Comparing Rituximab and Cyclophosphamide in Induction Therapy for Childhood-Onset ANCA-Associated Vasculitis: An ARChiVe registry-cohort study.

OBJECTIVE: Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are chronic life-threatening vasculitides requiring substantial immunotherapy. Adult trials identified rituximab (RTX) as an alternative to cyclophosphamide (CYC) for remission-induction of GPA/MPA. Disease rarity has limited feasibility of similar trials in pediatrics. We aim to evaluate the relative efficacy and toxicity of CYC and RTX for childhood GPA/MPA through registry-based comparative evaluation. METHODS: From A Registry of Childhood Vasculitis we identified GPA/MPA patients who received induction with RTX or CYC. Pediatric vasculitis activity score (PVAS) and pediatric vasculitis damage index (pVDI) evaluated disease activity and damage. Descriptive statistics summarized patient characteristics. RTX/CYC comparisons used logistic regression for primary outcomes of post-induction remission (PVAS=0) or low disease activity (PVAS<2). Hospital admission for adverse events and pVDI were compared using logistic regression and ordinal regression, respectively. RESULTS: Among 104 patients, 43% received RTX, 46% CYC, 11% both. Treatment groups did not significantly differ for diagnosis PVAS and onset age. There was no difference in remission between groups (63% overall; OR 1.07, 95% CI: 0.45, 2.52). Hospitalizations occurred in 22% of RTX patients versus 10% on CYC (OR 2.27, 95% CI: 0.73, 7.05). The median 12-month pVDI was one in both groups (OR 0.98, 95% CI 0.43, 2.22). CONCLUSION: This is the first study comparing CYC and RTX for induction in pediatric GPA/MPA. No significant differences were shown in rates of remission, severe adverse events, or organ damage. Limitations included lack of standardized treatment regimens, retrospectivity, and lack of longitudinal adverse drug-related event data.

Enrichment of Rare Variants of Hemophagocytic Lymphohistiocytosis Genes in Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: Our objective was to evaluate whether there is an enrichment of rare variants in familial hemophagocytic lymphohistiocytosis (HLH)-associated genes among patients with systemic juvenile idiopathic arthritis (sJIA) with or without macrophage activation syndrome (MAS). METHODS: Targeted sequencing of HLH genes (LYST, PRF1, RAB27A, STX11, STXBP2, UNC13D) was performed in patients with sJIA from an established cohort. Sequence data from control participants were obtained in silico (database of Genotypes and Phenotypes: phs000280.v8.p2). Rare variant association testing (RVT) was performed with sequence kernel association test package. Significance was defined as P < 0.05 after 100,000 permutations. RESULTS: Sequencing data from 524 sJIA cases were jointly called and harmonized with exome-derived target data from 3,000 controls. Quality control operations produced a set of 480 cases and 2,924 ancestrally matched control participants. RVT of cases and controls revealed a significant association with rare protein-altering variants (minor allele frequency [MAF] < 0.01) of STXBP2 (P = 0.020) and ultrarare variants (MAF < 0.001) of STXBP2 (P = 0.006) and UNC13D (P = 0.046). A subanalysis of 32 cases with known MAS and 90 without revealed a significant difference in the distribution of rare UNC13D variants (P = 0.0047) between the groups. Additionally, patients with sJIA more often carried two or more HLH variants than did controls (P = 0.007), driven largely by digenic combinations involving LYST. CONCLUSION: We identified an enrichment of rare HLH variants in patients with sJIA compared with controls, driven by STXBP2 and UNC13D. Biallelic variation in HLH genes was associated with sJIA, driven by LYST. Only UNC13D displayed enrichment in patients with MAS. This suggests that HLH variants may contribute to the pathophysiology of sJIA, even without MAS.

Proteomic mapping identifies serum marker signatures associated with MIS-C specific hyperinflammation and cardiovascular manifestation.

Multisystem inflammatory syndrome in children (MIS-C) shares several clinical and immunological features with Kawasaki Disease (KD) and pediatric hyperinflammation, but the immuno-phenotypic overlap among these clinical mimics is still incompletely understood. Here we analyzed serum samples from treatment-naïve patients with MIS-C (n = 31) and KD (n = 11), pediatric hyperinflammation (n = 13) and healthy controls (HC, n = 10) by proximity extension assay (PEA) to profile 184 blood biomarkers. Collectively, immunophenotypic overlap between MIS-C and hyperinflammation exceeds overlap with KD. Overexpression of IL-17A in MIS-C and KD could best separate these conditions from hyperinflammatory conditions, while those were hallmarked by overabundance of adenosin deaminase and IL-18. Depletion in serum TNF-related subfamily member 9 (TNFRSF9) and apoptosis inducing ligand (TRAIL) linked with cardiovascular manifestations and myocarditis in MIS-C. Altogether, our analysis highlights important differences in molecular marker signatures also across different MIS-C and KD cohorts and suggests several previously unidentified molecular associations in context of cardiovascular inflammation.

P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

A novel serum calprotectin (MRP8/14) particle-enhanced immuno-turbidimetric assay (sCAL turbo) helps to differentiate systemic juvenile idiopathic arthritis from other diseases in routine clinical laboratory settings.

BACKGROUND: Differential diagnosis in children with signs of unprovoked inflammation can be challenging. In particular, differentiating systemic juvenile idiopathic arthritis (SJIA) from other diagnoses is difficult. We have recently validated the complex of myeloid-related proteins 8/14 (MRP8/14, also known as S100A8/A9 complex or serum calprotectin) as a helpful biomarker supporting the diagnosis of SJIA. The results were subsequently confirmed with a commercial ELISA. However, further optimization of the analytical technology is important to ensure its feasibility for large-scale use in routine laboratory settings. METHODS: To evaluate the accuracy in identifying children with SJIA, the performance of a particle-enhanced immuno-turbidimetric assay for serum calprotectin (sCAL turbo) on an automated laboratory instrument was analyzed. Samples from 615 children were available with the diagnoses SJIA (n = 99), non-systemic JIA (n = 169), infections (n = 51), other inflammatory diseases (n = 126), and acute lymphoblastic leukemia (ALL, n = 147). In addition, samples from 23 healthy controls were included. RESULTS: The sCAL turbo assay correlated well with the MRP8/14 ELISA used in previous validation studies (r = 0.99, p < 0.001). It could reliably differentiate SJIA from all other diagnoses with significant accuracy (cutoff at 10,500 ng/ml, sensitivity 84%, specificity 94%, ROC area under curve 0.960, p < 0.001). CONCLUSIONS: Serum calprotectin analyses are a helpful tool supporting the diagnosis of SJIA in children with prolonged fever or inflammatory disease. Here, we show that an immuno-turbidimetric assay for detection of serum calprotectin on an automated laboratory instrument can be implemented in clinical laboratory settings to facilitate its use as a diagnostic routine test in clinical practice.

Incident psoriasis under treatment with tumor necrosis factor-α inhibitors in juvenile idiopathic arthritis patients-analysis of the BiKeR registry.

The efficacy of tumor necrosis factor inhibitors (TNFi) for the treatment of psoriasis is well established, but patients may develop psoriasis for the first time while on TNFi as a paradoxical effect. Limited data on this association in patients with juvenile idiopathic arthritis (JIA) are available. Safety data from patients registered to the German Biologics registry (BiKeR) were analyzed. Patients were grouped by treatment regime: single TNFi, multiple TNFi, non-TNFi biologics or bDMARD-naïve control group receiving methotrexate. TNFi-associated psoriasis was defined as incident diagnosis of psoriasis after starting TNFi treatment. Patients with a history of psoriasis or psoriasis arthritis prior to TNFi therapy were excluded. Event rates using AEs reported after first dose were compared by Wald's test. A total of 4149 patients were treated with a TNFi (etanercept, adalimumab, golimumab, infliximab), 676 with a non-TNFi biologic (tocilizumab, abatacept, anakinra, canakinumab) and 1692 with methotrexate only. A total of 31 patients were diagnosed with incident psoriasis while on one of the above treatments. Compared with methotrexate, psoriasis was more frequent in the TNFi cohorts (RR 10.8, p = 0.019), specifically in the subgroup of TNF antibodies (RR 29.8, p = 0.0009), whereas no significant signal was observed with etanercept. Also, non-TNFi-treated patients presented high incident psoriasis rates (RR 25.0, p = 0.003). Our findings indicate a higher rate of incident psoriasis in JIA patients treated with TNFi monoclonal antibodies or non-TNFi biologic treatment. JIA patients receiving monoclonal antibody TNFi or non-TNFi bDMARD should be monitored for incident psoriasis. Medication change, if topical skin treatment remains insufficient, may be considered.

Phagocyte-related S100 proteins and cytokines in acute lymphoblastic leukemia and their prognostic value.

In this retrospective cohort study, we evaluated the level of biomarkers of inflammation like phagocyte-related S100 proteins and a panel of cytokines in 128 children with pre-B ALL and 22 with T-ALL. The biomarkers were evaluated at diagnosis and during antileukemic therapy (day 29 and after six months) and we evaluated their correlation with basic laboratory values. Further, for the children with pre-B ALL, we evaluated whether the biomarkers could predict the outcome of ALL expressed as minimal residual disease (MRD), relapse, and death.The levels of S100A9, S100A12, IL-1beta, IL-12p70, IL-13, IL-17, IL-18, and MPO serum levels increased significantly as chemotherapy was initiated. The difference was most pronounced for S100A9 and S100A12, which had strong positive correlations with the neutrophil counts. In contrast, TNF-alpha, IL-6, IL-10, CCL-2, MMP-3, and CD25 serum levels decreased after chemotherapy. Although none of these biomarkers appear to be an independent predictor of outcomes, in predictive models with MRD as the outcome, AUC increased from 76% (95% CI 68-84%) when using initial risk group stratification alone to 83% (95% CI 73-91%) in a multivariate predictive model including initial risk group stratification and the biomarkers S100A12, TNF-alpha, and IL-10.

Inflammatory Biomarkers Can Differentiate Acute Lymphoblastic Leukemia with Arthropathy from Juvenile Idiopathic Arthritis Better Than Standard Blood Tests.

OBJECTIVE: To evaluate the predictive value of biomarkers of inflammation like phagocyte-related S100 proteins and a panel of inflammatory cytokines in order to differentiate the child with acute lymphoblastic leukemia (ALL) from juvenile idiopathic arthritis (JIA). STUDY DESIGN: In this cross-sectional study, we measured S100A9, S100A12, and 14 cytokines in serum from children with ALL (n = 150, including 27 with arthropathy) and JIA (n = 236). We constructed predictive models computing areas under the curve (AUC) as well as predicted probabilities in order to differentiate ALL from JIA. Logistic regression was used for predictions of ALL risk, considering the markers as the respective exposures. We performed internal validation using repeated 10-fold cross-validation and recalibration, adjusted for age. RESULTS: In ALL, the levels of S100A9, S100A12, interleukin (IL)-1 beta, IL-4, IL-13, IL-17, matrix metalloproteinase-3, and myeloperoxidase were low compared with JIA (P < .001). IL-13 had an AUC of 100% (95% CI 100%-100%) due to no overlap between the serum levels in the 2 groups. Further, IL-4 and S100A9 had high predictive performance with AUCs of 99% (95% CI 97%-100%) and 98% (95% CI 94%-99%), respectively, exceeding both hemoglobin, platelets, C-reactive protein, and erythrocyte sedimentation rate. CONCLUSIONS: The biomarkers S100A9, IL-4, and IL-13 might be valuable markers to differentiate ALL from JIA.

Biologics with or without methotrexate in treatment of polyarticular juvenile idiopathic arthritis: effectiveness, safety and drug survival.

OBJECTIVE: To investigate the impact of additionally given MTX on biologic treatment of polyarticular JIA in terms of effectiveness, safety and drug survival. METHODS: Patients suffering from polyarticular JIA and treated with either monotherapy with a first biologic or a combination of a biologic and MTX were selected from the BIKER registry. The TNF-α inhibitors (TNFi) adalimumab, etanercept and golimumab and the IL-6 inhibitor tocilizumab were considered. Upon a non-randomized study design, we adjusted the different cohorts using propensity score matching to improve comparability. RESULTS: A total of 2148 patients entered the analysis, who were treated by either combination therapy (n = 1464) or monotherapy (n = 684). Disease activity declined significantly more in patients upon combination therapy than upon biologic monotherapy. Comparison of adjusted cohorts revealed that patients who received TNFi gained more benefit from additionally given MTX than patients treated with tocilizumab. Median survival time of therapy with biologics was significantly longer upon combination therapy (3.1 years) than with monotherapy (2.7 years), as demonstrated by a Kaplan-Meier analysis (log rank test: P = 0.002). The safety profile was moderately affected by additional MTX due to increased incidence of gastrointestinal and hepatic adverse events. Serious adverse events occurred at an equal rate of 3.6 events per 100 patient-years in both cohorts. CONCLUSION: Additionally given MTX improves the effectiveness of biologic treatment in polyarticular JIA without seriously compromising treatment safety. Especially TNFi benefit from combination, while no improvement in outcome has been observed by combining tocilizumab with MTX.

An Immunological Axis Involving Interleukin 1ß and Leucine-Rich-α2-Glycoprotein Reflects Therapeutic Response of Children with Kawasaki Disease: Implications from the KAWAKINRA Trial.

PURPOSE: A recent phase II open-label study of the interleukin 1 (IL-1) receptor antagonist (IL-1Ra) anakinra in treating IVIG-resistant Kawasaki disease (KD) patients reported promising results. Here, we aimed to characterize the immunological impact of IL-1 blockade in this unique study population. METHODS: Patients' and control sera and supernatants of cells (whole blood, neutrophils, coronary artery endothelial cells) stimulated with recombinant IL-1ß were analyzed for single or multiple marker (n = 22) expression by ELISA or multiplexed bead array assay. Data were analyzed using unsupervised hierarchical clustering, multiple correlation, and multi-comparison statistics and were compared to retrospective analyses of KD transcriptomics. RESULTS: Inflammation in IVIG-resistant KD (n = 16) is hallmarked by over-expression of innate immune mediators (particularly IL-6 > CXCL10 > S100A12 > IL-1Ra). Those as well as levels of immune or endothelial cell activation markers (sICAM-1, sVCAM-1) declined most significantly in course of anakinra treatment. Prior as well as following IL-1R blockade, over-expression of leucine-rich-α2-glycoprotein 1 (LRG1) associated best with remnant inflammatory activity and the necessity to escalate anakinra dosage and separated inflammatory KD patients from sJIA-MAS (n = 13) and MIS-C (n = 4). Protein as well as retrospective gene expression analyses indicated tight association of LRG1 with IL-1ß signaling and neutrophilia, while particularly neutrophil stimulation with recombinant IL-1ß resulted in concentration-dependent LRG1 release. CONCLUSION: Our study identifies LRG1 as known trigger of endothelial activation and cardiac re-modeling to associate with IL-1ß signaling in KD. Besides a potential patho-mechanistic implication of these findings, our data suggest blood leukocyte and neutrophil counts to best predict response to IL-1Ra treatment in IVIG-resistant KD.

The 2021 EULAR/American College of Rheumatology Points to Consider for Diagnosis, Management and Monitoring of the Interleukin-1 Mediated Autoinflammatory Diseases: Cryopyrin-Associated Periodic Syndromes, Tumour Necrosis Factor Receptor-Associated Periodic Syndrome, Mevalonate Kinase Deficiency, and Deficiency of the Interleukin-1 Receptor Antagonist.

BACKGROUND: The interleukin-1 (IL-1) mediated systemic autoinflammatory diseases, including the cryopyrin- associated periodic syndromes (CAPS), tumour necrosis factor receptor-associated periodic syndrome (TRAPS), mevalonate kinase deficiency (MKD) and deficiency of the IL-1 receptor antagonist (DIRA), belong to a group of rare immunodysregulatory diseases that primarily present in early childhood with variable multiorgan involvement. When untreated, patients with severe clinical phenotypes have a poor prognosis, and diagnosis and management of these patients can be challenging. However, approved treatments targeting the proinflammatory cytokine IL-1 have been life changing and have significantly improved patient outcomes. OBJECTIVE: To establish evidence-based recommendations for diagnosis, treatment and monitoring of patients with IL-1 mediated autoinflammatory diseases to standardise their management. METHODS: A multinational, multidisciplinary task force consisting of physician experts, including rheumatologists, patients or caregivers and allied healthcare professionals, was established. Evidence synthesis, including systematic literature review and expert consensus (Delphi) via surveys, was conducted. Consensus methodology was used to formulate and vote on statements to guide optimal patient care. RESULTS: The task force devised five overarching principles, 14 statements related to diagnosis, 10 on therapy, and nine focused on long-term monitoring that were evidence and/or consensus-based for patients with IL-1 mediated diseases. An outline was developed for disease-specific monitoring of inflammation-induced organ damage progression and reported treatments of CAPS, TRAPS, MKD and DIRA. CONCLUSION: The 2021 EULAR/American College of Rheumatology points to consider represent state-of-the-art knowledge based on published data and expert opinion to guide diagnostic evaluation, treatment and monitoring of patients with CAPS, TRAPS, MKD and DIRA, and to standardise and improve care, quality of life and disease outcomes.

The 2021 EULAR/American College of Rheumatology points to consider for diagnosis, management and monitoring of the interleukin-1 mediated autoinflammatory diseases: cryopyrin-associated periodic syndromes, tumour necrosis factor receptor-associated periodic syndrome, mevalonate kinase deficiency, and deficiency of the interleukin-1 receptor antagonist.

BACKGROUND: The interleukin-1 (IL-1) mediated systemic autoinflammatory diseases, including the cryopyrin-associated periodic syndromes (CAPS), tumour necrosis factor receptor-associated periodic syndrome (TRAPS), mevalonate kinase deficiency (MKD) and deficiency of the IL-1 receptor antagonist (DIRA), belong to a group of rare immunodysregulatory diseases that primarily present in early childhood with variable multiorgan involvement. When untreated, patients with severe clinical phenotypes have a poor prognosis, and diagnosis and management of these patients can be challenging. However, approved treatments targeting the proinflammatory cytokine IL-1 have been life changing and have significantly improved patient outcomes. OBJECTIVE: To establish evidence-based recommendations for diagnosis, treatment and monitoring of patients with IL-1 mediated autoinflammatory diseases to standardise their management. METHODS: A multinational, multidisciplinary task force consisting of physician experts, including rheumatologists, patients or caregivers and allied healthcare professionals, was established. Evidence synthesis, including systematic literature review and expert consensus (Delphi) via surveys, was conducted. Consensus methodology was used to formulate and vote on statements to guide optimal patient care. RESULTS: The task force devised five overarching principles, 14 statements related to diagnosis, 10 on therapy, and nine focused on long-term monitoring that were evidence and/or consensus-based for patients with IL-1 mediated diseases. An outline was developed for disease-specific monitoring of inflammation-induced organ damage progression and reported treatments of CAPS, TRAPS, MKD and DIRA. CONCLUSION: The 2021 EULAR/American College of Rheumatology points to consider represent state-of-the-art knowledge based on published data and expert opinion to guide diagnostic evaluation, treatment and monitoring of patients with CAPS, TRAPS, MKD and DIRA, and to standardise and improve care, quality of life and disease outcomes.

Independent risk factors for myasthenic crisis and disease exacerbation in a retrospective cohort of myasthenia gravis patients.

BACKGROUND: Myasthenic crisis (MC) and disease exacerbation in myasthenia gravis (MG) are associated with significant lethality and continue to impose a high disease burden on affected patients. Therefore, we sought to determine potential predictors for MC and exacerbation as well as to identify factors affecting outcome. METHODS: We examined a retrospective, observational cohort study of patients diagnosed with MG between 2000 and 2021 with a mean follow-up of 62.6 months after diagnosis from eight tertiary hospitals in Germany. A multivariate Cox regression model with follow-up duration as the time variable was used to determine independent risk factors for MC and disease exacerbation. RESULTS: 815 patients diagnosed with MG according to national guidelines were included. Disease severity at diagnosis (quantitative MG score or Myasthenia Gravis Foundation of America class), the presence of thymoma and anti-muscle specific tyrosine kinase-antibodies were independent predictors of MC or disease exacerbation. Patients with minimal manifestation status 12 months after diagnosis had a lower risk of MC and disease exacerbation than those without. The timespan between diagnosis and the start of immunosuppressive therapy did not affect risk. Patients with a worse outcome of MC were older, had higher MGFA class before MC and at admission, and had lower vital capacity before and at admission. The number of comorbidities, requirement for intubation, prolonged mechanical ventilation, and MC triggered by infection were associated with worse outcome. No differences between outcomes were observed comparing treatments with IVIG (intravenous immunoglobulin) vs. plasma exchange vs. IVIG together with plasma exchange. CONCLUSIONS: MC and disease exacerbations inflict a substantial burden of disease on MG patients. Disease severity at diagnosis and antibody status predicted the occurrence of MC and disease exacerbation. Intensified monitoring with emphasis on the prevention of infectious complications could be of value to prevent uncontrolled disease in MG patients.

MRP8/14 serum levels as diagnostic markers for systemic juvenile idiopathic arthritis in children with prolonged fever.

OBJECTIVES: Differential diagnosis in children with prolonged fever is challenging. In particular, differentiating systemic-onset JIA (SJIA) from infectious diseases is difficult. Biomarkers are needed that support the diagnostic work-up. The aim of this study was to validate the usefulness of Myeloid-related protein 8/14 (MRP8/14) measurements in the diagnostic work-up of febrile children and to transfer it to clinical practice. METHODS: Data for 1110 paediatric patients were included and divided into two cohorts: (cohort A) for validation of MRP8/14 test performance with three different testing systems: the experimental ELISA, commercial ELISA and an innovative (point-of-care test) lateral flow immunoassay (LFIA); (cohort B) to validate the diagnostic accuracy with the two latter assays. RESULTS: In cohort A (n = 940), MRP8/14 was elevated in SJIA (12 110 ± 2650 ng/ml mean ± 95% CI) compared with 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 (19 740 ± 5080 ng/ml) were even higher compared with other diagnoses (4590 ± 1160 ng/ml) (P < 0.001, sensitivity 73%, specificity 90%). In group 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, soluble IL-2 receptor 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.

Patient parameters and response after administration of rituximab in pediatric mature B-cell non-Hodgkin lymphoma.

BACKGROUND: Mature aggressive B-cell lymphomas are heterogenous malignancies that make up more than half of all diagnosed non-Hodgkin lymphoma in children and adolescents. The overall survival rate increased over the last decades to 80%-90% due to fine tuning of polychemotherapy. However, new therapeutic implications are needed to further increase the overall survival. Current clinical trials analyze the therapeutic effect of rituximab in pediatric patients, while the mechanism of action in vivo is still not fully understood. METHODS: Effector molecules important for tumor defense were analyzed before and at day 5 after rituximab treatment via flow cytometry. Serum rituximab levels were measured with an ELISA. RESULTS: We evaluated patient parameters that may affect treatment response in relation to rituximab administration and serum rituximab levels. We indeed found a reduction of Fcγ receptor (FcγR) II levels after rituximab treatment in monocyte subtypes, whereas FcγRI expression was significantly increased. Serum levels of proinflammatory marker proteins S100A8/A9 and S100A12 significantly decreased after treatment to normal levels from an overall proinflammatory state before treatment. CD57, perforin, and granzyme B expression decreased after treatment, comprising a less cytolytic natural killer (NK) cell population. CONCLUSION: The highlighted effects of rituximab treatment on patient's immune response help in understanding the biology behind tumor defense mechanisms and effector function. After subsequent studies, these novel insights might be translated into patient care and could contribute to improve treatment of pediatric patients with mature aggressive B-cell lymphoma.

A myostatin-CCL20-CCR6 axis regulates Th17 cell recruitment to inflamed joints in experimental arthritis.

The interactions of fibroblast-like synoviocyte (FLS)-derived pro-inflammatory cytokines/chemokines and immune cells support the recruitment and activation of inflammatory cells in RA. Here, we show for the first time that the classical myokine myostatin (GDF-8) is involved in the recruitment of Th17 cells to inflammatory sites thereby regulating joint inflammation in a mouse model of TNFalpha-mediated chronic arthritis. Mechanistically, myostatin-deficiency leads to decreased levels of the chemokine CCL20 which is associated with less infiltration of Th17 cells into the inflamed joints. In vitro, myostatin alone or in combination with IL-17A enhances the secretion of CCL20 by FLS whereas myostatin-deficiency reduces CCL20 secretion, associated with an altered transmigration of Th17 cells. Thus, the communication between activated FLS and Th17 cells through myostatin and IL-17A may likely contribute to a vicious cycle of inflammation, accounting for the persistence of joint inflammation in chronic arthritis. Blockade of the CCL20-CCR6 axis by inhibition of myostatin may, therefore, be a promising treatment option for chronic inflammatory diseases such as arthritis.

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.

Discrimination of COVID-19 From Inflammation-Induced Cytokine Storm Syndromes Using Disease-Related Blood Biomarkers.

OBJECTIVE: Infection with the novel coronavirus SARS-CoV-2 triggers severe illness with high mortality in a subgroup of patients. Such a critical course of COVID-19 is thought to be associated with the development of cytokine storm, a condition seen in macrophage activation syndrome (MAS) and secondary hemophagocytic lymphohistiocytosis (HLH). However, specific data demonstrating a clear association of cytokine storm with severe COVID-19 are still lacking. The aim of this study was to directly address whether immune activation in COVID-19 does indeed mimic the conditions found in these classic cytokine storm syndromes. METHODS: Levels of 22 biomarkers were quantified in serum samples from patients with COVID-19 (n = 30 patients, n = 83 longitudinal samples in total), patients with secondary HLH/MAS (n = 50), and healthy controls (n = 9). Measurements were performed using bead array assays and single-marker enzyme-linked immunosorbent assay. Serum biomarker levels were assessed for correlations with disease outcome. RESULTS: In patients with secondary HLH/MAS, we observed pronounced activation of the interleukin-18 (IL-18)-interferon-γ axis, increased serum levels of IL-1 receptor antagonist, intercellular adhesion molecule 1, and IL-8, and strongly reduced levels of soluble Fas ligand in the course of SARS-CoV-2 infection. These observations appeared to discriminate immune dysregulation in critical COVID-19 from the well-recognized characteristics of other cytokine storm syndromes. CONCLUSION: Serum biomarker profiles clearly separate COVID-19 from MAS or secondary HLH in terms of distinguishing the severe systemic hyperinflammation that occurs following SARS-CoV-2 infection. These findings could be useful in determining the efficacy of drugs targeting key molecules and pathways specifically associated with systemic cytokine storm conditions in the treatment of COVID-19.

Impaired IFN-γ-dependent STAT3 Activation Is Associated With Dysregulation of Regulatory and Inflammatory Signaling in Monocytes of Ulcerative Colitis Patients.

BACKGROUND: The Janus kinase/signal transducer and activator of transcription (JAK/STAT) inhibitor tofacitinib has been recently approved for the treatment of ulcerative colitis (UC) but not Crohn's disease (CD). Systematic analysis of the JAK/STAT pathway in inflammatory bowel disease is still missing. The aim of this study was to investigate JAK/STAT activation and adjacent signaling in monocytes of patients with inflammatory bowel diseases, which are key players in inflammatory responses. METHODS: Blood samples of active UC (n = 28) and CD patients (n = 28) and healthy controls (n = 22) were collected for primary monocyte investigation. STAT phosphorylation (pSTAT), cytokine secretion, and surface marker expression ± prior tofacitinib blockade in addition to Th-17 and regulatory T cell induction in cocultures were analyzed upon interferon (IFN)-γ timulation. RESULTS: Baseline frequencies of pSTAT1+ and pSTAT3+ monocytes were significantly higher in UC, whereas IFN-γ-associated crosstalk induction of pSTAT3+ monocytes was missing in UC-derived monocytes compared with controls and CD. This coincided with decreased interleukin (IL)-10 and cluster of differentiation (CD)39 levels, diminished regulatory T cell (Treg) induction, and increased IL-12 and IL-23 secretion compared with controls, which was not observed in CD monocytes. Tofacitinib induced stronger inhibition of inflammatory cytokine release (IL-6, TNFα, IL-12, IL-23) in UC compared with CD monocytes. CONCLUSIONS: In UC monocytes, IFN-γ-associated activation of the JAK/STAT pathway is impaired with an imbalance between STAT1 and STAT3, coinciding with stronger induction of inflammatory monocytes by IFN-γ compared with controls or CD. The fact that tofacitinib had stronger regulatory impact on UC than on CD monocytes further underlines a stronger inflammatory involvement of the JAK/STAT pathway in UC pathogenesis, which might result from missing STAT3 activation to counteract STAT1-induced inflammation.

Definition and validation of serum biomarkers for optimal differentiation of hyperferritinaemic cytokine storm conditions in children: a retrospective cohort study.

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. FUNDING: German Research Foundation, the Center for Interdisciplinary Clinical Research at University Hospital Muenster, the EU's Horizon 2020 research and innovation programme, and the Deutsche Kinderkrebsstiftung.