Antiphospholipid antibodies and COVID-19 thrombotic vasculopathy: one swallow does not make a summer

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In vivo utjecaj imunosupresije u majki za vrijeme trudnoće na imunosni sustav novorođenčadi

When used in pregnancy, immunosuppressants can cross the placental barrier and enter foetal circulation, possibly affecting the immune system of the foetus. This study evaluated the immune function in eight children born by mothers with connective tissue diseases who received immunosuppressants (cyclosporine A or dexamethasone) during pregnancy and in six babies from mothers with similar diseases, but who did not receive any treatment. Judging by the cytokine production of interleukin-2 and interferon-γ in peripheral blood mononuclear cells stimulated by phorbol-myristate-acetate (PMA) and ionomycin, immunosuppressive drugs given for rheumatic disorders during pregnancy do not induce significant immunosuppression in babies.Imunosupresivni lijekovi davani za vrijeme trudnoće mogu proći placentalnu barijeru i ući u cirkulaciju fetusa, s mogućim utjecajem na njegov imunosni sustav. U radu je praćena imunosna funkcija kod osmero djece rođene od majki s bolestima vezivnog tkiva, koje su tretirane za vrijeme trudnoće imunosupresivnim lijekovima (ciklosporin A ili deksametazon) i kod osmero novorođenč adi rođene od majki sa sličnim bolestima, ali koje nisu bile tretirane. Imunosupresivni lijekovi primijenjeni za vrijeme trudnoće kod majki koje boluju od reumatskih bolesti ne izazivaju značajniju imunosupresiju u novorođenčadi praćenu nastajanjem citokina, interleukina 2 i interferona γ_ u perifernim mononuklearnim krvnim stanicama pod djelovanjem forbol-miristat-acetata (PMA) i ionomicina

8-Chloro-Cyclic AMP and Protein Kinase A I-Selective Cyclic AMP Analogs Inhibit Cancer Cell Growth through Different Mechanisms

Cyclic AMP (cAMP) inhibits the proliferation of several tumor cells. We previously reported an antiproliferative effect of PKA I-selective cAMP analogs (8-PIP-cAMP and 8-HA-cAMP) on two human cancer cell lines of different origin. 8-Cl-cAMP, another cAMP analog with known antiproliferative properties, has been investigated as a potential anticancer drug. Here, we compared the antiproliferative effect of 8-Cl-cAMP and the PKA I-selective cAMP analogs in three human cancer cell lines (ARO, NPA and WRO). 8-Cl-cAMP and the PKA I-selective cAMP analogs had similarly potent antiproliferative effects on the BRAF-positive ARO and NPA cells, but not on the BRAF-negative WRO cells, in which only 8-Cl-cAMP consistently inhibited cell growth. While treatment with the PKA I-selective cAMP analogs was associated with growth arrest, 8-Cl-cAMP induced apoptosis. To further investigate the actions of 8-Cl-cAMP and the PKA I-selective cAMP analogs, we analyzed their effects on signaling pathways involved in cell proliferation and apoptosis. Interestingly, the PKA I-selective cAMP analogs, but not 8-Cl-cAMP, inhibited ERK phosphorylation, whereas 8-Cl-cAMP alone induced a progressive phosphorylation of the p38 mitogen-activated protein kinase (MAPK), via activation of AMPK by its metabolite 8-Cl-adenosine. Importantly, the pro-apoptotic effect of 8-Cl-cAMP could be largely prevented by pharmacological inhibition of the p38 MAPK. Altogether, these data suggest that 8-Cl-cAMP and the PKA I-selective cAMP analogs, though of comparable antiproliferative potency, act through different mechanisms. PKA I-selective cAMP analogs induce growth arrest in cells carrying the BRAF oncogene, whereas 8-Cl-cAMP induce apoptosis, apparently through activation of the p38 MAPK pathway

Pathogenic Role of Complement in Antiphospholipid Syndrome and Therapeutic Implications

Antiphospholipid syndrome (APS) is an acquired autoimmune disease characterized by thromboembolic events, pregnancy morbidity, and the presence of antiphospholipid (aPL) antibodies. There is sound evidence that aPL act as pathogenic autoantibodies being responsible for vascular clots and miscarriages. However, the exact mechanisms involved in the clinical manifestations of the syndrome are still a matter of investigation. In particular, while vascular thrombosis is apparently not associated with inflammation, the pathogenesis of miscarriages can be explained only in part by the aPL-mediated hypercoagulable state and additional non-thrombotic effects, including placental inflammation, have been described. Despite this difference, evidence obtained from animal models and studies in APS patients support the conclusion that complement activation is a common denominator in both vascular and obstetric APS. Tissue-bound aPL rather than circulating aPL-beta2 glycoprotein I immune complexes seem to be responsible for the activation of the classical and the alternative complement pathways. The critical role of complement is supported by the finding that complement-deficient animals are protected from the pathogenic effect of passively infused aPL and similar results have been obtained blocking complement activation. Moreover, elevated levels of complement activation products in the absence of abnormalities in regulatory molecules have been found in the plasma of APS patients, strongly suggesting that the activation of complement cascade is the result of aPL binding to the target antigen rather than of a defective regulation. Placental complement deposits represent a further marker of complement activation both in animals and in patients, and there is also some suggestive evidence that complement activation products are deposited in the affected vessels. The aim of this review is to analyze the state of the art of complement involvement in the pathogenesis of APS in order to provide insights into the role of this system as predictive biomarker for the clinical manifestations and as therapeutic target

New insight into antiphospholipid syndrome: antibodies to \u3b22glycoprotein I-domain 5 fail to induce thrombi in rats

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of \u3b22glycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analysed the in vivo pro-coagulant effect of two groups of 5 serum IgG each reacting selectively with domain 1 or domain 5 in LPS-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy and vascular deposition of \u3b22glycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-\u3b22glycoproteinI antibodies served as controls. All the anti-domain 1 positive IgG exhibited potent pro-coagulant activity while the anti-domain 5 positive and the negative control IgG failed to promote blood clot and vessels occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound \u3b22glycoprotein I while able to interact with fluid-phase \u3b22glycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit in vivo thrombogenic effect and the interaction of these antibodies with circulating \u3b22glycoproteinI suggest their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining really at risk patients for more appropriate treatments to avoid recurrences and disability

Vandetanib versus cabozantinib in medullary thyroid carcinoma:A focus on anti‐angiogenic effects in zebrafish model

Medullary thyroid carcinoma (MTC) is a tumor deriving from the thyroid C cells. Vandetanib (VAN) and cabozantinib (CAB) are two tyrosine kinase inhibitors targeting REar-ranged during Transfection (RET) and other kinase receptors and are approved for the treatment of advanced MTC. We aim to compare the in vitro and in vivo anti‐tumor activity of VAN and CAB in MTC. The effects of VAN and CAB on viability, cell cycle, and apoptosis of TT and MZ‐CRC‐1 cells are evaluated in vitro using an MTT assay, DNA flow cytometry with propidium iodide, and An-nexin V‐FITC/propidium iodide staining, respectively. In vivo, the anti‐angiogenic potential of VAN and CAB is evaluated in Tg(fli1a:EGFP)y1 transgenic fluorescent zebrafish embryos by ana-lyzing the effects on the physiological development of the sub‐intestinal vein plexus and the tu-mor‐induced angiogenesis after TT and MZ‐CRC‐1 xenotransplantation. VAN and CAB exert comparable effects on TT and MZ‐CRC‐1 viability inhibition and cell cycle perturbation, and stimulated apoptosis with a prominent effect by VAN in MZ‐CRC‐1 and CAB in TT cells. Regard-ing zebrafish, both drugs inhibit angiogenesis in a dose‐dependent manner, in particular CAB shows a more potent anti‐angiogenic activity than VAN. To conclude, although VAN and CAB show comparable antiproliferative effects in MTC, the anti‐angiogenic activity of CAB appears to be more relevant.</p

Beta 2 glycoprotein I and neutrophil extracellular traps: Potential bridge between innate and adaptive immunity in anti-phospholipid syndrome

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by recurrent vascular thrombosis and miscarriages in the absence of known causes. Antibodies against phospholipid-binding proteins (aPL) are pathogenic players in both clotting and pregnancy APS manifestations. There is sound evidence that antibodies specific for beta2 glycoprotein I (β2GPI) trigger thrombotic and pregnancy complications by interacting with the molecule on the membranes of different cell types of the coagulation cascade, and in placenta tissues. In addition to the humoral response against β2GPI, both peripheral and tissue CD4+ β2GPI-specific T cells have been reported in primary APS as well as in systemic lupus erythematosus (SLE)-associated APS. While adaptive immunity plays a clear role in APS, it is still debated whether innate immunity is involved as well. Acute systemic inflammation does not seem to be present in the syndrome, however, there is sound evidence that complement activation is crucial in animal models and can be found also in patients. Furthermore, neutrophil extracellular traps (NETs) have been documented in arterial and venous thrombi with different etiology, including clots in APS models. Keeping in mind that β2GPI is a pleiotropic glycoprotein, acting as scavenger molecule for infectious agents and apoptotic/damaged body constituents and that self-molecules externalized through NETs formation may become immunogenic autoantigens, we demonstrated β2GPI on NETs, and its ability to stimulate CD4+β2GPI-specific T cells. The aim of this review is to elucidate the role of β2GPI in the cross-talk between the innate and adaptive immunity in APS

1079-102 Correlation between norepinephrine and epinephrine myocardial spillover and tumor necrosis factor-alpha in conventional versus off-pump coronary artery bypass surgery

Background: Complete revascularization obtained by coronary artery bypass surgery does not prevent long term left ventricular remodeling and heart failure development. Periprocedural events linked to different surgical techniques, such as cardiopulmonary bypass with cardioplegic arrest (CABG) versus off-pump procedures may trigger an irreversible microvascular dysfunction or myocytes necrosis and apoptosis. Methods: To test this hypothesis we measured norepinephrine and epinephrine coronary sinus and aortic spillover before and after surgery, simultaneously with Tumor Necrosis Factor-alpha (TNF-alpha) measurements in 30 patients randomized to CABG (n=15), or off-pump (n=15) coronary surgery. Plasma catecholamines were assessed by high performance liquid chromatography and TNF-alpha by ELISA. Results: Norepinephrine and epinephrine spillover was similar in the two groups before surgery, being 1.38\ub10.62 and 1.08\ub10.45, respectively. After surgery norepinephrine spillover was 1.43\ub10.56, 0.72\ub10.49 in CABG and off-pump, respectively (P<0.05 CABG versus off-pump, means \ub1SD ). Epinephrine spillover was 1.27\ub10.16 and 0.65\ub10.15 respectively (P<0.05, CABG versus off-pump). TNF-alpha significantly increased only in CABG patients being 22.17\ub16.79 and 35.4\ub15.98, pg/mL, before and after surgery (P<0.05), respectively. After surgery norepinephrine spillover correlated with TNF-alpha levels (P=0.01, R=0.553). Conclusions: Patients undergoing off-pump interventions showed significantly lower catecholamines spillover as compared to CABG, suggesting that the off-pump technique may result less invasive, not only for a lower local and whole body inflammatory response but also for a lower sympathetic drive. For the first time in humans we have detected an increase in epinephrine-spillover after cardiac surgery. Further studies are necessary to evaluate if the short-term advantages observed after off-pump coronary surgery translate into a long-term attenuation of left ventricular remodeling and in the prevention of heart failure progression

Blood Cell-Bound C4d as a Marker of Complement Activation in Patients With the Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is a chronic and disabling condition characterized by recurrent thrombosis and miscarriages mediated by antibodies against phospholipid-binding proteins (aPL), such as beta2glycoprotein I (β2GPI). Complement is involved in APS animal models and complement deposits have been documented in placenta and thrombotic vessels despite normal serum levels. Analysis of circulating blood cells coated with C4d displays higher sensitivity than the conventional assays that measure soluble native complement components and their unstable activation products in systemic lupus erythematosus (SLE). As C4d-coated blood cell count has been reported to be more sensitive than serum levels of complement components and their activation products in systemic lupus erythematosus (SLE) patients, we decided to evaluate the percentage of C4d positive B lymphocytes (BC4d), erythrocytes (EC4d), and platelets (PC4d) in primary APS patients and asymptomatic aPL positive carriers as marker of complement activation in APS. We assessed by flow cytometry the percentages of BC4d, EC4d, and PC4d in primary APS (PAPS; n. 23), 8 asymptomatic aPL positive carriers, 11 APS-associated SLE (SAPS), 17 aPL positive SLE, 16 aPL negative SLE, 8 aPL negative patients with previous thrombosis, 11 immune thrombocytopenia (ITP) patients, and 26 healthy subjects. In addition, we used an in vitro model to evaluate the ability of a monoclonal anti-β2GPI antibody (MBB2) to bind to normal resting or activated platelets and fix complement. EC4d and PC4d percentages were significantly higher in PAPS and aPL carriers as well as aPL positive SLE and SAPS than in aPL negative controls. The highest values were found in PAPS and in SAPS. The EC4d and PC4d percentages were significantly correlated with serum C3/C4 and anti-β2GPI/anti-cardiolipin IgG. In vitro studies showed that MBB2 bound to activated platelets only and induced C4d deposition. The detection of the activation product C4d on circulating erythrocytes and platelets supports the role of complement activation in APS. Complement may represent a new therapeutic target for better treatment and prevention of disability of APS patients