Miri Blank

Bacterial induction of autoantibodies to β2-glycoprotein-I accounts for the infectious etiology of antiphospholipid syndrome

The antiphospholipid syndrome (APS) is characterized by the presence of pathogenic autoantibodies against beta2-glycoprotein-I (beta2GPI). The factors causing production of anti-beta2GPI remain unidentified, but an association with infectious agents has been reported. Recently, we identified a hexapeptide (TLRVYK) that is recognized specifically by a pathogenic anti-beta2GPI mAb. In the present study we evaluated the APS-related pathogenic potential of microbial pathogens carrying sequences related to this hexapeptide. Mice immunized with a panel of microbial preparations were studied for the development of anti-beta2GPI autoantibodies. IgG specific to the TLRVYK peptide were affinity purified from the immunized mice and passively infused intravenously into naive mice at day 0 of pregnancy. APS parameters were evaluated in the infused mice on day 15 of pregnancy. Following immunization, high titers of antipeptide [TLRVYK] anti-beta2GPI Abs were observed in mice immunized with Haemophilus influenzae, Neisseria gonorrhoeae, or tetanus toxoid. The specificity of binding to the corresponding target molecules was confirmed by competition and immunoblot assays. Naive mice infused with the affinity-purified antipeptide Abs had significant thrombocytopenia, prolonged activated partial thromboplastin time and elevated percentage of fetal loss, similar to a control group of mice immunized with a pathogenic anti-beta2GPI mAb. Our study establishes a mechanism of molecular mimicry in experimental APS, demonstrating that bacterial peptides homologous with beta2GPI induce pathogenic anti-beta2GPI Abs along with APS manifestations.

Induction of primary antiphospholipid syndrome in mice by immunization with a human monoclonal anticardiolipin antibody (H-3).

Antiphospholipid syndrome (APLS) is characterized by thrombocytopenia, thromboembolic phenomena, and recurrent fetal loss, associated with anticardiolipin antibodies (ACA) and/or lupus anticoagulant. The syndrome may be primary or may be associated with other conditions such as systemic lupus erythematosus. We have previously shown the ability to induce APLS in naive mice following passive transfer of serum and monoclonal ACAs. Similarly we generated the secondary APLS in BALB/c mice following immunization with a pathogenic anti-DNA antibody. In the current study we report on the induction of primary APLS following immunization of BALB/c mice with a human monoclonal ACA (H-3). The mice developed high persistent titers of ACA. The APLS was characterized by prolonged activated partial thromboplastin time, low fecundity rate (21% vs. 48% of control immunized mice), high resorption index of fetuses (25% vs. 3%), and low weights of embryos and placentae. Our study points to the ability of inducing primary APLS in naive mice. The induction of various presentations of APLS by different ACA may explain the diversity of clinical manifestations seen in patients with APLS.

Induction of Early Atherosclerosis in LDL-Receptor–Deficient Mice Immunized With β2-Glycoprotein I

BACKGROUND Immunization with beta2-glycoprotein I (beta2GPI), the probable target of autoimmune anticardiolipin antibodies, results in experimental antiphospholipid syndrome in different mouse strains. The present study was undertaken to evaluate the effect of beta2GPI immunization on the progression of atherosclerosis. METHODS AND RESULTS In the first experiment, 3 groups of LDL receptor-deficient (LDL-RD) mice (n=15 per group) were immunized with either beta2GPI or ovalbumin or were not immunized and were fed a chow diet for 12 weeks. In a second experiment, 3 groups of LDL-RD mice (n=10 per group) were immunized similarly and fed an atherogenic diet for 6 weeks. All beta2GPI-immunized mice developed high titers of anti-beta2GPI antibodies as well as a specific lymph node proliferation to beta2GPI. The average cholesterol levels did not differ between the mice fed similar diets, regardless of the immunization protocol. Atherosclerosis was enhanced in the beta2GPI-immunized mice (mean aortic lesion, 26 000+/-5700 microm2) in comparison with their ovalbumin-immunized (mean, 3000+/-1099 microm2; P<0.01) and nonimmunized (mean, 2250+/-700 microm2; P<0.01) littermates. The average lesion size in the beta2GPI-immunized mice fed an atherogenic diet (mean, 98 000+/-8305 microm2) was larger than the ovalbumin-immunized mice (mean, 81 250+/-12 933 microm2; P=NS) or the nonimmunized controls (mean, 75 625+/-7281 microm2; P=NS). The atherosclerotic plaques in the beta2GPI-immunized mice appeared to be more mature, and denser infiltration of CD4 lymphocytes was present in the subendothelium of the aortic sinuses from this group of mice. CONCLUSIONS The results of the present study provide the first direct evidence for the proatherogenic effect of ss2GPI immunization and establish a new model for immune-mediated atherosclerosis.

Intravenous immunoglobulin therapy affects T regulatory cells by increasing their suppressive function.

Intravenous Ig therapy (IVIg) is reported to be a useful regimen in treating autoimmune diseases. In this study, we asked whether IVIg (in vitro) could increase the expression of TGF-β, IL-10, and the transcription factor FoxP3 in T regulatory (Treg) cells, and the idea that IVIg could enhance suppressive properties of these cells. CD4+ T cells from 12 healthy individuals were cultured in the presence or absence of IVIg vs human control IgG during 16, 24, and 36 h. Using FACS analysis and gating on CD4+CD25high Treg cells, we assessed the expression of intracellular TGF-β, IL-10, and FoxP3. In addition, the production of TNF-α by stimulated CD4+ T cells alone or in culture with CD25+ by itself or together with IVIg was also assessed. The presence of IVIg with Treg cells in culture significantly increased the intracellular expression of TGF-β (17.7 ± 8.5% vs 29.8 ± 13%; p = 0.02), IL-10 (20.7 ± 4.7% vs 34.2 ± 5.2%; p = 0.008) and FoxP3 (20.8 ± 5.2% vs 33.7 ± 5.9%; p = 0.0006) when compared with cells cultured alone or with control human IgG. The suppressive effect of CD4+CD25+ T cells presented as the decrease of TNF-α production by stimulated CD4+CD25− (effector T cells) was further increased by adding IVIg to cell culture. We hereby demonstrate an additional mechanism by which IVIg could maintain self-tolerance and decrease immune-mediated inflammation.

Adjuvants and autoimmunity

Some adjuvants may exert adverse effects upon injection or, on the other hand, may not trigger a full immunological reaction. The mechanisms underlying adjuvant adverse effects are under renewed scrutiny because of the enormous implications for vaccine development. In the search for new and safer adjuvants, several new adjuvants were developed by pharmaceutical companies utilizing new immunological and chemical innovations. The ability of the immune system to recognize molecules that are broadly shared by pathogens is, in part, due to the presence of special immune receptors called toll-like receptors (TLRs) that are expressed on leukocyte membranes. The very fact that TLR activation leads to adaptive immune responses to foreign entities explains why so many adjuvants used today in vaccinations are developed to mimic TLR ligands. Alongside their supportive role, adjuvants were found to inflict by themselves an illness of autoimmune nature, defined as ‘the adjuvant diseases’. The debatable question of silicone as an adjuvant and connective tissue diseases, as well as the Gulf War syndrome and macrophagic myofaciitis which followed multiple injections of aluminium-based vaccines, are presented here. Owing to the adverse effects exerted by adjuvants, there is no doubt that safer adjuvants need to be developed and incorporated into future vaccines. Other needs in light of new vaccine technologies are adjuvants suitable for use with mucosally delivered vaccines, DNA vaccines, cancer and autoimmunity vaccines. In particular, there is demand for safe and non-toxic adjuvants able to stimulate cellular (Th1) immunity. More adjuvants were approved to date besides alum for human vaccines, including MF59 in some viral vaccines, MPL, AS04, AS01B and AS02A against viral and parasitic infections, virosomes for HBV, HPV and HAV, and cholera toxin for cholera. Perhaps future adjuvants occupying other putative receptors will be employed to bypass the TLR signaling pathway completely in order to circumvent common side effects of adjuvant-activated TLRs such as local inflammation and the general malaise felt because of the costly whole-body immune response to antigen. Lupus (2009) 18, 1217—1225.

Differential Effects of Anti–β2-Glycoprotein I Antibodies on Endothelial Cells and on the Manifestations of Experimental Antiphospholipid Syndrome

BACKGROUND The antiphospholipid syndrome (APS) entails a prothrombotic state associated with the presence of anticardiolipin antibodies (aCL). aCL were shown to promote endothelial cell and platelet activation and to induce an APS-like syndrome in mice when administered intravenously. Recent data suggest that aCL target the plasma cofactor beta2-glycoprotein I (beta2GPI) rather than negatively charged phospholipids. However, it has not been determined whether different epitope-specific anti-beta2GPI antibodies obtained from one patient possess pathogenic properties. METHODS AND RESULTS Three beta2GPI-binding IgM monoclonal antibodies (mAbs) (ILA-1, ILA-3, and ILA-4) were cloned from a patient with APS. The three antibodies were shown to bind beta2GPI immobilized on irradiated plates, yet only ILA-1 bound beta2GPI coated onto nonirradiated plates. Furthermore, when using the anti-beta2GPI enzyme-linked immunosorbent assay, ILA-1 was the only mAb inhibited by fluid phase beta2GPI. ILA-1 and ILA-3, but not ILA-4, induced adherence of U937 cells to endothelial cells in vitro (reflecting activation of endothelial cells). mAbs ILA-1 and ILA-3 as opposed to ILA-4 induced significant expression of adhesion molecules when preincubated with human umbilical vein endothelial cells. Passive administration of ILA-1 and ILA-3 to pregnant BALB/c mice induced clinical findings consistent with APS (increased fetal resorptions, reduced platelet counts, and prolonged activated partial thromboplastin time), whereas both ILA-4 and the control human IgM did not produce similar effects. CONCLUSIONS The results of the study demonstrate the differential effects of various populations of anti-beta2GPI antibodies on endothelial cell activation and on experimental APS.

Antiphospholipid syndrome infectious origin.

Antiphospholipid syndrome (APS) is characterized by the presence of pathogenic autoantibodies against β2-glycoprotein-I (β2GPI). The factors causing production of anti-β2GPI remain unidentified, but an association with infectious agents has been reported. Studies on experimental APS models proved that molecular mimicry between β2GPI-related synthetic peptides and structures within bacteria, viruses, tetanus toxoid, and CMV are a cause for experimental APS. Any explanation of how microbial infections might set off APS must take into account the observation that all individuals appear to harbor potentially autoreactive lymphocytes, as well as natural antiphospholipid antibodies, but that these cells or antibodies remain innocuous unless somehow activated. Herein, we discuss the association of antiphospholipid antibodies in the infectious state, molecular mimicry as a proposed cause for development of APS, and the contribution of the database to this topic.

Neuronal-binding antibodies from patients with antiphospholipid syndrome induce cognitive deficits following intrathecal passive transfer

Antiphospholipid antibodies (aPL) have been suggested to play a role in causing cognitive and behavioral impairments. In the present study we investigated the pathogenic potential of aPL by intracerebro-ventricular (ICV) administration of immunoglobulins (IgG) from patients with antiphospholipid syndrome (APS). IgG, purified from the sera of four APS patients, was tested for binding to normal mouse brain by immunohistological staining. These IgG (7.5 m g) were injected ICV unilaterally to male C3H mice. Mice injected with IgG purified from pooled sera derived from healthy subjects served as controls. The mice were examined neurologically for motor function and coordination, and cognitively in a Morris water maze. The cognitive tests were performed with the experimenterblinded to the treatment. The performance of the mice in four separate experiments was compared by analysis of variance with repeated measures. IgG from one APS patient was found to bind best to neuronal structures in the hippocampus and cerebral cortex. Mice (n 43) injected with this IgG performed worse in the water maze compared to the controls(n 45) with significant effects of the aPL IgG on the overall performance of the mice (treatment, P < 0.03), on learning throughout the experiment (treatment ×day, P < 0.02) and on short term memory (treatment ×day ×trial, P < 0.002). IgG injected from two of the three other patients also bound specifically to mouse brain neurons and produced an impairment in performance of the water maze. These results support the hypothesis that aPL that gain access to the central nervous system may play a direct role in the pathogenesis of neurological manifestations of APS.

Vitamin D: an instrumental factor in the anti-phospholipid syndrome by inhibition of tissue factor expression

Background and aims Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterised by thrombosis, obstetric complications and the presence of anti-phospholipid antibodies such as anti-β2GPI-Abs. These antibodies may set off the coagulation cascade via several mechanisms, including the induction of tissue factor (TF) expression. Vitamin D has recently emerged as an immunomodulator that might exert an anti-thrombotic effect. Therefore, we studied serum vitamin D levels in a cohort of APS patients, as well as the effect of vitamin D in an in vitro model of APS-mediated thrombosis. Methods Serum vitamin D levels were measured in 179 European APS patients and 141 healthy controls using the LIAISON chemiluminescent immunoassay, and the levels were evaluated in conjunction with a wide spectrum of clinical manifestations. In an vitro model, anti-β2GPI antibodies were purified from four patients with APS to evaluate the expression of TF in activated starved human umbilical vein endothelial cells. The effect of vitamin D (1,25-dihydroxyvitamin D, 10 nm) on anti-β2GPI-Abs mediated TF expression was analysed by immunoblot. Results Vitamin D deficiency (serum level ≤15 ng/ml) was documented in 49.5% of our APS patients versus 30% of controls (p<0.001) and was significantly correlated with thrombosis (58% vs 42%; p<0.05), neurological and ophthalmic manifestations, pulmonary hypertension, livedo reticularis and skin ulcerations. In vitro vitamin D inhibited the expression of TF induced by anti-β2GPI-antibodies. Conclusions Vitamin D deficiency is common among APS patients and is associated with clinically defined thrombotic events. Vitamin D inhibits anti-β2GPI-mediated TF expression in vitro. Thus, vitamin D deficiency might be associated with decreased inhibition of TF expression and increased coagulation in APS. Evaluation of vitamin D status and vitamin D supplementation in APS patients should be considered.

Anti-vitamin D, vitamin D in SLE: preliminary results.

Abstract:  The aim of this study was to detect antibodies to vitamin D in systemic lupus erythematosus (SLE) and other autoimmune diseases. The results may shed light to a novel aspect of vitamin D deficiency in autoimmune diseases. Sera from 171 patients with SLE, 56 with antiphospholipid syndrome (APS), and 18 with pemphigus vulgaris (PV) were studied employing an enzyme‐linked immunosorbent assay for anti‐vitamin D antibodies along with 94 healthy blood donors. In parallel, vitamin D concentrations in the serum were determined by a DiaSorin commercial kit (LIAISON 25 OH vitamin D). Antibody‐positive and antibody‐negative individuals were compared with respect to demographic variables, SLE disease activity index (SLEDAI) score, autoantibodies profile, and serum vitamin D levels. Anti‐vitamin D antibodies were detected in 7 (4%) of 171 patients with SLE, in 2 (3.5%) of 56 of sera from patients with APS, and in 2 (11%) of 18 sera from patients with PV. Vitamin D levels were similar in both SLE groups with and without anti‐vitamin D antibodies. Demographic features, organ involvement, SLEDAI score, and autoantibodies did not differ between the groups. Except for anti‐dsDNA antibodies, in which anti‐vitamin D antibodies were strongly associated with these antibodies in sera from SLE patients (P= 0.0004). Anti‐vitamin D antibodies are observed in a subset of patients with SLE, APS, and PV, and are associated with anti‐dsDNA antibodies in SLE. Further studies are required to explore the potential diagnostic and prognostic role of these novel antibodies in SLE.