Background: Critically ill patients with coronavirus disease 2019 (COVID-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a relationship with anti-phospholipid antibodies (aPLs) has been proposed, but results are controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of C reactive protein. The presence of anticardiolipin (aCL), anti-beta2-glycoprotein I (anti-b 2 GPI), and anti-phosphatidylserine/ prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-b 2 GPI antibodies was not reported. Objective: To evaluate the prevalence and the clinical association of aPL in a large cohort of COVID-19 patients, and to characterize the epitope specificity of anti-b 2 GPI antibodies. Methods: ELISA and chemiluminescence assays were used to test 122 sera of patients suffering from severe COVID-19. Of them, 16 displayed major thrombotic events.

Natural killer (NK) cells have antitumor, antiviral, and antibacterial functions, and efforts are being made to manipulate them in immunotherapeutic approaches. However, their activation mechanisms remain poorly defined, particularly during bacterial infections. Here, we show that upon lipopolysaccharide or E. coli exposure, dendritic cells (DCs) produce three cytokines-interleukin 2 (IL-2), IL-18, and interferon β (IFN-β)-necessary and sufficient for NK cell activation. IFN-β enhances NK cell activation by inducing IL-15 and IL-15 receptor α not only in DCs but, surprisingly, also in NK cells. This process allows the transfer of IL-15 on NK cell surface and its cis presentation. cis-presented NK cell-derived and trans-presented DC-derived IL-15 contribute equally to optimal NK cell activation.

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Background. Critically ill patients with coronavirus disease 2019 (COVID-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a relationship with anti-phospholipid antibodies (aPL) has been proposed, but results are controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of C reactive protein. The presence of anti-cardiolipin (aCL), anti-beta2-glycoprotein I (anti-β2GPI) and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-β2GPI antibodies was not reported. Objective. To evaluate the prevalence and the clinical association of aPL in a large cohort of COVID-19 patients, and to characterize the epitope specificity of anti-β2GPI antibodies. Methods. ELISA and chemiluminescence assays were used to test 122 sera of patients suffering from severe COVID-19. Of them, 16 displayed major thrombotic events. Results. Anti-β2GPI IgG/IgA/IgM were the most frequent in 15.6/6.6/9.0% of patients, while aCL IgG/IgM were detected in 5.7/6.6% by ELISA. Comparable values were found by chemiluminescence. aPS/PT IgG/IgM were detectable in 2.5 and 9.8% by ELISA. No association between thrombosis and aPL was found. Reactivity against domain 1 and 4-5 of β2GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-β2GPI nor with thrombosis. Conclusions. aPL show a low prevalence in COVID-19 patients and are not associated with major thrombotic events. aPL in COVID-19 patients are mainly directed against β2GPI but display an epitope specificity different from antibodies in antiphospholipid syndrome.

BackgroundAntiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)).MethodsSixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (β2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-β2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human β2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human β2GPI or after CL-micelle absorption.ResultsComparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM aß2GPI and aCL. Anti-CL and anti-ß2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and ß2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized β2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-β2GPI humoAbs.ConclusionsThe LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1018-x) contains supplementary material, which is available to authorized users.

Background Consistently with their diagnostic and prognostic value, autoantibodies specific for systemic sclerosis (SSc) embedded in immune complexes (ICs) elicited a pro-inflammatory and pro-fibrotic cascade in healthy skin fibroblasts, engaging Toll-like receptors (TLRs) via their nucleic acid components. The objective of this study was to investigate the pathogenicity of SSc-ICs in endothelial cells. Methods ICs were purified from the sera of SSc patients bearing different autoantibody specificities (antibodies against DNA topoisomerase I, centromeric proteins, RNA polymerase, and Th/To), patients with systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS), or healthy controls (NHS) using polyethylene glycol precipitation. Human umbilical vein endothelial cells (HUVECs) were incubated with ICs, positive and negative controls. mRNA levels of endothelin-1 (et-1), collagenIα1 (colIα1), interferon (IFN)-α, and IFN-β were investigated by real-time PCR; et-1 and il-6 mRNA levels were assessed after pre-treatment with bafilomycin. ICAM-1 expression was evaluated by cell ELISA; secretion of IL-6, IL-8, and transforming growth factor (TGF)-β1 in culture supernatants was measured by ELISA. The expression of Fcγ receptors (CD64, CD32, and CD16) was assessed in endothelial cells at FACS analysis. Intracellular signaling pathways culminating with NFκB, p38MAPK, SAPK-JNK, and Akt were assessed by Western blotting. Healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with ICs, and TGF-β1 secretion and mRNA levels of colIα1 and matrix metalloproteinase (mmp)-1, protein expression of α smooth muscle actin (α-SMA), and IL-6 were evaluated by Western blotting; et-1 mRNA levels were assessed in fibroblasts pre-treated with IL-6 and TGF-β inhibitors and stimulated with ATA-ICs. Results All SSc stimulated IL-6 secretion; ACA-ICs and anti-Th/To-ICs increased ICAM-1 expression; all SSc-ICs but anti-Th/To-ICs augmented IL-8 levels; all SSc-ICs but ACA-ICs and ARA-ICs upregulated et-1, and all SSc-ICs but ARA-ICs affected TGF-β1 secretion. colIα1, IFN-α, and IFN-β mRNA levels were not affected by any SSc-IC. FcγRII (CD32) and FcγRIII (CD16) were not detectable on HUVECs, while FcγRI (CD64) was minimally expressed. A differential modulation of tlr expression was observed: tlr2, tlr3, and tlr4 were upregulated by ATA-ICs and ACA-ICs, while anti-Th/To-ICs resulted in tlr9 upregulation. Pre-treatment with bafilomycin did not affect the upregulation of et-1 and il-6 induced by ATA-ICs, ACA-ICs, and anti-Th/To-ICs; a 23% reduction in both genes was reported for ARA-ICs. All SSc-ICs activated p38MAPK and Akt, and all SSc-ICs but ARA-ICs yielded the activation of NFκB; ATA-ICs and ACA-ICs increased the activation rate of both subunits of SAPK-JNK. When healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with SSc-ICs, TGF-β1 secretion, colIα1, α-SMA, and IL-6 expression levels were significantly modulated. Pre-treatment with IL-6 and TGF-β inhibitors prevented et-1 upregulation induced by ATA-ICs by 85% and 77%, respectively. Conclusions These data provide the first demonstration of the pathogenicity of ICs from scleroderma patients with different autoantibodies on the endothelium. Endothelial activation induced by SSc-ICs ultimately led to a pro-fibrotic phenotype in healthy skin fibroblasts.

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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.