Elizabeth Papalardo

Markers of Oxidative and Nitrosative Stress in Systemic Lupus Erythematosus: Correlation with Disease Activity

OBJECTIVE Free radical-mediated reactions have been implicated as contributors in a number of autoimmune diseases, including systemic lupus erythematosus (SLE). However, the potential for oxidative/nitrosative stress to elicit an autoimmune response or to contribute to disease pathogenesis, and thus be useful when determining a prognosis, remains largely unexplored in humans. This study was undertaken to investigate the status and contribution of oxidative/nitrosative stress in patients with SLE. METHODS Sera from 72 SLE patients with varying levels of disease activity according to the SLE Disease Activity Index (SLEDAI) and 36 age- and sex-matched healthy controls were evaluated for serum levels of oxidative/nitrosative stress markers, including antibodies to malondialdehyde (anti-MDA) protein adducts and to 4-hydroxynonenal (anti-HNE) protein adducts, MDA/HNE protein adducts, superoxide dismutase (SOD), nitrotyrosine (NT), and inducible nitric oxide synthase (iNOS). RESULTS Serum analysis showed significantly higher levels of both anti-MDA/anti-HNE protein adduct antibodies and MDA/HNE protein adducts in SLE patients compared with healthy controls. Interestingly, not only was there an increased number of subjects positive for anti-MDA or anti-HNE antibodies, but also the levels of both of these antibodies were statistically significantly higher among SLE patients whose SLEDAI scores were > or = 6 as compared with SLE patients with lower SLEDAI scores (SLEDAI score <6). In addition, a significant correlation was observed between the levels of anti-MDA or anti-HNE antibodies and the SLEDAI score (r = 0.734 and r = 0.647, respectively), suggesting a possible causal relationship between these antibodies and SLE. Furthermore, sera from SLE patients had lower levels of SOD and higher levels of iNOS and NT compared with healthy control sera. CONCLUSION These findings support an association between oxidative/nitrosative stress and SLE. The stronger response observed in serum samples from patients with higher SLEDAI scores suggests that markers of oxidative/nitrosative stress may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.

Annexin A2 is involved in antiphospholipid antibody-mediated pathogenic effects in vitro and in vivo.

Antiphospholipid (aPL) antibodies recognize receptor-bound beta(2) glycoprotein I (beta(2)GPI) on target cells, and induce an intracellular signaling and a procoagulant/proinflammatory phenotype that leads to thrombosis. Evidence indicates that annexin A2 (A2), a receptor for tissue plasminogen activator and plasminogen, binds beta(2)GPI on target cells. However, whether A2 mediates pathogenic effects of aPL antibodies in vivo is unknown. In this work, we studied the effects of human aPL antibodies in A2-deficient (A2(-/-)) mice. A2(-/-) and A2(+/+) mice were injected with immunoglobulin G (IgG) isolated from either a patient with antiphospholipid syndrome (IgG-APS), a healthy control subject (IgG-normal human serum), a monoclonal anti-beta(2)GPI antibody (4C5), an anti-A2 monoclonal antibody, or monoclonal antibody of irrelevant specificity as control. We found that, after IgG-APS or 4C5 injections and vascular injury, mean thrombus size was significantly smaller and tissue factor activity was significantly less in A2(-/-) mice compared with A2(+/+) mice. The expression of vascular cell adhesion molecule-1 induced by IgG-APS or 4C5 in explanted A2(-/-) aorta was also significantly reduced compared with A2(+/+) mice. Interestingly, anti-A2 monoclonal antibody significantly decreased aPL-induced expression of intercellular cell adhesion molecule-1, E-selectin, and tissue factor activity on cultured endothelial cells. Together, these data indicate for the first time that A2 mediates the pathogenic effects of aPL antibodies in vivo and in vitro APS.

In vivo inhibition of antiphospholipid antibody‐induced pathogenicity utilizing the antigenic target peptide domain I of β2‐glycoprotein I: proof of concept

Summary.  Objectives: In the antiphospholipid syndrome (APS), the immunodominant epitope for the majority of circulating pathogenic antiphospholipid antibodies (aPLs) is the N‐terminal domain I (DI) of β2‐glycoprotein I. We have previously shown that recombinant DI inhibits the binding of aPLs in fluid phase to immobilized native antigen, and that this inhibition is greater with the DI(D8S/D9G) mutant and absent with the DI(R39S) mutant. Hence, we hypothesized that DI and DI(D8S/D9G) would inhibit aPL‐induced pathogenicity in vivo. Methods: C57BL/6 mice (n = 5, each group) were injected with purified IgG derived from APS patients (IgG‐APS, 500 μg) or IgG from normal healthy serum (IgG‐NHS) and either recombinant DI, DI(R39S), DI(D8S/D9G), or an irrelevant control peptide (at 10–40 μg). Outcome variables measured were femoral vein thrombus dynamics in treated and control groups following standardized vessel injury, expression of vascular cell adhesion molecule‐1 (VCAM‐1) on the aortic endothelial surface, and tissue factor (TF) activity in murine macrophages. Results: IgG‐APS significantly increased thrombus size as compared with IgG‐NHS. The IgG‐APS thrombus enhancement effect was abolished in mice pretreated with recombinant DI (P ≤ 0.0001) and DI(D8S/D9G) (P ≤ 0.0001), but not in those treated with DI(R39S) or control peptide. This inhibitory effect by DI was dose‐dependent, and at lower doses DI(D8S/D9G) was a more potent inhibitor of thrombosis than wild‐type DI (P ≤ 0.01). DI also inhibited IgG‐APS induction of VCAM‐1 on the aortic endothelial surface and TF production by murine macrophages. Conclusion: Our findings in this proof‐of‐concept study support the development of recombinant DI or the novel variant DI(D8S/D9G) as a potential future therapeutic agent for APS.

Effect of hydroxychloroquine treatment on pro-inflammatory cytokines and disease activity in SLE patients: data from LUMINA (LXXV), a multiethnic US cohort:

Objective: We sought to determine the effect of hydroxychloroquine therapy on the levels proinflammatory/prothrombotic markers and disease activity scores in patients with systemic lupus erythematosus (SLE) in a multiethnic, multi-center cohort (LUMINA). Methods: Plasma/serum samples from SLE patients (n = 35) were evaluated at baseline and after hydroxychloroquine treatment. Disease activity was assessed using SLAM-R scores. Interferon (IFN)-α2, interleukin (IL)-1β, IL-6, IL-8, inducible protein (IP)-10, monocyte chemotactic protein-1, tumor necrosis factor (TNF)-α and soluble CD40 ligand (sCD40L) levels were determined by a multiplex immunoassay. Anticardiolipin antibodies were evaluated using ELISA assays. Thirty-two frequency-matched plasma/serum samples from healthy donors were used as controls. Results: Levels of IL-6, IP-10, sCD40L, IFN-α and TNF-α were significantly elevated in SLE patients versus controls. There was a positive but moderate correlation between SLAM-R scores at baseline and levels of IFN-α (p = 0.0546). Hydroxychloroquine therapy resulted in a significant decrease in SLAM-R scores (p = 0.0157), and the decrease in SLAM-R after hydroxychloroquine therapy strongly correlated with decreases in IFN-α (p = 0.0087). Conclusions: Hydroxychloroquine therapy resulted in significant clinical improvement in SLE patients, which strongly correlated with reductions in IFN-α levels. This indicates an important role for the inhibition of endogenous TLR activation in the action of hydroxychloroquine in SLE and provides additional evidence for the importance of type I interferons in the pathogenesis of SLE. This study underscores the use of hydroxychloroquine in the treatment of SLE.

A prospective open-label pilot study of fluvastatin on proinflammatory and prothrombotic biomarkers in antiphospholipid antibody positive patients

Objective To determine if proinflammatory and prothrombotic biomarkers are differentially upregulated in persistently antiphospholipid antibody (aPL)-positive patients, and to examine the effects of fluvastatin on these biomarkers. Methods Four groups of patients (age 18–65) were recruited: (a) primary antiphospholipid syndrome; (b) systemic lupus erythematosus (SLE) with antiphospholipid syndrome (APS) (SLE/APS); (c) persistent aPL positivity without SLE or APS (Primary aPL); and (d) persistent aPL positivity with SLE but no APS (SLE/aPL). The frequency-matched control group, used for baseline data comparison, was identified from a databank of healthy persons. Patients received fluvastatin 40 mg daily for 3 months. At 3 months, patients stopped the study medication and they were followed for another 3 months. Blood samples for 12 proinflammatory and prothrombotic biomarkers were collected monthly for 6 months. Results Based on the comparison of the baseline samples of 41 aPL-positive patients with 30 healthy controls, 9/12 (75%) biomarkers (interleukin (IL)-6, IL1β, vascular endothelial growth factor (VEGF), tumour necrosis factor (TNF)-α, interferon (IFN)-α, inducible protein-10 (IP10), soluble CD40 ligand (sCD40L), soluble tissue factor (sTF) and intracellular cellular adhesion molecule (ICAM)-1) were significantly elevated. Twenty-four patients completed the study; fluvastatin significantly and reversibly reduced the levels of 6/12 (50%) biomarkers (IL1β, VEGF, TNFα, IP10, sCD40L and sTF). Conclusions Our prospective mechanistic study demonstrates that proinflammatory and prothrombotic biomarkers, which are differentially upregulated in persistently aPL-positive patients, can be reversibly reduced by fluvastatin. Thus, statin-induced modulation of the aPL effects on target cells can be a valuable future approach in the management of aPL-positive patients.

Value of isolated IgA anti-β2 -glycoprotein I positivity in the diagnosis of the antiphospholipid syndrome.

OBJECTIVE To examine the prevalence of isolated IgA anti-β2 -glycoprotein I (anti-β2 GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V of β2 GPI, with clinical manifestations of the antiphospholipid syndrome (APS) in 3 patient groups and to evaluate the pathogenicity of IgA anti-β2 GPI in a mouse model of thrombosis. METHODS Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture [LUMINA]) (n = 558), patients with SLE from the Hopkins Lupus Cohort (n = 215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n = 5,098) were evaluated. IgA anti-β2 GPI titers and binding to domain IV/V of β2 GPI were examined by enzyme-linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti-β2 GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity. RESULTS A total of 198 patients were found to be positive for IgA anti-β2 GPI isotype, and 57 patients were positive exclusively for IgA anti-β2 GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS-related clinical manifestation. Fifty-four percent of all the IgA anti-β2 GPI-positive serum samples reacted with domain IV/V of anti-β2 GPI, and 77% of those had clinical features of APS. Isolated IgA anti-β2 GPI positivity was associated with an increased risk of arterial thrombosis (P < 0.001), venous thrombosis (P = 0.015), and all thrombosis (P < 0.001). The association between isolated IgA anti-β2 GPI and arterial thrombosis (P = 0.0003) and all thrombosis (P = 0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti-β2 GPI antibodies induced significantly larger thrombi and higher TF levels compared to controls. CONCLUSION Isolated IgA anti-β2 GPI-positive titers may identify additional patients with clinical features of APS. Testing for these antibodies when other antiphospholipid tests are negative and APS is suspected is recommended. IgA anti-β2 GPI antibodies directed to domain IV/V of β2 GPI represent an important subgroup of clinically relevant antiphospholipids.

Statins for the treatment of antiphospholipid syndrome

Fluvastatin has been shown to revert proinflammatory/prothrombotic effects of antiphospholipid antibodies (aPL) in vitro and in mice. Here, we examined whether fluvastatin affects the levels of proinflammatory/prothrombotic markers in antiphospholipid syndrome (APS) patients. Vascular endothelial growth factor (VEGF), soluble tissue factor (sTF), tumor necrosis factor‐α (TNF‐α), soluble intercellular adhesion molecule‐1 (sICAM‐1), sE‐selectin (E‐sel), C‐reactive protein (CRP), and soluble vascular cell adhesion molecule (sVCAM‐1), were measured in the sera of 93 APS patients and 60 controls and in the sera of nine patients with APS before and after 30 days of treatment with fluvastatin. Elevated levels of VEGF, sTF, and TNF‐α were found in APS patients. Fluvastatin significantly reduced those markers in the majority of treated subjects. The data from this study show that statins may be beneficial in aPL‐positive patients and warrant larger clinical trials to confirm the efficacy of the drug for the treatment of APS clinical manifestations.

Isolated elevation of IgA anti-β2glycoprotein I antibodies with manifestations of antiphospholipid syndrome: a case series of five patients

Current diagnostic classification criteria recommend elevated titres of anti-cardiolipin (aCL) and/or anti-β2GPI antibody by ELISA IgG or IgM and/or lupus anticoagulant (LA) to confirm antiphospholipid syndrome (APS). Although IgA aPL antibodies have been shown to be pathogenic in animal models of APS, their clinical significance has remained elusive. We report four cases of exclusive IgA anti-β2GPI antibody sero-positivity with concomitant clinical manifestations associated with APS. Four of the five patients were LA negative. 1) Thirty-eight-year-old African-American female with SLE presented with resolving digital ulcers. Serum IgA anti-β2GPI antibody titres were 118.5 SAU (normal range: 0–20 SAU). 2) Twenty-seven-year-old African-American woman with SLE was evaluated for recent onset of severe headaches, unresponsive to analgesics and anti-migraine medications. MRI of the brain revealed hyper-intensities in the white matter in the frontal lobes. Serum IgA anti-β2GPI antibody titres were 29.1 Standard A Units (SAU). 3) Thirty-two-year-old Hispanic female with history of two unexplained miscarriages and negative serologies for SLE. Serum IgA anti-β2GPI antibody titres were 102.0 SAU. 4) Twenty-five-year-old white female with history of recent unexplained miscarriage in the 11th week of gestation and associated complaints of numbness and tingling in her hands. Her IgA anti-β2GPI antibody titre was 62.0 SAU. 5) Twenty-five-year-old African-American woman with SLE, positive for anti-Ro antibodies with a history of ischemic fingers, a pregnancy loss and recent pregnancy complicated due to pre-eclampsia. Her LA was positive and her IgA anti-β2GPI antibody titer was 186.0 SAU. This case series supports that elevated IgA anti-β2GPI antibody titres may identify additional patients who have clinical features of APS but who do not meet current diagnostic criteria.

C6 knock-out mice are protected from thrombophilia mediated by antiphospholipid antibodies

Background: Complement activation plays a role in pathogenesis of the antiphospholipid syndrome (APS), but the involvement of the C5b-9 membrane attack complex (MAC) is unknown. Here we studied the effects of human polyclonal antiphospholipid (aPL) antibodies on thrombosis and tissue factor (TF) up-regulation in C6 deficient (C6−/−) mice. Methods: C6−/− mice or the wild-type C3H/HeJ (C6+/+) mice were injected twice with IgG-APS (n = 2) or IgM-APS (n = 1) isolated from APS patients or with the corresponding control immunoglobulins (Igs) of normal human serum, (NHS) (IgG-NHS or IgM-NHS). Then, the sizes of induced thrombi in the femoral vein were determined 72 hours after the first injection. Tissue factor was determined in homogenates of carotid arteries and in peritoneal macrophages. Results: Thrombus sizes were significantly larger in C6+/+ treated with IgG-APS1 or with IgG-APS2 or with IgM-APS when compared with C6+/+ mice treated with IgG-NHS or with IgM-NHS, respectively. The sizes of thrombi were significantly smaller in the C6−/− mice injected with IgG-APS1, IgG-APS2 or IgM-APS (p < 0.001), compared to their C6+/+ counterparts showing an important abrogation of thrombus formation in mice lacking C6. The TF expression and activity in the C6−/− mice treated with IgG-APS or IgM-APS were diminished when compared to C3H/HeJ (C6+/+) mice treated with the same Igs. All mice injected with IgG-APS and IgM-APS had medium-high titers of anticardiolipin (aCL) and anti-β2glycoprotein I (aβ2GPI) antibodies. Conclusions: These data indicate that the C6 component of the complement system mediates aPL-thrombogenic effects, underscoring an important pathogenic mechanism and indicating the possibility of inhibiting complement to ameliorate APS-related manifestations.

A unique antiphospholipid assay recognizing phospholipid mixture compared with criteria antiphospholipid immunoassays in lupus patients

Background While essential for the classification of antiphospholipid syndrome (APS), anticardiolipin (aCL) assays lack specificity and anti-β2glycoproteinI (anti-β2GPI) assays lack sensitivity in this regard. Our aim was to perform a comparative analysis of the APhL ELISA assay (IgG/IgM) and criteria antiphospholipid (aPL) immunoassays in identifying APS-related clinical manifestations in a large group of patients with systemic lupus erythematosus (SLE). Methods Serum samples from 1178 patients from the Hopkins (n = 543), LUMINA (n = 588) and Jamaican SLE cohorts (n = 47) were examined for IgG/IgM positivity in aCL (in-house), anti-β2GPI (two commercial kits) and APhL (Louisville APL) ELISA assays. Correlation of assay positivity with clinical manifestations and sensitivity, specificity, positive and negative predictive values and likelihood ratios were evaluated. A case series analysis was also performed in patients for whom there was isolated positivity in the specific aPL assays. Results The prevalence of aCL positivity was 34.9%, anti-β2GPI kit A was 22.6%, APhL was 11.5% and anti-β2GPI kit B was 7.6% in the study population. Anti-β2GPI kit B, aCL and APhL assays were correlated with venous thrombosis, while only APhL was significantly correlated with arterial thrombosis and consistently correlated with pregnancy-related morbidity. No significant correlations were noted for anti-β2GPI kit A. Sensitivity was greatest for aCL assays followed by anti-β2GPI kit A, APhL and anti-β2GPI kit B, while specificity was greatest and equal for anti-β2GPI kit B and APhL assays. Conclusions Overall, APhL antibodies, especially IgG, represent a promising biomarker for the classification of APS patients in the context of autoimmunity and in risk assessment with regards to pregnancy morbidity and thrombotic manifestations.