Changes in membrane lipids drive increased endocytosis following Fas ligation

Once activated, some surface receptors promote membrane movements that open new portals of endocytosis, in part to facilitate the internalization of their activated complexes. The prototypic death receptor Fas (CD95/Apo1) promotes a wave of enhanced endocytosis that induces a transient intermixing of endosomes with mitochondria in cells that require mitochondria to amplify death signaling. This initiates a global alteration in membrane traffic that originates from changes in key membrane lipids occurring in the endoplasmic reticulum (ER). We have focused the current study on specific lipid changes occurring early after Fas ligation. We analyzed the interaction between endosomes and mitochondria in Jurkat T cells by nanospray-Time-of-flight (ToF) Mass Spectrometry. Immediately after Fas ligation, we found a transient wave of lipid changes that drives a subpopulation of early endosomes to merge with mitochondria. The earliest event appears to be a decrease of phosphatidylcholine (PC), linked to a metabolic switch enhancing phosphatidylinositol (PI) and phosphoinositides, which are crucial for the formation of vacuolar membranes and endocytosis. Lipid changes occur independently of caspase activation and appear to be exacerbated by caspase inhibition. Conversely, inhibition or compensation of PC deficiency attenuates endocytosis, endosome-mitochondria mixing and the induction of cell death. Deficiency of receptor interacting protein, RIP, also limits the specific changes in membrane lipids that are induced by Fas activation, with parallel reduction of endocytosis. Thus, Fas activation rapidly changes the interconversion of PC and PI, which then drives enhanced endocytosis, thus likely propagating death signaling from the cell surface to mitochondria and other organelles

Relationship between gender differences and clinical outcome in patients with the antiphospholipid syndrome

Antiphospholipid syndrome (APS), characterized by artherial and/or venous thrombosis, pregnancy morbidity and "antiphospholipid" antibodies (aPLs), is more common in women than in men, with a female to male ratio of about 3.5:1. Only few studies have investigated the clinical differences between male and female patients with APS. Therefore, this study was aimed to analyze the differences of clinical manifestations and laboratory tests, at diagnosis, between female and male APS patients and the clinical outcome. We enrolled 191 consecutive APS patients (125 with primary APS, PAPS, and 66 with secondary APS, SAPS) with a female predominant ratio of approximately 3:1 (142 vs 49). The prevalence of PAPS was higher in males than females (p<0.001). The analysis of aPL profile revealed that high IgM anti-cardiolipin (aCL) and high-medium IgG aCL titers were more frequent in males. In thrombotic APS peripheral arterial thrombosis was more common in male than female patients (p=0.049), as well as myocardial infarction (p=0.031). Multivariate analysis to correct for cardiovascular risk factors, high titer of aPLs and triple positivity for aPLs, revealed that the odds ratio for myocardial infarction in male was 3.77. Thus, APS may be considered as a disease in which serological (IgM titer) and clinical profiles are influenced by gender

Alarmin HMGB1 and Soluble RAGE as New Tools to Evaluate the Risk Stratification in Patients With the Antiphospholipid Syndrome

Antiphospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized by arterial and/or venous thrombosis, pregnancy morbidity in the presence of circulating “anti-phospholipid antibodies” (aPL). One of the main target antigens of aPL is β2-glycoprotein I (β2-GPI). APS may occur as a primary syndrome or associated with Systemic Lupus Erythematosus (SLE). High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different type of cells during activation and/or cell death and may act as a proinflammatory mediator through ligation to its receptors, including RAGE. There is accumulating evidence that HMGB1 contributes to the pathogenesis of inflammatory and autoimmune diseases, especially SLE. In a previous study we demonstrated increased serum levels of HMGB1 in both primary and secondary APS patients. In this work we analyzed: (i) in vitro whether anti-β2-GPI antibodies from APS patients may induce both a HMGB1 cellular relocation by activation of its putative receptor RAGE in platelets and monocytes and, (ii) ex vivo, serum levels of HMGB1/soluble RAGE (sRAGE) in APS patients and their possible correlation with clinical manifestations. Platelets and monocytes from healthy donors were incubated with affinity purified anti-β2-GPI antibodies. HMGB1 and RAGE expression were analyzed by Western Blot. Sera from 60 consecutive APS patients (primary or secondary), diagnosed according to the Sydney Classification Criteria, were enrolled. As a control, 30 matched healthy subjects were studied. Serum levels of HMGB1 and sRAGE were analyzed by Western Blot. In vitro results showed that anti-β2-GPI antibodies were able to induce RAGE activation and HMGB1 cellular relocation in both monocytes and platelets. HMGB1 and sRAGE serum levels were significantly increased in APS patients in comparison with healthy subjects (p<0.0001). Interestingly, APS patients with spontaneous recurrent abortion showed significantly higher levels of sRAGE; moreover, in APS patients a direct correlation between serum levels of HMGB1 and disease duration was detected. Our observations suggest that anti-β2-GPI antibodies may trigger RAGE activation and HMGB1 cellular relocation during APS. Monitoring these molecules serum levels may represent an useful tool to evaluate the pathogenesis and risk stratification of clinical manifestations in APS

Paracrine Diffusion of PrPC and Propagation of Prion Infectivity by Plasma Membrane-Derived Microvesicles

Cellular prion protein (PrPc) is a physiological constituent of eukaryotic cells. The cellular pathways underlying prions spread from the sites of prions infection/peripheral replication to the central nervous system are still not elucidated. Membrane-derived microvesicles (MVs) are submicron (0.1–1 µm) particles, that are released by cells during plasma membrane shedding processes. They are usually liberated from different cell types, mainly upon activation as well as apoptosis, in this case, one of their hallmarks is the exposure of phosphatidylserine in the outer leaflet of the membrane. MVs are also characterized by the presence of adhesion molecules, MHC I molecules, as well as of membrane antigens typical of their cell of origin. Evidence exists that MVs shedding provide vehicles to transfer molecules among cells, and that MVs are important modulators of cell-to-cell communication. In this study we therefore analyzed the potential role of membrane-derived MVs in the mechanism(s) of PrPC diffusion and prion infectivity transmission. We first identified PrPC in association with the lipid raft components Fyn, flotillin-2, GM1 and GM3 in MVs from plasma of healthy human donors. Similar findings were found in MVs from cell culture supernatants of murine neuronal cells. Furthermore we demonstrated that PrPSc is released from infected murine neuronal cells in association with plasma membrane-derived MVs and that PrPSc-bearing MVs are infectious both in vitro and in vivo. The data suggest that MVs may contribute both to the intercellular mechanism(s) of PrPC diffusion and signaling as well as to the process of prion spread and neuroinvasion

Detection of antiphospholipid antibodies by automated chemiluminescence assay

The laboratory diagnosis of antiphospholipid antibody syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL) by lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-beta 2-glycoprotein I IgG or IgM antibodies (anti-beta 2-GPI), usually detected by ELISA. In this study we tested aCL by a new automated system using the chemiluminescence principle. Our results showed that, while almost all the sera from APS patients, positive for IgG aCL and anti-beta 2-GPI by ELISA, were also positive for IgG aCI by chemiluminescence, only 30.13% of patients without clinical manifestations of APS, but positive for aCL and persistently negative for anti-beta 2-GPI (by ELISA) and LA, confirmed the positive test by chemiluminescence. This difference was highly significant (p<0.0001). Interestingly, this test also prompted to identify 20% of patients positive for LA, but persistently negative for both aCL and anti-beta 2-GPI IgG (ELISA). Thus, the new technology of automated chemiluminescence assay for measuring aPL may represent an useful tool to identify "true" APS patients. (C) 2012 Elsevier B.V. All rights reserved

In vivo and in vitro macrophage activationinduced by IFN gamma spontaneously released by spleencells from tumor bearing mice

Peritoneal macrophages obtained from Lewis Lung carcinoma (3LL) tumor bearing mice release high amounts of soluble factors such as C3,H2O2 and lysosomal enzymes but fail to exert cytotoxic activity on tumor cells. In the present work we show that they acquire this property and become fully activated after in vitro incubation with supernatants derived from cultures of splenocytes from tumor bearing syngeneic mice. The presence of IFN gamma in the above supernatants was detected by immunoblotting analysis and by bioassay. The role played by IFN gamma in macrophage activation was investigated

The Role of Autophagy as a Trigger of Post-Translational Modifications of Proteins and Extracellular Vesicles in the Pathogenesis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease, characterized by persistent joint inflammation, leading to cartilage and bone destruction. Autoantibody production is directed to post-translational modified (PTM) proteins, i.e., citrullinated or carbamylated. Autophagy may be the common feature in several types of stress (smoking, joint injury, and infections) and may be involved in post-translational modifications (PTMs) in proteins and the generation of citrullinated and carbamylated peptides recognized by the immune system in RA patients, with a consequent breakage of tolerance. Interestingly, autophagy actively provides information to neighboring cells via a process called secretory autophagy. Secretory autophagy combines the autophagy machinery with the secretion of cellular content via extracellular vesicles (EVs). A role for exosomes in RA pathogenesis has been recently demonstrated. Exosomes are involved in intercellular communications, and upregulated proteins and RNAs may contribute to the development of inflammatory arthritis and the progression of RA. In RA, most of the exosomes are produced by leukocytes and synoviocytes, which are loaded with PTM proteins, mainly citrullinated proteins, inflammatory molecules, and enzymes that are implicated in RA pathogenesis. Microvesicles derived from cell plasma membrane may also be loaded with PTM proteins, playing a role in the immunopathogenesis of RA. An analysis of changes in EV profiles, including PTM proteins, could be a useful tool for the prevention of inflammation in RA patients and help in the discovery of personalized medicine

“METHOD AND KIT FOR IN VITRO DIAGNOSIS OF ANTI-PHOSPHOLIPID SYNDROME”

Gli anticorpi “antifosfolipidi” sono un gruppo di autoanticorpi che, in associazione con trombosi (arteriose e/o venose) e aborti spontanei, caratterizzano la Sindrome da Anticorpi Antifosfolipidi (APS). Gli anticorpi anti-β2-Glicoproteina I (β2GPI) sono i principali autoanticorpi usati per la diagnosi di APS. Esistono pazienti con le manifestazioni cliniche dell'APS, ma negativi ai criteri diagnostici di laboratorio. E’ fondamentale identificare nuovi target antigenici per la diagnosi di APS. Dimostriamo che la β2GPI carbamilata è un nuovo autoantigene della Sindrome proponendo un metodo analitico in vitro, oggetto dell'invenzione, per rilevare anticorpi specifici per la β2GPI carbamilata.Antiphospholipid antibodies are a group of autoantibodies which, in association with thrombosis (arterial and/or venous) and recurrent abortions, characterize the Antiphospholipid Antibody Syndrome (APS). Anti-β2-Glycoprotein I (β2GPI) antibodies are the main autoantibodies used for the diagnosis of APS. There are patients with clinical manifestations of APS, but negative by laboratory diagnostic criteria. It is essential to identify new antigenic targets for the diagnosis of APS. We demonstrate that carbamylated β2GPI is a new autoantigen of the Syndrome by proposing an in vitro analytical method, object of the invention, to detect specific antibodies to carbamylated β2GPI