Neutrophil extracellular traps release in gout and pseudogout depends on the number of crystals regardless of leukocyte count.

OBJECTIVES: Microcrystal-induced arthritis is still an unresolved paradigm for medicine. Overt inflammation may be absent even when crystals occur in SF. Recently, the production of neutrophil extracellular traps (NETs) embedding MSU crystals has been proposed as a possible mechanism of the auto-resolution of the inflammatory phase during gout. We aimed to verify and quantify the release of NETs in SFs during gout and pseudogout attacks and to compare any differences with respect to crystals and neutrophils number, and to analyse activation of necroptosis pathway in SF from crystal-induced arthritis. METHODS: SF samples were obtained by arthrocentesis from 22 patients presenting acute crystal-induced arthritis, gout or pseudogout (n = 11 each group), and from 10 patients with acute non-crystal arthritis as controls. NETosis was quantified in SF by nucleic acid stain and by quantification of human neutrophil elastase. Activation of phosphorylated MLKL was assessed by western blot. RESULTS: We observed that SF neutrophils encountering MSU and CPPD crystals during episodes of gout and pseudogout release NETs in relation to the number of crystals in SF and irrespective of neutrophil density and type of crystal. This release was accompanied by necroptosis through the activation of the MLKL pathway. CONCLUSIONS: Our findings suggest that a role of NETs in crystal-induced arthritis is to 'trap extracellular particles', including microcrystals. Embedding crystals in aggregates of NETs may be the basis of tophi and CPPD deposition, and may have implications for disease evolution rather than for spontaneous resolution of the acute attack.

Hints for Genetic and Clinical Differentiation of Adult-Onset Monogenic Autoinflammatory Diseases.

Monogenic autoinflammatory diseases (mAIDs) are inherited errors of innate immunity characterized by systemic inflammation recurring with variable frequency and involving the skin, serosal membranes, synovial membranes, joints, the gastrointestinal tube, and/or the central nervous system, with reactive amyloidosis as a potential severe long-term consequence. Although individually uncommon, all mAIDs set up an emerging chapter of internal medicine: recent findings have modified our knowledge regarding mAID pathophysiology and clarified that protean inflammatory symptoms can be variably associated with periodic fevers, depicting multiple specific conditions which usually start in childhood, such as familial Mediterranean fever, tumor necrosis factor receptor-associated periodic syndrome, cryopyrin-associated periodic syndrome, and mevalonate kinase deficiency. There are no evidence-based studies to establish which potential genotype analysis is the most appropriate in adult patients with clinical phenotypes suggestive of mAIDs. This review discusses genetic and clinical hints for an ideal diagnostic approach to mAIDs in adult patients, as their early identification is essential to prompt effective treatment and improve quality of life, and also highlights the most recent developments in the diagnostic work-up for the most frequent hereditary periodic febrile syndromes worldwide.

Updated overview of molecular pathways involved in the most common monogenic autoinflammatory diseases.

An apparently unprovoked recurrent inflammation is the quintessential hallmark of autoinflammatory diseases (AIDs), a large and heterogeneous group of disorders in which there is poor regulation of the innate immune system with no clearly demonstrated autoimmune machinery involvement. Innate immunity pathways are diverse and our understanding of their molecular composition and function is continuously expanding. The impaired immune responses we observe in monogenic AIDs, mostly in the hereditary periodic fever syndromes, is officiated by target molecules of microbial origin (pathogen-associated molecular patterns) and also host molecules (danger-associated molecular patterns). Further crucial components of innate immune mechanisms that contribute differently in the deregulated inflammatory patterns of different AIDs include Toll-like receptors, Nod-like receptors, scaffolding proteins (such as the caspase recruitment domain of proteins), cytosolic DNA-sensing molecules, inflammatory multi-protein complexes (referred to as inflammasomes), complement system, and others. In recent years, the knowledge of protean molecular pathways responsible for the most common monogenic AIDs has expanded, in parallel with very recent extraordinary technological advances, allowing the identification and characterisation of some unknown aspects of the innate immunity. This review will list and describe the most common monogenic febrile syndromes belonging to AIDs and will focus on current insights dealing with their pathologic processes.

Serum immunoglobulin D levels in patients with Behçet's disease according to different clinical manifestations.

OBJECTIVES: Behçet's disease (BD) is an autoinflammatory disorders mainly characterised by recurrent oral aphthosis, genital ulcers, and uveitis. The involvement of immunoglobulin D (IgD) in BD physiopathology is still unclear. The aim of our study was to assess the role of IgD in BD by comparing circulating levels of IgD in a cohort of BD patients and healthy controls (HC), as well as by correlating IgD levels with BD activity and different clinical presentations. METHODS: Serum IgD and SAA levels were analysed by ELISA assay in ninety-nine serum samples collected from 72 BD patients and in 29 HC subjects. RESULTS: Serum concentration of IgD were higher in BD patients compared with HC (p=0.029), in patients with high serum amyloid A (SAA) levels compared with patients with normal SAA levels (p=0.035), and among subjects with active mucocutaneous involvement compared with other patients (p=0.036). No correlations were identified between IgD serum levels and disease activity assessed by the BD current activity form (BDCAF) (p=0.640). No differences were observed in the IgD serum levels between patients with and without specific disease manifestations. Increased SAA levels (Odds Ratio = 3.978, CI: 1.356 -11.676) and active mucocutaneous BD manifestations (Odds Ratio = 4.286, CI: 1.192 - 15.407) were associated with a high risk for increased IgD serum levels. CONCLUSIONS: Serum IgD levels are significantly increased in BD patients, especially among patients with active mucocutaneous manifestations, suggesting a possible role of IgD in BD pathogenesis and in the onset of mucosal and skin lesions.

Correlation of Serum Amyloid-A Levels, Clinical Manifestations, Treatment, and Disease Activity in Patients with Behçet's Disease.

BACKGROUND: Behçet's disease (BD) is an inflammatory disorder potentially leading to life- and sight-threatening complications. No laboratory marker correlating with disease activity or predicting the occurrence of disease manifestations is currently available. OBJECTIVES: To determine an association between serum amyloid-A (SAA) levels and disease activity via the BD Current Activity Form (BDCAF), to evaluate disease activity in relation to different SAA thresholds, to examine the association between single organ involvement and the overall major organ involvement with different SAA thresholds, and to assess the influence of biologic therapy on SAA levels. METHODS: We collected 95 serum samples from 64 BD patients. Related demographic, clinical, and therapeutic data were retrospectively gathered. RESULTS: No association was identified between SAA levels and BD disease activity (Spearman's rho = 0.085, P = 0.411). A significant difference was found in the mean BDCAF score between patients presenting with SAA levels < 200 mg/L and those with SAA levels > 200 mg/L (P = 0.027). SAA levels > 200 mg/L were associated with major organ involvement (P = 0.008). A significant association was found between SAA levels > 150 mg/dl and ocular (P = 0.008), skin (P = 0.002), and mucosal (P = 0.012) manifestations. Patients undergoing biologic therapies displayed more frequently SAA levels < 200 mg/L vs. patients who were not undergoing biologic therapies (P = 0.012). CONCLUSIONS: Although SAA level does not represent a biomarker for disease activity, it might be a predictor of major organ involvement and ocular disease relapse at certain thresholds in patients with BD.

Specific recycling receptors are targeted to the immune synapse by the intraflagellar transport system.

T cell activation requires sustained signaling at the immune synapse, a specialized interface with the antigen-presenting cell (APC) that assembles following T cell antigen receptor (TCR) engagement by major histocompatibility complex (MHC)-bound peptide. Central to sustained signaling is the continuous recruitment of TCRs to the immune synapse. These TCRs are partly mobilized from an endosomal pool by polarized recycling. We have identified IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, as a central regulator of TCR recycling to the immune synapse. Here, we have investigated the interplay of IFT20 with the Rab GTPase network that controls recycling. We found that IFT20 forms a complex with Rab5 and the TCR on early endosomes. IFT20 knockdown (IFT20KD) resulted in a block in the recycling pathway, leading to a build-up of recycling TCRs in Rab5(+) endosomes. Recycling of the transferrin receptor (TfR), but not of CXCR4, was disrupted by IFT20 deficiency. The IFT components IFT52 and IFT57 were found to act together with IFT20 to regulate TCR and TfR recycling. The results provide novel insights into the mechanisms that control TCR recycling and immune synapse assembly, and underscore the trafficking-related function of the IFT system beyond ciliogenesis.

Possible chondroprotective effect of canakinumab: an in vitro study on human osteoarthritic chondrocytes.

Canakinumab is a human IgGκ monoclonal antibody that neutralizes the activity of interleukin (IL)-1ß blocking interaction with IL-1ß receptors. Our study aimed to evaluate the in vitro effect of canakinumab on human osteoarthritic (OA) chondrocytes cultivated in the presence or absence of tumor necrosis factor (TNF)-α. Articular cartilage was obtained from the femoral heads of patients with osteoarthritis (OA). Chondrocytes were incubated with two concentrations (1µg/ml and 10µg/ml) of canakinumab alone or with TNF-α (10ng/ml) for 48h. We evaluated cell viability, release of proteoglycans (PG) and nitric oxide (NO) in culture medium, inducible nitric oxide synthase (iNOS) and metalloproteinanes (MMP)-1,3,13 gene expression, apoptosis, necrosis and morphological feature by transmission electron microscopy (TEM). Canakinumab alone did not have cytotoxic effect. Cell viability was reduced significantly (p<0.001) by TNF-α and restored by canakinumab at both concentrations used. TNF-α determined a significant decrease of PG (p<0.001) and an increase of NO (p<0.001) and MMP-1,3,13 gene expression. Canakinumab significantly increased the PG levels and decreased (1µg/ml, p<0.01; 10µg/ml, p<0.01) NO levels in cells cultured with TNF-α. The NO data were confirmed by the immunocytochemistry assay for iNOS. A significant reduction of MMP-1,3,13 gene expression was induced by canakinumab. Our experiments confirmed the pro-apoptotic effect of TNF-α and demonstrated a protective role of canakinumab. The results concerning biochemical data were further confirmed by the morphological findings obtained by TEM. We showed that canakinumab counteracts the negative effects of TNF-α on OA chondrocyte cultures and may have a potential chondroprotective role in OA.

S1P1 expression is controlled by the pro-oxidant activity of p66Shc and is impaired in B-CLL patients with unfavorable prognosis.

Although intrinsic apoptosis defects are causal to the extended survival of chronic lymphocytic leukemia (CLL) B cells, several lines of evidence support a contribution of the peripheral lymphoid organs and BM microenvironment to the extended lifespan of leukemic B cells. Lymphocyte trafficking is controlled by homing signals provided by stromal cell-derived chemokines and egress signals provided by sphingosine-1-phosphate (S1P). In the present study, we show that expression of S1P1, the S1P receptor responsible for lymphocyte egress, is selectively reduced in CLL B cells with unmutated IGHV. Expression of S1P2, which controls B-cell homeostasis, is also impaired in CLL B cells but independently of the IGHV mutational status. We provide evidence herein that p66Shc, a Shc adaptor family member the deficiency of which is implicated in the apoptosis defects of CLL B cells, controls S1P1 expression through its pro-oxidant activity. p66Shc also controls the expression of the homing receptor CCR7, which opposes S1P1 by promoting lymphocyte retention in peripheral lymphoid organs. The results of the present study provide insights into the regulation of S1P1 expression in B cells and suggest that defective egress caused by impaired S1P1 expression contributes to the extended survival of CLL B cells by prolonging their residency in the prosurvival niche of peripheral lymphoid organs.

Inhibition of interleukin-1 by canakinumab as a successful mono-drug strategy for the treatment of refractory Behçet's disease: a case series.

Recommendations related to ocular, mucosal and cutaneous involvement of Behçet's disease (BD) are mainly evidence-based, but in cases of vascular, neurological and gastrointestinal involvement there are no guidelines to define the best treatment strategy. We report three adult patients with BD, who received an interleukin-1ß inhibitor by subcutaneous injections, canakinumab (at the dosage of 150 mg every 6 weeks), after failure shown by corticosteroids and different combinations of immunosuppressant agents. The prompt and sustained clinical efficacy demonstrated by canakinumab as a monotherapy supports the opportunity of using this specific anti-interleukin-1ß agent as a valid therapeutic option for resistant or refractory BD. Open trials and observational studies should be performed to test canakinumab efficacy on a larger number of patients. The most appropriate dosage and intervals between administrations should be decided according to the individual patient, severity or recurrence of clinical manifestations and major organ involvement.

Untangling the web of systemic autoinflammatory diseases.

The innate immune system is involved in the pathophysiology of systemic autoinflammatory diseases (SAIDs), an enlarging group of disorders caused by dysregulated production of proinflammatory cytokines, such as interleukin-1ß and tumor necrosis factor-α, in which autoreactive T-lymphocytes and autoantibodies are indeed absent. A widely deranged innate immunity leads to overactivity of proinflammatory cytokines and subsequent multisite inflammatory symptoms depicting various conditions, such as hereditary periodic fevers, granulomatous disorders, and pyogenic diseases, collectively described in this review. Further research should enhance our understanding of the genetics behind SAIDs, unearth triggers of inflammatory attacks, and result in improvement for their diagnosis and treatment.

Biological treatments in Behçet's disease: beyond anti-TNF therapy.

Behçet's disease (BD) is universally recognized as a multisystemic inflammatory disease of unknown etiology with chronic course and unpredictable exacerbations: its clinical spectrum varies from pure vasculitic manifestations with thrombotic complications to protean inflammatory involvement of multiple organs and tissues. Treatment has been revolutionized by the progressed knowledge in the pathogenetic mechanisms of BD, involving dysfunction and oversecretion of multiple proinflammatory molecules, chiefly tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1ß, and IL-6. However, although biological treatment with anti-TNF-α agents has been largely demonstrated to be effective in BD, not all patients are definite responders, and this beneficial response might drop off over time. Therefore, additional therapies for a subset of refractory patients with BD are inevitably needed. Different agents targeting various cytokines and their receptors or cell surface molecules have been studied: the IL-1 receptor has been targeted by anakinra, the IL-1 by canakinumab and gevokizumab, the IL-6 receptor by tocilizumab, the IL12/23 receptor by ustekinumab, and the B-lymphocyte antigen CD-20 by rituximab. The aim of this review is to summarize all current experiences and the most recent evidence regarding these novel approaches with biological drugs other than TNF-α blockers in BD, providing a valuable addition to the actually available therapeutic armamentarium.

Working the endless puzzle of hereditary autoinflammatory disorders.

Hereditary autoinflammatory disorders encompass manifold dysfunctions of innate immunity caused by mutations in genes coding for the main characters of the inflammatory scene: most of these conditions have an early onset, ranging from the first days of life to the first decades, and include hereditary periodic fevers, NLRP-related diseases, granulomatous and pyogenic syndromes, which are basically characterized by upturned inflammasome activity and overproduction of bioactive interleukin (IL)-1ß and other proinflammatory cytokines. The discovery of a causative link between autoinflammation and IL-1ß release has improved our understanding of the intimate mechanisms of innate immunity, and has likewise led to the identification of extraordinary treatments for many of these disorders.

The most recent advances in pathophysiology and management of tumour necrosis factor receptor-associated periodic syndrome (TRAPS): personal experience and literature review.

Tumour necrosis factor-receptor associated periodic syndrome (TRAPS) is a rare autosomal dominant autoinflammatory disorder characterised by recurrent episodes of long-lasting fever and inflammation in different regions of the body, as musculo-skeletal system, skin, gastrointestinal tube, serosal membranes and eye. Inflammatory attacks usually start in the pediatric age with initial corticosteroid-responsiveness. Most reported cases of TRAPS involve patients of European ancestry and diagnosis can be formulated by the combination of genetic analysis and a compatible phenotype. Its prognosis is strictly dependent on the appearance of amyloidosis, secondary to uncontrolled relapsing inflammation. Thanks to a better understanding of its pathogenesis, the disease is now managed with anti-interleukin (IL)-1 antagonists, rather than corticosteroids or tumour necrosis factor (TNF) inhibitors. The aim of this review is to describe the current understanding and advances of TRAPS genetic basis, pathogenesis and management options by integrating the most recent data in the medical literature.

Leptin, adiponectin, resistin, visfatin serum levels and idiopathic recurrent pericarditis: biomarkers of disease activity? A preliminary report.

OBJECTIVES: Idiopathic recurrent acute pericarditis (IRAP) represents the most troublesome complication of acute pericarditis and is an autoimmune process. White adipose tissue produces more than 50 adipokines that participate in inflammation and autoimmunity. This study investigated whether serum leptin, resistin, visfatin and adiponectin are increased in IRAP versus healthy controls and if their levels correlate with parameters of disease activity. METHODS: Serum leptin, resistin, visfatin and adiponectin levels were assayed by enzyme-linked immunosorbent assay in 14 IRAP patients during recurrences (group 1), in 23 IRAP patients during symptom-free intervals (group 2) and in 18 healthy controls (group 3). Assessment parameters included demographic characteristics of patients and controls, clinical characteristics of patients and markers of inflammation. Comparisons between groups as well as reciprocal comparisons were evaluated. RESULTS: Group 1 showed serum leptin (p<0.008), visfatin (p<0.002), and adiponectin (p<0.04) significantly higher than group 2 and control group, whereas resistin serum levels did not significantly differ (p=0.69). Among IRAP patients, serum leptin significantly correlated with serum amyloid A (SAA) levels (rs=0.43, r2= 0.27, p<0.02). Other than this correlation, none of the considered adipokines significantly correlated with the other considered variables in univariate analysis. CONCLUSIONS: Leptin, adiponectin and visfatin are increased in IRAP patients versus healthy controls. Our data suggest that these adipokines might be involved in IRAP pathogenesis and that a possible increased cardiovascular risk in these patients, through an early onset atherosclerosis, should be kept in mind. SAA might be a link between IRAP and increased cardiovascular diseases.

Biological treatments: new weapons in the management of monogenic autoinflammatory disorders.

Treatment of monogenic autoinflammatory disorders, an expanding group of hereditary diseases characterized by apparently unprovoked recurrent episodes of inflammation, without high-titre autoantibodies or antigen-specific T cells, has been revolutionized by the discovery that several of these conditions are caused by mutations in proteins involved in the mechanisms of innate immune response, including components of the inflammasome, cytokine receptors, receptor antagonists, and oversecretion of a network of proinflammatory molecules. Aim of this review is to synthesize the current experience and the most recent evidences about the therapeutic approach with biologic drugs in pediatric and adult patients with monogenic autoinflammatory disorders.

Impaired expression of p66Shc, a novel regulator of B-cell survival, in chronic lymphocytic leukemia.

Intrinsic apoptosis defects underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL). Here, we show that the Shc family adapter p66Shc uncouples the B-cell receptor (BCR) from the Erk- and Akt-dependent survival pathways, thereby enhancing B-cell apoptosis. p66Shc expression was found to be profoundly impaired in CLL B cells compared with normal peripheral B cells. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, based on the mutational status of IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes implicated in apoptosis defects of CLL showed an alteration in the balance of proapoptotic and antiapoptotic members of the Bcl-2 family in patients with CLL. Reconstitution experiments in CLL B cells, together with data obtained on B cells from p66Shc(-/-) mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family toward proapoptotic members. The data identify p66Shc as a novel regulator of B-cell apoptosis which attenuates BCR-dependent survival signals and modulates Bcl-2 family expression. They moreover provide evidence that the p66Shc expression defect in CLL B cells may be causal to the imbalance toward the antiapoptotic Bcl-2 family members in these cells.

Long-term clinical course of patients carrying the Q703K mutation in the NLRP3 gene: a case series.

BACKGROUND: Cryopyrin-associated periodic syndromes (CAPS) comprise a spectrum of apparently distinct, rare, autosomal dominant autoinflammatory disorders of increasing severity caused by NLRP3 gene mutations. The Q703K allele is a variant of unknown pathogenetic significance, and has been considered to be both a clinically unremarkable polymorphism and a low- penetrance mutation. OBJECTIVES: To analyse the long-term clinical course in a cohort of patients presenting with periodic fever attacks and carrying the Q703K mutation in the NLRP3 gene. METHODS: Seven Caucasian patients (mean age 37.3±8.5 years, 2 males and 5 females) were identified as carriers of the Q703K mutation among 71 patients with CAPS-like symptoms. RESULTS: The mean age at disease onset was 25.58±16.08 years and the mean disease duration was 12.28±8.36. The mean number of febrile episodes was 7.56±6.48 and the mean duration of fever attacks was 6.66±4.71 days. Six out of 7 patients had a low grade fever, while 1 patient had no fever episodes. All patients were characterised by symptoms consistent with recurrent inflammatory syndrome. Six patients out of 7 presented skin lesions, 4/7 arthralgia, 4/7 myalgia, 4/7 conjunctivitis, 3/7 headache. All patients also complained of severe fatigue. In 4/7 patients symptoms were triggered or worsened by generalised cold exposure. CONCLUSIONS: We suggest that patients carrying the low-penetrance Q703K mutation in the NLRP3 gene may present with FCAS-like symptoms. However, given the high frequency of healthy carriers, the role of additional, still unknown genetic and/or environmental modifiers is conceivable.

Systemic-onset juvenile idiopathic arthritis complicated by early onset amyloidosis in a patient carrying a mutation in the MEFV gene.

Systemic juvenile idiopathic arthritis (SJIA) is a disorder characterized by arthritis in children starting before 16 years of age associated with daily high fever, persisting for more than 2 weeks, and at least one of the following clinical features: evanescent cutaneous rash, lymphadenopathy, serositis or hepatosplenomegaly. SJIA patients carry a significantly higher frequency of MEFV mutations, the gene responsible for familial Mediterranean fever, and may be characterized by a more aggressive disease. In this line, we describe a 9-year-old girl affected with SJIA who carried a heterozygous G196W mutation in MEFV. Our patient was characterized by an aggressive disease course, resistance to conventional immunosuppressive agents and developed renal amyloidosis just 2 years after the disease onset.