[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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
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.
[Show abstract][Hide abstract] 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
Annals of the Rheumatic Diseases 06/2014; 73(Suppl 2):365-365. DOI:10.1136/annrheumdis-2014-eular.4306 · 10.38 Impact Factor
[Show abstract][Hide abstract] 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
Annals of the Rheumatic Diseases 01/2014; 72(Suppl 3):A798-A798. DOI:10.1136/annrheumdis-2013-eular.2364 · 10.38 Impact Factor
[Show abstract][Hide abstract] 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
Annals of the Rheumatic Diseases 01/2014; 71(Suppl 3):644-644. DOI:10.1136/annrheumdis-2012-eular.117 · 10.38 Impact Factor
[Show abstract][Hide abstract] 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
Annals of the Rheumatic Diseases 01/2014; 71(Suppl 3):639-639. DOI:10.1136/annrheumdis-2012-eular.29 · 10.38 Impact Factor
[Show abstract][Hide abstract] 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
Annals of the Rheumatic Diseases 01/2014; 72(Suppl 3):A947-A947. DOI:10.1136/annrheumdis-2013-eular.2840 · 10.38 Impact Factor
[Show abstract][Hide abstract] 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
Annals of the Rheumatic Diseases 01/2014; 71(Suppl 3):370-370. DOI:10.1136/annrheumdis-2012-eular.2629 · 10.38 Impact Factor
[Show abstract][Hide abstract] 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
Annals of the Rheumatic Diseases 01/2014; 71(Suppl 3):321-322. DOI:10.1136/annrheumdis-2012-eular.2493 · 10.38 Impact Factor
[Show abstract][Hide abstract] 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 .
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  and sterilized by heating at 180°C for 2 h before each experiment. Human HDL were isolated from peripheral blood of healthy volunteers . 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.
Disclosure of Interest None Declared
Annals of the Rheumatic Diseases 01/2014; 72(Suppl 3):A370-A371. DOI:10.1136/annrheumdis-2013-eular.1135 · 10.38 Impact Factor
[Show abstract][Hide abstract] 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
Annals of the Rheumatic Diseases 01/2014; 71(Suppl 3):i-642. DOI:10.1136/annrheumdis-2012-eular.83 · 10.38 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate apolipoprotein (apo) A-I, apo B, lipoprotein (Lp) (a), HDL-cholesterol (C), LDL-C, triglycerides (TG) and total cholesterol (TC) values in the serum and synovial fluid (SF) of untreated rheumatoid arthritis (RA), psoriatic arthritis (PsA), and osteoarthritis (OA) patients.
Paired SF and serum samples were collected simultaneously from 14 patients with RA, 14 with PsA, and 16 with OA and tested for apo A-I, apo B, HDL-C, LDL-C, Lp(a), TC and TG. Serum C reactive protein (CRP) and amyloid A (SAA) levels were also determined.
The inflammatory arthritis patients had higher SF lipid levels with the exception of HDL. Reflecting increased synovial permeability, the lipid SF/serum ratio was always higher in RA and PsA with respect to OA patients. The positive correlation between serum and SF apo A-I, apo B, HDL-C, TG, and Lp(a) levels confirmed that there is lipoprotein diffusion into the SF. RA and PsA patients had lower concentrations of all serum lipids except for Lp(a) with respect to OA patients. The levels in the RA patients were similar to those in healthy matched controls, while the PsA patients had significantly lower apo A-I and HDL levels and higher apo B and LDL values.
Lipid diffusion into the joint cavity, which largely depends on the degree of inflammation, may contribute to modulating local inflammatory processes.
Clinica chimica acta; international journal of clinical chemistry 01/2012; 413(1-2):303-7. DOI:10.1016/j.cca.2011.10.019 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SUMMARY Osteonecrosis is a disease characterized by the death of marrow and bone tissues. All bones may be affected, most commonly those of the hip, knee, shoulder, ankle as well as the small bones of the hands and feet. When the disease involves a weight-bearing joint there is a significant risk that subarticular fracture may develop leading to disabling arthrosis and requiring, therefore, arthroplasty surgery. Osteonecrosis typically affects patients in their third, fourth and fifth decades of life and is associated with many factors including other diseases and co-morbidities. Multifocal osteonecrosis is defined according to the involvement of at least three separated anatomic sites. We describe the case of a young man with osteonecrosis of the shoulder and hip joints which required total arthroplasty. Among biochem- ical investigations, an increase in the plasminogen activator inhibitor type 1 (PAI-1) levels associated with mild hy- perhomocysteinemia was present. Another finding was the HLA B27, without signs of spondyloarthropathies. In pa- tients with osteonecrosis, especially if multifocal, a careful medical history, a complete physical examination and some biochemical investigations, particularly those related to thrombophilia and hypofibrinolysis, should be performed.
[Show abstract][Hide abstract] ABSTRACT: SUMMARY Nasopharyngeal carcinoma has long been reported as the predominant type of cancer associated with dermatomyositis in many several Asian countries, including Hong Kong, Singapore, and Southern-Cina. Dermatomyositis is one of the idiopathic inflammatory myopathies showing characteristic cutaneous manifestations. Reviews from the western lit- erature have demonstrated that certain cancers, such as ovarian and breast carcinoma in women and lung and prostate carcinoma in men, are highly associated with DM relative to the general population. We report the case of a Cau- casian Italian patient with nasopharyngeal carcinoma and dermatomyositis. Considering the rarity of nasopharyn- geal carcinoma among whites, both the detection and the report of each new case are noteworthy in defining the ge- ographic and ethnic distribution of this tumor.
[Show abstract][Hide abstract] ABSTRACT: We evaluated both the efficacy and safety of anakinra in daily routine rheumatoid arthritis clinical practice.
We studied 60 cases, including patients with previous anti-TNFalpha exposure, treated with anakinra (100 mg/daily s.c.) in combination with methotrexate (7.5-10 mg/week i.m.) or leflunomide (20 mg/die) in a two year observational study. Efficacy measures were assessed using the American College of Rheumatology (ACR) response criteria. Safety was evaluated according to a modified World Health Organization adverse reaction term dictionary.
At week 14, ACR 20% response criteria have been fulfilled by 53 (91.3%) out of 58 patients, 51 (87.9%) of them achieving also an ACR 50%and 15 (25.8%) an ACR 70%response. Thirteen patients touched 102 weeks of treatment: ACR 20% response was achieved in 92.3%, while ACR 50% and ACR 70% were respectively found in 84.6% and 38.4% of the cases. The mean decrease in HAQ score was 0.38, p<0.001. Of the 16 patients who were previously treated with anti-TNFalpha blockers, 81.2% responded to anakinra. There was no significant difference in the ACR response between groups with and without previous anti-TNFalpha exposure. Seventeen patients (28.3%) stopped anakinra because of side-effects (5%) or failure to respond (23.3%). Only 4 cases of pulmonitis, of which 2 have been hospitalised, and 1 case with tuberculosis (previously treated with infliximab) were observed.
Our clinical experience confirms that anakinra is effective and safe in the treatment of rheumatoid arthritis. Anakinra seems also useful in patients with previous anti-TNFalpha blockers failures. Even though major adverse events were rare, clinicians should be aware of such a possibility.