Complementary roles for scavenger receptor A and CD36 of human monocyte-derived macrophages in adhesion to surfaces coated with oxidized low-density lipoproteins and in secretion of H2O2

Oxidized low-density lipoprotein (oxLDL) is considered one of the principal effectors of atherogenesis. To explore mechanisms by which oxLDL affects human mononuclear phagocytes, we incubated these cells in medium containing oxLDL, acetylated LDL (acLDL), or native LDL, or on surfaces coated with these native and modified lipoproteins. The presence of soluble oxLDL, acLDL, or native LDL in the medium did not stimulate H2O2 secretion by macrophages. In contrast, macrophages adherent to surfaces coated with oxLDL secreted three- to fourfold more H2O2 than macrophages adherent to surfaces coated with acLDL or native LDL. Freshly isolated blood monocytes secreted little H2O2 regardless of the substrate on which they were plated. H2O2 secretion was maximal in cells maintained for 4–6 d in culture before plating on oxLDL-coated surfaces. Fucoidan, a known ligand of class A macrophage scavenger receptors (MSR-A), significantly reduced macrophage adhesion to surfaces coated with oxLDL or acLDL. Monoclonal antibody SMO, which blocks oxLDL binding to CD36, did not inhibit adhesion of macrophages to oxLDL-coated surfaces but markedly reduced H2O2 secretion by these cells. These studies show that MSR-A is primarily responsible for adhesion of macrophages to oxLDL-coated surfaces, that CD36 signals H2O2 secretion by macrophages adherent to these surfaces, and that substrate-bound, but not soluble, oxLDL stimulates H2O2 secretion by macrophages

LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1 -Rac1 pathway

Leucine-rich repeat-containing G protein–coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/β-catenin signaling in vitro; however, deletion of LGR5 in stem cells has little or no effect on Wnt/β-catenin signaling or cell proliferation in vivo. Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell–cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell–cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell–cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1–Rac1 pathway to strengthen cell–cell adhesion in normal adult crypt stem cells and colon cancer cells

EBV-gp350 Confers B-Cell Tropism to Tailored Exosomes and Is a Neo-Antigen in Normal and Malignant B Cells—A New Option for the Treatment of B-CLL

gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies

A Novel Multiplex Cell Viability Assay for High-Throughput RNAi Screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%–58.7±14.4% when two indicators were combined and 40–48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost

Multiplication végétative in vitro du chanvre (Cannabis sativa L.). Application à la conserva- tion des clones sélectionnés

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