Morphological and Mechanical Characterization of Extracellular Vesicles and Parent Human Synoviocytes under Physiological and Inflammatory Conditions.

The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This "gel-in" vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant.

Integrin alpha5 in human breast cancer is a mediator of bone metastasis and a therapeutic target for the treatment of osteolytic lesions.

Bone metastasis remains a major cause of mortality and morbidity in breast cancer. Therefore, there is an urgent need to better select high-risk patients in order to adapt patient's treatment and prevent bone recurrence. Here, we found that integrin alpha5 (ITGA5) was highly expressed in bone metastases, compared to lung, liver, or brain metastases.

Remodeling of the Actin Network Associated with the Non-Structural Protein 1 (NS1) of West Nile Virus and Formation of NS1-Containing Tunneling Nanotubes.

The cellular response to the recombinant NS1 protein of West Nile virus (NS1) was studied using three different cell types: Vero E6 simian epithelial cells, SH-SY5Y human neuroblastoma cells, and U-87MG human astrocytoma cells. Cells were exposed to two different forms of NS1: (i) the exogenous secreted form, sNS1, added to the extracellular milieu; and (ii) the endogenous NS1, the intracellular form expressed in plasmid-transfected cells. The cell attachment and uptake of sNS1 varied with the cell type and were only detectable in Vero E6 and SH-SY5Y cells.

miRNA-30 Family Members Inhibit Breast Cancer Invasion, Osteomimicry, and Bone Destruction by Directly Targeting Multiple Bone Metastasis-Associated Genes.

miRNAs are master regulators of gene expression that play key roles in cancer metastasis. During bone metastasis, metastatic tumor cells must rewire their biology and express genes that are normally expressed by bone cells (a process called osteomimicry), which endow tumor cells with full competence for outgrowth in the bone marrow. Here, we establish miR-30 family members miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e as suppressors of breast cancer bone metastasis that regulate multiple pathways, including osteomimicry.

Baculovirus-assisted Reovirus Infection in Monolayer and Spheroid Cultures of Glioma cells.

The mammalian orthoreovirus Type 3 Dearing has great potential as oncolytic agent in cancer therapy. One of the bottlenecks that hampers its antitumour efficacy in vivo is the limited tumour-cell infection and intratumoural distribution. This necessitates strategies to improve tumour penetration.

Alpha-helicoidal HEAT-like Repeat Proteins (αRep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation.

A new generation of artificial proteins, derived from alpha-helicoidal HEAT-like repeat protein scaffolds (αRep), was previously characterized as an effective source of intracellular interfering proteins. In this work, a phage-displayed library of αRep was screened on a region of HIV-1 Gag polyprotein encompassing the C-terminal domain of the capsid, the SP1 linker and the nucleocapsid. This region is known to be essential for the late steps of HIV-1 life cycle, Gag oligomerization, viral genome packaging and the last cleavage step of Gag, leading to mature, infectious virions.

Interference with the production of infectious viral particles and bimodal inhibition of replication are broadly conserved antiviral properties of IFITMs.

IFITMs are broad antiviral factors that block incoming virions in endosomal vesicles, protecting target cells from infection. In the case of HIV-1, we and others reported the existence of an additional antiviral mechanism through which IFITMs lead to the production of virions of reduced infectivity. However, whether this second mechanism of inhibition is unique to HIV or extends to other viruses is currently unknown.

Deciphering critical amino acid residues to modify and enhance the binding affinity of ankyrin scaffold specific to capsid protein of human immunodeficiency virus type 1.

Background: AnkGAG1D4 is an artificial ankyrin repeat protein which recognizes the capsid protein (CA) of the human immunodeficiency virus type 1 (HIV-1) and exhibits the intracellular antiviral activity on the viral assembly process. Improving the binding affinity of AnkGAG1D4 would potentially enhance the AnkGAG1D4-mediated antiviral activity.

Objective: To augment the affinity of AnkGAG1D4 scaffold towards its CA target, through computational predictions and experimental designs.

PUMA gene delivery to synoviocytes reduces inflammation and degeneration of arthritic joints.

In rheumatoid arthritis (RA), the proliferation of fibroblast-like synoviocytes (FLS) is the cause of chronic inflammation in joints and of joint damage. Delivery of the pro-apoptotic gene PUMA to FLS via human adenovirus type 5 (HAdV5) vectors has been tested as a therapeutic approach, but efficiency is hampered by low transduction, as FLS do not express HAdV5 receptors on the cell surface. Here we show that efficient transduction of PUMA in FLS can be achieved by conjugating HAdV5 to a baculovirus, which binds to the cell surface via the envelope glycoprotein Gp64.

Active and separate secretion of fiber and penton base during the early phase of Ad2 or Ad5 infection.

Fiber and penton base overproduced in adenovirus (Ad) infected cells can be secreted prior to progeny release and thereby regulate progeny spread. We aimed to investigate the mechanisms of fiber and penton base secretion in Ad2- or Ad5-infected A549 cells. Our flow cytometry analyses detected abundant surface fiber molecules, but little penton base molecules at 12h post infection.

Transfer of the Cystic Fibrosis Transmembrane Conductance Regulator to Human Cystic Fibrosis Cells Mediated by Extracellular Vesicles.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in a deficiency in chloride channel activity. In this study, extracellular vesicles (EVs), microvesicles, and exosomes were used as vehicles to deliver exogenous CFTR glycoprotein and its encoding mRNA (mRNA(GFP-CFTR)) to CF cells to correct the CFTR chloride channel function. We isolated microvesicles and exosomes from the culture medium of CFTR-positive Calu-3 cells, or from A549 cells transduced with an adenoviral vector overexpressing a GFP-tagged CFTR (GFP-CFTR).

Combined Antiviral Therapy Using Designed Molecular Scaffolds Targeting Two Distinct Viral Functions, HIV-1 Genome Integration and Capsid Assembly.

Designed molecular scaffolds have been proposed as alternative therapeutic agents against HIV-1. The ankyrin repeat protein (Ank(GAG)1D4) and the zinc finger protein (2LTRZFP) have recently been characterized as intracellular antivirals, but these molecules, used individually, do not completely block HIV-1 replication and propagation. The capsid-binder Ank(GAG)1D4, which inhibits HIV-1 assembly, does not prevent the genome integration of newly incoming viruses.

Crystal structure of an antiviral ankyrin targeting the HIV-1 capsid and molecular modeling of the ankyrin-capsid complex.

Ankyrins are cellular repeat proteins, which can be genetically modified to randomize amino-acid residues located at defined positions in each repeat unit, and thus create a potential binding surface adaptable to macromolecular ligands. From a phage-display library of artificial ankyrins, we have isolated Ank(GAG)1D4, a trimodular ankyrin which binds to the HIV-1 capsid protein N-terminal domain (NTD(CA)) and has an antiviral effect at the late steps of the virus life cycle. In this study, the determinants of the Ank(GAG)1D4-NTD(CA) interaction were analyzed using peptide scanning in competition ELISA, capsid mutagenesis, ankyrin crystallography and molecular modeling.

Pseudomonas aeruginosa-induced apoptosis in airway epithelial cells is mediated by gap junctional communication in a JNK-dependent manner.

Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1.

Human full-length coagulation factor X and a GLA domain-derived 40-mer polypeptide bind to different regions of the adenovirus serotype 5 hexon capsomer.

The interaction of human adenovirus (HAdV)-C5 and many other adenoviruses with blood coagulation factors (e.g., human factor X, FX) involves the binding of their GLA domain to the hexon capsomers, resulting in high levels of hepatotropism and potential hepatotoxicity.

A novel platform for virus-like particle-display of flaviviral envelope domain III: induction of Dengue and West Nile virus neutralizing antibodies.

CD16-RIgE is a chimeric human membrane glycoprotein consisting of the CD16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE (RIgE). Coexpression of CD16-RIgE and HIV-1 Pr55Gag polyprotein precursor (Pr55GagHIV) in insect cells resulted in the incorporation of CD16-RIgE glycoprotein into the envelope of extracellular virus-like particles (VLPs), a phenomenon known as pseudotyping. Taking advantage of this property, we replaced the CD16 ectodomain of CD16-RIgE by the envelope glycoprotein domain III (DIII) of dengue virus serotype 1 (DENV1) or West Nile virus Kunjin (WNVKun).

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells.

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells.

Neutrophil elastase degrades cystic fibrosis transmembrane conductance regulator via calpains and disables channel function in vitro and in vivo.

Rationale: Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride channel regulating fluid homeostasis at epithelial surfaces. Its loss of function induces hypohydration, mucus accumulation, and bacterial infections in CF and potentially other lung chronic diseases.

Objectives: To test whether neutrophil elastase (NE) and neutrophil-mediated inflammation negatively impact CFTR structure and function, in vitro and in vivo.

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein.

Background: Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.

Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay.

The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr).

Quantification of HIV-based lentiviral vectors: influence of several cell type parameters on vector infectivity.

A human immunodeficiency virus type (HIV-1)-based lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein and encoding the GFP reporter gene was used to evaluate different methods of lentiviral vector titration. GFP expression, viral DNA quantification and the efficiency of vector DNA integration were assayed after infection of conventional HIV-1-permissive cell lines and human primary adult fibroblasts with the vector. We found that vector titers based on GFP expression determined by flow cytometry may vary by more than 50-fold depending on the cell type and the promoter-cell combination used.

A fiber-modified adenoviral vector interacts with immunoevasion molecules of the B7 family at the surface of murine leukemia cells derived from dormant tumors.

Tumor cells can escape the immune system by overexpressing molecules of the B7 family, e.g. B7-H1 (PD-L1 or CD86), which suppresses the anti-tumor T-cell responses through binding to the PD-1 receptor, and similarly for B7.

Cell entry and trafficking of human adenovirus bound to blood factor X is determined by the fiber serotype and not hexon:heparan sulfate interaction.

Human adenovirus serotype 5 (HAdV5)-based vectors administered intravenously accumulate in the liver as the result of their direct binding to blood coagulation factor X (FX) and subsequent interaction of the FX-HAdV5 complex with heparan sulfate proteoglycan (HSPG) at the surface of liver cells. Intriguingly, the serotype 35 fiber-pseudotyped vector HAdV5F35 has liver transduction efficiencies 4-logs lower than HAdV5, even though both vectors carry the same hexon capsomeres. In order to reconcile this apparent paradox, we investigated the possible role of other viral capsid proteins on the FX/HSPG-mediated cellular uptake of HAdV5-based vectors.

Development of a recombinant CHO cell model for the investigation of CAR and DAF role during early steps of echovirus 6 infection.

The early steps of echovirus 6 (E6) infection remain poorly understood and the only described receptor for haemagglutinating E6 strains is the decay accelerating factor (DAF). There is, however, accumulating evidence suggesting that E6 interaction with DAF is necessary but not sufficient for infection. In this report, we investigated the role of the coxsackie-adenovirus-receptor (CAR) as a potential DAF co-receptor during E6 infection.

Baculovirus display of single chain antibody (scFv) using a novel signal peptide.

Background: Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication.

Results: Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag.