Capillary leak syndrome was associated with more severe multisystem inflammatory syndrome in children during the COVID-19 pandemic.

AIM: This population-based study investigated the occurrence of capillary leak syndrome (CLS) in children with multisystem inflammatory syndrome in children (MIS-C), associated with COVID-19. We also examined associations between CLS and MIS-C disease severity. METHODS: All eligible individuals aged 0-18 years, who were diagnosed with MIS-C in Skåne, southern Sweden, from 1 April 2020 to 31 July 2021, were studied. They were all included in the Pediatric Rheumatology Quality Register and clinical and laboratory data were compared between patients with and without CLS. RESULTS: We included 31 patients (61% male) with MIS-C in the study. The median age at diagnosis was 10.6 years (range 1.99-17.15) and 45% developed CLS. All six patients who required intensive care had CLS. Patients with CLS also had a higher incidence of reduced cardiac function, measured as low ejection fraction. The CLS group exhibited significantly higher C-reactive protein values (p < 0.001) and N-terminal pro-B-type natriuretic peptide levels (p < 0.001), as well as lower platelet counts (p = 0.03), during the first week of treatment. Individuals with CLS also received more intense immunosuppression. CONCLUSION: CLS was a common complication of MIS-C in our study and these patients had a more severe disease course that required more intensive treatment.

Synovial monocytes contribute to chronic inflammation in childhood-onset arthritis via IL-6/STAT signalling and cell-cell interactions.

Introduction: Monocytes are key effector cells in inflammatory processes. We and others have previously shown that synovial monocytes in childhood-onset arthritis are activated. However, very little is known about how they contribute to disease and attain their pathological features. Therefore, we set out to investigate the functional alterations of synovial monocytes in childhood-onset arthritis, how they acquire this phenotype, and whether these mechanisms could be used to tailorize treatment. Methods: The function of synovial monocytes was analysed by assays believed to reflect key pathological events, such as T-cell activation-, efferocytosis- and cytokine production assays using flow cytometry in untreated oligoarticular juvenile idiopathic arthritis (oJIA) patients (n=33). The effect of synovial fluid on healthy monocytes was investigated through mass spectrometry and functional assays. To characterize pathways induced by synovial fluid, we utilized broad-spectrum phosphorylation assays and flow cytometry, as well as inhibitors to block specific pathways. Additional effects on monocytes were studied through co-cultures with fibroblast-like synoviocytes or migration in transwell systems. Results: Synovial monocytes display functional alterations with inflammatory and regulatory features, e.g., increased ability to induce T-cell activation, resistance to cytokine production following activation with LPS and increased efferocytosis. In vitro, synovial fluid from patients induced the regulatory features in healthy monocytes, such as resistance to cytokine production and increased efferocytosis. IL-6/JAK/STAT signalling was identified as the main pathway induced by synovial fluid, which also was responsible for a majority of the induced features. The magnitude of synovial IL-6 driven activation in monocytes was reflected in circulating cytokine levels, reflecting two groups of low vs. high local and systemic inflammation. Remaining features, such as an increased ability to induce T-cell activation and markers of antigen presentation, could be induced by cell-cell interactions, specifically via co-culture with fibroblast-like synoviocytes. Conclusions: Synovial monocytes in childhood-onset arthritis are functionally affected and contribute to chronic inflammation, e.g., via promoting adaptive immune responses. These data support a role of monocytes in the pathogenesis of oJIA and highlight a group of patients more likely to benefit from targeting the IL-6/JAK/STAT axis to restore synovial homeostasis.

Risk factors for multisystem inflammatory syndrome in children - A population-based cohort study of over 2 million children.

Background: Although severe acute COVID-19 is rare in children, SARS-CoV-2 infection can trigger the novel post-infectious condition multisystem inflammatory syndrome in children (MIS-C). Increased knowledge on risk factors for MIS-C could improve our understanding of the pathogenesis of the condition and better guide targeted public health interventions. The aim of the study was to assess risk factors for MIS-C with the aim to identify vulnerable children. Methods: A register-based cohort study including all children and adolescents <19 years born in Sweden between March 1, 2001- December 31, 2020 was performed. Data on sociodemographic risk factors and comorbidities (sex, age, parental region of birth, parental education, asthma, autoimmune disease, chromosomal anomalies, chronic heart disease, chronic lung disease, obesity, life-limiting condition) were retrieved from national health and population registers. The outcome was MIS-C diagnosis according to the Swedish Pediatric Rheumatology Quality Register during March 1, 2020 - December 8, 2021.Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox regression analysis. Incidence rates per 100 000 person-years were calculated assuming a Poisson distribution. Findings: Among 2 117 443 children included in the study, 253 children developed MIS-C, corresponding to an incidence rate of 6·8 (95% CI: 6·0-7·6) per 100 000 person-years. Male sex (HR 1·65, 95% CI: 1·28-2·14), age 5-11 years (adjusted HR 1·44, 95% CI: 1·06-1·95 using children 0-4 years as reference), foreign-born parents (HR 2·53, 95% CI: 1·93-3·34), asthma (aHR 1·49, 95% CI: 1·00-2·20), obesity (aHR 2·15, 95% CI: 1·09-4·25) and life-limiting conditions (aHR 3·10, 95% CI: 1·80-5·33) were associated with MIS-C. Children 16-18 years had a reduced risk for MIS-C (aHR 0·45, 95% CI: 0·24-0·85). Interpretation: We report increased risks for MIS-C in children with male sex, age 5-11 years, foreign-born parents, asthma, obesity, and life-limiting condition. Knowing these risk populations might facilitate identification of children with MIS-C and potentially guide targeted public health interventions. Nevertheless, the absolute risks for MIS-C were very low. Funding: Financial support was provided from the Swedish Research Council (grant no 2018-02640), the Swedish Heart-Lung Foundation (grant no 20210416), the Asthma and Allergy Association, Ake Wiberg foundation, the Samariten Foundation, the Society of Child Care, and Region Stockholm.

Population-based study of multisystem inflammatory syndrome associated with COVID-19 found that 36% of children had persistent symptoms.

AIM: Our aim was to describe the outcomes of multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19. METHODS: This national, population-based, longitudinal, multicentre study used Swedish data that were prospectively collected between 1 December 2020 and 31 May 2021. All patients met the World Health Organization criteria for MIS-C. The outcomes 2 and 8 weeks after diagnosis are presented, and follow-up protocols are suggested. RESULTS: We identified 152 cases, and 133 (87%) participated. When followed up 2 weeks after MIS-C was diagnosed, 43% of the 119 patients had abnormal results, including complete blood cell counts, platelet counts, albumin levels, electrocardiograms and echocardiograms. After 8 weeks, 36% of 89 had an abnormal patient history, but clinical findings were uncommon. Echocardiogram results were abnormal in 5% of 67, and the most common complaint was fatigue. Older children and those who received intensive care were more likely to report symptoms and have abnormal cardiac results. CONCLUSION: More than a third (36%) of the patients had persistent symptoms 8 weeks after MIS-C, and 5% had abnormal echocardiograms. Older age and higher levels of initial care appeared to be risk factors. Structured follow-up visits are important after MIS-C.

Identification of novel autoantigens as potential biomarkers in juvenile idiopathic arthritis associated uveitis.

Background: Many children with juvenile idiopathic arthritis (JIA) have autoantibodies, targeting nuclear components (anti-nuclear antibodies, ANA). ANA in JIA is associated with uveitis, an eye inflammation which may cause permanent vision impairment if not detected and treated. However, ANA-testing is neither specific nor sensitive enough to be a clinically reliable predictor of uveitis risk, and the precise autoantigens targeted by ANA in JIA are largely unknown. If identified, specific autoantibodies highly associated with uveitis could be used as biomarkers to facilitate identification of JIA patients at risk. Methods: Antibodies from six ANA-positive, oligoarticular JIA patients, with and without uveitis, were explored by two large-scale methods: (1) screening against 42,100 peptides on an autoimmunity profiling planar array, and (2) immunoprecipitations from cell lysates with antigen identification by mass spectrometry. Three hundred thirty-five peptide antigens, selected from proteins identified in the large-scale methods and the scientific literature were investigated using a bead-based array in a cohort of 56 patients with oligoarticular- or RF-negative polyarticular JIA, eight of which were having current or previous uveitis. Results: In the planar array, reactivity was detected against 332 peptide antigens. The immunoprecipitations identified reactivity towards 131 proteins. Only two proteins were identified by both methods. In the bead-based array of selected peptide antigens, patients with uveitis had a generally higher autoreactivity, seen as higher median fluorescence intensity (MFI) across all antigens, compared to patients without uveitis. Reactivity towards 17 specific antigens was significantly higher in patients with uveitis compared to patients without uveitis. Hierarchical clustering revealed that patients with uveitis clustered together. Conclusion: This study investigated autoantigens in JIA and uveitis, by combining two exploratory methods and confirmation in a targeted array. JIA patients with current or a history of uveitis had significantly higher reactivity towards 17 autoantigens and a generally higher autoreactivity compared to JIA patients without uveitis. Hierarchical clustering suggests that a combination of certain autoantibodies, rather than reactivity towards one specific antigen, is associated with uveitis. Our analysis of autoantibodies associated with uveitis in JIA could be a starting point for identification of prognostic biomarkers useful in JIA clinical care.

The Vasculopathy of Juvenile Dermatomyositis: Endothelial Injury, Hypercoagulability, and Increased Arterial Stiffness.

OBJECTIVE: Vasculopathy is considered central to the pathogenesis of juvenile dermatomyositis (DM) and is associated with severe extramuscular manifestations. We undertook this study to investigate the hypothesis that the vasculopathy of juvenile DM can be noninvasively tracked by examining biomarkers of endothelial injury, subclinical inflammation, hypercoagulability, and vascular arterial stiffness. METHODS: The study population was a UK cohort of children with juvenile DM. Circulating endothelial cells (CECs) and microparticles (MPs) were identified using immunomagnetic bead extraction and flow cytometry, respectively. Plasma thrombin generation was determined using a fluorogenic assay. Cytokine and chemokine levels were measured by electrochemiluminescence. Arterial stiffness was assessed using pulse wave velocity (PWV). Results were expressed as the median and interquartile range (IQR), and statistical significance was assessed using nonparametric analyses. RESULTS: Ninety patients with juvenile DM and 79 healthy control subjects were included. The median age of the patients was 10.21 years (IQR 6.68-13.40), and the median disease duration was 1.63 years (IQR 0.28-4.66). CEC counts were higher in all patients with juvenile DM compared to controls (median 96 cells/ml [IQR (40-192] and 12 cells/ml [IQR 8-24], respectively; P < 0.0001). Circulating MP numbers were also significantly higher in patients with active juvenile DM compared to controls (median 204.7 × 103 /ml [IQR 87.9-412.6] and 44.3 × 103 /ml [IQR 15.0-249.1], respectively; P < 0.0001). MPs were predominantly of platelet and endothelial origin. Enhanced plasma thrombin generation was demonstrated in patients with active juvenile DM compared to those with inactive disease (P = 0.0003) and controls (P < 0.0001). Carotid-radial PWV adjusted for age was increased in patients with juvenile DM compared to controls (P = 0.003). CONCLUSION: We observed increased endothelial injury and increased levels of proinflammatory cytokines in patients with active juvenile DM. MP profiles reflected distinct disease activity status in juvenile DM and are markers of vascular pathology, platelet activation, and thrombotic propensity. Ongoing long-term vascular injury may result in increased arterial stiffness in patients with juvenile DM.

Neutrophils Lose the Capacity to Suppress T Cell Proliferation Upon Migration Towards Inflamed Joints in Juvenile Idiopathic Arthritis.

Neutrophils are highly abundant in synovial fluid of rheumatic inflamed joints. In oligoarticular juvenile idiopathic arthritis (JIA), synovial fluid neutrophils have impaired effector functions and altered phenotype. We hypothesized that these alterations might impact the immunoregulatory interplay between neutrophils and T cells. In this study we analyzed the suppressive effect of neutrophils, isolated from blood and synovial fluid of oligoarticular JIA patients, on CD4+ T cells activated by CD3/CD28 stimulation. JIA blood neutrophils suppressed T cell proliferation but synovial fluid neutrophils from several patients did not. The loss of T cell suppression was replicated in an in vitro transmigration assay, where healthy control neutrophils migrated into synovial fluid through transwell inserts with endothelial cells and synoviocytes. Non-migrated neutrophils suppressed proliferation of activated CD4+ T cells, but migrated neutrophils had no suppressive effect. Neutrophil suppression of T cells was partly dependent on reactive oxygen species (ROS), demonstrated by impaired suppression in presence of catalase. Migrated neutrophils had reduced ROS production compared to non-migrated neutrophils. A proteomic analysis of transwell-migrated neutrophils identified alterations in proteins related to neutrophil ROS production and degranulation, and biological processes involving protein transport, cell-cell contact and inflammation. In conclusion, neutrophils in synovial fluid of children with JIA have impaired capacity to suppress activated T cells, which may be due to reduced oxidative burst and alterations in proteins related to cell-cell contact and inflammation. The lack of T cell suppression by neutrophils in synovial fluid may contribute to local inflammation and autoimmune reactions in the JIA joint.

Polyclonal, newly derived T cells with low expression of inhibitory molecule PD-1 in tonsils define the phenotype of lymphocytes in children with Periodic Fever, Aphtous Stomatitis, Pharyngitis and Adenitis (PFAPA) syndrome.

PURPOSE: PFAPA syndrome is a benign, recurrent inflammatory disease of childhood. Tonsillectomy is one of the therapeutic options with a yet unexplained biological mechanism. We tested whether specific lymphocyte subsets recruited from blood to human tonsils participate in PFAPA pathogenesis. METHODS: Paired tonsils/peripheral blood (PB) samples were investigated (a) from children with PFAPA that successfully resolved after tonsillectomy (n=10) (b) from children with obstructive sleep apnoea syndrome as controls (n=10). The lymphocyte profiles were analysed using 8-colour flow cytometry, immunoglobulin (IGH) and T-cell receptor (TCR) gene rearrangements via PCR and next generation sequencing; a TREC/KREC analysis was performed using qPCR. RESULTS: The PFAPA tonsils in the asymptomatic phase had a lower percentage of B-lymphocytes than controls; T-lymphocyte counts were significantly higher in PB. The percentages of cytotoxic CD8pos T-lymphocytes were approximately 2-fold higher in PFAPA tonsils; the transitional B cells and naïve stages of both the CD4pos and CD8pos T-lymphocytes with a low expression of PD-1 molecule and high numbers of TREC were also increased. With the exception of elevated plasmablasts, no other differences were significant in PB. The expression levels of CXCL10, CXCL9 and CCL19 genes were significantly higher in PFAPA tonsils. The IGH/TCR pattern showed no clonal/oligoclonal expansion. DNA from the Epstein-Barr virus, Human Herpervirus-6 or adenovirus was detected in 7 of 10 PFAPA tonsils but also in 7 of 9 controls. CONCLUSIONS: Our findings suggest that the uninhibited, polyclonal response of newly derived lymphocytes participate in the pathogenesis of PFAPA. Because most of the observed changes were restricted to tonsils and were not present in PB, they partly explain the therapeutic success of tonsillectomy in PFAPA syndrome.

International periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis syndrome cohort: description of distinct phenotypes in 301 patients.

OBJECTIVES: The aims of this study were to describe the clinical features of periodic fever, aphthous stomatitis, pharyngitis and cervical adenitis (PFAPA) and identify distinct phenotypes in a large cohort of patients from different countries. METHODS: We established a web-based multicentre cohort through an international collaboration within the periodic fevers working party of the Pediatric Rheumatology European Society (PReS). The inclusion criterion was a diagnosis of PFAPA given by an experienced paediatric rheumatologist participating in an international working group on periodic fever syndromes. RESULTS: Of the 301 patients included from the 15 centres, 271 had pharyngitis, 236 cervical adenitis, 171 oral aphthosis and 132 with all three clinical features. A total of 228 patients presented with additional symptoms (131 gastrointestinal symptoms, 86 arthralgias and/or myalgias, 36 skin rashes, 8 neurological symptoms). Thirty-one patients had disease onset after 5 years and they reported more additional symptoms. A positive family history for recurrent fever or recurrent tonsillitis was found in 81 patients (26.9%). Genetic testing for monogenic periodic fever syndromes was performed on 111 patients, who reported fewer occurrences of oral aphthosis or additional symptoms. Twenty-four patients reported symptoms (oral aphthosis and malaise) outside the flares. The CRP was >50 mg/l in the majority (131/190) of the patients tested during the fever. CONCLUSION: We describe the largest cohort of PFAPA patients presented so far. We confirm that PFAPA may present with varied clinical manifestations and we show the limitations of the commonly used diagnostic criteria. Based on detailed analysis of this cohort, a consensus definition of PFAPA with better-defined criteria should be proposed.

Myeloid related protein induces muscle derived inflammatory mediators in juvenile dermatomyositis.

INTRODUCTION: The aetiopathogenesis of juvenile dermatomyositis (JDM) remains poorly understood. In particular the contribution of monocytes or macrophages, which are frequently observed to be an infiltrate within muscle tissue very early in the disease process, is unknown. We hypothesised that these cells secrete the pro-inflammatory myeloid related protein (MRP) 8/14 which may then contribute to muscle pathology in JDM. METHODS: In this study of 56 JDM patients, serum MRP8/14 levels were compared with clinical measures of disease activity. Muscle biopsies taken early in disease were assessed by immunohistochemistry to determine the frequency and identity of MRP-expressing cells. The effects of MRP stimulation and endoplasmic reticulum (ER) stress on muscle were tested in vitro. Serum or supernatant levels of cytokines were analyzed by multiplex immunoassay. RESULTS: Serum MRP8/14 correlated with physician's global assessment of disease activity in JDM (R = 0.65, p = 0.0003) and muscle strength/endurance, childhood myositis assessment score (CMAS, R = -0.55, p = 0.004). MRP8/14 was widely expressed by CD68+ macrophages in JDM muscle tissue. When cultured with human myoblasts, MRP8 led to the secretion of MCP-1 and IL-6, which was enhanced by ER stress. Both inflammatory mediators were detected in significantly higher levels in the serum of JDM patients compared to healthy controls. CONCLUSIONS: This study is the first to identify serum MRP8/14 as a potential biomarker for disease activity in JDM. We propose that tissue infiltrating macrophages secreting MRP8/14 may contribute to myositis, by driving the local production of cytokines directly from muscle.

PFAPA syndrome: clinical characteristics and treatment outcomes in a large single-centre cohort.

OBJECTIVES: This paper aims to describe clinical and laboratory features and disease outcome in a single-centre cohort of patients with PFAPA syndrome (Periodic Fever, Aphtous stomatitis, Pharyngitis, and Adenitis) and to test performance of diagnostic and therapeutic algorithms. METHODS: Patients fulfilling criteria were selected from the fever clinic population. Prospective follow-up together with recruitment of newly diagnosed patients followed pre-defined guidelines. Diagnostic and therapeutic algorithms and definitions of outcome and therapy response were formulated. Paired blood samples during febrile and afebrile periods were compared. RESULTS: Out of 176 patients referred for suspected periodic fever 125 children fulfilled criteria. Their age at onset was 23 months, median episode duration 3.5 days at 4-week intervals. Fever was associated with pharyngitis (91%), cervical adenitis (78%) and aphtae (41%). Among therapeutic options, episodic prednisone proved to be the most common first-line treatment. Administered to 77 patients, it reduced symptoms in 94%. Tonsillectomy led to the full symptom resolution in all 18 patients. Forty-six patients reached disease remission. CONCLUSIONS: Distribution of typical symptoms, response to therapies and disease outcome in a large patient cohort were documented. We offer diagnostic and therapeutic algorithms that have proven effective during this prospective trial. Our findings support the general belief of benign nature of this aetiologically unclear condition, despite proportion of patients having persistent disease for years. Maintenance of normal findings in afebrile intervals, striking response to a single dose of prednisone and normal growth and development together with spontaneous tendency towards prolongation of afebrile intervals are important confirmatory features of PFAPA syndrome.

Disease activity assessment in childhood vasculitis: development and preliminary validation of the Paediatric Vasculitis Activity Score (PVAS).

BACKGROUND: Rare chronic childhood vasculitides lack a reliable disease activity assessment tool. With emerging new treatment modalities such a tool has become increasingly essential for both clinical practice and therapeutic trials to reproducibly quantify change in disease state. OBJECTIVE: To develop and validate a paediatric vasculitis activity assessment tool based on modification of the Birmingham Vasculitis Activity Score (BVASv.3). METHODS: A paediatric vasculitis registry was reviewed to identify clinical features missing in the BVASv.3. A modified nominal group technique was used to develop a working version of the Paediatric Vasculitis Activity Score (PVAS). Prospective validation provided tool reliability, reproducibility and responsiveness to change. Training of assessors was done according to the BVAS principles. RESULTS: BVAS items were redefined (n=22) and eight paediatric items added in Cutaneous (n=4), Cardiovascular (n=3) and Abdominal (n=1) sections. The final PVAS has 64 active items in nine categories. The principles of new/worse and persistently active disease were retained as were the overall score and weighting of categories. The median PVAS in 63 children with systemic vasculitis was 4/63 (0-38/63). There was a high interobserver agreement for the overall as well as for subsystem scores (linear-weighted-κ ≥0.87). PVAS correlated with physician's global assessment (p<0.01); treatment decision (p=<0.01) and erythrocyte sedimentation rate (ESR) (p=0.01). In response to treatment, 15/19 patients assessed demonstrated a significant fall in PVAS (p=0.002), with good agreement among assessors for this change. CONCLUSIONS: The PVAS validity in children with systemic vasculitis was demonstrated. Like the BVAS, we anticipate that the PVAS will provide a robust tool to objectively define disease activity for clinical trials and future research.

A national registry for juvenile dermatomyositis and other paediatric idiopathic inflammatory myopathies: 10 years' experience; the Juvenile Dermatomyositis National (UK and Ireland) Cohort Biomarker Study and Repository for Idiopathic Inflammatory Myopathies.

OBJECTIVES: The paediatric idiopathic inflammatory myopathies (IIMs) are a group of rare chronic inflammatory disorders of childhood, affecting muscle, skin and other organs. There is a severe lack of evidence base for current treatment protocols in juvenile myositis. The rarity of these conditions means that multicentre collaboration is vital to facilitate studies of pathogenesis, treatment and disease outcomes. We have established a national registry and repository for childhood IIM, which aims to improve knowledge, facilitate research and clinical trials, and ultimately to improve outcomes for these patients. METHODS: A UK-wide network of centres and research group was established to contribute to the study. Standardized patient assessment, data collection forms and sample protocols were agreed. The Biobank includes collection of peripheral blood mononuclear cells, serum, genomic DNA and biopsy material. An independent steering committee was established to oversee the use of data/samples. Centre training was provided for patient assessment, data collection and entry. RESULTS: Ten years after inception, the study has recruited 285 children, of which 258 have JDM or juvenile PM; 86% of the cases have contributed the biological samples. Serial sampling linked directly to the clinical database makes this a highly valuable resource. The study has been a platform for 20 sub-studies and attracted considerable funding support. Assessment of children with myositis in contributing centres has changed through participation in this study. CONCLUSIONS: This establishment of a multicentre registry and Biobank has facilitated research and contributed to progress in the management of a complex group of rare muscloskeletal conditions.