Proteomes of umbilical vein and microvascular endothelial cells reflect distinct biological properties and influence immune recognition.

Human umbilical vein endothelial cells (HUVEC) are widely used as a source of endothelial cells (EC). However, HUVEC characteristics cannot be extrapolated to other types of EC, particularly microvascular ECs. Our objective was to compare the proteomes of microvascular ECs and HUVEC.

IgG from patients with pulmonary arterial hypertension and/or systemic sclerosis binds to vascular smooth muscle cells and induces cell contraction.

Objectives: Pulmonary arterial hypertension (PAH) is characterised by remodelling of pulmonary arteries with enhanced vascular smooth muscle cell (VSMC) contraction, migration and proliferation. The authors investigated the presence of antibodies to human VSMCs in the serum of patients with systemic sclerosis with or without PAH and idiopathic PAH (iPAH).

Methods And Results: Antibodies to VSMCs were detected by immunofluorescence in sera from healthy controls and patients with scleroderma without PAH, scleroderma-associated PAH and iPAH.

Targets of anti-endothelial cell antibodies in pulmonary hypertension and scleroderma.

Anti-endothelial cell antibodies (AECAs) have been identified in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and in patients with idiopathic pulmonary arterial hypertension (iPAH). However, their target antigens remain poorly identified. Sera from 24 patients with SSc without PAH, 20 patients with SSc with PAH, 30 with iPAH and 12 healthy controls were collected.

Identification of target antigens of anti-endothelial cell and anti-vascular smooth muscle cell antibodies in patients with giant cell arteritis: a proteomic approach.

Introduction: Immunological studies of giant cell arteritis (GCA) suggest that a triggering antigen of unknown nature could generate a specific immune response. We thus decided to detect autoantibodies directed against endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in the serum of GCA patients and to identify their target antigens.

Methods: Sera from 15 GCA patients were tested in 5 pools of 3 patients' sera and compared to a sera pool from 12 healthy controls (HCs).

Identification of new autoantibody specificities directed at proteins involved in the transforming growth factor β pathway in patients with systemic sclerosis.

Introduction: Antinuclear antibodies (ANAs), usually detected by indirect immunofluorescence on HEp-2 cells, are identified in 90% of patients with systemic sclerosis (SSc). Thus, approximately 10% of SSc patients have no routinely detectable autoantibodies, and for 20% to 40% of those with detectable ANAs, the ANAs do not have identified specificity (unidentified ANAs). In this work, we aimed to identify new target autoantigens in SSc patients.

The role of inflammation and autoimmunity in the pathophysiology of pulmonary arterial hypertension.

Pulmonary arterial hypertension is characterized by a remodeling of pulmonary arteries with endothelial cell, fibroblast, and vascular smooth muscle cell activation and proliferation. Since pulmonary arterial hypertension occurs frequently in autoimmune conditions such as systemic sclerosis, inflammation and autoimmunity have been suspected to play a critical role in both idiopathic pulmonary arterial hypertension and systemic sclerosis-associated pulmonary arterial hypertension. High levels of pro-inflammatory cytokines such as interleukin-1 and interleukin-6, platelet-derived growth factor, or macrophage inflammatory protein 1 have been found in lung samples of patients with pulmonary arterial hypertension, along with inflammatory cell infiltrates mainly composed of macrophages and dendritic cells, T and B lymphocytes.

Microsomal prostaglandin e2 synthase-1 modulates the response to vascular injury.

Background: Microsomal (m) prostaglandin (PG) E₂ synthase (S)-1 catalyzes the formation of PGE₂ from PGH₂, a cyclooxygenase product that is derived from arachidonic acid. Previous studies in mice suggest that targeting mPGES-1 may be less likely to cause hypertension or thrombosis than cyclooxygenase-2-selective inhibition or deletion in vivo. Indeed, deletion of mPGES-1 retards atherogenesis and angiotensin II-induced aortic aneurysm formation.

Pathogenesis of giant cell arteritis: More than just an inflammatory condition?

Giant cell arteritis (GCA) is characterized by intimal hyperplasia and luminal obstruction leading to ischemic manifestations involving extra-cranial branches of carotid arteries and aorta. Histopathological lesions involve all layers of the arterial wall and are associated with multinucleated giant cells, fragmented internal elastic lamina and polymorphic cellular infiltrates, including T lymphocytes and macrophages. The pathophysiology of GCA is still poorly understood.

IgG from patients with systemic sclerosis bind to DNA antitopoisomerase 1 in normal human fibroblasts extracts.

By using a semi-quantitative immunoblotting technique, we have analyzed serum immunoglobulin G (IgG) reactivities of patients with limited cutaneous systemic sclerosis and anticentromere antibodies, patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies, patients with diffuse systemic sclerosis without antitopoisomerase 1 or anticentromere antibodies and age- and gender-matched healthy controls with normal human skin fibroblasts and HEp-2 cells antigens. Serum IgG reactivities of patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies differed significantly from those of healthy controls or systemic sclerosis patients in other groups for reactivity with fibroblast proteins. IgG from patients with antitopoisomerase 1 antibodies bound to a 90 kDa fibroblast band and to a 100 kDa protein band in a HEp-2 cell protein extract.

Identification of target antigens of antifibroblast antibodies in pulmonary arterial hypertension.

Rationale: Pulmonary arterial hypertension (PAH) may be classified as idiopathic (IPAH) or familial (FPAH) or associated with various conditions and exposures such as dexfenfluramine intake (Dex-PAH) or systemic sclerosis (SSc-PAH). Because fibroblast dysfunction has been identified in SSc and IPAH and antifibroblast antibodies (AFAs) with a pathogenic role have been detected in the serum of SSc patients, we used a proteomic approach combining two-dimensional electrophoresis and immunoblotting to identify the target antigens of AFAs in such patients.

Objectives: To identify target antigens of antifibroblast antibodies in pulmonary arterial hypertension.

Identification of target antigens of antiendothelial cell antibodies in healthy individuals: A proteomic approach.

In order to identify target antigens of anti-endothelial cell (anti-EC) antibodies (AECA) in healthy individuals, we have used a proteomic approach combining 2-DE and immunoblotting. Whole cell protein extracts obtained from human umbilical vein EC (HUVEC) cultures were used as a source of antigens. Serum IgG from 12 healthy blood donors were tested at a concentration of 200 microg/mL.

Serum eosinophil cationic protein: a marker of disease activity in Churg-Strauss syndrome.

The cytotoxic proteins released by activated eosinophils should play a role in the development of Churg-Strauss syndrome (CSS). Eighteen patients (15 males and 3 females, age 41 +/- 13.3 years) with CSS according to the American College of Rheumatology criteria were included in the study.

A combined SDS-PAGE and proteomics approach to identify target autoantigens in healthy individuals and patients with autoimmune diseases.

We here present a method for the identification of antigens recognized by autoantibodies in healthy individuals and patients with autoimmune diseases. We have analyzed IgG reactivities from healthy individuals and patients with autoimmune diseases with endothelial cell antigens by combining a one-dimensional (1D) quantative immunoblotting technique and a 2D immunoblotting technique. Whole-cell protein extracts obtained from human umbilical vein endothelial cells (HUVEC) were used as a source of antigens.

Antitopoisomerase 1 antibodies in systemic sclerosis: how to improve the detection?

Among the multiple autoantibodies identified in the serum of systemic sclerosis (SSc) patients, three are disease-specific, mutually exclusive, and helpful to determine the prognosis: anticentromere antibodies, antitopoisomerase 1 antibodies (ATA), and anti-RNA-polymerase III antibodies. ATA can be identified through different techniques, including double immunodiffusion (DID) assay, enzyme-linked immunosorbent assay (ELISA), or immunoblot. Although all of them are commonly used, none of them can be considered as the reference.

Immunoblotting on HEp-2 cells increases the detection of antitopoisomerase 1 antibodies in patients with systemic sclerosis.

In order to improve the detection of antitopoisomerase 1 antibodies in a cohort of 111 systemic sclerosis patients, we have performed immunoblots on protein extracts of HEp-2 cells. Using indirect immunofluorescence and ELISA, 27 patients (24.3%) had antitopoisomerase 1 antibodies, 32 (28.

[Systemic sclerosis: pathophysiology of a multifaceted disease].

Systemic sclerosis is a rare disease characterized by vascular hyperreactivity and collagen deposition. Endothelial cell, fibroblast and lymphocyte abnormalities have been reported in systemic sclerosis. Fibroblast dysfunction is characterized by uncontrolled activation of the transforming growth factor-beta (TGF-beta) pathway and excess synthesis of both connective tissue growth factor (CTGF) and free radicals.

Anti-endothelial cell antibodies from patients with limited cutaneous systemic sclerosis bind to centromeric protein B (CENP-B).

By using a quantitative immunoblotting technique on protein extracts of human macrovascular and microvascular endothelial cells, we have analyzed the self-reactive repertoires of IgG from 20 patients with limited cutaneous SSc, 40 patients with diffuse SSc and 60 age- and sex-matched healthy controls. Serum IgG from 15/20 patients with limited cutaneous SSc and anti-centromere antibodies bound to at least one of the two 75- and 85-kDa protein bands in the different endothelial cell extracts, whereas IgG from healthy controls or patients with diffuse SSc did not. N-terminal sequencing of the 75- and 85-kDa bands identified CENP-B as the sole antigen in both bands.

Analysis of autoantibody repertoires in small- and medium-sized vessels vasculitides. Evidence for specific perturbations in polyarteritis nodosa, microscopic polyangiitis, Churg-Strauss syndrome and Wegener's granulomatosis.

In order to identify new antibody reactivities, we have used a quantitative immunoblotting technique on extracts of normal human tissues to analyze the repertoires of serum IgM, serum IgG and purified IgG autoantibodies of patients with systemic vasculitides. Patients fulfilled the American College of Rheumatology and Chapel Hill criteria for the diagnosis of polyarteritis nodosa (PAN) (n=8), PAN related to hepatitis B virus (HBV) infection (n=5), Wegener's granulomatosis (WG) (n=6), microscopic polyangiitis (MPA) (n=18) or Churg-Strauss syndrome (CSS) (n=8). Sera from patients with chronic HBV infection without PAN (n=5) and age- and gender-matched healthy individuals (n=45) were used as controls.

Anti-annexin V antibodies: are they prothrombotic?

Annexin V inhibits prothrombin activation and is able to prevent thrombus formation under normal venous and arterial blood flow conditions. Antibodies to annexin V have been identified in association with several pathological conditions, including systemic lupus erythematosus (SLE) with or without anti-phospholipid syndrome, recurrent spontaneous abortions and systemic sclerosis (SSc). These antibodies are suspected to exert a detrimental role and interfere with annexin V function.

[Immunomodulatory effects of intravenous immunoglobulins].

Intravenous immunoglobulins (IVIg) are therapeutic preparations of normal human IgG obtained from pools of more than 1000 healthy blood donors. They are currently used in the treatment of a wide range of auto-immune diseases, whether associated with auto-antibodies or auto-reactive T lymphocytes, as well as in the treatment of systemic inflammatory diseases. Several mechanisms of action have been identified during the last 20 years, including: (i) modulation of Fc receptors expression on leukocytes and endothelial cells; (ii) interaction with complement proteins; (iii) modulation of cytokines and chemokines synthesis and release; (iv) modulation of cell proliferation and apoptosis; (v) remyelinisation; (vi) neutralisation of circulating autoantibodies; (vii) selection of repertoires of B and T lymphocytes; (viii) interaction with other cell-surface molecules on lymphocytes and monocytes; (ix) corticosteroid sparing.

IgG reactivity with a 100-kDa tissue and endothelial cell antigen identified as topoisomerase 1 distinguishes between limited and diffuse systemic sclerosis patients.

We have analyzed antibody (Ab) reactivities of patients with limited systemic sclerosis (SSc) and anti-centromere Ab, patients with diffuse SSc and anti-topoisomerase 1 (anti-topo 1) Ab, patients with diffuse SSc without anti-topo 1 or anti-centromere Ab and age- and gender-matched healthy controls with normal human tissue and endothelial cell (EC) antigens. IgG reactivities with tissue antigens differed significantly between patients with anti-topo 1 Ab and patients with anti-centromere Ab. One 100-kDa band identified as topoisomerase 1 in macrovascular and microvascular EC extracts was recognized by IgG from patients with anti-topo 1 Ab and 50% of patients without specific Ab.

IgM and IgG autoantibodies from microscopic polyangiitis patients but not those with other small- and medium-sized vessel vasculitides recognize multiple endothelial cell antigens.

Using a quantitative immunoblotting technique on extracts of macrovascular and microvascular endothelial cells (EC), we analyzed serum IgM and IgG reactivities of patients with active disease fulfilling the ACR and Chapel Hill criteria for the diagnosis of polyarteritis nodosa (PAN) (n = 8), PAN related to hepatitis B virus (HBV) infection (HBV-PAN) (n = 5), Wegener's granulomatosis (n = 6), microscopic polyangiitis (MPA) (n = 18), Churg-Strauss syndrome (n = 8), and patients with chronic HBV infection without PAN (n = 5) and age- and gender-matched healthy individuals (n = 45). MPA patients' IgM bound to 200-, 105-, 80-, 65-, 45-, 35-, and 33-kDa major bands, whereas IgM from controls and other patients bound predominantly to the 65-kDa band in EC extracts. MPA patients' IgG reacted mainly with 105-, 70-, 55-, and 38-kDa protein bands, whereas IgG from controls and other patients did not.

New insights into the pathogenesis of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disorder characterized by vascular abnormalities and excessive collagen synthesis. Extracellular matrix overproduction by fibroblasts results from abnormal interactions among endothelial cells, mononuclear cells (lymphocytes and monocytes) and fibroblasts, in a setting of vascular hyperreactivity and tissue hypoxia. Many autoantibodies have been identified in the sera of SSc patients; some of them are specific to the disease, such as anti-centromere antibodies in limited SSc, anti-topoisomerase 1 and anti-RNA polymerase I/III antibodies in diffuse SSc.

[Pathogenesis of systemic scleroderma: immunological aspects].

Systemic sclerosis (SSc) is a connective tissue disorder that is characterized by excessive collagen synthesis by fibroblasts and by vascular hyperreactivity and obliteration phenomena. Excessive collagen production is the consequence of abnormal interactions between endothelial cells, fibroblasts and mononuclear cells. Immunological abnormalities are present very early in the development of SSc.