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Intra-articular injections of autologous platelet concentrates are considered capable to enhance the healing of cartilage lesions, alleviate joint inflammation, and relieve other musculoskeletal pathological conditions. The aim of this study was to analyze the soluble fractions obtained from platelet-rich plasma (pure- and leukocyte-PRP) to compare time- and preparation-dependent modifications of growth factor concentrations and the supporting activity of the two preparations on synovial fibroblast growth and hyaluronic acid (HA) production in vitro. The release kinetics of FGF-2, SDF-1, VEGF, HGF, EGF, PD GF-AB/BB, IGF-1, VCAM-1, and TGF-β isoforms were followed up to 168 h after PRP activation, and their amounts were determined by multiplex-beads immunoassay. Synovial cell growth and supernatant HA production were respectively analyzed by Alamar Blue assay and ELISA. Time-dependent modifications grouped molecules in three peculiar patterns: one reaching the highest concentrations within 18 h and decreasing afterwards, another progressively increasing up to 168 h, and the last peaking at the central time points. Synovial fibroblast growth in response to L-PRP and P-PRP revealed differences over time and among added concentrations. Both preparations displayed a preserved supporting capacity of HA synthesis.

Immune cell evaluation could be useful for clarifying etiopathogenesis, providing a support for formulating the diagnoses of clinically similar joint pathologies or guiding indications for possible therapeutic targets. To contribute to differential diagnosis in joint pathologies we performed an immunophenotypical profile analyzing different immune cells in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA).The Krenn and immunologic synovitis (IMSYC) scores, which include the evaluation of T lymphocytes (CD3 positive), B lymphocytes (CD20), endothelial cells (CD31), macrophages (CD68) and proliferating cells (Ki-67 positive) were used to analyze the synovial tissue samples. Moreover, to corroborate immune activation, neutrophils (CD15 positive), NK cells (CD56 positive), plasma cells (CD138 positive), IgG4 and IgG4 secreting-CD138 cells were analyzed using immunohistochemical techniques.We confirmed that all the samples had a high synovitis score according to both the Krenn and IMSYC scores. In both the RA and OA groups, we found similar scores for CD3 (T lymphocytes), CD20 (B lymphocytes), CD31 (endothelial cells), CD56 (NK cells), CD68 (macrophages) CD138 (plasma cells) and IgG4. In contrast, CD15 (neutrophils) was significantly higher in RA compared to OA. Interestingly, IgG4 secreting-CD138 cells were significantly higher in RA than OA, even if CD138 had the same score in both the RA and OA samples.This study found that the scores for different immune cells were similar in both RA and OA synovial tissue with a high synovitis score. CD15 and IgG4 secreting-CD138 were the only immune cells with a higher score in RA compared to OA, suggesting a potential use for discriminating among pathologies with a high synovitis score.

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Background Several studies have identified a central role for the activation of the complement system in the pathogenesis of OA, finding an abnormally high expression and activation of complement in human osteoarthritic joints and the specific contribution of complement (C) components 5 and 6 and the membrane attack complex (MAC) (1). The complement system consists of several proteins which contribute to an enzymatic cascade ending in lysis. Two main, different pathways converge into C3 factor activation. In the classical pathway C4 is involved, while in alternative pathways Factor-b (FB) is involved. Objectives The aim of this study was to investigate the expression of complement factors (C3, C4 and FB) and activation fragments (C3a, C5a, FBa, MAC) in OA joint tissues. Methods Osteochondral biopsies from 33 patients (16 OA, 12 Rheumatoid Arthritis-RA and 15 Postraumatic cases-PT) who underwent knee, hip or shoulder arthroplasty were collected. Immunohistochemistry and quantitative Imaging analysis were perfomed to analyze C3, C4 and CFB expression. Cartilage pathology was evaluated utilizing Mankin score. Chondrocytes, synoviocytes, cartilage and synovial biopsies were obtained from 11 OA patients undergoing joint replacement surgery. Cultures were set up with or without pro-inflammatory (IL-1beta) stimulus. Culture supernatants were analyzed for C3a, C5a, CFBa and C5b-9 production using enzyme-linked immuno-sorbent assay (ELISA). Statistical analysis was performed utilizing Wilcoxon matched pairs test and Mann Whitney’s test, using Graph Pad Prism software. Results In osteochondral biopsies, C factor expression was located in bone marrow and in a few subchondral bone cells. C3 was the most expressed factor in all patient subtypes while factor C4 had a very low expression, except for some sporadic cases. (OA: C3 vs FactorB p=0.0280, C3 vs C4 p=0.0005; RA: C3 vs FactorB p= 0.0269, C3 vs C4 p=0.0005; PT: C3 vs FactorB p=ns, C3 vs C4 p=0.0001) CFB was significantly augmented in PT and was not significantly different between OA and RA. C factors expression did not correlate with Mankin score or with the joint type (knee, hip or shoulder). Cell positivity was mainly present in the subchondral bone (bone marrow) whereas cartilage was almost negative. As to activation fragments, C3a was the most produced factor (vs C5a and CFBa p Conclusion Complement factors were expressed mainly in bone marrow and in a few subchondral bone cells, but not in cartilage. Complement activating fragments are produced by chondrocytes and synoviocytes. The dissimilar expression of activating fragments by different cell types could suggest various C activating mechanisms in joint tissues. Reference [1] Wang Q, et al. Nat Med 2011;17:1674-1679. Disclosure of Interests None declared

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500kHz to 5MHz) and for a low-frequency value (38kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biologicalin vitrotests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days.

Summary Introduction We describe an unusual case of left ventricular myxoma associated with deep vein thrombosis (DVT). Materials and methods A 79-year-old woman was admitted with bilateral proximal DVT that developed while she was on warfarin. The anticoagulant therapy had been started (after 2 weeks of low-molecular weight heparin) 1 month earlier in a Cardiologic Unit, where the patient had been treated for “left ventricular thrombosis associated with acute pericarditis”. After discharge, the patient continued to experience malaise, recurrent dizziness, dyspnea, flushing, and progressively severe leg swelling despite transthoracic echocardiography (TTE) findings of normal left ventricular function. She was finally admitted to our Unit for ultrasound studies for possible DVT. On admission the INR was in the therapeutic range with increased C-reactive protein (CRP), thrombocytopenia, mild anemia, anti-PF4 antibodies, lupus anticoagulant (LAC), a positive direct Coombs test, and mildly increased anti-platelet and anticardiolipin antibody levels. Malignancy, SLE, and inherited thrombophilia were excluded by a broad-spectrum instrumental and laboratory investigation. Since heparin-induced-thrombocytopenia could not be excluded, the warfarin was replaced with fondaparinux (7.5 mg/day). Given the persistence on TTE of a “small floating mass” at the left ventricular apex, we suspected the previously diagnosed thrombosis was actually an unusually located myxoma, and this diagnosis was confirmed by CT and MRI. The patient also had markedly increased serum levels of interleukin 6 (IL-6), regarded by many as a marker of cardiac myxoma and the source of the constitutional symptoms and immunologic features (“activity”) of these lesions. The patient is now well except for recurrent flushing episodes. The IL-6 level is still high although the ESR, CRP, LAC have normalized. Since isolated IL-6 increases are associated with recurrence in PMR patients, we consider our patient at risk for relapse of systemic illness. Results Three cases of cardiac myxoma associated with DVT have been reported. Myxoma production of IL-6 could explain the extracardiac symptoms and the inflammatory/autoimmune activation that caused the DVT. Discussion Although histologic confirmation is lacking, the features of this case strongly suggest an atypical-site myxoma, in particular the concordant findings generated by the various diagnostic exams and unchanging nature of the ventricular lesion during follow-up.

Objective To investigate the modulation of systemic levels of soluble interleukin-6 receptor (sIL-6R) and soluble gp130 (sgp130) in untreated and treated polymyalgia rheumatica (PMR) patients during a followup period of at least 24 months in order to evaluate the relationship of these molecules with clinical outcome and their feasibility to provide a prognostic tool in clinical practice. Methods We analyzed sIL-6R and sgp130 serum levels in 93 PMR patients, and 46 age-matched normal controls, at disease onset and at 1, 3, 6, 12, and 24 months of followup during corticosteroid therapy by enzyme-linked immunosorbent assay. Results No difference in sIL-6R and sgp130 levels was observed between PMR patients and normal controls at disease onset or during followup. A significant correlation was found between the number of relapses and sIL-6R concentrations at baseline and after 1, 3, and 12 months of therapy. No correlation was found between sgp130 levels and the number of relapses. Cox multivariate analysis indicated that the best model for predicting relapses was identified by sIL-6R levels and the hemoglobin value at baseline. We found that high sIL-6R levels combined with low hemoglobin values resulted in a 10.1-fold increased risk of relapse. Conclusion Our data support the identification of a potential prognostic marker of PMR outcome that might have important implications in clinical practice. Because targeting sIL-6R with blocking antibodies has proven useful in other rheumatic disorders, our results could suggest the opportunity to evaluate sIL-6R–blocking treatment in patients with PMR and elevated levels of sIL-6R at disease onset.

Background: The cell membrane is a primary and fundamental player in most cellular processes, and fatty acids form a major structural component of cell membranes. The aim of this study was to compare the membrane fatty acid profiles of different human blood leukocytes and selected cell lines, to identify the effects of in vitro culture on fatty acid profiles, and to test medium supplements for their effect on fatty acid profiles.Methods: Different classes of leukocytes were isolated from human blood and their membrane fatty acid profiles were analysed and compared. After culturing in vitro immortalised and primary leukocytes, membrane fatty acids were analysed and compared. Finally, different lipid formulations were developed and used for supplementing leukocytes in vitro in an effort to maintain the in vivo fatty acid profile. Descriptive and analytical tests were performed to compare the obtained fatty acid profiles.Results: Membrane fatty acid profiles of primary human CD4+ T-lymphocytes, CD8+ T-lymphocytes, B-lymphocytes and monocytes differed. Moreover, there were differences among Jurkat, Raji and THP-1 cell lines and the corresponding primary leukocyte classes, as well as between freshly prepared and in vitro cultured primary lymphocytes. A lipid supplement was able to maintain cultured Jurkat cells with a membrane fatty acid profile almost identical to that of the primary CD4+ T-lymphocytes. Finally, variations in the lipid supplement composition enabled the development of Jurkat cells with different membrane fatty acid profiles characterising different physiological or pathological human conditions.Conclusions: Each leukocyte class has its own specific membrane fatty acid profile in vivo. Cultured primary leukocytes and immortalized leukocytic cells display different membrane fatty acid profiles when compared to their respective in vivo counterparts. The membrane fatty acid composition of cultured cells can be restored to reflect that of the corresponding in vivo condition through use of optimised lipid supplementation. Typical physiological or pathological leukocyte membrane fatty acid profiles can be obtained by tuning in vitro fatty acid supplementation.

Serum interleukin-6 (sIL6) is an acknowledged predictor of all-cause mortality in older age. A common G/C polymorphism has been identified at position −174 of the IL6 gene promoter (IL6−174G>C), but its associations with sIL6 and mortality are still unclear. Data from a population-based elderly cohort (n = 824) were used to study the associations of baseline sIL6 with the IL6−174 C-allele (C+) carrier status and all-cause mortality at 4 years, in the presence and absence of preexisting major diseases (PMD). Analyses were adjusted for socio-demographic factors and body-mass-index. Three-hundred-eighty-eight participants (47.1%) had PMD. Compared to the bottom sIL6 quartile, mortality increased both in presence [Hazard Ratio (HR) = 3.04; 95% confidence interval (CI): 1.48–6.25] and absence of PMD [HR = 3.91; 95%CI: 1.42–10.72] for the third higher sIL6 quartile, but only in presence of PMD for the top sIL6 quartile [HR = 2.30; 95%CI: 1.09–4.83]. In absence of PMD, C+ carrier status did not affect both sIL6 and mortality. In presence of PMD, C+ carrier status was associated with increased baseline sIL6 [odds ratio 2.01; 95%CI: 1.25–3.22, for all sIL6 quartiles above the bottom] but not with increased mortality risk. A survival advantage was even found for C+ carriers with PMD and sIL6 in the top quartile [HR = 0.31, 95%CI: 0.13–0.76]. In conclusion, although associated with increased sIL6 levels in presence of major diseases, the IL6−174 C-allele does not seem to have direct detrimental effects on survival in older age.

Objective. To evaluate in vivo expression of chemokine receptors in cartilage tissue samples from healthy and diseased joints. Methods. Presence and distribution of several chemokine receptors in cartilage samples from patients with osteoarthritis (OA) or inflammatory arthritis (IA) and from multi-organ donors were assessed by immunohistochemistry. The expression of messenger RNA (mRNA) for chemokine receptors was also analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results. Normal and OA-affected cartilage showed a moderate to high expression of chemokine receptors, while staining of IA samples ranged from low to absent. Differences between OA and IA samples were present for all receptors but CCR2 and CXCR4. Moreover, mRNAs for CCR1, CCR5 and CXCR1 were found both in normal and pathological chondrocytes, suggesting that chemokine receptor down-modulation seen in IA samples could be a post-transcriptional event. Conclusion. Data on normal and pathological chondrocytes underline the role of chemokines in cartilage homeostasis and suggest an imbalance towards catabolic processes in inflammatory conditions.

While numerous previous studies have investigated age-related changes of cytokine production, little is known about chemokines, the importance of which in regulating immune response is becoming increasingly evident. In this study, a group of healthy subjects over 90 years old is compared to a group of young subjects, we evaluated the ability of monocytes, T lymphocytes and NK cells: (1) to produce RANTES and MIP-1α, either in basal conditions or after stimulation with, respectively, LPS, anti-CD3 MoAb and IL-2; (2) to express the corresponding chemokine receptors (CCR1, CCR3, CCR5). We demonstrate that: (a) monocytes, T lymphocytes and NK cells spontaneously produced detectable amounts of chemokines, both in young and old subjects; (b) monocyte-dependent RANTES and MIP-1α production induced by LPS was up-regulated in nonagenarian subjects as anti-CD3-induced secretion from T cells; (c) RANTES and MIP-1α production by IL-2 stimulated NK cells was reduced in elderly subjects; (d) CCR1, CCR3 and CCR5 were widely expressed on monocytes, but less expressed on T lymphocytes and NK cells. The diversity within PBMC might reflect their different states of activation and/or responsiveness, influencing the ability to develop rapid innate and long-lasting adaptive immune responses.

Osteoarthritis and rheumatoid arthritis are characterized by focal loss of cartilage due to an up-regulation of catabolic pathways, induced mainly by pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumour necrosis factor alpha (TNFalpha). Since reactive oxygen species are also involved in this extracellular-matrix-degrading activity, we aimed to compare the chondrocyte oxidative status responsible for cartilage damage occurring in primarily degenerative (osteoarthritis) and inflammatory (rheumatoid arthritis) joint diseases. Human articular chondrocytes were isolated from patients with osteoarthritis or rheumatoid arthritis, or from multi-organ donors, and stimulated with IL-1beta and/or TNFalpha. We evaluated the oxidative stress related to reactive nitrogen and oxygen intermediates, measuring NO(-)(2) as a stable end-product of nitric oxide generation and superoxide dismutase as an antioxidant enzyme induced by radical oxygen species. We found that cells from patients with osteoarthritis produced higher levels of NO(-)(2) than those from patients with rheumatoid arthritis. In addition, IL-1beta was more potent than TNFalpha in inducing nitric oxide in both arthritides, and TNFalpha alone was almost ineffective in cells from rheumatoid arthritis patients. We also observed that the intracellular content of copper/zinc superoxide dismutase (Cu/ZnSOD) was always lower in rheumatoid arthritis chondrocytes than in those from multi-organ donors, whereas no differences were found in intracellular manganese SOD (MnSOD) or in supernatant Cu/ZnSOD and MnSOD levels. Moreover, intracellular MnSOD was up-regulated by cytokines in osteoarthritis chondrocytes. In conclusion, our results suggest that nitric oxide may play a major role in altering chondrocyte functions in osteoarthritis, whereas the harmful effects of radical oxygen species are more evident in chondrocytes from patients with rheumatoid arthritis, due to an oxidant/antioxidant imbalance.

The function of chemokines in promoting and modulating leukocyte migration is essential for a prompt and efficacious inflammatory response and in host defence against infections. In order to investigate whether this important aspect of immunological response is influenced by ageing, we evaluated the basal levels as well as the ability of peripheral blood mononuclear cells from young and healthy elderly subjects to produce chemokines (IL-8, MCP-1, MIP-Ialpha, RANTES) in response to stimulation with anti-CD3 monoclonal antibody and lipopolysaccharide (LPS), a gram negative bacterial endotoxin. Our main findings are a spontaneous chemokine production; a 20% decrease of proliferative response to anti-CD3 monoclonal antibody accompanied by an age related increase of MIP-Ialpha and RANTES production and by a general increase of all chemokine production compared to unstimulated conditions; a proliferative defect of monocytes to LPS challenge associated with an increase of chemokine production compared to basal conditions with a progressive age-related increase of MIP-lalpha. In conclusion, this study suggests that chemokines could have a compensatory role in balancing the impaired mechanisms involved in 'specific' immune response during ageing. The successful activation of this strategy could contribute to the good performance of immune system so maintaining healthy status in elderly.

Background In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β. Methodology/Principal Findings The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13. Conclusions/Significance Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.

Un numero sempre crescente di evidenze sottolinea l’importante contributo dell’angiogenesi nell’ambito dei meccanismi che inducono le modificazioni articolari caratteristiche dell’osteoartrosi (OA). Nei pazienti con OA, alcune molecole correlate al processo angiogenetico, quali vascular endothelial growth factor (VEGF), vascular cell adhesion molecule-1 (VCAM-1) e fibroblast growth factor-2 (FGF-2) sono espresse a livello articolare e possono avere un ruolo non solo in relazione all’angiogenesi, ma anche all’omeostasi cartilaginea. Questo studio si propone di valutare i livelli circolanti di VEGF, VCAM-1 e FGF-2 in pazienti con OA della mano affetti da forma erosiva (OA-E) e non-erosiva (OA-NE) e di determinare le eventuali associazioni con il danno articolare valutato tramite analisi radiologica convenzionale. Sono stati analizzati, con metodo ELISA, i sieri di 146 pazienti con OA-E, di 148 pazienti con OA-NE e di 148 soggetti di controllo (CN). I livelli circolanti di sVCAM-1 nei pazienti con OA-E sono risultati più elevati rispetto a quelli evidenziati nei CN; non sono state dimostrate differenze tra i pazienti con OAE e i pazienti con OA-NE. L’analisi di VEGF e FGF-2 non ha evidenziato differenze significative tra pazienti con OA e CN, inoltre non sono emerse correlazioni tra i livelli circolanti di VEGF, sVCAM-1 e FGF-2 e parametri radiologici e clinici. I dati ottenuti, dimostrando un aumento dei livelli circolanti di sVCAM-1 nei pazienti con forma erosiva di OA della mano, caratterizzata da una peggiore decorso e maggiore aggressività, aggiungono nuove informazioni circa la possibilità di identificare sVCAM-1 come biomarcatore di severità di malattia.

Background Large vessel vasculitides (LVV) – Takayasu Arteritis (TA) and Giant Cell Arteritis (GCA) are inflammatory diseases of the aorta and its main branches. A cytokine cluster involving the IL-6 – IL-17 axis as well as another one centring on the IL-12-IFN-γ axis are involved in LVV pathogenesis. (1) Disease activity could be evaluated by measuring ESR and CRP as well as by using imaging techniques (Doppler Ultrasound, Computed Tomography scan, Magnetic Resonance Imaging or Positron Emission Tomography scan – CT/PET) and specific scoring systems (Indian Takayasu9s Arteritis Activity Score - ITAS and Kerr/National Institutes of Health index - Kerr / NIH) . To date, angiogenic factors have been evaluated but specific biomarkers for disease activity are still lacking (2) Objectives The aim of our study was to evaluate angiogenic and anti-angiogenic factor expression in LVV, in order to evaluate their role as biomarkers. Methods We enrolled 67 patients with LVV [TA (40, 59.7%) or GCA (27, 40.3%)] and 50 Normal Controls (NC). Angiogenic (angiopoietin-1, angiopoietin-2, Fibroblast Growth Factor2 – FGF-2, Vascular Cell Adesion Molecule1 – VCAM-1, Vascular Endothelial Growth Factor – VEGF) and anti-angiogenic (angiostatin, Endostatin-1, Pentraxin 3 – PTX3) factors were analysed using enzyme-linked immuno-sorbent assay (ELISA). Disease activity was evaluated according to Kerr (≥2) and ITAS (≥1) scores, only 47 (70.1%) patients underwent CT/PET. Serum levels of angiogenic and anti-angiogenic factors were compared among active LVV, inactive LVV and NCs. When evaluating disease activity according to CT/PET, it was considered active if SUV >2. Statistical analysis was performed utilizing Mann Whitney, Kruskal Wallis for multiple comparison and Dunn9s post hoc test, using Graph Pad Prism software. Results No significant differences were found between LVV and NCs in angiogenic factors. Conversely, anti-angiogenic factors were all significantly increased in LVV (both GCA and TA) in comparison with NCs (angiostatin: TA vs NC, p Conclusions Our data show an imbalance between angiogenic and anti-angiogenic factor levels in serum. No disease activity related changes were observed for these factors. References Weyand CM, Goronzy JJ. Immune mechanisms in medium and large-vessel vasculitis. Nat Rev Rheumatol 2013;9:731–740; Villa I, Bilbao MA, Martinez-Taboada VM. Advances in the diagnosis of large vessel vasculitis: identification of biomarkers and imaging studies. Rheumatol Clin 2011;7:S22-S27 Disclosure of Interest None declared

OBJECTIVES: To investigate the clinical associations of hand osteoarthritis (HOA) and their relationships with radiographic features. METHODS: A total of 446 patients with hand osteoarthritis (HOA; 233 with erosive HOA (EHOA) and 213 with non-EHOA) and 307 controls were evaluated. Demographic and clinical data from patients and controls were recorded based on medical records/clinical reports and an anamnesis of drug consumption. Posteroanterior radiographs of both hands were obtained from all HOA patients and were assessed using the Kellgren and Lawrence (K&L) and Kallman scoring systems. RESULTS: After adjustment for age, gender, and body mass index (BMI), HOA patients showed a significantly increased odds ratio (OR) for hypercholesterolaemia [OR 2.10, 95% confidence interval (CI) 1.39-3.16, p < 0.0005] and autoimmune thyroiditis (OR 4.85, 95% CI 1.77-13.29, p = 0.002), as well as for knee (OR 1.63, 95% CI 1.09-2.44, p = 0.018) and hip OA (OR 1.87, 95% CI 1.07-3.27, p = 0.029). No significant increase for systemic hypertension, ischaemic heart disease, and diabetes mellitus was found. Patients with EHOA and non-EHOA showed similar risks for the above-mentioned co-morbidities. A similar occurrence of clinical associations was also observed in patients with HOA alone and in those with generalized OA. No association between radiographic scores and clinical associations was observed. CONCLUSIONS: Patients with HOA present a direct association with hypercholesterolaemia (and autoimmune thyroiditis) but do not show increased ischaemic cardiovascular manifestations compared to controls. No significant association between radiographic scores and co-morbidities was found.

Objective To characterize the clinical and radiographic joint phenotype in erosive hand osteoarthritis (EHOA) and non-EHOA. Methods A total of 446 patients with HOA (233 with EHOA and 213 with non-EHOA) were evaluated. Demographic (sex and age at disease onset), clinical (body mass index and distribution of nodes), and radiographic features (Kellgren/Lawrence and Kallman's scores obtained from radiographs of both hands) from all patients were recorded. Results Patients with EHOA had a significantly earlier disease onset. Clinical and radiographic distribution of structural damage in the distal interphalangeal (DIP), proximal interphalangeal (PIP), and first carpometacarpal joints was similar in EHOA and non-EHOA. EHOA patients showed higher percentages of nodes and more severe radiographic scores; the more severe radiographic score of joints with nodes was due to both osteophytes and joint space narrowing (JSN). A direct correlation between osteophytes and JSN scores was observed. Central erosions (CE) were more prevalent in the DIP joints than in the PIP joints. Gull-wing pattern of CE was prevalent in the DIP joints, whereas saw-tooth pattern was prevalent in the PIP joints. Marginal erosions (ME) were present in 100% of EHOA patients and in 80% of non-EHOA patients. An ordinal correlation between the presence of ME and osteophyte score was found. Conclusion We found quantitative, but not topographic, differences in structural damage between EHOA and non-EHOA. Heberden's nodes, severe radiologic scores, and CE were concentrated in the second, third, and fifth DIP joints of both hands. ME were also present in the majority of non-EHOA patients.

Objective: IL-13/IL-4/IL-4R system has strong chondroprotective activity. We investigated polymorphisms in these genes as potential hand osteoarthritis (OA) susceptibility loci by performing a case-control association study. Methods: Eighteen common single nucleotide polymorphisms (SNPs) (nine in IL-4R, five in IL-4 and four in IL-13) were genotyped in 403 patients (380 females) with hand OA and 322 healthy controls (308 females). Results: Two SNPs (rs1805013 and rs1805015), mapping to the IL-4R gene, were associated with P-values of 0.0116 and 0.0305 respectively in the whole sample. As far as the non-erosive hand OA group (n= 159) is concerned, the significance level of association of SNP rs1805013 is increased. After correction for multiple testing (correction for the 54 tests) the significance was not retained.None of the IL-13 SNPs analyzed showed association with hand OA. Some of the analyzed SNP within the IL-4 gene showed significant association with hand OA only when considering subgroups of patients. With respect to the CMC1 OA group, two SNPs in IL-4 (rs2243250 and rs2243274) showed association with a P-value of 0.027 and 0.018 respectively. None of these associations remained after correction for multiple testing. Conclusions: The present study shows a trend to an association between non-erosive hand OA in Caucasian population and a genetic variant in the coding region of IL-4R gene. Our results, in keeping with previous data on hip OA, confirm the suggestion that IL-4/. IL-4R system plays a role in OA pathogenesis. Further confirmation studies on different populations are necessary. © 2010 Osteoarthritis Research Society International.

Background Osteoarthritis (OA) is characterized by the degradation and breakdown of cartilage and by chronic, low-grade inflammation. Even though multiple factors are probably active in this process, recent evidence has implicated the innate immune system as being principally responsible and, in particular, the roles of synovial macrophages and the complement system. Furthermore, a recent study has identified a central role for the activation of the complement system in the pathogenesis of OA, finding an abnormally high expression and activation of complement in human osteoarthritic joints and the specific contribution of complement components 5 and 6 (C5, C6) and of the membrane attack complex (MAC) (1). The complement system consists of several proteins which contribute to an enzymatic cascade ending in lysis. Two main, different pathways converge into C3 factor activation. In the classical pathway C4 is involved, while in alternative pathway Factor-b is involved. Objectives The aim of this study was to investigate complement involvement in OA articular tissues (cartilage and synovium), focusing in particular on the expression of factors involved in the classical or alternative pathway: complement component 3 (C3), C4 and complement factor-b. Methods Chondrocytes, cartilage biopsies and synoviocytes were obtained from OA patients undergoing joint replacement surgery. Chondrocyte high density, synoviocytes and cartilage biopsy cultures were set up with or without pro-inflammatory (IL-1β) stimulus. RNA was extracted from chondrocyte cultures, cartilage biopsies and synoviocytes and real time PCR analyses of factor-b, C3 and C4 genes were performed. Culture supernatants were analysed for C5b-9 complex production using enzyme-linked immuno-sorbent assay (ELISA). Results All complement factors analyzed were expressed both in OA chondrocytes and in OA synoviocytes. Regarding the chondrocytes (high density culture and cartilage explants n=11), factor-b gene expression was significantly higher than C3 and C4 (Wilcoxon matched pairs test p=0.0046 and p=0.0010, respectively). No difference between C3 and C4 expression was found. Furthermore, factor-b and C3, but not C4 gene expression was significantly induced by IL-1β stimulation (p=0.0425 and p=0.0010, respectively). In cultured synoviocytes (n=6), also, Factor-b and C3, but not C4, were significantly induced by IL-1β stimulation (p=0.056 and p=0.0313, respectively). Preliminary data on C5b-9 ELISA quantification showed no presence of the protein in synoviocyte culture supernatants. On the other hand, a large amount of this complex was detected in the supernatants of OA cartilage biopsies. Conclusions Complement factors are expressed by OA articular tissues and their expression is further increased by inflammatory stimulus. Moreover, the enhanced basal production of factor-b and the different modulation by IL-1β of the production of the complement factors suggests a major involvement of the alternative pathway of complement activation. References Wang Q. et al, Identification of a central role for complement in osteoarthritis, nature medicine 2011 Disclosure of Interest None declared

Background A role for interleukin-6 (IL-6) in the pathogenesis of large-vessel vasculitides (LVV) has been suggested by previously reported results indicating increased serum IL-6 levels in patients compared to healthy controls (1,2). In addition, IL-6 relevance in LVV is also supported by genetic evidence, as highlighted by a very recent study reporting an association of specific IL-6 gene polymorphism with Takayasu9s arteritis (TA) susceptibility (3). IL-6 mediates its functions through two membrane proteins: the IL-6 receptor (IL-6R) and gp130, the signal transducing element. Soluble form of IL-6R (sIL-6R), after binding with IL-6, is able to associate to membrane gp130 and mediate intracellular signalling in IL-6R negative cells. The biological activity of IL-6 strictly depends on the interrelationship occurring among these molecules and the role of sIL-6R as an inflammatory biomarker has been recognized in several diseases. Objectives Since, to date, reliable biomarkers for assessing clinical activity in LVV are lacking, we aimed to investigate serum levels of IL- 6 and IL-6R in patients with Giant Cell Arteritis (GCA) and TA, in order to evaluate their relationship with disease activity assessed by 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG PET/CT) and by clinical indices including National Institutes of Health-NIH/Kerr criteria and Indian Takayasu Activity Score (ITAS). Methods Sera were obtained from 72 patients with LVV (33 GCA and 39 TA) who underwent a PET/CT scan and from 60 age-matched normal controls (NC). PET/CT scans were reviewed by a nuclear medicine physician who had no knowledge of the clinical information. Vascular uptake was graded using a 4 point semiquantitative scale. ITAS, Kerr/NIH scores and sera for serum biomarker investigations were obtained within 20 days of the PET/CT scans.IL-6 and sIL-6R serum levels were evaluated using commercial DuoSet ELISA kits (R&D Systems) following the manufacturer9s instruction. Results IL-6 and sIL-6R serum levels were significantly higher in both GCA and TA patients compared to NC (IL-6: GCA vs NC p Conclusions IL-6 and sIL-6R were elevated in large-vessel vasculitis (TA and GCA) compared to NC, but neither soluble factor appeared to be associated with disease activity assessed both by PET/CT scan and clinical scores (Kerr-NIH and ITAS scores). References Park MC et al., Rheumatology 2006. 2. Alibaz-Oner F et al., Clinical and Experimental Rheumatology, in press. 3. Renauer P et al, Arthritis Rheumatol 2015 Disclosure of Interest None declared

Background In large-vessel vasculitides (LVV), the hypoxic enviroment subsequent to stenosis of the vascular lumen is a potential signal for the generation of new blood vessels. Angiogenesis, the formation of newly-formed capillaries from pre existing vessels, may be a compensatory response to ischemia and to increased metabolic activity. Furthermore endothelial cells of newly formed vessels can cause inflammation due to the production of colony stimulation factors and chemokines for leukocyte recruitment. Angiogenesis also plays a key role in the patogenetic events leading to cronic inflammation. Angiogenic factors activate endothelial cells promoting their proliferation and migration, they also induce capillary tube formation. The balance between angiogenic and antiangiogenic factors induces and regulates the angiogenic process (1). Objectives The purpose of this study was to analyze the amount of the angiogenic factors Vascular Endotelial Growth Factor (VEGF), FGF-2, Vascular Adesion Molecules 1 (VCAM-1), angiopoietin-1, anti-angiogenic factor endostatin and pentraxin-3 (PTX3) in the serum of patients with Giant Cell Arteritis (GCA) and Takayasu9s arteritis (TA) and to correlate biomarker serum levels to: PET/TC findings, clinical indices including Indian Takayasu activity score (ITAS) and Kerr/National Institute of Health, age-matched normal control (NC) biomarker serum levels. Methods Sera were obtained from 72 patients with LVV (33 GCA patients and 39 TA) who underwent a PET/CT scan and from 60 age-matched normal controls (NC). PET/CT scans were reviewed by a nuclear medicine physician who has no knowledge of the clinical information. Vascular uptake was graded using a 4 point semiquantitative scale. ITAS, Kerr/NIH scores and sera for serum biomarkers investigations were obtained within 20 days of the PET/CT scans. Angiogenic factor serum levels were evaluated using commercial DuoSet kits (R&D Systems) following the manufacturer9s instructions. Results Endostatin and PTX-3 serum levels were significantly higher compared to age-matched NC (Endostatin: GCA vs NC p Conclusions The results highlight an imbalance in the production of positive and negative regulatory factors in the angiogenic response in LVV. In addition, VEGF seems to differentially contribute to the definition of the GCA and TA angiogenic profiles. References Maruotti N et al, Clin Exp Rheumatol 2008 Disclosure of Interest None declared

Summary Objective To test the importance of the interleukin-4 (IL-4)/IL-4 receptor (IL-4R) system in osteoarthritis (OA) we evaluated soluble IL-4R (sIL-4R) levels in sera of patients with different forms of OA and healthy individuals. Methods We recruited: 141 patients with hand OA, 70 with nodal and 71 with erosive hand OA; 64 patients undergoing total joint replacement, 34 with hip and 30 with knee OA; and 38 ethnically and geographically age-matched healthy individuals [normal controls (NC)]. Results Serum sIL-4R concentration was found to be significantly higher in all OA patients than that in NC. When patients were divided into four subgroups (nodal, erosive, hip and knee OA) significant differences were present when comparing NC with each subgroup. This was true also when small-joint OA groups were compared with large-joint OA groups, the latter being associated with higher IL-4R levels. Conclusions We found increased levels of sIL-4R in OA patients compared with healthy individuals. We speculate that this reduces availability of IL-4, and its effects on chondrocytes.

Evaluation of the role of VEGF in cartilage pathophysiology.VEGF release from chondrocytes in the presence of IL-1beta, TGFbeta and IL-10 was detected by immunoassay. VEGF receptor -1 and -2 expression and VEGF ability to modulate caspase -3 and cathepsin B expression were detected by immunohistochemistry on cartilage biopsies and cartilage explants. VEGF effects on chondrocyte proliferation was analysed by a fluorescent dye that binds nucleic acids.VEGF production by osteoartritis (OA) chondrocytes was significantly reduced by IL-1beta while it was increased in the presence of TGFbeta. Cartilage VEGFR-1 immunostaining was significantly downregulated in 'early' OA patients compared to normal controls (NC). VEGFR-2 expression was negligible both in OA and in NC. VEGF decreased the expression of caspase-3 and cathepsin B, whereas it did not affect proliferation.VEGF is able to down-modulate chondrocyte activities related to catabolic events involved in OA cartilage degradation.

To test a hypothesis of compartmentalized pathogenesis of different types of arthritis, namely inflammatory arthritis (IA) and osteoarthritis (OA), synovial and cartilage biopsies were examined for the expression of TNF and IL-1 receptors. In cartilage, we found constitutive expression of all receptors in normal tissues, and decreased expression of signal-transducing receptors in pathological chondrocytes. In synovium, there was a lower expression of signal-transducing receptors in cases of OA compared to those of IA. In OA, the three signal-transducing receptors were more abundantly expressed in cartilage, while in IA they were mainly present in synovial tissue (TNFRp75 being expressed more than p55). IL-1 decoy receptor type II was low or absent in synovial tissues, but present in cartilage. The increased expression of TNFRp75 and IL-1RI in OA cartilage, compared to IA, in addition to the abundant local cytokine production, strengthens the hypothesis of autocrine/paracrine action by inflammatory cytokines in the pathogenesis of cartilage damage.

Recently, novel members of the TNF/TNF receptor superfamily, receptor activator of nuclear factor- kappa B ligand (RANKL), its receptor RANK, and the decoy receptor osteoprotegerin (OPG), have been identified as paracrine mediators of both the immune system and bone functions. The balance of RANK/RANK-L and OPG is critical for osteoclastogenesis modulation and physiological bone remodeling. In order to evaluate whether RANKL/OPG balance is modified by ageing, we analyzed, by imunoassay, systemic levels of OPG and sRANKL in healthy elderly subjects (age range from 70 to over 90 years) and in patients affected by two age-related diseases, osteoarthritis (OA) and polymyalgia rheumatica (PMR), characterized by bone metabolism alteration and involvement of the immune system. We demonstrated that (a) plasma concentrations of OPG increased significantly with age; (b) conversely, sRANKL significantly declined in the group of subjects aged between 81 and 90 years, being similar to the young controls in the other age groups; (c) in OA and PMR, circulating OPG did not differ from plasma levels found in age-matched control groups, while sRANKL concentration was significantly increased compared to controls. Hence, in ageing, the sRANKL/OPG system appears to be modified, with prominent changes in circulating OPG levels; in OA and PMR, the sRANKL/OPG balance alteration was shown to be mainly due to the increase of plasma sRANKL concentration.

A gradual decline in the functional activity of the immune system is described with advancing age. The adaptive immune system seems the most severely affected, but some age-associated modifications also occurs in NK cells. Several studies investigated the age related changes of cytokine production, while little is known about chemokines, whose importance in regulating immune-response becomes even more evident. In this study we investigated whether the ability of T lymphocytes and NK cells to produce IL-8, either spontaneously or after activation, respectively with anti-CD3 monoclonal antibody or interleukin 2 (IL-2) was affected by age. We demonstrated that: (a) T lymphocytes and NK cells spontaneously produced detectable amounts of IL-8; (b) anti-CD3 stimulation of T lymphocytes significantly increased IL-8 production and the increment was more evident in the nonagenarian subjects; (c) similarly, IL-2 stimulation of NK cells rose the production of IL-8 but the amount produced by the old was lower than the one produced by the young group. Because of the co-stimulatory role of chemokines on NK responses and given the demonstrated importance of NK cells in defence against viral infections, the decreased production of IL-8 can be involved in the defective functional activity of NK cells from old subjects.

Objective. To evaluate peripheral production and synovial expression of vascular endothelial growth factor (VEGF) in polymyalgia rheumatica (PMR). Methods. Circulating levels of VEGF in PMR (serum concentration and in vitro release by peripheral blood mononuclear cells [PBMC]) were investigated by enzyme-linked immunosorbent assay. Local expression of VEGF in shoulder synovial tissue was investigated by immunohistochemical analysis. Investigations were performed in patients with active, untreated disease and in patients treated with corticosteroids. Results. VEGF serum concentrations were significantly higher in untreated PMR patients than in normal control subjects. During steroid treatment, VEGF serum concentrations reached their lowest level after the sixth month of treatment. PBMC isolated from untreated PMR patients spontaneously secreted a higher amount of VEGF compared with PBMC from control subjects. Corticosteroid therapy did not affect the ability of PBMC to produce VEGF. Immunohistochemical staining performed on shoulder synovial tissue showed VEGF expression in both the lining layer and the sublining area. In 3 of 4 treated patients, no VEGF staining was found in synovial tissue during corticosteroid therapy. VEGF expression correlated with vessel density, but was not associated with avb3 and avb5 integrin expression. Conclusion. Peripheral and local VEGF releases have different responses to steroid treatment in PMR. The lack of response to corticosteroids by peripheral VEGF production supports the hypothesis that systemic involvement is dominant in PMR. At the synovial level, VEGF production is linked to vascular proliferation and is thus directly involved in the pathogenesis of synovitis.

Objective. To investigate systemic in-flammation and autoimmune response to citrullinated peptides in patients with erosive and non erosive “lone” hand osteoarthritis (HOA) with no hip/knee involvement and their relationship with radiographic structural damage. Methods. Sera were obtained from a total of 99 patients with HOA (52 pa-tients with erosive HOA and 47 pa-tients with non-erosive HOA) and from 50 control subjects (NC). Hand radio-graphs were obtained from all patients and scored for joint damage according to the Kellgren-Lawrence and the Kall-man scores. Serum levels of high-sen-sitivity CRP (hsCRP), IL-6, pentraxin-3 (PTX-3), anti-CCP and anti-modified citrullinated vimentin (MCV) antibodies were evaluated by a sandwich ELISA.Results. Circulating levels of inflam-matory biomarkers hsCRP, IL-6 and PTX3 were not significantly different in the two groups of patients with erosive and non-erosive HOA compared to NC and no significant difference was seen between non-erosive and erosive HOA.Anti-CCP positivity was detected re-spectively in 1 patient (2.1%) with non-erosive HOA and 1 patient (1.9%) with erosive HOA. Anti-MCV antibod-ies were present in 4 patients (8.5%) with non-erosive HOA, and 4 patients (7.7%) with erosive HOA. In the con-trol group, one subject (2%) was posi-tive for anti-CCP and 2 subjects (4%) had anti-MCV antibodies.Significant correlation was obtained only between body mass index and hsCRP concentration (r=0.4071; p

BACKGROUND & AIMS: Different amino acid sequences of hepatitis B virus surface antigen (HBsAg) are involved in the activation of CD4+ lymphocytes needed to induce an optimal antiviral function. The aim of this study was to characterize the CD4-mediated response to immunodominant HBsAg epitopes in hepatitis B virus (HBV) vaccine recipients by defining minimal sequences recognized by T cells, cytokine profiles, and HLA restriction of peptide recognition. METHODS: T-lymphocyte lines and clones specific for HBsAg were isolated from the peripheral blood of subjects immunized with recombinant HBsAg and stimulated in vitro with synthetic peptides spanning the whole HBsAg sequence. RESULTS: Four immunodominant epitopes (sequences 21-40, 136- 155, 156-175, and 211-226) were identified. Using panels of truncated peptides of different length, sequences 21-28, 165-172, and 215-223 were shown to correspond to the minimal epitopes recognized by T cells. The antigen-specific T-lymphocyte proliferation was HLA class II restricted, and each peptide could be presented in association with different HLA class II determinants. Th0/Th2 cytokine patterns were induced on peptide stimulation. CONCLUSIONS: These results indicate the presence of at least four immunodominant epitopes within HBsAg that represent potential candidates for the design of anti-HBV synthetic vaccines. (Gastroenterology 1997 Jun;112(6):2017-27)

To assess whether a different IgG subclass distribution was elicited in “low” and “high responders” after vaccination with recombinant hepatitis B virus surface antigen, we selected from 360 vaccine recipients 30 “lowresponder” subjects, with anti-HBs levels of 10–160 mIU/ml, and 40 “high-responder” subjects, with anti-HBs levels greater than 10,000 mIU/ml. In both groups all IgG subclasses were elicited in the anti-HBs response and the greatest contribution was that of IgG 1, followed by IgG2. IgG l was significantly less represented after the second (58%) and third doses (61%) of vaccine in “low responders” compared with “high responders” (65% and 69%). The relative percentage of IgG2 was significantly higher after the second (33%) and third (30%) doses of vaccine in “low responders” than in “high responders” (29% and 26%). In “low responders” the age of vaccine recipients significantly influenced the anti-HBs IgG subclass distribution: IgG2 and IgG4 production was positively correlated with age, whereas the opposite was observed for IgGl. These data support the evidence that: (1) IgGl and IgG2 subclasses are mainly involved in the specific anti-HBs response both in “high” and “low responders”; (2) the relative contribution of specific IgG2 to vaccination is higher in low responders and progressively increases with age.