Anti-phosphatidylserine/prothrombin antibodies and thrombosis associate positively with HLA-DRB1*13 and negatively with HLA-DRB1*03 in SLE.

OBJECTIVES: Emerging evidence demonstrates that aPS-PT associate with thrombotic events. Genetic predisposition, including HLA-DRB1 alleles, is known to contribute to the occurrence of conventional aPL [anti-ß2glycoprotein-I (anti-ß2GPI) and aCL]. We investigated associations between aPS-PT and HLA-DRB1* alleles and thrombosis in SLE. Conventional aPL were included for comparison. METHODS: We included 341 consecutive SLE patients, with information on general cardiovascular risk factors, including blood lipids, LA and thrombotic events. aPS/PT, anti-ß2GPI and aCL of IgA/G/M isotypes and LA were quantified. RESULTS: aPS/PT antibodies associated positively with HLA-DRB1*13 [odds ratio (OR) 2.7, P = 0.002], whereas anti-ß2GPI and aCL antibodies associated primarily with HLA-DRB1*04 (OR 2.5, P = 0.0005). These associations remained after adjustment for age, gender and other HLA-DRB1* alleles. HLA-DRB1*13, but not DRB1*04, remained as an independent risk factor for thrombosis and APS after adjustment for aPL and cardiovascular risk factors. The association between DRB1*13 and thrombosis was mediated by aPS-PT positivity. HLA-DRB1*03, on the other hand, associated negatively with thrombotic events as well as all aPL using both uni- and multivariate analyses. HLA-DRB1*03 had a thrombo-protective effect in aPL-positive patients. Additionally, HLA-DRB1*03 was associated with a favourable lipid profile regarding high-density lipoprotein and triglycerides. CONCLUSIONS: HLA-DRB1*13 confers risk for both aPS-PT and thrombotic events in lupus. The association between HLA-DRB1*13 and thrombosis is largely, but not totally, mediated through aPS-PT. HLA-DRB1*03 was negatively associated with aPL and positively with favourable lipid levels. Thus, HLA-DRB1*03 seems to identify a subgroup of SLE patients with reduced vascular risk.

Anti-protein arginine deiminase antibodies are distinctly associated with joint and lung involvement in rheumatoid arthritis.

OBJECTIVES: RA is a chronic inflammatory disease in which possible interstitial lung disease (ILD) is an extra-articular manifestation that carries significant morbidity and mortality. RF and ACPA are included in the RA classification criteria but prognostic and diagnostic biomarkers for disease endotyping and RA-ILD are lacking. Anti-protein arginine deiminase antibodies (anti-PAD) are a novel class of autoantibodies identified in RA. This study aimed to assess clinical features, ACPA and anti-PAD antibodies in RA patients with articular involvement and ILD. METHODS: We retrospectively collected joint erosions, space narrowing, clinical features and lung involvement of a cohort of 71 patients fulfilling the 2010 ACR/EULAR RA classification criteria. Serum samples from these patients were tested for ACPA IgG (QUANTA Flash CCP3), and anti-PAD3 and anti-PAD4 IgG, measured with novel assays based on a particle-based multi-analyte technology (PMAT). RESULTS: Anti-PAD4 antibodies were significantly associated with radiographic injury (P = 0.027) and erosions (P = 0.02). Similarly, ACPA levels were associated with erosive disease (P = 0.014). Anti-PAD3/4 double-positive patients displayed more joint erosions than patients with anti-PAD4 antibodies only or negative for both (P = 0.014 and P = 0.037, respectively). RA-ILD (15.5%, 11/71 patients) was associated with older age (P < 0.001), shorter disease duration (P = 0.045) and less erosive disease (P = 0.0063). ACPA were elevated in RA-ILD, while anti-PAD4 were negatively associated (P = 0.043). CONCLUSION: Anti-PAD4 and anti-PAD3 antibodies identify RA patients with higher radiographic injury and bone erosions. In our cohort, ILD is associated with lower radiographic and erosive damage, as well as low levels of anti-PAD4 antibodies.

Clinical utility of circulating calprotectin to assist prediction and monitoring of COVID-19 severity: An Italian study.

BACKGROUND: Calprotectin (S100A8/A9) has been identified as a biomarker that can aid in predicting the severity of disease in COVID-19 patients. This study aims to evaluate the correlation between levels of circulating calprotectin (cCP) and the severity of COVID-19. METHODS: Sera from 245 COVID-19 patients and 110 apparently healthy individuals were tested for calprotectin levels using a chemiluminescent immunoassay (Inova Diagnostics). Intensive care unit (ICU) admission and type of respiratory support administered were used as indicators of disease severity, and their correlation with calprotectin levels was assessed. RESULTS: Samples from patients in the ICU had a median calprotectin concentration of 11.6 µg/ml as compared to 3.5 µg/ml from COVID-19 patients who were not in the ICU. The median calprotectin concentration in a cohort of healthy individuals collected before the COVID-19 pandemic was 3.0 µg/ml (95% CI: 2.820-2.969 µg/ml). Patients requiring a Venturi mask, continuous positive airway pressure, or orotracheal intubation all had significantly higher values of calprotectin than controls, with the increase of cCP levels proportional to the increasing need of respiratory support. CONCLUSION: Calprotectin levels in serum correlate well with disease severity and represent a promising serological biomarker for the risk assessment of COVID-19 patients.

Ultrasound subclinical synovitis in anti-CCP-positive at-risk individuals with musculoskeletal symptoms: an important and predictable stage in the rheumatoid arthritis continuum.

OBJECTIVES: To investigate whether anti-CCP2-positive at-risk individuals with musculoskeletal (MSK) symptoms but without clinical synovitis (CCP2+ at-risk) develop US subclinical synovitis before inflammatory arthritis and if US subclinical synovitis can be predicted. METHODS: First, US scans of CCP2+ at-risk individuals who developed inflammatory arthritis ('progressors') were reviewed for subclinical synovitis prior to inflammatory arthritis development. Patients in whom the pre-progression US scan was negative but the scan was conducted >6 months before progression were excluded. Subsequently, regression analyses were performed to identify predictors of US synovitis in CCP2+ at-risk individuals without baseline US abnormalities who had one or more longitudinal US scan and a complete dataset. RESULTS: US subclinical synovitis was detected in one or more scan in 75 of 97 progressors (77.3%) {median time to inflammatory arthritis development from first evidence of US synovitis 26.5 weeks [interquartile range (IQR) 7-60]}, in whom one or more scan was available, excluding those with a negative scan >6 months from inflammatory arthritis development (n = 38). In 220 CCP2+ at-risk individuals with normal baseline US scans, who had one or more longitudinal US scan and a complete dataset, US synovitis was detected in 69/220 (31.4%) [median time to first developing US synovitis 56.4 weeks (IQR 33.0-112.0)]. In the multivariable analysis, only anti-CCP3 antibodies were predictive for the development of US synovitis [odds ratio 4.75 (95% CI 1.97, 11.46); P < 0.01]. CONCLUSIONS: In anti-CCP2+ at-risk individuals, a stage of subclinical synovitis usually precedes the development of inflammatory arthritis. Anti-CCP2+/CCP3+ individuals without clinical or US subclinical synovitis may represent the optimal window of opportunity for intervention to prevent joint disease.

Predictive value of anti-CarP and anti-PAD3 antibodies alone or in combination with RF and ACPA for the severity of rheumatoid arthritis.

OBJECTIVES: The objective of this study was to analyse the predictive value of anti-carbamylated protein (anti-CarP) and anti-peptidyl-arginine deiminase type-3 (anti-PAD3) antibodies, alone or in combination with RF and ACPA, to identify patients at high risk of developing severe RA outcomes. METHODS: Patients within the Swiss Clinical Quality Management registry with a biobank sample were tested for RF, ACPA, anti-CarP, and anti-PAD3 antibodies. We examined the association of each autoantibody with DAS28, HAQ and radiographic damage (Ratingen) at baseline and longitudinally. RESULTS: Analyses included 851 established RA patients and 516 disease controls [axial spondyloarthritis (axSpA = 320) and PsA (196)]. Anti-CarP and anti-PAD3 antibodies were, respectively, present in 22.4% and 10.7% of the whole RA population, and in 13.2% and 3.8% of the RF and ACPA double seronegative patients. At baseline, RA patients with anti-PAD3 had higher DAS28 (4.2 vs 3.7; P= 0.005) and significantly more radiographic damage (14.9 vs 8.8; P= 0.02) than anti-PAD3-negative patients. In the ACPA-negative subgroup, baseline Ratingen scores were significantly higher in anti-PAD3-positive patients (P= 0.01). The combination of anti-PAD3, RF IgM, and ACPA was associated with significantly higher baseline radiographic scores than the double seropositive group (P= 0.04). The presence of any two of the previous autoantibodies was associated with significantly greater radiographic progression over 10 years than if all were absent (P= 0.02). There were no differences in RA outcome measures with regards to anti-CarP. CONCLUSIONS: Anti-PAD3 antibodies are associated with higher disease activity and joint damage scores in RA patients.

Anti-RNPC-3 antibody predicts poor prognosis in patients with interstitial lung disease associated to systemic sclerosis.

OBJECTIVE: To analyse the prevalence, the clinical characteristics, the overall survival and the event-free survival (EFS) of SSc patients who express anti-U11/U12 RNP (RNPC-3) antibodies. METHODS: A total of 447 SSc patients from Barcelona (n = 286) and Milan (n = 161) were selected. All samples were tested using a particle-based multi-analyte technology. We compared anti-RNPC-3 positive and negative patients. Epidemiological, clinical features and survival were analysed. End-stage lung disease (ESLD) was defined if the patient developed forced vital capacity <50% of predicted, needed oxygen therapy or lung transplantation. EFS was defined as the period of time free of either ESLD or death. RESULTS: Nineteen of 447 (4.3%) patients had anti-RNPC-3 antibodies and interstitial lung disease (ILD) was more frequent (11, 57.9% vs 144, 33.6%, P =0.030) in individuals with anti-RNPC-3 antibodies. More patients reached ESLD in the positive group (7, 36.8% vs 74, 17.3%, P = 0.006), and a higher use of non-glucocorticoid immunosuppressive drugs was observed (11, 57.9% vs 130, 30.4%, P = 0.012). Anti-RNPC-3 positive patients had lower EFS, both in the total cohort (log-rank P =0.001), as well as in patients with ILD (log-rank P = 0.002). In multivariate Cox regression analysis, diffuse cutaneous subtype, age at onset, the presence of ILD or pulmonary arterial hypertension and the expression of anti-RNPC-3 positivity or anti-topo I were independently associated with worse EFS. CONCLUSION: The presence of anti-RNPC-3 was associated with higher frequency of ILD and either ESLD or death. These data suggest anti-RNPC-3 is an independent poor prognosis antibody in SSc, especially if ILD is also present.

Infusion of anti-DFS70 antibodies prolonged survival of lupus-prone mice.

BACKGROUND: Systemic-lupus-nephritis is a chronic autoimmune disease characterized by immune complex deposition and a flare of autoantibodies and leading to renal injury. OBJECTIVES: To expose anti-Dense-Fine-Speckled-70 (DFS70)-antibodies to genetically-prone-lupus-mice. METHODS: NZBXW/F1 female mice were monitored for the onset of glomerulonephritis by proteinuria upon infusion of anti-DFS70 (40 µg/mouse), commercial-human-IgG (cIgG) or phosphate-buffered-saline (PBS) as controls. The survival time was detected by mice death. Circulating anti-dsDNA were tested by ELISA. Proteinuria, was defined by a standard semi-quantitative-Bayer-Multistix-dipstick. Kidney histology was analyzed by periodic-acid-Schiff-PAS staining. RESULTS: A significantly higher percentage of anti-DFS70-infused mice exhibited prolonged survival time as compared with cIgG and PBS-subjected mice (p < 0.022). One mouse out of 10 mice injected with anti-DFS70-antibodies died at week 36, whereas, 6 out of 10 mice subjected with PBS found dead at this time. Eighty percent of anti-DFS70 injected mice did not show severe glomerulonephritis by histology. CONCLUSIONS: anti-DFS70 attenuated the progression of glomerulonephritis and prolonged the survival time. Circulating anti-DFS70-autoantibodies may confer a protective role against renal injury in murine-lupus-nephritis. Our data may propose a novel therapy approach for lupus patients.

Serum calprotectin (S100A8/9), clinical and ultrasound assessment in patients with juvenile idiopathic arthritis.

OBJECTIVES: To explore the association between serum S100A8/9 (calprotectin), clinical and ultrasound (US) assessment in juvenile idiopathic arthritis (JIA) patients. METHODS: A total of 30 well-characterised consecutive patients (18 female) with non-systemic JIA and 20 age-matched healthy controls were included. Serum and plasma samples obtained the same day of the clinical and sonographical assessment were tested for calprotectin levels by ELISA. Clinical status was defined using Wallace criteria. Ultrasonographic B-mode and power Doppler (PD) assessment of 44 joints for each subject was performed. RESULTS: Clinically active disease was present in 14 patients, while 16 patients were active according to US evaluation. We found no differences in the serum/plasma calprotectin levels in clinically active disease group [29.6 (5.4-198.1) ng/ml; 12.6 (2.8-65.8) ng/ml] as compared with inactive disease group [24.8 (14.1-204.3); 12.7 (3.4-65.1)] (p=0.73; p=0.29). There was also no difference between US active disease [29.8 (5.4-204.3); 12.9 (2.8-65.8)] and US inactive disease [24.8 (12.1-197.1); 11.7 (3.4-44.2)] with regard to the serum/plasma calprotectin levels (p=0.83; p=1.0). Serum/plasma calprotectin levels correlated moderately with C-reactive protein (CRP) (Spearman r=0.44, p=0.01; Spearman r=0.56, p=0.0021). CONCLUSIONS: To our knowledge, this is the first study to simultaneously examine the correlation between serum/plasma calprotectin levels, clinical and US assessment in JIA. Calprotectin was not associated with the disease status in JIA patients with low number of active joints and its levels were moderately correlated with CRP. Our preliminary study needs to be extended with a larger number of patients.

Novel Anti-Hexokinase 1 Antibodies Are Associated With Poor Prognosis in Patients With Primary Biliary Cholangitis.

INTRODUCTION: Antibodies to hexokinase 1 (HK1) and kelch-like 12 (KLHL12) have been identified as potential biomarkers in primary biliary cholangitis (PBC), and this study assesses changes of these antibodies over time and if they are associated with clinical outcomes. METHODS: Two hundred fifty-four PBC patients (93.3% female, 51 ± 12.3 years old) were tested for anti-HK1 and anti-KLHL12, antimitochondrial (AMA), anti-gp210, and anti-sp100 antibodies. One hundred sixty-nine patients were tested twice and 49 three times within 4.2 (0.8-10.0) years. Biochemistry and clinical features at diagnosis, response to therapy, events of decompensation, and liver-related death or transplantation were evaluated. RESULTS: Anti-HK1 and anti-KLHL2 were detected in 46.1% and 22.8% patients, respectively. AMA were positive in 93.7%, anti-sp100 in 26.4%, and anti-gp210 in 21.3% of patients. Anti-HK1 and anti-KLHL12 positivity changed over time in 13.3% and 5.5% of patients, respectively. Anti-HK1 or anti-KLHL12 were present in 37.5% of AMA-negative patients, and in 40% of AMA, anti-gp210, and anti-sp100 negative. No significant differences were observed between those with or without HK1 and KLHL12 antibodies, but transplant-free survival and time to liver decompensation were significantly lower in patients anti-HK1 positive (P = 0.039; P = 0.04) and in those anti-sp100 positive (P = 0.01; P = 0.007). No changes in survival and events of liver decompensation were observed according to the positivity of AMA, anti-KLHL12, or anti-gp210 antibodies. DISCUSSION: HK1 and KLHL12 antibodies are present in 40% of PBC patients who are seronegative by the conventional PBC-specific antibodies. The novel antibodies remain rather steady during the course of the disease, and HK1 antibodies are associated with unfavourable outcomes.

Two Novel Technologies for the Detection of Anti-cardiolipin and Anti ß2-Glycoprotein Antibodies in the Real Life: Chemiluminescent in Comparison to the Addressable Laser Bead Immunoassays.

In the present study, we evaluated two novel technologies, the chemiluminescent immunoassay (CIA) QUANTA Flash on BIO-FLASH (Inova Diagnostics, San Diego, CA, USA) and the addressable laser bead immunoassay (ALBIA) on BioPlex™ 2200 (Bio-Rad, Hercules, CA, USA) for the detection of anti-cardiolipin IgG/IgM (aCL) and anti-ß2-glycoprotein IgG/IgM (aß2GPI) antibodies. The study was performed on 134 samples from consecutive patients (59 males and 75 females, mean age 54 ± 10 years) who consulted a rheumatologist because thrombosis and/or pregnancy complications were present or another immunological disease (Sjogren's syndrome, inflammatory arthritis). Fourteen patients of the total fulfilled 25the Sydney criteria for APS and for these patients previous results of aPLs were available. Sera were tested for aCL and aß2GPI of IgG and IgM isotypes using CIA (BIO-FLASH) and ALBIA (BioPlex™ 2200). Overall agreement between CIA and ALBIA ranged from 88.1% (aCL IgG) to 97.8% (aß2GPI IgG). Cohen's kappa coefficient ranged from 0.53 to 0.91, implying moderate to almost perfect agreement. Almost perfect agreement was found between BioPlex™ 2200 and BIO-FLASH aß2GPI IgG and aCL IgM with Cohen's kappa of 0.91 and 0.88, respectively. On the other hand, moderate agreement was found between BioPlex™ 2200 and BIO-FLASH aCL IgG and ß2GPI IgM assays with Cohen's kappa of 0.57 and 0.53, respectively. The two novel technologies look promising and comparable but further studies with larger cohorts are needed to contribute to the better understanding of the new aPLs antibodies assays performance.

Evaluation of a novel extended automated particle-based multi-analyte assay for the detection of autoantibodies in the diagnosis of primary biliary cholangitis.

Background: Anti-mitochondrial autoantibodies (AMA) detected by indirect immunofluorescence (IIF) on rodent tissues are the diagnostic marker of primary biliary cholangitis (PBC). However, up to 15% of patients with PBC are AMA-negative by IIF. In the effort to close the serological gap and improve the diagnostic sensitivity of PBC testing, recently, novel autoantibodies specific for PBC, such as kelch-like 12 (KLHL12, KLp epitope) and hexokinase 1 (HK1) have been described. In this study, we evaluated the autoantibody profile in a large cohort of PBC patients and in patients with other liver disease, including anti-HK1 and anti-KLp autoantibodies. Methods: Sera of 194 PBC patients (126 AMA-IIF-positive and 68 AMA-IIF-negative) and 138 disease controls were tested for a panel of PBC-specific antibodies (MIT3, sp100, gp210, HK1, KLp) using a new automated particle-based multi-analyte technology (PMAT) assay on the Aptiva instrument (Inova). Results: Selecting a cutoff yielding a specificity of >95% for all the markers, the sensitivity for anti-MIT3, anti-sp100, anti-gp210, anti-HK1 and anti-KLp in the PBC AMA-IIF-negative cohort was 20.6%, 16.2%, 23.5%, 22.0%, 17.6 and 13.2%, respectively. Six out of the 68 (8.8%) AMA-IIF negative sera were positive for anti-HK1 or anti-KLp alone. Using these new markers in addition to anti-MIT3, anti-sp100 and anti-gp210, the overall sensitivity in this cohort of AMA-IIF-negative patients increased from 53% to 61.8%, reducing the serological gap in AMA-negative PBC patients. Conclusions: PBC antibody profiling, made possible by the new Aptiva-PMAT technology, allows recognition of a higher number of AMA-negative PBC patients than conventional immunoassays and may represent a useful tool to evaluate the prognostic significance of autoantibody association in PBC patients.

In vivo pathogenicity of IgG from patients with anti-SRP or anti-HMGCR autoantibodies in immune-mediated necrotising myopathy.

OBJECTIVES: In autoimmunity, autoantibodies (aAb) may be simple biomarkers of disease or true pathogenic effectors. A form of idiopathic inflammatory myopathy associated with anti-signal recognition particle (SRP) or anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) aAb has been individualised and is referred to as immune-mediated necrotising myopathy (IMNM). The level of aAb correlates with IMNM activity and disease may respond to immunosuppression, suggesting that they are pathogenic. We aimed to evaluate the pathogenicity of IgG from patients with anti-SRP or anti-HMGCR aAb in vivo by developing the first mouse model of IMNM. METHODS: IgG from patients suffering from anti-SRP or anti-HMGCR associated IMNM were passively transferred to wild-type, Rag2-/- or complement C3-/- mice. Muscle deficiency was evaluated by muscle strength on electrostimulation and grip test. Histological analyses were performed after haematoxylin/eosin staining or by immunofluorescence or immunohistochemistry analysis. Antibody levels were quantified by addressable laser bead assay (ALBIA). RESULTS: Passive transfer of IgG from patients suffering from IMNM to C57BL/6 or Rag2-/- mice provoked muscle deficiency. Pathogenicity of aAb was reduced in C3-/- mice while increased by supplementation with human complement. Breakage of tolerance by active immunisation with SRP or HMGCR provoked disease. CONCLUSION: This study demonstrates that patient-derived anti-SRP+ and anti-HMGCR+ IgG are pathogenic towards muscle in vivo through a complement-mediated mechanism, definitively establishing the autoimmune character of IMNM. These data support the use of plasma exchanges and argue for evaluating complement-targeting therapies in IMNM.

Autoantibodies to a novel Rpp38 (Th/To) derived B-cell epitope are specific for systemic sclerosis and associate with a distinct clinical phenotype.

OBJECTIVE: Detection of antinuclear antibodies and specific autoantibodies is important in the diagnosis and classification of SSc. Several proteins of the Th/To complex, including Rpp25, Rpp38 and hPop1 are the target of autoantibodies in SSc patients. However, very little is known about the epitope distribution of this autoantigen. Consequently, we screened Rpp25, Rpp38 and hPop1 for B cell epitopes and evaluated their clinical relevance. METHODS: Serum pools with (n = 2) and without (n = 1) anti-Th/To autoantibodies were generated and used for epitope discovery. Identified biomarker candidate sequences were then utilized to synthesize synthetic, biotinylated, soluble peptides. The peptides were tested to determine reactivity with sera from SSc cohorts (n = 202) and controls (n = 159) using a chemiluminescence immunoassay. Additionally, samples were also tested for antibodies to full-length recombinant Rpp25 antibodies by chemiluminescence immunoassay. RESULTS: Several immunodominant regions were found on the three proteins. The strongest reactivity was observed with an Rpp38 peptide (aa 229-243). Autoantibodies to the Rpp38 peptide were detected in 8/149 (5.4%) limited cutaneous SSc patients, but not in any of 159 controls (P = 0.003 by two-sided Fisher's exact probability test). Although reactivity to the novel antigenic peptide was correlated with the binding to Rpp25 (rho = 0.44; P < 0.0001), subsets of patient sera either reacted strongly with Rpp25 or with the novel Rpp38-derived peptide. CONCLUSION: A novel Rpp38 epitope holds promise to increase the sensitivity in the detection of anti-Th/To autoantibodies, thus enhancing the serological diagnosis of SSc.

Autoantibodies to Mi-2 alpha and Mi-2 beta in patients with idiopathic inflammatory myopathy.

OBJECTIVES: The objective of this study was to compare the results obtained from different assays for the detection of anti-Mi-2 antibodies, which are important markers in the diagnosis of DM. METHODS: The study included 82 patients (68 females/14 males), most of whom had DM (n = 57), followed by PM (n = 16) and juvenile DM (n = 9). All samples were tested using a novel particle-based multi-analyte technology (PMAT) (Inova Diagnostics, research use only) in parallel with a line immunoassay (LIA: Euroimmun). To assess clinical specificity for the PMAT assay, a total of 775 disease and healthy controls were tested. RESULTS: 29 samples were positive by at least one test for anti-Mi-2 antibodies. Of those, 24 were Mi-2ß LIA+, five were Mi-2α LIA+ and 23 Mi-2 PMAT+. The comparison shows varying agreement between the different methods (kappa 0.27-0.77). When LIA results were used as reference for receiver operating characteristics analysis, high area under the curve values were found for both PMAT vs LIA Mi-2α and LIA Mi-2ß. When analysing the results in the context of the myositis phenotype, PMAT associated closest with the DM phenotype. In the control group, 3/775 controls (all low levels) were anti-Mi-2+ resulting in a sensitivity and specificity of 28.1% and 99.6%, respectively. CONCLUSION: Overall, good agreement was found between LIA and PMAT for anti-Mi-2 antibodies, which is important for the standardization of autoantibodies. Anti-Mi-2ß antibodies measured by PMAT tend be more highly associated with the clinical phenotype of DM.

Interleukin-17/Interleukin-21 and Interferon-γ producing T cells specific for ß2 Glycoprotein I in atherosclerosis inflammation of systemic lupus erythematosus patients with antiphospholipid syndrome.

Systemic lupus erythematosus is frequently associated with antiphospholipid syndrome. Patients with lupus-antiphospholipid syndrome are characterized by recurrent arterial/venous thrombosis, miscarriages, and persistent presence of autoantibodies against phospholipid-binding proteins, such as ß2-Glycoprotein I. We investigated the cytokine production induced by ß2-Glycoprotein I in activated T cells that infiltrate in vivo atherosclerotic lesions of lupus-antiphospholipid syndrome patients. We examined the helper function of ß2-Glycoprotein I-specific T cells for tissue factor production, as well as their cytolytic potential and their helper function for antibody production. Lupus-antiphospholipid syndrome patients harbor in vivo activated CD4+ T cells that recognize ß2-Glycoprotein I in atherosclerotic lesions. ß2-Glycoprotein I induces T-cell proliferation and expression of both Interleukin-17/Interleukin-21 and Interferon-γ in plaque-derived T-cell clones. ß2-Glycoprotein I-specific T cells display strong help for monocyte tissue factor production, and promote antibody production in autologous B cells. Moreover, plaque-derived ß2-Glycoprotein I-specific CD4+ T lymphocytes express both perforin-mediated and Fas/FasLigand-mediated-cytotoxicity. Altogether, our results indicate that ß2-Glycoprotein I is able to elicit a local Interleukin-17/Interleukin-21 and Interferon-γ inflammation in lupus-antiphospholipid syndrome patients that might lead, if unabated, to plaque instability and subsequent arterial thrombosis, suggesting that the T helper 17/T helper 1 pathway may represent a novel target for the prevention and treatment of the disease.

Only monospecific anti-DFS70 antibodies aid in the exclusion of antinuclear antibody associated rheumatic diseases: an Italian experience.

Background The dense fine speckled (DFS) is one of the most common patterns that can be observed as a result of the anti-nuclear antibodies (ANA) test on HEp-2 cells and is mostly caused by antibodies to DFS70 as the main antigenic target. As was recently demonstrated, isolated anti-DFS70 positivity can be used as an aid in the exclusion of ANA associated rheumatic diseases (AARD) due to the opportunity to better interpret unexplained positive IIF ANA results. Methods Our study included 333 subjects with AARD, 51 undifferentiated connective tissue disease (UCTD) patients, 235 disease controls and 149 healthy blood donors from an Italian cohort. All samples were tested for anti-DFS70 and anti-ENA antibodies using QUANTA Flash assays (Inova Diagnostics, San Diego, CA, USA). Results No differences in the prevalence of anti-DFS70 antibodies were seen among AARD, non-AARD and UCTD (2.1% [7/333] vs. 2.3% [9/384] vs. 5.9% [3/51], respectively; p-value = 0.188). AARD patients positive for anti-DFS70 antibodies showed in all cases an accompanying anti-ENA specificity. In contrast, monospecific anti-DFS70 antibodies showed a significantly different distribution with a clear trend across the main groups (AARD vs. non-AARD vs. UCTD: 0% [0/7] vs. 22% [2/9] vs. 100% [3/3], p = 0.007). Anti-DFS70 antibody levels among AARD, non-AARD and UCTD patients were not significantly different (p = 0.094). Within the anti-DFS70 antibody positive cases, AARD cohort showed a higher variability (median [min-max]: 3.2 [3.2-450.8] CU) compared to non-AARD (median [min-max]: 3.2 [3.2-75.7] CU) and UCTD patients (median [min-max]: 3.2 [3.2-59.0] CU). Conclusions Our preliminary data showed a similar frequency of anti-DFS70 antibodies in AARD, UCTD and non-AARD cohorts. Monospecificity of anti-DFS70 antibodies but not their mere presence is the key element in the diagnostic algorithm. Mono-specific anti-DFS70 antibodies might be a helpful biomarker to discriminate individuals with AARD from non-AARD presenting with a positive ANA.

Increased prevalence of anti-DFS70 antibodies in young females: experience from a large international multi-center study on blood donors.

Background Isolated antibodies to DFS70 have been described in healthy individuals and are rarely found in patients with antinuclear antibody-associated autoimmune rheumatic diseases (AARD). However, no data is available on geographic differences in the prevalence of anti-DFS70 antibodies. We aimed to study the prevalence of anti-DFS70 antibodies in blood donor samples from several countries representing various ethnical backgrounds and geographic regions in the world. Methods Sera from apparently healthy blood donors (n≥300 per site) were collected in seven countries (USA, Italy, Spain, Germany, UK, Belgium and Brazil). All samples (n=2628) were tested for anti-DFS70 antibodies by QUANTA Flash DFS70 (Inova Diagnostics, Inc., San Diego, CA, USA). Results The prevalence of anti-DFS70 antibodies varied from 4/321 (1.2%, Italy) to 42/497 (8.5%, USA). Consequently, the prevalence of the antibodies was significantly higher in USA compared to all other countries (p<0.05). In addition, the prevalence in the combined cohort (all sites) was higher in young blood donors (<35 years; 5.0% vs. 2.7%; p=0.0017) and among females (4.5% vs. 3.0%; p=0.0446). However, when cohorts from different countries were corrected for age and gender, no significant difference between the countries were found. Conclusions This is the first study to analyze the prevalence of anti-DFS70 antibodies in different geographic areas using a standardized assay. Our findings show that the antibodies are most prevalent in young females.

ß2 Glycoprotein I Recognition Drives Th1 Inflammation in Atherosclerotic Plaques of Patients with Primary Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is characterized by recurrent arterial/venous thrombosis and miscarriages in the persistent presence of autoantibodies against phospholipid-binding proteins (aPLs), such as ß2 glycoprotein I (ß2GPI). In addition to the aPL thrombophilic effect, arterial thrombosis was related to accelerated atherosclerosis in animal models; however, contrasting findings were reported in primary APS patients with regard to the increased number of plaques or abnormal arterial wall thickness. We investigated the cytokine production induced by ß2GPI in activated T cells that infiltrate in vivo atherosclerotic lesions of primary APS patients with atherothrombosis. We also examined the helper function of ß2GPI-specific T cells for monocyte matrix metalloproteinase-9 and tissue factor production, as well as their cytolytic potential and their helper function for Ab production. APS patients with atherothrombosis harbor in vivo-activated CD4+ T cells that recognize ß2GPI in atherothrombotic lesions. ß2GPI induces T cell proliferation and IFN-γ expression in plaque-derived T cell clones. ß2GPI-specific T cells display helper function for monocyte matrix metalloproteinase-9 and tissue factor production and promote Ig production in autologous B cells. Moreover, plaque-derived ß2GPI-specific CD4+ T lymphocytes express perforin-mediated and Fas/Fas ligand-mediated cytotoxicity. ß2GPI, and especially the DI domain, drive a local Th1 inflammatory response, with subsequent plaque instability that eventually favors atherothrombosis. This finding may explain the association between aPLs and arterial thrombosis, despite the lack of evidence of surrogate markers for atherosclerosis in primary APS.

Beyond thrombosis: Anti-ß2GPI domain 1 antibodies identify late pregnancy morbidity in anti-phospholipid syndrome.

Antibodies against ß2 glycoprotein I (anti-ß2GPI) have been identified as the main pathogenic autoantibody subset in anti-phospholipid syndrome (APS); the most relevant epitope is a cryptic and conformation-dependent structure on ß2GPI domain (D) 1. Anti-ß2GPI domain profiling has been investigated in thrombotic APS, leading to the identification of antibodies targeting D1 as the main subpopulation. In contrast, scarce attention has been paid to obstetric APS, hence this study aimed at characterizing the domain reactivity with regards to pregnancy morbidity (PM). To this end, 135 women with persistently positive, medium/high titre anti-ß2GPI IgG, without any associated systemic autoimmune diseases and at least one previous pregnancy were included: 27 asymptomatic carriers; 53 women with obstetric APS; 20 women with thrombotic APS; and 35 women with both thrombotic and obstetric complications. Anti-D1 and anti-D4/5 antibodies were tested using a chemiluminescent immunoassay and a research ELISA assay, respectively (QUANTA Flash® ß2GPI Domain 1 IgG and QUANTA Lite® ß2GPI D4/5 IgG, Inova Diagnostics). Positivity for anti-D1 antibodies, but not anti-D4/5 antibodies, was differently distributed across the 4 subgroups of patients (p < 0.0001) and significantly correlated with thrombosis (χ2 = 17.28, p < 0.0001) and PM (χ2 = 4.28, p = 0.039). Patients with triple positivity for anti-phospholipid antibodies displayed higher anti-D1 titres and lower anti-D4/5 titres compared to women with one or two positive tests (p < 0.0001 and p = 0.005, respectively). Reactivity against D1 was identified as a predictor for PM (OR 2.4, 95% confidence interval [CI] 1.2-5.0, p = 0.017); in particular, anti-D1 antibodies were predictive of late PM, conveying an odds ratio of 7.3 (95% CI 2.1-25.5, p = 0.022). Positivity for anti-D1 antibodies was not associated with early pregnancy loss. Anti-D4/5 antibodies were not associated with clinical APS manifestations. As a whole, our data suggest that anti-D1 antibodies are significantly associated not only with thrombosis, but also with late PM, while positive anti-D4/5 antibodies are not predictive of thrombosis or PM.

Prevalence of anti-DFS70 antibodies in patients with and without systemic autoimmune rheumatic diseases.

OBJECTIVES: Autoantibodies to the dense fine speckled 70 (DFS70) antigen are common among antinuclear antibodies (ANA) positive healthy individuals (HI). We assessed the prevalence of anti-DFS70 antibodies in patients with and without ANA-associated rheumatic diseases (AARDs) by two methods: chemiluminescent immunoassay (CIA) and an indirect immunofluorescence (IIF) assay based on immunoadsorption for DFS70. METHODS: Fifty-one ANA-positive sera samples from patients with confirmed clinical diagnosis of AARD, 92 samples from HI and 85 samples submitted to a reference laboratory for routine ANA testing were evaluated for the presence of anti-DFS70 antibodies. The samples were evaluated by QUANTA Flash DFS70 CIA using BIO-FLASH instrument and by NOVA Lite selected HEp-2 kit on NOVA View - an automated IIF system. Sera with DFS positive pattern were pre-absorbed with highly purified human DFS70 antigen, and then tested again. RESULTS: Twenty-four samples (10.5%) tested by QUANTA Flash DFS70 CIA were positive for anti-DFS70 antibodies. The prevalence of monospecific anti-DFS70 antibodies was significantly higher in healthy subjects than in patients with AARDs (10.9% vs. 1.9%, p=0.02). The frequency of anti-DFS70 antibodies in samples submitted for routine ANA testing was 15.2%. A very good agreement was found between CIA and the DFS pattern identified by the automated HEp-2 IIF (kappa=0.97). In 80% of the samples obtained from patients without AARDs, immunoadsorption effectively inhibited the anti-DFS70 antibodies. CONCLUSIONS: The data confirm that mono-specific anti-DFS70 antibodies are a strong discriminator between ANA positive HI and AARD patients, and their evaluation should be included in ANA testing algorithms.