Objectives Differential diagnosis in children with prolonged fever is challenging. In particular, differentiating systemic-onset juvenile idiopathic arthritis (SJIA) from infectious diseases is difficult. Biomarkers are needed supporting the diagnostic work-up. The aim of this study was to validate the usefulness of MRP8/14 measurements in the diagnostic wok-up of febrile children and transfer it to clinical practice. Methods Data of 1,110 paediatric patients were included and divided into two cohorts: (A) For validation of MRP8/14 test performance with 3 different testing systems: the experimental enzyme-linked immunosorbent sandwich assay (ELISA), commercial ELISA and an innovative (POCT) lateral flow immunoassay (LFIA); (B) to validate the diagnostic accuracy with the two latter assays. Results In cohort A (n = 940), MRP8/14 was elevated in SJIA (12110±2650 ng/ml mean ± 95% CI) compared to other diagnoses (including infections and autoinflammatory diseases; 2980±510 ng/ml) irrespective of fever and anti-inflammatory treatment (p < 0.001). In untreated patients with fever (n = 195) MRP8/14 levels in SJIA (19740±5080 ng/ml) were even higher compared to other diagnoses (4590±1160 ng/ml) (p < 0.001, sensitivity 73%, specificity 90%). In cohort B1, the performance of the tests was confirmed in untreated patients with fever (n = 170): commercial ELISA (sensitivity 79%, specificity 89%) and LFIA (sensitivity 84%, specificity 81%). Compared with ferritin, IL-18, ESR, sIL2-R, and procalcitonin, MRP8/14 showed the best accuracy. Conclusion MRP8/14 serum analyses have been validated as a helpful tool supporting the diagnosis of SJIA in febrile children. The results could be confirmed with commercial ELISA and LFIA enabling a rapid diagnostic point-of-care screening test.

Background Cytokine storm syndromes are life-threatening complications that can occur in children with rheumatic conditions (macrophage activation syndrome [MAS]), inherited cytotoxicity defects (ie, primary haemophagocytic lymphohistiocytosis [HLH]), or as a result of infection or malignancies (ie, secondary HLH). To adequately steer treatment, an early and clear discrimination of these entities is essential. We aimed to define and validate serum biomarker profiles that can differentiate between primary HLH, secondary HLH (predominantly infection-associated), and MAS associated with systemic juvenile idiopathic arthritis (systemic JIA-MAS).Methods In this multicentre, retrospective, cohort study, serum samples from patients (0-18 years) with a clinical diagnosis of primary HLH, secondary HLH, or systemic JIA-MAS were analysed by immunoassays for 55 cytokines and chemokines. Serum samples were collected from patients treated at seven clinical centres in Europe and North America. 15 serum biomarkers were validated using an independent commercial assay, and the diagnostic accuracy of the best performing biomarkers was tested in an independent validation cohort.Findings Serum samples were collected between Dec 7, 2010, and Jan 26, 2018. In the discovery cohort of 43 patients (24 girls and 19 boys) multi-marker analyses revealed distinct serum biomarker profiles associated with primary or secondary HLH versus systemic JIA-MAS. Ten biomarkers were identified that were differentially elevated in either HLH or systemic JIA-MAS and distinguished between these clinical entities, six of which were tested in an independent validation cohort of 79 patients (34 girls and 45 boys). Serum concentrations of S100A12 and interleukin-18, as well as ratios of both S100A12 and IL-18 with chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 were identified as the most promising candidates for differential diagnostics.Interpretation At initial presentation, when it is unclear whether a patient with excessive hyperferritinaemic inflammation has primary HLH, infection-associated secondary HLH, or MAS, high serum concentrations of S100A12 indicate an initial differential diagnosis of systemic JIA-MAS, thus helping to guide subsequent treatment decisions. We therefore suggest the inclusion of serum S100A12 and IL-18 in the diagnostic investigations for hyperferritinaemic syndromes; however, the definition and introduction of universially applicable cutoff values are still required.

Objective. Systemic juvenile idiopathic arthritis (JIA) features characteristics of autoinflammation and autoimmunity, culminating in chronic arthritis. In this study, we hypothesized that aberrant or incomplete polarization of T helper cells contributes to disease pathology.Methods. Cells or serum samples were obtained from healthy controls (n = 72) and systemic JIA patients (n = 171). Isolated naive T helper cells were cultured under Th1, Th17, and T follicular helper (Tfh) or T peripheral helper (Tph)-polarizing conditions and were partly cocultured with allogenic memory B cells. Cell samples were then analyzed for surface marker, transcription factor, and cytokine expression, as well as plasmablast generation. Serum samples were subjected to multiplexed bead and self-antigen arrays and enzyme-linked immunosorbent assays, and all data were compared to retrospective RNA profiling analyses.Results. Differentiation of systemic JIA-naive T helper cells toward Th1 cells resulted in low expression levels of interferon-γ (IFNγ) and eomesodermin, which was associated in part with disease duration. In contrast, developing Th1 cells in patients with systemic JIA were found to produce elevated levels of interleukin-21 (IL-21), which negatively correlated with cellular expression of IFNγ and eomesodermin. In both in vitro and ex vivo analyses, IL-21 together with programmed cell death 1 (PD-1), inducible T cell costimulator (ICOS), and CXCR5 expression induced naive T helper cells from systemic JIA patients to polarize toward a Tfh/Tph cell phenotype. Retrospective analysis of whole-blood RNAsequencing data demonstrated that Bcl-6, a master transcription factor in Tfh/Tph cell differentiation, was overexpressed specifically in patients with systemic JIA. Naive T helper cells from systemic JIA patients which were stimulated in vitro promoted B cellular plasmablast generation, and self-antigen array data indicated that IgG reactivity profiles of patients with systemic JIA differed from those of healthy controls.Conclusion. In the pathogenesis of systemic JIA, skewing of naive T helper cell differentiation toward a Tfh/Tph cell phenotype may represent an echo of autoimmunity, which may indicate the mechanisms driving progression toward chronic destructive arthritis.

Objective: Systemic juvenile idiopathic arthritis (sJIA) features characteristics of autoinflammation and autoimmunity, culminating in chronic arthritis. Previous work indicated decreased IFNg-expression by T helper (Th) cells in sJIA. Here, we hypothesized this to result from aberrant or incomplete Th cell polarization. Methods: Cells or sera were obtained from healthy controls (HC, n=26) and sJIA patients (n=61). Isolated naive Th cells were cultured under Th1, Th17, and T follicular or T peripheral helper (Tf/ph) polarizing conditions and were partly co-cultured with allogenic memory B cells. Surface marker, transcription factor, and/or cytokine expression in peripheral or polarized Th cells or sera as well as B cellular plasma blast generation were studied by flow cytometry, multiplexed bead array assays, ELISA and retrospective RNA profiling analyses. Results: SJIA naive Th cell differentiation towards Th1 resulted in low IFNg; and Eomesodermin expression. Instead, developing sJIA Th1 cells revealed elevated IL-21 release that negatively correlated with cellular IFNg; and Eomesodermin expression. Both In vitro and ex vivo, IL-21 together with PD-1, ICOS and CXCR5 expression indicated naive sJIA Th cell differentiation to rather polarize towards a Tf/ph phenotype. Retrospective analysis of whole blood RNA sequencing data demonstrated sJIA-specific overexpression of Bcl-6 as respective master transcription factor. Compared to controls, in vitro generated sJIA Tf/ph cells promoted enhanced B cellular plasma-blast generation. Conclusion: In sJIA pathogenesis skewing of sJIA naive Th cell differentiation towards a Tf/ph phenotype may represent an echo of autoimmunity, which could shed light on the mechanisms driving the progression towards chronic destructive arthritis.