Low O2 and high CO2 in LLC-PK1 cells culture mimics renal ischemia-induced apoptosis

Ischemia, absence or loss of blood flow in organs always presents as a dual phenomenon: tissue oxygen deficit and CO2 excess (hypercapnia). Commonly hypoxic cell culture models kept CO2 at normal nonischemic values. We report a study of apoptosis in an in vitro model of renal hypoxia that mimics in vivo tissue gas atmosphere composition determined during experimental ischemia in rat kidney (low O2 plus high CO 2). Renal tubular LLC-PK1 cell were transiently exposed to hypoxia, to hypercapnia or to both conditions (simulated ischemia). Exposure to simulated ischemic atmosphere, but not to low O2 or high CO2 alone, induced cell apoptosis in vitro. This suggests that ischemia-induced apoptosis in vivo would be dependent on the natural, joint action of hypoxia and hypercapnia. This should be taken into account in cell culture studies that would like to mimic in vivo ischemic conditions. © 2004 USCAP, Inc. All rights reserved.This work was supported by EU Grant QLK6-CT-2000-00064, SAF 2000/3090-CE0057 and FISS 01/1691Peer Reviewe

Coming Back For More: Exploring Three Theoretical Perspectives Influencing IS Resumption

IS resumption describes ex-users’ returning to an information system (IS) after quitting it beforehand. IS research has conceptualized, operationalized, and defined this behavior and explained some initial causes, such as nonuse-related dissatisfaction, that lead ex-users to IS resumption. We aim to complement that understanding by discussing the influence of three theoretical perspectives: reasoned action, experiential responses, and habitual responses. We use those perspectives to develop hypotheses and offer a comprehensive understanding of causes leading to IS resumption intention. We chose a quantitative approach to evaluate our research model, conducting an online survey with 190 ex-users of video-on-demand services (VoD). Our findings show that subjective norm and attitude influence IS resumption intention, while higher satisfaction with prior usage influences ex-users’ attitude. We contribute to IS research by carving out how the three theoretical perspectives influence ex-users’ IS resumption intention, providing a solid theoretical basis for future studies on IS resumption

Loss of Nef-mediated CD3 down-regulation in the HIV-1 lineage increases viral infectivity and spread

Nef is an accessory protein of primate lentiviruses that is essential for efficient replication and pathogenesis of HIV-1. A conserved feature of Nef proteins from different lentiviral lineages is the ability to modulate host protein trafficking and down-regulate a number of cell surface receptors to enhance replication and promote immune evasion. Notably, the inability of Nef to down-regulate CD3 from infected T cells distinguishes HIV-1 Nef and its direct simian precursors from other primate lentiviruses. Why HIV-1 does not employ this potential immune evasion strategy is not fully understood. Using chimeric HIV-1 constructs expressing lentiviral Nef proteins that differ in their ability to down-modulate CD3, we show that retaining CD3 on the surface of infected primary T cells results in increased viral replication and cell-to-cell spread. We identified increased expression of envelope (Env) trimers at the cell surface and increased Env incorporation into virions as the determinants for the Nef- and CD3-dependent enhancement of viral infectivity. Importantly, this was independent of Nef-mediated antagonism of the host restriction factor SERINC5. CD3 retention on the surface of infected primary T cells also correlated with increased T cell signaling, activation, and cell death during cell-to-cell spread. Taken together, our results show that loss of an otherwise conserved function of Nef has a positive effect on HIV-1 replication, allowing for more efficient replication while potentially contributing to HIV-1 pathogenesis by triggering T cell activation and cell death during viral spread

Macrophage-derived Lipocalin-2 contributes to ischemic resistance mechanisms by protecting from renal injury

Renal ischemia-reperfusion injury triggers an inflammatory response associated to infiltrating macrophages which determines the further outcome of disease. Brown Norway rats are known to show endogenous resistance to ischemia-induced renal damage. By contrast, Sprague Dawley rats exhibit a higher susceptibility to ischemic injury. In order to ascertain cytoprotective mechanisms, we focused on the implication of lipocalin-2 protein in main resistance mechanisms in renal ischemia/reperfusion injury by using adoptive macrophage administration, genetically modified ex vivo either to overexpress or to knockdown lipocalin-2. In vitro experiments with bone marrow-derived macrophages both from Brown Norway rats and from Sprague Dawley rats under hypoxic conditions showed endogenous differences regarding cytokine and lipocalin-2 expression profile in the two strains. Most interestingly, we observed that macrophages of the resistant strain express significantly more lipocalin-2. In vivo studies showed that tubular epithelial cell apoptosis and renal injury significantly increased and reparative markers decreased in Brown Norway rats after injection of lipocalin-2-knockdown macrophages, while the administration of lipocalin-2-overexpressing cells significantly decreased Sprague Dawley susceptibility. These data point to a crucial role of macrophage-derived lipocalin-2 in endogenous cytoprotective mechanisms. We conclude that expression of lipocalin-2 in tissue-infiltrating macrophages is pivotal for kidney-intrinsic cytoprotective pathways during ischemia reperfusion injury.The authors would like to thank M Ángeles Muñoz for her excellent technical support. This work was supported by grants from FIS PI12/00720 from Instituto de Salud Carlos III cofunded by FEDER funds/European Regional Development Fund (ERDF)-a way to buid Europe- and SENEFRO (awarded to AS) and SAF 2012-39947-02 (awarded to GH). MJ was supported by a grant from Fritz Thyssen Stiftung (Az.10.12.2.156) and a grant from University of Frankfurt (Focus Line B) and AS is supported by Miguel Servet II contracting system (CPII 14/00026).Peer Reviewe

CPT1a gene expression reverses the inflammatory and anti-phagocytic effect of 7-ketocholesterol in RAW264.7 macrophages

Background: macrophage are specialized cells that contributes to the removal of detrimental contents via phagocytosis. Lipid accumulation in macrophages, whether from phagocytosis of dying cells or from circulating oxidized low-density lipoproteins, alters macrophage biology and functionality. It is known that carnitine palmitoyl transferase 1-a (CPT1a) gene encodes an enzyme involved in fatty acid oxidation and, therefore, lipid content. However, the potential of CPT1a to activate macrophage phagocytic function have not been elucidated. Methods: using a murine macrophage cell line, RAW264.7, we determine if intracellular accumulation of 7-ketocholesterol (7-KC) modulates macrophage phagocytic function through CPT1a gene expression. In addition, the effects of CPT1a genetic modification on macrophage phenotype and phagocytosis has been studied. Results: our results revealed that CPT1a gene expression decreased by the accumulation of 7-KC at the higher dose of 7-KC. This was concomitant with an impair ability to phagocytize bioparticles and an inflammatory phenotype. GW3965 treatment, which have shown to facilitate the efflux of cholesterol, eliminated the intracellular lipid droplets of 7-KC-laden macrophages, increased the gene expression of CPT1a, diminished the gene expression of the inflammatory marker iNOS and restored macrophage phagocytosis. Furthermore, CPT1a Knockdown per se was detrimental for macrophage phagocytosis whereas transcriptional activation of CPT1a heightened the uptake of bioparticles. Conclusions: altogether, our findings indicate that downregulation of CPT1a by lipid content modulates macrophage phagocytosis and inflammatory phenotype