Apolipoprotein E delivery by peritoneal implantation of encapsulated recombinant cells improves the hyperlipidaemic profile in apoE-deficient mice

Plasma apolipoprotein E (apoE) is a 34-kDa polymorphic protein which has atheroprotective actions by clearing remnant lipoproteins and sequestering excess cellular cholesterol. Low or dysfunctional apoE is a risk factor for hyperlipidaemia and atherosclerosis, and for restenosis after angioplasty. Here, in short-term studies designed to establish proof-of-principle, we investigate whether encapsulated recombinant Chinese hamster ovary (CHO) cells can secrete wild-type apoE3 protein in vitro and then determine whether peritoneal implantation of the microcapsules into apoE-deficient (apoE(-/-)) mice reduces their hypercholesterolaemia.Recombinant CHO-E3 cells were encapsulated into either alginate poly-L-lysine or alginate polyethyleneimine/polybrene microspheres. After verifying stability and apoE3 secretion, the beads were then implanted into the peritoneal cavity of apoE(-/-) mice; levels of plasma apoE3, cholesterol and lipoproteins were monitored for up to 14 days post-implantation.Encapsulated CHO-E3 cells continued to secrete apoE3 protein throughout a 60-day study period in vitro, though levels declined after 14 days. This cell-derived apoE3 was biologically active. When conditioned medium from encapsulated CHO-E3 cells was incubated with cultured cells pre-labelled with [H-3]-cholesterol, efflux of cholesterol was two to four times greater than with normal medium (at 8 h, for example, 7.4+/-0.3% vs. 2.4+/-0.2% of cellular cholesterol; P<0.001). Moreover, when secreted apoE3 was injected intraperitoneally into apoE(-/-) mice, apoE3 was detected in plasma and the hyperlipidaemia improved. Similarly, when alginate polyethyleneimine/polybrene capsules were implanted into the peritoneum of apoE(-/-) mice, apoE3 was secreted into plasma and at 7 days total cholesterol was reduced, while atheroprotective high-density lipoprotein (HDL) increased. In a second study, apoE was detectable in plasma of five mice treated with alginate poly-L-lysine beads, 4 and 7 days post-implantation, though not at day 14. Furthermore, their hypercholesterolaemia was reduced, while HDL was clearly elevated in all mice at days 4 and 7 (from 18.4+/-6.2% of total lipoproteins to 31.1+/-6.8% at 7 days; P<0.001); however, these had rebounded by day 14, possibly due to the emergence of anti-apoE antibodies.We conclude that microencapsulated apoE-secreting cells have the potential to ameliorate the hyperlipidaemia of apoE deficiency, but that the technology must be improved to become a feasible therapeutic to treat atherosclerosis. (C) 2004 Elsevier B.V. All rights reserved

QCD and strongly coupled gauge theories : challenges and perspectives

We highlight the progress, current status, and open challenges of QCD-driven physics, in theory and in experiment. We discuss how the strong interaction is intimately connected to a broad sweep of physical problems, in settings ranging from astrophysics and cosmology to strongly coupled, complex systems in particle and condensed-matter physics, as well as to searches for physics beyond the Standard Model. We also discuss how success in describing the strong interaction impacts other fields, and, in turn, how such subjects can impact studies of the strong interaction. In the course of the work we offer a perspective on the many research streams which flow into and out of QCD, as well as a vision for future developments.Peer reviewe

Correlative SICM-FCM reveals changes in morphology and kinetics of endocytic pits induced by disease-associated mutations in dynamin

Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a wholeclass of dynamin-dependent endocytosis, including clathrin-mediated endocytosis bycaveoli. It has been suggestedthat mutations in theDNM2gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactlymutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used livecorrelative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study howdisease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly followingtheir dynamics of formation, maturation, and internalizationinskinfibroblastsfrompatients with centronuclearmyopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we havedeveloped, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, andsignificantly increases the lifetime of CCPs. Differently,p.R522H slows down the formation of CCPs without affectingtheir internalization. We also found that CNM mutations inDNM2affect the distribution of caveoli and reduce dorsalruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electronmicroscopy data,argue for the impairment of several forms of endocytosis inDNM2-linked CNM.—Ali,T.,Bednarska,J.,Vassilopoulos,S.,Tran,M.,Diakonov,I.A.,Ziyadeh-Isleem,A.,Guicheney,P.,Gorelik,J.,Korchev,Y.E.,Reilly,M.M.,Bitoun,M.,Shevchuk,A.CorrelativeSICM-FCMreveals changes in morphology and kinetics of endocytic pitsinduced by disease-associated mutations in dynamin