Elevated anticardiolipin antibodies in autoimmune haemolytic anaemia irrespective of underlying systemic lupus erythematosus

In patients with systemic lupus erythematosus (SLE) and concomitant Coombs positive autoimmune haemolytic anaemia (AIHA) anticardiolipin antibodies (aCL) are found more frequently and at higher titres than in SLE patients without AIHA. In order to assess if aCL elevation is primarily associated with the underlying SLE, or with AIHA itself, we examined AIHA patients with and without underlying SLE for the presence of aCL. aCL (IgG and IgM) were determined by ELISA in 74 SLE patients without AIHA, 22 SLE patients with AIHA, 50 patients with idiopathic AIHA (warm-reactive autoantibodies), 52 patients with idiopathic AIHA (cold-reactive autoantibodies) and 50 healthy controls. The mean IgG and IgM aCL titres in SLE patients without AIHA (IgG 37.0 U/ml, IgM 8.9 U/ml) were significantly elevated compared with the values in healthy controls (IgG 9.1 U/ml, IgM 3.2 U/ml; P < 0.005). The mean aCL levels in SLE patients with AIHA (IgG 53.2 U/ml, IgM 28.2 U/ml) were higher than in SLE patients without AIHA (P = 0.09 for IgG, P < 0.005 for IgM). Interestingly, the mean aCL levels of patients with idiopathic AIHA (warm-reactive autoantibody type: IgG 29.2 U/ml, IgM 19.3 U/ml; cold-reactive autoantibody type: IgG 27.4 U/ml, IgM 18.9 U/ml) were also significantly elevated compared with healthy controls P < 0.001). As aCL are elevated not only in SLE (with and without concomitant AIHA) but also in idiopathic AIHA it can be speculated that aCL are involved in the pathomechanism of autoantibody-induced erythrocyte destruction per se irrespective of an underlying SLE.     

Association between antiphospholipid antibodies and arterial or venous thrombosis/thrombocytopenia

The relationship between thrombotic or thrombocytopenic complications and the existence of anticardiolipin antibodies (aCL) and/or lupus anticoagulant (LA) was studied in 146 patients with systemic lupus erythematosus (SLE). The prevalence of arterial thrombosis was obviously higher in patients who had both aCL and LA than in patients with either aCL or LA alone or in those with neither. Since a substantial fraction of the former group of patients with arterial thrombosis also had thrombocytopenia, there is a possibility that aCL and LA might enhance platelet activation and aggregation. To test this hypothesis, we studied the in vitro effects of aCL and LA on the enhancement of platelet activation by flow cytometric analysis using anti-CD62P and anti-CD41 monoclonal antibodies directed against platelet activation-dependent granule-external membrane (PADGEM) protein and platelet glycoprotein IIb (GPIIb). The IgG fraction purified from aCL+.LA+ plasma apparently enhanced platelet activation induced by adenosine diphosphate (ADP) at a low concentration, but IgG fractions from aCL+.LA- or aCL-.LA+ plasma did not cause enhancement of platelet activation. These results suggest that aCL and LA may cooperate to promote platelet activation, and may be involved, at least partially, in the pathogenesis of arterial thrombosis in patients with SLE.     

Antibodies to beta 2-glycoprotein I and clinical manifestations in patients with systemic lupus erythematosus

OBJECTIVE: To investigate whether anticardiolipin antibodies (aCL) in patients with systemic lupus erythematosus (SLE) bind to beta 2-glycoprotein I (beta 2GPI), and to search for a relationship between the presence of IgG and/or IgM anti-beta 2GPI antibody and clinical manifestations in SLE patients. METHODS: IgG and IgM anti-beta 2GPI in 308 Japanese SLE patients were measured using phospholipid-independent enzyme immunoassays. Relationships to clinical histories and to various laboratory data were examined. RESULTS: The values of anti-beta 2GPI and aCL, as measured by conventional enzyme immunoassay, showed a strong correlation, but the anti-beta 2GPI assay was more useful in distinguishing beta 2GPI-dependent aCL from beta 2GPI-independent aCL. The presence of IgG anti-beta 2GPI was associated with an increased frequency of a history of thrombosis. Comparisons of various laboratory data suggested that the titer of anti-beta 2GPI may fluctuate with disease activity. CONCLUSION: The results suggest that pathogenic aCL is directed against structurally altered beta 2GPI and that enzyme immunoassay for anti-beta 2GPI may prove useful in evaluating the risk of thrombosis and monitoring the clinical course in patients with SLE.     

Antibodies to beta2-glycoprotein I: a potential marker for clinical features of antiphospholipid antibody syndrome in patients with systemic lupus erythematosus

OBJECTIVE: To clarify risk factors for the development of clinical features of antiphospholipid syndrome (APS) in patients with anticardiolipin antibodies (aCL) in systemic lupus erythematosus (SLE). METHODS: We studied 65 SLE patients, all with positive IgG and/or IgM aCL. Patients were divided into 2 groups; I: 29 SLE patients with features of APS (SLE/APS) and II: 36 aCL positive SLE patients without any feature of APS (SLE/aCL). Serum samples were collected from our serum bank. Anti-beta2-glycoprotein I (anti-beta2-GPI) were tested by ELISA using irradiated plates in the absence of cardiolipin. Anti-dsDNA antibodies were tested by standard Farr assay. RESULTS: There were no major differences between SLE clinical manifestations in both groups. However, the frequency of IgG anti-beta2-GPI was markedly increased in SLE/APS (18/29, 62%) than in SLE/aCL (4/36, 11%) (chi-squared 18.6, p=0.0001). The levels of anti-dsDNA antibodies in the same samples were slightly lower in SLE/APS. CONCLUSION: Our data suggest that increased levels of IgG anti-beta2-GPI may be a specific feature of SLE/APS patients rather than reflecting a polyclonal B cell activation.     

Altered platelet magnesium and plasma and urinary soluble form of intercellular adhesion molecule I (sICAM-1) concentrations in insulin dependent diabetes mellitus (IDDM) patients with microalbuminuria

To examine the association between anticardiolipin (aCL) antibodies and epilepsy, we investigated the serum titers of aCL antibodies in a total 252 systemic lupus erythematosus (SLE) patients recruited in a prospective study. Twenty-one cases with epilepsy which were not attributable to any causes other than SLE were identified after being followed-up for five years. The clinical manifestations were recorded and blood samples were tested for the presence of aCL antibodies (IgG, IgM and IgA isotypes). Among 21 patients with epilepsy, 12 (57.1%), 2 (9.5%) and 2 (9.5%), respectively, had elevated baseline serum levels of IgG, IgM and IgA aCL antibodies. There was a dose-response relationship between risk of seizure and the baseline serum level of aCL antibodies (P < 0.01). The odds ratio of developing seizure were 3.7 for those who had a high level of aCL antibodies compared with those without a detectable level of aCL antibodies as the referent. Our results indicate that epilepsy as a primary neuropsychiatric event among lupus patients is associated with a high titer of aCL antibodies.     

Anticardiolipin antibodies in systemic lupus erythematosus: clinical correlates, HLA associations, and impact on survival

OBJECTIVE: To determine the frequency and clinical and HLA associations of anticardiolipin (aCL) antibodies in patients with systemic lupus erythematosus (SLE), as well as their impact on survival. METHODS: We studied 139 patients with SLE seen at a university based practice. We tested for clinical, laboratory, and HLA associations with levels of aCL antibody isotypes either in sera available in the bank (distant past) or in 2 samples. Demographic, clinical, laboratory, and HLA data were subjected to univariate survival analysis; variables of importance were entered into Cox multivariate regression analyses. RESULTS: aCL antibodies (any isotype) were present in 57 (41.0%) of the 139 patients tested in the distant past sample, and in 23 (32.3%) as a persistent event in the 71 patient subgroup tested twice. IgG aCL were significantly associated with deep venous thrombosis (DVT) (p = 0.04). No other clinical or HLA association was found with aCL positivity. In the survival analyses, older age at diagnosis, presence of major infections, endstage renal disease, and IgM aCL antibody positivity in the distant past emerged as important independent factors adversely affecting survival. In the subgroup tested twice for aCL antibodies (n = 71), persistent IgM aCL antibody positivity (n = 10) emerged as an important independent factor. Among the subgroup of patients that had HLA data available (n = 88), HLA-DQw7 and thromboembolic events also adversely affected survival. CONCLUSION: We confirmed the association of IgG aCL antibody positivity with DVT, and the impact on survival of endstage renal disease, major infections, and older age at diagnosis. IgM aCL antibody positivity present either as an isolated event in the distant past or as a persistent finding, thromboembolic events, and HLA-DQw7 emerged as important prognostic factors.     

Expression and immune response to hepatitis C virus core DNA-based vaccine constructs

Primary antiphospholipid syndrome (pAPS) can be experimentally induced in mice by immunization with anti-cardiolipin (aCL) antibodies (Abs). Recently, we have pointed to the pathogenic role of antiphosphatidylserine (aPS) Abs by inducing an experimental model of pAPS in naive mice following passive transfer of human aPS to the tail vein of ICR mice. The aim of the present study was to induce experimental pAPS in mice following active immunization with aPS. Mice were immunized with IgG and IgM aPS, purified from two patients with pAPS. The sera of the mice were examined for the presence of different antiphospholipid Abs, and the beta 2 GPI dependency of aPS, using an enzyme-linked immunosorbent assay (ELISA). Inhibition ELISA studies, with silica beads coated with phospholipids, were used to detect cross-reactivity of aPS or aCL to other phospholipids. The mice were also tested for the presence of thrombocytopenia, prolonged activated partial thromboplastin time (APTT), and the percentage of fetal resorptions. The purified IgG aPS but not IgM aPS had a strong lupus anticoagulant activity. Both IgG and IgM aPS were beta 2 GPI dependent and did not bind PS in the absence of the glycoprotein. The mice immunized with IgG aPS, but not IgM aPS, developed high titers of mouse aPS. The mice generated polyspecific Abs, cross-reacting with aCL and aPS, as well as individual aPS or aCL Abs. Only the mice immunized with IgG aPS developed clinical parameters of pAPS: prolonged APTT, thrombocytopenia, and an increased fetal resorption rate. In conclusion, active immunization with IgG, but not IgM, aPS induces experimental pAPS in naive mice, thus pointing to the participation of aPS in the idiotypic network.     

Lupus anticoagulants in patients with systemic lupus erythematosus

Lupus anticoagulants (LA) and anticardiolipin antibodies (aCL) are known as thrombosis-related antiphospholipid antibodies. LA is not as well characterized as aCL, and the relation between LA and aCL is not clarified. Since standardized method for the detection of LA has not been established, we measured LA activities in outpatients with SLE by using two different methods (KCT and dRVVT), and analyzed the characteristics of LA in SLE. LA was detected in 29.8% of all samples (14.3% in both methods, 15.5% in one method). IgG-aCL and IgM-aCL was detected in 38% and 20%, respectively, of all LA positive samples. Though a good correlation was observed between LA activities and IgG-aCL levels, a considerable number of LA positive samples were negative for aCL. This indicated the presence of factors with LA activity other than aCL. On the contrary there was also a high percentage of LA negative samples with positive aCL (42.4% in IgG-aCL, 47.4% in IgM-aCL), suggesting the presence of aCL with poor or low LA activity. These findings showed the heterogeneity of antiphospholipid antibodies both in LA and in aCL. The platelet function tests showed increased platelet adhesiveness and normal platelet aggregation in LA positive patients with SLE even in the inactive phase. The serum levels of factors such as protein C, protein S, antithrombin III and thrombomodulin were within normal range. Clinical features such as hemolytic anemia, thrombosis and abortion were more frequently observed in LA positive population than in LA negative population. The clinical features tend to be different between patients with dRVVT-LA and those with KCT-LA, though not significant. Because of the heterogeneity in LA, a combination of more than two different methods including dRVVT was recommended for the detection and the evaluation of LA.     

Prevalence of antiphospholipid antibodies in patients with ankylosing spondylitis

OBJECTIVE: To establish the prevalence of antiphospholipid antibodies (aPL) in a group of patients with ankylosing spondylitis (AS). The relation of the antibodies with different clinical and analytical features was studied. METHODS: Eighty-four patients with AS (71 men) and 40 age and sex matched controls were studied. aPL determinations included: anticardiolipin antibodies (aCL) of the IgM, IgG, and IgA classes, the presence of lupus anticoagulant (LAC), and a false positive serologic test for syphilis. Comparisons between variables were done by Student t test, Mann-Whitney test and Chi squared test. Correlations between aPL and clinical variables were performed by Pearson coefficients. RESULTS: Twenty-four patients with AS (29%) has positive IgG aCL, compared with only 2 cases in the control group (5%) (p < 0.002). There were no differences in other aPL determinations between patients and controls. There was no correlation between the presence of aCL (IgG, IgM, or IgA) and LAC and the different aspects of the disease. Two patients fulfilled the criteria for antiphospholipid syndrome. CONCLUSION: Our results indicate the presence of IgG aCL in patients with AS higher than in the normal population but their relation with thrombosis and other systemic manifestations seems weak.     

Changes in levels of soluble T-cell activation markers, sIL-2R, sCD4 and sCD8, in relation to disease exacerbations in patients with systemic lupus erythematosus: a prospective study

OBJECTIVES: To assess serial activation of T-cell subsets in relation to auto-antibody production and the occurrence of disease exacerbations in patients with systemic lupus erythematosus (SLE). METHODS: To study the possible role of T-cells in the pathophysiology of the disease, 16 consecutive exacerbations were prospectively studied in a cohort of patients with SLE, and serial plasma levels of sIL-2R, sCD4, and sCD8 preceding and during these exacerbations were determined. Levels of these molecules were related to total IgM and IgG, and anti-dsDNA. RESULTS: During major disease exacerbations (n = 6), levels of sIL-2R increased significantly (p < 0.001). Levels of sCD4 were predominantly in the normal range, whereas levels of sCD8 were frequently increased. No change in levels of both molecules could be detected in the period before the exacerbation. During minor exacerbations (n = 10), levels of sIL-2R remained stable. Levels of sCD4, however, tended to drop, whereas levels of sCD8 tended to rise. No correlations were found between sIL-2R, sCD4 or sCD8 on the one hand, and total IgM, IgG, or anti-dsDNA on the other. CONCLUSIONS: Levels of sIL-2R are increased, and rise before major exacerbations of SLE. Levels of sCD4 and sCD8, however, are not related to levels of sIL-2R, and do not reflect B-cell activation, nor disease activity during exacerbations of SLE. Thus for the clinical follow up of SLE measurement of levels of sCD4 or sCD8 is of limited value.     

Different anticoagulant and immunological properties of anti-prothrombin antibodies in patients with antiphospholipid antibodies

Lupus anticoagulant (LA) antibodies are acquired inhibitors of coagulation belonging-together with anticardiolipid (aCL) antibodies-to the family of antiphospholipid antibodies. Since LA antibodies affect coagulation reactions via recognition of the complex of lipid-bound prothrombin, they may be better named anti-prothrombin antibodies. We studied their immunological properties in the plasma of 59 patients with antiphospholipid antibodies by means of specific ELISA systems that allowed the characterization of the interaction of these antibodies with human prothrombin and anionic phospholipids. The mode of presentation of prothrombin was found to greatly influence the reactivity of anti-prothrombin antibodies. In fact, when plain polystyrene plates were used to immobilize prothrombin, virtually no binding was observed. Conversely, when prothrombin was coated on high-activated PVC ELISA plates, 34 samples (58%) contained antibodies that recognize human prothrombin in solid phase. In particular, IgG antibodies were found in 21 plasmas and IgM in 22; both IgG and IgM isotypes were present in 9 of these cases. A higher prevalence was observed in the ELISA for the detection of the antibodies directed at the calcium-mediated complex of phosphatidylserine (PS)-bound prothrombin: 53 samples (90%), preadsorbed with cardiolipin liposomes to remove aCL antibodies, showed the presence of IgG and/or IgM anti-prothrombin antibodies. When the results were analyzed according to the immunoglobulin isotypes, 44 (75%) and 39 (66%) samples were found to contain IgG and IgM anti-prothrombin antibodies, respectively. Both IgG and IgM were present in the plasma of 30 patients. Only half of these samples reacted also with PVC-bound prothrombin. Apparently, the higher rate of positivity of the ELISA for the detection of antibodies to the complex of PS-bound prothrombin was not due to differences in the amount of antigen available in the 2 systems, as judged by binding experiments performed with a rabbit polyclonal anti-human prothrombin antiserum. Finally, the anticoagulant properties of 14 total IgG preparations (12 of them contained anti-prothrombin antibodies positive in both ELISA systems, whereas the other 2 cases reacted either with PVC-bound prothrombin only or with PS-bound prothrombin only) were evaluated by diluted Russell's Viper Venom Time and by diluted activated Partial Thromboplastin Time. To rule out the beta 2-glycoprotein I (beta 2-GPI)-dependent anticoagulant effect of the aCL antibodies contained in the preparations, the coagulation tests were performed in beta 2-GPI deficient plasma. Six preparations failed to show anticoagulant activity in both assay systems, suggesting that 2 types of IgG anti-prothrombin antibodies exist, that differ with respect to their anticoagulant properties. These findings suggest that anti-prothrombin antibodies resemble aCL antibodies with respect to the behaviour in "in vitro" coagulation reactions and underline the wide heterogeneity of antiphospholipid antibodies.     

Extreme leukoreduction of major histocompatibility complex class II positive B cells enhances allogeneic platelet immunity

In a murine model of platelet alloimmunization, we examined the definitive role that mononuclear cells (MC) have in modulating platelet immunity by using platelets from severe combined immunodeficient (SCID) mice. CB.17 (H-2(d)) SCID or BALB/c (H-2(d)) mouse platelets were transfused weekly into fully allogeneic CBA (H-2(k)) mice and antidonor antibodies measured by flow cytometry. MC levels in BALB/c platelets were 1.1 +/- 0.6/microL and SCID mouse platelets could be prepared to have significantly lower (<0. 05/microL) MC numbers. Transfusions with 10(8) BALB/c platelets (containing approximately 100 MC/transfusion) stimulated IgG antidonor antibodies in 100% of the recipients by the fifth transfusion, whereas 10(8) SCID mouse platelets (containing approximately 5 MC/transfusion) stimulated higher-titered IgG alloantibodies by the second transfusion. When titrations of BALB/c peripheral blood MC were added to the SCID mouse platelets, levels approaching 1 MC/microL reduced SCID platelet immunity to levels similar to BALB/c platelets. Characterization of the alloantibodies showed that the low levels of MC significantly influenced the isotype of the antidonor IgG; the presence of 1 MC/microL was associated with induction of noncomplement fixing IgG1 antidonor antibodies, whereas platelet transfusions, devoid of MC (<0. 05/microL), were responsible for complement-fixing IgG2a production. When magnetically sorted defined subpopulations of MC were added to the SCID platelets, major histocompatability complex (MHC) class II positive populations, particularly B cells, were found to be primarily responsible for the reduced SCID mouse platelet immunity. The presence of low numbers of MC within the platelets was also associated with an age-dependent reduction in platelet immunogenicity; this relationship however, was not observed with SCID mouse platelets devoid of MC. The results suggest that a residual number of MHC class II positive B cells within allogeneic platelets are required for maximally reducing alloimmunization.     

Radiodiagnosis of elbow and shoulder--questions by the clinician

Anti-double stranded(ds) DNA antibody is one of markers of systemic lupus erythematosus (SLE). Two human monoclonal anti-DNA antibody-producing cell lines were established from two SLE patients. One cell line secreted IgG isotype antibody (KSUG) and the other secreted IgM isotype antibody (KSUN). The light chains of the two immunoglobulins were lambda chains. The nucleotide sequences for the immunoglobulin variable region genes of the two antibodies were determined and compared to germline sequences. The heavy and lambda light chains of KSUG were VH3 family and V lambda IIIb, respectively. The heavy and lambda light chains of KSUN were VH4 family and V lambda IX, respectively. Antibody KSUG, IgG isotype, showed somatic mutations, whereas KSUN, IgM isotype, used the germline gene without mutation. These findings reconfirm the current paradigms that IgM anti-DNA antibodies are produced by utilizing germline genes whereas IgG anti-DNA antibodies are produced by somatic mutations.     

Platelet density and size: the interpretation of heterogeneity

Platelets have been separated according to buoyant density using a colloidal silica-polyvinylpyrrolidone system and subjected to electronic sizing. All density populations were found to be heterogeneous in size, the most dense platelets ranging from less than 3 fl to greater than 21 fl in both man and rat. Light platelet fractions contained no platelets greater than 13 fl in either species. Cohort labeling with [75Se]selenomethionine showed no indication of significant change in platelet buoyant density with ageing; greater specific activity found in young, dense platelets appears to be related to increased protein synthetic activity shown in vitro and likely to occur also in their precursor megakaryocytes. It is postulated that dense, intermediate and light platelets are released synchronously by the three different ploidy classes of megakaryocyte, that varying density indicates differing structural characteristics and presumably differences in function. The present findings do not deny the possibility that platelets decrease in size with ageing but if such occurs, it is not associated with a significant change in platelet buoyant density.     

Expression of alphav integrins and vitronectin receptor identity in breast cancer cells

BACKGROUND: In light of recent reports of diminished platelet serotonin concentration and increased plasma serotonin levels in patients with Alzheimer disease (AD), we hypothesized that a state of heightened platelet activation might be present in AD. OBJECTIVE: To compare baseline activation of unstimulated platelets in patients with AD with that in control subjects. PATIENTS AND METHODS: Flow cytometry was used to measure platelet activation in 91 patients with probable AD and 40 age-matched control subjects. Groups were compared for percentage of circulating platelet aggregates, expression of CD62p, formation of leukocyte-platelet complexes, and presence of circulating platelet microparticles, controlling for effects of demographic, clinical, physiological, and logistical factors. RESULTS: Multiple analysis of covariance on ranked data revealed a 39.5% increase in percentage of platelet aggregates (P=.0001), a 59.3% increase in expression of CD62p (P=.001), and a 53.3% increase in leukocyte-platelet complexes (P=.0001) in the group with AD but no differences in the number of platelet microparticles, overall platelet count, plasma fibrinogen level, or plasma platelet factor 3. Activation was weakly correlated with sex, but was independent of age, severity of disease, duration of disease, depression, agitation, and family history of dementia. CONCLUSIONS: Platelets of patients with AD exhibit greater unstimulated activation than those of controls. Potential causes of such activation include possible stimulation of platelets by damaged cerebral endothelial cells or platelet activation induced by membrane abnormalities previously reported to be present in platelets of patients with AD. In light of recent evidence that platelets are the principal source of both amyloid precursor protein and beta-amyloid peptide in human blood, it is possible that AD platelet activation may reflect or even contribute to the pathogenesis of the disease.     

Immunochemical study of incomplete platelet autoantibodies by the anti-globulin consumption test (AGCT) with specific anti-immunoglobulin sera

The indirect anti-globulin consumption test (AGCT) with specific immunoglobulin antisera (anti-IgG and anti-IgM) has been applied to the immunochemical characterization of incomplete platelet auto-antibodies in 33 patients with idiophatic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE) and autoimmune hemolytic anemia (AHA). In these cases indirect AGCT on platelets was positive with polyvalent gamma antiserum. The test with anti-IgG was positive in all cases except two, while always negative with anti-IgM, with no relation to the presence of complete platelet antibodies, the type of disease and the immunochemical type of erythrocyte autoantibodies in AHA patients. These results indicate that the incomplete platelets auto-antibodies were of the IgG class.     

Interleukin-6 and small intestinal luminal immunoglobulins

Our aim was to determine the relationships between interleukin-6 and immunoglobulin levels within small intestinal luminal secretions. Twenty adult subjects with small intestinal bacterial overgrowth (N = 13), irritable bowel syndrome (N = 4), and nonulcer dyspepsia (N = 3) underwent endoscopic aspiration of secretions from the small intestinal mucosal surface for assessment of IL-6, IgA1, IgA2, IgM, IgG1, IgG2, IgG3, and IgG4 concentrations. Serum immunoglobulin concentrations and small intestinal histology were also determined. IgA2 and IgG3 were the predominant IgA and IgG subclasses in luminal secretions in 19/20 (95%) and 20/20 (100%) subjects, respectively. IgA1 and IgG1 predominated in serum in all subjects. No subject had villous atrophy. Luminal IL-6 concentrations correlated significantly with luminal IgA2, IgM, and IgG3 concentrations but not with IgA1 or any other IgG subclass levels. Conversely, luminal IL-6 or immunoglobulin concentrations did not correlate significantly with levels of any immunoglobulin isotype in serum. These observations suggest that important relationships exist between local IL-6 and IgA2, IgM, and IgG3 responses in human small intestinal luminal secretions. Local investigation is mandatory when assessing intestinal immune activity.     

Anti-DNA, anti-deoxyribonucleoprotein and rheumatoid factor measured by ELISA in patients with systemic lupus erythematosus, Sj?gren's syndrome and rheumatoid arthritis

Serum concentrations of anti-DNA and anti-deoxyribonucleoprotein (NP) antibodies were measured in parallel by standardized ELISA methods with a polyvalent anti-immunoglobulin conjugate in patients with systemic lupus erythematosus (SLE), Sj?gren's syndrome (SS) and rheumatoid arthritis (RA). High levels of these antibodies predominated in systemic lupus erythematosus. While an appreciable incidence of antibodies also occurred in SS and RA, they were mostly at lower levels. By using heavy chain-specific anti-immunoglobulin conjugates, IgG antibodies to both DNA and NP were found in SLE more frequently and at higher levels than were IgM antibodies. In contrast, IgM antibodies to DNA and NP predominated in SS and RA. The immunoglobulin class of the anti-DNA and anti-NP responses in a given SLE patient were not infrequently different. For example, a patient might show a very high IgG but low IgM anti-DNA value, with the reverse being true for anti-NP. IgG anti-DNA antibodies were significantly associated with depressions of C3. During changes in SLE serology, normalization of DNA binding by Farr radioimmunoassay and/or complement was most frequently associated with normalization of the IgG anti-DNA antibody concentrations. In patients simultaneously possessing elevated levels of anti-DNA, anti-NP and rheumatoid factor (RF), absorption with aggregated human IgG usually decreased only the RF activity. In some, however, such absorption decreased all three antibody values simultaneously. The latter findings support observations that some RF possess antinuclear properties.     

Human anticardiolipin monoclonal autoantibodies cause placental necrosis and fetal loss in BALB/c mice

OBJECTIVE: To analyze the structure, specificity, and in vivo pathogenetic potential of 2 human anticardiolipin (aCL) monoclonal antibodies (MAb). METHODS: Human aCL IgG MAb were generated from hybridized Epstein-Barr virus-induced B cell lines from a healthy subject (MAb 519) and from a patient with primary antiphospholipid syndrome (MAb 516). Studies of antigen-binding specificity and analysis of Ig V-gene mutations were carried out. The MAb were independently injected into mated female BALB/c mice, and their effect on pregnancy outcome was compared with that of MAb 57, a highly mutated and antigen-selected human IgG1lambda rabies virus antibody. RESULTS: Both MAb 519 and MAb 516 utilized minimally mutated V(H)DJ(H) and VkappaJkappa gene segments and bound cardiolipin and other anionic phospholipids in the absence of beta2-glycoprotein I (beta2-GPI). The mice injected with aCL MAb displayed a significantly higher rate of fetal resorption and a significant reduction in fetal and placental weight as compared with those injected with MAb 57. These findings were accompanied by a finding of placental human IgG deposition and necrosis in the aCL MAb-treated animals. CONCLUSION: The results of this study indicate that human aCL IgG that are beta2-GPI independent can induce pathology.     

Beta2-glycoprotein I is necessary to inhibit protein C activity by monoclonal anticardiolipin antibodies

OBJECTIVE: To clarify mechanisms of the thrombosis associated with anticardiolipin antibodies (aCL), we examined the effects on activated protein C (APC) of monoclonal aCL and beta2-glycoprotein I (beta2GPI), which is required for formation of the epitopes of aCL. METHODS: We developed the chromogenic assay, in which the degradation of coagulation factor Va by APC is reflected in the reduced generation of thrombin from prothrombin, using soybean trypsin inhibitor to inhibit APC. APC activities were measured in the presence and absence of 3.4 microM beta2GPI and/or 2.5 microg/ml of IgM monoclonal aCL (EY2C9 and EY1C8) established from peripheral blood lymphocytes obtained from a patient with aCL. RESULTS: Without APC, the formed thrombin activity decreased by the addition of 3.4 microM beta2GPI. When 12.8 nM APC was added, beta2GPI partially reversed the APC-induced inhibition of thrombin generation in a concentration-dependent manner. With 3.4 microM beta2GPI, the thrombin generation in monoclonal aCL (2.5 microg/ml) decreased to 77.1-80.2% by the addition of 12.8 nM APC, but the values were above that in the control IgM (72.7%). Without beta2GPI, the APC activity was unaffected by the addition of monoclonal aCL. CONCLUSION: Beta2-glycoprotein I exhibits procoagulant activity by inhibiting APC activity and anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL and only in the presence of beta2GPI.