Background: Antiphospholipid antibodies (aPL) are associated with thrombosis and pregnancy loss in patients with Antiphospholipid Syndrome (APS). aPL and bacterial lipopolysaccharide (LPS) bind to and activate endothelial cells (EC) through NFκB and p38 MAPK pathways. Recent studies suggest that aPL might interact with toll-like receptor-4 (TLR-4), the receptor for LPS.

Despite the discovery of anti-endothelial cell antibodies (AECA) in a heterogeneous group of disorders characterized by endothelial damage, their pathogenic role is still debated. Experimental in vitro models indicate that they can either damage endothelial cells or trigger cell signaling by reacting with as yet undefined surface molecules. However, clinical studies suggest that, in addition to AECA, other pathogenic mechanisms are involved in the vasculitic process. Recently, antibodies specific for beta 2 glycoprotein I, the phospholipid-binding protein targeted by anti-phospholipid antibodies, have been shown to display anti-endothelial activity. These autoantibodies recognize beta 2 glycoprotein I adhered to the endothelium and induce a cell perturbation that might underlie the thrombophilic state of the anti-phospholipid syndrome.

Antibodies (Abs) against the structure specific recognition protein 1 (SSRP1) were reported in a small systemic lupus erythematosus (SLE) series but not in other systemic autoimmune diseases. The aim of the study was to confirm the selective presence of anti-SSRP1 Abs in a larger SLE series and to evaluate their relationship with disease activity and other immune markers. Anti-SSRP1 Abs were investigated by a 'home made' ELISA in: 120 SLE, 65 rheumatoid arthritis ( RA), 51 systemic sclerosis (SSc), 23 Churg-Strauss syndrome (CSS) and 40 idiopathic autoimmune urticaria (IAU) patients and 190 healthy controls. Sera from MRL lpr/lpr and Balb-c mice were also tested. Anti-SSRP1 Abs were detected in 43 SLE (35.8%), nine SSc (17.6%), eight RA (12.3%), six IAU (15%), three CSS (13%) patients and five healthy controls (2.6%). Antibody prevalence and titers were significantly higher in SLE patients than in sera from both normal and disease controls. Anti-SSRP1 Ab activity was also detected in sera from MRL lpr/lpr but not Balb-c mice. The antibodies did not correlate with the disease activity evaluated as the ECLAM index score and were more prevalent in patients without renal involvement. No correlation was found with other serum autoantibodies. Our results confirm that anti-SSRP1 Abs are associated with but not specific for the lupus disease.

Antiphospholipid antibodies are mainly directed against beta 2 glycoprotein I (beta2GPI), a plasma phospholipid-binding protein expressed on endothelial cells of different anatomical localizations. Anti-beta2GPI antibodies recognize the molecule on endothelial monolayers in vitro, and, once bound, might activate the cells both in vitro and in vivo experimental models inducing a proinflammatory and a procoagulant phenotype. Cell activation is associated with nuclear factor-kappaB (NF-kappaB) translocation and with a signaling cascade comparable to that triggered by the toll-like receptors (TLRs)-4. The cell membrane receptor(s) for beta2GPI adhesion is still under investigation. It has been suggested that beta2GPI might adhere through electrostatic interaction between its cationic phospholipid binding site and anionic structures on the cell membrane; however, binding to annexin II-the endothelial cell receptor for tissue plasminogen activator-plays also a role. Because annexin II does not display any transmembrane protein, it has been suggested that it requires a yet unknown "adaptor" protein to signal the cells. Because of the molecular mimicry between beta2GPI and viral/bacterial structures-the natural ligands for TLRs-antibodies might cross-link the molecule associated to annexin II and TLR-4 eventually triggering the signaling.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antiphospholipid antibodies (aPLs) and recurrent thrombosis or fetal loss. The thrombophilic state has been partially related to the induction of a proinflammatory and procoagulant endothelial cell (EC) phenotype induced by anti-beta(2)-glycoprotein I (beta(2)-GPI) antibodies that bind beta(2)-GPI expressed on the EC surface. Anti-beta(2)-GPI antibody binding has been shown to induce nuclear factor-kappa B (NF-kappa B) translocation leading to a proinflammatory EC phenotype similar to that elicited by interaction with microbial products (lipopolysaccharide [LPS]) and proinflammatory cytokines (interleukin 1 beta [IL-1 beta], tumor necrosis factor alpha [TNF-alpha]). However, the upstream signaling events are not characterized yet. To investigate the endothelial signaling cascade activated by anti-beta(2)-GPI antibodies, we transiently cotransfected immortalized human microvascular endothelial cells (HMEC-1) with dominant-negative constructs of different components of the pathway (Delta TRAF2, Delta TRAF6, Delta MyD88) together with reporter genes (NF-kappa B luciferase and pCMV-beta-galactosidase). Results showed that both human anti-beta(2)-GPI IgM monoclonal antibodies as well as polyclonal affinity-purified anti-beta(2)-GPI IgG display a signaling cascade comparable to that activated by LPS or IL-1. Delta TRAF6 and Delta MyD88 significantly abrogate antibody-induced as well as IL-1- or LPS-induced NF-kappa B activation, whereas Delta TRAF2 (involved in NF-kappa B activation by TNF) does not affect it. Moreover, anti- beta(2)-GPI antibodies and LPS followed the same time kinetic of IL-1 receptor-activated kinase (IRAK) phosphorylation, suggesting an involvement of the toll-like receptor (TLR) family. Our findings demonstrate that anti-beta(2)-GPI antibodies react with their antigen likely associated to a member of the TLR/IL-1 receptor family on the EC surface and directly induce activation.

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.

HIV type 1 expression was significantly up-regulated in chronically infected promonocytic cell line (U1) co-cultured with human umbilical vein endothelial cells (HUVEC). Virus replication, evaluated as supernatant p24 release, was higher when U1 were co-cultured with IL-1beta-activated HUVEC than with unstimulated HUVEC. When non-adherent U1 were removed from co-cultures, the remaining U1 cells adherent to the endothelial monolayer still showed enhanced HIV replication in comparison with an equal number of U1 cultured alone. While addition of adhesion molecule blocking antibodies (anti-intercellular adhesion molecule-1 (ICAM-1), -vascular cell adhesion molecule-1 (VCAM-1), -CD18 and -very late antigen-4 (VLA-4)) strongly inhibited adherence of U1 cells to endothelial monolayers, such treatment resulted in only a partial reduction in p24 release. Furthermore, HIV replication in U1 cells was enhanced on culture in HUVEC-conditioned media. Such data suggest that soluble mediators secreted by endothelial monolayers may modulate HIV-1 expression. Indeed, addition of cytokine and chemokine antagonists to both U1/HUVEC co-cultures and to U1 cultured in HUVEC-conditioned media clearly down-regulated p24 release. Anti-IL-6, anti-tumour necrosis factor-alpha (TNF-alpha) and, particularly, anti-MCP-1 MoAbs reduced p24 release, while anti-IL-8 polyclonal antiserum and IL-1 receptor antagonist (IL-1Ra) had no significant effect. Thus, the interaction between HUVEC and infected monocytic cells up-regulates HIV-1 replication predominantly through production of endothelium-derived soluble factors including MCP-1, TNF-alpha and IL-6. This phenomenon may influence the passage of HIV-1 from latency to productive replication and enhance virus spreading during physiological and/or pathological contact of monocytes with endothelium.

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.

We investigated the in vitro effect of human recombinant interleukin 11 (hrIL-11) on HIV-I expression in the human chronically infected promonocytic cell lines U1 and OM10 and in the T lymphoblastoid one ACH2. Exogenous IL-II induced a marked reduction in p24 release and RT activity by IL-6- or IL-la-stimulated U1 and OM10. A similar, but less evident, effect was observed on unstimulated promonocytic cells, while no variations in virus expression were seen in TNF alpha-activated cultures. The T lymphoblastoid cells ACH2 were not significantly affected by IL-11, either in unstimulated or IL-6- and IL-1 beta-stimulated conditioned. TNF alpha-activated cells showed a reduction in HIV-I replication, even if lower than that observed in promonocytic cells. Our results indicate for the first time that IL-11 may inhibit HIV-1 expression in chronically infected cell lines, acting selectively on monocyte compartment.

Beta2-glycoprotein I (beta2GPI) is a cofactor for anti-phospholipid (aPL) binding to cardiolipin (CL)-coated plates. Beta2GPI is also able to bind to endothelial cell (EC) membranes as supported by in-vivo as well as by in-vitro studies. The PL-binding site in the fifth domain of the molecule is involved in the adhesion to endothelium. Actually, specific mutations in this molecular portion abolish endothelium binding and a synthetic peptide spanning the sequence Glu274-Cys288 of the CL-binding site displays comparable adhesion to EC monolayers. Heparan sulphate appears to be one of the anionic EC membrane structures with which cationic beta2GPI interacts, as supported by studies with heparitinase-treated EC. Beta2GPI binding to EC might be related to its activity as endothelial growth factor or as a lipid-carrying glycoprotein. Adhesion of beta2GPI to endothelial membranes offers suitable epitopes for circulating aPL that, once bound, can induce cell activation

Objective: to investigate the relationship between disease activity and in vitro cytokine, soluble(s)CD23 and polyclonal and anti-DNA antibody production by PBMC from patients with active systemic lupus erythematosus (SLE).Methods: cytokines, sCD23 and immunoglobulins were estimated by ELISA in unstimulated and polyclonal mitogen-stimulated culture supernatants.Results: PHA-induced IL-2 and IFN-gamma production were decreased, whereas spontaneous and PHA-induced IL-6 and IL-10 production were increased in cultures of SLE lymphocytes. Conversely, spontaneous and PHA-stimulated IL-4 and sCD23 production was comparable between patients and controls. Finally, we found an increase in in vitro spontaneous polyclonal and anti-DNA IgG secretion.Conclusions: We demonstrated an expanded type-2 cytokine profile with no correlation with parameters of disease activity.

A lot of attention has recently been paid to antiphospholipid and antiendothelial cell antibodies as serological markers for systemic autoimmune diseases. Antiphospholipid antibodies appear to display a prognostic value for recurrent thrombosis and fetal loss both in a subset of systemic lupus erythematosus and in patients with the primary antiphospholipid syndrome. The range of diagnostic tests for the antiphospholipid syndrome is now becoming larger with the inclusion of assays suitable for identifying antibodies against phospholipids other than cardiolipin and especially against phospholipid-binding proteins. Antiendothelial cell antibodies have been suggested as serological markers for immune-mediated vascular damage. In vitro and in vivo experimental models as well as epidemiological studies seem to support this. However, the assays for their detection still lack standardization.

Objective To study type 1 and type 2 cytokine patterns in HIV-negative high-risk intravenous drug users (IVDU). Design We 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 high-risk IVDU, HIV-negative controls and HIV-positive subjects. Methods Cytokine production was measured in supernatants using enzyme-linked immunosorbent assay (ELISA) in 10 HIV-negative high-risk IVDU, 25 HIV-negative controls, and 12 HIV-positive IVDU. We also determined spontaneous in vitro immunoglobulin (Ig) G and IgM production. Results HIV-negative high-risk IVDU showed increased IFN-γ and decreased IL-4, IL-10 and IL-2, although the latter was not significant compared with HIV-negative controls. Further, HIV-negative high-risk IVDU had reduced IgG production and impaired IgM-IgG switch. Conclusions The reduced IL-2 and IL-4 production suggest an impaired CD4+ T-cell function in HIV-negative high-risk IVDU. The increased IFN-γ production along with the decreased type 2 cytokine profile is consistent with the hypothesis that protective immunity against HIV may reside in type 1 responses and cell-mediated immunity.

The effect of the new immunomodulating synthetic dipeptide pidotimod on some human immune functions was studied after ex vivo administration in healthy elderly subjects, as a model of acquired immune deficit. Results showed that treatment with pidotimod enhanced mitogen-induced lymphocyte proliferation and gamma-IFN production. The drug slightly reduced IL-6 content in supernatants from adherent monocytes, while it did not significantly affect polyclonal Ig synthesis. These findings further support the biological response modifying activity of pidotimod in human subjects.

The effects of the novel immunosuppressant Deoxyspergualin (DSP) on the development of experimental autoimmune thyroiditis (EAT) in CBA mice were studied. For EAT induction, the mice were immunized with 100 micrograms of porcine thyroglobulin (p Tg) emulsified in CFA on day 0 and in IFA, for boosting, on day 14. Twenty-eight days after primary immunization, histological and serological signs of EAT occurred in control mice treated with PBS which showed marked lymphoid infiltration of the thyroid glands along with increased serum titres of anti-pTg antibodies. Development of both these EAT features was significantly suppressed when the mice were treated with 2.5 mg/kg body weight DSP, given daily, five times a week, from day -2 to day +28 after immunization. The effect appeared to be dose-dependent and DSP was ineffective when given under the same experimental conditions at the dose of 0.5 mg/kg body weight. No DSP-toxic effects could be observed during the experiment. These results provide further evidence for the powerful immunosuppressive properties of DSP and suggest that this drug may be used in the treatment of autoimmune thyroid diseases and other T-cell mediated autoimmune disorders in humans.