Induction Of Antiphospholipid Syndrome Research Articles

Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development

AbstractAntiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) are a major cause for acquired thrombophilia, but specific interventions preventing autoimmune aPL development are an unmet clinical need. Although autoimmune aPL cross react with various coagulation regulatory proteins, lipid-reactive aPL, including those derived from patients with COVID-19, recognize the endolysosomal phospholipid lysobisphosphatidic acid presented by the cell surface–expressed endothelial protein C receptor. This specific recognition leads to complement-mediated activation of tissue factor (TF)–dependent proinflammatory signaling and thrombosis. Here, we show that specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) prevents the prothrombotic effects of aPL derived from patients with COVID-19 in mice and the aPL–induced proinflammatory and prothrombotic activation of monocytes. The induction of experimental APS is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, and NAPc2 suppresses monocyte endosomal reactive oxygen species production requiring the TF cytoplasmic domain and interferon-α secretion from dendritic cells. Latent infection with murine cytomegalovirus causes TF cytoplasmic domain–dependent development of persistent aPL and circulating phospholipid-reactive B1 cells, which is prevented by short-term intervention with NAPc2 during acute viral infection. In addition, treatment of lupus prone MRL-lpr mice with NAPc2, but not with heparin, suppresses dendritic-cell activation in the spleen, aPL production and circulating phospholipid-reactive B1 cells, and attenuates lupus pathology. These data demonstrate a convergent TF-dependent mechanism of aPL development in latent viral infection and autoimmune disease and provide initial evidence that specific targeting of the TF initiation complex has therapeutic benefits beyond currently used clinical anticoagulant strategies.

Antigenicity analysis of human parvovirus B19-VP1u protein in the induction of anti-phospholipid syndrome

ABSTRACT Mounting evidence suggests a connection between human parvovirus B19 (B19) and autoimmune diseases, and especially an association between the B19-VP1 unique region (VP1u) and anti-phospholipid syndrome (APS). However, little is known about the antigenicity of B19-VP1u in the induction of APS-like syndrome. To elucidate the antigenicity of B19-VP1u in the induction of APS, N-terminal truncated B19-VP1u (tVP1u) proteins were prepared to immunize Balb/c mice to generate antibodies against B19-tVP1u proteins. The secreted phospholipase A2 (sPLA2) activities and binding specificity of mice anti-B19-tVP1u antibodies with cardiolipin (CL) and beta-2-glycoprotein I (β2GPI) were evaluated by performing immunoblot, ELISA and absorption experiments. A mice model of passively induced APS was adopted. Although sPLA2 activities were identified in all B19-tVP1u proteins, only amino acid residues 61–227 B19-tVP1u exhibited a higher sPLA2 activity. Autoantibodies against CL and β2GPI exhibited binding activities with all B19-tVP1u proteins. IgG that was purified from mice that had been immunized with amino acid residues 21–227 to 121–227 B19-tVP1u proteins exhibited significantly higher binding activity with CL. IgG that was purified from mice that had been immunized with amino acid residues 21–227, 31–227, 82–227 and 91–227 B19-tVP1u proteins exhibited significantly higher binding activity with β2GPI. Accordingly, significantly higher binding inhibition of CL was detected in the presence of amino acid residues 61–227 and 101–227 B19-tVP1u. Significantly higher binding inhibition of β2GPI was detected in the presence of amino acid residues 21–227, 31–227, 82–227 and 91–227 B19-tVP1u. The mice that received amino acid residues 31–227 or 61–227 anti-tB19-VP1u IgG revealed significant thrombocytopenia and those that received amino acid residues 21–227, 31–227, 61–227, 71–227, 82–227, 91–227, 101–227 or 114–227 anti-tB19-VP1u IgG exhibited significantly prolonged aPTT. These findings provide further information concerning the role of B19-VP1u antigenicity in APS-like autoimmunity.

Possible Association of Etanercept, Venous Thrombosis, and Induction of Antiphospholipid Syndrome

Tumor necrosis factor α (TNF α) inhibitors are commonly used for treatment of aggressive rheumatoid arthritis and other rheumatic diseases. Etanercept is one of the medications approved for treatment of rheumatoid arthritis. Though many studies have documented the safety and efficacy of these medications, evidence for adverse effects is emerging including cancer, infections, and cardiovascular disease. There have been studies showing that these medications induce autoantibody production, including antinuclear antibodies and anti-dsDNA antibodies. Limited data exists, however, regarding induction of antiphospholipid antibodies (APLs) by TNF α inhibitors, including anticardiolipin antibodies (ACLs), lupus anticoagulant (LAC), and anti-β 2-glycoprotein I (anti-β 2 GPI), or an association between antibody development and clinical manifestations. In this case series, we describe five patients who developed venous thromboembolism (VTE) and APLs while receiving etanercept therapy. All five of our patients met the criteria for diagnosis of APS after receiving etanercept. Our case series supports the association between etanercept, APLs, and VTE. We believe that testing for APLs prior to initiation of anti-TNF therapy is reasonable, given this relationship and the risks associated with VTE.

Sulfasalazine-Induced Hypersensitivity Syndrome Associated With Human Herpesvirus 6 Reactivation and Induction of Antiphospholipid Syndrome

Drug-induced hypersensitivity syndrome is a toxicoderma with systemic involvement. Suspicion of this disorder obliges rapid withdrawal of the suspected drug, which may have been introduced up to 3 months earlier. Screening for human herpesvirus (HHV) 6 reactivation is important both for its diagnostic value and for its association with a poor prognosis. Reactivation of this virus is not a contraindication for systemic corticosteroid treatment, which should be tapered slowly in order to avoid recurrence. The possible appearance of antiphospholipid antibodies must also be considered in those cases associated with thrombocytopenia, altered hemostasis, or thrombotic events. Autoimmune disorders may also develop as a sequela of the condition. Medium-to-long-term follow-up is required even after complete resolution of the condition. We describe a new case of sulfasalazine-induced hypersensitivity syndrome associated with HHV-6 reactivation and the induction of anticardiolipin and anti-thyroid peroxidase antibodies.

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization.

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity.

Induction of APS after TTd Hyper-Immunization has a Different Outcome in BALB/c and C57BL/6 Mice

The antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by vascular thrombosis and/or pregnancy complications (lower fecundity and lower litter size), as well as by an increase in anti-β(2) glycoprotein I (β(2) GPI)-specific autoantibody titer. We have investigated how the genetic background of the immune system [T helper (Th) prevalence] and the type of animal model of APS influence the induced pathology. Antiphospholipid syndrome induced by tetanus toxoid (TTd) hyper-immunization and by intravenous application of monoclonal anti-β(2) GPI-specific antibody 26 was compared in C57BL/6 (Th1 prone) and BALB/c (Th2 prone) mice. Tetanus toxoid hyper-immunization of BALB/c mice led to reduction in fertility, but in C57BL/6 mice a decrease in fecundity occurred. In both cases, pathology was caused by anti-β(2) GPI antibodies, the production of which was adjuvant and strain dependent. We conclude that TTd immunization and i.v. application of monoclonal antibody 26 induced the same reproductive pathology and that the type of pathology is strain dependent.

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity.

Antiphospholipid syndrome induction exacerbates a transgenic Alzheimer disease model on a female background

The antiphospholipid syndrome (APS) is characterized by antiphospholipid antibodies (aPL) and vascular brain disease which is often associated with dementia. We examined the neurodegenerative pathological processes underlying APS by inducing APS in a transgenic animal model of Alzheimer's disease. Female C57B6/SJL mice carrying the APP(695)SWE mutation (Tg2576) and wild-type (wt) controls were immunized with β₂-glycoprotein-I (APS mice) or adjuvant alone (controls) at 4 months of age. At the age of 8 months the APP-APS mice developed high levels of aPL associated with motor hypoactivity in a staircase test (p<0.03 by t-test) and impaired performance in the cognitive T-maze (p<0.02 for main effect of treatment by repeated measures ANOVA) relative to APP-CFA mice and controls. wt-APS and wt-control did not differ significantly in their behavior or cognition. Histological studies revealed mature plaques only in the APP-APS group which also had higher amyloid load and number of activated microglia compared to all other groups. The results indicate a significant interaction between APP genotype and the induction of APS on a female background. The mechanisms involved may also be important in human APS-AD co-morbidity.

Up-regulation of adhesion molecule expression and induction of TNF-α on vascular endothelial cells by antibody against human parvovirus B19 VP1 unique region protein

Background Human parvovirus B19 infection has been frequently described as a cause or trigger of various autoimmune diseases. In previous studies, we have postulated the association among human parvovirus B19 (B19)-VP1 unique region (VP1u), production of anti-beta2-glycoprotein I (anti-β2GPI) antibody and anti-phospholipid syndrome (APS)-like autoimmunity. However, the precise role of B19-VP1u in induction of APS is still obscure. Methods To further elucidate the pathogenic roles of VP1u in B19 infection and autoimmunity, we examined the effect of anti-B19-VP1u IgG antibodies on endothelial cells that is recognized to play crucial roles in APS. Human vascular endothelial cells, ECV-304, were incubated with various preparations of purified human or rabbit IgG. The activation of endothelial cells and production of cytokines were assessed by flow cytometry and ELISA, respectively. Results Purified IgG from rabbits immunized with recombinant B19-VP1u proteins can up-regulate ICAM-1 (CD54), VCAM-1 (CD106), E-selectin (CD62E), MHC class II (HLA-DR, DP, DQ) molecule expression, and TNF-α production in endothelial cells as compared to those endothelial cells cultured with control IgG. Additionally, significantly increased phosphorylated-P38 mitogen-activated protein kinase (P38 MAPK) and iNOS were observed in both human anti-β2GPI IgG and rabbit anti-B19-VP1u IgG treated-ECV-304 cells, respectively. Conclusions These experimental results imply that antibodies against B19-VP1u play important roles in the immunopathological processes as well as human anti-β2GPI IgG that leads to development of APS by involving p38 phosphorylation and iNOS activation. It could provide a clue in understanding the role of anti-B19-VP1u antibodies in APS manifestations.

Genetic and immunological factors interact in a mouse model of CNS antiphospholipid syndrome

The antiphospholipid syndrome (APS) includes systemic and central nervous system (CNS) pathology associated with antibodies to a complex of phospholipids and β 2-glycoprotein I (β 2-GPI). We have recently reported the induction of APS associated with behavioral and cognitive deficits in BALB/c female mice that developed 4–5 months after immunization with β 2-GPI. In the present study, we examined the influence of genetic factors on the ability to induce experimental APS with CNS involvement by testing several mouse strains immunized with β 2-GPI. Female mice from five strains were immunized once with β 2-GPI in complete Freund's adjuvant (CFA) or with CFA alone (controls). Autoantibody levels were examined at 1 and 5 months after immunization. Neurological assessment in a staircase test was performed 4–5 months following the immunization. Induction of APS resulted in elevated levels of antibodies against negatively charged phospholipids and β 2-GPI in all five mouse strains. Autoantibody levels were significantly higher in Balb/c, ICR, and C57BL/6 mouse strains compared to AKR and C3H. aPL levels dropped significantly more in the C57BL/6 compared to Balb/c mice over a period of 4 months. Hyperactivity reflected by higher number of stairs climbed in 3 min, was induced by APS in the Balb/c and ICR, mouse strains. Exploratory behavior reflected by more frequent rears, was seen in the APS-Balb/c and AKR mice. Hypoactivity and less exploration were seen in the APS-C57BL/6 and C3H mice. The study supports a link between high levels of aPL and behavioral changes in a mouse APS model. Qualitative differences in behavioral patterns may be due to nervous system as well as immune genetic factors. The minimal effect of APS in C57BL/6 mice may provide a suitable background for the study of transgenes in these mice.

Induction and treatment of the antiphospholipid syndrome--lessons from animal models.

The diagnosis of antiphospholipid syndrome (APS) is based on a combination of clinical and serological criteria. This review summarizes the literature regarding animal models of APS. Animal models of this syndrome help to elucidate pathogenic roles of the characteristic antiphospholipid antibodies in pregnancy morbidity, vascular thrombosis, neurologic disease and atherosclerosis. Furthermore, these models also provide measures to evaluate novel therapies for APS. These include bone-marrow transplantation, intravenous immunoglobulin, anticoagulation, interleukin-3, in addition to other therapies. Experimental induction and treatment of APS largely contributes to our understanding of the disease and to better management of its clinical characteristics.

Antiphospholipid antibody levels in intravenous immunoglobulin (IVIg) preparations.

The antiphospholipid syndrome (APS) is characterized by various clinical manifestations and by elevated levels of antiphospholipid antibodies. Passive induction of APS by infusion of these antibodies has been demonstrated in animal models. Intravenous immunoglobulin (IVIg) is one of the therapeutic options in APS. In this study, five commercially used preparations of IVIg were tested for the presence of elevated levels of cardiolipin, beta-glycoprotein-I, phosphatidylserine, antinuclear, and double-stranded DNA autoantibodies, as well as for lupus anticoagulant activity. The absence of abnormal elevated levels of any of these autoantibodies in five different IVIg preparations provides additional evidence for the safety of IVIg use in APS.

Suppression of experimental antiphospholipid syndrome and systemic lupus erythematosus in mice by anti-CD4 monoclonal antibodies.

To investigate whether anti-CD4 antibodies can suppress experimental antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE) induced by an anti-DNA monoclonal antibody (MAb). BALB/c mice were treated with anti-CD4 MAb either before or 2 months after induction of experimental APS and SLE. Control mice were treated with rat IgG or phosphate buffered saline. Serologic and clinical manifestations of the disease were determined. Treatment of mice with anti-CD4 before or 2 months after disease induction prevented the development of experimental APS and SLE. The treated mice did not develop leukopenia or proteinuria, and had fewer episodes of fetal resorption. Similarly, the treated mice did not develop elevated erythrocyte sedimentation rate, prolonged activated partial thromboplastin time, or thrombocytopenia, and had significantly lower levels of antibodies to double-stranded DNA, histones, MIV-7, cardiolipin, and phosphatidylserine. Levels of CD4+ cells in the lymph nodes declined temporarily after the treatment and then returned to normal. Anti-CD4 antibodies can prevent experimental APS and SLE. These results may suggest a role for anti-CD4 treatment in human autoimmune diseases.

Induction of anti-phospholipid syndrome in naive mice with mouse lupus monoclonal and human polyclonal anti-cardiolipin antibodies.

The primary anti-phospholipid syndrome is characterized by recurrent venous and arterial thromboembolic phenomena, recurrent fetal loss, thrombocytopenia, and serological evidence of anti-cardiolipin (aCL) antibodies or/and the presence of lupus anticoagulant (prolonged activated partial thromboplastin time). The exact role of aCL antibodies in pathogenesis is not clear and the mechanism by which the antibodies may induce the various manifestations is unknown. In the current study we evaluated the effect of passive transfer of aCL antibodies (to the tail vein of naive mice) on fecundity, fetal loss (fetal resorption), and the weight of embryos and placentae. Two types of aCL antibodies were employed: (i) mouse monoclonal aCL antibodies derived from a BALB/c mouse in which experimental systemic lupus erythematosus was induced by a pathogenic idiotype (idiotype 16/6) of anti-DNA antibodies and (ii) polyclonal IgG and IgM aCL antibodies derived from serum of a patient with primary anti-phospholipid syndrome. After infusion of either antibody (10 micrograms per mouse) we could demonstrate lower fecundity rate, increased resorption index of embryos (equivalent to recurrent fetal loss), lower number of embryos per pregnancy, and lower mean weights of embryos and placentae in comparison to mice infused with appropriate control immunoglobulins. We conclude that the aCL antibodies may have direct effects on fecundity and on the outcome of pregnancy.