Autoantibodies testing in autoimmunity: Diagnostic, prognostic and classification value.

Diagnosis of autoimmune diseases is in most cases challenging for clinicians as there is not a single specific laboratory or histological marker to diagnose or exclude the presence of the conditions. This review focused on the current knowledge of the role of autoantibodies' testing in various diseases, such as systemic lupus erythematosus, rheumatoid arthritis, antiphospholipid syndrome, undifferentiated connective tissues disease, primary biliary cholangitis and primary sclerosing cholangitis. Similarly, the prognostic and diagnostic values of autoantibodies testing in patients with interstitial lung disease have been reviewed. In-depth research on the molecular action of these autoantibodies on immune regulation and diseases pathogenesis has been explored beyond their correlation with disease phenotypes, highlighting the impact of autoantibodies targeting on disease outcomes and etiopathogenesis.

Low-Level Radon Activity Concentration-A MetroRADON International Intercomparison.

An international comparison of continuous monitors measuring radon activity concentration was performed to validate the traceability of the European radon calibration facilities. It was carried out by comparing the secondary standards used by these previous facilities, ranging from 100 Bq·m-3 to 300 Bq·m-3. Secondary standards were individually compared to a secondary reference device previously calibrated in a reference radon atmosphere traceable to a primary standard. The intercomparison was organized by the National Institute for Nuclear, Chemical, and Biological Protection (SUJCHBO) in the period from October 2019 to April 2020 within the European Metrology Program for Innovation and Research (EMPIR), JRP-Contract 16ENV10 MetroRADON. Eight European laboratories participated in this study. The results of the experiment are presented and discussed.

Intercomparison of Radon Flux Monitors at Low and at High Radium Content Areas under Field Conditions.

Interlaboratory exercises are a good tool to compare the response of different systems to the same quantity and to identify possible inconsistencies between them. One of the main goals of the EMPIR 19ENV01 traceRadon project is to harmonize radon flux measurements based on different systems and methodologies. In the framework of the traceRadon Project, two radon flux intercomparison campaigns were carried out in October 2021 at high and at low radon source areas. Four institutions participated in the field intercomparison exercises with their own systems. Every system was based on a specific radon monitor (diffusion or pump mode) and an accumulation chamber (with manual or automatic opening). Radon fluxes were calculated by each participant using both exponential and linear fittings of the radon activity concentration measured over time within the accumulation chambers. The results of this study show mainly: (i) the exponential approach is not advisable due to the variability of the radon flux and the leakage of the systems during long-time measurements; (ii) the linear approach should be applied to minimize the measurement period in agreement with the time response and sensitivity of the monitors; (iii) radon flux measured at high radon source areas (radium content of about 800 Bq kg-1) risks being underestimated because of the influence of advective effects; (iv) radon flux measured at low radon source areas (radium content of about 30 Bq kg-1) may present large uncertainties if sensitive radon monitors with pump mode are not used.

Beta 2 glycoprotein I and neutrophil extracellular traps: Potential bridge between innate and adaptive immunity in anti-phospholipid syndrome.

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by recurrent vascular thrombosis and miscarriages in the absence of known causes. Antibodies against phospholipid-binding proteins (aPL) are pathogenic players in both clotting and pregnancy APS manifestations. There is sound evidence that antibodies specific for beta2 glycoprotein I (ß2GPI) trigger thrombotic and pregnancy complications by interacting with the molecule on the membranes of different cell types of the coagulation cascade, and in placenta tissues. In addition to the humoral response against ß2GPI, both peripheral and tissue CD4+ ß2GPI-specific T cells have been reported in primary APS as well as in systemic lupus erythematosus (SLE)-associated APS. While adaptive immunity plays a clear role in APS, it is still debated whether innate immunity is involved as well. Acute systemic inflammation does not seem to be present in the syndrome, however, there is sound evidence that complement activation is crucial in animal models and can be found also in patients. Furthermore, neutrophil extracellular traps (NETs) have been documented in arterial and venous thrombi with different etiology, including clots in APS models. Keeping in mind that ß2GPI is a pleiotropic glycoprotein, acting as scavenger molecule for infectious agents and apoptotic/damaged body constituents and that self-molecules externalized through NETs formation may become immunogenic autoantigens, we demonstrated ß2GPI on NETs, and its ability to stimulate CD4+ß2GPI-specific T cells. The aim of this review is to elucidate the role of ß2GPI in the cross-talk between the innate and adaptive immunity in APS.

The Metrological Traceability, Performance and Precision of European Radon Calibration Facilities.

An interlaboratory comparison for European radon calibration facilities was conducted to evaluate the establishment of a harmonized quality level for the activity concentration of radon in air and to demonstrate the performance of the facilities when calibrating measurement instruments for radon. Fifteen calibration facilities from 13 different European countries participated. They represented different levels in the metrological hierarchy: national metrology institutes and designated institutes, national authorities for radiation protection and participants from universities. The interlaboratory comparison was conducted by the German Federal Office for Radiation Protection (BfS) and took place from 2018 to 2020. Participants were requested to measure radon in atmospheres of their own facilities according to their own procedures and requirements for metrological traceability. A measurement device with suitable properties was used to determine the comparison values. The results of the comparison showed that the radon activity concentrations that were determined by European calibration facilities complying with metrological traceability requirements were consistent with each other and had common mean values. The deviations from these values were normally distributed. The range of variation of the common mean value was a measure of the degree of agreement between the participants. For exposures above 1000 Bq/m3, the variation was about 4% for a level of confidence of approximately 95% (k=2). For lower exposure levels, the variation increased to about 6%.

ß2 glycoprotein I participates in phagocytosis of apoptotic neurons and in vascular injury in experimental brain stroke.

Beta-2 Glycoprotein I (ß2-GPI) is the main target of anti-phospholipid antibodies (aPL) in the autoimmune anti-phospholipid syndrome, characterized by increased risk of stroke. We here investigated the antibody independent role of ß2-GPI after ischemia/reperfusion, modeled in vivo by transient middle cerebral artery occlusion (tMCAo) in male C57Bl/6J mice; in vitro by subjecting immortalized human brain microvascular endothelial cells (ihBMEC) to 16 h hypoxia and 4 h re-oxygenation. ApoH (coding for ß2-GPI) was upregulated selectively in the liver at 48 h after tMCAo. At the same time ß2-GPI circulating levels increased. ß2-GPI was detectable in brain parenchyma and endothelium at all time points after tMCAo. Parenchymal ß2-GPI recognized apoptotic neurons (positive for annexin V, C3 and TUNEL) cleared by CD68+ brain macrophages. Hypoxic ihBMEC showed increased release of IL-6, over-expression of thrombomodulin and IL-1α after re-oxygenation with ß2-GPI alone. ß2-GPI interacted with mannose-binding lectin in mouse plasma and ihBMEC medium, potentially involved in formation of thrombi. We show for the first time that brain ischemia triggers the hepatic production of ß2-GPI. ß2-GPI is present in the ischemic endothelium, enhancing vascular inflammation, and extravasates binding stressed neurons before their clearance by phagocytosis. Thus ß2-GPI may be a new mediator of brain injury following ischemic stroke.

Complement activation and endothelial perturbation parallel COVID-19 severity and activity.

BACKGROUND: Animal models and few clinical reports suggest the involvement of the complement system in the onset of severe manifestations of coronavirus disease-2019 (COVID-19). However, complement contribution to endotheliopathy and hypercoagulability has not been elucidated yet. OBJECTIVE: To evaluate the association among complement activation, endothelial damage and disease severity or activity in COVID-19 patients. METHODS: In this single-centre cohort study, 148 patients with COVID-19 of different severity were evaluated upon hospital admission and 30 days later. Markers of complement activation (SC5b-9 and C5a) and endothelial perturbation (von Willebrand factor [vWF], tissue-type plasminogen activator [t-PA], plasminogen activator inhibitor-1 [PAI-1], soluble thrombomodulin [sTM], and soluble endothelial selectin [sE-selectin]) were measured in plasma. RESULTS: The patients had high plasma levels of SC5b-9 and C5a (p = 0.0001 for both) and vWF, t-PA and PAI-1 (p = 0.0001 for all). Their SC5b-9 levels correlated with those of vWF (r = 0.517, p = 0.0001) and paralleled disease severity (severe vs mild p = 0.0001, severe vs moderate p = 0.026 and moderate vs mild p = 0.001). The levels of sE-selectin were significantly increased only in the patients with severe disease. After 30 days, plasma SC5b-9, C5a and vWF levels had significantly decreased (p = 0.0001 for all), and 43% of the evaluated patients had normal levels. CONCLUSIONS: Complement activation is boosted during the progression of COVID-19 and dampened during remission, thus indicating its role in the pathophysiology of the disease. The association between complement activation and the biomarkers of endothelial damage suggests that complement may contribute to tissue injury and could be the target of specific therapy.

Anti-Phospholipid Antibodies in COVID-19 Are Different From Those Detectable in the Anti-Phospholipid Syndrome.

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 anti-cardiolipin (aCL), anti-beta2-glycoprotein I (anti-ß2GPI), and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-ß2GPI 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-ß2GPI 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-ß2GPI IgG/IgA/IgM was the most frequent in 15.6/6.6/9.0% of patients, while aCL IgG/IgM was 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 ß2GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-ß2GPI 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 ß2GPI but display an epitope specificity different from antibodies in antiphospholipid syndrome.

Complement Activation and Thrombin Generation by MBL Bound to ß2-Glycoprotein I.

ß2-Glycoprotein I (ß2-GPI) is an abundant plasma glycoprotein with unknown physiological function and is currently recognized as the main target of antiphospholipid Abs responsible for complement activation and vascular thrombosis in patients with antiphospholipid syndrome (APS). In this study, we provide evidence that mannose-binding lectin (MBL) binds to ß2-GPI in Ca++ and a dose-dependent manner and that this interaction activates complement and promotes complement-dependent thrombin generation. Surprisingly, a significant binding was observed between MBL and isolated domains II and IV of ß2-GPI, whereas the carbohydrate chains, domain I and domain V, were not involved in the interaction, documenting a noncanonical binding mode between MBL and ß2-GPI. Importantly, this interaction may occur on endothelial cells because binding of MBL to ß2-GPI was detected on the surface of HUVECs, and colocalization of MBL with ß2-GPI was observed on the endothelium of a biopsy specimen of a femoral artery from an APS patient. Because ß2-GPI-mediated MBL-dependent thrombin generation was increased after priming the endothelium with TNF-α, our data suggests that this mechanism could play an important yet unrecognized role under physiological conditions and may be upregulated in pathological situations. Moreover, the complement activation and the procoagulant effects of the ß2-GPI/MBL complex may contribute to amplify similar activities of anti-ß2-GPI Abs in APS and possibly act independently of Abs, raising the issue of developing appropriate therapies to avoid recurrences and disability in patients at risk for these clinical conditions.

Anti-phospholipid antibodies in COVID-19 are different from those detectable in the anti-phospholipid syndrome.

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-ß2GPI) and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-ß2GPI 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-ß2GPI 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-ß2GPI 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 ß2GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-ß2GPI 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 ß2GPI but display an epitope specificity different from antibodies in antiphospholipid syndrome.

Personalized medicine in rheumatoid arthritis: How immunogenicity impacts use of TNF inhibitors.

Up to 40% of patients treated with tumor necrosis factor alpha inhibitors (TNFi) do not respond to therapy. Testing drug bioavailability and/or anti-drug antibody (ADAb) levels may justify dosage adjustment or switch to different drugs, enabling a personalized medicine approach. We report a multicenter cross-sectional study on different methods [ELISA and a cell based functional assay (reporter gene assay - RGA)] for drug/ADAb detection, and on the relationship between drug bioavailability and ADAb. 163 patients with rheumatoid arthritis (RA) treated with infliximab (IFX; n = 67), adalimumab (ADL; n = 49) or etanercept (ETA; n = 47) were tested for drug and ADAb levels. Furthermore, we report prospective data from additional 70 patients (59 RA and 11 juvenile idiopathic arthritis - JIA) tested for drug and ADAb levels at baseline (T0) and after 3 (T3) and 6 months (T6) of treatment with ADL or ETA only. IFX-treated patients were not included because of the increasing use of IFX biosimilars. Stringent inclusion criteria were used in order to avoid unwanted variables in both studies; none of the patients used TNFi before the study, and TNFi was used only in combination with methotrexate. Clinical response was defined according to EULAR response criteria. The two assays performed comparably in the comparison study. Accordingly, ELISA was selected for the prospective study because of its feasibility in the clinical setting. The cross-sectional study found ADAb in IFX and ADL treated groups only, that were associated with a decrease in pharmacological drug availability in the blood. Comparable results were found for the ADL-treated group in the prospective study which also showed a relationship between drug/ADAb levels and the loss of clinical response. Altogether our findings support drug and anti-drug Ab monitoring in the real-world clinical setting thus enabling individualized treatment and reducing disability in chronic inflammatory arthritis.

New insight into antiphospholipid syndrome: antibodies to ß2glycoprotein I-domain 5 fail to induce thrombi in rats.

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of ß2glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analyzed the in vivo pro-coagulant effect of two groups of 5 sera IgG each reacting selectively with domain 1 or domain 5 in lipopolysaccharide (LPS)-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy, and vascular deposition of ß2glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-ß2glycoproteinI antibodies served as controls. All the anti-domain 1-positive IgG exhibited potent pro-coagulant activity while the anti-domain 5-positive and the negative control IgG failed to promote blood clot and vessel occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound ß2glycoproteinI while being able to interact with fluid-phase ß2glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit a thrombogenic effect in vivo, and the interaction of these antibodies with circulating ß2glycoproteinI suggests their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining "really at risk" patients for more appropriate treatments to avoid recurrences and disability.

Gene expression profiling identifies distinct molecular signatures in thrombotic and obstetric antiphospholipid syndrome.

Antiphospholipid antibodies (aPL) cause vascular thrombosis (VT) and/or pregnancy morbidity (PM). Differential mechanisms however, underlying the pathogenesis of these different manifestations of antiphospholipid syndrome (APS) are not fully understood. Therefore, we compared the effects of aPL from patients with thrombotic or obstetric APS on monocytes to identify different molecular pathways involved in the pathogenesis of APS subtypes. VT or PM IgG induced similar numbers of differentially expressed (DE) genes in monocytes. However, gene ontology (GO) analysis of DE genes revealed disease-specific genome signatures. Compared to PM, VT-IgG showed specific up regulation of genes associated with cell response to stress, regulation of MAPK signalling pathway and cell communication. In contrast, PM-IgG regulated genes involved in cell adhesion, extracellular matrix and embryonic and skeletal development. A novel gene expression analysis based on differential variability (DV) was also applied. This analysis identified similar GO categories compared to DE analysis but also uncovered novel pathways modulated solely by PM or VT-IgG. Gene expression analysis distinguished a differential effect of VT or PM-IgG upon monocytes supporting the hypothesis that they trigger distinctive physiological mechanisms. This finding contributes to our understanding of the pathology of APS and may lead to the development of different targeted therapies for VT or PM APS.

Obstetric and vascular antiphospholipid syndrome: same antibodies but different diseases?

Recurrent thrombosis and miscarriages are the main clinical manifestations of antiphospholipid syndrome (APS). Although most patients display both clinical signs, some patients can have isolated vascular or obstetric variants. Emerging data raise the question of whether obstetric and vascular APS are the same or different diseases. An important difference between the two conditions is that a thrombophilic state is a common feature in vascular APS, whereas clot occlusions of the decidual spiral arteries are seldom observed in obstetric APS, and infarctions are found in only one-third of APS placentae. Conversely, inflammation, which is undetectable in vascular APS, is frequently observed in the placentae of patients with obstetric APS and has been documented in the placentae of pregnant mice with fetal loss mediated by antiphospholipid antibodies. Attempts to identify different antibodies or epitopes responsible for the two clinical manifestations of APS have so far been unsuccessful. Possible mechanisms exist that might explain the development of the two clinical presentations, including the tissue distribution and expression level of the main target antigen of antiphospholipid antibodies, ß2 glycoprotein I (ß2GPI). The identification of the factors that promote the onset of either obstetric or vascular APS has important diagnostic and therapeutic implications.

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.

Fetal outcome and recommendations of pregnancies in lupus nephritis in the 21st century. A prospective multicenter study.

The aim of this multicenter study was to assess the present risk of fetal complications and the inherent risk factors in pregnant women with lupus nephritis. Seventy-one pregnancies in 61women (59 Caucasians and 2 Asians) with lupus nephritis were prospectively followed between October 2006 and December 2013. All patients received a counselling visit within 3 months before the beginning of pregnancy and were followed by a multidisciplinary team. At baseline mild active nephritis was present in 15 cases (21.1%). Six pregnancies (8.4%) resulted in fetal loss. Arterial hypertension at baseline (P = 0.003), positivity for lupus anticoagulant (P = 0.001), anticardiolipin IgG antibodies (P = 0.007), antibeta2 IgG (P = 0.018) and the triple positivity for antiphospholipid antibodies (P = 0.004) predicted fetal loss. Twenty pregnancies (28.2%) ended pre-term and 12 newborns (16.4%) were small for gestational age. Among the characteristics at baseline, high SLE disease activity index (SLEDAI) score (P = 0.027), proteinuria (P = 0.045), history of renal flares (P = 0.004), arterial hypertension (P = 0.009) and active lupus nephritis (P = 0.000) increased the probability of preterm delivery. Odds for preterm delivery increased by 60% for each quarterly unit increase in SLEDAI and by 15% for each quarterly increase in proteinuria by 1 g per day. The probability of having a small for gestational age baby was reduced by 85% in women who received hydroxychloroquine therapy (P = 0.023). In this study, the rate of fetal loss was low and mainly associated with the presence of antiphospholipid antibodies. Preterm delivery remains a frequent complication of pregnancies in lupus. SLE and lupus nephritis activity are the main risk factors for premature birth. Arterial hypertension predicted both fetal loss and preterm delivery. Based on our results the key for a successful pregnancy in lupus nephritis is a multidisciplinary approach with close medical, obstetric and neonatal monitoring. This entails: a) a preconception evaluation to establish and inform women about pregnancy risks; b) planning pregnancy during inactive lupus nephritis, maintained inactive with the lowest possible dosage of allowed drugs; c) adequate treatment of known risk factors (arterial hypertension, antiphospholipid and antibodies); d) close monitoring during and after pregnancy to rapidly identify and treat SLE flares and obstetric complications.

Maternal outcome in pregnant women with lupus nephritis. A prospective multicenter study.

Retrospective studies reported a high incidence of maternal complications in pregnant women with lupus. In this paper we prospectively assessed the rate of risk and the risk factors of maternal outcome in women with stable lupus nephritis who received pre-pregnancy counseling. This prospective multicenter study includes 71 pregnancies in 61 women with lupus nephritis who became pregnant between 2006 and 2013. Complete renal remission was present before pregnancy in 56 cases (78.9%) and mild active nephritis in 15 cases. All women underwent a screening visit before pregnancy and were closely monitored by a multidisciplinary team. Lupus anticoagulant, serum C3 and C4 complement fractions, anti-DNA antibodies, anti-C1q antibodies, anticardiolipin IgG and IgM antibodies, anti-beta2 IgG and IgM antibodies were tested at screening visit, at first, second, third trimester of pregnancy, and one year after delivery. Renal flares of lupus during or after pregnancy, pre-eclampsia, and HELLP syndrome were defined as adverse maternal outcomes. Fourteen flares (19.7%), six cases of pre-eclampsia (8.4%) and two cases of HELLP (2.8%) occurred during the study period. All flares responded to therapy and the manifestations of pre-eclampsia and HELLP were promptly reversible. Low C3, high anti-DNA antibodies and predicted all renal flares. High anti-C1q antibodies and low C4 predicted early flares. The body mass index (BMI) was associated with increased risk of late flares. History of previous renal flares and the presence of clinically active lupus nephritis at conception did not increase the risk of renal flares during pregnancy. History of renal flares before pregnancy, arterial hypertension, and longer disease predicted pre-eclampsia/HELLP. In pregnant women with lupus nephritis adverse maternal outcomes were relatively common but proved to be reversible when promptly diagnosed and treated. Immunological activity, arterial hypertension and BMI may predispose to maternal complications.

Antiphospholipid antibodies detected by line immunoassay differentiate among patients with antiphospholipid syndrome, with infections and asymptomatic carriers.

BACKGROUND: Antiphospholipid 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)). METHODS: Sixty-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 (ß2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-ß2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human ß2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human ß2GPI or after CL-micelle absorption. RESULTS: Comparison 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 ß2GPI 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-ß2GPI humoAbs. CONCLUSIONS: The 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.

Antiphospholipid Antibodies and Infertility: A Gene Expression Study in Decidual Stromal Cells.

BACKGROUND: Antiphospholipid antibodies (aPL) have been advocated as potential mediators of unexplained female infertility, but no evidence has yet been raised to support such an association. OBJECTIVES: To test the hypothesis that aPL might interfere with uterine decidualization, a gene expression study was performed on decidual stromal cells treated with different aPL preparations. METHODS: Decidual stromal cells were isolated from first-trimester deciduas obtained from two women undergoing elective abortion, and treated with: (i) a ß2GPI-dependent aPL monoclonal antibody (IS3); (ii) IS3 plus TIFI, a synthetic peptide mimicking PL-binding region of ß2GPI; and (iii) IgG from healthy subjects (NHS). Gene expression data were acquired using human HT-12 v3 beadchip arrays (Illumina). Differential expression analysis was performed by fitting a gene-wise linear model using the treatment group and decidual source as covariates. RESULTS: In the comparison of IS3 versus IgG NHS-treated decidual cells, gene ontology (GO) enrichment was expressed in terms relating to well-characterized aPL-mediated cellular effects: "inflammatory response," "immune response," "response to stress," "oxydoreductase activity," "metalloendopeptidase activity," and "cytokine/chemokine activity." As expected, almost all genes were up-regulated by IS3 treatment. The same GO categories appeared to be differentially expressed when IS3 treatment was compared to IS3 + TIFI, but with most genes being down-regulated. CONCLUSIONS: Given the inflammatory response evinced on gene expression analysis of decidual stromal cells treated with a ß2GPI -dependent aPL monoclonal antibody, it is feasible that aPL might interfere with uterine decidualization, affecting the early stages of implantation and ultimately resulting in female infertility.

Detection of red blood cell antibodies in mitogen-stimulated cultures from patients with hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by defects in red blood cell (RBC) membrane proteins leading to premature RBC clearance in the spleen. The presence of RBC autoantibodies has never been extensively investigated in HS. STUDY DESIGN AND METHODS: RBC antibody-bound immunoglobulin (Ig)G was investigated in 91 consecutive HS patients by mitogen-stimulated direct antiglobulin test (MS-DAT), a sensitive method able to magnify latent RBC antibody autoimmunity and related with hemolytic variables, previous splenectomy, and type of membrane defect. RESULTS: A total of 61% of HS cases had RBC antibodies by MS-DAT (29 Band 3, 17 spectrin deficiency, and nine no defined defect). The amount of RBC-bound IgG was greater in HS compared with controls (236 ± 192 ng/mL vs. 52 ± 29 ng/mL, p < 0.0001), although lower than that observed in autoimmune hemolytic anemia (AIHA; 634 ± 371 ng/mL vs. 236 ± 192 ng/mL, p < 0.0001). Western blot experiments showed that purified IgG fraction from MS-DAT-positive patients bind to α- and ß-spectrin, Band 3, and Band 4.9. Positive cases displayed increased reticulocytosis and slightly reduced hemoglobin (Hb) values compared to negative ones. Patients displaying RBC-bound IgG of more than 250 ng/mL (the positive threshold of AIHA) showed increased number of spherocytes and mainly had spectrin deficiency. RBC-bound IgG and free Hb increased over time after storage at 4°C, a surrogate of ex vivo aging, more evidently in HS than controls, and particularly in Band 3 deficiency. CONCLUSION: RBC autoantibodies were detected by MS-DAT in more than a half of HS patients. Positive cases showed a more evident hemolytic pattern suggesting a pathogenic role of these autoantibodies in RBC opsonization and splenic removal.