Human CARMIL2 deficiency underlies a broader immunological and clinical phenotype than CD28 deficiency.

Patients with inherited CARMIL2 or CD28 deficiency have defective T cell CD28 signaling, but their immunological and clinical phenotypes remain largely unknown. We show that only one of three CARMIL2 isoforms is produced and functional across leukocyte subsets. Tested mutant CARMIL2 alleles from 89 patients and 52 families impair canonical NF-κB but not AP-1 and NFAT activation in T cells stimulated via CD28. Like CD28-deficient patients, CARMIL2-deficient patients display recalcitrant warts and low blood counts of CD4+ and CD8+ memory T cells and CD4+ TREGs. Unlike CD28-deficient patients, they have low counts of NK cells and memory B cells, and their antibody responses are weak. CARMIL2 deficiency is fully penetrant by the age of 10 yr and is characterized by numerous infections, EBV+ smooth muscle tumors, and mucocutaneous inflammation, including inflammatory bowel disease. Patients with somatic reversions of a mutant allele in CD4+ T cells have milder phenotypes. Our study suggests that CARMIL2 governs immunological pathways beyond CD28.

A signalome screening approach in the autoinflammatory disease TNF receptor associated periodic syndrome (TRAPS) highlights the anti-inflammatory properties of drugs for repurposing.

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease caused by mutations in TNF Receptor 1 (TNFR1). Current therapies for TRAPS are limited and do not target the pro-inflammatory signalling pathways that are central to the disease mechanism. Our aim was to identify drugs for repurposing as anti-inflammatories based on their ability to down-regulate molecules associated with inflammatory signalling pathways that are activated in TRAPS. This was achieved using rigorously optimized, high through-put cell culture and reverse phase protein microarray systems to screen compounds for their effects on the TRAPS-associated inflammatory signalome. 1360 approved, publically available, pharmacologically active substances were investigated for their effects on 40 signalling molecules associated with pro-inflammatory signalling pathways that are constitutively upregulated in TRAPS. The drugs were screened at four 10-fold concentrations on cell lines expressing both wild-type (WT) TNFR1 and TRAPS-associated C33Y mutant TNFR1, or WT TNFR1 alone; signalling molecule levels were then determined in cell lysates by the reverse-phase protein microarray. A novel mathematical methodology was developed to rank the compounds for their ability to reduce the expression of signalling molecules in the C33Y-TNFR1 transfectants towards the level seen in the WT-TNFR1 transfectants. Seven high-ranking drugs were selected and tested by RPPA for effects on the same 40 signalling molecules in lysates of peripheral blood mononuclear cells (PBMCs) from C33Y-TRAPS patients compared to PBMCs from normal controls. The fluoroquinolone antibiotic lomefloxacin, as well as others from this class of compounds, showed the most significant effects on multiple pro-inflammatory signalling pathways that are constitutively activated in TRAPS; lomefloxacin dose-dependently significantly reduced expression of 7/40 signalling molecules across the Jak/Stat, MAPK, NF-κB and PI3K/AKT pathways. This study demonstrates the power of signalome screening for identifying candidates for drug repurposing.

A pro-inflammatory signalome is constitutively activated by C33Y mutant TNF receptor 1 in TNF receptor-associated periodic syndrome (TRAPS).

Mutations in TNFRSF1A encoding TNF receptor 1 (TNFR1) cause the autosomal dominant TNF receptor-associated periodic syndrome (TRAPS): a systemic autoinflammatory disorder. Misfolding, intracellular aggregation, and ligand-independent signaling by mutant TNFR1 are central to disease pathophysiology. Our aim was to understand the extent of signaling pathway perturbation in TRAPS. A prototypic mutant TNFR1 (C33Y), and wild-type TNFR1 (WT), were expressed at near physiological levels in an SK-Hep-1 cell model. TNFR1-associated signaling pathway intermediates were examined in this model, and in PBMCs from C33Y TRAPS patients and healthy controls. In C33Y-TNFR1-expressing SK-Hep-1 cells and TRAPS patients' PBMCs, a subtle, constitutive upregulation of a wide spectrum of signaling intermediates and their phosphorylated forms was observed; these were associated with a proinflammatory/antiapoptotic phenotype. In TRAPS patients' PBMCs, this upregulation of proinflammatory signaling pathways was observed irrespective of concurrent treatment with glucocorticoids, anakinra or etanercept, and the absence of overt clinical symptoms at the time that the blood samples were taken. This study reveals the pleiotropic effect of a TRAPS-associated mutant form of TNFR1 on inflammatory signaling pathways (a proinflammatory signalome), which is consistent with the variable and limited efficacy of cytokine-blocking therapies in TRAPS. It highlights new potential target pathways for therapeutic intervention.

Role of interleukin-6 in a patient with tumor necrosis factor receptor-associated periodic syndrome: assessment of outcomes following treatment with the anti-interleukin-6 receptor monoclonal antibody tocilizumab.

In this report, we describe treatment outcomes in the first case of a patient with tumor necrosis factor receptor-associated periodic syndrome (TRAPS) treated with the anti-interleukin-6 (anti-IL-6) receptor monoclonal antibody tocilizumab. Since IL-6 levels are elevated in TRAPS, we hypothesized that tocilizumab might be effective. The patient, a 52-year-old man with lifelong TRAPS in whom treatment with etanercept and anakinra had failed, was administered tocilizumab for 6 months, and the therapeutic response was assessed by measurement of monocyte CD16 expression and cytokine levels. Following treatment, the evolving acute attack was aborted and further attacks of TRAPS were prevented. The patient did not require corticosteroids and showed significant clinical improvement in scores for pain, stiffness, and well-being. Moreover, the acute-phase response diminished significantly with treatment. Monocyte CD16 expression was reduced and the numbers of circulating CD14+CD16+ and CD14++CD16- monocytes were transiently decreased. However, cytokine levels were not reduced. This case supports the notion of a prominent role for IL-6 in mediating the inflammatory attacks in TRAPS, but blockade of IL-6 did not affect the underlying pathogenesis. These preliminary findings require confirmation.

A novel NEMO gene mutation causing osteopetrosis, lymphoedema, hypohidrotic ectodermal dysplasia and immunodeficiency (OL-HED-ID).

Genetic conditions are increasingly recognised as a cause of multisystem diseases in children. We report a 6-year-old boy with hypohidrotic ectodermal dysplasia, immunodeficiency, osteopetrosis and lymphoedema, associated with a novel mutation in the NF-κß essential modulator (NEMO) gene. He is the longest surviving of three reported boys with these clinical features. Hypohidrotic ectodermal dysplasia, a congenital disorder of teeth, hair and eccrine sweat glands is most commonly inherited as an X-linked recessive trait. Associated immunodeficiency (HED-ID) may give rise to serious infections in early life. Mutations in the NEMO gene give rise to a heterogeneous group of disorders, including the X-linked dominant disorder incontinentia pigmenti. This is characterised by typical skin changes leading to linear pigmentary change and variable associated features; in males, prenatal death usually occurs. Our patient, like one if the previous cases and all of their mothers, demonstrates features of incontinentia pigmenti.

The role of high-dose intravenous immunoglobulin in rheumatology.

For many years, non-steroidal anti-inflammatory agents, steroids and immunosuppressive drugs have been the mainstay of treatment for rheumatological disorders. Over the last few years, the emergence of biologic treatments has dramatically changed the management of numerous rheumatological diseases. However, immunoglobulin treatment has been used for decades and its use has still not been superseded in certain rheumatological diseases. In fact, despite the introduction of newer immunomodulatory drugs, there has been an ever-increasing number of clinical indications for which intravenous immunoglobulin (IVIG) has been tried. Immunoglobulins are plasma proteins secreted by plasma cells, forming a major component of the adaptive immune system. IVIG is a blood product prepared from plasma, each batch prepared from a pool of 10,000-20,000 donations. Multiple purification steps during the manufacturing process aim to eliminate all known transmissible pathogens, but cannot completely exclude the risk from unknown pathogens. It should be noted that there has been the transmission of hepatitis C in one batch of immunoglobulin, reported in 1994, resulting in more than 200 patients in the USA and Europe being affected. Nevertheless, IVIG remains relatively safe compared with other immunosuppressive drugs. Headaches and fatigue are common side effects but fortunately the more severe problems such as aseptic meningitis, venous thromboembolism and acute renal failure remain rare. High-dose immunoglobulin when administered i.v. has immunomodulatory properties. The precise mechanism of action of IVIG is complex and not yet fully understood.

Novel markers of inflammation identified in tumor necrosis factor receptor-associated periodic syndrome (TRAPS) by transcriptomic analysis of effects of TRAPS-associated tumor necrosis factor receptor type I mutations in an endothelial cell line.

OBJECTIVE: To analyze the effects of tumor necrosis factor receptor-associated periodic syndrome (TRAPS)-associated mutant tumor necrosis factor receptor type I (TNFRI) expression in a cell type directly relevant to the inflammation in TRAPS, and to identify novel markers associated with mutant TNFRI expression. METHODS: Transcriptome analysis on 30,000 human genes was performed on SK-Hep-1 human endothelial cells transfected with either wild-type (WT) or TRAPS-associated mutant TNFRI. Quantitative reverse transcriptase-polymerase chain reaction and protein expression levels measured by enzyme-linked immunosorbent assay verified transcriptional changes for selected genes both in supernatants from cells expressing mutant TNFRI and in patient plasma. RESULTS: Cells expressing mutant TNFRI showed up-regulation of multiple proinflammatory genes relative to WT transfectants, including genes for pentraxin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, CCL2, and CCL5, which were also expressed as proteins. In addition, the expression of most of these markers was increased in the plasma and peripheral blood mononuclear cells from TRAPS patients relative to those from healthy controls. The cysteine mutations (C33Y and C52F), which are associated with a more severe clinical phenotype, induced more genes than the low-penetrance mutation R92Q, which is associated with a milder phenotype. The expression of most genes was induced by a death domain (DD)-dependent mechanism, since they were not induced by expression of TNFRI mutants with an inactivated DD. CONCLUSION: TRAPS-associated TNFRI mutants induce the expression of multiple genes encoding inflammatory molecules, cellular receptors, transcription factors, and regulators of apoptosis in endothelial cells that require the cytoplasmic signaling properties of the receptor. Different mutants have specific expression profiles, indicating mutation-specific effects. The expression of some of these markers was also elevated in samples from TRAPS patients.

Heterogeneity among patients with tumor necrosis factor receptor-associated periodic syndrome phenotypes.

OBJECTIVE: To investigate the prevalence of tumor necrosis factor receptor-associated periodic syndrome (TRAPS) among outpatients presenting with recurrent fevers and clinical features consistent with TRAPS. METHODS: Mutational screening was performed in affected members of 18 families in which multiple members had symptoms compatible with TRAPS and in 176 consecutive subjects with sporadic (nonfamilial) "TRAPS-like" symptoms. Plasma concentrations of soluble tumor necrosis factor receptor superfamily 1A (sTNFRSF1A) were measured, and fluorescence-activated cell sorter analysis was used to measure TNFRSF1A shedding from monocytes. RESULTS: Eight novel and 3 previously reported TNFRSF1A missense mutations were identified, including an amino acid deletion (Delta D42) in a Northern Irish family and a C70S mutation in a Japanese family, both reported for the first time. Only 3 TNFRSF1A variants were found in patients with sporadic TRAPS (4 of 176 patients). Evidence for nonallelic heterogeneity in TRAPS-like conditions was found: 3 members of the "prototype familial Hibernian fever" family did not possess C33Y, present in 9 other affected members. Plasma sTNFRSF1A levels were low in TRAPS patients in whom renal amyloidosis had not developed, but also in mutation-negative symptomatic subjects in 4 families, and in 14 patients (8%) with sporadic TRAPS. Reduced shedding of TNFRSF1A from monocytes was demonstrated in vitro in patients with the T50M and T50K variants, but not in those with other variants. CONCLUSION: The presence of TNFRSF1A shedding defects and low sTNFRSF1A levels in 3 families without a TNFRSF1A mutation indicates that the genetic basis among patients with "TRAPS-like" features is heterogeneous. TNFRSF1A mutations are not commonly associated with nonfamilial recurrent fevers of unknown etiology.

The TNF receptor-associated periodic syndrome (TRAPS): emerging concepts of an autoinflammatory disorder.

The present report describes and expands the clinical and genetic spectrum of the autoinflammatory disorder, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS). A total of 20 mutations have been identified since our initial discovery of 6 missense mutations in TNF receptor super family 1A (TNFRSF1A) in 1999. Eighteen of the mutations result in amino acid substitutions within the first 2 cysteine-rich domains (CRDs) of the extracellular portion of the receptor. A single splicing mutation also affects the first CRD by causing the insertion of 4 amino acids. Haplotype analysis of the most commonly occurring and ethnically heterogeneous mutation, R92Q, demonstrates an ancient founder; however, analysis of the T50M mutation, another commonly occurring mutation in Irish and Scottish families, does not, suggesting that T50M is a recurring mutation. Mutations that result in cysteine substitutions demonstrate a higher penetrance of the clinical phenotype (93% versus 82% for noncysteine residue substitutions), and also increase the probability of developing life-threatening amyloidosis (24% versus 2% for noncysteine residue substitutions). Retrospective and prospective evaluation of more than 50 patients, representing 10 of the 20 known mutations, allows us to expand and better define the clinical spectrum of TRAPS. Recurrent episodes of fever, myalgia, rash, abdominal pain, and conjunctivitis that often last longer than 5 days are the most characteristic clinical features of TRAPS. Defective shedding of TNFRSF1A can only partially explain the pathophysiologic mechanism of TRAPS, since some mutations have normal shedding. Consequently, other mechanisms may be mediating the observed phenotype. We are currently investigating other possible mechanisms using stable and transiently transfected cell systems in vitro, as well as developing a knockin mouse model. Preliminary data suggest that etanercept may be effective in decreasing the severity, duration, and frequency of symptoms in TRAPS patients. Additionally, it provides a viable therapeutic alternative to glucocorticoid therapy, which has numerous serious, long-term adverse effects. Two clinical trials are being conducted to evaluate the efficacy of etanercept in decreasing the frequency and severity of symptoms in TRAPS. Lastly, we have summarized data that R92Q and P46L, and probably as yet undiscovered substitutions, represent very low penetrance mutations that may play a much larger role in more broadly defined inflammatory diseases such as rheumatoid arthritis. Our laboratories are currently undertaking both clinical and basic research studies to define the role of these mutations in more common inflammatory diseases.