Pier Luigi Meroni

Accelerated Atherosclerosis in Autoimmune Rheumatic Diseases

Received October 16, 2004; revision received June 4, 2005; accepted June 7, 2005. Atherosclerosis is a multifactorial process that commences in childhood but manifests clinically later in life. Atherosclerosis is increasingly considered an immune system–mediated process of the vascular system. The presence of macrophages and activated lymphocytes within atherosclerotic plaques supports the concept of atherosclerosis as an immune system–mediated inflammatory disorder.1,2 Inflammation can aggravate atherosclerosis via different mechanisms secondary to autoimmunity, infectious diseases, and other proatherogenic changes that occur during the inflammatory state. Autoimmune rheumatic diseases (AIRDs) are associated with higher rates of cardiovascular morbidity and mortality, primarily secondary to accelerated atherosclerosis. This phenomenon can be attributed to traditional risk factors for atherosclerosis and use of specific drugs, such as corticosteroids, but also might be the result of other autoimmune and inflammatory mechanisms that are aggravated in AIRDs. Several AIRDs exhibit increased overt cardiovascular disease (CVD) prevalence as well as findings of advanced subclinical atherosclerosis, which may precede the appearance of a clinical disease and thus be a target of early identification and preventive therapy. Cells of the immune system can be found within atherosclerotic plaques, which suggests that they have a role in the atherogenic process. Their migration and activation within the plaques can be secondary to various stimuli, including infectious agents.3 These cells probably aggravate atherosclerosis, because CD4+ and CD8+ T-cell depletion reduced fatty streak formation in C57BL/6 mice. In addition, after crossing of apolipoprotein E (ApoE)-knockout mice with immunodeficient scid/scid mice, the offspring had a 73% reduction in aortic fatty streak lesions compared with the immunocompetent apoE mice. Moreover, when CD4+ T cells were transferred from the immunocompetent to the immunodeficient mice, they increased lesion area in the latter by 164%.4 It is therefore not surprising that as in autoimmune diseases, the cellular components …

Anti-inflammatory and immunosuppressive drugs and reproduction

Rheumatic diseases in women of childbearing years may necessitate drug treatment during a pregnancy, to control maternal disease activity and to ensure a successful pregnancy outcome. This survey is based on a consensus workshop of international experts discussing effects of anti-inflammatory, immunosuppressive and biological drugs during pregnancy and lactation. In addition, effects of these drugs on male and female fertility and possible long-term effects on infants exposed to drugs antenatally are discussed where data were available. Recommendations for drug treatment during pregnancy and lactation are given.

Risk of congenital complete heart block in newborns of mothers with anti-Ro/SSA antibodies detected by counterimmunoelectrophoresis:. A prospective study of 100 women.

OBJECTIVE To assess the true prevalence of congenital complete heart block (CCHB) in infants of anti-Ro/SSA-positive women known to have connective tissue disease (CTD) and, secondarily, to evaluate the prevalence of other electrocardiographic abnormalities in these newborns at birth. METHODS A prospective study was conducted in 4 referral hospitals. One hundred anti-Ro/SSAA-positive mothers were followed up before they became pregnant and during the index pregnancy. Counterimmunoelectrophoresis and immunoblotting were used to test for antibodies to extractable nuclear antigens. RESULTS Of the 100 women with anti-Ro/SSA antibodies, 2 had infants who developed CCHB in utero (2%). The CCHB was detected at 22 weeks and 20 weeks, respectively. One of the 2 mothers had primary Sjögrens syndrome (SS), and the other had undifferentiated CTD (UCTD). No case of CCHB occurred among the infants of 53 mothers with systemic lupus erythematosus (SLE). No fetal death occurred due to CCHB. In 2 centers, electrocardiography was recorded in 24 unselected newborns, and 4 were found to have sinus bradycardia. CONCLUSION The prevalence of CCHB in newborns of prospectively followed up women already known to be anti-Ro/SSA positive and with known CTD was 2%. This finding is useful with regard to preconception counseling of these women. The risk of delivering an infant with CCHB may be higher in mothers with primary SS or UCTD than in those with SLE. Additional electrocardiographic abnormalities such as sinus bradycardia and prolongation of the QT interval may be present in their children.

ANA screening: an old test with new recommendations

The impact of autoimmune diseases is growing from both a clinical and a laboratory point of view. Diagnostic assays are now being transferred from dedicated specialised laboratories into high-throughput service laboratories. The increasing number of available methods has raised the variability among the laboratories, making their reproducibility a critical problem. On the other hand, reliable tests are needed for early diagnosis and prompt treatment, and the cost of repeated confirmatory tests should be reduced and unnecessary further investigations avoided. New methodologies, particularly for antinuclear antibodies (ANAs), have been applied to autoantibody detection in order to screen and process larger volumes of clinical specimens more quickly and at less cost than the traditional methods. However, because of the lack of their sensitivity to detect all ANAs and standardisation, inaccuracies (including false positives and negatives) in the results have been reported. A committee of the American College of Rheumatology was formed to address this issue. Specific recommendations have been suggested that represent a realistic first step in the standardisation of diagnostic tests for autoimmune diseases.

International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies

Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1–13), anti-double stranded DNA antibodies (14–18), specific antibodies (19–23) and validation of methods (24–25) were created. Significant differences between experts were observed regarding recommendations 24–25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

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.

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 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.

Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6

Fetal loss in animals and humans is frequently associated with inflammatory conditions. D6 is a promiscuous chemokine receptor with decoy function, expressed in lymphatic endothelium, that recognizes and targets to degradation most inflammatory CC chemokines. Here, we report that D6 is expressed in placenta on invading extravillous trophoblasts and on the apical side of syncytiotrophoblast cells, at the very interface between maternal blood and fetus. Exposure of D6−/− pregnant mice to LPS or antiphospholipid autoantibodies results in higher levels of inflammatory CC chemokines and increased leukocyte infiltrate in placenta, causing an increased rate of fetal loss, which is prevented by blocking inflammatory chemokines. Thus, the promiscuous decoy receptor for inflammatory CC chemokines D6 plays a nonredundant role in the protection against fetal loss caused by systemic inflammation and antiphospholipid antibodies.