ABSTRACT
Immune responses to factor VIII remain the greatest complication in the treatment of severe hemophilia A. Recent epidemiological evidence has highlighted that recombinant factor VIII produced in baby hamster kidney cells is more immunogenic than factor VIII produced in Chinese hamster ovary cells. Glycosylation differences have been hypothesized to influence the immunogenicity of these synthetic concentrates. In two hemophilia A mouse models, baby hamster kidney cell-derived factor VIII elicited a stronger immune response compared to Chinese hamster ovary cell-derived factor VIII. Furthermore, factor VIII produced in baby hamster kidney cells exhibited accelerated clearance from circulation independent of von Willebrand factor. Lectin and mass spectrometry analysis of total N-linked glycans revealed differences in high-mannose glycans, sialylation, and the occupancy of glycan sites. Factor VIII desialylation did not influence binding to murine splenocytes or dendritic cells, nor surface co-stimulatory molecule expression. We did, however, observe increased levels of immunoglobulin M specific to baby hamster kidney-derived factor VIII in naïve hemophilia A mice. De-N-glycosylation enhanced immunoglobulin M binding, suggesting that N-glycan occupancy masks epitopes. Elevated levels of immunoglobulin M and immunoglobulin G specific to baby hamster kidney-derived factor VIII were also observed in healthy individuals, and de-N-glycosylation increased immunoglobulin G binding. Collectively, our data suggest that factor VIII produced in baby hamster kidney cells is more immunogenic than that produced in Chinese hamster ovary cells, and that incomplete occupancy of N-linked glycosylation sites leads to the formation of immunoglobulin M- and immunoglobulin G-factor VIII immune complexes that contribute to the enhanced clearance and immunogenicity in these mouse models of hemophilia A.
Subject(s)
Factor VIII , Hemophilia A , Animals , CHO Cells , Cricetulus , Disease Models, Animal , Factor VIII/immunology , Factor VIII/pharmacology , Female , Glycosylation , Hemophilia A/drug therapy , Hemophilia A/genetics , Hemophilia A/immunology , Hemophilia A/pathology , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Male , Mice , Mice, Knockout , Recombinant Proteins/immunology , Recombinant Proteins/pharmacologyABSTRACT
The DNA compacting properties of polyamines (especially spermine) are well-known, hence the use of spermine as the cationic part in several synthetic DNA carriers. Here, we describe the synthesis of modified spermines, with a "lipophosphoramidate" as the lipidic part, and their use for efficient in vitro transfection. Physicochemical measurements (particle size, zeta potentials, pKa determination) and gel retardation assays were also performed. Theoretical membrane-disrupting ability was established by FRET. Taken together, our results indicate that lipophosphoramidates constitute an interesting alternative to "classical" lipidic parts of cationic lipids used as DNA carriers.
Subject(s)
DNA/metabolism , Drug Carriers/chemical synthesis , Gene Transfer Techniques , Liposomes/chemistry , Nanostructures/chemistry , Phosphatidylethanolamines/chemistry , Spermine/chemistry , Cations , DNA/administration & dosage , DNA/genetics , Drug Carriers/pharmacology , Fluorescence Resonance Energy Transfer , Models, Chemical , Particle Size , Transfection/methodsABSTRACT
Lipophosphoramidates with two different permanent cations as polar heads were synthesized and evaluated for their gene transfer activity. Physicochemical measurements (particle size, zeta potentials) and gel retardation assays were also performed. In vitro biological evaluation was conducted with A542 and HeLa cell lines, and cytotoxicity determined by a chemiluminescent assay. The set of results indicates that, on the whole, dicationic lipophosphoramidates constitute an interesting alternative to their monocationic analogues.
Subject(s)
Amides/chemical synthesis , DNA/metabolism , Drug Carriers/chemical synthesis , Gene Transfer Techniques , Phospholipids/chemical synthesis , Phosphoric Acids/chemical synthesis , Animals , Cations , Cell Line, Tumor , Cytotoxicity Tests, Immunologic , DNA/administration & dosage , DNA/genetics , HeLa Cells , Humans , Luminescent Measurements , Phosphoramides , Time FactorsABSTRACT
A series of 'retinoid-like chalcones' and diverse derivatives relative to licochalcone A were synthesized from a new enaminone synthon. These syntheses occurred via a new aromatic annelation. These new derivatives have been tested in vitro as potential antimalarial agents. The 4-hydroxy-chalcone-like (compound 6a, derived from beta-ionone) exhibits a good and reproducible inhibitory effect on the in vitro culture of Plasmodium falciparum, with an IC 50 lower than 10 microM for inhibition of 3H-hypoxanthine uptake by parasites (respectively, 4.93 and 8.47 microM for strains K1 and Thaï).
Subject(s)
Antimalarials/chemical synthesis , Chalcone/chemical synthesis , Erythrocytes/parasitology , Plasmodium falciparum/drug effects , Retinoids/chemical synthesis , Animals , Antimalarials/chemistry , Antimalarials/pharmacology , Chalcone/analogs & derivatives , Chalcone/pharmacology , Humans , Inhibitory Concentration 50 , Malaria/blood , Malaria/drug therapy , Malaria/parasitology , Parasitemia/drug therapy , Plasmodium falciparum/growth & development , Plasmodium falciparum/metabolism , Retinoids/chemistry , Retinoids/pharmacologyABSTRACT
Two new families of cationic lipids were designed and synthesized for gene delivery, namely "lipophosphoramidates" and "lipophosphoguanidines", whose efficiency was noteworthy. The most efficient have an arsonium cation as the polar head, and the unsaturated lipidic tails (e.g. oleyl) gave the better in vivo results (mice lungs).
Subject(s)
Amides/chemistry , DNA/administration & dosage , Gene Transfer Techniques/instrumentation , Guanidines/chemistry , Lipids/chemistry , Phosphoric Acids/chemistry , Animals , Cations/chemistry , Cell Line , Cricetinae , DNA/genetics , Genes, Reporter/genetics , Genetic Vectors/chemistry , Humans , Mice , Molecular Structure , Phosphoramides , PhosphorylationABSTRACT
New structure-activity relationships of a series of methylene or side chain modified retinoids on NB4 acute promyelocytic leukemia cells are investigated. The differentiation- and apoptosis-inducing potential of these compounds is analyzed on the basis of their selective retinoic acid receptor binding profile.