Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell–independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the VH3+ BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A–bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scidPrkdc/γc−/− mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell–derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell–mediated immune tolerance.

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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.

ObjectivesThe International League of Associations for Rheumatology classification criteria define systemic juvenile idiopathic arthritis (SJIA) by the presence of fever, rash and chronic arthritis. Recent initiatives to revise current criteria recognise that a lack of arthritis complicates making the diagnosis early, while later a subgroup of patients develops aggressive joint disease. The proposed biphasic model of SJIA also implies a ‘window of opportunity’ to abrogate the development of chronic arthritis. We aimed to identify novel SJIA biomarkers during different disease phases.MethodsChildren with active SJIA were subgrouped clinically as systemic autoinflammatory disease with fever (SJIA syst ) or polyarticular disease (SJIA poly ). A discovery cohort of n=10 patients per SJIA group, plus n=10 with infection, was subjected to unbiased label-free liquid chromatography mass spectrometry (LC-MS/MS) and immunoassay screens. In a separate verification cohort (SJIA syst , n=45; SJIA poly , n=29; infection, n=32), candidate biomarkers were measured by multiple reaction monitoring MS (MRM-MS) and targeted immunoassays.ResultsSignatures differentiating the two phenotypes of SJIA could be identified. LC-MS/MS in the discovery cohort differentiated SJIA syst from SJIA poly well, but less effectively from infection. Targeted MRM verified the discovery data and, combined with targeted immunoassays, correctly identified 91% (SJIA syst vs SJIA poly ) and 77% (SJIA syst vs infection) of all cases.ConclusionsMolecular signatures differentiating two phenotypes of SJIA were identified suggesting shifts in underlying immunological processes in this biphasic disease. Biomarker signatures separating SJIA in its initial autoinflammatory phase from the main differential diagnosis (ie, infection) could aid early-stage diagnostic decisions, while markers of a phenotype switch could inform treat-to-target strategies.

SummaryImmunoglobulin E-mediated allergy and certain autoimmune diseases are characterized by the presence of a T helper type 2 (Th2) immune response and allergen-specific or self-reactive IgE. Soluble CD23 (sCD23) is a B-cell factor that fosters IgE class-switching and synthesis, suggesting that sCD23 may be a therapeutic target for these pathologies. We produced a recombinant protein, CTLA4Fce, by fusing the ectodomain of the immunoregulatory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) with a fragment of the IgE H-chain constant region. In SDS-PAGE/inmunoblot analyses, CTLA4Fce appeared as a 70 000 MW polypeptide that forms homodimers. Flow cytometry showed that CTLA4Fce binds to IgE receptors FceRI and FceRII/CD23, as well as to CTLA-4 counter-receptors CD80 and CD86. Binding of CTLA4Fce to FceRII/CD23 appeared stronger than that of IgE. Since the cells used to study CD23 binding express CD80 and CD86, simultaneous binding of CTLA4Fce to CD23 and CD80/CD86 seems to occur and would explain this difference. As measured by a human CD23-specific ELISA, CTLA4Fce -but not IgE -induced a concentration-dependent reduction of sCD23 in culture supernatants of RPMI-8866 cells. Our results suggest that the simultaneous binding of CTLA4Fcɛ to CD23-CD80/CD86 may cause the formation of multi-molecular complexes that are either internalized or pose a steric hindrance to enzymatic proteolysis, so blocking sCD23 generation. CTLA4Fce caused a concentration-dependent reduction of lymphocyte proliferation in human peripheral blood mononuclear cell samples stimulated in vitro with concanavalin A. The ability to bind IgE receptors on effector cells, to regulate the production of sCD23 and to inhibit lymphocyte proliferation suggests that CTLA4Fcɛ has immunomodulatory properties on human Th2 responses.

ObjectivesTo investigate the ability of high-sensitivity C-reactive protein (hsCRP) and S100A12 to serve as predictive biomarkers of successful drug withdrawal in children with clinical remission of juvenile idiopathic arthritis (JIA).MethodsThis multicentre trial (PREVENT-JIA) enrolled 119 patients with JIA in clinical remission, and 100 patients reached the intervention phase in which the decision whether to continue or stop treatment was based on S100A12 and hsCRP levels. Patients were monitored for 12 months after stopping medication for flares of disease. Results were compared with withdrawal of therapy without biomarker-based stratification in patients from the German Biologika in der Kinderrheumatologie (BiKeR) pharmacovigilance registry.ResultsIn the PREVENT-JIA group, 49 patients had a flare, and 45% of patients stopping medication showed flares within the following 12 months. All patients (n=8) continuing therapy due to permanently elevated S100A12/hsCRP at more than one visit flared during the observation phase. In the BiKeR control group, the total flare rate was 62%, with 60% flaring after stopping medication. The primary outcome, time from therapy withdrawal to first flare (cumulative flare rate after therapy withdrawal), showed a significant difference in favour of the PREVENT-JIA group (p=0.046; HR 0.62, 95% CI 0.38 to 0.99). As additional finding, patients in the PREVENT-JIA trial stopped therapy significantly earlier.ConclusionBiomarker-guided strategies of therapy withdrawal are feasible in clinical practice. This study demonstrates that using predictive markers of subclinical inflammation is a promising tool in the decision-making process of therapy withdrawal, which translates into direct benefit for patients.Trial registration numberISRCTN69963079.

BackgroundSystemic juvenile idiopathic arthritis (SJIA) is a childhood rheumatic auto-inflammatory disorder of largely unknown pathogenesis. The presence of fever, rash and arthritis support a diagnosis of SJIA, though early in disease arthritis may be minimal, complicating the exclusion of alternative diagnoses such as infection. Furthermore, two clinical phenotypes of SJIA can be identified, a chronic articular-dominant (ART_SJIA) and a classical auto-inflammatory phenotype (AID_SJIA).ObjectivesTo identify novel serum protein biomarkers that may discriminate ART_SJIA from AID_SJIA and distinguish AID_SJIA from infection.MethodsPatients with active SJIA (joint activity plus or minus fever and elevated laboratory inflammation markers) were sub-grouped into the two clinical phenotypes (ART_SJIA and AID_SJIA). Serum from patients with SJIA or confirmed infection was analysed for the standard laboratory markers: C-reactive protein (CRP), white cell count (WCC) and erythrocyte sedimentation rate (ESR). A “discovery cohort” (n=10 per group) of patient serum samples was subjected to unbiased label-free proteomics using liquid chromatography mass spectrometry (LC-MS/MS) and in a separate “verification cohort” (AID_SJIA, n=48; ART_SJIA, n=29; infection, n=32) candidate biomarkers were measured by multiple reaction monitoring MS (MRM-MS; Agilent 6490 and 6495) and microsphere bead-based immunoassay (Luminex). Serum concentrations of S100A12 and MRP8/14 were also measured in all samples using enzyme linked immunoabsorbant assays (ELISAs).ResultsThe routine laboratory markers CRP, WCC, ESR, as well as ELISA-measured S100A12 and MRP8/14 serum concentrations were highest in AID_SJIA, followed by infection and were lowest in patients with ART_SJIA. Proteins identified and quantified by LC-MS/MS could differentiate patients with ART_SJIA from those with AID_SJIA (area under the curve, AUC: 0.97). The discrimination between AID_SJIA and infection performed less well, AUC: 0.58. Targeted MRM measurement of novel protein candidate biomarkers verified the discovery cohort data. A combined biomarker panel consisting of MRM, ELISA and Luminex analysis which was evaluated using a Random forest model, indicated the correct overall identification of the clinical groups to be as follows: ART_SJIA: 24/29 (83%), AID_SJIA: 37/45 (82%) and infection: 20/32 (63%).ConclusionsSignificant differences in the serum protein signature between two phenotypes of SJIA suggest that different immunological processes may underlie these phenotypes. Serum protein profiles also distinguished patients with SJIA and infection. Distinguishing these two groups remains an important goal of research and therefore this study could help inform future prospective studies.Disclosure of InterestNone declared

Background Unavailability of vaccines endangers the overall goal to protect individuals and whole populations against infections. Methods The German notification system includes the publication of vaccine supply shortages reported by marketing authorisation holders (MAH), information on the availability of alternative vaccine products, guidance for physicians providing vaccinations and an unavailability reporting tool to monitor regional distribution issues. Aim This study provides a retrospective analysis of supply issues and measures in the context of European and global vaccine supply constraints. Results between October 2015 and December 2020, the 250 notifications concerned all types of vaccines (54 products). Most shortages were caused by increased demand associated with immigration in Germany in 2015 and 2016, new or extended vaccine recommendations, increased awareness, or changes in global immunisation programmes. Shortages of a duration up to 30 days were mitigated using existing storage capacities. Longer shortages, triggered by high demand on a national level, were mitigated using alternative products and re-allocation; in a few cases, vaccines were imported. However, for long lasting supply shortages associated with increased global demand, often occurring in combination with manufacturing issues, few compensatory mechanisms were available. Nevertheless, only few critical incidents were identified: (i) shortage of hexavalent vaccines endangering neonatal immunisation programmes in 2015;(ii) distribution issues with influenza vaccines in 2018; and (iii) unmet demand for pneumococcal and influenza vaccines during the coronavirus disease (COVID)-19 pandemic. Conclusion Vaccine product shortages in Germany resemble those present in neighbouring EU states and often reflect increased global demand not matched by manufacturing capacities.