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1.
Blood Rev ; 39: 100610, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31471128

RESUMO

Beta-2-Glycoprotein I (ß2GPI) plays a number of essential roles throughout the body. ß2GPI, C-reactive protein and thrombomodulin are the only three proteins that possess the dual capability to up and down regulate the complement and coagulation systems depending upon external stimulus. Clinically, ß2GPI is the primary antigen in the autoimmune condition antiphospholipid syndrome (APS), which is typically characterised by pregnancy morbidity and vascular thrombosis. This protein is also capable of adopting at least two distinct structural forms, but it has been argued that several other intermediate forms may exist. Thus, ß2GPI is a unique protein with a key role in haemostasis, homeostasis and immunity. In this review, we examine the genetics, structure and function of ß2GPI in the body and how these factors may influence its contribution to disease pathogenesis. We also consider the clinical implications of ß2GPI in the diagnosis of APS and as a potentially novel therapeutic target.

2.
Rheumatology (Oxford) ; 59(1): 146-152, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31257438

RESUMO

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.

3.
Sci Rep ; 9(1): 1283, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718722

RESUMO

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.

4.
Front Immunol ; 9: 2413, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405613

RESUMO

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.


Assuntos
Síndrome Antifosfolipídica/etiologia , Síndrome Antifosfolipídica/metabolismo , Coagulação Sanguínea , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Domínios e Motivos de Interação entre Proteínas , beta 2-Glicoproteína I/metabolismo , Adulto , Animais , Anticorpos Antifosfolipídeos/sangue , Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/diagnóstico , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Ligação Proteica , Domínios Proteicos , Trombose/sangue , Trombose/etiologia , Trombose/metabolismo , beta 2-Glicoproteína I/química
5.
Front Immunol ; 9: 2244, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30323817

RESUMO

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.


Assuntos
Anticorpos Antifosfolipídeos/sangue , Síndrome Antifosfolipídica/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Domínios Proteicos/imunologia , beta 2-Glicoproteína I/sangue , Adulto , Análise de Variância , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , beta 2-Glicoproteína I/imunologia
6.
J Autoimmun ; 93: 114-123, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30033000

RESUMO

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.


Assuntos
Síndrome Antifosfolipídica/genética , Imunoglobulina G/farmacologia , Monócitos/imunologia , Complicações na Gravidez/genética , Trombose/genética , Transcriptoma/imunologia , Adulto , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/patologia , Estudos de Casos e Controles , Adesão Celular , Comunicação Celular , Matriz Extracelular/química , Matriz Extracelular/imunologia , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Anotação de Sequência Molecular , Monócitos/efeitos dos fármacos , Monócitos/patologia , Gravidez , Complicações na Gravidez/imunologia , Complicações na Gravidez/patologia , Cultura Primária de Células , Trombose/imunologia , Trombose/patologia
7.
PLoS One ; 12(10): e0186513, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29049363

RESUMO

Domain I (DI) of beta-2-glycoprotein I (ß2GPI) contains the immunodominant epitope for pathogenic antiphospholipid antibodies (aPL). DI is exposed in the linear form of the molecule but not in the circular form that comprises 90% of serum ß2GPI. The majority of circulating ß2GPI is biochemically reduced with two free thiols in Domain V. However, increased levels of oxidised ß2GPI are found in patients with antiphospholipid syndrome (APS). It is not known whether oxidation of ß2GPI favours the linear form of the molecule and thus promotes development of anti-DI antibodies. We investigated whether the proportion of oxidised ß2GPI associates with the presence of anti-DI in APS patients. Serum samples from 44 APS patients were screened for IgG, IgM and IgA anti-DI, anti-ß2GPI, anti-cardiolipin (anti-CL) and biochemically reduced ß2GPI. A negative correlation was found between the proportion of ß2GPI in the biochemically reduced form and IgG anti-DI levels (r = -0.54, p = 0.0002), but not with IgM or IgA anti-DI. Moreover, the proportion of ß2GPI in the reduced form was lower in IgG anti-DI positive than anti-DI negative APS patients (p = 0.02). The relative amount of reduced ß2GPI was no different between patients who were positive or negative for IgG, IgM and IgA anti-ß2GPI or anti-CL. This study demonstrates that oxidised ß2GPI lacking free cysteine-thiol groups most closely associates with IgG anti-DI positivity compared to IgG anti-CL and anti-ß2GPI. Future studies are required to ascertain the directionality of this association to define causation.


Assuntos
Síndrome Antifosfolipídica/imunologia , Autoanticorpos/imunologia , Imunoglobulina G/imunologia , beta 2-Glicoproteína I/metabolismo , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução
8.
Sci Rep ; 7(1): 10788, 2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28883515

RESUMO

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.


Assuntos
Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Cálcio/metabolismo , Células Endoteliais/metabolismo , Fator Xa/metabolismo , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Biomarcadores , Estudos de Casos e Controles , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Espaço Intracelular/metabolismo , Masculino , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos
9.
Cell Death Dis ; 8(1): e2549, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-28079888

RESUMO

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.


Assuntos
Anticorpos Antifosfolipídeos/genética , Infarto do Miocárdio/genética , beta 2-Glicoproteína I/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Adulto , Animais , Anticorpos Antifosfolipídeos/metabolismo , Apoptose/genética , Hipóxia Celular/genética , Modelos Animais de Doenças , Humanos , Imunoglobulina G/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologia , beta 2-Glicoproteína I/genética
10.
PLoS One ; 11(6): e0156407, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27253369

RESUMO

INTRODUCTION: Currently available clinical assays to detect antiphospholipid antibodies (aPL) test for IgG and IgM antibodies to cardiolipin (aCL) and ß2-glycoprotein I (aß2GPI). It has been suggested that testing for IgA aPL and for antibodies to Domain I (DI), which carries the key antigenic epitopes of ß2GPI, could add value to these current tests. We performed an observational, multicenter cohort study to evaluate the utility of IgG, IgM and IgA assays to each of CL, ß2GPI and DI in APS. METHODS: Serum from 230 patients with APS (n = 111), SLE but not APS (n = 119), and 200 healthy controls were tested for IgG, IgM and IgA aCL, aß2GPI and aDI activity. Patients with APS were further classified into thrombotic or obstetric APS. Logistic regression and receiver operator characteristic analyses were employed to compare results from the nine different assays. RESULTS: All assays displayed good specificity for APS; IgG aCL and IgG aß2GPI assays however, had the highest sensitivity. Testing positive for IgA aß2GPI resulted in a higher hazard ratio for APS compared to IgM aß2GPI. Positive IgG, IgM or IgA aDI were all associated with APS, and in subjects positive for aCL and/or aß2GPI, the presence of aDI raised the hazard ratio for APS by 3-5 fold. IgG aCL, aß2GPI, aDI and IgA aDI were associated with thrombotic but not obstetric complications in patients with APS. CONCLUSION: Measuring IgG aDI and IgA aß2GPI and aDI may be useful in the management of patients with APS, particularly thrombotic APS.


Assuntos
Anticorpos Anticardiolipina/sangue , Anticorpos Antifosfolipídeos/isolamento & purificação , Síndrome Antifosfolipídica/sangue , Trombose/sangue , beta 2-Glicoproteína I/isolamento & purificação , Adulto , Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/patologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Gravidez , Testes Sorológicos , Trombose/imunologia , beta 2-Glicoproteína I/imunologia
11.
Mol Immunol ; 75: 161-7, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27289032

RESUMO

Antiphospholipid antibodies (aPL) occur in patients with multiple sclerosis (MS) with a number of studies reporting elevated levels; their exact prevalence and pathogenic role remain unclear. Epidemiological studies associate MS with an increased risk of deep venous thromboembolism and stroke; overlapping clinical features with APS. Antibodies against the first domain - Domain I (DI) - of ß2glycoprotein I (ß2GPI), show the most clinical significance and evidence for pathogenicity in the antiphospholipid syndrome (APS), but have not yet been investigated in MS. Serum from a well-defined cohort of 127 MS patients and 92 healthy controls were tested for IgM and IgG antibodies against cardiolipin (CL), ß2GPI and DI. Higher frequency of IgM and IgG anti-CL were found in MS patients (18.1% and 21.3%), compared to controls (1.1% in both cases, p<0.0001). We report that anti-DI antibodies were associated with MS patients, with 6.3% and 7.1% positive for IgM and IgG, respectively, compared to controls, 1.1% (p<0.05). IgM anti-CL antibodies were elevated in secondary progressive MS and primary progressive MS compared to relapse-remitting MS, (p<0.005). This study enrolled the largest number of patients with definite MS for studying the association with aPL. Although we confirmed IgM and IgG anti-CL antibodies occur in patients with MS, this is the first study that identified anti-DI antibodies in MS patients. This new finding may prove valuable and future studies are required to evaluate its role as a potential risk factor of thromboembolic phenomena in MS.


Assuntos
Anticorpos Anticardiolipina/imunologia , Autoanticorpos/imunologia , Cardiolipinas/imunologia , Esclerose Múltipla/imunologia , beta 2-Glicoproteína I/imunologia , Adulto , Idoso , Anticorpos Anticardiolipina/sangue , Autoanticorpos/sangue , Autoantígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
12.
PLoS One ; 10(12): e0143771, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26636577

RESUMO

An increasing number of investigations including human studies demonstrate that pharmacological ischaemic preconditioning is a viable way to protect the heart from myocardial ischaemia/reperfusion (I/R) injury. This study investigated the role of hydroxychloroquine (HCQ) in the heart during I/R injury. In vitro and in vivo models of myocardial I/R injury were used to assess the effects of HCQ. It was found that HCQ was protective in neonatal rat cardiomyocytes through inhibition of apoptosis, measured by TUNEL and cleaved caspase-3. This protection in vitro was mediated through enhancement of ERK1/2 phosphorylation mediated by HCQ in a dose-dependent fashion. A decrease in infarct size was observed in an in vivo model of myocardial I/R injury in HCQ treated animals and furthermore this protection was blocked in the presence of the ERK1/2 inhibitor U0126. For the first time, we have shown that HCQ promotes a preconditioning like protection in an in vivo simulated rat myocardial I/R injury model. Moreover, it was shown that HCQ is protective via enhanced phosphorylation of the pro-survival kinase ERK1/2.


Assuntos
Hidroxicloroquina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Musculares/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Animais , Butadienos/farmacologia , Caspase 3/metabolismo , Células Cultivadas , Humanos , Masculino , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Musculares/antagonistas & inibidores , Traumatismo por Reperfusão Miocárdica/enzimologia , Nitrilos/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
13.
BMC Biotechnol ; 15: 104, 2015 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-26576675

RESUMO

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.


Assuntos
Síndrome Antifosfolipídica/tratamento farmacológico , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , beta 2-Glicoproteína I/isolamento & purificação , beta 2-Glicoproteína I/metabolismo , Adulto , Automação Laboratorial , Escherichia coli/genética , Feminino , Humanos , Corpos de Inclusão , Masculino , Pessoa de Meia-Idade , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , beta 2-Glicoproteína I/genética , beta 2-Glicoproteína I/uso terapêutico
14.
Rheumatology (Oxford) ; 54(10): 1876-81, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26044042

RESUMO

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.


Assuntos
Antirreumáticos/farmacologia , Antirreumáticos/uso terapêutico , Apoptose/efeitos dos fármacos , Linfócitos B/patologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Rituximab/farmacologia , Rituximab/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/efeitos dos fármacos , Contagem de Células , Ciclofosfamida/farmacologia , Ciclofosfamida/uso terapêutico , Quimioterapia Combinada , Feminino , Seguimentos , Humanos , Incidência , Modelos Logísticos , Londres , Lúpus Eritematoso Sistêmico/patologia , Linfopenia/epidemiologia , Masculino , Metilprednisolona/farmacologia , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento
15.
Arthritis Res Ther ; 17: 47, 2015 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-25890027

RESUMO

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.


Assuntos
Síndrome Antifosfolipídica/imunologia , Autoanticorpos/sangue , Coagulação Sanguínea , Fator Xa/imunologia , Adulto , Síndrome Antifosfolipídica/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
16.
Am J Reprod Immunol ; 73(5): 390-401, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25469631

RESUMO

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.


Assuntos
Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Imunoglobulina G/imunologia , Complicações na Gravidez/imunologia , Trofoblastos/imunologia , Adulto , Anticorpos Antifosfolipídeos/sangue , Anticorpos Antifosfolipídeos/isolamento & purificação , Síndrome Antifosfolipídica/sangue , Síndrome Antifosfolipídica/patologia , Linhagem Celular , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/isolamento & purificação , Gravidez , Complicações na Gravidez/sangue , Complicações na Gravidez/patologia , Trofoblastos/patologia
17.
Rheumatology (Oxford) ; 54(4): 722-7, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25273993

RESUMO

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.


Assuntos
Anticorpos Antifosfolipídeos/farmacologia , Síndrome Antifosfolipídica/induzido quimicamente , Modelos Animais de Doenças , Imunoglobulina G/farmacologia , Camundongos , Trombose/induzido quimicamente , beta 2-Glicoproteína I/imunologia , Animais , Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/complicações , Síndrome Antifosfolipídica/imunologia , Imunoglobulina G/imunologia , Masculino , Estrutura Terciária de Proteína , Trombose/complicações , Trombose/imunologia
19.
Arthritis Res Ther ; 16(1): R48, 2014 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-24502558

RESUMO

INTRODUCTION: Circulating nucleosomes released from apoptotic cells are important in the pathogenesis of systemic lupus erythematosus (SLE). Both nucleosomes and anti-nucleosome antibodies are deposited in inflamed tissues in patients with SLE. Active inflammation promotes nitration of tyrosine residues on serum proteins. Our hypothesis was that levels of nitrated nucleosomes would be elevated in patients with SLE and could be associated with disease activity. We therefore carried out a retrospective longitudinal study to investigate factors affecting levels of nitrated nucleosomes (NN) in patients with SLE. METHODS: A novel serum ELISA was developed to measure serum NN and modified to measure serum nitrated albumin (NA). Levels of both NN and NA were measured in 397 samples from 49 patients with SLE followed through periods of disease flare and remission for a mean of 89 months. Anti-nucleosome antibody (anti-nuc) levels were measured in the same samples. The effects of 24 different clinical, demographic and serological variables on NN, NA and anti-nuc levels were assessed by univariable and multivariable analysis. RESULTS: Patients with SLE had higher mean NN than healthy controls or patients with other autoimmune rheumatic diseases (P =0.01). Serum samples from 18 out of 49 (36.7%) of SLE patients were never positive for NN. This group of 18 patients was characterized by lower anti-double stranded DNA antibodies (anti-dsDNA), disease activity and use of immunosuppressants. In the remaining 63.3%, NN levels were variable. High NN was significantly associated with anti-Sm antibodies, vasculitis, immunosuppressants, hydroxychloroquine and age at diagnosis. NN levels were raised in neuropsychiatric flares. NN levels did not completely parallel NA results, thus providing additional information over measuring nitration status alone. NN levels were not associated with anti-nuc levels. CONCLUSIONS: NN are raised in a subset of patients with SLE, particularly those who are anti-Sm positive. Elevated NN may be a marker of vascular activation and neuropsychiatric flares in these patients.


Assuntos
Autoanticorpos/sangue , Lúpus Eritematoso Sistêmico/sangue , Nucleossomos/imunologia , Adulto , Autoanticorpos/imunologia , Autoantígenos/imunologia , Biomarcadores/sangue , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Estudos Longitudinais , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Estudos Retrospectivos
20.
Methods Mol Biol ; 1134: 221-35, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24497366

RESUMO

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.


Assuntos
Síndrome Antifosfolipídica/diagnóstico , Testes Diagnósticos de Rotina/métodos , Anticorpos Anticardiolipina/imunologia , Síndrome Antifosfolipídica/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Inibidor de Coagulação do Lúpus/imunologia , beta 2-Glicoproteína I/imunologia
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