N ter Haar

Evidence-based recommendations for genetic diagnosis of familial Mediterranean fever

Familial Mediterranean fever (FMF) is a disease of early onset which can lead to significant morbidity. In 2012, Single Hub and Access point for pediatric Rheumatology in Europe (SHARE) was launched with the aim of optimising and disseminating diagnostic and management regimens for children and young adults with rheumatic diseases. The objective was to establish recommendations for FMF focusing on provision of diagnostic tools for inexperienced clinicians particularly regarding interpretation of MEFV mutations. Evidence-based recommendations were developed using the European League against Rheumatism standard operating procedure. An expert committee of paediatric rheumatologists defined search terms for the systematic literature review. Two independent experts scored articles for validity and level of evidence. Recommendations derived from the literature were evaluated by an online survey and statements with less than 80% agreement were reformulated. Subsequently, all recommendations were discussed at a consensus meeting using the nominal group technique and were accepted if more than 80% agreement was reached. The literature search yielded 3386 articles, of which 25 were considered relevant and scored for validity and level of evidence. In total, 17 articles were scored valid and used to formulate the recommendations. Eight recommendations were accepted with 100% agreement after the consensus meeting. Topics covered were clinical versus genetic diagnosis of FMF, genotype–phenotype correlation, genotype–age at onset correlation, silent carriers and risk of amyloid A (AA) amyloidosis, and role of the specialist in FMF diagnosis. The SHARE initiative provides recommendations for diagnosing FMF aimed at facilitating improved and uniform care throughout Europe.

House dust mite reduction in the management of atopic dermatitis. A critically appraised topic

AD is an itchy, inflammatory, chronic relapsing eczematous skin disease and it is among the most common dermatological conditions. The prevalence of AD has increased steadily in recent decades and now varies between 1% and 3% in adults and up to 20% in children. Topical treatment with corticosteroids and calcineurin inhibitors, such as tacrolimus and pimecrolimus, is often used in combination with emollients to treat the signs and symptoms of AD. Unfortunately, topical therapies are sometimes insufficient and therefore other therapies like ultraviolet therapy or systemics are needed. Besides a genetic predisposition, environmental factors such as exposure to HDM might play a role in the course of AD. The allergens of HDM (Euroglyphus maynei) are thought to play a role in the sensitization and induction of clinical symptoms of AD. The mites that most frequently induce AD are Dermatophagoides pteronyssinus and Dermatophagoides farinae. Reduction of these mites, by covering mattresses and pillows, frequent or high-filtration vacuum cleaning or the use of acaricidal sprays, has been shown to reduce the severity of asthma. As this disease is closely related to AD, HDM reduction may play a role in the treatment of established AD.

PReS-FINAL-2335: Preliminary analysis of 85 patients with mevalonate kinase deficiency from the eurofever registry

Mevalonate kinase deficiency (MKD) is a rare autoinflammatory disease, caused by mutations in the isoprenoid pathway that lead to fever episodes. Approximately 300 MKD patients are known.

A1.06 Phagocyte involvement in systemic onset juvenile idiopathic arthritis

Background Systemic onset Juvenile Idiopathic Artritis (sJIA) is a systemic autoinflammatory disease, characterised by arthritis, spiking fever and rash and elevation of serum S100-proteins and interleukin (IL)-18. The role of monocytes and neutrophils in the inflammatory cascade of sJIA is still unclear. Objective To study the role of monocytes and neutrophils in the inflammatory cascade of sJIA. Methods We determined neutrophil activation ex vivo (phenotype and cell membrane markers) and after stimulation (ROS-production and degranulation) of cells derived from sJIA with disease onset or in remission, compared to healthy donors (HDs). To investigate the role of monocytes, we assessed cytokine production of PBMCs from sJIA patients and HDs after stimulation with TLR-4 activating S100-proteins (+/- ATP) or other TLR-ligands. In a cohort of sJIA patients at onset and during inactive disease, we evaluated cell counts and serum levels of cytokines, chemokines and other analytes. Cytokine concentrations in supernatant and serum were determined by multiplex immunoassay. Results Twenty-one of 23 patients with onset sJIA had elevated neutrophil counts, while monocyte counts were elevated in only 5/23 patients. Many inflammatory markers were significantly elevated in serum of onset sJIA patients, among which several neutrophil specific proteins indicating the importance of this cell type. Neutrophils from onset sJIA patients showed an activated phenotype, reflected by higher ex vivo cell membraneexpression of FC-gamma receptors (CD32 and CD64), markers of secretory vesicles (CD35) and specific granules (CD66b). ROS production and degranulation were also enhanced in onset sJIA. Neutrophil phenotypenormalized when patients were in remission. In contrast to the hyperactivated status of neutrophils in active sJIA, PBMCs from these patients produced less Il-1b, IL-18, IL-6 and TNF-a upon TLR-stimulation compared to PBMCs from remission patients or HDs, suggesting tolerance after exposure to high TLR4 stimulating S100-levels in vivo. Conclusions We show here that monocytes from onset sJIA patients produce less cytokines upon stimulation, while the neutrophils are hyperactivated, reflected by increased cell membrane activation markers, ROS production and degranulation. The exact role of each cell type and activity and their interaction in sJIA pathology is currently under investigation.

Genetic and phenotypic characteristics of 114 patients with mevalonate kinase deficiency

Mevalonate kinase deficiency (MKD) is a rare autoinflammatory syndrome, characterized by febrile episodes and generalized inflammation.

Monocytes and neutrophils in the inflammatory cascade of systemic onset Juvenile Idiopathic Arthritis

8th International Congress of Familial Mediterranean Fever and Systemic Autoinflammatory Diseases

A2.7 Interleukin-18 production upon S100 stimulation is reduced in systemic-onset juvenile idiopathic arthritis

Background Systemic onset Juvenile Idiopathic Artritis (sJIA), also known as Still’s disease, is characterised by arthritis with symptoms of systemic inflammation such as spiking fever, rash and serositis. It is considered an autoinflammatory disease with a major role for the innate immune system. The S100-proteins S100A8, S100A9 and S100A12 interleukin (IL)-18 in plasma are extremely elevated in sJIA patients and have been proposed to be useful biomarkers for diagnosis, disease activity and response to therapy. However, the relationship between S100-proteins and IL-18 in sJIA and the role of S100 proteins in pathogenesis is still unknown. Objective To study the relation between S100-proteins and IL-18 in sJIA. Materials and methods Peripheral blood mononuclear cells (PBMCs) from sJIA patients during active disease and during remission and PBMCs from healthy controls were stimulated with S100A8, S100A9, S100A12 or LPS (positive control) for four hours. As a second signal for NLRP3 inflammasome activation, ATP was added during the last 30 min. Cytokine levels in supernatant were measured by Luminex; cell frequencies and TLR-expression were analysed by flow cytometry. Results Upon stimulation with S100-proteins, PBMCs from healthy donors produced inflammasome dependent IL-1b, IL-18 as well as inflammasome independent IL-6 and TNF-a; IL-18 and IL-1b production was further enhanced when ATP was added. S100 proteins are known to bind to TLR4 and RAGE; blocking TLR4 by adding CLI-095 decreased cytokine production to near normal levels, while blocking RAGE did not have an effect on cytokine production. When compared to healthy controls, PBMCs from sJIA patients produced less IL-18 upon S100 stimulation. The addition of ATP enhanced this differential effect. PBMCs from patients with active disease were less responsive to S100 stimulation than PBMCs from the same patient in remission. The same trend of hyporesponsiveness was seen when PBMCs from sJIA patients were stimulated with TLR2 or TLR7/8 ligands, suggesting cross-tolerance in these patient cells. Conclusions S100-proteins serve as a first signal via TLR4 to establish NLRP3 inflammasome activation and thereby induce production of IL-1b and IL-18. Interestingly, PBMCs from sJIA patients with active disease are hyporesponsive to S100-proteins compared to sJIA patients in remission and healthy controls. We are currently investigating whether this hyporesponsiveness can be explained by stress tolerance of PBMCs after prolonged elevation of serum S100-proteins. Acknowledgments for providing S100 proteins Holzinger D1, Vogl T2, Foell D1, Roth J2 1University Hospital Muenster and University Children’s Hospital Muenster, Muenster, Germany 2 University of Muenster, Institute of Immunology, Muenster, Germany

OP0196 The Role of Neutrophils in the Inflammatory Cascade of Systemic Onset Juvenile Idiopathic Arthritis

Background Systemic onset Juvenile Idiopathic Artritis (sJIA) is considered an autoinflammatory disease, with increased circulating neutrophil counts and extremely high serum levels of S100 proteins and interleukin (IL)-18. We have previously shown that upon stimulation with S100 proteins and ATP, peripheral blood mononuclear cells (PBMCs) of healthy donors produce high levels of IL-1β and IL-18; however this cytokine production is decreased in PBMCs of sJIA patients with active disease and partly restored when patients are in remission.1 Given the observed hyporesponsiveness of PBMCs from sJIA patients, we hypothesized that neutrophils might play an important role in the inflammatory cascade of sJIA. Objectives Our objective was to investigate the role of neutrophils in sJIA, with a focus on the relation between neutrophils, S100 proteins and IL-18. Methods We determined ex vivo cell frequencies of patients at disease onset, patients with inactive disease and healthy controls by flow cytometric analysis, using fluorescent beads for absolute counts. Neutrophils from healthy donors were sorted and subsequently primed with S100 proteins in a time series up to 20 hours with or without Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) for the last 10 minutes. Elastase and cytokine levels in supernatant were measured by Luminex, neutrophil activation markers were analysed by flow cytometry. Results Patients with new onset sJIA had significantly elevated neutrophil counts compared to healthy controls and patients with inactive disease. Immature neutrophils (CD16dimCD62L+) and normally matured neutrophils (CD16+CD62L+) were significantly higher in patients with new onset sJIA compared to healthy controls or patients with inactive disease, while hypersegmented neutrophils (CD16+CD62Ldim) were comparable between patients and controls. Given the high serum levels of S100 proteins in active sJIA, we investigated if neutrophils could be activated by these proteins. Priming with S100A9 before stimulation with fMLP increased activation and degranulation of specific granules and secretory vesicles compared to unprimed cells. This was reflected by an increased expression of CD11b, CD66b and CD35. Upon stimulation with S100A9, we observed an increase in elastase secretion in the culture supernatant, as well as a minor increase in IL-6, IL-18 and TNF-α. Conclusions Patients with new onset sJIA have increased neutrophil counts. S100 proteins prime neutrophils and enhance degranulation upon a second stimulus such as fMLP. We are now exploring whether IL-18 or other pro-inflammatory mediators elevated in sJIA plasma can also prime neutrophils. Further, we will investigate whether ex vivo neutrophils from sJIA patients are more activated or respond differently to stimulation compared to patients with inactive disease or healthy controls. References N. Ter Haar, D. Holzinger, W. de Jager, R. Scholman, S. de Roock, B. Vastert. IL-18 production upon s100 stimulation is reduced in active sJIA patients compared to sJIA patients in remission and healthy controls. Pediatric Rheumatology 2014, 12(Suppl 1):O24 Acknowledgements We would like to acknowledge D. Holzinger, T. Vogl, D. Foell and J. Roth for providing the S100 proteins. Disclosure of Interest None declared

Therapy of autoinflammatory diseases: a review of the literature

Results RCTs provide evidence for rilonacept and canakinumab in CAPS, colchicine in FMF and (adeno)tonsillectomy in PFAPA syndrome. For Behcet disease, RCTs have been conducted on the effect of several drugs, including colchicine, azathioprine, cyclosporine A, interferon alfa, etanercept, sucralfate suspension and pimecrolimus. Descriptive studies suggest that NSAIDs and corticosteroids are highly effective in respectively CRMO and PFAPA and moderately effective in the other diseases. Etanercept and anakinra appear to induce a complete or partial response in most patients with MKD, TRAPS, PAPA and colchicine-resistant FMF. Anakinra appears to induce a complete response in the majority of the CAPS and DIRA patients, but seems to be less effective in NLRP12-mediated periodic fever. Complete remission by infliximab is described in cases with Blau’s syndrome, CRMO, PAPA, refractory FMF and Behcet disease, but the effect of infliximab in TRAPS patients seems to be disappointing.

Therapy of autoinflammatory diseases: results from an international registry

Methods A web-based registry collecting baseline and clinical information of autoinflammatory diseases was located at the member area of the PRINTO website (www.printo. it). Participating hospitals included pediatric rheumatology centers of the PRINTO network and adult centers with a specific interest in autoinflammatory diseases. Data was collected on Blau’s syndrome, Behcet’s disease, CAPS, CRMO, DIRA, FMF, MKD, NLRP12-mediated periodic fever, PAPA, PFAPA TRAPS and undefined periodic fevers. In total, 704 patients were included in this retrospective study on therapy.