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Biomedical subjects

G Balestrieri

Publications and source records attributed to G Balestrieri.

At least 19 recordsLinked to original sources

Pregnancy complications of the antiphospholipid syndrome.

Starting from their first description, antiphospholipid antibodies (aPL) were associated with repeated miscarriages and fetal losses. Other complications of pregnancy like preterm birth,with pre-eclampsia or severe placental insufficiency were also frequently reported and are included in the current classification criteria of the antiphospholipid syndrome (APS). The titre, the isotype of the antibodies or their antigen specificity may be important in the risk level determination. Some of the difference in the reported results can be explained by the poor standardization achieved in aPL testing or by the not univocal classification of pregnancy complications. The pathogenesis of pregnancy failures is linked to the thrombophilic effect of aPL but also to different mechanisms including a direct effect of antibodies on the throphoblast differentiation and invasion. The study of experimental animal models provided sound evidence of the pathogenic role of aPL both in lupus prone and naive mice. The definition of APS as a condition linked to high obstetric risk and the application of an effective therapy have completely changed the prognosis of pregnancy in these patients. In fact, despite the high number of complications and preterm delivery, today a successful outcome can be achieved in the large majority of the cases.

Abortion, Spontaneous↗

Immunization of naive BALB/c mice with human beta2-glycoprotein I breaks tolerance to the murine molecule.

OBJECTIVE: Immunization of naive mice with beta2-glycoprotein I (beta2GPI) leads to the generation of pathogenic anticardiolipin antibodies associated with clinical manifestations of the antiphospholipid syndrome (APS). The aim of this study was to determine whether immunization of naive mice with human beta2GPI, which shares homology with mouse beta2GPI molecules, breaks tolerance to murine beta2GPI and leads to the generation of anti-mouse beta2GPI. METHODS: Twenty-four female BALB/c mice were immunized in the footpads with 10 microg of human beta2GPI. Twelve age- and sex-matched BALB/c mice were immunized in the same manner with Freund's complete adjuvant and served as controls. The reactivity of whole sera, polyclonal IgG, and affinity-purified anti-beta2GPI IgG antibodies against human, bovine, and mouse beta2GPI was evaluated by enzyme-linked immunosorbent assay. RESULTS: High titers of anti-human beta2GPI IgG antibodies were detected 1 month after immunization. Progressively increasing titers against murine and bovine beta2GPI were recorded 1-4 months after injection. CONCLUSION: Immunization of mice with human beta2GPI resulted in the generation of antibodies reacting with human, bovine, and murine beta2GPI. The loss of tolerance to mouse beta2GPI is attributable to the high interspecies homology of beta2GPI. These results may point to molecular mimicry as a possible cause of APS.

Animals↗

Prevalence and pattern of cognitive impairment in systemic lupus erythematosus patients with and without overt neuropsychiatric manifestations.

The prevalence and pattern of cognitive impairment in systemic lupus erythematosus (SLE) patients with (NPSLE) and without (nSLE) overt neuropsychiatric manifestations were investigated. Fifty-two nSLE patients, 23 NPSLE patients and 27 healthy controls were evaluated with a battery of standardized neuropsychological and psychological tests. Disease duration, disease activity index, and current corticosteroid therapy were collected. Cognitive impairment was identified in 14 (26.9%) and in 12 (52.2%) of subjects with nSLE and NPSLE, respectively. Both SLE groups showed a significant impairment compared with controls on tasks assessing verbal and non-verbal long-term memory, and visuoconstructional abilities. In addition, NPSLE patients reported worse performances than both nSLE patients and controls on task evaluating short-term visuospatial memory. NPSLE subjects were significantly more anxious and depressed compared to both nSLE subjects and controls. By multivariate analysis, only depression levels, among clinical variables, significantly predicted cognitive performance. This study shows that cognitive impairment occurs frequently in both nSLE and NPSLE subjects. The higher frequency in NPSLE may be related to coexisting depressive disturbances.

Adrenal Cortex Hormones↗

Statins prevent endothelial cell activation induced by antiphospholipid (anti-beta2-glycoprotein I) antibodies: effect on the proadhesive and proinflammatory phenotype.

OBJECTIVE: To investigate the ability of statins, the inhibitors of the hydroxymethylglutaryl-coenzyme A reductase enzyme, to affect endothelial cell activation induced by anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in vitro. METHODS: Human umbilical vein endothelial cell (HUVEC) activation was evaluated as U937 monocyte adhesion, E-selectin, and intercellular adhesion molecule I (ICAM-1) expression by cell enzyme-linked immunosorbent assay and as interleukin-6 (IL-6) messenger RNA (mRNA) expression by RNA protection assay. E-selectin-specific nuclear factor kappaB (NF-kappaB) DNA-binding activity was evaluated by the gel-shift assay. HUVECs were activated by polyclonal affinity-purified IgG, human monoclonal IgM anti-beta2GPI antibodies, human recombinant IL-1beta, tumor necrosis factor alpha, or lipopolysaccharide (LPS). RESULTS: Fluvastatin reduced, in a concentration-dependent manner (1-10 microM), the adhesion of U937 to HUVECs and the expression of E-selectin and ICAM-1 induced by anti-beta2GPI antibodies as well as by cytokines or LPS. Another lipophilic statin, simvastatin, displayed similar effects but to a lesser extent than fluvastatin. The inhibition of E-selectin expression exerted by fluvastatin was related to the impairment of NF-kappaB binding to DNA. Moreover, the drug attenuated the expression of IL-6 mRNA in HUVEC exposed to anti-beta2GPI antibodies or cytokines. Incubation of HUVECs with mevalonate (100 microM), concomitantly with fluvastatin, greatly prevented the inhibitory effect of statin. CONCLUSION: Endothelial activation mediated by anti-beta2GPI antibody can be inhibited by statins. Because of the suggested role of endothelial cell activation in the pathogenesis of antiphospholipid syndrome (APS), our data provide, for the first time, a rationale for using statins as an additional therapeutic tool in APS.

Antibodies, Antiphospholipid↗

Antiphospholipid antibodies and the endothelium.

The interaction between aPL (particularly anti-beta 2GPI antibodies) and endothelium does represent a potential pathogenetic mechanism for the thrombotic manifestations of the syndrome. The autoantibody-mediated EC activation probably plays a role in sustaining the appearance of a proadhesive, proinflammatory, and procoagulant phenotype. The heterogeneity of the APS clinical manifestations is likely linked to the varied effects that aPL can induce on ECs and to the different functions that ECs display depending on the anatomic localization.

Antibodies, Antiphospholipid↗

Anti-C1q antibodies may help in diagnosing a renal flare in lupus nephritis.

It is still uncertain which, if any, immunologic parameters may help diagnose a renal flare of lupus nephritis. Anti-C1q antibody (Ab) titers have been elevated in patients with lupus with renal involvement, but little information is available on whether the titers are different in quiescent and active phases of lupus nephritis. In this study, we compared anti-C1q Ab titers with other serological test results in 48 patients with biopsy-proven lupus nephritis to assess which parameter could offer the best reliability for differentiating between quiescent and active phases of lupus nephritis. Serum C3 and C4 levels, as well as anti-double-stranded DNA, antiendothelial cell, anti-C1q, and antiphospholipid Ab titers, were evaluated in patients with quiescent renal disease (38 samples) and those with clinical evidence of renal activity (23 samples). Only anti-C1q Ab titers correlated with active renal disease in both univariate (P < 0.0001) and multivariate analysis (P < 0.0001), with a sensitivity of 87% and a specificity of 92%. In six patients, immunologic parameters were measured serially. In all patients, the high anti-C1q Ab titers returned to normal values after treatment-induced remission. The other serological parameters did not show a significant association with renal disease activity. In patients with biopsy-proven lupus nephritis, anti-C1q Ab titers appear to be strongly related to renal disease activity. Their measurement may be useful for confirming the diagnosis of renal flares of lupus nephritis.

Adult↗

Anticardiolipin antibody assay: a methodological analysis for a better consensus in routine determinations--a cooperative project of the European Antiphospholipid Forum.

Despite the widely recognized practical importance of anticardiolipin (aCL) ELISA, the reliability of this test has been recently discussed. In order to investigate this area on European scale, we sent to 30 experienced centers a questionnaire focusing on the diagnostic procedures applied to patients with antiphospholipid syndrome (APS) and on the detailed protocols used to perform aCL. Anticardiolipin ELISA was found to be the most frequently performed test in patients with suspected APS, but significant difference was shown among the various protocols. The cross-laboratory multiple examination of ten serum samples evaluated independently by the 24 centers pointed out the difficulty in getting comparable results. Therefore a "consensus" protocol was derived from the aCL methods giving the best performance. The materials and reagents necessary to perform the "consensus" method, including, as putative standards, one IgG and one IgM monoclonal antibody (HCAL and EY2C9) were distributed to 19 Centers. The results of one IgG and one IgM aCL high positive sera measured in serial dilutions were compared. A progressive decrease in the variability of the values obtained for a given sample appeared evident when all the laboratories used the same standard, in their own in-house ELISA and even more in the "consensus" ELISA. Our data show that aCL ELISA standardization is necessary in order to obtain comparable results in different laboratories.

Adult↗

Anti-beta2 glycoprotein I antibodies cause inflammation and recruit dendritic cells in platelet clearance.

Scavenger phagocytes are mostly responsible for the in vivo clearance of activated or senescent platelets. In contrast to other particulate substrates, the phagocytosis of platelets does not incite proinflammatory responses in vivo. This study assessed the contribution of macrophages and dendritic cells (DCs) to the clearance of activated platelets. Furthermore, we verified whether antibodies against the beta2 Glycoprotein I (beta2GPI), which bind to activated platelets, influence the phenomenon. DCs did not per se intemalise activated platelets. In contrast, macrophages efficiently phagocytosed platelets. In agreement with the uneventful nature of the clearance of platelets in vivo, phagocytosing macrophages did not release IL-1beta, TNF-alpha, or IL-10, beta2GPI bound to activated platelets and was required for their recognition by anti-beta2GPI antibodies. DCs internalised platelets opsonised by anti-beta2GPI antibodies. The phagocytosis of opsonised platelets determined the release of TNF-alpha and IL-1beta by DCs and macrophages. Phagocytosing macrophages, but not DCs, secreted the antiinflammatory cytokine IL-10. We conclude that anti-beta2GPI antibodies cause inflammation during platelet clearance and shuttle platelet antigens to antigen presenting DCs.

Antibodies↗

Antiphospholipid antibodies affect trophoblast gonadotropin secretion and invasiveness by binding directly and through adhered beta2-glycoprotein I.

OBJECTIVE: To investigate the in vitro ability of antiphospholipid antibodies (aPL) to bind human trophoblast cells and to affect gonadotropin secretion and invasiveness. METHODS: Antiphospholipid antibody IgG from women with recurrent miscarriages, beta2-glycoprotein I (beta2GPI)-independent IgG aPL human monoclonal antibody (mAb) (519), and IgM anti-beta2GPI human mAb (TMIG2) were investigated for their binding to trophoblasts cultured for various amounts of time, their ability to affect invasiveness of Matrigel-coated filters, and their release of human chorionic gonadotropin (hCG). RESULTS: Polyclonal IgG aPL, as well as mAb 519 and TMIG2, bound to trophoblasts, the highest binding being found when cells displayed the greatest amount of syncytium formation. TM1G2 binding was found to be betaGPI dependent. Both polyclonal and monoclonal aPL, but not the controls, significantly reduced hCG release and Matrigel invasiveness. CONCLUSION: These findings suggest that aPL recognition of both anionic PL and adhered beta2GPI on trophoblast cell structures might represent a potential pathogenetic mechanism for defective placentation in women with the antiphospholipid syndrome.

Anions↗

Anti-beta2 glycoprotein I antibodies prevent the De-activation of platelets and sustain their phagocytic clearance.

Exposure to phosphatidylserine (PS) tags dying and senescent cells for removal and identifies activated platelets. In this study we followed the fate of PS-exposing platelets in the presence of antibodies purified from Systemic Lupus Erythematosus (SLE) and primary Anti-phospholipid Syndrome (APS) patients' sera by beta2GPI affinity chromatography. Thrombin-activated platelets exposed PS and associated to beta2GPI. Both events were required for recognition by antibodies. Human monocyte-derived macrophages phagocytosed activated platelets only. Each macrophage internalized an average of 3.16+/-0.2 platelets after 60 min at 37 degrees C. Phagocytosis did not increase after longer incubations (4.65+/-0.26 platelets internalized by each macrophage after 300 min). Recognition of platelets by anti-beta2GPI antibodies significantly increased phagocytosis (P< 0.01). Upon withdrawal of thrombin, platelets downregulated PS (PS exposure t(1/2): 242 min) and the ability to be recognized by macrophages. Purified beta2GPI bound to PS-exposing platelets (association t(1/2): 250 min). Phosphatidyl serine exposure and beta2GPI association had virtually identical kinetics. Antibody binding prolonged the exposure of the beta2GPI/PS complex (t(1/2): >1200 min). The ability to phagocytose opsonized platelets was accordingly sustained (5.3+/-0.2 opsonized platelets were internalized by each macrophage after 60 min and 9.4+/-0.3 after 300 min). Anti-beta2GPI antibodies therefore poise activated platelets in a PS-exposing status, preventing the recycling of their function and favoring their phagocytic clearance.

Antibodies↗

Dendritic cell presentation of antigens from apoptotic cells in a proinflammatory context: role of opsonizing anti-beta2-glycoprotein I antibodies.

OBJECTIVE: To verify whether opsonization of apoptotic cells skews the outcome of apoptotic cell antigen presentation by dendritic cells (DCs). METHODS: RMA cells, which were engineered with a mutant ovalbumin (OVA) protein and were devoid of the leader secretory sequence (OVA-RMA), underwent ultraviolet irradiation to induce apoptosis. Binding of anti-beta2-glycoprotein I antibodies (anti-beta2GPI) and phagocytosis of apoptotic cells were assessed by flow cytometry and confocal microscopy. Presentation of processing antigens and major histocompatibility complex (MHC) class II-restricted or MHC class I-restricted antigens was assessed using OVA-specific T cell hybridomas. RESULTS: Anti-beta2GPI facilitated presentation of epitopes from internalized apoptotic cells to MHC class II-restricted, but not to class I-restricted, T lymphocytes. DCs challenged with supernatants of apoptotic cells did not activate OVA-specific T cells, making it unlikely that anti-beta2GPI complexed with antigen released from dying cells plays a role in antigen presentation. DCs challenged with low numbers of anti-beta2GPI-opsonized apoptotic cells secreted interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-10 in an autocrine/paracrine manner. CONCLUSION: Opsonization influences the outcome of the disposal of low numbers of apoptotic cells by DCs. This implies that soluble factors bound to apoptotic cells modulate their immunogenicity.

Antibodies↗

Anti-endothelial cell IgG fractions from systemic lupus erythematosus patients bind to human endothelial cells and induce a pro-adhesive and a pro-inflammatory 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.

Adult↗

Human beta 2-glycoprotein I binds to endothelial cells through a cluster of lysine residues that are critical for anionic phospholipid binding and offers epitopes for anti-beta 2-glycoprotein I antibodies.

Beta 2-Glycoprotein I (beta 2GPI) is a phospholipid-binding protein recognized by serum autoantibodies from the anti-phospholipid syndrome both in cardiolipin- and beta 2GPI-coated plates. We found that: 1) recombinant wild-type beta 2GPI bound to HUVEC and was recognized by both human monoclonal IgM and affinity-purified polyclonal IgG anti-beta 2GPI anti-phospholipid syndrome Abs; and 2) a single amino acid change from Lys286 to Glu significantly reduced endothelial adhesion. Double and triple mutants (from Lys284,287 to Glu284,287, from Lys286,287 to Glu286,287, and from Lys284,286,287 to Glu284,286,287) completely abolished endothelial binding. A synthetic peptide (P1) spanning the sequence Glu274-Cys288 of the beta 2GPI fifth domain still displayed endothelial adhesion. Another peptide (P8), identical with P1 except that Cys281 and Cys288 were substituted with serine residues, did not bind to HUVEC. Anti-beta 2GPI Abs, once bound to P1 adhered to HUVEC, induced E-selectin expression and up-regulated IL-6 secretion. Control experiments conducted with irrelevant Abs as well as with the P8 peptide did not show any endothelial Ab binding nor E-selectin and IL-6 modulation. Our results suggest that: 1) beta 2GPI binds to endothelial cells through its fifth domain; 2) the major phospholipid-binding site that mediates the binding to anionic phospholipids is also involved in endothelial binding; 3) HUVEC provide a suitable surface for beta 2GPI binding comparable to that displayed by anionic phospholipids dried on microtiter wells; and 4) the formation of the complex between beta 2GPI and the specific Abs leads to endothelial activation in vitro.

Amino Acid Sequence↗

Apoptotic cell clearance in systemic lupus erythematosus. I. Opsonization by antiphospholipid antibodies.

OBJECTIVE: To verify whether antiphospholipid antibodies (aPL) recognize and opsonize apoptotic human cells. METHODS: Apoptosis was induced via CD95 crosslinking or ultraviolet irradiation. IgG and anti-beta2-glycoprotein I (anti-beta2-GPI) antibodies were purified from patient sera by affinity chromatography. The aPL that bound to apoptotic cells were assessed by flow cytometry, and the subdomains recognized were identified by confocal microscopy. Human macrophages were derived from monocytes, and their ability to phagocytose 3H-labeled apoptotic bodies, whether opsonized or not opsonized by aPL, was assessed. Tumor necrosis factor alpha (TNF alpha) secretion was evaluated by enzyme-linked immunosorbent assay. RESULTS: The aPL, but not control Ig or Ig from aPL-negative patients, bound to apoptotic cells, but not to viable cells. Nuclear antigens were not recognized. Opsonization of apoptotic cells by aPL substantially enhanced recognition and binding by scavenger macrophages, with massive TNF alpha secretion. CONCLUSION: Antiphospholipid antibodies facilitate apoptotic cell clearance by macrophages and trigger TNF alpha release, possibly enhancing the immunogenicity of the autoantigens they contain.

Antibodies, Antiphospholipid↗

Apoptotic cell clearance in systemic lupus erythematosus. II. Role of beta2-glycoprotein I.

OBJECTIVE: To analyze the contribution of beta2-glycoprotein I (beta2-GPI) to apoptotic cell recognition by antiphospholipid antibodies (aPL) and macrophages from patients with autoimmune disease. METHODS: Phosphatidylserine expression by Jurkat cells undergoing apoptosis upon CD95 crosslinking or ultraviolet irradiation was verified by confocal microscopy of cells stained with fluorescein isothiocyanate-labeled annexin V. Beta2-GPI was purified by heparin/cationic-exchange chromatography and was biotinylated or used to purify beta2-GPI-specific antibodies by affinity chromatography. Binding to apoptotic cells was assessed by flow cytometry. The clearance of 3H-labeled, apoptotic cells by macrophages was assessed by beta counting. RESULTS: The array of epitopes generated by beta2-GPI association with apoptotic cells specifically targets their recognition and is required for their opsonization by human aPL. Nevertheless, beta2-GPI is not required for apoptotic cell clearance by human macrophages in the absence of aPL. CONCLUSION: The proinflammatory clearance of aPL-opsonized apoptotic cells, but not the nonphlogistic clearance of apoptotic cells by scavenger macrophages, depends on beta2-GPI.

Antibodies, Antiphospholipid↗