Adult-onset Still's disease: analysis of a monocentric cohort of patients.

OBJECTIVE: Adult-onset Still's disease (AOSD) is a multigenic autoinflammatory disease with a severe systemic involvement. Because of the rarity of the disease, most published cohorts are multicentric. The aim of this report is to describe a monocentric cohort of AOSD patients, reporting clinical features and response to therapy in a long follow-up. METHOD: Thirty-eight patients, attending the Clinical Immunology Unit and fulfilling Yamaguchi, Fautrel, or Daghor-Abbaci classification criteria for AOSD, were recruited for this study. In all patients, clinical and serological data were collected at diagnosis and every 6 months thereafter. The Pouchot score was calculated at every visit. RESULTS: Fever, arthromyalgia, and skin rash were the most frequent manifestations, followed by lymphadenopathy, sore throat, arthritis, splenomegaly, hepatic involvement, pleuropericarditis, and weight loss. As far as the disease course is concerned, 25% presented a monocyclic and 35% a polycyclic pattern, and 40% developed chronic articular involvement. Severe complications were observed at disease onset in 21% of the patients. All of the patients were treated with steroids; 74% also received conventional synthetic disease-modifying anti-rheumatic drugs (methotrexate in most cases) and 71% biological disease-modifying anti-rheumatic drugs (interleukin-1 inhibitors in most cases). Therapeutic switching for lack/loss of efficacy or adverse drug reactions was necessary in 66%. CONCLUSION: The analysis of this cohort confirms that AOSD is a complex, severe, and heterogeneous disease. However, despite long-term treatment and comorbidities, therapies are effective and well tolerated. The therapeutic armamentarium now available allows long-lasting remission with low immunosuppression to be achieved in most patients.

Increased production of inflammatory cytokines by circulating monocytes in mesial temporal lobe epilepsy: A possible role in drug resistance.

We analyzed peripheral blood mononuclear cells (PBMCs) and serum inflammatory biomarkers in patients with mesial temporal lobe epilepsy (drug-resistant - DR, vs. drug-sensitive - DS). Patients with epilepsy showed higher levels of serum CCL2, CCL3, IL-8 and AOPP, and lower levels of FRAP and thiols compared to healthy controls (HC). Although none of the serum biomarkers distinguished DR from DS patients, when analysing intracellular cytokines after in vitro stimulation, DR patients presented higher percentages of IL-1ß and IL-6 positive monocytes compared to DS patients and HC. Circulating innate immune cells might be implicated in DR epilepsy and constitute potential new targets for treatments.

Evaluation of a novel particle-based assay for detection of autoantibodies in idiopathic inflammatory myopathies.

BACKGROUND: Myositis specific antibodies (MSA) represent not only important diagnostic tools for idiopathic inflammatory myopathies (IIM), but also help to stratify patients into subsets with particular clinical features, treatment responses, and disease outcome. Consequently, standardization of MSA is of high importance. Although many laboratories rely on protein immunoprecipitation (IP) for the detection of MSA, IP standardization is challenging and therefore reliable alternatives are mandatory. Recently, we identified significant variation between IP and line immunoassay (LIA) for the detection of MSA and myositis associated antibodies. In this study we aimed to compare the results from our previous study to the results obtained with a novel fully automated particle-based technology for the detection of MSA and MAA. METHODS: A total of 54 sera from patients with idiopathic inflammatory myopathy (IIM) were tested using three methods: IP, LIA (Euroimmun, Germany) and a novel particle-based multi-analyte technology (PMAT, Inova Diagnostics, US, research use only). The analysis focused on antibodies to EJ, SRP, Jo-1, NXP-2, MDA5, TIF1-γ, and Mi-2. RESULTS: Significant variations were observed among all methods. Overall, the novel PMAT assays showed slightly better correlation with IP, but the kappa agreement was strongly dependent on the antibody tested. When the results obtained from IP were used as reference for receiver operating characteristic (ROC) curve analysis, good discrimination and a high area under the curve (AUC) value were found for PMAT (AUC = 0.83, 95% confidence interval, CI 0.70-0.95) which was significantly higher (p = .0332) than the LIA method (AUC = 0.70, 95% CI 0.56-0.84). CONCLUSION: The novel PMAT used to detect a spectrum of MSA in IIM represents a potential alternative to IP and other diagnostic assays. Additional studies based on larger cohorts are needed to fully assess the performance of the novel PMAT system for the detection of autoantibodies in myositis.

Cytokines and soluble receptors of the interleukin-1 family in Schnitzler syndrome.

OBJECTIVES: Schnitzler syndrome (SchS) is an autoinflammatory disorder characterized by chronic urticaria, fever, and monoclonal gammopathy. The success of interleukin-1 (IL-1) blocking therapies suggests a crucial role for IL-1 in disease induction. The aim of this study is to perform a comprehensive analysis of IL-1 family cytokines and soluble receptors in a group of SchS patients. METHOD: Three patients fulfilling the criteria for the diagnosis of SchS were recruited; 80 blood donors formed the control group. IL-1 family cytokines (IL-1α, IL-1ß, IL-33, IL-18), soluble receptors (sIL-1R1, sIL-1R2, sIL-1R3, sIL-1R4), and antagonists [IL-1Ra, IL-18 binding protein (IL-18BP)] were measured by a multiarray enzyme-linked immunosorbent assay. Free IL-18 was calculated as the amount of IL-18 not inhibited by IL-18BP. Cytokine levels were compared by the Mann-Whitney test. RESULTS: IL-18 and free IL-18 were increased in patients compared with controls (p = 0.005 and p = 0.0082, respectively), while IL-18BP levels were not different. IL-1α, IL-1ß, and IL-33 were undetectable in both patients and controls. The soluble receptors sIL-1R1, sIL-1R2, and ST2/sIL-1R4, and the IL-1 antagonist IL-1Ra were all within normal ranges; sIL-1R3 was significantly lower in patients than in controls (p = 0.039). CONCLUSIONS: The data indicate that SchS is characterized by increased circulating levels of free IL-18, possibly leading to a higher activation of innate/inflammatory effector cells. At variance with other inflammatory diseases, the lack of increase in sIL-1R1 and sIL-1R2 and the decreased levels of sIL-R3 imply a failure in the counterbalancing mechanism aimed at inhibiting excessive IL-1ß in tissues.

Association between anti-citrullinated alpha enolase antibodies and clinical features in a cohort of patients with rheumatoid arthritis: a pilot study.

In recent years several antibodies against citrullinated peptides (ACPAs) have been identified in patients with rheumatoid arthritis (RA) and their pathogenic, diagnostic and prognostic significance is under intense investigation. Among ACPAs, those targeting citrullinated alpha enolase (anti-CEP1) have been identified in RA but data about their ability to predict the development of erosive disease are conflicting. Furthermore, no data are currently available concerning their possible association with extra-articular manifestations (EAMs) in RA. The aim of this study was to investigate the prevalence and significance of anti-CEP1 from a prognostic point of view. In this pilot study we confirmed that anti-CEP1 Abs are associated with higher prevalence of bone erosions, but we also provided the first evidence of an association between anti-CEP1 Abs and RA interstitial lung disease (ILD). These results provide the basis to investigate the association between anti-CEP1 Abs and EAMs in larger cohorts of RA patients to possibly confirm its role as biomarker for RA-ILD.

Label-free detection of immune complexes with myeloid cells.

The aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins.

Fingerprinting of anti-citrullinated protein antibodies (ACPA): specificity, isotypes and subclasses.

Anti-citrullinated protein antibodies (ACPA) are a family of rheumatoid arthritis (RA)-specific autoantibodies that recognize the amino acid citrulline, resulting from the post-translational modification of arginine. Peptidyl arginine deiminase, the enzyme responsible for citrullination, is present in humans in different isoforms with different tissue distribution, enzymatic activity and target specificity; nonetheless, the number of proteins citrullinated in physiological or pathological conditions is wide, but not every citrullinated protein is a target for antibodies. In pre-RA patients the immune response to citrullinated antigens is initially restricted, expands with time and, after the onset of the disease, is relatively stable. ACPA are heterogeneous in terms of not only fine specificity but also isotype and IgG subclasses usage. This heterogeneity may be relevant for the immunopathogenesis of RA, conditioning the interaction of antibodies with complement and Fc receptors.

Two Takayasu arteritis patients successfully treated with rituximab.

Takayasu arteritis (TA) is a rare form of chronic large vessel vasculitis of unknown origin involving the aorta and its major branches. Recently, the involvement of B lymphocytes in TA has been suggested, and active refractory TA patients were successfully treated with B cell depletion therapy (BCDT). We report two cases of patients with TA successfully treated with anti-CD20 monoclonal antibody (rituximab). The favorable outcome of rituximab treatment in our patients also support the view that BCDT can be a useful option for refractory TA, and its potential should be evaluated in controlled trials.

Free IL-18 and IL-33 cytokines in chronic spontaneous urticaria.

Overproduction of IL-18 has been described in chronic urticaria. To evaluate free IL-18 and IL-33 in chronic spontaneous urticaria (CSU). IL-18, its inhibitor IL-18BP, IL-33 and its soluble receptor ST2 (sST2) were measured (ELISA) in the sera of 73 CSU patients. Free IL-18 was calculated (law of mass action). Autologous serum skin test (ASST) was performed in all patients. Total IL-18, IL-18BP and free IL-18 serum levels were significantly higher in CSU than in controls. IL-18 and IL-18BP increased significantly in both ASST-positive and negative subgroups. Free IL-18 resulted significantly higher in the ASST-negative, but not in the ASST-positive subgroup. No differences in IL-33/sST2 levels were detected between CSU and controls. Increased levels of free IL-18 and IL-18BP, but not IL-33, was detected in CSU. Whether IL-18 up-regulation is a consequence of inflammation or one of the causes of the pathology needs to be addressed.

Cell surface expression of activating receptors and co-receptors on peripheral blood NK cells in systemic autoimmune diseases.

OBJECTIVES: A defined role for natural killer (NK) cells and their activating receptors in autoimmunity has not been clearly established. The aim of this study was to evaluate the levels of the CD3-CD56+ NK cells and their expression of receptors and co-receptors in the peripheral blood of patients with systemic autoimmune disorders. METHODS: Thirty-four subjects with systemic sclerosis (SSc), 14 with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), 14 with systemic lupus erythematosus (SLE), and 14 healthy donors were studied. The activating receptors NKp46, NKp44, NKp30, NKG2D, and DNAM-1 and the co-receptors NTB-A and 2B4 were analysed by flow cytometry on peripheral blood NK cells. RESULTS: In SSc, AAV, and SLE we detected a significant decrease in the percentage of CD3-CD56+ NK cells compared to healthy controls. No differences in the expression of NKp46, NKp44, and NKp30 were identified. On the contrary, NKG2D and DNAM-1 expression was decreased in SLE, but not in SSc and AAV, NTB-A was decreased in SLE, and 2B4 in both SLE and SSc. No differences were detected between active and inactive SLE patients. In SSc, only patients affected by pulmonary arterial hypertension (PAH) and interstitial lung disease (ILD) had a low expression of DNAM-1, 2B4, and NKp30. CONCLUSIONS: These data demonstrate that patients with different systemic autoimmune diseases differ in the expression of activating receptors and co-receptors on CD3-CD56+ NK cells. The down-regulation of receptors and co-receptors in SSc with lung involvement suggests their possible role in this manifestation of the disease.

Multicentric evaluation of a second generation assay to detect antiviral citrullinated peptide antibodies: a collaborative study by the Forum Interdisciplinare per la Ricerca nelle Malattie Autoimmuni.

AIMS: A novel immunoenzymatic assay using viral citrullinated peptides derived from Epstein-Barr virus-encoded proteins (viral citrullinated peptide 2 (VCP2)) has been developed and evaluated by means of a multicentre collaborative study. METHODS: Three hundred nine sera from patients with established rheumatoid arthritis (RA), 36 with early arthritis, 12 with juvenile arthritis and 453 controls were tested for VCP2 and cyclic citrullinated peptide (CCP) antibodies. RESULTS: The VCP2 assay showed 78.3% sensitivity and 97.1% specificity. VCP2 and CCP had a high concordance rate in patients with RA (88%) and controls (97%). However, 36 RA sera were positive in the CCP assay but negative on VCP2, and two RA sera reacted only on VCP2. CONCLUSIONS: The new VCP2 assay is endowed with high sensitivity and specificity. VCP2-positive RA sera are mostly but not completely contained in the CCP-positive population. Studies are in progress to establish whether the VCP2 assay can detect clinically distinct subsets of patients with RA.

Antibodies to a new viral citrullinated peptide, VCP2: fine specificity and correlation with anti-cyclic citrullinated peptide (CCP) and anti-VCP1 antibodies.

Anti-citrullinated protein/peptide antibodies (ACPA) are a hallmark of rheumatoid arthritis (RA) and can be measured using different citrullinated substrates. In this paper we describe a new viral citrullinated peptide - VCP2 - derived from the Epstein-Barr virus-encoded protein EBNA-2 and analyse its potential as substrate for ACPA detection. Analysing sera from 100 RA patients and 306 controls, anti-VCP2 immunoglobulin (Ig)G were found in 66% of RA sera, IgM in 46% and IgA in 39%, compared with less than 3% of control sera. Anti-VCP2 IgG was associated with erosive arthritis, the presence of rheumatoid factor and anti-VCP1 and anti-cyclic citrullinated peptide (CCP) antibodies. Anti-VCP2 antibodies were detected in 1% and anti-VCP1 antibodies in 4% of CCP-negative RA sera; conversely, 3% of the VCP-negative sera were CCP-positive. Taken together, these data suggest that VCP2 could offer a valuable tool for ACPA detection. Inhibition assays showed that two non-overlapping epitopes - a citrulline-glycine stretch shared between VCP1 and VCP2 and the N-terminal portion of the VCP2 sequence - were targeted by anti-VCP2 antibodies. Moreover, in some RA sera that tested positive in CCP and VCP2 assays, preincubation with VCP2 inhibited binding to CCP, whereas in other sera the binding was unaffected. Thus, the reactivity with more than one ACPA substrate might be due in some RA patients to antibody populations with different specificities, and in others to cross-reactive antibody populations. Finally, affinity-purified anti-VCP2 antibodies immunoprecipitated deiminated Epstein-Barr virus nuclear antigen (EBNA-2) from an EBNA-2-transfected cell line, suggesting that viral sequences may be involved in the generation of the ACPA response.

Mortality rate and outcome factors in mixed cryoglobulinaemia: the impact of hepatitis C virus.

OBJECTIVES: Mixed cryoglobulinaemia (MC) is a chronic small-vessel vasculitis. Shortly after the discovery of hepatitis C virus (HCV) in 1989, an association between HCV infection and MC was being increasingly reported, suggesting the potential pathogenetic implication of HCV in most of the cases that had been previously diagnosed as essential MC. A number of studies have pointed out prognostic factors linked to mortality in this disorder. None of them, however, have clarified the impact of HCV discovery on the natural history of the disease. The aim of the present study was to evaluate mortality in MC after the discovery of HCV infection. METHODS: We retrospectively collected clinical and serological data in 70 unselected HCV-positive patients being followed up at our unit from 1990. Clinical and prognostic factors linked to poor outcome were evaluated. RESULTS: Chronic hepatitis, renal involvement, and intestinal vasculitis were the most frequent causes of death. CONCLUSION: Compared to other series, the outcome in our MC seemed to be better. Factors linked to a poor outcome were renal involvement, widespread vasculitis, male sex, and cryocrit.

Replication of the tumor necrosis factor receptor-associated factor 1/complement component 5 region as a susceptibility locus for rheumatoid arthritis in a European family-based study.

OBJECTIVE: We recently showed, using a candidate gene approach in a case-control association study, that a 65-kb block encompassing tumor necrosis factor receptor-associated factor 1 (TRAF1) and C5 is strongly associated with rheumatoid arthritis (RA). Compared with case-control association studies, family-based studies have the added advantage of controlling potential differences in population structure and are not likely to be hampered by variation in population allele frequencies, as is seen for many genetic polymorphisms, including the TRAF1/C5 locus. The aim of this study was to confirm this association in populations of European origin by using a family-based approach. METHODS: A total of 1,356 western European white individuals from 452 "trio" families were genotyped for the rs10818488 polymorphism, using the TaqMan allelic discrimination assay. RESULTS: We observed evidence for association, demonstrating departure from Mendel's law, with an overtransmission of the rs10818488 A allele (A = 55%; P = 0.036). By taking into consideration parental phenotypes, we also observed an increased A allele frequency in affected versus unaffected parents (A = 64%; combined P = 0.015). Individuals carrying the A allele had a 1.2-fold increased risk of developing RA (allelic odds ratio 1.24, 95% confidence interval 1.04-1.50). CONCLUSION: Using a family-based study that is robust against population stratification, we provide evidence for the association of the TRAF1/C5 rs10818488 A allele and RA in populations of European descent, further substantiating our previous findings. Future functional studies should yield insight into the biologic relevance of this locus to the pathways involved in RA.

IgG, IgA, IgM antibodies to a viral citrullinated peptide in patients affected by rheumatoid arthritis, chronic arthritides and connective tissue disorders.

OBJECTIVES: Anti-citrullinated protein/peptide antibodies (ACPA), a family of antibodies with overlapping specificities, represent a specific marker of rheumatoid arthritis (RA). The aim of the present study is to investigate the prevalence and clinical significance of IgG, IgA and IgM ACPA by a newly described assay employing a viral citrullinated peptide (VCP). METHODS: IgG, IgA and IgM anti-VCP antibodies have been measured in sera from 146 patients affected by RA and 404 controls, including 204 chronic arthritides, 111 connective tissue disorders and 89 healthy subjects. The affinity of the different isotypes for VCP was analysed by liquid phase inhibition assays. RESULTS: Among RA patients, 40 were single positive for IgG anti-VCP, five for IgA and 11 for IgM. Ten patients were double positive for IgG and IgA, four for IgG and IgM, six for IgA and IgM. In 15 RA patients IgG, IgA and IgM anti-VCP antibodies were detected. No correlation could be found between the isotype and the clinical manifestations or duration of the disease. IgA anti-VCP were strongly associated with RA, whereas IgM anti-VCP were detected also in a low percentage of systemic lupus erythematosus, psoriatic arthritis and mixed cryoglobulinaemia (MC) patients. IgG anti-VCP displayed a higher affinity for the antigen than IgA or IgM. CONCLUSIONS: These data show that anti-VCP of IgG and IgA isotype discriminate RA from other chronic arthritides and disease controls and suggest an independent production of each isotype.

Antibodies directed against ribosomal P proteins cross-react with phospholipids.

Anti-ribosomal P protein (anti-P) antibodies are marker antibodies in systemic lupus erythematosus (SLE). Their association with psychiatric or neurological manifestations has been proposed, but remains controversial. Anti-phospholipid antibodies are the hallmark of a syndrome that may comprise a number of neurological manifestations. Thus, anti-P and anti-phospholipid antibodies have both been associated with central nervous system involvement and their co-existence in the same sera was reported. We verified the ability of purified anti-P antibodies to bind different phospholipids and phospholipid-binding proteins in solid-phase assays. Anti-P antibodies from five of eight patients bound cardiolipin (CL) when saturated with fetal calf serum (FCS); in three cases anti-CL antibodies were also detected in the flow-through. No anti-P eluate, nor any corresponding flow-through, bound beta(2)-glycoprotein I alone or prothrombin. Moreover, no anti-P eluate bound CL when the plates were blocked with bovine serum albumin in the absence of FCS. Anti-P antibodies with anti-CL activity bound both ssDNA and dsDNA and also nucleosomes in three patients. Our data indicate a great heterogeneity of anti-P antibodies that appear to be overlapped partially with the other autoantibody populations detected frequently in SLE.

Anti-C1q antibodies in pregnant patients with systemic lupus erythematosus.

OBJECTIVE: To study anti-C1q antibodies in pregnant patients with systemic lupus erythematosus (SLE) and to evaluate their prognostic significance for the occurrence of disease flares or pregnancy complications. METHODS: Twenty-one pregnancies in 19 SLE patients prospectively followed were analyzed. Disease activity was evaluated on the basis of the physician's intention to treat and a modified version of the ECLAM index. Anti-C1q and anti-dsDNA antibodies were detected in the sera by an ELISA assay. Antinuclear antibodies, anti-ENA antibodies, anticardiolipin antibodies and lupus anticoagulant were also performed. RESULTS: In all the patients the disease was inactive at the beginning of the pregnancy. Four flares of disease activity were observed in 4 pregnancies (19%) and obstetric complications were encountered in 7 pregnancies (43%). Anti-C1q antibodies were positive in 4 (19%) pregnancies and anti-dsDNA antibodies in 8 (38%). The presence of anti-phospholipid antibodies at the first assessment was correlated with the occurrence of obstetric complications (p<0.05). The presence of anti-C1q and anti-dsDNA antibodies at the first assessment had no prognostic significance for the occurrence of flares or obstetric complications during the course of pregnancy. Although the small number of patients studied did not allow for statistically significant analysis, flares appeared to be more likely to occur in patients presenting with anti-dsDNA or anti-C1q antibodies during pregnancy compared to patients with no changes in these antibody titers (43% vs 8% respectively). CONCLUSIONS: The presence of anti-C1q and anti-dsDNA antibodies does not seem to be prognostic for the occurrence of flares during pregnancy. Further studies are warranted to explore this possibility.