RESUMEN
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
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/inmunologíaRESUMEN
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
The effects of immunoglobulin G (IgG) from patients with the antiphospholipid syndrome (APS) upon monocyte activation have not been fully characterized. We carried out a comprehensive proteomic analysis of human monocytes treated with IgG from patients with different manifestations of the APS. Using 2-dimensional differential gel electrophoresis (2D DiGE), 4 of the most significantly regulated proteins (vimentin [VIM], zinc finger CCH domain-containing protein 18, CAP Gly domain-containing linker protein 2, and myeloperoxidase) were differentially regulated in monocytes treated with thrombotic or obstetric APS IgG, compared with healthy control (HC) IgG. These findings were confirmed by comparing monocytes isolated from APS patients and HC. Anti-VIM antibodies (AVAs) were significantly increased in 11 of 27 patients (40.7%) with APS. VIM expression on HC monocytes was stimulated more strongly by APS IgG from patients with higher-avidity serum AVA. We further characterized the proteome of thrombotic APS IgG-treated monocytes using a label-free proteomics technique. Of 12 proteins identified with the most confidence, 2 overlapped with 2D DiGE and many possessed immune response, cytoskeletal, coagulation, and signal transduction functions which are all relevant to APS and may therefore provide potential new therapeutic targets of this disease.
Asunto(s)
Síndrome Antifosfolípido/inmunología , Inmunoglobulina G/inmunología , Monocitos/inmunología , Proteoma/inmunología , Proteómica/métodos , Adulto , Síndrome Antifosfolípido/sangre , Western Blotting , Células Cultivadas , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Proteoma/genética , Proteoma/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Células U937RESUMEN
Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial redox-driven proton pump that couples the production of NADPH to the mitochondrial metabolic rate. In this study, we demonstrated for the first time that NNT has a significant effect in the modulation of the immune response and host defense against pathogens. We found that NNT mRNA is enriched in immune system-related tissues and regulated during macrophage activation. Overexpression of NNT in a macrophage cell-line resulted in decreased levels of reactive oxygen species (ROS) and nitric oxide upon induction of the macrophage inflammatory responses. These cells failed to fully activate MAPK signaling pathways, resulting in defective secretion of proinflammatory cytokines in response to LPS, and were inefficient in clearance of intracellular bacteria. We have shown that C57BL/6J mice, which have a deletion in the Nnt gene, exhibited greater resistance to acute pulmonary infection with Streptococcus pneumoniae. Macrophages from these mice generated more ROS and established a stronger inflammatory response to this pathogen. Our results demonstrate a novel role for NNT as a regulator of macrophage-mediated inflammatory responses.
Asunto(s)
Inflamación/fisiopatología , Macrófagos/inmunología , NADP Transhidrogenasas/fisiología , Animales , Línea Celular , Sistema Inmunológico/enzimología , Pulmón/patología , Macrófagos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fagocitosis/fisiología , Infecciones Neumocócicas/patología , Infecciones Neumocócicas/fisiopatología , Neumonía Bacteriana/etiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The KY protein underlies a form of muscular dystrophy in the mouse but its role in muscle remains elusive. Immunodetection of endogenous KY protein in C2C12-derived myotubes and expression of a recombinant form in neonatal cardiomyocytes indicated that KY is a Z-band associated protein. Moreover, characterization of a KY interacting protein fragment led to the identification of Igfn1 (Immunoglobulin-like and fibronectin type 3 domain containing 1). Igfn1 is a transcriptionally complex locus encoding many protein variants. A yeast two-hybrid screen identified the Z-band protein filamin C (FLNC) as an interacting partner. Consistent with this, expression of an IGFN1 recombinant fragment showed that the three N-terminal globular domains, common to at least five IGFN1 variants, are sufficient to provide Z-band targeting. Taken together, the yeast two-hybrid, biochemical and immunofluorescence data support the notion that KY, IGFN1 and FLNC are part of a Z-band associated protein complex likely to provide structural support to the skeletal muscle sarcomere.
Asunto(s)
Proteínas Portadoras/química , Proteínas Contráctiles/química , Proteínas de Microfilamentos/química , Proteínas Musculares/química , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Proteínas Contráctiles/genética , Proteínas Contráctiles/aislamiento & purificación , Cartilla de ADN/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Filaminas , Técnicas In Vitro , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/aislamiento & purificación , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Fibras Musculares Esqueléticas/química , Proteínas Musculares/genética , Proteínas Musculares/aislamiento & purificación , Músculo Esquelético/química , Miocitos Cardíacos/metabolismo , Péptido Hidrolasas , Mapeo de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sarcómeros/química , Distribución Tisular , Técnicas del Sistema de Dos HíbridosRESUMEN
Beta-2-glycoprotein I (ß2GPI) is a blood protein and the major antigen in the autoimmune disorder antiphospholipid syndrome (APS). ß2GPI exists mainly in closed or open conformations and comprises of 11 disulfides distributed across five domains. The terminal Cys288/Cys326 disulfide bond at domain V has been associated with different cysteine redox states. The role of this disulfide bond in conformational dynamics of this protein has not been investigated so far. Here, we report on the enzymatic driven reduction by thioredoxin-1 (recycled by Tris(2-carboxyethyl)phosphine; TCEP) of ß2GPI. Specific reduction was demonstrated by Western blot and mass spectrometry analyses confirming majority targeting to the fifth domain of ß2GPI. Atomic force microscopy images suggested that reduced ß2GPI shows a slightly higher proportion of open conformation and is more flexible compared to the untreated protein as confirmed by modelling studies. We have determined a strong increase in the binding of pathogenic APS autoantibodies to reduced ß2GPI as demonstrated by ELISA. Our study is relevant for understanding the effect of ß2GPI reduction on the protein structure and its implications for antibody binding in APS patients.
Asunto(s)
Autoanticuerpos/química , Conformación Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , beta 2 Glicoproteína I/química , Autoanticuerpos/inmunología , Cisteína/química , Cisteína/metabolismo , Disulfuros/química , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Microscopía de Fuerza Atómica , Modelos Moleculares , Unión Proteica/inmunología , Relación Estructura-Actividad , beta 2 Glicoproteína I/inmunología , beta 2 Glicoproteína I/metabolismoRESUMEN
Resident macrophages are an integral component of many tissues and are important in homeostasis and repair. This study examines the contribution of resident tissue macrophages to bone physiology. Using immunohistochemistry, we showed that a discrete population of resident macrophages, OsteoMacs, was intercalated throughout murine and human osteal tissues. OsteoMacs were distributed among other bone lining cells within both endosteum and periosteum. Furthermore, OsteoMacs were coisolated with osteoblasts in murine bone explant and calvarial preparations. OsteoMacs made up 15.9% of calvarial preparations and persisted throughout standard osteoblast differentiation cultures. Contrary to previous studies, we showed that it was OsteoMacs and not osteoblasts within these preparations that responded to pathophysiological concentrations of LPS by secreting TNF. Removal of OsteoMacs from calvarial cultures significantly decreased osteocalcin mRNA induction and osteoblast mineralization in vitro. In a Transwell coculture system of enriched osteoblasts and macrophages, we demonstrated that macrophages were required for efficient osteoblast mineralization in response to the physiological remodeling stimulus, elevated extracellular calcium. Notably, OsteoMacs were closely associated with areas of bone modeling in situ, forming a distinctive canopy structure covering >75% of mature osteoblasts on diaphyseal endosteal surfaces in young growing mice. Depletion of OsteoMacs in vivo using the macrophage-Fas-induced apoptosis (MAFIA) mouse caused complete loss of osteoblast bone-forming surface at this modeling site. Overall, we have demonstrated that OsteoMacs are an integral component of bone tissues and play a novel role in bone homeostasis through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics.
Asunto(s)
Huesos/fisiología , Macrófagos/fisiología , Osteoblastos/fisiología , Animales , Huesos/citología , Calcificación Fisiológica , Calcio/metabolismo , Diferenciación Celular , Células Cultivadas , Expresión Génica , Humanos , Lipopolisacáridos/inmunología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoblastos/citología , Osteocalcina/genética , Osteocalcina/metabolismo , OsteogénesisRESUMEN
Neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ILD) particularly in the most severe form, idiopathic pulmonary fibrosis (IPF), although the underlying mechanisms remain unclear. Neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. Hypoxia has an important role in inflammation and may also contribute to pulmonary disease. We aimed to determine how neutrophil activation occurs in ILD and the relative importance of hypoxia. Using lung biopsies and bronchoalveolar lavage (BAL) fluid from ILD patients we investigated the extent of hypoxia and neutrophil activation in ILD lungs. Then we used ex vivo neutrophils isolated from healthy volunteers and BAL from patients with ILD and non-ILD controls to further investigate aberrant neutrophil activation in hypoxia and ILD. We demonstrate for the first time using intracellular staining, HIF-1α stabilization in neutrophils and endothelial cells in ILD lung biopsies. Hypoxia enhanced both spontaneous (+1.31-fold, p < 0.05) and phorbol 12-myristate 13-acetate (PMA)-induced (+1.65-fold, p < 0.001) neutrophil extracellular trap (NET) release, neutrophil adhesion (+8.8-fold, <0.05), and trans-endothelial migration (+1.9-fold, p < 0.05). Hypoxia also increased neutrophil expression of the αM (+3.1-fold, p < 0.001) and αX (+1.6-fold, p < 0.01) integrin subunits. Interestingly, NET formation was induced by αMß2 integrin activation and prevented by cation chelation. Finally, we observed NET-like structures in IPF lung sections and in the BAL from ILD patients, and quantification showed increased cell-free DNA content (+5.5-fold, p < 0.01) and MPO-citrullinated histone H3 complexes (+21.9-fold, p < 0.01) in BAL from ILD patients compared to non-ILD controls. In conclusion, HIF-1α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of ß2 integrins. Our results identify a novel HIF-1α- αMß2 integrin axis in NET formation for future exploration in therapeutic approaches to fibrotic ILD.
Asunto(s)
Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Trampas Extracelulares/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Enfermedades Pulmonares Intersticiales/metabolismo , Pulmón/patología , Neutrófilos/inmunología , Adulto , Anciano , Células Cultivadas , Femenino , Fibrosis , Humanos , Masculino , Persona de Mediana Edad , Activación Neutrófila , Transducción de Señal , Migración Transendotelial y TransepitelialRESUMEN
The importance of neutrophils in the pathogenesis of autoimmune rheumatic diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), is increasingly recognised. Generation of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs) by activated neutrophils are both thought to contribute to pathology; although the underlying mechanisms, particularly the effects of IgG autoantibodies upon neutrophil function, are not fully understood. Therefore, we determined whether purified IgG from patients with SLE or RA have differential effects upon neutrophil activation and function. We found that SLE- and RA-IgG both bound human neutrophils but differentially regulated neutrophil function. RA- and SLE-IgG both increased PMA-induced ß1 integrin-mediated adhesion to fibronectin, whilst only SLE-IgG enhanced αMß2 integrin-mediated adhesion to fibrinogen. Interestingly, only SLE-IgG modulated neutrophil adhesion to endothelial cells. Both SLE- and RA-IgG increased ROS generation and DNA externalisation by unstimulated neutrophils. Only SLE-IgG however, drove DNA externalisation following neutrophil activation. Co-culture of neutrophils with resting endothelium prevented IgG-mediated increase of extracellular DNA, but this inhibition was overcome for SLE-IgG when the endothelium was stimulated with TNF-α. This differential pattern of neutrophil activation has implications for understanding SLE and RA pathogenesis and may highlight avenues for development of novel therapeutic strategies.
Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Inmunoglobulina G/inmunología , Integrina beta1/inmunología , Lupus Eritematoso Sistémico/inmunología , Activación Neutrófila , Neutrófilos/inmunología , Artritis Reumatoide/patología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Lupus Eritematoso Sistémico/patología , Neutrófilos/patologíaRESUMEN
Microphthalmia transcription factor (MITF) regulates bone homeostasis by inducing expression of critical genes associated with osteoclast function. Gpnmb is a macrophage-enriched gene that has also been shown to be expressed in osteoblasts. Here, we have shown gpnmb to be highly induced in maturing murine osteoclasts. Microarray expression profile analysis identified gpnmb as a potential target of MITF in RAW264.7 cells, subclone C4 (RAW/C4), that overexpress this transcription factor. Electrophoretic mobility shift assays identified a MITF-binding site (M-box) in the gpnmb promoter that is conserved in different mammalian species. Anti-MITF antibody supershifted the DNA-MITF complex for the promoter site while MITF binding was abolished by mutation of this site. The gpnmb promoter was transactivated by co-expression of MITF in reporter gene assays while mutation of the gpnmb M-box prevented MITF transactivation. The induction of gpnmb expression during osteoclastogenesis was shown to exhibit similar kinetics to the known MITF targets, acp5 and clcn7. GPNMB expressed in RAW/C4 cells exhibited distinct subcellular distribution at different stages of osteoclast differentiation. At days 5 and 7, GPNMB protein co-localised with the osteoclast/macrophage lysosomal/endocytic marker MAC-3/LAMP-2, suggesting that GPNMB resides in the endocytic pathway of mature macrophages and is possibly targeted to the plasma membrane of bone-resorbing osteoclasts. The inclusion of gpnmb in the MITF regulon suggests a role for GPNMB in mature osteoclast function.
Asunto(s)
Proteínas del Ojo/genética , Glicoproteínas de Membrana/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Osteoclastos/metabolismo , Fosfatasa Ácida/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Diferenciación Celular , Línea Celular , Células Cultivadas , Canales de Cloruro/genética , Secuencia Conservada , ADN/genética , ADN/metabolismo , Cartilla de ADN/genética , Endocitosis , Humanos , Isoenzimas/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Regiones Promotoras Genéticas , Ligando RANK/farmacología , Proteínas Recombinantes/farmacología , Regulón , Homología de Secuencia de Ácido Nucleico , Fosfatasa Ácida Tartratorresistente , Activación TranscripcionalRESUMEN
Antiphospholipid antibodies (aPL), the serological hallmark of antiphospholipid syndrome (APS), are a heterogeneous group of autoantibodies raised against circulating blood proteins. Of these proteins, the phospholipid-binding b2-glycoprotein I (ß2GPI) is considered to be the main autoantigen in APS. Indeed, IgG antibodies targeting b2GPI (ab2GPI) directly cause both thrombosis and pregnancy morbidity in several mouse models. While antibodies raised against all five domains of b2GPI have been reported, a subgroup of IgG ab2GPI raised against the first domain (DI) of b2GPI (aDI), strongly correlate with thrombotic APS, and drive thrombosis and pregnancy loss in vivo. Few studies have focused on determining the type of IgG subclass(es) for aPL. The subclass of an antibody is important as this dictates the potential activity of an antibody; for example, IgG1 and IgG3 can fix complement better and are able to cross the placenta compared to IgG2 and IgG4. It is unknown what subclass IgG aDI are, and whether they are the same as ab2GPI. To determine IgG subclass distribution for ab2GPI and aDI, we purified total IgG from the serum of 19 APS patients with known ab2GPI and aDI activity. Using subclass-specific conjugated antibodies, we modified our established in-house ab2GPI and aDI ELISAs to individually measure IgG1, IgG2, IgG3, and IgG4. We found that while IgG1, IgG2, and IgG3 ab2GPI levels were similar, a marked difference was seen in IgG subclass aDI levels. Specifically, significantly higher levels of IgG3 aDI were detected compared to IgG1, IgG2, or IgG4 (p < 0.05 for all comparisons). Correlation analysis of subclass-specific ab2GPI vs. aDI demonstrated that IgG3 showed the weakest correlation (r = 0.45, p = 0.0023) compared to IgG1 (r = 0.61, p = 0.0001) and IgG2 (r = 0.81, p = 0.0001). Importantly, total subclass levels in IgG purified from APS and healthy serum (n = 10 HC n = 12 APS) did not differ, suggesting that the increased IgG3 aDI signal seen in APS-derived IgG is antigen-specific. To conclude, our data suggests that aDI show a different IgG subclass distribution to ab2GPI. Our results highlight the importance of aDI testing for patient stratification and may point toward differential underlying aPL-driven pathogenic processes that may be subclass restricted.
Asunto(s)
Anticuerpos Antifosfolípidos/sangre , Síndrome Antifosfolípido/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/sangre , Dominios Proteicos/inmunología , beta 2 Glicoproteína I/sangre , Adulto , Análisis de Varianza , Femenino , Humanos , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , beta 2 Glicoproteína I/inmunologíaRESUMEN
APS is an autoimmune disease in which antiphospholipid antibodies (aPL) cause vascular thrombosis and pregnancy morbidity. In patients with APS, aPL exert pathogenic actions by binding serum beta-2-glycoprotein I (ß2GPI) via its N-terminal domain I (DI). We previously showed that bacterially-expressed recombinant DI inhibits biological actions of IgG derived from serum of patients with APS (APS-IgG). DI is too small (7 kDa) to be a viable therapeutic agent. Addition of polyethylene glycol (PEGylation) to small molecules enhances the serum half-life, reduces proteolytic targeting and can decrease immunogenicity. It is a common method of tailoring pharmacokinetic parameters and has been used in the production of many therapies in the clinic. However, PEGylation of molecules may reduce their biological activity, and the size of the PEG group can alter the balance between activity and half-life extension. Here we achieve production of site-specific PEGylation of recombinant DI (PEG-DI) and describe the activities in vitro and in vivo of three variants with different size PEG groups. All variants were able to inhibit APS-IgG from: binding to whole ß2GPI in ELISA, altering the clotting properties of human plasma and promoting thrombosis and tissue factor expression in mice. These findings provide an important step on the path to developing DI into a first-in-class therapeutic in APS.
Asunto(s)
Síndrome Antifosfolípido/etiología , Síndrome Antifosfolípido/metabolismo , Coagulación Sanguínea , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Dominios y Motivos de Interacción de Proteínas , beta 2 Glicoproteína I/metabolismo , Adulto , Animales , Anticuerpos Antifosfolípidos/sangre , Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/diagnóstico , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Unión Proteica , Dominios Proteicos , Trombosis/sangre , Trombosis/etiología , Trombosis/metabolismo , beta 2 Glicoproteína I/químicaRESUMEN
Factor (F) Xa reactive IgG isolated from patients with antiphospholipid syndrome (APS) display higher avidity binding to FXa with greater coagulant effects compared to systemic lupus erythematosus (SLE) non APS IgG. FXa signalling via activation of protease-activated receptors (PAR) leads to increased intracellular calcium (Ca2+). Therefore, we measured alterations in Ca2+ levels in human umbilical vein endothelial cells (HUVEC) following FXa-mediated PAR activation and investigated whether FXa reactive IgG from patients with APS or SLE/APS- alter these responses. We observed concentration-dependent induction of Ca2+ release by FXa that was potentiated by APS-IgG and SLE/APS- IgG compared to healthy control subjects' IgG, and FXa alone. APS-IgG and SLE/APS- IgG increased FXa mediated NFκB signalling and this effect was fully-retained in the affinity purified anti-FXa IgG sub-fraction. Antagonism of PAR-1 and PAR-2 reduced FXa-induced Ca2+ release. Treatment with a specific FXa inhibitor, hydroxychloroquine or fluvastatin significantly reduced FXa-induced and IgG-potentiated Ca2+ release. In conclusion, PAR-1 and PAR-2 are involved in FXa-mediated intracellular Ca2+ release in HUVEC and FXa reactive IgG from patients with APS and/or SLE potentiate this effect. Further work is required to explore the potential use of IgG FXa reactivity as a novel biomarker to stratify treatment with FXa inhibitors in these patients.
Asunto(s)
Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/metabolismo , Calcio/metabolismo , Células Endoteliales/metabolismo , Factor Xa/metabolismo , Inmunoglobulina G/inmunología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Biomarcadores , Estudios de Casos y Controles , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Espacio Intracelular/metabolismo , Masculino , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
A significant amount of myocardial damage during a myocardial infarction (MI) occurs during the reperfusion stage, termed ischaemia/reperfusion (I/R) injury, and accounts for up to 50% of total infarcted tissue post-MI. During the reperfusion phase, a complex interplay of multiple pathways and mechanisms is activated, which ultimately leads to cell death, primarily through apoptosis. There is some evidence from a lupus mouse model that lupus IgG, specifically the antiphospholipid (aPL) antibody subset, is pathogenic in mesenteric I/R injury. Furthermore, it has previously been shown that the immunodominant epitope for the majority of circulating pathogenic aPLs resides in the N-terminal domain I (DI) of beta-2 glycoprotein I (ß2GPI). This study describes the enhanced pathogenic effect of purified IgG derived from patients with lupus and/or the antiphospholipid syndrome in a cardiomyocyte H/R in vitro model. Furthermore, we have demonstrated a pathogenic role for aPL containing samples, mediated via aPL-ß2GPI interactions, resulting in activation of the pro-apoptotic p38 MAPK pathway. This was shown to be inhibited using a recombinant human peptide of domain I of ß2GPI in the fluid phase, suggesting that the pathogenic anti-ß2GPI antibodies in this in vitro model target this domain.
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Anticuerpos Antifosfolípidos/genética , Infarto del Miocardio/genética , beta 2 Glicoproteína I/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Adulto , Animales , Anticuerpos Antifosfolípidos/metabolismo , Apoptosis/genética , Hipoxia de la Célula/genética , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina G/inmunología , Masculino , Ratones , Persona de Mediana Edad , Infarto del Miocardio/fisiopatología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ratas , Daño por Reperfusión/genética , Daño por Reperfusión/fisiopatología , beta 2 Glicoproteína I/genéticaRESUMEN
INTRODUCTION: The aim of this study was to examine the prevalence and functional effects of antibodies directed against Factor (F)Xa and other serine proteases (SP) in patients with antiphospholipid syndrome (APS). METHODS: Serum from patients with APS (n=59), systemic lupus erythematosus (SLE; n=106), other autoimmune rheumatic disease (ARD; n=63) and 40 healthy controls (HC) were tested for IgG activity against thrombin (Thr), FXa, FVIIa, phosphatidylserine (PS)/FXa and antithrombin (AT)-III by enzyme-linked immunosorbent assay (ELISA). Anti-FXa positive IgG were purified to measure their avidity by chaotropic ELISA and functional effects upon clotting time (FXa-ACT) and FXa enzymatic activity (±AT-III). RESULTS: Anti-FXa IgG were found in patients with SLE (49.1%) and APS (33.9%) (P<0.05) but not in ARD controls and HC. In contrast, anti-Thr and anti-PS/FXa IgG were identified in other ARD and anti-FVIIa IgG were low in all groups. The avidity of APS-IgG to FXa was significantly higher than SLE-IgG (P<0.05). Greatest prolongation of FXa-ACT was observed with APS-IgG and greatest inhibitory effect upon FXa enzymatic activity was found with APS-IgG followed by SLE-IgG compared to HC-IgG. ATIII inhibition of FXa was significantly reduced by APS-IgG compared with HC and SLE (P<0.05) and did not correlate with binding to AT-III. CONCLUSION: APS anti-FXa IgG have higher avidity to FXa and greater effects upon the enzymatic and coagulant activity of FXa compared with SLE anti-FXa IgG. Further studies of anti-FXa antibodies in APS, SLE and other non-autoimmune thrombotic disease cohorts are now required to evaluate whether targeting FXa with selective inhibitors in patients bearing anti-FXa antibodies may be an effective treatment strategy.
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Síndrome Antifosfolípido/inmunología , Autoanticuerpos/sangre , Coagulación Sanguínea , Factor Xa/inmunología , Adulto , Síndrome Antifosfolípido/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
PROBLEM: Some patients with antiphospholipid syndrome (APS) suffer pregnancy morbidity (PM) but not vascular thrombosis (VT), whilst others suffer VT only. Therefore, we compared the effects of IgG from VT+/PM- and VT-/PM+ subjects on human first-trimester trophoblast (HTR8) cells. METHOD OF STUDY: HTR-8 cells were incubated with APS VT+/PM-, APS VT-/PM+ or healthy control (HC) IgG. We measured trophoblast invasion by cell invasion assay; mRNA expression of TLR4 and adaptor proteins; phosphorylation of p38 MAPK, NFκB and ERK; and expression of interleukin (IL)-8 and IL-6. RESULTS: VT-/PM+ IgG, but not VT+/PM- IgG significantly reduced HTR-8 invasion. The effects on invasion were blocked by TLR-4 inhibition. Neither VT+/PM- nor VT-/PM+ IgG altered MyD88 mRNA expression, phosphorylation of signalling molecules or cytokine expression. CONCLUSIONS: VT-/PM+ IgG exert functionally relevant effects on human trophoblast cells but VT+/PM- IgG do not.
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Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/inmunología , Inmunoglobulina G/inmunología , Complicaciones del Embarazo/inmunología , Trofoblastos/inmunología , Adulto , Anticuerpos Antifosfolípidos/sangre , Anticuerpos Antifosfolípidos/aislamiento & purificación , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/patología , Línea Celular , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/aislamiento & purificación , Embarazo , Complicaciones del Embarazo/sangre , Complicaciones del Embarazo/patología , Trofoblastos/patologíaRESUMEN
Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by recurrent vascular thrombosis (VT) and/or pregnancy morbidity (PM) in the presence of persistent antiphospholipid antibodies (aPL), detected by lupus anticoagulant (LA), anticardiolipin (aCL) antibody, and/or anti-ß2 glycoprotein I (aß2GPI) antibody assays. These aPL, considered to be diagnostic markers and pathogenic drivers of APS, are a heterogeneous group of antibodies directed against anionic phospholipids, phospholipid-binding plasma proteins, and phospholipid-protein complexes. Although APS is currently considered as a single disease, it presents with a wide range of clinical symptoms and biological characteristics. The clinical diagnosis of APS in a patient with symptoms and signs is dependent upon the presence of a persistently positive result in an aPL assay. The tests recommended for detecting aPL are the standardized enzyme-linked immunosorbent assay (ELISA) to detect aCL and aß2GPI and clotting assays for LA performed according to the guidelines of the International Society on Thrombosis and Haemostasis. This chapter describes the standard laboratory test for the diagnosis of APS discussing the clinical and theoretical aspects of LA, aCL, and aß2GPI assays.
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Síndrome Antifosfolípido/diagnóstico , Pruebas Diagnósticas de Rutina/métodos , Anticuerpos Anticardiolipina/inmunología , Síndrome Antifosfolípido/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Inhibidor de Coagulación del Lupus/inmunología , beta 2 Glicoproteína I/inmunologíaRESUMEN
Deciphering the contribution of individual genes and in turn pathways to cellular processes can be complicated and is often based on prior knowledge or assumptions of gene function. Phenotype-driven mutagenesis screens based around n-ethyl-n-nitrosurea (ENU) have been successful in a wide range of physiological systems in identifying novel genes that contribute to a given phenotype. Here, we describe methodologies we have employed in analysing cellular phenotypes in pipelines of mutagenised mice. Examples of primary screens to identify outliers, and secondary screens to provide a more detailed characterisation are outlined.
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Etilnitrosourea/toxicidad , Leucocitos/efectos de los fármacos , Mutagénesis , Fenotipo , Animales , Recuento de Células Sanguíneas/instrumentación , Recuento de Células Sanguíneas/métodos , Células de la Médula Ósea/metabolismo , Cruzamiento , Citocinas/análisis , Citocinas/metabolismo , Etilnitrosourea/administración & dosificación , Femenino , Citometría de Flujo/métodos , Leucocitos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
In a mouse model of pneumonia caused by Streptococcus pneumoniae, differences in the timing and vigor of the host inflammatory response have been associated with susceptibility to invasive disease. BALB/c and CBA/Ca mice are known to be resistant and susceptible to acute pneumococcal disease, respectively. In this study, we have demonstrated that BMM from BALB/c and CBA/Ca mice differ in their expression and regulation of TLR9 in response to S. pneumoniae. We have also shown that BMM from CBA/Ca mice failed to fully activate p38, NF-kappaB, and ERK 1/2 signaling pathways, resulting in reduced secretion of TNF-alpha and CCL5 in response to this pathogen. In addition, we have established that S. pneumoniae induced significant cell death in BMM from CBA/Ca mice. These findings indicate that variations between the two strains are likely to reflect differences in macrophage responses to the pathogen.