Measurement of antinuclear antibodies and their fine specificities: time for a change in strategy?

OBJECTIVES: The current strategy for antinuclear antibody (ANA) analysis involves screening for presence with a subsequent detailed analysis of their specificity. The aim of this study is to compare the clinical and financial efficacy of this strategy between different commercial tests in a large cohort of unselected patients. METHODS: In all consecutive 1030 patients associations were defined between results from different ANA test systems and the pre-test probability for connective tissue disease (CTDs). Test systems were used for screening (ANA-IIF vs. CTD screen) and definition of their fine specificity (profile 3 line blot vs. CTD single analytes). RESULTS: Positive ANA-IIF and/or CTD screen results were found in 304 sera. Further analysis for ANA-specificity by profile 3 line blot and CTD single analytes showed 86 discrepant results of which more than a third are clinically relevant, with the CTD single analyte assay performing better than the line blot in supporting or confirming the presence of a CTD. Autoantigens present in one test but absent in the other were of minor practical use. The ANA screening and identification strategies currently employed are not cost-effective as 83% of tests were performed in order to find specific autoantibodies in patients without the fitting clinical signs or symptoms. This causes many unexpected positive results and subsequent confusion with regard to interpretation. CONCLUSIONS: We advocate that some autoantigens should be excluded from the line blot and CTD assays and propose the use of a cost-effective and selective ANA specificity testing purely based on clinical guidance.

Apolipoprotein E receptor 2 is involved in the thrombotic complications in a murine model of the antiphospholipid syndrome.

Antiphospholipid (aPL)/anti-ß(2) glycoprotein I (anti-ß(2)GPI) antibodies stimulates tissue factor (TF) expression within vasculature and in blood cells, thereby leading to increased thrombosis. Several cellular receptors have been proposed to mediate these effects, but no convincing evidence for the involvement of a specific one has been provided. We investigated the role of Apolipoprotein E receptor 2 (ApoER2') on the pathogenic effects of a patient-derived polyclonal aPL IgG preparation (IgG-APS), a murine anti-ß(2)GPI monoclonal antibody (E7) and of a constructed dimeric ß(2)GPI I (dimer), which in vitro mimics ß(2)GPI-antibody immune complexes, using an animal model of thrombosis, and ApoER2-deficient (-/-) mice. In wild type mice, IgG-APS, E7 and the dimer increased thrombus formation, carotid artery TF activity as well as peritoneal macrophage TF activity/expression. Those pathogenic effects were significantly reduced in ApoER2 (-/-) mice. In addition, those effects induced by the IgG-APS, by E7 and by the dimer were inhibited by treatment of wild-type mice with soluble binding domain 1 of ApoER2 (sBD1). Altogether these data show that ApoER2 is involved in pathogenesis of antiphospholipids antibodies.

Long-term follow-up of a randomised controlled trial of azathioprine/methylprednisolone versus cyclophosphamide in patients with proliferative lupus nephritis.

OBJECTIVES: The objectives of this study are to analyse the long-term follow-up of a randomised controlled trial of induction treatment with azathioprine/methylprednisolone (AZA/MP) versus high-dose intravenous cyclophosphamide (ivCY) in patients with proliferative lupus nephritis (LN) and to evaluate the predictive value of clinical, laboratory and renal biopsy parameters regarding renal outcome. METHODS: 87 patients with biopsy-proven proliferative LN were treated with either AZA/MP (n=37) or ivCY (n=50), both with oral prednisone. After 2 years, renal biopsy was repeated, and all patients continued with AZA/oral prednisone. The primary study end point was sustained doubling of serum creatinine. Secondary end points included renal relapse, end-stage renal disease and mortality. RESULTS: After a median follow-up of 9.6 years, no significant differences between AZA/MP versus ivCY groups were found in the proportion of patients with sustained doubling of serum creatinine (n=6 (16%) vs n=4 (8%); p=0.313), end-stage renal disease (n=2 (5%) vs n=2 (4%); p=1.000) or mortality (n=6 (16%) vs n=5 (10%); p=0.388). Renal relapses occurred more often in the AZA/MP group (n=14 (38%) vs n=5 (10%); p=0.002, HR: 4.5). Serum creatinine, proteinuria and immunosuppressive treatment regimens at the last follow-up were comparable. Clinical and laboratory parameters at baseline and after 2 years, and renal biopsy parameters (only) at baseline predicted renal outcome. CONCLUSION: Induction treatment with ivCY was superior to AZA/MP in preventing renal relapses, but other parameters for renal function did not differ. AZA/MP can therefore serve as an alternative in patients with proliferative LN who wish to avoid gonadal toxicity of CY. Several prognostic factors of long-term renal outcome were identified.

Beta2-glycoprotein I can exist in 2 conformations: implications for our understanding of the antiphospholipid syndrome.

The antiphospholipid syndrome is defined by the presence of antiphospholipid antibodies in blood of patients with thrombosis or fetal loss. There is ample evidence that beta(2)-glycoprotein I (beta(2)GPI) is the major antigen for antiphospholipid antibodies. The autoantibodies recognize beta(2)GPI when bound to anionic surfaces and not in solution. We showed that beta(2)GPI can exist in at least 2 different conformations: a circular plasma conformation and an "activated" open conformation. We also showed that the closed, circular conformation is maintained by interaction between the first and fifth domain of beta(2)GPI. By changing pH and salt concentration, we were able to convert the conformation of beta(2)GPI from the closed to the open conformation and back. In the activated open conformation, a cryptic epitope in the first domain becomes exposed that enables patient antibodies to bind and form an antibody-beta(2)GPI complex. We also demonstrate that the open conformation of beta(2)GPI prolonged the activated partial thromboplastin time when added to normal plasma, whereas the activated partial thromboplastin time is further prolonged by addition of anti-beta(2)GPI antibodies. The conformational change of beta(2)GPI, and the influence of the autoantibodies may have important consequences for our understanding of the antiphospholipid syndrome.

Association between beta2-glycoprotein I plasma levels and the risk of myocardial infarction in older men.

von Willebrand factor (VWF) serves as adhesive surface for platelets to adhere to the vessel wall. We have recently found that beta2-glycoprotein I is able to inhibit platelet binding to VWF, indicating a role in the pathophysiology of arterial thrombosis. In the present study, we investigated whether differences in beta2-glycoprotein I plasma levels influence the risk of myocardial infarction. We have measured beta2-glycoprotein I and VWF antigen levels in 539 men with a first myocardial infarction and in 611 control subjects. Although we did not find a profound effect of beta2-glycoprotein I plasma levels on myocardial infarction in the overall population, we found a dose-dependent protective effect of increasing beta2-glycoprotein I plasma levels on myocardial infarction in men 60 years and older. In this age group, we found an odds ratio of 0.41 (95% confidence interval, 0.22-0.74) for high beta2-glycoprotein I levels compared with low levels. High plasma levels of beta2-glycoprotein I remained protective for myocardial infarction despite high levels of VWF. To conclude, high circulating levels of beta2-glycoprotein I appeared to be associated with a reduced risk of myocardial infarction in elderly men. In vivo experiments are needed to investigate the exact contribution of beta2-glycoprotein I on the pathophysiology of myocardial infarction.

Twenty-two years of failure to set up undisputed assays to detect patients with the antiphospholipid syndrome.

The antiphospholipid syndrome is defined by the persistent presence of antiphospholipid antibodies in plasma of patients with a history of thrombosis and/or pregnancy morbidity. From the definition in 1985 onwards, confusion has arisen concerning who has the syndrome and who has not. Although the clinical criteria are well defined, there is ongoing discussion regarding serologic criteria. Lack of standardization of the assays that define the serologic criteria, notably phospholipids-dependent coagulation assays, and enzyme-linked immunosorbent assays (ELISAs) for anticardiolipin and anti-beta2 glycoprotein I, have led to heated arguments regarding which population(s) of antibodies should be measured to detect a patient at risk for (recurrent) thrombosis or pregnancy complications. Everybody agrees on the need to better standardize the assays, but different views are held on how this should be achieved, and commercial interests have hampered consensus on which assays should be applied, how they should be performed, and the cutoff values that discriminate between pathologic and nonpathologic results. New prospective cohort studies to reevaluate the clinical significance of the available assays are essential, but the lack of sufficient patient numbers visiting single hospital facilities frustrates progress. This review discusses shortcomings of the current serologic assays, provides strategies to solve these shortcomings, and discusses new developments/assays to improve the specificity of such assays for thrombosis and pregnancy complications.

The development of offspring from mothers with systemic lupus erythematosus. A systematic review.

OBJECTIVE: To analyze published data on the influence of maternal systemic lupus erythematosus (SLE) on different aspects of child development. METHODS: A systematic review was conducted using PubMed and Embase searches for SLE or SLE-related antibodies and physical, neurocognitive, psychiatric or motor development outcomes in children. RESULTS: In total 24 cohort and 4 case-control studies were included after initial screening of 1853 hits. Learning disorders (LD) were reported in 21.4-26% of SLE offspring, exceeding the prevalence in the general population. Four studies reported that dyslexia and reading problems were present in 14.3-21.6% of lupus offspring with a clear male predominance. Furthermore, a twofold increased rate of autism spectrum disorders (ASD) (n=1 study) and a two- to threefold increased risk for speech disorders (n=3 studies) were reported in lupus offspring compared to controls, although the latter was not statistically significant. More divergent results were found for attention deficit (n=5 studies) and behavior disorders (n=3 studies). In two large controlled studies attention disorders were more prevalent and a trend towards more behavior disorders was reported in 2 of 3 studies analyzing this subject. Finally, IQ and motor skills were not affected in respectively 7 and 5 studies. Cardiopulmonary functioning and mood disorders were scarcely investigated (both n=1). Maternal anti-SSA antibodies were associated with LD in offspring in one study. Other SLE-related antibodies were rarely studied. CONCLUSION: This systematic review suggests that maternal SLE is associated with LD (specifically dyslexia), ASD, attention deficit and probably speech problems in offspring. However, over half of the studies were assigned a low or moderate evidence level. Therefore, further research is necessary to substantiate the found evidence and expand the scope to lesser researched areas such as cardiopulmonary functioning.

Delineating the deranged immune system in the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is a systemic autoimmune disease that is characterized serologically by the presence of antiphospholipid antibodies (aPL) and clinically by vascular thrombosis and obstetric complications. The protein ß2 glycoprotein I (ß2GPI) is identified as the most important autoantigen in this syndrome. Activation of endothelial cells, thrombocytes and placental tissue by anti-ß2GPI antibodies relates to the clinical manifestations of APS. This review describes genetic and environmental factors in relation to APS and summarizes the current knowledge on abnormalities in components of both the innate and adaptive immune system in APS. The role of dendritic cells, T-cells, B-cells, monocytes, neutrophils and NK-cells as well as the complement system in APS are discussed. Several gaps in our knowledge on the pathophysiology of APS are identified and a plea is made for future extensive immune cell profiling by a systems medicine approach in order to better unravel the pathogenesis of APS, to gain more insight in the role of the immune system in APS as well as having the potential to reveal biomarkers or novel therapeutic targets.

Newer drugs for the treatment of lupus nephritis.

This article first reviews the current treatment of lupus nephritis, with a focus on the most serious forms, that is, the proliferative subtypes. Current standards for treatment have been developed empirically. Corticosteroids form the basis of all regimens. Cyclophosphamide given intravenously for prolonged periods is the current gold standard. Azathioprine can be regarded as an effective drug for maintenance treatment of lupus nephritis. Studies on its efficacy in schedules for remission induction are in progress. It has been learned from studies on 'conventional' immunosuppression that randomised, clinical trials should comprise large numbers of patients and a follow-up of many years to elucidate differences between effective strategies. These requirements are not met by any of the 'new' treatments we discuss in this review. There is only limited experience in patients with lupus nephritis with drugs that are currently used for immunosuppression in other autoimmune diseases, such as methotrexate, cyclosporin and high-dose intravenous gammaglobulins, nor with new immunosuppressive drugs that have been developed for immunosuppression in organ transplantation (mycophenolate mofetil, tacrolimus, fludarabine and cladribine). Hormonal therapy with the weak androgen prasterone (dehydroepiandrosterone; DHEA) has no role in treatment of active lupus nephritis. There are interesting experiences with agents that have evolved from progress in immunobiology and in our understanding of immunological processes. These modalities enable more specific immunosuppression and include monoclonal antibodies directed at immune cells, cytokines and components of the complement system, constructs developed to induce tolerance in pathogenic B cells, and gene therapy. Finally, we review data on autologous bone marrow transplantation in patients with systemic lupus erythematosus. We conclude that some strategies (like mycophenolate mofetil) are good candidates for further investigation in large-scale, prospective, randomised trials with prolonged follow-up (which are almost by definition hard to perform). Most new biological agents still are in a pre-clinical phase.

Beta2-glycoprotein I and LDL-receptor family members.

The presence of antiphospholipid antibodies in plasma is a risk factor for thrombo-embolic complications. In vitro, however, the same antibodies can prolong clotting times in coagulation assays, a classic marker for a bleeding tendency. For years this contradiction puzzles many scientists.We now know that the term antiphospholipid antibodies comprises a heterogeneous population of antibodies and there is growing evidence that only subpopulations of antiphospholipid antibodies are relevant for the clinical complication. In combination with new information on the complex interaction between antiphospholipid antibodies, the protein beta2-Glycoprotein I, and cellular surfaces have opened new avenues for the understanding of the pathology of this syndrome.

Optimisation of lupus anticoagulant tests: should test samples always be mixed with normal plasma?

Coagulation factor deficiencies are thought to interfere with the detection of the phospholipid-dependent coagulation inhibitor known as lupus anticoagulant (LA). Treatment with vitamin K antagonists (VKA) in particular, is thought to preclude accurate LA assessment. For this reason, the procedure to detect LA includes a mixing test, in which coagulation factor deficiencies are corrected by mixing samples with an equal volume of normal plasma. Despite these mixing tests, interpretation of LA test results is considered difficult in patients receiving high intensity VKA treatment. As a result, VKA treatment is often temporarily discontinued to allow LA assessment. However, whether coagulation factor deficiencies influence LA test results is unclear. We found that neither deficiency of a single coagulation factor, nor a functional coagulation factor deficiency due to high intensity VKA treatment, resulted in false positive dRVVT- or APTT-based (silica clotting time; SCT) LA test results. LA was readily detected in unmixed samples from VKA-treated LA-positive patients with both dRVVT and SCT reagents. VKA treatment caused an underestimation of the strength of the LA with SCT reagents, but did not lead to misclassification of LA status. Although mixing with normal plasma during both screen and confirm tests allowed more accurate assessment of the strength of the LA with SCT reagents in samples with an international normalised >2.5, the mixing procedure itself lead to misclassification of LA in weakly positive samples from patients not treated with VKA. Based on these findings, we conclude that mixing studies are not necessary during LA-assessment.

Ligation of signal inhibitory receptor on leukocytes-1 suppresses the release of neutrophil extracellular traps in systemic lupus erythematosus.

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of systemic Lupus erythematosus (SLE), since netting neutrophils release potentially immunogenic autoantigens including histones, LL37, human neutrophil peptide (HNP), and self-DNA. In turn, these NETs activate plasmacytoid dendritic cells resulting in aggravation of inflammation and disease. How suppression of NET formation can be targeted for treatment has not been reported yet. Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is a surface molecule exclusively expressed on phagocytes. We recently identified SIRL-1 as a negative regulator of human neutrophil function. Here, we determine whether ligation of SIRL-1 prevents the pathogenic release of NETs in SLE. Peripheral blood neutrophils from SLE patients with mild to moderate disease activity and healthy donors were freshly isolated. NET release was assessed spontaneously or after exposure to anti-neutrophil antibodies or plasma obtained from SLE patients. The formation of NETs was determined by microscopic evaluation using DNA dyes and immunostaining of NET components, as well as by live cell imaging. We show that SLE neutrophils spontaneously release NETs. NET formation is enhanced by stimulation with antibodies against LL37. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and MEK-ERK signaling prevents NET release in response to these antibodies. Signaling via the inhibitory receptor SIRL-1 was induced by ligation with anti-SIRL-1 specific antibodies. Both spontaneous and anti-neutrophil antibody-induced NET formation is suppressed by engagement of SIRL-1. Furthermore, NET release by healthy neutrophils exposed to SLE plasma is inhibited by SIRL-1 ligation. Thus, SIRL-1 engagement can dampen spontaneous and anti-neutrophil antibody-induced NET formation in SLE, likely by suppressing NAPDH oxidase and MEK-ERK activity. Together, these findings reveal a regulatory role for SIRL-1 in NET formation, potentially providing a novel therapeutic target to break the pathogenic loop in SLE.

The use of glucocorticoids in systemic lupus erythematosus. After 60 years still more an art than science.

Systemic Lupus Erythematosus (SLE) is a clinically diverse, chronic autoimmune disease with inflammation in several organ systems. Its pathogenesis is complex, but includes many factors that can be influenced by glucocorticoids (GCs). Indeed, GCs constitute the corner-stone in SLE-treatment. However, guidelines for GC-treatment of the different disease manifestations are lacking and not every patient responds (sufficiently). The focus of this systematic review is to evaluate the differential glucocorticoid treatment of various SLE manifestations. In addition, some relevant mechanisms of glucocorticoid action as well as resistance are discussed.

Cortisol during the day in patients with systemic lupus erythematosus or primary Sjogren's syndrome.

OBJECTIVE: To compare the level and change of cortisol during the day of patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) with low and high erythrocyte sedimentation rate (ESR). METHODS: Saliva was collected in the real-life environment of 21 women with SLE, 16 women with pSS, and 30 age-matched healthy women at 9 fixed timepoints during 2 consecutive days. Repeated measures ANOVA was performed to examine whether cortisol levels during the day were different for the patients with low ESR (≤ 20 mm/h) versus those with high ESR (> 20 mm/h). RESULTS: The groups with low and high ESR showed the characteristic change of cortisol during the day (time-of-day effect, F = 124.9, p < 0.001). The cortisol awakening level was lower for patients with high ESR than for patients with low ESR (group*time effect, F = 3.1, p = 0.02). CONCLUSION: The cortisol awakening level differs for patients with low and high ESR, which indicates the usefulness of further studies of hypothalamic-pituitary-adrenal axis dynamics in patients with SLE and pSS.

Lack of association of C-C chemokine receptor 5 Δ32 deletion status with rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, and disease severity.

OBJECTIVE: C-C chemokine receptor 5 (CCR5) plays an important role in inflammation. A 32 base-pair (Δ32) deletion in the CCR5 gene leads to a nonfunctional receptor. This deletion has been reported to have a protective effect on the development and progression of several autoimmune diseases. We investigated whether the Δ32 deletion is associated with disease susceptibility in a population of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and lupus nephritis (LN); and whether it is associated with disease severity. METHODS: DNA samples from 405 RA patients, 97 SLE patients, 113 LN patients, and 431 healthy controls were genotyped for the CCR5 Δ32 deletion. Differences in genotype frequencies were tested between patients and controls. Association of genotypes with disease severity was analyzed. RESULTS: Genotype frequencies of each group were in Hardy-Weinberg equilibrium. The genotype frequencies of patients did not differ significantly from controls (CCR5/Δ32, Δ32/Δ32: RA 18.3% and 1.2%, respectively; SLE 17.5% and 2.1%; LN 13.3% and 1.8%; controls 20.0% and 2.8%). However, there was a trend for lower Δ32 deletion allele frequency in LN patients compared to controls (p = 0.08). There was no significant association between the CCR5 status and disease severity in RA, SLE, or LN. CONCLUSION: Although an association with LN cannot be excluded, the CCR5 Δ32 deletion does not seem to be a disease susceptibility genotype for RA, SLE, or LN. No significant effect of the Δ32 deletion on disease severity was demonstrated.

Rapidly fatal HTLV-1-associated T-cell leukemia/lymphoma in a patient with SLE.

BACKGROUND: A 57-year-old Afro-Jamaican woman with an 8-year history of systemic lupus erythematosus, including lupus nephritis, was admitted to hospital with intractable back pain accompanied by fever and severe malaise. At the time of presentation she was receiving immunosuppressive treatment with glucocorticoids and azathioprine. She also had gout, hypertension and type II diabetes. INVESTIGATIONS: Physical and neurological examination and laboratory analyses, including biochemical, hematological and electrophoresis tests, X-ray of the lumbar spine, pelvis and chest, mammography, MRI of the lumbar spine, thoracic and abdominal CT, and biopsy of a peripheral lymph node and bone marrow with immunohistochemistry and serology for human T-cell lymphotrophic virus (HTLV) 1 and 2. DIAGNOSIS: HTLV-1-associated acute adult T-cell leukemia/lymphoma with bone marrow infiltration and hypercalcemia. Reaching the correct diagnosis was difficult and only possible through close collaboration with the pathologist and with consideration of the patient's ethnic and geographical background. MANAGEMENT: Chemotherapy with high-dose prednisone and adjusted doses of cyclophosphamide and doxorubicin. The patient developed tumor lysis syndrome and died 3 weeks after the diagnosis was made.