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

Objective: To evaluate whether there is an enrichment of rare variants in familial hemophagocytic lymphohistiocytosis (HLH) genes and 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 sJIA subjects from an established cohort. Sequence data from control subjects were obtained in silico (dbGaP:phs000280.v8.p2). Rare variant association testing (RVT) was performed with sequence kernel association test (SKAT) 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 3000 controls. Quality control operations produced a set of 481 cases and 2924 ancestrally-matched control subjects. RVT of sJIA cases and controls revealed a significant association with rare protein-altering variants (minor allele frequency [MAF]<0.01) of STXBP2 (p=0.020), and ultra-rare variants (MAF<0.001) of STXBP2 (p=0.007) and UNC13D (p=0.045). A subanalysis of 32 cases with known MAS and 90 without revealed significant association of rare UNC13D variants (p=0.0047). Additionally, sJIA patients more often carried ≥2 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 sJIA patients compared with healthy 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.

Novel chemotype NLRP3 inhibitors that target the CRID3-binding pocket with high potency.

The NLRP3 inflammasome plays a central role in various human diseases. Despite significant interest, most clinical-grade NLRP3 inhibitors are derived from sulfonylurea inhibitor CRID3 (also called MCC950). Here, we describe a novel chemical class of NLRP3-inhibiting compounds (NIC) that exhibit potent and selective NLRP3 inflammasome inhibition in human monocytes and mouse macrophages. BRET assays demonstrate that they physically interact with NLRP3. Structural modeling further reveals they occupy the same binding site of CRID3 but in a critically different conformation. Furthermore, we show that NIC-11 and NIC-12 lack the off-target activity of CRID3 against the enzymatic activity of carbonic anhydrases I and II. NIC-12 selectively reduces circulating IL-1ß levels in the LPS-endotoxemia model in mice and inhibits NLRP3 inflammasome activation in CAPS patient monocytes and mouse macrophages with about tenfold increased potency compared with CRID3. Altogether, this study unveils a new chemical class of highly potent and selective NLRP3-targeted inhibitors with a well-defined molecular mechanism to complement existing CRID3-based NLRP3 inhibitors in pharmacological studies and serve as novel chemical leads for the development of NLRP3-targeted therapies.

Whipple Disease Presenting as Isolated Transverse Myelitis with Permanent Neurological Damage in a Patient with Systemic Lupus Erythematosus: A Case Report of a Difficult Diagnosis with a Literature Review.

We describe an atypical case of Whipple disease exclusively involving the spinal cord in an adolescent receiving immunosuppressive therapy for systemic lupus erythematosus. The diagnosis was particularly difficult since lupus and Whipple disease can present similar clinical features and the patient's prolonged contact with sewage was initially not mentioned. A literature review of the clinical, imaging, diagnostic, and therapeutic challenges of Whipple disease is also performed.

HSP90ß controls NLRP3 autoactivation.

Gain-of-function mutations in NLRP3 are linked to cryopyrin-associated periodic syndromes (CAPS). Although NLRP3 autoinflammasome assembly triggers inflammatory cytokine release, its activation mechanisms are not fully understood. Our study used a functional genetic approach to identify regulators of NLRP3 inflammasome formation. We identified the HSP90ß-SGT1 chaperone complex as crucial for autoinflammasome activation in CAPS. A deficiency in HSP90ß, but not in HSP90α, impaired the formation of ASC specks without affecting the priming and expression of inflammasome components. Conversely, activating NLRP3 with stimuli such as nigericin or alum bypassed the need for SGT1 and HSP90ß, suggesting the existence of alternative inflammasome assembly pathways. The role of HSP90ß was further demonstrated in PBMCs derived from CAPS patients. In these samples, the pathological constitutive secretion of IL-1ß could be suppressed using a pharmacological inhibitor of HSP90ß. This finding underscores the potential of SGT1-HSP90ß modulation as a therapeutic strategy in CAPS while preserving NLRP3's physiological functions.

Interface Gain-of-Function Mutations in TLR7 Cause Systemic and Neuro-inflammatory Disease.

TLR7 recognizes pathogen-derived single-stranded RNA (ssRNA), a function integral to the innate immune response to viral infection. Notably, TLR7 can also recognize self-derived ssRNA, with gain-of-function mutations in human TLR7 recently identified to cause both early-onset systemic lupus erythematosus (SLE) and neuromyelitis optica. Here, we describe two novel mutations in TLR7, F507S and L528I. While the L528I substitution arose de novo, the F507S mutation was present in three individuals from the same family, including a severely affected male, notably given that the TLR7 gene is situated on the X chromosome and that all other cases so far described have been female. The observation of mutations at residues 507 and 528 of TLR7 indicates the importance of the TLR7 dimerization interface in maintaining immune homeostasis, where we predict that altered homo-dimerization enhances TLR7 signaling. Finally, while mutations in TLR7 can result in SLE-like disease, our data suggest a broader phenotypic spectrum associated with TLR7 gain-of-function, including significant neurological involvement.

Pyrin Inflammasome Activation Defines Colchicine-Responsive SURF Patients from FMF and Other Recurrent Fevers.

Syndrome of undifferentiated recurrent fever (SURF) is characterized by recurrent fevers, a lack of confirmed molecular diagnosis, and a complete or partial response to colchicine. Despite the clinical similarities to familial Mediterranean fever (FMF), the underlying inflammatory mechanisms of SURF are not yet understood. We here analyzed the in vitro activation of the pyrin inflammasome in a cohort of SURF patients compared to FMF and PFAPA patients. Peripheral blood mononuclear cells (PBMC) were collected from SURF (both colchicine-treated and untreated), FMF, PFAPA patients, and healthy donors. PBMC were stimulated ex vivo with Clostridium difficile toxin A (TcdA) and a PKC inhibitor (UCN-01), in the presence or absence of colchicine. The assembly of the pyrin inflammasome was evaluated by measuring the presence of apoptosis-associated Speck-like protein containing caspase recruitment domain (ASC) specks in monocytes using flow cytometry. IL-1ß secretion was quantified using an ELISA assay. No differences in TcdA-induced activation of pyrin inflammasome were observed among FMF, PFAPA, and healthy donors. Untreated SURF patients showed a reduced response to TcdA, which was normalized after colchicine treatment. In contrast to FMF, SURF patients, similar to PFAPA patients and healthy donors, did not exhibit pyrin inflammasome activation in response to UCN-01-mediated pyrin dephosphorylation. These data demonstrate that in vitro functional analysis of pyrin inflammasome activation can differentiate SURF from FMF and PFAPA patients, suggesting the involvement of the pyrin inflammasome in the pathophysiology of SURF.

Microvascular status in juvenile Sjögren's disease: the first nailfold videocapillaroscopy investigation.

INTRODUCTION: Juvenile Sjögren's disease (jSjD) is a rare autoimmune disease characterized by exocrine gland involvement and systemic manifestations, including small vessel vasculitis and Raynaud's phenomenon (RP). We aimed to investigate the microvascular status in jSjD patients by nailfold videocapillaroscopy (NVC) and the potential correlations with clinical and serological features. METHODS: Clinical data from thirteen consecutive jSjD patients (11 females and 2 males), with a mean age of 16 ± 4 years, diagnosed before 16 years of age (mean age at diagnosis 12 ± 3) according to the 2016 American College of Rheumatology/EULAR criteria for adult SjD, were collected including age- and sex-matched healthy controls (HCs). Clinical, laboratory, and instrumental data were collected, together with NVC examination. Non-specific and specific NVC parameters were investigated, such as capillary density, capillary dilations, giant capillaries, microhaemorrhages and abnormal shapes. Associations between NVC findings and clinical/serological features were explored and analysed using parametrical and non-parametrical tests. RESULTS: Capillary density reduction correlated significantly with articular involvement (arthralgias) (p = 0.024). Microhaemorrhages correlated with lower C3 levels (p = 0.034). No specific NVC pattern for jSjD was identified, whereas abnormal capillary shapes were significantly higher in jSjD patients than HCs (p = 0.005). NVC abnormalities were not associated with SjD-specific instrumental tests (biopsy, imaging, Schirmer's test). RP was present in 8% of jSjD patients. CONCLUSIONS: The reduction of capillary density, as well as microhaemorrhages at NVC analysis, are significantly associated with some clinical aspects like articular involvement and serum biomarkers (C3 reduction). The NVC is suggested as safe and further analysis in jSjD patients.

Long-Term Efficacy and Safety of Canakinumab in Patients With Tumor Necrosis Factor Receptor-Associated Periodic Syndrome: Results From a Phase III Trial.

OBJECTIVE: We aimed at assessing efficacy, safety, and tolerability of canakinumab in patients with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) during a 72-week long-term, open-label extension of the CLUSTER study. METHODS: Patients received open-label canakinumab 150 or 300 mg, either every 4 weeks (q4w) or every 8 weeks, with up-titration permitted after on-treatment flares (maximum dose: 300 mg q4w). Efficacy assessments included physician global assessment of disease activity, number of flares, and serum C-reactive protein (CRP) and serum amyloid A protein (SAA) levels. Adverse events were also reported. Results are described for the overall population and according to the cumulative dose of canakinumab adjusted for body weight (<36 mg/kg or ≥36 mg/kg). RESULTS: Of 53 patients entering the final phase (epoch 4) of CLUSTER, 51 completed the treatment. At the end of epoch 4, >94% of patients achieved no or minimal disease activity. Most patients had either no (69.8%) or one flare (24.5%), whereas at baseline, the median number of flares was 9.0 per year. Median CRP levels remained at <10 mg/L. Median SAA concentrations were largely unchanged, with medians of 11.5 mg/L and 14.5 mg/L in the <36 mg/kg and ≥36 mg/kg groups, respectively, at the end of the study. No unexpected safety findings were identified. CONCLUSION: Control of disease activity, with low flare incidence, was maintained with long-term canakinumab treatment in patients with TRAPS during the 72-week final epoch of the CLUSTER study, with no new safety findings.

Early anakinra treatment improves cardiac outcome of multisystem inflammatory syndrome in children, regardless of disease severity.

OBJECTIVE: The main aim of this study was to define the best treatment option for multisystem inflammatory syndrome in children (MIS-C) and to analyse the role of anakinra. METHODS: This is a multicentre retrospective cohort study. Patients were treated according to the attending physician's decision. The patients were divided into four groups on the basis of the first treatment at time of admittance: (i) IVIG, (ii) IVIG and methylprednisolone (≤2 mg/kg/day), (iii) IVIG with high-dose methylprednisolone (>2 mg/kg/day) and (iv) anakinra with or without IVIG and/or methylprednisolone. Primary outcomes were defined as the presence of at least one of the following features: death, the failure of initial treatment, meaning the need for additional treatment for clinical worsening and cardiac involvement at the end of follow-up. RESULTS: Two hundred thirty-nine patients were recruited. At univariate analysis, persistent heart involvement at discharge was more frequent in those not receiving anakinra as initial treatment (3/21 vs 66/189; P = 0.047). After comparisons between the four treatment regimens, adjusting for the propensity score, we observed that early treatment with anakinra was associated with a lower probability of developing persistent heart disease at the end of follow-up (odds ratio: 0.6; 95% CI: 0.4-1.0). CONCLUSION: We report that early treatment with anakinra is safe and very effective in patients with severe MIS-C. In addition, our study suggests that early treatment with anakinra is the most favourable option for patients with a higher risk of developing a severe disease outcome.

A Novel LC-MS/MS Method for Therapeutic Drug Monitoring of Baricitinib in Plasma of Pediatric Patients.

BACKGROUND: Janus kinase inhibitors are antirheumatic immunosuppressive drugs that target intracellular Janus kinases (JAKs). Baricitinib is a selective and reversible orally administered JAK1/JAK2 inhibitor approved for treating rheumatoid arthritis, atopic dermatitis, and alopecia areata in adult patients. Expanded access to baricitinib has been approved for treating pediatric patients affected by rare Mendelian autoinflammatory diseases with type I interferon-mediated damage. Knowledge of the pharmacokinetic properties and target plasma levels of baricitinib in pediatric patients is limited. In this study, a novel LC-MS/MS method for measuring baricitinib in plasma, validated according to the ICH M10 guidelines, is presented. METHODS: Sample preparation was performed by adding 10 µL of IS working solution (150 ng/mL) and 200 µL of MeOH to each plasma sample. Chromatographic separation was conducted using a Thermo Scientific Accucore Polar Premium column (50 mm × 2.1 mm, i.d. 2.6 m). This method was applied to 7 real anonymous plasma samples obtained from pediatric patients treated with baricitinib at IRCCS Istituto Giannina Gaslini (Genoa, Italy). Patients of both sexes had a median age of 14 years (range, 10-17 years). RESULTS: The LC-MS/MS method resulted linear over wide concentration ranges (1.024-100 ng/mL) and was accurate and reproducible in the absence of matrix effects, allowing for robust, specific, and rapid quantification of baricitinib from a low amount of plasma (50 µL). The plasma concentration of baricitinib in the samples of the patients, expressed as mean ± SD, was 11.25 ± 10.86 ng/mL. CONCLUSIONS: This novel LC-MS/MS method is suitable for the therapeutic drug monitoring of baricitinib and can help guide therapy optimization in pediatric patients.

Epidemiological and clinical evolution of multisystem inflammatory syndrome in children throughout the SARS-CoV-2 pandemic in a tertiary Italian children's hospital.

Multisystem inflammatory syndrome in children (MIS-C) is a potentially life-threatening disease temporally linked to SARS-CoV-2 whose incidence and clinical presentation may have been altered by the different SARS-CoV-2 variants and by vaccination. METHODS: We retrospectively collected the data of all MIS-C cases admitted to the Gaslini Children's Hospital, the hub for SARS-CoV-2 related diseases in Liguria region, Italy, from 01 October 2020, to 30 November 2022, evaluating the ratio between MIS-C cases and (1) COVID-19 paediatric cases in our region, (2) emergency department admissions and (3) emergency department febrile patients. We also compared MIS-C incidence in pre- post-vaccination periods. RESULTS: We observed a significant global decline in the incidence of MIS-Cover the four variant periods and after the starting of vaccination whereas clinical features, therapeutic management and severity did not significantly vary. CONCLUSIONS: In our setting, we demonstrated a significant decrease of MIS-C incidence according to the predominant variant and including not vaccinated children. Regardless of variant type, the patients showed similar phenotypes and severity throughout the pandemic. SARS-CoV-2 variants as well as immune protection after previous infections and/or vaccination may have interacted by playing different roles and reducing the incidence of MIS-C.

Novel heterozygous TREX1 mutation in a juvenile systemic lupus erythematosus patient with severe cutaneous involvement treated successfully with Jak-inhibitors: a case report.

Juvenile systemic lupus erythematosus (jSLE) is a complex inflammatory autoimmune disorder. In the last decades, genetic factors and activation pathways have been increasingly studied to understand their potential pathogenetic role better. Genetic and transcriptional abnormalities directly involved in the type I interferon (IFN) signaling cascade have been identified through family-based and genome-wide association studies. IFNs trigger signaling pathways that initiate gene transcription of IFN-stimulated genes through the activation of JAK1, TYK2, STAT1, and STAT2. Thus, the use of therapies that target the IFN pathway would represent a formidable advance in SLE. It is well known that JAK inhibitors have real potential for the treatment of rheumatic diseases, but their efficacy in the treatment of SLE remains to be elucidated. We report the case of a 13-year-old girl affected by jSLE, carrying a novel heterozygous missense variant on Three prime Repair EXonuclease 1 (TREX1), successfully treated with baricitinib on top of mofetil mycophenolate. The TREX1 gene plays an important role in DNA damage repair, and its mutations have been associated with an overproduction of type 1 interferon. This report underlines the role of translational research in identifying potential pathogenetic pathways in rare diseases to optimize treatment.

Comparison of EULAR/PRINTO/PReS Ankara 2008 and 2022 ACR/EULAR Classification Criteria for Granulomatosis with Polyangiitis in Children.

OBJECTIVE: Granulomatosis with polyangiitis (GPA) is an antineutrophil cytoplasmic antibody-associated vasculitis. The 2022 American College of Rheumatology/European Alliance of Associations for Rheumatology (ACR/EULAR)-endorsed classification criteria for GPA was derived using data only from adult patients. We aimed to assess the performance of the ACR/EULAR classification criteria for GPA in pediatric patients and compare it with the EULAR/Pediatric Rheumatology International Trials Organization (PRINTO)/Pediatric Rheumatology European Society (PReS)-endorsed Ankara 2008 criteria for GPA. METHODS: Retrospective data of pediatric patients with GPA in 20 centers from 9 countries were evaluated. The diagnosis of GPA was made according to the expert opinion. The sensitivity, specificity, positive predictive value, and negative predictive value of the criteria sets were evaluated. RESULTS: The study included 77 patients with GPA and 108 controls (immunoglobulin A vasculitis (n = 44), Takayasu's arteritis (n = 20), microscopic polyangiitis (n = 16), polyarteritis nodosa (n = 14), Behçet's disease (n = 12), eosinophilic granulomatosis with polyangiitis (n = 1), and Cogan's syndrome (n = 1)) with a median age of 17.8 and 15.2 years, respectively. Of patients with GPA, constitutional symptoms (85.7%) and ear-nose-throat involvement (79.2%) were the most common presentations. In the GPA group, 73 patients fulfilled the Ankara 2008 criteria and 69 the ACR/EULAR classification criteria. Sensitivities of the Ankara 2008 criteria and the ACR/EULAR classification criteria were 94.8% and 89.6%, while specificities were 95.3% and 96.3%, respectively. No significant difference was found between sensitivities and specificities of both classification criteria (p= 0.229 and p= 0.733, respectively). CONCLUSION: In children, both the ACR/EULAR and EULAR/PRINTO/PReS Ankara 2008 classification criteria for GPA perform well and similarly.

ARF1 prevents aberrant type I interferon induction by regulating STING activation and recycling.

Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.