistant peripheral spondyloarthritis (SpA). Methods Twenty seven resistant SpA patients with knee joint synovitis, included in an Intra-articular (IA) TNF alpha blocking open-label study1, were treated with biweekly four IA Etanercept injections (12.5 mg) in a single knee joint. The primary outcome (Thompson’s knee index: THOMP) and secondary outcomes were assessed at baseline and at week 8 of the study. The secondary outcomes are: C-reactive protein; Knee Joint Articular Index (KJAI), Health Assessment Questionnaire disability index (HAQ-DI), maximal synovial thickness by gray-scale ultrasonography (US-MST), synovial tissue cluster differentiation CD45+ mononuclear cell (ST-CD45+), synovial tissue-CD31+ vessels (ST-CD31+), and along with levels of synovial fluid (SF) soluble inflammation makers such as Interleukin 1ß (SF-IL 1ß), Interleukin 1 Receptor antagonist (SF-IL 1Ra) and Interleukin 6 (SF-IL 6). Results At the end of the study, composite clinical indexes, US-MST, ST and SF biological markers were significantly reduced compared to baseline. There was a significant correlation between CRP and THOMP, or KJAI, or HAQ-DI, or SF-IL 1Ra; between KJAI and THOMP or US-MST; between ST-CD45+ and THOMP, or KJAI or ST-CD31+, or SF-IL 1ß; between SF-IL 6 and THOMP, or KJAI, or SF-IL 1ß, or SF-IL 1Ra; between SF-IL 1 Ra and SF-IL 1ß. Comparing pre- versus post-IA Etanercept injection changes (Δ), we found a significant correlation between ΔCRP with ΔSF-IL 1ß, ΔKJAI with ΔTHOMP and ΔSF-IL 6; Δ HAQ with ΔSF-IL 6; ΔST-CD-45+ with ΔSF-IL 1ß; ΔSF-IL 6 and ΔSF-IL 1ß; ΔSF-IL 1Ra with ΔSF-IL 1ß and ΔSF-IL 6. Conclusions The significant association at single joint level of composite clinical indexes to inflammatory soluble markers and to synovial tissue marker expression, as well as between clinical and synovial biomarkers changes following IA-anti TNF-alpha blockers treatment, suggest that CD45+ in synovial tissue and IL-6 and IL-1β in SF may be considered potential biomarkers of the peripheral SpA response to IA TNF- alpha blocking. Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background Pharmaco-epidemiological studies on TNF-inhibitors (TNF-I) in rheumatoid arthritis (RA) and spondyloarthritis (SpA) are providing useful data about effectiveness and safety in clinical practice. Objectives To compare drug survival in RA and SpA according to TNF-I drug using data from a multicentre Italian cohort study. Methods The patients were selected from the Monitornet database, a prospective cohort study to monitor the long-term safety of biologic therapy involving 27 rheumatology centres across Italy, supported by the Italian regulatory agency AIFA. For the purpose of these analyses we included RA or SpA patients, who started a first course infliximab (INF), etanercept (ETA) or adalimumab (ADA). Drug survival was primarily defined from start until first discontinuation (overall drug survival) and secondarily due to ineffectiveness or adverse events (AEs). A first set of analyses assessed the relationship between diagnosis and drug survival using RA as reference category, adjusting for age, gender and comorbidities. A second set of analyses stratified by diagnosis explored the relationship between TNF-I and drug survival using INF as reference category, adjusting for age, gender, comorbidities, disease duration, previous DMARDs, calendar, baseline CRP, disease activity (DAS28 or BASDAI) and severity (HAQ score or BASFI). Drug survival was analyzed using Cox proportional hazards models. Results are presented as hazard ratios (HR) and 95% confidence intervals (CI). Results 1992 RA patients (79.8% women, mean age 55.5 yrs (SD 12.3), mean disease duration 9.6 yrs (SD 8.0), mean baseline DA28 4.4 (SD 1.9), baseline HAQ 1.4 (SD 0.7), median number of previous DMARDs 2 (IQR 1-3) and 993 SpA patients (55.2% men, mean age 49.6 yrs (SD 12.5), mean disease duration 6.9 yrs (7.5), mean baseline BASDAI 4.7 (SD 2.4), mean baseline BASFI 4.6 (SD 2.5) were included in the analyses. 44.2% RA patients started ETA, 21.4% INF, 34.4% ADA, while 45.8% SpA patients ETA, 22.3% INF, 31.9% ADA. Using RA as reference, SpA patients showed a lower drug discontinuation (HR 0.92 [95%CI 0.87, 0.99]), due to a lower withdrawal for inefficacy (HR 0.90 [95%CI 0.82, 0.99]). Both in RA and in SpA, compared to INF, ETA showed a slightly better survival on treatment, also due to lower discontinuation for inefficacy (see table). Conclusions These data support a better survival on treatment for SpA over RA and for ETA as compared with INF, mainly due to lower discontinuation for ineffectiveness rather than for AEs. Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background SIRT1 is an NAD-dependent protein deacetylase that targets both histones and non-histone proteins including transcription factors. It has been shown to play an important role in a variety of age-related diseases demonstrating a broad anti-inflammatory function in different tissues (1). Naturally occurring dietary polyphenols, such as catechins, have antioxidant and anti-inflammatory properties via modulating different signaling pathways and have been shown to activate SIRT1 directly or indirectly. Little is known of the role that SIRT1 plays in osteoarthritis (OA) and in particular there is no evidence suggesting that mediators or modulators of inflammation can interfere with SIRT1 function in OA. Objectives In this study, we explored the idea that the epigallocatechin-3-gallate (EGCG), the most active catechin found in green tea, modulates the expression of SIRT1 in an in vitro model which use synthetic calcium pyrophosphate (CPP) crystals to reproduce some inflammatory aspects of OA. Methods Synthetic CPP crystals were used at 0.025mg/ml to stimulate a monocytic cell line (THP-1) in presence or absence of EGCG (10-50μM) and 2% of fetal calf serum. The IkappaB kinase (IKK) inhibitor, EF-24, was used to suppress NFkB activity. The production of IL-1β and IL-8 were determined in the supernatants along with the chemoattractant activity on polymorphonuclear cells. SIRT1 expression was determined by real-time quantitative PCR after 24 and 48h stimulation. Results IL-8 and IL-1β production induced by crystals was suppressed in presence of the IKK inhibitor confirming the activation of NFkB in CPP crystal-induced inflammation. EGCG inhibited both cytokines production and the chemoattractant activity of culture supernatants in a dose dependent manner (e.g. IL-8 from 500 pg/ml to 100 and 45 pg/ml). The modulation of SIRT1 expression was more pronounced after 48h treatment and its trend corresponded to that observed for cytokine production. While CPP crystals provoked a slight diminution of SIRT1, in presence of EGCG SIRT1 expression was 1.6 time higher respect to control values. EGCG alone activated SIRT1 even if to a smaller extent. Conclusions With this study we showed, for the first time, evidence that SIRT1 expression is affected by CPP crystals and is modulated by EGCG. It has been demonstrated that SIRT1 is able to deacetylate the p65 subunit of NF-kB, blocking its ability to bind DNA, thereby inhibiting transcription of proinflammatory genes (2). As CPP crystal stimulation acts through the activation of NFkB and EGCG inhibits cytokines release and lead to enhanced SIRT1 mRNA, it is possible that EGCG inhibits CPP crystal-induced inflammation through the activation of SIRT1. To explore this hypothesis activity of SIRT1 in presence of EGCG will be evaluated as well as cytokine production after inhibition of SIRT1 by means of nicotinamide or RNA silencing. Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background A characteristic feature of crystal-induced inflammation is that acute attacks are self-limiting even without treatment. Many factors are involved in this spontaneous resolution. Among them, plasma proteins and lipoproteins identified in synovial fluids may modulate crystal-induced inflammation [1,2]. We demonstrated that high-density lipoproteins (HDL) down-regulate chemokine production induced by monosodium urate (MSU) crystals in vitro [3]. Objectives To evaluate the effects and mechanisms of action of HDL in a murine air-pouch model of MSU crystal-induced acute inflammation. Methods MSU crystals were prepared by Denko’s method [4] and sterilized by heating at 180°C for 2 h before each experiment. Human HDL were isolated from peripheral blood of healthy volunteers [5]. Air pouches were raised on the backs of CD1 mice (n=7 per condition). Various amounts of MSU crystals in 1 ml of PBS were injected into the pouch in the presence or absence of 0.1 mg of HDL for 3h. A group of mice was injected with MSU crystals pretreated with HDL for 1 h, centrifuged and resuspended in PBS. Negative control pouches received 1 ml of PBS. Pouch fluids were recovered by washing with 2 ml of PBS after the animals were sacrificed. Leukocyte count in lavage fluids was obtained using a hemocytometer and the percentage of neutrophils was determined by May-Grünwald-Giemsa staining. IL-1β, IL-6, CXCL1, CCL2 and TNF levels were measured in exudates by ELISA. Simultaneously, air pouch membranes were carefully dissected from adjacent subcutaneous and paraspinal tissues. IL-1β, IL-6, CCL2 and CXCL1 mRNA was isolated from exudate cells and membranes and analyzed by quantitative RT-PCR (qPCR). Results MSU crystals induced leukocytes infiltration (9.75 ± 1.73 x 105 cells/ml) comprising 68.57 ± 6.48 % of neutrophils. IL-1β (34.46 ± 11.03 pg/ml), IL-6 (692.61 ± 235.20 pg/ml), CXCL1 (493.73 ± 32.5 pg/ml) and CCL2 (2035.25 ± 2238.66 pg/ml) were measured in pouch fluids, while TNF levels remained under detection limit. Measurements of cytokine mRNA demonstrate that MSU crystals triggered IL-1β, IL-6 and CXCL1 expression in both pouch exudates and membranes whereas CCL2 mRNA was not modulated. The co-injection of MSU crystals and HDL inhibited leukocyte influx by ≈59% and neutrophil infiltration by ≈83% and, in turn, both protein and mRNA levels of all assessed cytokines were reduced. Similarly, injection of MSU crystals pretreated with HDL diminished leukocyte influx into the pouches and the production and expression of the inflammatory factors tested. Per se, PBS or HDL did not induce cell accumulation and cytokine production in pouches. Conclusions This study demonstrates that HDL limit the inflammatory response induced by MSU crystals in vivo by inhibiting leukocyte recruitment and cytokine release and expression. HDL inhibit MSU-induced inflammatory cytokine production by both infiltrating cells and pouch tissue cells. By analogy, HDL may represent an important factor modulating gouty inflammation by acting on both synovial tissue and synovial fluid cells. References Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Background Intra-articular injections of hyaluronic acid (HA) are widely used in the treatment of inflammatory and degenerative joint diseases. A novel slightly modified hyaluronic acid, HYADD4 (Hymovis®), has recently been tested in an ovine meniscectomy model of osteoarthritis, causing a reduction of vascularity and intimal hyperplasia, and an increase in the synthesis of high molecular weight HA by synovial fibroblasts (1). HYADD4 is a modified hyaluronate where the polysaccharidic backbone is derivatized with hexadecylic (C-16) side chains, through amide bonds, with a 1–3 mol % degree of substitution of repeating units. At a concentration as low as 0.3% (w/v) HYADD4 forms a gel, while unsubstituted hyaluronic acid forms viscous solutions at concentrations that are ten times higher. Objectives The purpose of this study is to investigate the effect of HYMOVIS on an in vitro models which reproduces some inflammatory aspects of the osteoarthritis process. Methods Synthetic calcium pyrophosphate (CPP) crystals (0.025mg/ml) were used to stimulate a monocytic cell line (THP-1), pre-activated with phorbol myristate acetate 50ng/ml, in presence or absence of Hymovis® (0.5 mg/ml). The production of IL-1β and IL-8 were determined in the supernatants along with the chemoattractant activity on polymorphonuclear cells. The results were compared with those obtained using 2 other types of HA (Hyalubrix® and Hyalgan®). The scavenger effect of Hymovis® on cytokines were investigated through the quantification of the trapping of these proteins to the chemical structure of HYADD4 and the others HA. The stimulation with LPS was used as control. Results Hymovis® has shown a strong effect on the inhibition of the production of IL-1β (from 1750±230 pg/ml to 120±52 pg/ml). Although of smaller intensity, this effect was observed also using the other HA (Hymovis>Hyalubrix>Hyalgan). The anti-inflammatory property of Hymovis was also evident on the release of IL-8 (from 2300±315 pg/ml to 150±35 pg/ml) and on the migration of polymorphonuclear cells. The inhibitory effect of IL-8 release was confirmed after stimulation with LPS. All the 3 types of HA have shown to possess a little capacity to trap IL-1β (about 20%) while any scavenger effect was observed towards IL-8. Conclusions The hexadecylic derivative of HA Hymovis® has shown a potent anti-inflammatory action on the released of pro-inflammatoy mediators upon stimulation with CPP crystals, frequently associated with more severe and advanced OA. Due to the physical-chemical property of HA preparations, it was likely that its coating on crystals might determine a lower inflammatory activity of crystals themselves. We were able to exclude this hypothesis testing a different stimuli as LPS. The little scavenger effect of all HA towards IL-1β does not account for the observed anti-inflammatory effect as well. It is possible that Hymovis® is able to protect cells from the extracellular compartments where inflammatory stimuli are present. The investigation of IL-1β and IL-8 at the transcriptional level will be evaluated to confirm this hypothesis. Disclosure of Interest None Declared
    No preview · Article · Jan 2014 · Annals of the Rheumatic Diseases

  • No preview · Article · Aug 2013 · Clinical Drug Investigation
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    ABSTRACT: Objective. Coeliac disease (CD) is a systemic autoimmune condition induced by gluten consumption in genetically predisposed people, affecting ∼1% of the general population. In the literature, there are many studies that report the association between CD and different kinds of arthritis. The aim of this study was to investigate the presence of entheseal abnormalities by US in patients with CD without clinical signs of articular involvement as compared with healthy control subjects. Methods. Sixty patients with CD attending the gastroenterology outpatient clinic of the University Federico II of Naples and 60 healthy control subjects matched for age and sex were enrolled in this study. Coeliac patients and healthy controls underwent clinical and US examination. Results. Among 60 CD patients, 24 (40%) presented at least one entheseal alteration as compared with 6 (10%) control subjects (P < 0.01). In CD patients, the entheseal site more frequently involved was patellar (distal and proximal), while in the healthy controls the enthesopathies were all localized at the Achilles tendon. Conclusion. In conclusion, the results of this study underline the ability of US to detect signs of subclinical enthesopathy and indicate the presence of a higher prevalence of subclinical enthesopathies in asymptomatic CD patients.
    No preview · Article · May 2013 · Rheumatology (Oxford, England)