Dorotea Natuzzi

myo-Inositol transport in rat intestinal brush border membrane vesicles, and its inhibition by d-glucose

The uptake of myo-inositol into rat intestinal brush border membrane vesicles (BBMV) has been investigated. It is demonstrated that myo-inositol is transported into the vesicles by a secondary active process, specifically using the sodium gradient as the driving force. In the absence of sodium gradient, the transport reaction is still sodium dependent, and rheogenic, indicating that a myo-inositol/sodium cotransport is likely to occur. A kinetic analysis shows an hyperbolic saturation process with a Km of 0.16 +/- 0.02 mM with respect to myo-inositol and Vmax of 68.5 +/- 21.2 pmol/min per mg protein. The transport is inhibited by D-glucose, phloridzin and few other sugars. The mechanism of D-glucose inhibition appears to be of the mixed type. Finally, the myo-inositol transport is trans-activated by myo-inositol itself, but not by D-glucose. It is concluded that myo-inositol is transported into rat intestine BBMV by a specific transport system, which is also able to bind D-glucose, but not efficiently transport it across the membrane.

Correlation between serum levels of IL-15 and IL-17 in patients with idiopathic inflammatory myopathies

Objectives: To assess the serum levels of interleukin (IL)-15 and IL-17 in patients with idiopathic inflammatory myopathies (IIM) and correlate them with levels of IL-1 receptor antagonist (IL-1ra), IL-6, IL-10, interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and MIP-1β. Possible correlations with disease activity parameters were also evaluated. Method: Sera from 14 patients with new-onset polymyositis (PM), 10 with dermatomyositis (DM), seven with anti-synthetase syndrome (ASS) and 19 healthy controls (HC) were analysed by multiplex immunoassay. Sera from 19 patients were analysed after a median follow-up of 5 months. All patients underwent physical examination, manual muscle testing (MMT) using the five-point MMT scales, the Health Assessment Questionnaire (HAQ), and serum creatine kinase (CK) measurement. All patients received glucocorticoids, and 13 were taking immunosuppressive therapy. Results: At baseline, serum levels of IL-15, IL-17, MCP-1, and MIP-1β were significantly higher in IIM patients than in HC. IL-17 serum levels were directly correlated (r = 0.39, p = 0.02) with disease duration while a significant inverse correlation was detected between IL-17 levels and MMT scores (r = –0.4, p = 0.02). The highest IL-15 levels were present in DM patients (p = 0.02 vs. PM). The most striking finding was the strong correlation between IL-15 and IL-17 levels (r = 0.60, p = 0.0001), and this correlation was even stronger in DM patients (r = 0.82, p = 0.006). Conclusions: The strong correlation between IL-15 and IL-17 in IIM patients, and especially in DM, suggests that there may be an interplay between the two cytokines in the pathogenesis of myositis. Further studies of larger patient cohorts and of muscle biopsies are needed to confirm these preliminary data.

Inactivation of the Reconstituted Oxoglutarate Carrier from Bovine Heart Mitochondria by Pyridoxal 5′-Phosphate

The effect of pyridoxal 5′-phosphate and some other lysine reagents on the purified,reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition ofoxoglutarate/oxoglutarate exchange by pyridoxal 5′-phosphate can be reversed by passing theproteoliposomes through a Sephadex column but the reduction of the Schiffs base by sodiumborohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal5′-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutaratetransport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutaratecarrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal5′-phosphate indicated that modification of a single amino acid residue/carrier molecule wassufficient for complete inhibition of oxoglutarate transport. After reduction with sodiumborohydride [3H]pyridoxal 5′-phosphate bound covalently to the oxoglutarate carrier. Incubation ofthe proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivationand no radioactivity was found associated with the carrier protein. In contrast, glutarate andsubstrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl,which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier againstinhibition by pyridoxal 5′-phosphate. These results indicate that pyridoxal 5′-phosphateinteracts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminalglycine residue) which is essential for substrate translocation and may be localized at or nearthe substrate-binding site.

Possible interplay between interleukin-15 and interleukin-17 into the pathogenesis of idiopathic inflammatory myopathies.

The aim of this study was to assess the serum levels of interleukin (IL)-15 and IL-17 in patients with idiopathic inflammatory myopathies (IIM) and correlate them with IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), MIP-1β levels. Possible correlations with disease activity parameters were also evaluated. Sera from 14 polymyositis (PM), 10 dermatomyositis (DM), 7 anti-synthetase syndrome new onset patients and 19 healthy controls (HCs) were analyzed by multiplex immunoassay. Sera from 19 patients were analyzed after 5 months median follow-up. All patients underwent physical examination, the 5-points manual muscle test (MMT), the health assessment questionnaire and serum creatine kinase measurement. All patients received glucocorticoids, and 13 were taking also immunosuppressive therapy. At baseline, serum levels of IL-15, IL-17, MCP-1 and MIP-1β were significantly higher in IIM patients than in HCs. IL-17 serum levels were directly correlated with disease duration (r=0.39, P=0.02), while a significant inverse correlation was detected between IL-17 levels and MMT scores (r=-0.4, P=0.02). The highest IL-15 levels were present in DM patients (P=0.02 vs PM). The most striking finding was the strong correlation between IL-15 and IL-17 levels (r=0.60, P=0.0001), and this correlation was even stronger in DM patients (r=0.82, P=0.006). The strong correlation between IL-15 and IL-17 in IIM patients, and especially in DM, suggests that there may be a interplay between the two cytokines in the pathogenesis of myositis. Further studies of larger patient cohorts and muscle biopsies are needed to confirm these preliminary data.

Photoaffinity labeling of the mitochondrial oxoglutarate carrier by azido-phthalonate

The effect of azido-phthalonate, a photoreactive analogue of oxoglutarate, on the transport of oxoglutarate was investigated in proteoliposomes reconstituted with the purified oxoglutarate carrier. In the dark, azido-phthalonate inhibits the reconstituted oxoglutarate/oxoglutarate exchange in a competitive manner with a Ki of 0.38 mM. Upon photoirradiation, the inhibition of the oxoglutarate exchange by azido-phthalonate is not removed by passing the proteoliposomes through a Sephadex column. The light-induced inhibition of the oxoglutarate/oxoglutarate exchange activity by azido-phthalonate is time- and concentration-dependent. The kinetic analysis of transport inhibition by azido-phthalonate reveals that one molecule of this substrate analogue bound to the functional carrier molecule is responsible for complete inhibition of the carrier function. Azido-[3H]phthalonate binds to the oxoglutarate carrier covalently. Incubation of the proteoliposomes with oxoglutarate during photoirradiation in the presence of azido-phthalonate protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the carrier protein. It is concluded that azido-phthalonate can be used for photoaffinity labeling of the mitochondrial oxoglutarate carrier at the substrate-binding site.

Possible role of adipocytokines in systemic sclerosis–associated small pericardial effusion:

Introduction: Pericardial effusion is a common manifestation of systemic sclerosis, but its pathogenesis has been poorly investigated. Adipokines and interleukins may play a role in the pathophysiology of pericardial effusion. This study aimed at evaluating serum levels of adipokines and interleukins in systemic sclerosis patients with and without pericardial effusion. Methods: A total of 87 systemic sclerosis patients (age 52.6 ± 14 years; disease duration 8.2 ± 6.7 years) were recruited in this study. Demographics, body mass index, and clinical characteristics were recorded in each patient. Pericardial effusion was considered pathologic when ≥50 mL was detected by echocardiography. Serum levels of adiponectin, leptin, resistin, visfatin, tumor necrosis factor-α, interferon-γ, interlueukin-2, interlueukin-10, and interlueukin-17 were measured using Multiplex Immunoassay (Bioplex 200 System). Results: In all, 11 (13%) systemic sclerosis patients had pericardial effusion. Systemic sclerosis patients with and without pericardial effusion did not differ in age, sex, and body mass index. Systemic sclerosis patients with pericardial effusion had significantly higher levels of visfatin (median/interquartile range: 1546 pg/mL (interquartile range: 8590) vs 388 pg/mL (interquartile range: 103), p = 0.03) and interlueukin-17 (1.33 pg/mL (interquartile range: 3.5) vs 0.05 pg/mL (interquartile range: 0.56), p = 0.04), but lower levels of adiponectin (2,845,000 pg/mL (interquartile range: 4,132,900) vs 5,272,100 pg/mL (interquartile range 8,243,600), p = 0.02) than patients without pericardial effusion. Interstitial lung disease, pulmonary arterial hypertension, and “limited” or “diffuse” cutaneous subset did not correlate to adipokines or interleukin levels. Conclusion: Visfatin and adiponectin may play an important role in the pathogenesis of systemic sclerosis–related pericardial effusion. Further longitudinal studies are needed to unravel a possible role of these molecules as biomarkers of pericardial effusion in systemic sclerosis patients.

AB0037 Serum cytokine signature in mucocutaneous and ocular behÇet's disease

Background Behçets disease (BD) is a multi-systemic inflammatory disorder consisting of recurrent oral aphthosis, genital ulcers, and chronic relapsing bilateral uveitis. However, many other organs including the vascular, gastrointestinal, neurological, and musculoskeletal systems can be affected. Pathogenetically, both innate and adaptive immunity have shown to play a pivotal role, and several proinflammatory cytokines derived from Th1 and Th17 lymphocytes seem to be involved in different pathogenic pathways leading to development of the clinical manifestations. Objectives The primary aim of our study was to compare a core set of proinflammatory cytokines between patients with BD and healthy control (HC). The secondary aim was to evaluate potential correlations between these putative circulating biomarkers, the status of disease activity, and the specific organ involvement at the time of sample collection. Methods Fifty-four serum samples were collected from 46 BD patients (17 males, 29 females, mean age 45,5±11,3 years), and 19 HC (10 males, 9 females, mean age 43±8.3 years). Twenty-five serum cytokines (APRIL/TNFS13, BAFF/TNFSF13B, sCD30/TNFRSF8, sCD163, Chitinase3-like1, gp130/sIL-6Rb, IFNb, sIL-6Ra, IL-10, IL-11, IL-19, IL-20, IL-26, IL-27 (p28), IL-28A/IFN-lambda2, IL-29/IFN-lambda1, IL-32, IL-34, IL-35, LIGHT/TNFSF-14, Pentraxin-3, sTNF-R1, sTNF-R2, TSLP and TWEAK/TNFSF-12) were simultaneously quantified using a Bio-Rad cytokine bead arrays. Results Serum levels of Chitinase3-like1, gp130/sIL-6Rb, IL-11, IL-26, sTNF-R1, sTNF-R2 were significantly higher in BD patients than in HC. Specifically, serum concentration of sTNF-R1 (p<0.01) and sTNF-R2 (p<0.01) resulted higher in both active- and inactive-BD than HC, whilst Chi-tinase3-like1 (p<0.05) and gp130/sIL-6Rb (p<0.01) serum levels were significantly higher in in-active-BD, and IL-26 (p<0.01) in active-BD than HC. No differences were observed between inactive- and active- BD group. In addition, comparing cytokines levels in patients affected by mucocutenous manifestations with (MO-BD) or without (M-BD) ocular involvement we observed that gp130/sIL-6Rb, sIL-6Ra, IL-35, and TSLP serum levels were significant enhanced in MO-BD compared to M-BD subgroup. Conclusions Our findings showed a signature of IL-6, TNF-α as well as of Th17 response in BD patients due to increased levels of gp130/sIL-6Rb, sTNF-R1, sTNF-R2, IL-26 respectively. This evidence could contribute to improve the knowledge regarding the role of these citokines in the induction of specific BD clinical features References Lopalco G, Lucherini OM, Vitale A, Talarico R, Lopalco A, Galeazzi M, et al. Putative Role of Serum Amyloid-A and Proinflammatory Cytokines as Biomarkers for Behcets Disease. Medicine (Baltimore) (2015) 94(42):e1858. Lucherini OM, Lopalco G, Cantarini L, Vitale A, Rotondo C, Lopalco A, et al. Correlation between serum amyloid-A and serum levels of proinflammatory cytokines in patients with Behçets disease. Pediatric Rheumatology Online Journal (2015) 13(Suppl 1):P6. Turan B, Pfister K, Diener PA, Hell M, Möller B, Boyvat A, et al. Soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 are produced at sites of inflammation and are markers of arthritis activity in Behçets disease. Scand J Rheumatol (2008) 37(2):135–41. Disclosure of Interest None declared

SAT0212 Pericardial Effusion Related To Systemic Sclerosis: A Possible Contribution of The Serum Levels of Adipokines and Interleukines

Background Pericardial effusion (PE) is a common clinical manifestation in systemic sclerosis (SSc). Few data are available about its pathogenesis. The role of adipokines and interleukins produced by the adipose tissue and the immune system in PE related to SSc had never been investigated. Objectives This study aimed at evaluating the differences in serum levels of adipokines and interleukins (IL) in systemic sclerosis (SSc) patients with and without PE. Methods A total of 87 outpatients (84 female, mean age of 52,6±14,2 ys, and disease duration of 8,2±6,7 ys), who fulfilled the ACR/EULAR 2013 SSc classification criteria, were recruited in this study. The anthropometric measures, as body mass index (BMI), demographic, clinical and laboratoristic characteristics and SSc related manifestations were assessed in each patient. The presence of PE was evaluated by means of echocardiographic techniques (the presence of fluid ≥5 cc was considered pathologic). Sera levels of adiponectin, leptin, resistin, visfatin, tumor necrosis factor α (TNF-α), interferon g (INF-g), IL-2, IL-10 and IL-17 were measured in SSc patients, using Multiplex Immunoassay (Bioplex 200 System), by means of two kits (Bioplex ProTM Cytokine/Chemokine and Growth Factor Assay e Bioplex Pro Diabetes Assay). The data normality was verified using Kolmogorov-Smirnov Test; the comparisons between SSc patients groups were evaluated by Mann-Whitney U test and t-student test, where appropiate. Statistic significance was set at p≤0,05. The results are expressed as median and interquartile range (IQR) or means±1standard deviation. The data analysis were assessed using IBM SPSS statistic 20. Results 11 SSc patients had PE. The SSc patients groups (with and without PE) did not differ in age, sex and BMI. 11 SSc patients were obese (BMI≥30) (2 with PE and 9 without PE). We found significant differences between SSc patients with PE and without PE in sera levels of visfatin (1546,9 (8590,9) vs 388,8 (103); p=0,036), adiponectin (2845000 (4132900,0) vs 5272100,0 (8243600,0); p=0,027) and IL-17 (1,33 (3,5) vs 0,05 (0,56); p=0,45). Moreover higher adiponectin/leptin ratio was found in patients with PE than SSc patients without PE (569,2 (1415,3) vs 166,9 (504,9) p=0,032). The presence of interstitial lung disease, pulmonary arterial hypertension, limited or diffuse skin subset, the different nail fold videocapillaroscopy pattern and the modified Rodnan skin score did not influenced the serum levels of adipokines and IL. In the SSc patients with PE there is no differences in visfatin and adiponectin serum level and adiponectine/leptin ratio between patients that are in menopause and those who are not. Conclusions The visfatin and adiponectin could play an important role in pathogetic mechanism in pericardial effusion related to SSc. Further study are necessary to unravel a role of visfatin and adiponectin as biomarkers of SSc and a role of the adipose tissue and innate immune system in PE related to SSc pathology. Disclosure of Interest None declared

AB0041 Serum Amyloid A Stimulates The Induction of Inflammatory Mediators in Monocytes from Behçet's Disease Patients: A Proof of Concept Study

Background Behcets disease (BD) is a complex inflammatory disorder characterised by an abnormal innate and adaptive immune response leading to hyper-activation of pro-inflammatory mediators. Recurrent oral aphthous, genital ulcers and chronic relapsing bilateral uveitis are the hallmarks of the disease. Serum amyloid-A (SAA) is a biomarker of inflammation recently associated to BD, and pro-inflammatory stimuli including IL-6, IL-1 and TNF are known to be involved in the SAA production. Moreover, several innate immune cells such as neutrophils, monocytes and macrophages have been reported to produce pro-inflammatory cytokines also through inflammasome activation after SAA stimulation. Objectives The purpose of this study was to investigate the involvement of SAA in the pathogenesis of Behçets disease Methods Monocytes obtained from heparinised venous blood of Behçets disease patients (BD, n=14) and healthy controls (HC, n=7) were stimulated or not with SAA, and serum cytokine levels of IL-1β, IL-18, IL-6 and TNF-α were analysed in the medium using a multiplex bead analysis. Statistical approaches included two-tailed Mann-Whitney test (for two non-parametric groups) and Students t-test (for two parametric groups) were used for statistical comparisons between groups. Correlations were calculated using Spearmans correlation (two-tailed p-value) analysis. Results BD monocytes showed an increased IL-1β (p=0.0017), TNF-α (p=0.0003) and IL-6 (p=0.0003) secretion after SAA stimulation. No differences were observed in the amounts of pro-inflammatory cytokines production between HC and BD. We also found that IL-1β levels positively correlated with IL-6 (r=0.842, p<0.001), and TNF-α (r=0.889, p<0.001), whilst TNF-α levels positively correlated with IL-6 (r=0.894, p<0.001) levels. Moreover, also IL-18 showed a positive trend with no significant differences between the two groups Conclusions These findings suggest that SAA can promote inflammatory mediators production thereby contributing to the inflammatory manifestations typically observed in BD. References Hatemi G, Yazici Y, Yazici H. Behçets syndrome. Rheum Dis Clin North Am 2013; 39:245–611. Vitale A, Rigante D, Lopalco G, Brizi MG, Caso F, Franceschini R, Denaro R, Galeazzi M, Punzi L, Iannone F, Lapadula G, Simpatico A, Marrani E, Costa L, Cimaz R, Cantarini L. Serum amyloid-A in Behçets disease. Clin Rheumatol. 2014. Lopalco G, Lucherini OM, Vitale A, Talarico R, Lopalco A, Galeazzi M, Lapadula G, Cantarini L, Iannone F. Putative Role of Serum Amyloid-A and Proinflammatory Cytokines as Biomarkers for Behcets Disease. Medicine (Baltimore). 2015 Oct;94(42):e1858. Uhlar CM, Grehan S, Steel DM, Steinkasserer A, Whitehead AS. Use of the acute phase serum amyloid A2 (SAA2) gene promoter in the analysis of pro- and anti-inflammatory mediators: differential kinetics of SAA2 promoter induction by IL-1 beta and TNF-alpha compared to IL-6. J Immunol Methods 1997;203:123–30. Disclosure of Interest None declared

OP0050 Serum Levels of Adipokines in The Categories of Body Mass Index (BMI) in Patients with Systemic Sclerosis

Background Adipose tissue is a source of factors stimulating the pro-oxidative-antioxidant system, such as adipokines, whose functions are closely associated with metabolic abnormalities and damage by oxidative stress. There are only few reports in literature about the correlation between body mass index (BMI), serum levels of adipokines and clinical manifestations of SSc. Objectives We evaluated serum levels of adipokines (leptin, resistin, visfatin, adiponectin) and cytokines (TNFα, IFNγ, IL-2, IL-10, IL-17) in SSc and controls (HD) and their pattern according the clinical and laboratory SSc features. Methods 89 SSc pts satisfying ACR EULAR criteria (age 52 ± 14.4 years and disease duration of 8.14 ± 6.6) and 26 HD were enrolled. Serum levels of Adiponectin, Leptin, Resistin, Visfatin, TNFα, IFNγ, IL-2, IL-10, IL-17A were measured using Bio-Rad kits and Multiplex Immunoassay tool. In all participants we collected clinical and laboratory features, such as visceral involvement, BMI, WHR (Waist to Hip Ratio), index of cardiovascular risk, acute phase reactants, cholesterol, triglycerides, smoking status, comorbidity and therapy. Data were analysed using IBM-SPSS Statistics 20 software. Mann-Whitney U-test or t-Student for unpaired data or Kruskal-Wallis test or ANOVA were used for comparison between groups. Spearmans or Pearsons test were used for correlations. A p-value ≤0.05 was considered statistically significant. Results The weight of SSc and HD differed (p<0,05) which was higher in HD. Serum levels of Leptin, Resistin, Visfatin and IL-10, IL-17 were increased in SSc compared to HD (p<0.05). In SSc, TNFα serum levels were not different from HD. Leptin levels positively correlated with body weight and BMI that in turn correlated with the values of ESR, CRP, WHR, triglycerides and cholesterol serum levels. Increased levels of IL-2, IL-10, IL-17, IFNγ, Leptin and Visfatin were found in overweight/obese SSc pateints (p<0.05) in respect to HD. TNFα was significantly increased in underweight patients (p=0.03) and there was a significant difference compared with normal weight (p=0.018). TNFα levels correlated with IL-2, IL-10, IL-17 and IFNγ levels and with esophageal involvement. There were no differences in cytokine levels between groups when correlated to skin subset, disease duration (Early/Late) and different class of cardiovascular risk. In HD, no statistically significant correlation between weight, TNFα and Leptin levels was detected. Conclusions Adipokines may play a role in inflammatory and immune pathways in SSc pathogenesis. The present data suggest a role for Leptin and TNFα as new potential SSc related circulating biomarker. Categories of modified BMI (underweight and obesity) seem to be associated to a different and a significant organ involvement in SSc. In underweight subjects, the increase of TNFα may be involved in the cachectic status, thus the role of TNFα needs be confirmed in further prospective longitudinal studies investigating also its correlation with disease severity and activity. References Pehlivan Y et al. Serum leptin, resistin and TNFa levels in patients with systemic sclerosis: the role of adipokines in scleroderma, Int J of Rheum Dis 15, 374–379, 2012 Disclosure of Interest None declared