Thomas McDonnell

Measuring IgA Anti-β2-Glycoprotein I and IgG/IgA Anti-Domain I Antibodies Adds Value to Current Serological Assays for the Antiphospholipid Syndrome

Introduction Currently available clinical assays to detect antiphospholipid antibodies (aPL) test for IgG and IgM antibodies to cardiolipin (aCL) and β2-glycoprotein I (aβ2GPI). It has been suggested that testing for IgA aPL and for antibodies to Domain I (DI), which carries the key antigenic epitopes of β2GPI, 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, β2GPI 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, aβ2GPI 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 aβ2GPI assays however, had the highest sensitivity. Testing positive for IgA aβ2GPI resulted in a higher hazard ratio for APS compared to IgM aβ2GPI. Positive IgG, IgM or IgA aDI were all associated with APS, and in subjects positive for aCL and/or aβ2GPI, the presence of aDI raised the hazard ratio for APS by 3–5 fold. IgG aCL, aβ2GPI, aDI and IgA aDI were associated with thrombotic but not obstetric complications in patients with APS. Conclusion Measuring IgG aDI and IgA aβ2GPI and aDI may be useful in the management of patients with APS, particularly thrombotic APS.

PEGylated drugs in rheumatology—why develop them and do they work?

Lack of efficacy and drug-related adverse effects are important reasons for the discontinuation of treatment in patients with rheumatic diseases. The development of new biologic therapies seeks to address these problems by specifically targeting the pathogenic mechanisms of disease. Most current biologics are proteins (particularly antibodies and enzymes) administered parenterally. It is important to optimize properties such as serum half-life, immunogenicity and solubility. Companies have thus begun to modify the drugs by conjugate chemistry, binding inert molecules such as polyethylene glycol (PEG) to biologic molecules to improve their pharmacodynamic properties. The use of PEG to alter these properties has to be weighed against the negative aspects of PEGylation, such as decreased activity and heterogeneity. This review focuses on the currently available PEGylated drugs used in rheumatological diseases, their efficacy, drawbacks and the current clinical trial evidence supporting their use.

A sestrin-dependent Erk-Jnk-p38 MAPK activation complex inhibits immunity during aging

Mitogen-activated protein kinases (MAPKs) including Erk, Jnk and p38 regulate diverse cellular functions and are thought to be controlled by independent upstream activation cascades. Here we show that the sestrins bind to and coordinate simultaneous Erk, Jnk and p38 MAPK activation in T lymphocytes within a new immune-inhibitory complex (sestrin–MAPK activation complex (sMAC)). Whereas sestrin ablation resulted in broad reconstitution of immune function in stressed T cells, inhibition of individual MAPKs allowed only partial functional recovery. T cells from old humans (>65 years old) or mice (16–20 months old) were more likely to form the sMAC, and disruption of this complex restored antigen-specific functional responses in these cells. Correspondingly, sestrin deficiency or simultaneous inhibition of all three MAPKs enhanced vaccine responsiveness in old mice. Thus, disruption of sMAC provides a foundation for rejuvenating immunity during aging.

Antiphospholipid antibodies enhance rat neonatal cardiomyocyte apoptosis in an in vitro hypoxia/reoxygenation injury model via p38 MAPK

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 (β2GPI). 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–β2GPI 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 β2GPI in the fluid phase, suggesting that the pathogenic anti-β2GPI antibodies in this in vitro model target this domain.

Antibodies against TFPI and protein C are associated with a severe thrombotic phenotype in patients with and without antiphospholipid syndrome

BACKGROUND Tissue factor pathway inhibitor (TFPI) antibodies, which have been reported in patients with antiphospholipid syndrome (APS), may impair TFPI activity and contribute to hypercoagulability, but their role in APS and in thrombosis remains undefined. OBJECTIVE/METHODS We assessed the presence and avidity of TFPI IgG antibodies, associations with protein C IgG antibodies and associations with clinical disease severity, in 50 patients with thrombotic APS and 50 thrombotic control patients, on long term anticoagulation with warfarin. RESULTS Thrombotic APS patients had a significantly higher prevalence of TFPI IgG antibodies (40%; 20/50) compared to thrombotic controls (18%; 9/50). TFPI antibodies were predominantly high avidity in APS (50%, 10/20 of positive patients) and strongly associated with a severe thrombotic phenotype (venous and arterial thromboembolism or recurrent thromboembolic episodes despite therapeutic anticoagulation) (odds ratio (OR): 12.0, 95%CI: 2.2-66.1, p = 0.004), while thrombotic control patients mainly showed low avidity antibodies (78%, 7/9 of positive patients). Coexistence of TFPI and protein C IgG antibodies, regardless of their avidity, was strongly associated with a more severe thrombotic phenotype in APS patients (OR: 20.2, 95%CI: 2.0-47.0, p < 0.0001) and also in thrombotic controls (OR: 75.0, 95%CI 1.2-195, p = 0.02). CONCLUSIONS Coexistent TFPI and protein C IgG antibodies, irrespective of their avidity, may be a useful marker for a severe thrombotic phenotype in thrombotic patients. This suggests a possibly pathophysiological relationship between the two antibodies, predisposing to thrombosis with a possibly more general role in the development of thrombotic complications.

Going viral in rheumatology: using social media to show that mechanistic research is relevant to patients with lupus and antiphospholipid syndrome

Abstract Objectives There is a lack of published data regarding patient interaction in basic scientific research, including methodologies for simple, cost-effective interactions and the outcomes of such studies. Therefore, we aimed to evaluate the ease of generating patient opinion data on specific scientific research projects whilst establishing a template for other groups to follow. Our secondary objective was to assess which research topics are of most interest to patients with SLE and/or APS. Methods Through patient-based interactions, we developed a lay summary of a mechanistic research proposal and a set of associated questions to assess patient opinion on this research topic. We disseminated the questions as an online survey with associated lay summary through patient-based charity websites and social media. The survey was open for 3 weeks. Results Of 527 respondents, 520 reported having SLE or APS. The patient response to the research proposal was overwhelmingly positive, with the majority expressing strong interest in the mechanistic aspect of the project. Analysis of free text box responses confirmed that the most popular research topics for patients were as follows: treatment, genetics, triggers, diagnosis and mechanistic research. Interestingly, patient interest in disease mechanisms featured more frequently than clinical topics, such as management of disease flares. Conclusion It is possible to conduct short-term, valuable patient engagement at low cost, using an online survey and social media. This methodology may form a good template for future patient engagement. The volume and distribution of positive response shows that patients are interested in mechanistic research.

PS5:99 Examining the modulatory effects of anti-serine protease antibodies upon factor xa, thrombin and complement interactions

Purpose Serine protease (SP) enzymes play a critical role in both the coagulation and complement cascades. Anti-SP antibodies are found in approximately 40% of patients systemic lupus erythematosus (SLE) and antiphospholipid Syndrome (APS). Complement activation is important in SLE and APS. However, little is known about the effects of anti-SP antibodies on complement activation in these diseases. We sought to investigate whether affinity-purified antibodies to the SP Factor(F)Xa and Thrombin (Thr) alter the effects of these SP on complement cleavage in the presence or absence of the physiological inhibitor anti-thrombin (AT). Methods Serum was obtained by informed consent from patients with SLE (n=10) and/or APS (n=2) under long-term follow-up at University College London Hospital. A novel method was developed to affinity purify anti-FXa and anti-Thr IgG separately. We incubated FXa (2 µM) and Thr (2.7 µM) separately with complement component C3 to determine baseline C3 cleavage. We then repeated the experiment in the presence of AT3, the appropriate anti-SP (anti-FXa or anti-Thr) or both. Finally we reviewed medical records of 40 patients with SLE to determine whether low C3 was associated with seropositivity for anti-FXa and/or anti-Thr in these patients. Results Both FXa and Thr cleaved C3 into C3a/C3 b. This was inhibited by AT, though both cleavage and inhibition by AT were stronger for Thr than FXa. Conversely anti-SP antibodies enhanced C3 cleavage by SP. Anti-FXa IgG (n=3) increased FXa-mediated cleavage of C3 by 1.3-fold. Anti-Thr IgG (n=8) increased Thr-mediated C3 cleavage by 1.8-fold. AT mediated inhibition was prevented by addition of anti-FXa IgG (n=2) but not anti-Thr IgG. Analysis of clinical serology records for the last 10 consecutive clinic visits of 28 anti-SP-positive patients showed a lower level of C3 (0.92 g/L) in the patients double positive for anti-FXa and anti-Thr than for anti-Thr alone (1.12 g/L) or anti-FXa alone (1.16 g/L). Conclusions Anti-FXa and anti-Thr enhance cleavage of C3 by FXa and Thr respectively. Presence of these antibodies in-vivo in patients with SLE and/or APS may promote increased complement activation and disease activity. This finding may have potential translational implications for future treatment of these diseases.Abstract PS5:99 Figure 1 Data showing relative C3 cleavage of Thr and FXa in the presence and absence of antibodies and inhibitors

S7D:6 Going viral in rheumatology: a rapid, cost-effective method of obtaining patient opinion about research in sle and aps

Purpose It is important to access opinions from patients in designing research into systemic lupus erythematous (SLE) and/or antiphospholipid syndrome (APS). It is difficult to obtain useful information from large numbers of unselected patients in a short period of time. There is a lack of published research about how to achieve this objective. On-line surveys and use of social media offer a potential method to address this challenge. We developed a novel approach to access patient opinion regarding key objectives for mechanistic research in SLE and APS. Methods We developed a one-page lay summary of a research project concerning investigation of serine proteases in patients with APS and SLE. This is a mechanistic laboratory project with potential future relevance to management of these diseases. Both the lay summary and an accompanying 9-question survey were refined with the help of an expert patient and patients’ charities, then disseminated as an online survey. The survey was open for four weeks, and was publicised via social media (Twitter, Facebook) and through the websites of LUPUS UK and APS Support UK. The survey data were then analysed and total project cost was £26. Results Of 527 respondents, 520 confirmed having been diagnosed with SLE and/or APS. The majority of respondents were very positive about the research, expressing strong interest in its mechanistic basis. We provided a free text box for respondents to express their opinions about the most important research topics in SLE and APS. 277 respondents provided free text comments. The most popular research topics were Treatment (mentioned by n=86 respondents), Genetics (n=56), Triggers (n=50), Diagnosis (n=38) and Mechanistic Research (n=28). Interestingly, patients expressed more interest in disease mechanisms than in clinical topics such as management of disease flares. Conclusions It is possible to conduct short term, valuable patient engagement at low cost, using an online survey and social media. This methodology may form a good template for future patient engagement. Our results suggest that patients are interested in mechanistic research.Abstract S7D:6 Figure 1 Patients responses grouped for subject matter, this demonstrates the most mentioned and least mentioned research topics

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

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

Antiphospholipid Antibodies to Domain I of Beta-2-Glycoprotein I Show Different Subclass Predominance in Comparison to Antibodies to Whole Beta-2-glycoprotein I

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 (β2GPI) 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.