RESUMEN
Antiphospholipid syndrome (APS) is an autoimmune prothrombotic disease characterized by thrombosis and/or pregnancy complications caused by antiphospholipid antibodies (aPL). The history of APS can be traced back to observations made during screening programs for syphilis conducted in the mid-20th century, with identification of patients with the so-called biological false-positive serological reactions for syphilis. Initial observation linking aPL with recurrent miscarriages was first reported more than 40 years ago. Since then, our understanding of the pathogenesis and management of APS has evolved markedly. Although APS is an autoimmune disease, anticoagulation mainly with vitamin K antagonists (VKAs) rather than immunomodulation, is the treatment of choice for thrombotic APS. Direct acting oral anticoagulants are inferior to VKAs, especially those with triple-positive APS and arterial thrombosis. Inflammation, complement activation, and thrombosis in the placenta may contribute to pathogenesis of obstetric APS. Heparin, mainly low-molecular-weight heparin, and low-dose aspirin represent the treatments of choice for women with obstetric complications. Increasingly, immunomodulatory agents such as hydroxychloroquine for thrombotic and obstetric APS are being used, especially in patients who are refractory to present standard treatment.
Asunto(s)
Síndrome Antifosfolípido , Enfermedades Autoinmunes , Sífilis , Trombosis , Embarazo , Humanos , Femenino , Síndrome Antifosfolípido/diagnóstico , Síndrome Antifosfolípido/tratamiento farmacológico , Síndrome Antifosfolípido/complicaciones , Sífilis/complicaciones , Sífilis/tratamiento farmacológico , Anticuerpos Antifosfolípidos , Anticoagulantes/uso terapéutico , Trombosis/tratamiento farmacológico , Trombosis/etiología , Trombosis/prevención & control , Enfermedades Autoinmunes/complicacionesRESUMEN
OBJECTIVE: aPL are found in the blood of 20-30% of patients with SLE. Although aPL cause vascular thrombosis in the antiphospholipid syndrome, it is not clear whether positive aPL levels in early SLE increase risk of subsequent vascular events (VE). In a previous analysis of 276 patients with SLE, we found that early positivity for ≥2 of IgG anti-cardiolipin (anti-CL), IgG anti-ß2-glycoprotein I (anti-ß2GPI) and anti-domain I of ß2-glycoprotein I (anti-DI) showed a possible association with VE. Here we have extended that analysis. METHODS: Serum samples taken from 501 patients with SLE early in their disease had been tested for IgG anti-CL, anti-ß2GPI and anti-DI by ELISA. Complete VE history was available for 423 patients of whom 23 were excluded because VE occurred before the diagnosis of SLE. For the remaining 400 patients we carried out Kaplan-Meier survival analysis to define groups at higher risk of VE. RESULTS: Of 400 patients, 154 (38.5%) were positive for one or more aPL, 27 (6.8%) were double/triple-positive and 127 (31.8%) were single-positive. There were 91 VE in 77 patients, of whom 42 were aPL-positive in early disease. VE were significantly increased in aPL-positive vs aPL-negative patients (P = 0.041) and in double/triple-positive vs single-positive vs aPL-negative patients (P = 0.0057). Omission of the IgG anti-DI assay would have missed 14 double/triple-positive patients of whom six had VE. CONCLUSION: Double/triple-positivity for IgG anti-CL, anti-ß2GPI and anti-DI in early SLE identifies a population at higher risk of subsequent VE.
Asunto(s)
Síndrome Antifosfolípido , Lupus Eritematoso Sistémico , Humanos , Anticuerpos Antifosfolípidos , Síndrome Antifosfolípido/complicaciones , Síndrome Antifosfolípido/diagnóstico , beta 2 Glicoproteína I , Cardiolipinas , Inmunoglobulina GRESUMEN
Antiphospholipid syndrome (APS) is an acquired highly prothrombotic disorder in which thrombo-inflammatory antiphospholipid antibodies (aPL) cause thrombosis via multiple mechanisms, including endothelial damage and activation. Obstetric complications in APS are caused by placental thrombosis, inflammation and complement activation. Anticoagulation is poorly effective in some patients especially those with triple positive aPL who are at ~30% risk of thrombosis recurrence within 10 years. Increasing therapeutic anticoagulation intensity may be beneficial but leads to excess bleeding with serious complications, such as intracerebral haemorrhage. Nonetheless, anticoagulation is still the mainstay of treatment despite the autoimmune nature of APS. The antimalarial immunomodulatory drug hydroxychloroquine (HCQ) has been used for many years for the treatment of inflammatory rheumatic diseases. HCQ has complex pleiotropic mechanisms of action upon multiple cell types. The proposed biological processes that HCQ regulates support the hypothesis that it may be a successful adjunctive treatment in the prevention of recurrent thrombosis and pregnancy complications.
Asunto(s)
Antimaláricos , Síndrome Antifosfolípido , Trombosis , Humanos , Femenino , Embarazo , Síndrome Antifosfolípido/complicaciones , Síndrome Antifosfolípido/tratamiento farmacológico , Hidroxicloroquina/farmacología , Hidroxicloroquina/uso terapéutico , Fibrinolíticos/uso terapéutico , Placenta , Antimaláricos/uso terapéutico , Trombosis/tratamiento farmacológico , Trombosis/etiología , Trombosis/prevención & control , Anticoagulantes/uso terapéuticoRESUMEN
Cellular senescence contributes to the pathophysiology of chronic obstructive pulmonary disease (COPD) and cardiovascular disease. Using endothelial colony-forming-cells (ECFC), we have demonstrated accelerated senescence in smokers and patients with COPD compared with non-smokers. Subgroup analysis suggests that ECFC from patients with COPD on inhaled corticosteroids (ICS) (n=14; eight on ICS) exhibited significantly reduced senescence (Senescence-associated-beta galactosidase activity, p21CIP1), markers of DNA damage response (DDR) and IFN-γ-inducible-protein-10 compared with patients with COPD not on ICS. In vitro studies using human-umbilical-vein-endothelial-cells showed a protective effect of ICS on the DDR, senescence and apoptosis caused by oxidative stress, suggesting a protective molecular mechanism of action of corticosteroids on endothelium.
Asunto(s)
Células Progenitoras Endoteliales , Enfermedad Pulmonar Obstructiva Crónica , Administración por Inhalación , Corticoesteroides/farmacología , Corticoesteroides/uso terapéutico , Senescencia Celular , HumanosRESUMEN
OBJECTIVE: To establish the demographic characteristics, laboratory findings and clinical outcomes in patients with autoimmune disease (AD) compared with a propensity-matched cohort of patients without AD admitted with COVID-19 to hospitals in the UK. METHODS: This is a multicentre observational study across 26 NHS Trusts. Data were collected both retrospectively and prospectively using a predesigned standardized case record form. Adult patients (≥18 years) admitted between 1 April 2020 and 31 July 2020 were included. RESULTS: Overall, 6288 patients were included to the study. Of these, 394 patients had AD prior to admission with COVID-19. Of 394 patients, 80 patients with SLE, RA or aPL syndrome were classified as severe rheumatologic AD. A higher proportion of those with AD had anaemia [240 (60.91%) vs 206 (52.28%), P = 0.015], elevated LDH [150 (38.08%) vs 43 (10.92%), P < 0.001] and raised creatinine [122 (30.96%) vs 86 (21.83%), P = 0.01], respectively. A significantly higher proportion of patients with severe rheumatologic AD had elevated CRP [77 (96.25%) vs 70 (87.5%), P = 0.044] and LDH [20 (25%) vs 6 (7.5%), P = 0.021]. Patients with severe rheumatologic AD had significantly higher mortality [32/80 (40%)] compared with propensity matched cohort of patients without AD [20/80 (25%), P = 0.043]. However, there was no difference in 180-day mortality between propensity-matched cohorts of patients with or without AD in general (P = 0.47). CONCLUSIONS: Patients with severe rheumatologic AD had significantly higher mortality. Anaemia, renal impairment and elevated LDH were more frequent in patients with any AD while elevated CRP and LDH were more frequent in patients with severe rheumatologic AD both of which have been shown to associate with increased mortality in patients with COVID-19.
Asunto(s)
Artritis Reumatoide , Enfermedades Autoinmunes , COVID-19 , Adulto , Humanos , COVID-19/epidemiología , Estudios Retrospectivos , Enfermedades Autoinmunes/complicaciones , Enfermedades Autoinmunes/epidemiología , Reino Unido/epidemiologíaRESUMEN
OBJECTIVES: Risk factors for thromboembolism in SLE are poorly understood. We hypothesized a possible role for protein C, based on its dual activity in inflammation and haemostasis and on the evidence of an association between acquired activated protein C (APC) resistance (APCR) and high-avidity anti-protein C antibodies (anti-PC) with a severe thrombotic phenotype in venous thrombosis APS patients. METHODS: In a cross-sectional study of 156 SLE patients, the presence and avidity of IgG anti-PC was established by in house-ELISA, and APCR to exogenous recombinant human APC (rhAPC) and Protac (which activates endogenous protein C) was assessed by thrombin generation-based assays. Associations with aPL profile, thrombotic history and disease activity (BILAG and SLEDAI-2K) were also established. RESULTS: Anti-PC were detected in 54.5% of patients and APCR in 59%. Anti-PC positivity was associated with APCR to both rhAPC (P <0.0001) and Protac (P =0.0001). High-avidity anti-PC, detected in 26.3% of SLE patients, were associated with APCR in patients with thrombosis only (P <0.05), and with the development of thrombosis over time (range: 0-52 years; P =0.014). High-avidity anti-PC levels correlated with SLEDAI-2K (P =0.033) and total BILAG (P =0.019); SLEDAI-2K correlated inversely with APCR to Protac (P =0.004). CONCLUSION: Anti-PC occur in patients with SLE, independently of aPL profile, and are associated with APCR. High-avidity anti-PC are associated with thrombosis and with active disease and might prove a novel marker to monitor the risk of thrombosis and disease progression in SLE.
Asunto(s)
Resistencia a la Proteína C Activada/inmunología , Autoanticuerpos/inmunología , Lupus Eritematoso Sistémico/inmunología , Proteína C/inmunología , Tromboembolia/inmunología , Resistencia a la Proteína C Activada/sangre , Resistencia a la Proteína C Activada/complicaciones , Resistencia a la Proteína C Activada/etiología , Biomarcadores/sangre , Estudios Transversales , Femenino , Humanos , Lupus Eritematoso Sistémico/complicaciones , Masculino , Persona de Mediana Edad , Factores de Riesgo , Trombina/metabolismo , Tromboembolia/etiologíaRESUMEN
OBJECTIVES: aPL are present in between 20 and 30% of patients with SLE. They can cause vascular events (VE) or pregnancy morbidity. aCL and anti-beta-2-glycoprotein I (anti-ß2GPI) are measured in clinical practice. Domain I (DI) of ß2GPI is the main site for aPL binding. We investigated the prevalence of IgG anti-DI, aCL and anti-ß2GPI antibodies in early SLE and their association with mortality and development of VE. METHODS: Samples from 501 patients with SLE that had been obtained and stored early during their disease were tested for IgG anti-DI, aCL and anti-ß2GPI antibodies by ELISA. LA status and history of VE were obtained by reviewing medical records. Kaplan-Meier analysis was used to investigate mortality and occurrence of VE, comparing groups with and without aPL in early disease. RESULTS: Of 501 patients, 190 (38%) had at least one of these aPL, of whom 112 had anti-DI alone. Of 276 patients with complete vascular history, 83 had experienced VE. The 39 patients who were double or triple-ELISA-positive for any combination of the three aPL were more likely to have or develop lupus anticoagulant (P<0.0001) than those who were single-ELISA-positive or negative. In Kaplan-Meier analysis, they showed a trend towards developing more VE (P = 0.06). CONCLUSION: IgG anti-DI antibodies were present in early serum samples from 29% of patients and were more common than IgG aCL or anti-ß2GPI. There was some evidence suggesting that double or triple-ELISA-positivity for these antibodies identified a group with worse outcomes.
Asunto(s)
Anticuerpos Antifosfolípidos/sangre , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/mortalidad , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/mortalidad , Adolescente , Adulto , Anciano , Anticuerpos Anticardiolipina/sangre , Niño , Preescolar , Femenino , Humanos , Inmunoglobulina G/inmunología , Lactante , Lupus Eritematoso Sistémico/sangre , Masculino , Persona de Mediana Edad , Embarazo , Adulto Joven , beta 2 Glicoproteína I/inmunologíaAsunto(s)
Síndrome Antifosfolípido , Humanos , Relevancia Clínica , Reino Unido , Protrombina , FosfatidilserinasRESUMEN
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.
Asunto(s)
Síndrome Antifosfolípido/genética , Inmunoglobulina G/farmacología , Monocitos/inmunología , Complicaciones del Embarazo/genética , Trombosis/genética , Transcriptoma/inmunología , Adulto , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/patología , Estudios de Casos y Controles , Adhesión Celular , Comunicación Celular , Matriz Extracelular/química , Matriz Extracelular/inmunología , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Humanos , Masculino , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/inmunología , Anotación de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/patología , Embarazo , Complicaciones del Embarazo/inmunología , Complicaciones del Embarazo/patología , Cultivo Primario de Células , Trombosis/inmunología , Trombosis/patologíaRESUMEN
BACKGROUND: In this paper we describe a novel method to achieve high yield bacterial expression of a small protein domain with considerable therapeutic potential; Domain I of Beta-2-glycoprotein I (ß2GPI). ß2GPI is intrinsic to the pathological progression of the Antiphospholipid Syndrome (APS). Patients develop autoantibodies targeting an epitope located on the N-terminal Domain I of ß2GPI rendering this domain of interest as a possible therapeutic. RESULTS: This new method of production of Domain I of ß2GPI has increased the production yield by ~20 fold compared to previous methods in E.coli. This largely scalable, partially automated method produces 50-75 mg of pure, folded, active Domain I of ß2GPI per litre of expression media. CONCLUSION: The application of this method may enable production of Domain I on sufficient scale to allow its use as a therapeutic.
Asunto(s)
Síndrome Antifosfolípido/tratamiento farmacológico , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , beta 2 Glicoproteína I/aislamiento & purificación , beta 2 Glicoproteína I/metabolismo , Adulto , Automatización de Laboratorios , Escherichia coli/genética , Femenino , Humanos , Cuerpos de Inclusión , Masculino , Persona de Mediana Edad , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/uso terapéutico , beta 2 Glicoproteína I/genética , beta 2 Glicoproteína I/uso terapéuticoRESUMEN
OBJECTIVE: To report on the long-term follow-up, clinically and serologically, of 98 patients with SLE treated with B cell depletion (BCD) over a 12 year period, focusing on the duration of the depletion. METHODS: A retrospective review of clinical and serological features of all SLE patients treated with BCD from January 2000 until December 2012 in the Centre for Rheumatology, University College London Hospital. Clinical activity was assessed by the classic BILAG score at baseline and 6 and 12 months after the treatment. RESULTS: The period of depletion is extremely variable between patients and within the same patient on different occasions. The patients were divided into two groups according to the duration of depletion and a defined threshold of 12 months was utilized. The group with longer duration of depletion was associated with a better outcome, with a decrease in BILAG score at 6 and 12 months. This group was also associated with lymphopenia present at any time during the course of the patient's disease. No other clinical or serological feature was associated with longer duration of BCD. CONCLUSION: Cycles of BCD that induce longer duration of BCD are associated with better outcome. Lymphopenia may help to predict longer duration of the depletion and better outcome, although the mechanism is unclear.
Asunto(s)
Antirreumáticos/farmacología , Antirreumáticos/uso terapéutico , Apoptosis/efectos de los fármacos , Linfocitos B/patología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Rituximab/farmacología , Rituximab/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/efectos de los fármacos , Recuento de Células , Ciclofosfamida/farmacología , Ciclofosfamida/uso terapéutico , Quimioterapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Modelos Logísticos , Londres , Lupus Eritematoso Sistémico/patología , Linfopenia/epidemiología , Masculino , Metilprednisolona/farmacología , Metilprednisolona/uso terapéutico , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Tiempo , Resultado del TratamientoRESUMEN
OBJECTIVE: IgG aPL against domain I of ß2-glycoprotein I (ß2GPI) [anti-DI (aDI)] is associated with the pathogenesis of APS, an autoimmune disease defined by thrombosis and pregnancy morbidity. To date, however, no study has demonstrated direct pathogenicity of IgG aDI in vivo. In this proof-of-concept study, we designed a novel system to affinity purify polyclonal aDI aPL in order to assess its prothrombotic ability in a well-characterized mouse microcirculation model for APS. METHODS: Two polyclonal IgG fractions were isolated from serum of a patient with APS, both with high aPL activity but differing in aDI activity (aDI-rich and aDI-poor). These IgG fractions were tested for their pathogenic ability in an in vivo mouse model of thrombosis. Male CD1 mice were injected intraperitoneally with either aDI-rich or aDI-poor IgG; as a control, IgG isolated from healthy serum was used. A pinch injury was applied to the right femoral vein and thrombus dynamics and tissue factor activity in isolated tissue were evaluated. RESULTS: Both aDI-rich and aDI-poor IgG retained aCL and anti-ß2GPI activity, while only aDI-rich IgG displayed high aDI activity, as defined by our in-house cut-offs for positivity in each assay. aDI-rich IgG induced significantly larger thrombi in vivo compared with aDI-poor IgG (P < 0.0001). Similarly, aDI-rich IgG significantly enhanced the procoagulant activity of carotid artery endothelium and peritoneal macrophages isolated from experimental animals (P < 0.01). CONCLUSION: These data directly demonstrate that the ability to cause thrombosis in vivo is concentrated in the aDI fraction of aPL.
Asunto(s)
Anticuerpos Antifosfolípidos/farmacología , Síndrome Antifosfolípido/inducido químicamente , Modelos Animales de Enfermedad , Inmunoglobulina G/farmacología , Ratones , Trombosis/inducido químicamente , beta 2 Glicoproteína I/inmunología , Animales , Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/complicaciones , Síndrome Antifosfolípido/inmunología , Inmunoglobulina G/inmunología , Masculino , Estructura Terciaria de Proteína , Trombosis/complicaciones , Trombosis/inmunologíaRESUMEN
Objectives: The disease-modifying anti-rheumatic drug methotrexate (MTX) is recognized to reduce cardiovascular risk in patients with systemic inflammatory diseases. However, the molecular basis for these cardioprotective effects remains incompletely understood. This study evaluated the actions of low-dose MTX on the vascular endothelium. Methods: Human endothelial cells (EC) were studied under in vitro conditions relevant to inflammatory arthritis. These included culture in a pro-inflammatory microenvironment and exposure to fluid shear stress (FSS) using a parallel plate model. Respectively treated cells were analyzed by RNA sequencing and quantitative real-time PCR for gene expression, by immunoblotting for protein expression, by phosphokinase activity arrays, by flow cytometry for cell cycle analyses and by mass spectrometry to assess folate metabolite levels. Results: In static conditions, MTX was efficiently taken up by EC and caused cell cycle arrest concurrent with modulation of cell signaling pathways. These responses were reversed by folinic acid (FA), suggesting that OCM is a predominant target of MTX. Under FSS, MTX did not affect cell proliferation or pro-inflammatory gene expression. Exposure to FSS downregulated endothelial one carbon metabolism (OCM) as evidenced by decreased expression of key OCM genes and metabolites. Conclusion: We found that FSS significantly downregulated OCM and thereby rendered EC less susceptible to the effects of MTX treatment. The impact of shear stress on OCM suggested that MTX does not directly modulate endothelial function. The cardioprotective actions of MTX likely reflect direct actions on inflammatory cells and indirect benefit on the vascular endothelium.
Asunto(s)
Antirreumáticos , Metotrexato , Humanos , Metotrexato/uso terapéutico , Células Endoteliales , Antirreumáticos/efectos adversos , Ácido Fólico , CarbonoRESUMEN
BACKGROUND: Assessing inflammatory disease activity in large vessel vasculitis (LVV) can be challenging by conventional measures. OBJECTIVES: We aimed to investigate somatostatin receptor 2 (SST2) as a novel inflammation-specific molecular imaging target in LVV. METHODS: In a prospective, observational cohort study, in vivo arterial SST2 expression was assessed by positron emission tomography/magnetic resonance imaging (PET/MRI) using 68Ga-DOTATATE and 18F-FET-ßAG-TOCA. Ex vivo mapping of the imaging target was performed using immunofluorescence microscopy; imaging mass cytometry; and bulk, single-cell, and single-nucleus RNA sequencing. RESULTS: Sixty-one participants (LVV: n = 27; recent atherosclerotic myocardial infarction of ≤2 weeks: n = 25; control subjects with an oncologic indication for imaging: n = 9) were included. Index vessel SST2 maximum tissue-to-blood ratio was 61.8% (P < 0.0001) higher in active/grumbling LVV than inactive LVV and 34.6% (P = 0.0002) higher than myocardial infarction, with good diagnostic accuracy (area under the curve: ≥0.86; P < 0.001 for both). Arterial SST2 signal was not elevated in any of the control subjects. SST2 PET/MRI was generally consistent with 18F-fluorodeoxyglucose PET/computed tomography imaging in LVV patients with contemporaneous clinical scans but with very low background signal in the brain and heart, allowing for unimpeded assessment of nearby coronary, myocardial, and intracranial artery involvement. Clinically effective treatment for LVV was associated with a 0.49 ± 0.24 (standard error of the mean [SEM]) (P = 0.04; 22.3%) reduction in the SST2 maximum tissue-to-blood ratio after 9.3 ± 3.2 months. SST2 expression was localized to macrophages, pericytes, and perivascular adipocytes in vasculitis specimens, with specific receptor binding confirmed by autoradiography. SSTR2-expressing macrophages coexpressed proinflammatory markers. CONCLUSIONS: SST2 PET/MRI holds major promise for diagnosis and therapeutic monitoring in LVV. (PET Imaging of Giant Cell and Takayasu Arteritis [PITA], NCT04071691; Residual Inflammation and Plaque Progression Long-Term Evaluation [RIPPLE], NCT04073810).
Asunto(s)
Aterosclerosis , Arteritis de Células Gigantes , Infarto del Miocardio , Arteritis de Takayasu , Humanos , Receptores de Somatostatina , Estudios Prospectivos , Fluorodesoxiglucosa F18 , Inflamación/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Imagen por Resonancia Magnética , Vasos Coronarios/patología , Aterosclerosis/diagnóstico por imagen , Radiofármacos/farmacologíaRESUMEN
OBJECTIVE: To characterize the interaction between procoagulant and/or anticoagulant serine proteases and human monoclonal IgG antiphospholipid antibodies (aPL) and polyclonal IgG derived from patients with the antiphospholipid syndrome (APS). METHODS: Five human monoclonal IgG with small differences in their sequences were tested for binding to protein C, activated protein C, plasmin, factor VIIa (FVIIa), FIX, FIXa, and FXII. Serum levels of antithrombin and anti-activated protein C were compared in 32 patients with APS, 29 patients with systemic lupus erythematosus (SLE), and 22 healthy controls. Purified polyclonal IgG derived from APS patients with elevated levels of serum antithrombin antibodies was also tested for its functional effects on thrombin and antithrombin activity. RESULTS: Studies of monoclonal antibodies showed that sequence changes in human aPL are important in determining their ability to bind procoagulant and anticoagulant/fibrinolytic serine proteases. Mean IgG antithrombin levels were significantly elevated in patients with APS and in SLE patients with aPL but no APS (SLE/aPL+) compared to healthy controls, but anti-activated protein C levels were not increased in these patients. Moreover, IgG purified from patients with APS displayed higher avidity for thrombin and significantly inhibited antithrombin inactivation of thrombin compared with IgG from SLE/aPL+ patients. CONCLUSION: High-avidity antithrombin antibodies, which prevent antithrombin inactivation of thrombin, distinguish patients with APS from SLE/aPL+ patients, and thus may contribute to the pathogenesis of vascular thrombosis in APS.
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Anticuerpos Antifosfolípidos/inmunología , Anticuerpos Monoclonales/inmunología , Síndrome Antifosfolípido/inmunología , Hemostasis/inmunología , Lupus Eritematoso Sistémico/inmunología , Serina Proteasas/inmunología , Adulto , Femenino , Humanos , Masculino , Trombosis/inmunologíaRESUMEN
OBJECTIVE: Beta-2-glycoprotein I (ß2 GPI) constitutes the major autoantigen in the antiphospholipid syndrome (APS), a common acquired cause of arterial and venous thrombosis. We recently described the novel observation that ß2 GPI may exist in healthy individuals in a free thiol (biochemically reduced) form. The present study was undertaken to quantify the levels of total, reduced, and posttranslationally modified oxidized ß2 GPI in APS patients compared to various control groups. METHODS: In a retrospective multicenter analysis, the proportion of ß2 GPI with free thiols in serum from healthy volunteers was quantified. Assays for measurement of reduced as well as total circulating ß2 GPI were developed and tested in the following groups: APS (with thrombosis) (n=139), autoimmune disease with or without persistent antiphospholipid antibodies (aPL) but without APS (n=188), vascular thrombosis without APS or aPL (n=38), and healthy volunteers (n=91). RESULTS: Total ß2 GPI was significantly elevated in patients with APS (median 216.2 µg/ml [interquartile range 173.3-263.8]) as compared to healthy subjects (median 178.4 µg/ml [interquartile range 149.4-227.5] [P<0.0002]) or control patients with autoimmune disease or vascular thrombosis (both P<0.0001). The proportion of total ß2 GPI in an oxidized form (i.e., lacking free thiols) was significantly greater in the APS group than in each of the 3 control groups (all P<0.0001). CONCLUSION: This large retrospective multicenter study shows that posttranslational modification of ß2 GPI via thiol-exchange reactions is a highly specific phenomenon in the setting of APS thrombosis. Quantification of posttranslational modifications of ß2 GPI in conjunction with standard laboratory tests for APS may offer the potential to more accurately predict the risk of occurrence of a thrombotic event in the setting of APS.
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Anticuerpos Antifosfolípidos/sangre , Síndrome Antifosfolípido/sangre , Trombosis/etiología , beta 2 Glicoproteína I/sangre , beta 2 Glicoproteína I/inmunología , Adulto , Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Trombosis/sangre , Trombosis/inmunologíaRESUMEN
A major mechanism of hypercoagulability in the antiphospholipid syndrome (APS) is antiphospholipid Ab-mediated upregulation of tissue factor (TF) on monocytes via activation of TLRs, p38 MAPK, and NF-kappaB pathways. We examined whether monocyte signaling pathways are differentially activated by IgG from patients with vascular thrombosis (VT) alone compared with IgG from patients with pregnancy morbidity (PM) alone. We purified IgG from 49 subjects. A human monocyte cell line and ex vivo healthy monocytes were treated with 100 microg/ml IgG for 6 h, and cell extracts were examined by immunoblot using Abs to p38 MAPK and NF-kappaB. To further investigate intracellular signaling pathways induced by these IgGs, specific inhibitors of p38 MAPK, NF-kappaB, TLR4, and TLR2 were used to determine their effect on TF activity. Only IgG from patients with VT but no PM (VT+/PM-) caused phosphorylation of NF-kappaBand p38 MAPK and upregulation of TF activity in monocytes. These effects were not seen with IgG from patients with PM alone (VT-/PM+), anti-phospholipid Ab-positive patients without APS, or healthy controls. TF upregulation caused by the VT+/PM- samples was reduced by inhibitors of p38 MAPK, NF-kappaB, and TLR4. The effects of VT+/PM- IgG on signaling and TF upregulation were concentrated in the fraction that bound beta-2-glycoprotein I. Our findings demonstrate that IgGs from patients with diverse clinical manifestations of APS have differential effects upon phosphorylation of NF-kappaB and p38 MAPK and TF activity that may be mediated by differential activation of TLR4.
Asunto(s)
Síndrome Antifosfolípido/inmunología , Inmunoglobulina G/inmunología , Monocitos/inmunología , Transducción de Señal/inmunología , Tromboplastina/inmunología , Adulto , Síndrome Antifosfolípido/metabolismo , Western Blotting , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosforilación , Embarazo , Complicaciones del Embarazo/inmunología , Tromboplastina/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Antiphospholipid syndrome (APS) is an autoimmune disorder in which autoantibodies cause clinical effects of vascular thrombosis and pregnancy morbidity. The only evidence-based treatments are anticoagulant medications such as warfarin and heparin. These medications have a number of disadvantages, notably risk of haemorrhage. Therefore, there is a pressing need to develop new, more focused treatments that target the actual pathogenic disease process in APS. The pathogenic antibodies exert their effects by interacting with phospholipid-binding proteins, of which the most important is beta-2-glycoprotein I. This protein has five domains, of which the N-terminal Domain I (DI) is the main site for binding of pathogenic autoantibodies. We previously demonstrated bacterial expression of human DI and showed that this product could inhibit the ability of IgG from patients with APS (APS-IgG) to promote thrombosis in a mouse model. Since DI is a small 7kDa protein, its serum half-life would be too short to be therapeutically useful. We therefore used site-specific chemical addition of polyethylene glycol (PEG) to produce a larger variant of DI (PEG-DI) and showed that PEG-DI was equally effective as the non-PEGylated DI in inhibiting thrombosis caused by passive transfer of APS-IgG in mice. In this paper, we have used a mouse model that reflects human APS much more closely than the passive transfer of APS-IgG. In this model, the mice are immunized with human beta-2-glycoprotein I and develop endogenous anti-beta-2-glycoprotein I antibodies. When submitted to a pinch stimulus at the femoral vein, these mice develop clots. Our results show that PEG-DI inhibits production of thromboses in this model and also reduces expression of tissue factor in the aortas of the mice. No toxicity was seen in mice that received PEG-DI. Therefore, these results provide further evidence supporting possible efficacy of PEG-DI as a potential treatment for APS.
Asunto(s)
Síndrome Antifosfolípido , Trombosis , Animales , Anticuerpos Antifosfolípidos , Autoanticuerpos , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina G , Ratones , Polietilenglicoles/farmacología , Trombosis/etiología , beta 2 Glicoproteína IRESUMEN
OBJECTIVES: The significance of antibodies directed against activated factor X (FXa) and thrombin (Thr) in patients with SLE and/or antiphospholipid syndrome (APS) is unknown. FXa and Thr are coregulated by antithrombin (AT) and activate complement. Therefore, we studied the ability of anti activated factor X (aFXa) and/or anti-(a)Thr IgG from patients with SLE±APS to modulate complement activation. METHODS: Patients with SLE±APS were selected on the basis of known aThr and/or aFXa IgG positivity, and the effects of affinity-purified aFXa/aThr IgG on FXa and Thr-mediated C3 and C5 activation were measured ±AT. Structural analyses of FXa and Thr and AT-FXa and AT-Thr complexes were analysed in conjunction with the in vitro ability of AT to regulate aFXa-FXa and aThr-Thr-mediated C3/C5 activation. RESULTS: Using affinity-purified IgG from n=14 patients, we found that aThr IgG increased Thr-mediated activation of C3 and C5, while aFXa IgG did not increase C3 or C5 activation. Structural analysis identified potential epitopes and predicted a higher likelihood of steric hindrance of AT on FXa by aFXa IgG compared with the AT-Thr-aThr IgG complex that was confirmed by in vitro studies. Longitudinal analysis of 58 patients with SLE (±APS) did not find a significant association between positivity for aFXa or aTHr IgG and C3 levels or disease activity, although there was a trend for patients positive for aFXa IgG alone or both aFXa and aThr IgG to have lower levels of C3 compared with aThr IgG alone during clinical visits. CONCLUSIONS: We propose a novel method of complement regulation in patients with SLE±APS whereby aFXa and aThr IgG may have differential effects on complement activation.