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The aim of this study was to evaluate the effect of an oral preparation containing a naturally occurring matrix of hydrolyzed collagen type II, chondroitin sulfate (CS), and hyaluronic acid (HA), and bioactive oligopeptides of natural hydrolyzed keratin (K) in patients affected by knee OA through the evaluation of synovial fluid (SF) and clinical changes before and after treatment. Thirty patients with knee OA and swollen joint were included in the study and submitted to arthrocentesis. Patients were randomized in two groups: 1) the treatment group (N.15) took a dietary supplement containing 120 mg HA, 240 mg CS and 300 mg K once a day for 4 weeks; 2) the control group (N.15) was only submitted to arthrocentesis. Patient symptoms were evaluated at the beginning and at the end of the study by the WOMAC self-assessment questionnaire, the Lequesne algofunctional index, and the VAS forms. SF changes were evaluated by measuring local inflammatory indices, cytokines IL-1β, IL-8, IL-6, IL-10 and GM-CSF. The group of patients treated with the oral supplement showed an improvement in the clinical indices WOMAC (p<0.01), Lequesne (p=0.014) and VAS pain (p<0.01). On the contrary, no significant changes were found in the control group. The SF collected from the treated group showed a reduction of IL-8 (p=0.015), IL-6 and IL-10 levels, while no changes in cytokines were observed in the control group. This pilot study suggests that an oral administration of a preparation containing a combination of HA, CS and K can improve some clinical parameters and affect cytokine concentrations in SF in patients with knee OA.

Background:The health benefits of plant polyphenols have stimulated a growing interest in the scientific community. Their ability to influence various pathological processes has been demonstrated in several in vitro and in vivo disease models [1]. We have recently shown that polydatin, a stilbenoid and precursor of resveratrol, is capable of preventing calcium pyrophosphate (CPP) crystal-induced arthritis in mice [2].Objectives:The aim of this study was to investigate some potential mechanisms of action associated with this anti-inflammatory effect.Methods:Acute arthritis was induced by injection of a suspension of sterile CPP crystals into the ankle joint of Balb/c mice. Animals were randomised to receive polydatin or colchicine (the control drug) according to a prophylactic protocol. Ankle swelling (primary outcome) was measured at different time points before and after CPP injection. Both joints and muscles were harvested at sacrifice. Histological parameters were assessed by H&E, safranin and Masson’s trichrome staining. Muscle damage was evaluated by H&E staining, while Kondziela’s inverted test was used to assess muscle strength. A cytokine antibody array (Ray-Biotech) was performed on the joint tissue.Results:Injection of CPP crystals into the ankle resulted in oedema with evident swelling (primary outcome), moderate areas of leukocyte infiltration, loss of homogeneity of synovial membrane structure, moderate to severe loss of proteoglycan in the superficial hyaline layer of cartilage with damage extending into the underlying region. Mice pretreated with polydatin showed reduced ankle swelling 48 hours after crystal injection, similar to that seen after colchicine pretreatment. This was associated with very limited inflammatory damage, with moderate and focal zones of proteoglycan loss and significant preservation of cartilage and bone structures. Intact cartilage and bone surfaces were observed in untreated joints. Regarding the effect on gastrocnemius muscle, CPP crystals induced leukocyte infiltration, loss of muscle fibres and eosinophilic degenerative fibres with enlarged interstitial areas due to increased muscle oedema. In PD- and colchicine-treated mice, muscle damage was limited and the musculoskeletal structure was generally preserved. The cytokine array showed the activation of different inflammatory pathways after CPP injection. PD showed to strongly influence leukocyte migration (MIP-1G, L-selectin, CXCL16, BLC), angiogenesis (VEGF, VEGF-R2, VEGF-R3, MMP-3) and resolution of inflammation (sTNF-RI, sTNF-RII).Conclusion:PD effectively prevents the acute inflammatory response to CPP crystals in mice, preserving both articular and muscular structures. The most affected pathways involve leukocyte migration and the angiogenic process.REFERENCES:[1] Oliviero F, Scanu A, Zamudio-Cuevas Y, Punzi L, Spinella P. Anti-inflammatory effects of polyphenols in arthritis. J Sci Food Agric. 2018;98:1653-1659. doi: 10.1002/jsfa.8664.[2] Oliviero F, Galozzi P, Scanu A, et al. Polydatin Prevents Calcium Pyrophosphate Crystal-Induced Arthritis in Mice. Nutrients. 2021;13:929. doi: 10.3390/nu13030929.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Dietary recommendations for patients with rheumatic diseases include adopting the Mediterranean diet and weight loss in overweight patients. The very low-calorie ketogenic diet (VLCKD) is characterized by a restrictive dietary plan, low in carbohydrates and high in fat.Objectives:To assess the impact of a 9-week VLCKD through a monocentric study on disease activity and inflammation indices in patients with psoriatic arthritis (PsA) who are moderately overweight or have first-degree obesity.Methods:PsA Patients, meeting inclusion criteria of stable treatment and disease activity according to DAPsA in the 6 months preceding baseline (W0) with BMI ≥27 and <35, were offered a 9-week VLCKD treatment. Assessment occurred every 3 weeks by an independent nutritionist and at W0 and after 9 weeks (W9) by a rheumatologist. Collected data included anthropometric measurements, clinical indices, and patient-reported outcomes. Laboratory data comprised highly sensitive CRP, ESR, IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, fecal calprotectin. Statistical analysis, was conducted with non-parametric tests.Results:Twenty patients were included from April 2022 to March 2023. Baseline characteristics and their evolution throughout the study period are reported in the table. All patients exhibited a significant reduction of approximately 3 BMI points. Indices of joint and skin disease activity, patient-reported pain, and impact on working ability showed significant decreases at W9. No significant differences were observed in inflammatory markers, enthesitis, and axial disease measures. Improvement in the lipid profile and insulin levels was also noted. Relevant significant associations were observed between BMI reduction and DAPSA reduction (rho 0.516, p 0.02), as well as between the reduction of BMI and weight and the impact on working ability, as measured with WPAI (rho 0.495, p 0.043 and rho 0.551, p 0.022, respectively). No inflammatory marker appeared to be associated with anthropometric changes. Abdominal circumference did show only a few slight associations with clinical and laboratory indices.Among baseline variables, older patients presented a lower reduction in abdominal circumference (rho 0.449. p 0.047). Patients with metabolic syndrome seemed more prone to obtaining BMI, weight and abdominal circumference reductions. Patients with IL-6≥7.0 ng/L at W0 presented a lower reduction in all anthropometric measures. Baseline TNF-alpha levels were associated with a higher probability of weight loss and BMI reduction (rho 0.458. p 0.042 and rho 0.469. p 0.037, respectively). Patients with a higher DAPSA at W0 seemed less prone to obtain weight loss, BMI and abdominal circumference reduction (rho -0.577. p 0.008, rho -0.669. p 0.001 and rho -0.457. p 0.043, respectively).Conclusion:VLCKD is well-tolerated and effective in patients with PsA, resulting in significant weight loss and improvements in the lipid profile, joint, and skin disease activity. Inflammatory markers do not vary significantly after weight loss and abdominal circumference reduction. Patients with higher disease activity at baseline obtain minor results on weight loss with VLCKD. Also, higher baseline TNF-alpha seems a promising indicator in predicting those who may benefit less from VLCKD. Patients with comorbidity could be more motivated in adhering to VLCKD and obtaining weight loss. REFERENCES:NIL.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

Low back pain (LBP) is a common condition with profound effects on well-being. We aimed to define the prevalence and the characteristics of LBP and to investigate its impact on the quality of life (QoL) of 409 students (265 females and 144 males), all high-school adolescents from the Veneto region. LBP was measured with a structured, self-report questionnaire, while the SF-36 questionnaire was used to measure physical and mental QoL. 253 students (61.3%) reported one or more episodes of LBP, with female predominance. Adolescents with LBP treated with drugs and rehabilitation cares have significantly poor belief in pain resolution (p=0.005), but more belief in a prevention program (p=0.006) than the others. After adjustment for sex, a significant association between the SF-36 dimension of vitality and the presence of LBP in males was observed. All SF-36 domains except mental health were significantly higher in females with LBP. Our study confirmed that LBP is frequent in Italian scholar adolescents and has an impact on QoL. Strategies for reducing the effects of LBP on QoL should be an important purpose for clinicians and health policy makers.

BackgroundBactericidal/permeability-increasing protein (BPI) is an antibacterial glycoprotein produced by polymorphonuclear cells (PMN). Although BPI has been detected in synovial fluid of patients with different type of arthritis, its effects in these diseases remain unexplored.ObjectivesTo investigate the effects of BPI in mouse models of crystal-induced inflammation and collagen-induced arthritis (CIA).MethodsAir pouches were raised on the backs of CD1 mice (n=14 per condition). 2 mg of calcium pyrophosphate (CPP) crystals in 1 ml of PBS were injected into the pouch in the presence or absence of 0.1 mg of BPI for 3 hour. Controls received 1 ml of PBS. After the sacrifice, pouch fluids were recovered by washing with 2 ml of PBS. Leukocyte count in lavage fluids was obtained using a hemocytometer and the PMN percentage was determined by May-Grünwald-Giemsa staining. CIA was induced in C57Bl/6 mice (n=12 per condition) by immunisation with collagen/complete Freund’s adjuvant emulsion at days 0 and 21. Arthritis was monitored by measuring paw swelling with a calliper and scored (0–5) every 2 days. Arthritic mice (paw score=2) were intraperitoneally injected with 200 µl of BPI (50 µg/ml) or vehicle only (PBS). Treatment and arthritis evaluation were carried out twice a week for 2 month. At the experiment endpoints, mice were subjected to knee and ankle ultrasound (US) and then euthanized. Hind paws were processed for histological analysis to assess inflammation, pannus formation, cartilage and bone destruction in knee and ankle joints (score 0–5). IL-1β, IL-6, CXCL1 and TNF levels were measured by ELISA in pouch fluids and serum from collected blood.ResultsThe injection of CPP crystals into the pouches induced leukocyte infiltration (26.27±4.14 × 105 cells/ml) comprising 73.5%±2.12% of PMN. IL-1β (80.99±3.65 pg/ml), IL-6 (892.90±28.14 pg/ml), CXCL1 (762.82±50.08 pg/ml) and TNF (52.70±0.49 pg/ml) were measured in lavage fluids. The co-injection of crystals and BPI inhibited leukocyte influx by 67% and PMN infiltration by 55% and, in turn, the levels of all assessed cytokines were reduced (46% IL-1β, 35% IL-6, 60% CXCL1, 64% TNF). CIA mice reached an arthritis score of 2 after 28–33 days and showed a progressive worsening of clinical signs that picked at day 49–56, and then improved only in BPI group. At the end of the experiment, hind paw swelling and scores were lower in BPI-mice (paw thickness=4.76±0.33 mm; score=4±0.5) than in controls (paw thickness=6.39±0.95 mm; score=5). US revealed synovial hypertrophy in all joints considered, but the presence of effusion only in knees of untreated animals. Power Doppler was positive in controls but not in BPI group. BPI decreased histological scores for pannus formation and inflammation by 3-fold, and for cartilage and bone destruction by 2-fold as compared to PBS-mice. CIA induction led to high levels of IL-1β (656.80±45.6 pg/ml), IL-6 (376.04±46.33 pg/ml), CXCL1 (834.08±68.8 pg/ml) and TNF (277.72±15.24 pg/ml), which were reduced with BPI treatment by 66%, 46%, 47% and 93%, respectively.ConclusionsThis study shows inhibitory effects of BPI on crystal-induced inflammation and CIA, suggesting a therapeutic potential of this protein for arthritis by down-regulating inflammatory process.Disclosure of InterestNone declared

Rheumatic diseases (RD) are among the most frequent disorders in the population and the major causes of chronic pain and disability. The resulting consequences are catastrophic, leading to a significant socio-economic burden, which includes significant reductions in quality of life (QoL) and limitations in regular work and daily activities of patients. In spite of this, rheumatic diseases are often misunderstood or diagnosed late, probably due to their characteristics of silent diseases, sometimes unrecognizable to unaffected or unskilled people. Actually, it is surprising that, despite their consequences on QoL and on individual impact, rheumatic diseases are underestimated by the public opinion, which is probably more attracted by other major diseases causing death. This silent perception can even be seen in some among the most recent psycho-social approaches to population needs in the fields of Health Psychology and Environmental Psychology. The latter, also known as Architectural Psychology, is a branch of Psychology that analyses the effects of the built environment on humans, including those affected by diseases. Paradoxically, in many cases, some components of the environments created to protect individuals and/or the population may represent barriers and subsequently causes of disability and suffering in patients with rheumatic diseases. In order to increase awareness about this particular aspect of social life, HEMOVE Onlus, a non-profit association, has promoted the creation of a multidisciplinary Task Group, which included mainly rheumatologists, psychologists and architects, with the aim of applying also for the benefit of rheumatic patients the most modern technical skills available in the context of Environmental Psychology, including in particular design and information technology.

BackgroundBlau syndrome (BS) is a rare autosomal dominant autoinflammatory syndrome that primarily affects skin, joints, and eyes, with varying severity of inflammation, and results from mutations in the NOD2 gene1. NOD2 bears highly conserved residues and structural similarities to the NLRP3 protein, a key component of the inflammasome which mutations underlie some autoinflammatory disorders, named cryopyrinopathies.ObjectivesThis study assessed the potential contribution of NLRP3 mutations to the inflammatory BS phenotype in an Italian affected family carrying p.E383K NOD2 mutation (Fig. 1).MethodsGenomic DNA was isolated from blood samples of the four affected family members and sequence analyses of NLRP3 were performed by an ABI 3730XL DNA sequencer (Applied Biosystems). A genotype/phenotype correlation of the carriers of NLRP3 variations was also assessed.To explain the differences in inflammatory phenotype severity, peripheral blood mononuclear cells (PBMC) were isolated from patients and healthy subjects and their ability to activate NF-κB and inflammasome pathways was measured by an array-based assay (reverse phase protein array).ResultsA heterozygous genetic variant of NLRP3 was found only in the proband's sample. The p.V198M mutation was known in literature as a low-penetrance mutation associated with cryopyrinopathies in symptomatic patients (Infevers database data). The proband with combined mutations presented the worsened BS phenotype (severe chronic bilateral uveitis with glaucoma, cataracts and visual impairment), whereas the three subjects with only p.E383K variant had a much milder disease.Concerning the activation of NF-κB pathway, all BS patients have raised levels of signalling key molecules (phosphorylated NF-κB p<0.05; phosphorylated IkB-α p<0.05) compared to the controls. However, the components of inflammasome pathway presented a pattern of activation related to individual patients rather than to the whole disease. Enhanced level of cleaved caspase-1, the key component of inflammasome, was noticed only in proband PBMCs (p=0.03), potentially promoted by the additional presence of p.V198M NLRP3 mutation.ConclusionsOur data strongly suggest that severe phenotype in BS may result from co-existence with mutations in genes associated with other autoinflammatory diseases, and are consistent with recently reported findings in hereditary periodic fever syndromes2. The synergistic effect of dual mutations can also explain the differential activation of the inflammatory pathways and may lead to appropriate targeted drug development.ReferencesSfriso P, Autoimm Rev. 2012Touitou I, J Med Genet. 2013Disclosure of InterestNone declared

BackgroundBactericidal/Permeability-Increasing Protein (BPI) is an antibacterial and antiinflammatory protein stored in polymorphonuclear cells (PMN). BPI has been detected in synovial fluid (SF) of patients with reactive arthritis, rheumatoid arthritis (RA), psoriatic arthritis (PsA) and osteoarthritis (OA).ObjectivesThe aims of this study were to evaluate the presence of BPI in SF of patients with crystal-induced arthritis and to compare its levels with those of other arthropathies. Furthermore, we tested whether BPI exerts antiinflammatory effects in in vitro models of sterile inflammation.MethodsSF was collected from the knees of 50 untreated patients: 9 with gout, 9 with calcium pyrophosphate (CPP) crystal-induced arthritis (CPP-IA), 8 with OA, 8 with OA and CPP crystals in SF (OA+CPP), 8 with RA and 8 with PsA. SF was examined under optical light microscopy. White cell count (WBC) and the PMN percentage were determined in SF according to standard procedures. SF BPI levels were assayed by ELISA.As regard the study in vitro, the THP-1 cell line was primed for 3h with phorbole myristate acetate (300 ng/ml), reincubated overnight, and treated with sterile synthetic monosodium urate (MSU) (0.5 mg/ml) or CPP (0.1 mg/ml) crystals for 24h in presence or absence of BPI (1 or 5 μg/ml). Cell supernatants were tested by ELISA for IL-1β and IL-8 production.ResultsThe results of SF analysis and BPI levels are reported in the table.No. casesWBC, ×103/mm3PMN, %BPI, ng/mlTotal group5012.40±12.6859.03±36.6243.78±53.17Gout925.32±10.1387.75±15.2831.07±7.31CPP-IA925.32±10.1387.75±15.2823.53±9.47OA80.3±0.540.33±0.5212.21±8.40OA+CPP80.82±0.7028.80±25.1615.87±14.73RA821.35±8.9868.00±22.63146.02±32.10PsA89.16±3.5663.00±24.1763.79±31.87BPI levels were similar and the lowest in SFs from OA and OA+CPP. Although the BPI concentrations in gout and CPP-IA SFs were higher than in OA and OA+CPP, the differences did not reach statistical significance. RA SF showed the highest BPI levels (p<0.001 vs gout, CPP-IA, OA, OA+CPP, PsA). In the total group of patients, we found a correlation between BPI and WBC count (r=0.474; p=0.004) and PMN (r=0.354; p=0.037).Exposure of THP-1 cells to MSU or CPP crystals increased the production of IL-1β (MSU: 655.5±28.28 pg/ml; CPP: 496.5±9.19 pg/ml) and IL-8 (MSU: 4538.5±37.48 pg/ml; CPP: 4424.7±9.55 pg/ml). The treatment with BPI decreased the release of both cytokines induced by crystals in a dose dependent manner. Indeed, in the presence of BPI 1 or 5 μg/ml, the IL-1β secretion was reduced by 19% and 40%, respectively. IL-8 crystal-induced production was inhibited in the presence of BPI 1 μg/ml (MSU: 20%; CPP: 27%), and abolished when THP-1 cells were treated with BPI 5 μg/ml.ConclusionsThis study is the first to define the presence of BPI in SF of crystal-induced arthritis. Our results demonstrate that although SFs from gout and CPP-IA contained the WBC and PMN highest levels, BPI was present at significantly lower concentrations than in RA and PsA. This could be explained by the lack of PMN activators able to induce the release of BPI in crystal-induced arthritis.The use of BPI in our in vitro models of crystal-induced inflammation leads to a significant inhibition of the proinflammatory cytokine release, suggesting an antiinflammatory activity of this protein also in sterile inflammation.Disclosure of InterestNone declared

BackgroundAlthough in vivo studies have demonstrated that periodontitis aggravates experimental arthritis, there are no animal models that mimic the co-occurrence of these diseases.ObjectivesTo investigate the arthritogenic effect of lipopolysaccharide (LPS) in a mouse model of periodontal disease.MethodsPeriodontitis was induced in CD1 mice by injection of 0.01 or 0.05 µg of LPS in 5 µl of PBS every 48 hour into the vestibular gingiva of the second molar on the left maxilla. Untreated mice or injected with LPS at the tail were used as controls. Mice (n=10 per condition) were monitored daily and arthritis was estimated by conventional visual scoring method (scale 0–5) and recording the paw swelling with a calliper. 2 weeks after the 9th injection mice were sacrificed to collect blood, maxilla and paw samples. The left maxilla was analysed by microCT and the alveolar bone loss was assessed measuring the distance between the cementum-enamel junction (CEJ) and the alveolar bone crest (ABC) of each molar. Ultrasound (US) was performed to measure the ankle joint space. Periodontal and paw tissues were processed for histological analysis. Inflammation, vascular proliferation and bone resorption were scored (0–3) in maxilla. Inflammation, pannus formation, cartilage and bone destruction were scored (0–5) in ankle joints. CXCL1, IL-1β, IL-6 and TNF serum levels were determined by ELISA.ResultsAnkle swelling and inflammation were noted after the 5th periodontal injection of 0.05 µg of LPS, picked at day 18 and continued for the next 15 days with paw swelling and score higher than those of untreated mice (at the sacrifice p<0.001). 0.01 µg of LPS did not induce paw changes. Therefore, the subsequent assessments were conducted only in mice injected with 0.05 µg of LPS. The CEJ-ABC distance was greater in the inoculated (0.29±0.08 mm) than in the control (0.17±0.05 mm) mice (p<0.001). Histological analysis showed that LPS induced a mild vascular proliferation (score 0.8±0.42) in periodontal tissue and a substantial alveolar bone resorption (score 1.8±0.42), but not inflammation. US revealed the presence of effusion and a 1.5-fold higher joint space in the ankle of mice with periodontitis than in controls (p<0.05). Leukocyte infiltration (score 2.36±1.56) and synovial proliferation (score 2.09±1.54) were observed after histology in ankle joints of mice injected orally. The same sections had slight cartilage (score 1.32±1.21) and bone destruction (score 0.68±0.72). Animals that received LPS tail injection did not show any clinical and histological signs of arthritis. CXCL1 and TNF were higher in arthritic mice (CXCL1:2226.87±264.38 pg/ml; TNF:24.55±7.0 pg/ml), than in controls (CXCL1:445.97±92.09 pg/ml; TNF:3.22±1.04 pg/ml). Although there was no statistical difference, IL-1β and IL-6 were highest in LPS-mice (IL-1β:79.49±11.99 pg/ml; IL-6:196.02±40.62 pg/ml).ConclusionsThis study shows that experimental arthritis and periodontal disease can co-occur after LPS oral injection in mice. Our model may be useful to improve the understanding of the mechanisms underlying the link between periodontitis and arthritis.Disclosure of InterestNone declared

BackgroundAdult-onset Still’s disease (AOSD) is a rare autoinflammatory disease characterised by fever, arthritis, and multi-organ involvement. Inflammation in AOSD is mediated by interleukin (IL)−1β, as confirmed by the dramatic clinical efficacy of selective blockers of this cytokine. The genetic predisposition to this rampant IL-1-driven inflammation remains nevertheless elusive. Previous studies failed to identify associations between polymorphisms in the genes encoding IL-1 and AOSD, thus pointing at more complex genetic mechanisms. This ‘missing heritability’ cannot be adequately investigated with traditional techniques for genetic partitioning, such as GWAS, which only assess common variants and polymorphisms. Studies focusing on highly penetrant rare variants or different types of mutations (i.e. small copy-number variations; insertions/deletions) are warranted.ObjectivesWe hypothesised that genetically determined changes in IL-1-related pathways resulting in excessive IL-1β activity lead to the development of autoinflammation in AOSD. Scope of this study was to unravel the combined mutational variation of a network of IL-1-related receptors, pathways, counter-regulators, and cellular processes possibly involved in the pathogenesis of AOSD and IL-1-mediated inflammation in general.MethodsWe collected clinical, demographic, and genetic data from a large cohort of 76 AOSD patients and developed an innovative platform based on molecular inversion probes (MIP) technology for performing highly multiplexed targeted-resequencing. This allows efficient sequencing of the coding sequence of 48 genes related to the IL-1-pathway, and allows studying rare and common variants in one assay. We have also screened 500 healthy controls, and 1000s of samples with other disorders using the same assay.ResultsWe identified rare and unique (i.e. private variants) in the IL1 pathway in several individuals with AOSD. Whether any these are involved in a strong predisposition to AOSD is currently followed-up. Rare genetic variants have been identified in six IL-1-pathway ‘clusters’:Deregulated activation of the inflammasome and release of IL–1β and IL–18.IL–1 family receptors and intracellular signalling mediators.Other pro–inflammatory cytokines and receptors.Regulatory molecules, including IL–1Ra or IL–37.Cellular processes regulating production of IL–1 and IL–18 (i.e. autophagy).Production of ROS, which function as markers of cellular damage and trigger inflammation.ConclusionsUnravelling the genetic bases of inflammation in AOSD deepens our understanding of the human innate immunome. Of note, this study platform may serve for the genetic analysis of other IL-1-mediated conditions, including gout and other autoinflammatory diseases, whose genetic predisposition remains elusive. Equally important, the identification of pathways amenable to targeting with small molecules or biologics may translate into remarkable clinical implications.Disclosure of InterestNone declared