E. Nigel Harris

Antiphospholipid Antibodies From Antiphospholipid Syndrome Patients Activate Endothelial Cells In Vitro and In Vivo

BACKGROUND Antiphospholipid (aPL) antibodies are associated with thrombosis in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. Although aPL antibodies have been shown to inhibit protein C activation and activate endothelial cells (ECs) in vitro, no study has examined whether these antibodies activate ECs in vivo. Therefore, human affinity-purified aPL (ap aPL) antibodies from APS patients were tested in a mouse model of microcirculation using the cremaster muscle that allows direct microscopic examination of thrombus formation and adhesion of white blood cells (WBCs) to ECs as an indication of EC activation in vivo. Adhesion molecule expression on human umbilical vein endothelial cells (HUVECs) after aPL exposure was performed to confirm EC activation in vitro. METHODS AND RESULTS All 6 ap aPL antibodies significantly increased the expression of VCAM-1 (2.3- to 4.4-fold), with one of the antibodies also increasing the expression of E-selectin (1.6-fold) on HUVECs in vitro. In the in vivo experiments, each ap aPL antibody except for 1 preparation increased WBC sticking (mean number of WBCs ranged from 22.7 to 50.6) compared with control (14.4), which correlated with enhanced thrombus formation (mean thrombus size ranged from 1098 to 6476 versus 594 microm2 for control). CONCLUSIONS Activation of ECs by aPL antibodies in vivo may create a prothrombotic state on ECs, which may be the first pathophysiological event of thrombosis in APS.

Hydroxychloroquine Reverses Thrombogenic Properties of Antiphospholipid Antibodies in Mice

BACKGROUND Previous studies have demonstrated that human monoclonal and polyclonal anticardiolipin antibodies have thrombogenic properties in vivo. Using such a model in which these antibodies have been shown to increase both the size of an induced thrombus and the duration of time in which such a clot lasts, we investigated whether hydroxychloroquine alters the dynamics of such thrombus formation. METHODS AND RESULTS Three groups of nine mice were injected with purified immunoglobulin G (IgG) from a patient with the antiphospholipid syndrome (IgG-APS) and then fed with hydroxychloroquine at various doses (100, 6, and 3 mg/kg body wt). Three control groups of mice were also studied, including mice injected with IgG-APS and then fed with placebo, as well as two other groups injected with IgG from normal human serum and fed either hydroxychloroquine or placebo. A standardized thrombogenic injury was subsequently induced in the femoral vein of each mouse and the area (size) of thrombus measured as well as the total period of time that thrombus was present. Mice treated with hydroxychloroquine and IgG-APS showed significantly smaller thrombi that persisted for a shorter period of time compared with animals treated with IgG-APS and placebo. CONCLUSIONS Hydroxychloroquine significantly diminished both thrombus size and total time of thrombus formation in mice previously injected with IgG-APS.

The prevalence and clinical associations of anticardiolipin antibodies in a large inception cohort of patients with connective tissue diseases

PURPOSE To determine the prevalence and clinical associations of anticardiolipin antibodies (aCL) in a blinded, controlled study of patients with a variety of connective tissue diseases (CTD) using a standardized aCL testing system. PATIENTS AND METHODS Anticardiolipin antibodies (IgG, IgM, and IgA) were measured by direct enzyme-linked immunosorbent assay (ELISA) in the baseline serum samples of patients enrolled in a Cooperative Study of Systematic Rheumatic Diseases (CSSRD), National Institutes of Health (NIH) supported, 5-year inception-cohort, prospective study of early rheumatic diseases: rheumatoid arthritis (RA, n = 70), systemic lupus erythematosus (SLE, n = 70), scleroderma (PSS, n = 45), myositis (PM/DM, n = 36), and early undifferentiated connective tissue disease (EUCTD, n = 165). Diagnosis was based on standardized criteria and determined at the last study visit. A nested group of patients with Sjögrens syndrome (SJ, n = 44) was also defined. Serum from 200 blood donors (BB) served as controls. Additional patients with known antiphospholipid syndrome (APS, n = 33) and ANCA-related renal vasculitis (ANCA, n = 52) were also studied. Laboratory personnel were blinded to sample diagnostic group. RESULTS The prevalence of either IgG or IgM aCL among each diagnostic group was RA 15.7%, SLE 15.76%, PSS 6.7%, PM/DM 8.3%, EUCTD 9.1%, SJ 6.8%, ANCA 3.8%, and BB controls 4.0%. Prevalence of aCL was significantly different for both the RA and SLE groups versus BB controls (P < 0.01) but not among other diagnostic groups. Only 2 study patients had positive tests for IgA aCL (1 with PM/DM and 1 with EUCTD) versus 15% of APS with positive IgA aCL. Study patients positive for IgG or IgM aCL were significantly more likely to have hemolytic anemia or a positive serologic test for syphilis and less likely to have Raynauds phenomenon. However, no associations were found between aCL positivity and thrombocytopenia, seizures, renal insufficiency, presence of a positive antinuclear antibody or rheumatoid factor, subcutaneous nodules or digital ulcers. CONCLUSIONS Based on results from this large CSSRD inception cohort, anticardiolipin antibodies are present in approximately 16% of patients with RA or SLE but are less common in patients with PSS, PM/DM, EUCTD, SJ, and ANCA vasculitis, where their prevalence approaches that in the normal population. Few consistent clinical association can be found among patients with CTD who are aCL positive. The complete diagnostic and prognostic importance and specificity of these antibodies remains to be fully determined.

Clinical and laboratory findings in patients with antiphospholipid antibodies and cerebral ischemia

We reviewed the clinical and laboratory data of 128 patients with cerebrovascular disease and antiphospholipid antibodies. Cases were evenly divided between men and women, and the mean age of the study group was 46 years. Cerebral infarction occurred in 97 patients, and transient hemispheric ischemic attacks without stroke were recorded in 19; 12 suffered ocular ischemia. Systemic lupus erythematosus was diagnosed in 16% of all cases. Histories of systemic thromboembolic events and recurrent miscarriages were noted in 14% of the patients and in 19% of the women, respectively. Evidence of cerebral infarction preceding the index event was present in 30% of cases. During a mean follow-up of 16 months, nine of 96 (9%) patients sustained new cerebral infarctions. Of 72 echocardiographic studies, 16 (22%) showed valvular abnormalities. Cerebral angiography detected intracranial lesions in 24 of 49 patients (49%). These data indicate that antiphospholipid antibodies can be identified in stroke patients without known autoimmune disorders. They also suggest that antiphospholipid antibody-associated cerebrovascular ischemia may be recurrent and often occurs in patients with systemic thromboembolic events. Our findings should help design a prospective clinical trial that will assess the risk of recurrent thromboembolism in this population, identify stroke risk factors, and address therapy.

Should anticardiolipin tests be performed in otherwise healthy pregnant women

Positive anticardiolipin and lupus anticoagulant tests are not confined to patients with the antiphospholipid syndrome, and the usefulness of these tests in healthy pregnant women is uncertain. This study sought to determine the prevalence of anticardiolipin antibodies and correlation with pregnancy outcome in 1449 pregnant women. Results were compared with 40 patients with the antiphospholipid syndrome. Persistence of positive anticardiolipin antibody tests was also ascertained. Twenty-six of 1449 sera (1.79%) were immunoglobulin G anticardiolipin positive, and 63 (4.3%) were immunoglobulin M anticardiolipin positive. Twenty-three of 26 positive for immunoglobulin G anticardiolipin and 55 of 63 positive for immunoglobulin M anticardiolipin results were low. Anticardiolipin positivity did not correlate with complications or outcome. Immunoglobulin G isotype and level distinguished patients with antiphospholipid syndrome from otherwise healthy women with positive anticardiolipin tests. In healthy pregnant women positive anticardiolipin tests occur infrequently, at low levels, and are rarely associated with adverse pregnancy outcome. This test should be requested only when the antiphospholipid syndrome is suspected.

Antiphospholipid Antibodies and Reproduction

ABSTRACT: Antiphospholipid antibodies (APAs) may be identified in the laboratory by using either coagulation studies or solid‐phase immunologic assays (ELISA; RIA). These methodologies do not necessarily evaluate the same antibody; consequently, it is appropriate to screen a patients plasma by utilizing both assays. APAs have been associated with a variety of obstetrical complications including recurrent spontaneous abortion, intrauterine fetal death, early onset preeclampsia, deep vein thrombosis, and postpartum serositis syndrome. The Kaolin Clotting Time appears to be the most sensitive coagulation test for identifying the lupus anticoagulant. However, preliminary studies would suggest the presence of anticardiolipin antibodies as detected by solid‐phase assays are more sensitive and predictive of the clinical course. Although there are no prospective trials to analyze treatment of patients with APA, preliminary data suggest the use of prednisone in combination with aspirin significantly improves the probability of delivery of a viable infant. In addition, heparin, intravenous gammaglobulin, and exchange plasmapheresis have all been tried with varying degrees of success in individual patients in small series.

Antiphospholipid antibodies and the antiphospholipid syndrome

SummaryThe study of aPL antibodies and the APS has appealed to large numbers of investigators over the last decade. This accounts, in part, for the great degree of apparently contradictory data being published. What seems certain is that these antibodies are associated with thrombosis and recurrent pregnancy loss, and animal data suggest a direct role in pathogenesis. In vitro studies demonstrate that these antibodies have a variety of functional effects on the hemostatic system, giving further credence to an antibody role in thrombosis. Further studies will doubtless give better insight into the mechanisms of antibody action and this will provide a more rational basis for treatment.

A Quarter of a Century in Anticardiolipin Antibody Testing and Attempted Standardization Has Led Us to Here, which Is?

The anticardiolipin (aCL) test has been widely used by physicians since the mid-1980s for diagnosing patients with antiphospholipid syndrome (APS). Establishment of this diagnosis has enabled effective management of patients with recurrent thrombosis and recurrent pregnancy losses. The test was first established in 1983 as a radioimmunoassay and soon thereafter converted into an enzyme-linked immunosorbent assay (ELISA). The other test commonly used in the diagnosis of APS is the lupus anticoagulant (LA) test. The aCL ELISA is sensitive for the diagnosis of APS but lacks specificity. On the other hand, the LA assay, although more specific, is not as sensitive as the aCL ELISA. More specific tests are now available such as the anti-beta2 glycoprotein I (anti-beta2GPI) assay, the antiprothrombin assay, and other ELISAs that use negatively charged phospholipids instead of cardiolipin to coat the plates. In the past 25 years, there have been numerous efforts to standardize aCL, LA, and anti-beta2GPI tests but there are still reports of significant intra- and interlaboratory variation in results for all three assays. This article discusses in detail the clinical value of these tests, technical problems associated with their use, the current laboratory classification criteria for diagnosis of APS, and possible new and better assays that will be available in the near future for diagnosis of APS.

Diastolic dysfunction is a feature of the antiphospholipid syndrome

Recurrent thrombi, thrombocytopenia, pregnancy loss, and stroke in association with medium to high concentrations of anticardiolipin antibodies are well-recognized features of antiphospholipid syndrome. Cardiac manifestations of primary antiphospholipid syndrome (PAPS) also have been documented but involve structural and valvular heart disease. Diastolic dysfunction in PAPS has not been well described. Therefore, 10 patients with PAPS (nine women and one man) of mean age 30 +/- 7 years (range 20 to 46 years) and 10 healthy age-, sex-, weight-, and height-matched control subjects were studied by echocardiography. Anticardiolipin antibody concentrations of patients with PAPS were > 80 immunoglobulin G phospholipid units as determined by enzyme-linked immunosorbent assay. Doppler-derived parameters of left ventricular filling showed a significant association between PAPS and diastolic dysfunction compared with control, as evidenced by a decrease in peak early filling velocity (52 +/- 10 cm/sec vs 67 +/- 12 cm/sec; p < 0.01), a decrease in the ratio of peak early to peak atrial filling velocities (1.03 +/- 0.40 vs 1.52 +/- 0.28; p < 0.005), a decrease in the mean deceleration rate of early filling (338 +/- 75 cm/sec2 vs 590 +/- 227 cm/sec2; p < 0.005), and an increase in the percentage of atrial contribution to filling and deceleration time. Left ventricular mass, diastolic filling time, and heart rate did not differ between groups. Left ventricular systolic function was normal and ejection fraction did not differ between patients with PAPS and control subjects (63% +/- 2% vs 65% +/- 7%; p not significant).(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenic antibodies in women with obstetric features of antiphospholipid syndrome who have negative test results for lupus anticoagulant and anticardiolipin antibodies

OBJECTIVE Our goal was to determine whether women with clinical features of antiphospholipid syndrome but negative test results for lupus anticoagulant and anticardiolipin antibodies have pathogenic antibodies not identified by currently used methods. STUDY DESIGN Sera were obtained from women with clinical features associated with antiphospholipid antibodies who had negative test results for lupus anticoagulant and anticardiolipin antibodies (antiphospholipid syndrome-like). We studied (1) the effect of passive immunization with their purified immunoglobulin G fraction on murine pregnancy (n = 35) and (2) the presence of antiphospholipid antibodies other than lupus anticoagulant or anticardiolipin antibodies (n = 39). Sera were also retested for anticardiolipin antibodies and lupus anticoagulant. RESULTS Fetal loss occurred in 235 of 1088 (22%) pups in 137 mice immunized with immunoglobulin G fraction from antiphospholipid syndrome-like women compared with 23 of 402 (6%) pups in 53 control mice. Immunoglobulin G from 11 study patients resulted in the loss of at least one third of the exposed pups. Five women had positive levels of antiphosphatidylserine antibodies (>99th percentile). All levels were low positive, and three women also had low-positive levels of anticardiolipin antibodies on repeat testing. Five of the 11 (45%) women whose immunoglobulin G fractions caused at least 33% fetal loss also had positive test results for antiphospholipid antibodies. CONCLUSIONS A subset of women with clinical disorders suspicious for antiphospholipid syndrome but who had negative test results for lupus anticoagulant and anticardiolipin antibodies by current methods have serum immunoglobulin G that is pathogenic to murine pregnancy. Testing for pathogenic immunoglobulin G may provide additional means to identify women with an as yet uncharacterized immune condition. The clinical relevance of low levels of antiphospholipid antibodies in these women remains unproved.