Maria Orietta Borghi

Toll‐like receptor and antiphospholipid mediated thrombosis: in vivo studies

Objective: A study was undertaken to investigate the in vivo pathogenic role of Toll-like receptor 4 (TLR-4) in the antiphospholipid syndrome (APS) by studying the thrombogenic antiphospholipid (aPL) activity in lipopolysaccharide (LPS) non-responsive (LPS–/–) mice and the association between tlr4 gene polymorphisms and APS in patients. Methods: IgGs from two patients with APS, one with aPL negative systemic lupus erythematosus (SLE) and one with normal human serum (NHS), were evaluated for thrombosis, tissue factor (TF) activity and endothelial cell activation in LPS–/– mice displaying a tlr4 spontaneous mutation vs LPS responsive (LPS+/+) mice. Human tlr4 Asp299Gly and Thr399Ile polymorphisms were evaluated by allele-specific PCR in 110 patients with APS with arterial/venous thrombosis and in 220 controls of the same ethnic origin. Results: IgG-APS produced significantly larger thrombi and more leucocytes (WBC) adhering to endothelial cells in the cremaster muscle microcirculation of LPS+/+ mice than IgG-NHS or aPL negative SLE-IgG. These effects were abrogated after absorption of the anti-β2glycoprotein I activity by an affinity column. The two IgG-APS induced significantly smaller thrombi and fewer WBC adhering to endothelial cells in LPS−/− mice than in LPS+/+ mice. IgG-APS induced higher TF activity in carotid artery homogenates of LPS+/+ mice than in LPS−/− mice. The prevalence of Asp299Gly and Thr399Ile tlr4 polymorphisms was significantly lower than in controls. Conclusions: These findings in LPS−/− mice and the reduction in the “protective” polymorphism in patients with APS with thrombosis suggest that TLR-4 is involved in the interaction of aPL with endothelial cells in vivo.

Antiphospholipid Antibodies and the Antiphospholipid Syndrome: Pathogenic Mechanisms

Antiphospholipid antibodies (Abs) are associated with thrombosis and are a risk factor for recurrent pregnancy loss and obstetric complications in patients with the antiphospholipid syndrome. It is generally accepted that the major autoantigen for aPL Abs is beta (2) glycoprotein I, which mediates the binding of aPL Abs to target cells (i.e., endothelial cells, monocytes, platelets, trophoblasts, etc.) leading to thrombosis and fetal loss. This article addresses molecular events triggered by aPL Abs on endothelial cells, platelets, and monocytes and complement activation, as well as a review of the current knowledge with regard to the putative receptor(s) recognized by aPL Abs on target cells as well as novel mechanisms that involve fibrinolytic processes. A section is devoted to the description of thrombotic and inflammatory processes that lead to obstetric complications mediated by aPL Abs. Based on experimental evidence using in vitro and in vivo models, new targeted therapies for treatment and/or prevention of thrombosis and pregnancy loss in antiphospholipid syndrome are proposed.

TH1 and TH2 cytokine production by peripheral blood mononuclear cells from HIV-infected patients

ObjectiveTo study the TH1->TH2 cytokine switch, thought to occur during the progression of HIV infection. DesignWe investigated interleukin (IL)-2, interferon (IFN)-γ, IL-4, IL-6 and IL-10 production by phytohaemagglutinin (PHA)-stimulated and unstimulated peripheral blood mononuclear cell (PBMC) cultures from HIV-negative controls and HIV-positive subjects, stratified according to the Centers for Disease Control and Prevention (CDC) criteria. We correlated the above parameters with markers of disease progression. MethodsCytokine production was measured in supernatants using enzyme-linked immunosorbent assay (ELISA) in 40 patients and 17 controls. To evaluate disease progression, we also determined CD4+ cell counts, PHA-induced proliferative response, p24 release and spontaneous immunoglobulin (Ig) G and IgM production. ResultsIn agreement with the TH1->TH2 switch hypothesis, we found that in the course of HIV disease mitogen-stimulated IL-2 production decreased, spontaneous and stimulated IL-6 production and spontaneous IL-10 secretion increased; IL-4 showed an increasing trend, although it was reduced in HIV-positive subjects. Finally, immunoglobulin production increased over time. In contrast, mitogen-stimulated IFN-γ and IL-10 production did not change among the CDC categories, although the former decreased and the latter increased in comparison with HIV-negative controls. ConclusionsOur data partially agree with the TH1->TH2 switch hypothesis. Since IL-6 and IL-10 are produced by different cell types, whose proportions and functional features vary in the course of the disease, further experiments with purified lymphocyte subsets and monocytes are required. Nevertheless, as already suggested, we believe that a switch from a type 1 to a type 2 response occurs in HIV infection.

Anti-endothelial cell antibodies in patients with Wegener's granulomatosis and micropolyarteritis.

Anti‐endothelial cell antibodies (AECA) have been detected by cell surface radioimmunoassay innine out of 15 patients with micropolyarteritis (MPA) and in two out of five patients with Wegenersgranulomatosis. AECA mostly belonged to the IgG isotype and were present in the active phase ofthe diseases. These antibodies were not detectable in 10 sera from patients with essential mixedcryoglobulinaemia. suggesting that they were not a mere epiphenomenon consequent to theinflammatory vascular injury. The binding activity was not related to ABH antigens or to HLA class Iantigens displayed by resting human endothelial cells in culture and was not influenced by removingimmune complexes. Absorption of the anti‐neutrophil cytoplasmic antibodies (ANCA). present inMPA and Wegeners granulomatosis sera, did not affect the endothelial binding. AECA‐positive seradid not display lytic activity against endothelial cells, neither alone nor after addition of freshcomplement or normal human peripheral blood mononuelear cells. Although AECA arc notcytolytic for endothelial cell monolayers in vitro, the reactivity against intact endothelial cells suggeststheir possible involvement in in vivo pathological processes affecting vascular structures in smallvessel primary vasculitides.

Anti-endothelial cell IgG fractions from systemic lupus erythematosus patients bind to human endothelial cells and induce a pro-adhesive and a pro-infiammatory phenotype in vitro

Affinity purified immunoglobulin G (IgG) fractions from systemic lupus erythematosus (SLE) patients positive for anti-endothelial cell antibodies (AECA) bind human umbilical vein endothelial cell (HUVEC) monolayers. In vitro incubation of serial protein concentrations of SLE AECA IgG induces a dose-dependent endothelial activation: i) increase of functional adhesion of the monocytic cell line U937; ii) upregulation of E-Selectin, ICAM-1, VCAM-1 expression evaluated by a cell solid-phase enzyme linked immunoassay; and iii) increased secretion of interleukin (IL)-6 in the culture supernatants. Control experiments carried out with HUVEC monolayers incubated with IgG fractions from normal healthy controls or from AECA negative SLE sera do not affect at all endothelial adhesion molecule expression or pro-inflammatory cytokine secretion. The AECA IgG effects are not related to both anti-phospholipid or anti-DNA activities. Taken together the findings suggest that these autoantibodies might be important in recruiting and in activating mononuclear leukocytes responsible for vessel wall infiltration and raise the possibility that AECA might display a pathogenic role in SLE vessel damage.

Antibodies to endothelial cells in primary vasculitides mediate in vitro endothelial cytotoxicity in the presence of normal peripheral blood mononuclear cells

Twenty-eight out of 62 patients with Wegeners granulomatosis and micropolyarteritis display circulating antiendothelial cell antibodies (AECA) detectable by a cell surface radioimmunoassay. These antibodies do not induce an in vitro endothelial damage either alone or in the presence of fresh complement; however, 50% of IgG-AECA positive sera can be cytotoxic in the presence of human normal peripheral blood mononuclear cells (PBM) at high effector/target ratios. The specificity of the PBM-mediated cytotoxicity is supported by the absence of the phenomenon in AECA negative sera, by the disappearance of the lytic effect after absorption of AECA, and by the finding that cellular-mediated cytotoxicity can be reproduced by purified IgG-AECA positive fractions. On the contrary, polymorphonuclear leukocytes or adherent mononuclear cells are not involved in such a cytotoxic activity. AECA seem to be directed against determinants consitutively expressed on the endothelial surface since the activation of endothelial cells by interleukin-1 beta or interferon-gamma affects neither the antibody binding nor their ability to mediate 51Cr release in the presence of PBM. These findings favor the hypothesis for a possible direct pathogenetic role of circulating AECA in the in vivo vascular damage.

A non–complement-fixing antibody to β2 glycoprotein I as a novel therapy for antiphospholipid syndrome

A single-chain fragment variable (scFv) recognizing β2-glycoprotein 1 (β2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against β2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-β2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-β2GPI antibodies from APS patients and displaced β2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy.

Prevalence and clinical significance of anti-cyclic citrullinated peptide antibodies in juvenile idiopathic arthritis

Background: Antibodies against cyclic citrullinated peptide (anti-CCP) are considered to be specific for rheumatoid arthritis (RA). Objective: To assess the clinical significance of anti-CCP in a cohort of patients with juvenile idiopathic arthritis (JIA). Methods: Anti-CCP were tested by an enzyme linked immunosorbent assay (ELISA) in serum samples from 109 patients with JIA (30 boys, 79 girls), with a mean age of 8.7 years (range 0.6–20.3) and mean disease duration of 3.6 years (range 3 months to 15.6 years). As control groups, anti-CCP were also tested in sera of 30 healthy children, 25 patients with juvenile onset systemic lupus erythematosus (SLE), and 50 adult patients (30 with RA, 20 with SLE). Results: Positive anti-CCP values were found in sera of two patients with JIA (2%), one with polyarthritis, and one with oligoarthritis. Statistical analysis showed that anti-CCP were not associated with the presence of antinuclear antibodies, raised erythrocyte sedimentation rate, or erosions. In the control groups, none of the patients with juvenile onset SLE and only one of 20 adults with SLE were positive for anti-CCP, but 19/30 (63%) adults with RA showed anti-CCP positivity. Conclusions: Anti-CCP can be detected in children with JIA, but are less frequently present than in adults with RA.

Patients with antiphospholipid syndrome display endothelial perturbation

BACKGROUND There is strong evidence that antiphospholipid antibodies (aPL) perturb endothelium both in vitro and in experimental animal models. by inducing a vasculopathy and an endothelial pro-inflammatory/coagulant phenotype. However, few contrasting studies raised the issue about the possibility to detect a comparable endothelial perturbation in anti-phospholipid syndrome (APS) patients. The aim of this observational case-control study was to evaluate several parameters of endothelial perturbation in patients with APS and without any other atherosclerosis risk factor. PATIENTS AND METHODS We investigated plasma levels of soluble adhesion molecules (s-ICAM-1, s-VCAM-1, s-E-selectin), soluble thrombomodulin (sTM), von Willebrand factor (vWF) and tissue plasminogen activator (t-PA) by solid-phase assays in 40 selected APS patients and 40 age- and sex-matched healthy subjects. In addition, we evaluated circulating endothelial cells by flow cytometry and brachial artery flow-mediated vasodilation. Patients and controls were free of conditions known to affect both the biological and the functional endothelial parameters. RESULTS Plasma levels of sTM, s-E-selectin and s-VCAM-1 did not differ from controls, while a significant increase in s-ICAM-1 (P = 0.029), t-PA (P = 0.003) and vWF titres (P = 0.002) was found. Circulating mature endothelial cells were also significantly higher in patients than in controls (P = 0.05) and decreased during both vitamin K antagonists (P = 0.001) and antiplatelet (P = 0.032) treatments. Mean brachial artery flow-mediated vasodilation responses were significantly impaired compared to healthy subjects (P = 0.0001). CONCLUSIONS As a whole these findings indicate that APS patients display an endothelial perturbation in the absence of other detectable traditional risk factors for atherosclerosis.

In vivo distribution of β2 glycoprotein I under various pathophysiologic conditions

In vitro studies have documented β2 glycoprotein I (β2GPI) binding to endothelial cells (ECs) and trophoblast using antiphospholipid antibodies. The in vivo binding of β2GPI to these cells and the conditions that favor their interaction have not been investigated. We analyzed the in vivo distribution of cyanine 5.5-labeled β2GPI in mice and evaluated the effect of pregnancy and circulating antibodies on its tissue localization. The signal was detected in the liver by whole body scan and ex vivo analysis. The β2GPI failed to bind to the vascular endothelium and reacted only with the ECs of uterine vessels. In pregnant mice the protein was localized on ECs and trophoblast at the embryo implantation sites. Immunized mice showed a similar β2GPI biodistribution to naive mice but the immunized pregnant animals exhibited a significant increase in fetal loss associated with C3 and C9 deposition at the implantation sites. Treatment of mice with LPS after β2GPI-Cy5.5 injection promoted protein localization on gut and brain ECs associated with IgG, C1q, and C9 deposition in immunized mice. These findings indicate that β2GPI binding to EC requires priming with pro-inflammatory factors which is not needed for uterine and placental localization probably dependent on hormonal changes.