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Background:Rituximab (RTX) is the mainstay of granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) treatment for both remission induction and maintenance. Evidence regarding treatment extension beyond the standard 18-month maintenance period is scarce. The latest EULAR guidelines for the management of ANCA-associated vasculitis (AAV) confirm optional RTX extension after maintenance according to relapse risk. Whether to change immunosuppressant (IS), continue biannual RTX, increase timing between doses or tailor infusions according to B cell repopulation is still an object of debate.Objectives:To assess the effectiveness and safety of a pilot strategy based on the individual relapse risk profile for AAV maintenance of remission following the 18-month fixed 6-monthly maintenance.Methods:GPA and MPA patients who completed 18 months of RTX maintenance (4 doses, 6 months apart) followed up at Policlinico San Matteo, Pavia, were assigned to a personalised maintenance continuation regimen based on the risk of future relapse. Patients with history of severe relapse were considered at high risk of relapse and continued 6-monthly RTX (RTX6m), independently from AAV subtype and ANCA specificity at baseline. GPA and/or PR3-positive had intermediate risk and received spaced 12-monthly RTX. MPO-positive MPA patients were at low risk and stopped RTX to commence another IS. A group of patients who stopped RTX upon maintenance completion without starting another IS was included for comparison.Results:Twenty-two patients were enrolled: 13 were females (59.1%), mean age 57±13 years. Fourteen (63.6%) had GPA, 12 (54.5%) were PR3+. Upon 18-month maintenance completion, patients continued treatment according to relapse risk: 7 (31.8%) remained on RTX6m, 9 (40.9%) underwent RTX spacing, 3 (13.6%) stopped RTX in favour of azathioprine (2) or mycophenolate (1), and 3 stopped RTX without starting any IS.Median individualised treatment duration was 23 months (IQR 13-36), with 12 patients being followed up for ≥24 months. Outcomes along time are displayed in Table 1 and Figure 1. None of the RTX6m patients relapsed (Figure 1A). Indeed, they experienced severe infections and hypogammaglobulinemia more often than the other groups (Figure 1B,C). One patient with hypogammaglobulinemia progressed to severe hypogammaglobulinemia at 4 months. There was no significant difference in ANCA seroconversion between the groups, but RTX6m patients showed a delayed CD19+ repopulation compared to others (Figure 1D). Notably, RTX6m median daily glucocorticoid (GC) dose was the highest at both 12 and 24 months (Table 1). In contrast, patients on RTX spacing had early GC<5mg/day tapering. Only one patient relapsed with nasal crusting at 20 months. Spacing patients had the lowest infection rates among all groups, although one patient developed severe hypogammaglobulinemia at 16 months. The IS group had no AAV relapses, intermediate GC doses and infectious events. Two treatment-free patients had a minor relapse (arthralgia, 28 months; proteinuria and arthralgia, 21 months). There were no differences in terms of VDI increase based on the treatment group. No severe relapses, deaths or progression to end-stage renal disease occurred.Conclusion:Tailoring maintenance treatment according to individual relapse risk seems effective in balancing disease activity control with the risk of infection. Spacing can be offered to patients with intermediate relapse risk to minimise the degree of immunosuppression and its drawbacks, while biannual RTX continuation beyond the standard maintenance period should be reserved to cases at highest risk. Efficacy and safety of spaced RTX infusions for AAV maintenance should be assessed in larger cohort studies and randomised controlled trials.Table 1. Outcomes at 12 and 24 months of individualised maintenance treatment groups. REFERENCES: NIL.Figure 1.Kaplan-Meier curves of the outcomes according to maintenance treatment.Acknowledgements:NIL.Disclosure of Interests:None declared.

Despite the clinical differences between EGPA and the other AAVs, treatment guidelines are mostly based on the evidence available for patients with MPA and GPA, due to the lack of enough randomized controlled trials on patients with EGPA [1]. The aim of our study is to analyse the efficacy, safety and steroid-sparing effect of azathioprine (AZA) and methotrexate (MTX) as induction and maintenance therapy in EGPA. We collected data from 57 retrospective patients divided in 4 groups according to the treatment received (MTX/AZA as 1st line agents in patients with non-severe EGPA or as 2nd line agents in patients with severe EGPA previously treated with cyclophosphamide or rituximab). We analysed the data collected during five years of follow up from the initiation of AZA/MTX and compared the patient groups according to time to first relapse, percentage of patients in remission (defined as R1: BVAS=0, R2: BVAS=0 with prednisone ≤5mg/day, R3: BVAS=0 with prednisone ≤ 3,75mg/day – following the MIRRA definition(2)), persistence on therapy, percentage of patients with side effects and average cumulative steroid dose. There were no significant differences in the time-to-first relapse. No statistically significant differences were found in R1 remission analysis; a higher percentage of patients receiving MTX as 1st line therapy obtained R2 remission in the first 6 months (54% vs 12%, p=0,04), R3 remission wasn't achieved by any patient receiving AZA as 1st line treatment in the first 18 months of follow up (vs 35% in the MTX group, p=0.07). MTX was correlated with more adverse events (68% vs 33%, p=0.0049) still, persistence on therapy was higher for MTX (median time of suspension was >60months vs 12 months, p=0.03). The average cumulative steroid dose was lower in patients receiving MTX as a maintenance therapy compared to those receiving AZA (6g vs 10g at 5 years-follow up, p=0,039). This study shows that MTX as 2nd line therapy has a better steroid-sparing effect and allows a better persistence on therapy, while as 1st line therapy it allows an earlier remission on lower prednisone dose. [1]Chung SA et al. 2021 ACR/Vasculitis Foundation Guideline for the Management of Antineutrophil Cytoplasmic Antibody–Associated Vasculitis. Arthritis Care Res. 2021;73(8):1088–105. [2]Wechsler ME et al. Mepolizumab or Placebo for Eosinophilic Granulomatosis with Polyangiitis. N Engl J Med. 2017;376(20):1921–32. NIL. None Declared. [Display omitted] Table 1Baseline characteristic of the population and statistical differences between therapy groupsTotalMTX 1st lineAZA 1st lineP-valueMTX 2nd lineAZA 2nd lineP-valueANCANegativeMPOUnknown31 (54%)21 (37%)5 (9%)20 (80%)5 (20%)03 (38%)4 (50%)1 (12%)0.013 (28%)4 (36%)4 (36%)5 (38%)8 (62%)00.6Creatinine (mg/dl)1.44 (SD 1.69; min 0.48; max 8.62)0.77 (SD 0.13; min 0.48; max 0,99)1.05 (SD 0.5; min 0.53; max 1.8)0.022.94 (SD 1.32; min 0.79; max 8.72)1.85 (SD 1.77; min 0.55; max 6)0.2Eosinophil count (x109/l)8.33 (SD 7.1; min 0.18; max 27.67)7.32 (SD 9.33; min 0.18, max 27.62)6.34 (SD 5.64, min 0.53; max 11.8)0.89.89 (SD 4.81; min 3.8; max 14.4)9.94 (SD 4.32; min 5.46; max 17)1C-reactive protein (mg/dl)5.67 (SD 5.81; min 0; max 17.4)3.94 (SD 5.92; min 0; max 14)4.08 (SD 5.51; min 0.02; max 14.1)0.96.77 (SD 6.27; min 1.17; max 17.4)9.53 (SD 4.66; min 0.62; max 13.16)0.3BVAS/6313.59 (SD 7.3; min 0; max 29)9.16 (SD 4.23; min 4; max 20)13.12 (SD 4.39; min 8; max 21)ns18.55 (SD 6.39; min 5; max 25)19.69 (SD 6.01; min 8; max 29)nsAsthma41 (72%)21 (84%)6 (75%)ns5 (45%)9 (69%)nsSinusitis37 (65%)19 (76%)6 (75%)ns7 (63%)5 (38%)nsPulmonary nodules24 (42%)9 (36%)2 (25%)ns6 (54%)7 (53%)nsCardiomyopathy16 (28%)6 (24%)0ns4 (36%)6 (46%)nsMononeuritis multiplex12 (21%)3 (12%)3 (37%)ns2 (18%)4 (30%)nsPurpura9 (16%)1 (4%)4 (50%)0.00191 (9%)3 (23%)nsBiopsy proven glomerulonephritis6 (11%)003 (27%)3 (25%)nsPeritonitis1 (2%)00ns01 (7%)ns

A large variety of snake toxins evolved from PLA₂ digestive enzymes through a process of 'accelerated evolution'. These toxins have different tissue targets, membrane receptors and mechanisms of alteration of the cell plasma membrane. Two of the most commonly induced effects by venom PLA₂s are neurotoxicity and myotoxicity. Here, we will discuss how these snake toxins achieve a similar cellular lesion, which is evolutionarily highly conserved, despite the differences listed above. They cause an initial plasma membrane perturbation which promotes a large increase of the cytosolic Ca²⁺ concentration leading to cell degeneration, following modes that we discuss in detail for muscle cells and for the neuromuscular junction. The different systemic pathophysiological consequences caused by these toxins are not due to different mechanisms of cell toxicity, but to the intrinsic anatomical and physiological properties of the targeted tissues and cells.

Background:In patients with rheumatoid arthritis (RA), sustained remission improves physical function, virtually arrests radiographic progression, and reduces mortality. Several definitions of disease remission have been proposed and validated in randomised controlled trials, but their applicability in clinical practice remains poorly explored.Objectives:To analyse the frequency of achievement of disease remission and sustained remission, and the drivers of loss of remission according to different definitions in patients with early RA treated with conventional synthetic DMARDs.Methods:Data were retrieved from a prospective monocentric cohort of patients with early RA (<12 months of symptoms at inclusion) treated with methotrexate (82% of the cases) (±low dose glucocorticoids) with the target of low disease activity. Patients were evaluated for the achievement of different definitions of remission after 6 months of treatment: SDAI (≤3.3), Boolean2.0 (SJC28, TJC28, CRP all ≤1; PGA ≤2), Boolean1.0 (SJC28, TJC28, CRP and PGA all ≤1). Maintainance of remission according to each set of criteria was evaluated after 6 and 12 months, and reasons for remission loss were classified into inflammatory (increase in SJC28 and/or CRP) or subjective (isolated increase in PGA and/or TJC28).Results:Of a total cohort of 816 patients newly diagnosed with RA, 751 (92%) had at least 6 months of follow-up, 712 (87.3%) 12 months, and 656 (80.4%) 24 months. Collectively, after 6 months, SDAI, Boolean2.0 and Boolean1.0 remission were achieved by 21%, 19.2% and 14.4% of the patients, respectively. Among patients in SDAI remission at T6, remission was maintained in 68.3% of the cases for 6 months, and in 49.6% for 12 months. The corresponding rates of 6-months and 12-months sustained remission were 52.6% and 35.8% for Boolean2.0, and 47.6% and 29.1% for Boolean1.0, without significant differences between the two (Figure 1A). Despite comparable proportions of disease relapses, the variables leading to loss of the remission status varied between Boolean2.0 and Boolean1.0 remission. In Boolean2.0, nearly 80% of the patients failed to maintain remission because of an increase of SJC28 and/or CRP >1. In contrast, in Boolean1.0, an isolated increase of the PGA >1 was responsible for 37% of remission losses (Figure 1B). As a result, 55.3% of the patients in Boolean1.0 remission after 6 months were able to maintain 3vBoolean remission (SJC28, TJC28 and CRP all ≤1) in the following 12 months, compared with 35.8% of the patients who reached Boolean2.0 at T6 (p=0.004).Figure 1.Conclusion:In the early stages of RA, the risk of relapse after achieving Boolean2.0 point remission is high and mostly driven by inflammatory flares. In contrast, stringent remission according to the original Boolean1.0 definition leads to more prolonged suppression of inflammation, at the expense of a significant number of subjective flares. The advantages and limitations of different sets of criteria in clinical practice need to be further analysed.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:Serena Bugatti Pfizer, Ludovico De Stefano: None declared, Bernardo D’Onofrio: None declared, Emanuele Cassione Bozzalla: None declared, Antonio Manzo: None declared, Carlomaurizio Montecucco Galapagos.

Following the introduction of effective immunosuppressive treatments, ANCA-associated vasculitides (AAV) have become chronic diseases with a remitting-relapsing course. Therefore, preventing chronic damage accrual during follow-up is critical, as relapses, treatment-related side effects, and comorbidities may significantly affect the long-term outcomes of AAV patients. At present, no study specifically evaluated the burden of damage in patients with eosinophilic granulomatosis with polyangiitis (EGPA). To describe short-term (6 months) and long-term (5 years) damage accrual in patients with newly diagnosed EGPA. Patients diagnosed with EGPA, according to ACR criteria and/or Chapel Hill definitions and regularly followed-up in our vasculitis center for ≥5 years were included. Damage accrual was assessed with the Vasculitis Damage Index (VDI). Short-term and long-term damage accrual was defined by VDI at 6 months and at 5 years, respectively, and categorized as related to vasculitis or its treatment. VDI data at 6 months were available for 45 EGPA patients: 24 (53.3%) female, mean age at diagnosis 51.6±13.0 years. ANCA were positive in 17 patients (37.8%), with MPO being the only detected enzyme immunoassay (EIA)-specificity. At 6 months mean VDI was 2.8±1.3; 25/45 (55.6%) and 6/45 patients (13.3%) presented ≥3 and ≥5 items, respectively, whilst only 1 patient (2.2%) showed no items of damage. VDI data at 5 years were available for 32/45 EGPA patients (71.1%): 16 (50%) female, mean age at diagnosis 51.5±13.1 years. MPO-ANCA were positive in 13 patients (40.6%). At 5 years mean VDI was 3.5±1.3, with 26/32 (81.3%) and 7/32 patients (21.9%) presenting ≥3 and ≥5 items, respectively; notably, no patients presented a VDI=0 at 5 years. The most frequent disease-related VDI items at 6 months and at 5 years were asthma, chronic sinusitis, peripheral neuropathy, cardiomyopathy, pulmonary function tests abnormalities and nasal blockage (Figure 1). Osteoporotic fractures, diabetes and systemic hypertension were the most commonly reported treatment-related items at 6 months and at 5 years (Figure 1). Damage accrual progressively rose during the 5-year follow-up (P=0.023), mainly due to disease-related items rather than treatment-related items both at 6 months (disease related VDI 2.6±1.2, treatment-related VDI 0.3±0.6) and at 5 years (disease related VDI 2.9±1.2, treatment-related VDI 0.6±0,7). No significant difference in terms of damage accrual was observed between ANCA-positive and ANCA-negative patients (P >0.5). In our cohort of EGPA patients damage accrual occurs early, with more than half of the patients displaying ≥3 VDI items already at 6 months. Poor control of previous disease activity, particularly ENT and respiratory manifestations, contributes to progressive damage accrual more than treatment side effects. None declared [Display omitted]

BackgroundThe management of rheumatoid arthritis (RA) has substantially improved during the last few decades; despite that, a significant proportion of patients remain refractory to conventional synthetic disease modifying anti-rheumatic drugs (csDMARDs), as well as to one or more biologic/target-synthetic (b/ts) DMARDs. It is estimated the overall proportion of refractory RA is around 3-17%. Refractory disease has been defined among patients with established, long-standing, RA. However, at present time, it is not known whether early diagnosis, prompt institution of DMARD within the window of opportunity and treatment steered-to-target are efficacious to prevent multidrug failure in patients with new-diagnosed RA.ObjectivesFirst, to assess if an Early Arthritis Clinic (EAC) background – early diagnosis and treatment within the “window of opportunity”, disease management according to treat-to-target (T2) strategy – reduces the risk of being DMARDs refractory; secondly, to detect possible predictors of refractoriness in patients escalated to b/tsDMARDs.MethodsData were retrieved from a prospective monocentric cohort of 810 patients with early RA (symptoms duration <12 months at inclusion) diagnosed between 2005-2017 and treated with csDMARDs (methotrexate in 90.4% of the cases) according to a T2T strategy in the setting of an EAC. The population of interest included all patients escalated to b/tsDMARDs because of inefficacy/intolerance to csDMARDs. The frequency and factors associated with failure to first and second b/tsDMARDs were analysed by logistic regression.ResultsA total of 135/816 (16.5%) early RA patients required treatment escalation to a b/tsDMARDs after a median (IQR) of 18.5 (11-39) months from diagnosis; of them, 117 had follow-up visits available for a median (IQR) of 96 (37-122.5) months after treatment initiation. Fifty-six patients (50.4%) failed the first b/tsDMARD after a median (IQR) of 14 (7.3-44.3) months due to inefficacy/side effects. The rates of failure of the second and third b/tsDMARD were 20.5% and 19.7%. Thirteen patients (11.1%) could be defined D2T, having failed a median (IQR) number of 2 (2-3) b/tsDMARDs with different mechanisms of action, and 3 (2.8-4) different b/tsDMARDs in general. By logistic regression, significant predictors of first b/tsDMARD failure were disease duration ≤24 months and autoantibody-negativity (respectively HR 1.89, 1.01-4.12, p=0.03; HR 1.66, 1.00-3.15, p=0.04). The latter confirms to be a significant predictor of second b/tsDMARD failure as well (HR 2.13, 1.08-4.17, p=0.03). At univariable analysis, significant predictors of D2T were concomitant diagnosis of fibromyalgia, autoantibody-negative status and lower doses of methotrexate. At bivariate logistic regression, fibromyalgia and autoantibody-negative RA maintained independent association with adjusted ORs (95% CI) of 3.6 (1.02-16.4) and 4.3 (1.01-24.1), respectively.ConclusionEarly diagnosis, prompt initiation of methotrexate and management according to T2T strategy have substantially improved RA outcomes, and only about 16% of patients require escalation to second-line therapies. However, in those refractory to csDMARD, EAC setting seems not to ameliorate treatment persistence, as retention rates of b/tsDMARDs are similar to those observed in established disease. Early methotrexate inefficacy and autoantibody-negative disease appear to be predictive of first b/tsDMARD failure; moreover, patients with autoantibody-negative disease maintain a higher risk of failure of subsequent lines of therapies. Collectively, our findings suggest the need for a more tailored approach to refractory RA patients, especially to autoantibody-negative ones.Reference[1]Nagy G, Roodenrijs NMT, Welsing PM, et al. EULAR definition of difficult-to-treat rheumatoid arthritis. Ann Rheum Dis. 2021;80(1):31-35. doi:10.1136/annrheumdis-2020-217344.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

C-X-C motif chemokine ligand 13 (CXCL13) is a chemokine involved in the recruitment of B cells in secondary lymphoid organs and ectopic inflamed sites. Recent studies in animals models and in healthy individuals have shown that serum levels of CXCL13 correlate with GC activity after immunization, pointing at their role as an early biomarker of the adaptive immune response. In rheumatoid arthritis (RA) CXCL13 systemic expression has been shown to be upregulated. However, the factors involved in CXCL13 systemic upregulation and its significance as putative biomarker in patients with autoimmune diseases are currently unclear. To analyze the dynamics of CXCL13 systemic expression over time in RA after controlled induction of an adaptive immune response. Fifty-five autoantibody-positive RA patients with established disease under stable treatment with b/tsDMARDs and with no evidence of previous SARS-CoV-2 infection were included in the study. All patients received two doses of the BNT162b2 vaccine at a three weeks interval. Serum samples were collected at the time of the first BNT162b2 dose (baseline - BL), after three weeks at the time of the second dose (post dose 1 – PD1) and after two further weeks (post dose 2 – PD2). At each time point CXCL13 (pg/mL) and SARS-CoV-2 anti-spike protein antibody titres were measured. The majority of patients were female (n=42, 76%) and both ACPA and RF positive (n=42, 76%). All patients were receiving stable b/tsDMARD treatment (anti-CTLA4: 23 patients [41.8%], JAKi: 16 [29%], anti-IL6: 8 [14.5%] and anti-TNF alpha: 8 [14.5%]). At baseline the median (IQR) CXCL13 values were 81.1 (58.6-125) and all patients were negative for SARS-CoV-2 anti-spike protein antibody. Effective immunization was confirmed in all patients, in particular in 22/55 (40%) after the first dose and in 55/55 (100%) after the second dose, with a significant increase in SARS-CoV-2 anti-spike protein IgG antibody titer at PD2 (median [IQR], 142 [95.4-224]). Despite the effective induction of an adaptive immune response, no significant changes in the median (IQR) values of CXCL13 were observed at PD1 (three weeks after induction: 92.2 [61.8-141]) and PD2 (two weeks after booster: 74.2 [56-126]) with respect to each other and compared to baseline (Friedman p-value=0,947). Sub-analyses based on patients' stratification according to the b/tsDMARD treatment, confirmed a non-significant variation in CXCL13 titers between baseline, PD1 and PD2 in all groups (Friedman p-value= 0,911). Further sub-analyses limited to patients with baseline-PD1 value of CXCL13<100 pg/ml (to exclude potential biases derived from high starting value of CXCL13 titers on the delta of response) confirmed the absence of significant changes (Friedman p-value= 0.237). At the time-point analysed, the induction of an adaptive immune response per se does not appear sufficient to impact on of the expression dynamics of serum CXCL13 in established autoantibody-positive RA. NIL. NIL. None Declared.

Background:Crystal arthropaties, including gout and calcium pyrophosphate deposition disease (CPPD), are prevalent and burdensome conditions, significantly impacting the quality of life. The prevalence of these conditions in males and females are different, implying a differential approach to the diagnosis of acute oligoarthritis.Objectives:Our primary aim was to characterise the clinical presentation of crystal arthropaties in female patients and define the diagnostic approach to these conditions in a multicentre Italian population.Methods:Adult patients presenting with acute mono-oligoarthritis attributed to gout, calcium pyrophosphate deposition (CPPD), or unspecified crystal arthropathy were systematically enrolled in five Italian rheumatologic centres. Clinical, demographic, and imaging data, encompassing conventional radiography and ultrasound findings were gathered through a cross-sectional online survey conducted between January 1, 2023, and November 15, 2023. Individuals with a pre-existing diagnosis of any other inflammatory arthropathy were excluded from the analysis. Consequently, a comparative analysis between female and male patients was conducted. Statistical evaluations were executed using GraphPad, employing the Mann-Whitney test to compare variables while maintaining a significance level of 0.05. A multiple logistic regression was applied to ascertain predictors for each diagnostic test.Results:A total of 399 patients presenting with mono-oligoarthritis and diagnosed with crystal arthropathy were enrolled. Among them, 136 (34%) were females, and 263 were males. Female patients were found to be significantly older (mean age 67.1 vs. 63.8 years, p=0.008), had a lower BMI (mean BMI 24.3 vs. 27.4, p<0.001), and exhibited a higher prevalence of comorbidities (60.3% vs. 17.2%, p<0.001). Differences in dietary habits, smoking habits, and alcohol consumption were observed between the two groups. At presentation, women more frequently exhibited involvement of the knee (36.7%) and hand (21.3%), while men more often presented with involvement of the foot (55.6%) and knee (22.6%). Female patients reported significantly worse levels of pain and global health status. Women were also more likely to undergo synovial fluid examination (36.8% vs. 18.4%, p=0.001) and conventional radiography (43.4% vs. 28.7% p=0.004) to obtain a definite diagnosis. However, in 37.5% of cases, female patients did not receive a definitive diagnosis, whereas the proportion was significantly lower in males. The predominant diagnosis in female patients was CPPD (45.5%), while in males, the diagnosis was acute or chronic gout (20.3% and 50.2%, respectively). Predictors of synovial fluid examination, ultrasound, and conventional radiography examinations were comorbidities and pain, regardless of sex.Conclusion:In this multicentre cohort, female patients with crystal arthropathy are frequently complex elderly individuals with comorbidities and a low quality of life. Despite undergoing various examinations including synovial fluid analysis, ultrasound scans and radiography, nearly 40% of cases do not receive a definite diagnosis.REFERENCES:[1] Lorenzin M, et al; SIR Study Group ATTACk. Predictors of disease activity in gout: a 12-month analysis of the ATTACk (Achieving improvement in the management of crystal-induced arthritis) multicentre cohort study. Clin Exp Rheumatol. 2023 Mar;41(3):628-633.[2] Ramonda R, et al. Reumatismo. 2014 Jun 6;66(1):48-56.[3] Lorenzin M, et al; SIR Study Group ATTACk. Impact of disease duration and gender on the sensitivity and specificity of 2015 ACR/EULAR classification criteria for gout. Cross-sectional results from an Italian multicentric study on the management of crystal-induced arthritis (ATTACk). Clin Exp Rheumatol. 2022 Jul;40(7):1368-1377.Acknowledgements:Prof. Annamaria Iagnocco, Prof. Marcello Govoni, Dr. Enrico Selvi.Disclosure of Interests:None declared.

Background:Raman spectroscopy (RS) is a sensitive molecular technique that allows capturing the vibrational modes of chemical bonds in materials through the emission of photons at specific wavelengths and recording their inelastic scattering properties. These properties have attracted considerable interest in the exploitation of RS for the analysis of biological tissues, as a tool to dissect sensitively widespread changes within the cells and in the extracellular matrix, relying on limited amount of tissue. Despite initial successful results in situ and in vivo in the context of degenerative and neoplastic conditions, the analytical potential of RS in inflammatory disease remains poorly defined.Objectives:Here, we performed a proof-of-concept study evaluating the applicability of synovial tissue analysis at chemical level by RS in the context of human chronic inflammatory arthritis. We hypothesize that RS might be a tool to delineate sub-clinical and sub-histological inflammatory changes based on extremely limited amount of tissue specimens, thus potentially suitable for micro-invasive tissue sampling in difficult to target joints and disease states.Methods:Five patients with rheumatoid arthritis (RA) undergoing ultrasound-guided synovial biopsy and five patients undergoing orthopaedic non-prosthetic surgery (controls, CTRL) were enrolled. Synovial tissue from each patient was snap frozen in OCT. Parallel, 10 um tick sections were used for H&E staining and for RS. In each tissue 5 randomly selected maps were drawn within areas characterized by preserved lining layer. Each map consisted of about 450 spectra. To achieve the best spectral quality, each spectrum was acquired using a confocal Raman microscope in the fingerprint region (350-1900 cm−1) by setting an exposure time of 10 seconds, focusing the green laser source (532 nm) at 50% power, magnifying with a 20× objective and selecting 1800 L/mm grating. Raman data analysis was performed after cosmic rays removal. Savitzky–Golay filter, baseline subtraction and Asymmetric Least Squared Smoothing Baseline algorithm were selected for the pre-processing workflow. Spectra were normalized applying SNV and then cleared from OCT and noise through K-Means clustering. The resulting organic tissue-related spectra were then subjected to principal component analysis (PCA).Results:PC analysis was performed on 19732 Raman spectra over 53 maps. Less than 5% of the spectra acquired were considered not suitable for subsequent analysis. The full dataset was well separated using PCA. PC1 incorporates the highest variance (69%) and the corresponding loading plot spectrum showed well-defined positive peaks at 749, 1130 and 1585 cm-1 porphyrin groups typical molecular vibrations (myeloperoxidase) and negative peaks at 855, 1450 and 1670 cm-1 attributable to proteins. PC4 showed predominantly a lipid character as evidenced by its positive peaks at 1004, 1156, 1520 and 1657 typical of carotenoids and triglycerides, while the negative peaks express a protein character being attributable to Amide I and Amide III (Figure 1). Average PC1 per map and per patient showed a highly discriminative power between RA and CTRL with an AUC of 0.903 and 0.93, respectively. No significant correlations were observed with the Krenn’s score (KS) (rho=0.061, p=0.85). Oppositely, PC4 showed a significant correlation with the KS (rho=0.928, p<0.01) despite a lower discriminative power for diagnosis. Restriction of the analysis to the samples with KS<2 (no inflammation, n=2 RA and n=2 CTRL) confirmed a significant difference in PC1 between RA and CTRL (p<0.001). The spectral variations between the CTRL and RA were further highlighted by plotting loading vectors PC1 and PC4 (Figure 2).Conclusion:Results of the current study demonstrate the applicability of RS for chemical analysis of frozen synovial tissue in human chronic inflammatory arthritis. Pilot analyses provide evidence of the potential of RS to highlight chemical differences between RA and CTRL based on the evaluation of a very limited amount of tissue over and above the histopathologic level.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

The rheumatoid arthritis (RA) treatment landscape is diverse, with multiple therapies available that differ in several attributes such as mode of administration and benefit-risk profile. Patients and prescribers face challenging trade-offs during treatment selection to accommodate patients' circumstances in order to ensure comprehensive disease management. EULAR recommendations for RA management emphasize the need to recognize patient preferences in shared decision-making (SDM). Therefore, it is essential to understand how preferences differ in the RA patient population. This study elicited trade-offs that RA patients were willing to make during treatment selection while accounting for preference heterogeneity. An online discrete choice experiment (DCE) was conducted from September to October 2021 in which RA patients were required to elicit their preferences for attributes of treatments for RA (Figure 1) and make trade-offs between them. Attributes were selected and defined based on literature review and qualitative patient interviews, and were then tested in a quantitative pilot. Main data collection consisted of an online survey in which participants were asked to repeatedly choose between hypothetical treatments. Eligible patients were ≥18 years old, diagnosed with RA, currently received systemic disease-modifying anti-rheumatic drug therapy for RA, and were residents of France, Germany, Italy, Spain, United Kingdom, or United States. Male patients were oversampled to support subgroup analysis of preferences for effects on sperm parameters. Data were analyzed using a correlated mixed logit model and differences in preferences between sex and age were explored. Relative attribute importance (RAI) scores and maximum acceptable risk (MAR) measures were derived from the estimates. A total of 2,090 patients participated; 42% were female with predefined oversampling of male patients, with a mean age of 45.2 years (range 18–83). Estimated effects were significant for all attributes (p<0.001), implying that they all influenced treatment choices and suggesting preferences differed between participants. Average RAI scores revealed different priorities (p<0.001) between males and females (Figure 1). While reducing pain and negative effect on semen parameters was most important to male patients, female patients were most concerned by risk of blood clots and serious infections. The remaining attributes were of lower importance but were still relevant. However, no single attribute explained treatment preferences by more than 30%. Preferences were also affected by patients' age: patients aged 18-44 years placed less importance on frequency and mode of treatment administration (p<0.05) than older age groups. Patients were willing to make benefit-risk trade-offs; they accepted extra risks of blood clots (male: 1.8%; female: 0.8%), serious infections (male: 2.5%; female: 1.0%), or negative effects on sperm (male: 7.4%) for an oral pill every day instead of injection once a week. They also accepted extra risks of blood clots (male: 2.3%; female: 1.2%), serious infections (male: 3.2%; female: 1.6%), or negative effects on sperm (male: 10.4%) for reducing amount of pain from 30% to 10%. Similar observations were made for improved performance of daily activities. However, acceptable trade-offs varied between patients (p<0.05). Preferences of RA patients were driven by benefits and risks of RA treatments, with no single attribute dominating the decision making. Patients were willing to accept higher risk of serious infections and blood clots in exchange for improvements in pain, daily activities, or administration convenience. These findings emphasize the importance of considering the entire treatment profile, including benefits, risks, and administration to support SDM between providers and patients. Preference drivers: males – pain, blood clots; females – blood clots, infections, pain. Sperm risk data are based on male responses only. This study was funded by Galapagos NV (Mechelen, Belgium). Publication coordination was provided by Fabien Debailleul, PhD, of Galapagos NV. Medical writing support was provided by Brooke Middlebrook (Evidera) and publications management was provided by Aspire Scientific Ltd (Bollington, UK), funded by Galapagos NV. Rieke Alten Consultant of: AbbVie, Amgen, Biogen, BMS, Celltrion, Gilead, Janssen, Lilly, Medac, MSD, Mylan, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, UCB, Viatris, Juan Carlos Nieto González Speakers bureau: AbbVie, Amgen, Biogen, BMS, Celgene, FAES Farma, Gebro, Janssen, Lilly, MSD, Nordic Pharma, Novartis, Pfizer, Roche, Sandoz, Sanofi, UCB Pharma, Consultant of: AbbVie, Amgen, GSK, Galapagos, Janssen, Lilly, MSD, Peggy Jacques: None declared, Carlomaurizio Montecucco Speakers bureau: AbbVie, BMS, Boehringer, Eli Lilly, Galapagos, Pfizer, Roche, Sanofi, Consultant of: AbbVie, BMS, Gilead, Robert Moots Speakers bureau: Amgen, Galapagos, Consultant of: Ferring, Grant/research support from: Novartis, Helga Radner Speakers bureau: Gilead Sciences, Janssen, MSD, Pfizer Corporation Austria, Sebastian Heidenreich Employee of: Evidera Inc, which is part of Thermo Fisher Scientific's Clinical Research Group. Evidera received payment for conducting the work outlined in this work., Chiara Whichello Employee of: Evidera Inc, which is part of Thermo Fisher Scientific's Clinical Research Group. Evidera received payment for conducting the work outlined in this work., Nicolas Krucien Employee of: Evidera Inc, which is part of Thermo Fisher Scientific's Clinical Research Group. Evidera received payment for conducting the work outlined in this work., Monia Zignani Shareholder of: Galapagos, Employee of: Galapagos, Harald Vonkeman Speakers bureau: AbbVie, Boehringer Ingelheim, Galapagos, Janssen, Novartis, Pfizer, UCB, Grant/research support from: AbbVie, Boehringer Ingelheim, Galapagos, Janssen, Novartis, Pfizer, UCB, Katrien Van Beneden Shareholder of: Galapagos, Employee of: Galapagos. [Display omitted]