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Key Points A 'cytokine storm' is the final pathophysiological pathway in macrophage activation syndrome (MAS), and blocking various cytokines could be an attractive therapeutic strategy Standard doses of anti-IL-1 and anti-IL-6 biologic therapies do not have a major effect on MAS rates even if the underlying disease responds well to the treatment Several case reports suggest that anakinra might be effective at least in some patients with systemic juvenile idiophatic arthritis (sJIA)-associated MAS, particularly when used in high doses Findings from several studies support IFN-γ blockade as a novel therapy for haemophagocytic lymphohistiocytosis (HLH); increasing evidence suggests the same approach could be beneficial in MAS presenting as a complication of rheumatic diseases The exact mechanism of predisposition to MAS in sIJA is yet to be defined, but might be independent of underlying sJIA activity and similar to infection-associated secondary HLH Whole-exome/genome sequencing approaches exploring hypomorphic mutations that affect the cytolytic pathway to support this theory might reveal promising therapeutic alternatives Macrophage activation syndrome (MAS) is a potentially fatal complication of rheumatic disease, most notably systemic juvenile idiopathic arthritis. Findings from studies in animal models and from clinical observations, particularly in relation to the effects of anticytokine biologic therapies, have led to new concepts of the pathophysiology of this phenomenon. Macrophage activation syndrome (MAS) refers to acute overwhelming inflammation caused by a 'cytokine storm'. Although increasingly recognized as a life-threatening complication of various rheumatic diseases, clinically, MAS is strikingly similar to primary and secondary forms of haemophagocytic lymphohistiocytosis (HLH). Not surprisingly, many rheumatologists prefer the term secondary HLH rather than MAS to describe this condition, and efforts to change the nomenclature are in progress. The pathophysiology of MAS remains elusive, but observations in animal models, as well as data on the effects of new anticytokine therapies on rates and clinical presentations of MAS in patients with systemic juvenile idiopathic arthritis (sJIA), provide clues to the understanding of this perplexing clinical phenomenon. In this Review, we explore the latest available evidence and discuss potential diagnostic challenges in the era of increasing use of biologic therapies.

Nephropathic cystinosis is a rare genetic disorder characterized by cystine accumulation in lysosomes that causes early renal dysfunction and progressive chronic kidney disease. Although several metabolic pathways, including oxidative stress and inflammation, have been implicated in the progression of renal parenchyma damage, the precise mechanisms driving its progression are not fully understood. Recent studies suggest that epigenetic modifications, particularly DNA methylation (DNAm), play a critical role in the development of chronic kidney disease. We hypothesized that epigenetic dysregulation may contribute to the progression of kidney disease in cystinosis. To investigate this, we conducted genome-wide DNAm analyses on kidneys harvested from 6-month-old wild type (WT) and mice, a well-established model of cystinosis. Our analysis revealed extensive DNAm alterations in cystinotic kidneys, characterized by a significant hypermethylation profile. Interestingly, the majority of differentially methylated CpG sites were located within gene bodies and to a lesser extent in promoter and enhancer regions. Methylation changes were primarily found in genes and pathways crucial for kidney function, particularly those related to the physiology of the proximal tubules. Importantly, DNAm changes correlated with changes in gene expression, as validated by qPCR analyses of key genes. Furthermore, treatment of human proximal tubular epithelial cells with the demethylating agent decitabine resulted in the upregulation of critical transporter genes, suggesting a potential therapeutic approach. These findings underscore the role of epigenetic regulation in the progression of kidney damage in cystinosis and suggest that DNAm could serve as a promising target for novel therapeutic strategies.

The anti–interleukin-1 antibody canakinumab was effective at controlling and preventing recurrence of flares in autoimmune inflammatory diseases: familial Mediterranean fever, mevalonate kinase deficiency, and the TNF receptor–associated periodic syndrome.

BackgroundMAS is a severe, life-threatening form of secondary hemophagocytic lymphohistiocytosis associated with rheumatic diseases such as Still’s disease (systemic juvenile idiopathic arthritis or adult-onset Still’s disease) and systemic lupus erythematosus (SLE). IFNγ has been implicated in the underlying pathology of both conditions. High-dose glucocorticoids (GCs) are the standard first-line treatment for MAS; however, there is no standardized approach to treating patients who are refractory to high-dose GCs. In a pilot study of 9 patients with an inadequate response to high-dose GCs, IFNγ was rapidly neutralized, and MAS controlled, following emapalumab administration (NCT03311854). Emapalumab also had a favorable safety profile.ObjectivesTo present the design of a study enrolling patients with MAS in Still’s disease and SLE with the aim of evaluating the efficacy and safety of emapalumab.MethodsEMERALD (NCT05001737) is an open-label, single-arm, multicenter, phase 2/3 interventional study enrolling pediatric and adult patients with MAS and an inadequate response to high-dose GCs into two cohorts: MAS in Still’s disease (n=25) and MAS in SLE (n=16). Key eligibility criteria vary by cohort and phase (Table 1). Each cohort is a single-arm study comprising two phases: an optional run-in phase and an interventional phase. Patients are treated with high-dose GCs during the run-in phase and followed until MAS remission, inadequate response, or 12 weeks (whichever occurs first). Eligible patients enrolled in the interventional phase are treated with emapalumab intravenously for 4 weeks (6 mg/kg on Day 1; 3 mg/kg every 3 days from Days 4–16, then 3 mg/kg twice weekly from Days 17–28) or until protocol-defined complete response (CR). Patients will be followed up for 1 year after study completion.Key inclusion criteriaKey exclusion criteriaRun-in phase• Informed consent• Male and female patients aged 6 months to 80 years at diagnosis of MAS• MAS defined per the MAS diagnostic criteria and requiring treatment with GCsInterventional phase• Informed consent• Male and female patients aged 6 months to 80 years at diagnosis of MAS• Patients with a diagnosis of MAS having shown an inadequate response to high dose GCs as per local standard clinical practice• Primary hemophagocytic lymphohistiocytosis• Confirmed malignancy• Evidence of Leishmania infection, clinically active mycobacteria (typical and atypical), Histoplasma capsulatum, salmonella infections, or latent tuberculosis• History of hypersensitivity or allergy to any component of the study drug• A bacillus Calmette-Guérin vaccine within 12 weeks prior to screening• Live or attenuated live (other than bacillus Calmette-Guérin) vaccine within 4 weeks prior to Screening• Pregnancy or lactation• At time of emapalumab initiation, treatment with: – Canakinumab, Janus kinase inhibitors, tumor necrosis factor inhibitors, tocilizumab – Anakinra at a dose >4 mg/kg• Use of etoposide for MAS within 1 month prior to treatment with emapalumabMAS diagnostic criteria• Febrile patient presenting with ferritin >684 ng/mL• And any two of: – Platelet count ≤181 x109/L – Aspartate aminotransferase levels >48 U/L – Triglycerides >156 mg/dL – Fibrinogen levels ≤360 mg/dLResultsProtocol-defined CR at Week 8 after first emapalumab administration is the primary endpoint of the EMERALD study. Secondary efficacy endpoints include GC tapering, survival, time to first CR, overall response (CR and partial response), time to first overall response, MAS recurrence, pharmacokinetic/pharmacodynamic profile of emapalumab, and patient-reported outcomes. Adverse events, abnormal laboratory parameters, and anti-drug antibodies will be monitored as safety endpoints.ConclusionThe ongoing EMERALD study is designed to address the unmet need for efficacious and safe therapies for the treatment of MAS, particularly for patients who are refractory to high-dose GCs.AcknowledgementsMedical writing assistance was provided by Blair Hesp PhD CMPP of Kainic Medical Communications Ltd. (Dunedin, New Zealand), which was funded by Sobi AG (Basel, Switzerland).Disclosure of InterestsFabrizio De Benedetti Consultant of: Consultant for AbbVie, Sobi, Pfizer, Roche, Sanofi, Novartis, Novimmune, Grant/research support from: Research grants from AbbVie, Sobi, Pfizer, Roche, Sanofi, Novartis, Novimmune, Rafael Krmar Employee of: Employee of Swedish Orphan Biovitrum AB, Radmila Kanceva Employee of: Employee of Swedish Orphan Biovitrum AG, Alexei Grom Consultant of: Consultant for Novartis, AB2 Bio, Novimmune, Sobi.

BackgroundPediatric Antiphospolipid Syndrome (APS) is an autoimmune disease characterized by venous and/or arterial thrombotic events (TE) associated with 2 consecutive positive determinations (at least 12 weeks apart) of antiphospholipid antibodies (aPL), IgG/IgM anticardiolipin (aCL), IgG/IgM β2-glycoprotein I (aβ2GPI) and/or lupus anticoagulant (LA). Recent data suggests that aPL levels may decrease over time due to the natural history of the disease or to treatments. Therefore, monitoring of aPL levels may represent a strategy to evaluate disease activity and response to therapies.ObjectivesTo investigate the trend over time of aPL titers in children with APS, comparing patients under immunomodulatory therapies and those without them.MethodsA descriptive, observational, cross-sectional study was carried out in children with APS. aPLs testing was carried out in all patients from diagnosis every 3-4 months for 2 years. Interferon Gene Signature (IGS) was assessed as described by Crow[1]. Laboratory parameters, clinical and demographic data was retrieved and analyzed. Statistical analysis was performed with software R_v. 4.0.3.ResultsSixteen children with a diagnosis of APS were included in this study. The median age at disease onset was 11.5 years (range: 6 months – 17 years) and 62.5% were female; 87.5% Caucasians. Thirteen patients (81.2%) had a diagnosis of primary APS and 3 (18.8%) out of 16 of secondary APS.Regarding clinical manifestations, 11 children developed at least one TE (7 arterial and 5 venous). Cerebral territory was the most frequently involved with 5 thrombosis, followed by 3 deep vein thrombosis, 1 pulmonary thromboembolism and 2 arterial renal thrombosis. Most of the patients received an antiaggregant and/or anticoagulant therapy (13 and 8 patients, respectively). However, 1 patient presented a new TE after the antiaggregant withdrawal.A total of 12 (75%) children developed at least one non-criterion manifestation: 7 patients (44%) cardiac (Libman-Sacks endocarditis or valvular heart disease), 7 patients (44%) neurological (chorea or white matter lesions), 6 patients (37.5%) hematological (thrombocytopenia, hemolytic anemia or Evans syndrome) and 1 patient (3.8%) a renal thrombotic microangiopathy.Related to immunological parameters, 4 (25%) were positive for only 1 aPL, 5 (42%) for 2 aPL subtypes and 7 (44%) for all 3 aPL subtypes, showing the highest rates IgG aβ2GP (87.5%) and IgG aCL (81.25%). Lower rates were identified for LA (44%), IgM aCL (18.7%) and IgM aB2GP (12.5%). Four (25%) had ANA positivity (3 secondary and 1 primary APS).Regarding treatment, 13 children (81.25%) received at least one immunomodulatory drug (13 patients mycophenolate; 4 rituximab) and 3 (18.7%) were not treated with them.During the 2-year-follow-up, 11 patients (68.8%) showed a reduction of aPL titers compared to the onset, with becoming 9 negative (56.3%) at the end of follow-up (Figure 1A). Of those who were negative, 8 children were under immunomodulatory therapies and only 1 did not receive any treatment. Five patients (31.2%) showed stable titers of aPLs during follow-up. Of them, 2 were not treated with immunomodulatory therapies and 2 were under mycophenolate but with poor compliance (reduction of titers with the restart of therapy were observed).We also evaluated the presence of the IGS in 11 patients with APS for whom a whole blood RNA sample was available: the IGS was positive in 9 (81.8%) of 11 children (7 with primary APS and 2 with secondary APS).ConclusionOur data suggest the possible effect of immunomodulatory therapies in reducing antibody titers in APS. Therefore, it may represent a strategy to control the disease activity leading to a better prognosis. Further studies are needed to confirm and expand our data.Reference[1]Rice GI, et al. Lancet Neurol 2013; 12:1159-69.Figure 1.A. Trend of IgG aB2GP during 2 years of follow-up. B. Trend of IgG aCL during 2 years of follow-up.Red line (0): children without immunomodulatory (IMM) therapies; blue line (1): children with IMM therapies.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

Background:Methotrexate (MTX) is the most used drug for the treatment of children and adults suffering from arthritis although its intake is burdened by well-known side effects. The Methotrexate Intolerance Severity Score (MISS) was originally developed in English [1] to identify patients intolerant to MTX.Objectives:Methotrexate (MTX) is the most used drug to treat children and adults with arthritis and its use is burdened by adverse effects. The MTX intolerance severity score (MISS) was developed in English to identify patients who are intolerant to MTX. The aim of this study was to translate and validate the MISS in Italian.Methods:The Italian version of the MISS was developed following the “guidelines for process of cross-cultural adaptation of self-reported measures”. The Italian version of the MISS was validated in 125 patients with juvenile idiopathic arthritis (JIA) followed at the Rheumatology Unit of Bambino Gesù Children Hospital. We assessed the construct validity and calculated the internal consistency of the Italian MISS. We performed ROC analysis to assess the overall performance of the Italian MISS.Results:Of the study cohort (83% female), 71 patients had oligoarthritis, 43 had RF-negative polyarthritis, 4 had enthesitis-related arthritis (ERA), and 3 had psoriatic arthritis. The mean age (±IQR) at diagnosis was approximately 4 years, with a “disease duration” on the MISS questionnaire of 6 years (IQR 3.3-10.5). The mean (± SD) VAS and c-JADAS scores were 0.7 and 2.4 respectively, with 65% of patients having inactive disease. The majority (98%) of patients received MTX subcutaneously and the average duration of use of the drug was approximately 5 years. We translated and adapted the MISS to the Italian language. The Italian MISS showed a very good internal consistency as shown by a Cronbach a of 0.87 (95% CI, 0.84-0.90) and a composite reliability of 0.89 (95% CI, 0.83-0.91). A threshold of 6 to define intolerant patients, showed a sensitivity of 98.3% and specificity of 81.2%.Conclusion:We developed the Italian version of the MISS and showed its validity and reliability to identify patients intolerant to MTX in clinical practice and in a research setting.REFERENCES:[1] M. Bulatović et al., “High prevalence of methotrexate intolerance in juvenile idiopathic arthritis: development and validation of a methotrexate intolerance severity score.” Arthritis Rheum 63, 2007-2013 (2011).[2] D. Wild et al., “Principles of Good Practice for the Translation and Cultural Adaptation Process for Patient-Reported Outcomes (PRO) Measures: report of the ISPOR Task Force for Translation and Cultural Adaptation.” Value Health 8, 94-104 (2005).Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Juvenile Idiopathic Arthritis (JIA) is the most common form of chronic arthritis in childhood. The adaptive branch of the immune system plays a crucial role in the development of JIA. We have showed that switched memory B cells are expanded in patients with oligoarticular- and polyarticular-JIA.Objectives:To characterize the antigen-experienced immunoglobulin repertoire, we performed deep sequencing of the switched repertoire in peripheral blood (PB) of oligo-/ploy-JIA patients(n=14) and age matched controls(n=4), and in matched synovial fluid (SF) of oligo-/ploy-JIA patients(n=7).Methods:We employed a cDNA-based-5′RACE approach with unique molecular identifiers. De-multiplexing, UMI extraction, and UMI-based consensus assembling were performed using the MIGEC software. Clonotype assembly were performed using MiXCR. SHM analysis was performed with SHazaM. B cell tolerance checkpoints were analyzed with a validated flow cytometry-based system (Malkiel S, Arthritis Rheumatol. 2016).Results:Immunoglobulin heavy chain variable gene segment usage frequency was similar between patients and controls, and PB and SF. Thus, we did not observe clustering of patients from controls. Analysis of averaged complementarity-determining region 3 (CDR3) repertoire characteristics revealed that CDR3 length was similar among the three groups, whereas physico-chemical characteristics of amino acid residues were significantly different in the IgG repertoire, but not in the IgA repertoire. IgG from SF showed higher charge and polarity and an increased hydrophobicity index compared to PB of controls and patients. We then analyzed the frequency of somatic hypermutation (SHM) and the selection pressure with SHazaM. We found a significant reduction in the frequency of SHM and a higher frequency of sequences with less than 10 mutations per Ig molecule in patients (in both PB and SF) than controls. The IgG repertoire in SF exhibited decreased positive selection in CDRs. To further characterize the immunoglobulin repertoire, we performed a flow cytometry-based staining to assess the frequency of autoreactive B cells in the transitional, naïve and memory B cell subsets in PB and also of memory B cells in SF. We found that central tolerance checkpoints are effective in both JIA patients and controls: the frequency of autoreactive B cells decreased from transitional, to naïve B cells in both JIA patients and controls. However, the frequency of autoreactive B cells was higher in memory B cells of JIA patients compared to controls, with the highest frequency observed in memory B cells of SF.Conclusion:Altogether, the observed changes in the IgG repertoire in PB and SF establish an altered peripheral selection process of IgG+ B cells in oligo-/ploy-JIA with a decreased load of SHM. Our data show for the first time that an altered GC response, leading to the accumulation of autoreactive B cells in the memory compartment, is a feature of oligo-/ploy-JIA.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:Emiliano Marasco: None declared, Angela Aquilani: None declared, Ivan Caiello: None declared, Rebecca Nicolai: None declared, Giusyda Tarantino: None declared, Silvia Magni-Manzoni: None declared, Rita Carsetti: None declared, Fabrizio De Benedetti Novartis, SOBI.

Background:Juvenile Idiopathic Arthritis (JIA) is the most common form of chronic arthritis in childhood. The adaptive branch of the immune system plays a crucial role in the development of JIA. We have showed that switched memory B cells are expanded in patients with oligoarticular- and polyarticular-JIA.Objectives:The aim of this study is to investigate abnormalities in the switched memory B cell compartment and characterize the receptor signaling machinery in B cells of patients with oligo- and poly-JIA.Methods:We enrolled patients with a diagnosis of oligoarticular and polyarticular JIA (n=15) and age matched controls (n=15). Surface staining of lymphocytes was carried out to identify B cell and T cell subsets in peripheral blood (PB) and synovial fluid (SF). Immunoglobulins levels were retrieved from electornic records. Intracellular levels of phospho-proteins were evaluated in PB by flow cytometry. PBMC were stimulated in vitro with an anti-Ig and stained for intracellular phospho-proteins. Calcium mobilization after anti-Ig stimulation was evaluated with Indo-1 by flow cytometry.Results:The ratio of IgG to IgA switched memory B cells was higher in JIA patients than controls, indicating and expansion of IgG+ memory B cells over mucosal IgA+ memory B cells in JIA (Figure 1A). IgG+ memory B cells were the most abundant B cell subset in SF (Figure 1A). Serum levels of IgG, but not IgA, were higher in JIA patients than controls.To investigate if the hyperactivation of B cells is due to an henanced B cell receptor (BCR) signaling, we analyzed the basal phosphorylation levels of 3 key enzymes (Syk, BLNK, ERK1/2) in B cells of JIA patients. We observed a higher frequency of phosphorylation of BLNK, but not of Syk and ERK1/2 in both naïve and memory B cells compared to controls (Figure 1B). Next, we evaluated the surface expression of CD22, a negative regulator of B cell signaling in all B cell subsets. Transitional, naïve and IgM memory B cells of patients with JIA showed reduced levels of CD22 compared to controls; no significant differences were observed in switched memory B cells (Figure 1C). As BLNK and CD22 regulate the calcium mobilization following BCR stimulation, we analyzed calcium mobilization. Upon in vitro stimulation of BCR, we observed that naïve B cells of patients with JIA had a faster calcium response to BCR activation, with a shorter peak time compared to controls (Figure 1D).Conclusion:Our data confirm the expansion of IgG memory B cells and the hyperproduction of IgG in patients with JIA. We also show that naïve B cells of JIA patients exhibit a reduced expression of CD22 and an increased basal phosphorylation of BLNK. Upon activation of BCR, naïve B cells respond more promptly to BCR activation with faster calcium mobilization.Figure 1.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:Emiliano Marasco: None declared, Angela Aquilani: None declared, Matteo Trevisan: None declared, Ivan Caiello: None declared, Giusyda Tarantino: None declared, Rebecca Nicolai: None declared, Silvia Magni-Manzoni: None declared, Rita Carsetti: None declared, Fabrizio De Benedetti Novartis, SOBI

BackgroundSystemic lupus erythematosus (SLE) is a systemic autoimmune disease which leads to inflammation and organ damage caused by immune complex deposition. Classically, childhood SLE has been considered as a polygenic autoimmune disease; however, a pediatric monogenic lupus-like phenotype (LL) is emerging due to the recent recognition of several related novel high-penetrance gene variants. This fact associated to the high degree of concordance among monozygotic twins, supports the importance of genetic background in cSLE pathogenesis.ObjectivesTo identify the presence of variants in gene related to monogenic lupus and their relationship with clinical manifestations in cSLE or LL.MethodsA descriptive, observational, cross-sectional study was carried out in children with cSLE or LL. The genetic analysis (Sanger/Clinical Exome Sequencing) was performed from isolated DNA obtained from blood sample.ResultsForty-five children were included in the study. The genetic analysis detected at least one variant in 14 (31.1%) children, 5 (35.7%) with cSLE and 9 (64.3%) with LL. Of those who carry a genetic variant, the median age at disease onset was 11 years (range: 2-16) and 85.7% were female; most of them Caucasians (85.7%). Seven (50%) and 3 (21.4%) out of 14 patients had a positive family and/or a personal history for autoimmune diseases, respectively. Regarding clinical manifestations at onset, musculoskeletal were the most frequent (10 patients, 71.4%), followed by hematological (9 patients, 64.3%), cutaneous (9 patients, 64.3%), constitutional with fever (8 patients, 57.1%), neurological (7 patients, 50%), renal (5 patients, 35.7%), cardiac (3 patients, 21.4%) and pulmonary (2 patients, 14.3%) manifestations. Related to immunological parameters, 13 (92.8%) were ANA positive, 7 (50%) anti-dsDNA, 4 (28.6%) ENA and 3 (21.4%) aPL positive. Both C3 and C4 were low in 8 (57.1.4%) children and isolated C3 levels were low in 4 (28.6%) patients. Among the variants, we found that only two patients who carry a TREX variant showed normal C3 and C4 levels; one of them presented with lupus pernio as reported in literature. Also, we identified the same RNASEH2B (c.868G>A) variant in two siblings with similar phenotypes and TLR7 (c.1520T>C) variant in two siblings of other family. The patient who carried the SHOC2 variant presented polyarthritis and serositis, while the patient with the TNFRSF13B variant onset with a glomerulonephritis. Those manifestations have already been described related to these genes. Variants and phenotypes are detailed in Table 1.ConclusionAround 30% pediatric patients with cSLE or LL showed at least one variant in gene related to monogenic-lupus and some of them with similar phenotypes to those already described. This fact may suggest the genetics potential contribution to the cSLE pathogenesis. Further studies are necessary to confirm these data.Reference[1]Alperin JM, Ortiz-Fernández L, Sawalha AH. Monogenic Lupus: A Developing Paradigm of Disease. Front Immunol. 2018;9:2496.Table 1.GeneDiagnSkin N=6MSK N=8Hematological N=6Fever N=7Lung N=2Cardiac N=3NRL N=7Renal N=5C3/C4 N=12ANA N= 13 DNA N=7ADAR c.16-8 T>CSLExArthralgia↓ Hb, L, N+xxxx↓/↓+/+TNFAIP3 c.2170A>CSLERashx↓ Hb, L, N+xConduction disorderxLN↓/↓+/+RNASEH2Bc.105 107delAATSLExArthralgia↓ PLT, L+xSerositisxx↓/N+/-SHOC2SLERashArthritis↓ Hb, PLT, L+SerositisSerositisxLN type III↓/↓+/+IFIH1 c.2807 + 1G>ASLEOral ulcersArthralgia↓ PLT-xxxx↓/↓+/+TREX c.797A>G; TNFAIP3 c.1405C>GLLRash, oral ulcers, lupus pernioArthralgiax-xxHeadachexN/N+/-DNASE1 c.105G>CLLRashArthritis↓ Hb, L, N, PLT-xxxx↓/N+/-RNASEH2B c.868G>A;C1S c.619C>TLLxArthralgiax-xxHeadachex↓/N-/-RNASEH2B c.868G>A;TLR7 c.3094G>A; STAT5A c.1248C>GLLxArthralgiax+xxHeadachex↓/N+/-TREX1c.341G>ALLxxx-ILDxWMIxN/N+/-TNFRSF13B c.41G>ALLRashxx+xxxMP GMN↓/↓+/+TLR7 c.1520T>CLLRashArthritis↓ PLT+xxTEMP GMN↓/↓++TLR7 c.1520T>CLLRashx↓ N+xxSNC calcificationsx↓/↓+/-C2 c.719G>TPEPD c.703G>ACYBB c.593G>ALLRashArthritis↓ Hb, L+xxSNC calcificationsMP GMN↓/↓+/+Acknowledgements:NIL.Disclosure of InterestsNone Declared.

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinuclear antibodies (ANA). Monitoring of anti-DNA antibody levels may reflect disease activity, by contrast a single anti-RBP antibody determination is thought to suffice for clinical purposes. Recent data suggests that ANA levels may decrease over time secondary to the natural history or to treatments.ObjectivesTo investigate the trend of ANA and anti-dsDNA titers over time in children with a diagnosis of SLE.MethodsWe enrolled 15 children with SLE. ANA and anti-dsDNA testing were carried out in all patients from diagnosis every 3-4 months for 2 years. ANA were defined as negative for titers < 1:80. Laboratory parameters, clinical and demographic data was retrieved and analyzed. Interferon Gene Signature (IGS was assessed by the expression of 6 interferon-induced genes (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1) in whole blood RNA. Statistical analysis was performed with SW R_v. 4.0.3.ResultsFollowing 2 years of follow-up, all patients had ANA titers significantly lower than at time of the onset (MWW, p=0.0002) (Figure 1A). After two years of follow-up, 11 patients (73%) remained ANA positive (group 1), while 4 patients (26%) became negative (group 0). At time of diagnosis no significant differences in ANA titers (MWW, p=0.74) nor in disease activity, measured by SLEDAI, (MWW, p=0.88) were observed (table 1; Figure 1E). No significant differences in organ involvement were observed (Table 1). Assessing the change over time in ANA titers, the 2 groups of patients showed 2 different patterns: in group 0, ANA titers quickly declined and disappeared in the first 6 months after diagnosis; in group 1, ANA titers declined more slowly, remaining positive at 2 years (Figure 1C). ANA pattern (by IFA) was also evaluated, changes from homogenous pattern to speckled was observed during follow up (Figure 1B). Both C3 and C4 increased, with no different patterns between the 2 groups (Figure 1D). Similarly, anti-dsDNA antibodies titers declined over time with no clear different patterns between the groups (Figure 1C). We also analyzed the levels of IGS at last of follow-up, observing significant differences between the groups (MWW, p=0.018) with higher levels of IGS in ANA+ patients (Figure 1F).ConclusionOur analysis showed 2 different patterns in the reduction of ANA titers over time in children with SLE, with 26% of them becoming ANA negative after 6 months from diagnosis and remaining persistently negative during follow-up. Our data have important implications, specifically for the recruitment of patients into clinical trials, where the latest classification criteria of SLE require ANA positivity as entry criterion. A seronegative state may represent a different subcategory of patients with SLE with specific pathogenetic pathways, possibly independently from autoantibodies. Therefore, further studies are needed to confirm our data.Reference[1] Pisetsky DS, Nat Rev Rheumatol. 2020.Table 1.ANA- at 2yANA+ at 2yPatients, n411Female, n (%)3 (25)10 (9)Age, mean ± SD (years)13.80 ± 1.9113.45 ± 2.72Disease duration, mean ± SD (years)6.47 ± 4.226.17 ± 2.71EULAR/ACR 2019 criteriaFever, n (%)3 (75)8 (73)Acute cutaneous, n (%)4 (100)6 (55)Chronic cutaneous, n (%)0 (0)0 (0)Non-scarring alopecia, n (%)0 (0)1 (9)Oral/nasal ulcers, n (%)2 (50)7 (64)Joint involvement, n, (%)3 (75)4 (36)Serositis, n (%)0 (0)2 (18)Renal, n (%)1 (25)2 (18)Neurological, n (%)0 (0)0 (0)Hemolytic anemia, n (%)1 (25)4 (36)Leukopenia, n (%)4 (100)9 (82)Thrombocytopenia, n (%)3 (75)5 (45)Anti-dsDNA, n (%)4 (100)11 (100)Anti-Sm, n (%)1 (25)2 (18)LA, n (%)0 (0)1 (9.1)aCL, n (%)2 (50)2 (18)aB2GPI, n (%)2(50)2 (18)Low complement, n (%)4 (100)11 (100)C3, mean ± SD (mg/dl)40.25 ± 5.5152.73 ± 20.81C4, mean ± SD (mg/dl)3.67 ± 2.894.60 ± 2.72SLEDAI at diagnosis, mean ± SD12.75 ± 4.5012.18 ± 7.14SLEDAI at last follow-up, mean ± SD0.00 ± 0.001.36 ± 1.43Damage (SDI at last follow-up >0), n (%)0 (100)0 (100)TreatmentPDN, n (%)3 (75)11 (100)HCQ, n (%)4 (100)11 (100)MMF, n (%)4 (100)11 (100)RTX, n (%)0 (0)2 (18)Figure 1.Acknowledgements:NIL.Disclosure of InterestsNone Declared.