RESUMEN
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
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
There is limited evidence relating to the impact of disease modifying anti-rheumatic drugs (DMARDs) upon male fertility and peri-conception paternal exposure in men with rheumatic disease. Therefore, we conducted a systematic review of available evidence to update information on this subject and guide paternal counselling. A systematic search of PubMed and Embase was carried out up to September 2017, to find relevant peer-reviewed papers, using keywords for fertility/spermatogenesis/conception, men, and disease modifying or biologic drugs commonly prescribed in patients with rheumatic disease. The search yielded 724 papers, and the titles/abstracts were screened independently by 2 authors, duplicates removed and 233 potentially relevant papers selected for full text review. A total of 84 papers were included in the final analysis which covered the impact on fertility of over 611 male exposures to relevant drugs, and over 5986 pregnancies conceived during paternal exposure to (or within 3 months of stopping) these drugs. Aside from the known adverse impact of cyclophosphamide and sulfasalazine on spermatogenesis, overall there was no firm evidence of harm to fertility or pregnancy outcomes with paternal exposure to anti-TNF therapies, abatacept, rituximab, azathioprine, cyclosporine A, hydroxychloroquine, leflunomide, methotrexate or mycophenolate mofetil. There was no evidence found pertaining to the effects of male exposure to IVIG, tacrolimus, golimumab, anakinra or belimumab on fertility or pregnancy outcomes. These results provide further reassurance as to the safety of many DMARDs for men trying to conceive and will be useful when counselling men about risks of anti-rheumatic drugs to fertility and pregnancies, and following accidental conception.
Asunto(s)
Antirreumáticos/efectos adversos , Productos Biológicos/efectos adversos , Infertilidad Masculina/inducido químicamente , Exposición Paterna/efectos adversos , Resultado del Embarazo , Estudios de Cohortes , Femenino , Humanos , Masculino , Embarazo , Efectos Tardíos de la Exposición Prenatal/etiología , Enfermedades Reumáticas/tratamiento farmacológicoRESUMEN
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
This paper numerically and experimentally investigates the performance of free-space optical receiver using modes diversity coherent receipt under moderate-to-strong turbulence. By utilizing a three-mode photonic lantern with digital maximum ratio combining, a 40 Gbps QPSK optical signal is received. The turbulence strength is measured by the ratio of beam diameter to atmospheric coherence length, D/r0. The larger the D/r0, the stronger the turbulence is, and vice versa. Compared with conventional single mode fiber based receipt, the required transmitted power can reduce by 4.6 dB and 4 dB at 10% interruption probability under turbulence strength D/r0 = 8 and 16. The required transmitted power at bit error ratio of 2.2 × 10-2 can relax by 4.2 dB and 5 dB under turbulence strength D/r0 = 8 and 16.
RESUMEN
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
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
In this Letter, we theoretically and experimentally demonstrate a new method to generate tunable orbital angular momentum (OAM) by continuously changing the angle of linear polarization of the input light. We use the Fourier series of left- and right-hand projections to prove that the average OAM smoothly varied from l=-1 to l=1 with the angle of LP of input light changing from 0 to π, which is fulfilled by an electrical polarization controller.
RESUMEN
OBJECTIVE: To investigate whether rituximab, an anti-B cell therapy, improves symptoms of fatigue and oral dryness in patients with primary Sjögren's syndrome (SS). METHODS: We conducted a multicenter, randomized, double-blind, placebo-controlled, parallel-group trial that included health economic analysis. Anti-Ro-positive patients with primary SS, symptomatic fatigue, and oral dryness were recruited from 25 UK rheumatology clinics from August 2011 to January 2014. Patients were centrally randomized to receive either intravenous (IV) placebo (250 ml saline) or IV rituximab (1,000 mg in 250 ml saline) in 2 courses at weeks 0, 2, 24, and 26, with pre- and postinfusion medication including corticosteroids. The primary end point was the proportion of patients achieving a 30% reduction in either fatigue or oral dryness at 48 weeks, as measured by visual analog scale. Other outcome measures included salivary and lacrimal flow rates, quality of life, scores on the European League Against Rheumatism (EULAR) Sjögren's Syndrome Patient Reported Index and EULAR Sjögren's Syndrome Disease Activity Index, symptoms of ocular and overall dryness, pain, globally assessed disease activity, and cost-effectiveness. RESULTS: All 133 patients who were randomized to receive placebo (n = 66) or rituximab (n = 67) were included in the primary analysis. Among patients with complete data, 21 of 56 placebo-treated patients and 24 of 61 rituximab-treated patients achieved the primary end point. After multiple imputation of missing outcomes, response rates in the placebo and rituximab groups were 36.8% and 39.8%, respectively (adjusted odds ratio 1.13 [95% confidence interval 0.50, 2.55]). There were no significant improvements in any outcome measure except for unstimulated salivary flow. The mean ± SD costs per patient for rituximab and placebo were £10,752 ± 264.75 and £2,672 ± 241.71, respectively. There were slightly more adverse events (AEs) reported in total for rituximab, but there was no difference in serious AEs (10 in each group). CONCLUSION: The results of this study indicate that rituximab is neither clinically effective nor cost-effective in this patient population.
Asunto(s)
Antirreumáticos/uso terapéutico , Fatiga/tratamiento farmacológico , Rituximab/uso terapéutico , Síndrome de Sjögren/tratamiento farmacológico , Xerostomía/tratamiento farmacológico , Adulto , Anciano , Antirreumáticos/economía , Análisis Costo-Beneficio , Método Doble Ciego , Fatiga/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Medición de Resultados Informados por el Paciente , Calidad de Vida , Años de Vida Ajustados por Calidad de Vida , Rituximab/economía , Síndrome de Sjögren/complicaciones , Resultado del Tratamiento , Reino Unido , Escala Visual Analógica , Xerostomía/etiologíaAsunto(s)
Antirreumáticos/uso terapéutico , Artritis Psoriásica/diagnóstico , Complicaciones del Embarazo/diagnóstico , Adulto , Artritis Psoriásica/tratamiento farmacológico , Femenino , Humanos , Embarazo , Complicaciones del Embarazo/tratamiento farmacológico , Resultado del Embarazo , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: To develop a novel method for capturing the discrepancy between objective tests and subjective dryness symptoms (a sensitivity scale) and to explore predictors of dryness sensitivity. METHODS: Archive data from the UK Primary Sjögren's Syndrome Registry (n = 688) were used. Patients were classified on a scale from -5 (stoical) to +5 (sensitive) depending on the degree of discrepancy between their objective and subjective symptoms classes. Sensitivity scores were correlated with demographic variables, disease-related factors, and symptoms of pain, fatigue, anxiety, and depression. RESULTS: Patients were on average relatively stoical for both types of dryness symptoms (mean ± SD ocular dryness -0.42 ± 2.2 and -1.24 ± 1.6 oral dryness). Twenty-seven percent of patients were classified as sensitive to ocular dryness and 9% to oral dryness. Hierarchical regression analyses identified the strongest predictor of ocular dryness sensitivity to be self-reported pain and that of oral dryness sensitivity to be self-reported fatigue. CONCLUSION: Ocular and oral dryness sensitivity can be classified on a continuous scale. The 2 symptom types are predicted by different variables. A large number of factors remain to be explored that may impact symptom sensitivity in primary Sjögren's syndrome, and the proposed method could be used to identify relatively sensitive and stoical patients for future studies.
Asunto(s)
Autoevaluación Diagnóstica , Síndrome de Sjögren/diagnóstico , Xeroftalmia/diagnóstico , Xerostomía/diagnóstico , Anciano , Fatiga/diagnóstico , Fatiga/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dolor/diagnóstico , Dolor/epidemiología , Sistema de Registros , Síndrome de Sjögren/epidemiología , Reino Unido/epidemiología , Xeroftalmia/epidemiología , Xerostomía/epidemiologíaRESUMEN
OBJECTIVE: To systematically review and establish the prevalence of antibody positivity in assays not currently included in the APS classification criteria to detect antibodies directed against other phospholipids (PLs), PL binding proteins, coagulation factors and a mechanistic test for resistance of Annexin A5 (AnxA5) anticoagulant activity in APS and control populations. METHODS: We searched PubMed and EMBASE using the key words APS, antiphospholipid antibodies, non-criteria, new assays, IgA anticardiolipin antibodies, lupus anticoagulant, anti-Domain I, IgA anti-ß2-glycoprotein I antibodies, antiphosphatidylserine, anti-phosphatidylethanolamine, anti-phosphatidic acid, antiprothrombin, antiphosphatidylserine-prothtombin, anti-vimentin/cardiolipin complex and Annexin A5 resistance. Studies that met inclusion criteria to describe prevalence of non-criteria aPLs in APS patients (n > 10), disease and healthy control subjects were systematically examined. RESULTS: We selected 16 retrospective studies of 1404 APS patients, 1839 disease control and 797 healthy controls. The highest prevalence of non-criteria aPLs in the largest number of patients with APS was found in IgA anti-ß2GPI studies (129/229, 56.3%), AnxA5R (87/163, 53.4%) and IgG anti-Domain I (241/548, 44.0%). CONCLUSION: Our finding of a significantly high prevalence of all non-criteria aPLs studied in patients with APS compared with controls was tempered by wide variation in sample size, retrospective collection, assay methodology and different determination of positivity. Therefore, prospective studies of sufficient size and appropriate methodology are required to evaluate the significance of these assays and their utility in the management of patients with APS.
Asunto(s)
Anticuerpos Antiidiotipos/sangre , Síndrome Antifosfolípido/sangre , Síndrome Antifosfolípido/inmunología , Fosfolípidos/inmunología , Anexina A5/sangre , Anexina A5/inmunología , Estudios de Casos y Controles , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Prevalencia , Estudios Retrospectivos , beta 2 Glicoproteína I/sangre , beta 2 Glicoproteína I/inmunologíaRESUMEN
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.
Asunto(s)
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
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.
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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
IgG antiphospholipid antibodies (aPL) exert direct effects on various cell types, contributing to the pathogenesis of thrombosis and pregnancy morbidity in patients with the antiphospholipid syndrome (APS). Some IgG samples from these patients activate endothelial cells (EC) in vitro as judged by surface expression of adhesion molecules such as E-selectin, which can promote thrombosis. Endothelial microparticles (EMP), which themselves are potentially prothrombotic, are released by activated EC. Though elevated circulating EMP levels have been reported in patients with APS, it is not known whether these EMP are released due to a direct effect of aPL on the cells. We tested the effect of purified polyclonal IgG from patients with APS (APS-IgG) and healthy controls (HC-IgG) upon cultured human umbilical vein EC (HUVEC). HUVEC exposed to APS-IgG produced significantly more EMP than those exposed to HC-IgG (p=0.0036) and a greater proportion of these EMP carried surface E-selectin (6.2% ± 4.0 for APS-IgG vs. 3.4% ± 2.0 for HC IgG, p=0.0172). This study therefore demonstrates that purified polyclonal APS-IgG can drive EMP release. We propose that EMP generation may be a useful measure of aPL-mediated pathogenic effects upon EC.
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Anticuerpos Antifosfolípidos/sangre , Síndrome Antifosfolípido/inmunología , Micropartículas Derivadas de Células/inmunología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Inmunoglobulina G/sangre , Adulto , Síndrome Antifosfolípido/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Selectina E/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Persona de Mediana Edad , Factores de TiempoRESUMEN
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
Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to approximately 10 microg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1alpha and MIP-1beta. The release of these beta-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes.