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Cationic liposomes are commonly used as a transfection reagent for DNA, RNA or proteins and as a co-adjuvant of antigens for vaccination trials. A high density of positive charges close to cell surface is likely to be recognized as a signal of danger by cells or contribute to trigger cascades that are classically activated by endogenous cationic compounds. The present review provides evidence that cationic liposomes activate several cellular pathways like pro-apoptotic and pro-inflammatory cascades. An improved knowledge of the relationship between the cationic lipid properties (nature of the lipid hydrophilic moieties, hydrocarbon tail, mode of organization) and the activation of these pathways opens the way to the use and design of cationic tailored for a specific application (e.g. for gene transport or as adjuvants).

In this work, we investigated the properties of a fusogenic cationic lipid, diC14-amidine, and show that this lipid possesses per se the capacity to adopt either an interdigitated structure (below and around its transition temperature) or a lamellar structure (above the transition temperature). To provide experimental evidence of this lipid bilayer organization, phospholipids spin-labeled at different positions of the hydrocarbon chain were incorporated into the membrane and their electron spin resonance (ESR) spectra were recorded at different temperatures. For comparison, similar experiments were performed with dimyristoyl phosphatidylcholine, a zwitterionic lipid (DMPC) which adopts a bilayer organization over a broad temperature range. Lipid mixing between diC14-amidine and asolectin liposomes was more efficient below (10-15 °C) than above the transition temperature (above 25 °C). This temperature-dependent "fusogenic" activity of diC14-amidine liposomes is opposite to what has been observed so far for peptides or virus-induced fusion. Altogether, our data suggest that interdigitation is a highly fusogenic state and that interdigitation-mediated fusion occurs via an unusual temperature-dependent mechanism that remains to be deciphered.

Adjuvant efficiency is critical for inducing a protective and long-lasting immune response against weak immunogenic antigens. Discovered more than 70 years ago, aluminum salts remain the most widely used adjuvant in human vaccine. Prone to induce a strong humoral response, alum fails to drive a cell-mediated immunity, which is essential to fight against intracellular pathogens. Adjuvant systems that contain more than one component may represent an excellent alternative for completing the lack of T cell immunity associated with the injection of alum-based vaccine. In this work, we demonstrated that the adjuvant effects of alum strongly benefited from combining with a cationic lipid, the diC14 amidine. Indeed, we measured a significant improvement of alum-driven IL-1β release when human macrophages were co-cultured with a mixed suspension of alum and the diC14 amidine. Morphological analysis suggested that diC14 amidine improved the alum uptake by phagocytes. Furthermore, the addition of diC14 amidine to alum efficiently enhanced antigen processing and cross-presentation by antigen presenting cells. The biological relevance of these in vitro data was assessed by measuring the in vivo development of a cytotoxic activity and the enhanced synthesis of antigen-specific immunoglobulins after immunization with alum combined to diC14 amidine. Mechanistically, we demonstrated that diC14 amidine supported the alum adjuvanticity independently of the TLR-4 and caspase-1 agonist activities of the cationic lipid. Based on our findings, we conclude that diC14 amidine works synergistically with alum to achieve higher immune protection after vaccination.

Peritoneal macrophages were isolated from wild type (WT) mice and from mice invalidated for the P2X(7) receptor (KO) which had been pretreated with thioglycolate. In cells from WT mice, 1 mM ATP increased the intracellular concentration of calcium ([Ca(2+)](i)), the uptake of ethidium bromide, the production of reactive oxygen species (ROS), the secretion of IL-1beta, the release of oleic acid and of lactate dehydrogenase; it decreased the intracellular concentration of potassium ([K(+)](i)). In KO mice, ATP transiently increased the [Ca(2+)](i) confirming that the P2X(7) receptor is a major receptor of peritoneal macrophages. WKYMVm, an agonist of receptors for formylated peptides (FPR) also increased the [Ca(2+)](i) in murine macrophages. The slight increase of the [Ca(2+)](i) was strongly potentiated by ivermectin confirming the expression of functional P2X(4) receptors by murine peritoneal macrophages. CRAMP, the unique antimicrobial peptide derived from cathelin in mouse inhibited all the responses coupled to P2X(7) receptors in macrophages from WT mice. Agonists for FPR had no effect on the increase of the [Ca(2+)](i) in response to ATP. CRAMP had no effect on the increase of the [Ca(2+)](i) evoked by a combination of ATP and ivermectin in macrophages from P2X(7)-KO mice. In summary CRAMP inhibits the responses secondary to the activation of the murine P2X(7) receptors expressed by peritoneal macrophages. This inhibition is not mediated by FPR receptors and is specific since CRAMP has no effect on the response coupled to P2X(4) receptors. It can thus be concluded that the interaction between P2X(7) receptors and cathelin-derived antimicrobial peptides is species-specific, in some cases (man) positive in others (mouse) negative.

Addition of co-lipids into cationic lipid formulations is considered as promoting cell delivery of DNA by enhancing fusion processes with cell membranes. Here, by combining FRET and confocal microscopy, we demonstrate that some cationic lipids do not require a co-lipid to fuse efficiently with cells. These cationic lipids are able to self-organize into bilayers that are stable enough to form liposomes, while presenting some destabilizing properties reminiscent of the conically shaped fusogenic co-lipid, DOPE. We therefore analyzed the resident lipid structures in cationic bilayers by molecular dynamics simulations, clustering the individual lipid structures into populations of similarly shaped molecules, as opposed to the classical approach of using the static packing parameter to define the lipid shapes. Comparison of fusogenic properties with these lipid populations suggests that the ratio of cylindrical versus conical lipid populations correlates with the ability to fuse with cell membranes.

DiC(14)-amidine (amidine) is a nonphysiological, cationic lipid that forms stable liposomes under physiological pH and temperature. Cationic lipids have been proposed as delivery vector for DNA, proteins, and drugs. Furthermore, amidine carries at present a particular interest due to its immunomodulatory properties. (1-3) Molecular dynamics simulations reveal a remarkable fluidity in the hydrophobic bilayer core, with a tendency for strong surface curvature, in agreement with the relatively small size of experimentally formed liposomes. The amidine bilayer shows an interdigitated, nonlamellar bilayer phase, with a bilayer thickness of only 2.7 nm and an average area per lipid of 0.83 nm(2). A cluster analysis of the individual lipid structures shows a thermally accessible population of V-shaped lipids, indicative of fusion capabilities with the plasma membrane. Fusion experiments confirm this hypothesis. The results are compared to the zwitterionic DMPC (dimyristoylphosphocholine), which also carries two saturated C(14) tails.

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence TP1R.

Cationic lipids are positively charged amphiphilic molecules which, for most of them, form positively charged liposomes, sometimes in combination with a neutral helper lipid. Such liposomes are mainly used as efficient DNA, RNA or protein carriers for gene therapy or immunization trials. Over the past decade, significant progress has been made in the understanding of the cellular pathways and mechanisms involved in lipoplex-mediated gene transfection but the interaction of cationic lipids with cell components and the consequences of such an interaction on cell physiology remains poorly described. The data reported in the present review provide evidence that cationic lipids are not just carriers for molecular delivery into cells but do modify cellular pathways and stimulate immune or anti-inflammatory responses. Considering the wide number of cationic lipids currently available and the variety of cellular components that could be involved, it is likely that only a few cationic lipid-dependent functions have been identified so far.

The present study determined alpha-tocopherol mass transfer from an alpha-tocopherol-rich emulsion to LDL and HDL, and assessed the potential of different mechanisms to modulate alpha-tocopherol transfers. Emulsion particles rich in alpha-tocopherol were incubated in vitro with physiological concentrations of LDL or HDL. The influence of plasma proteins was assessed by adding human lipoprotein poor plasma (LPP) fraction with intact vs heat inactivated PLTP, or with a specific cholesteryl ester transfer protein (CETP) inhibitor, or by adding purified PLTP or pig LPP which lacks CETP activity. After 4 h incubation in absence of LPP, alpha-tocopherol content was increased by ~80% in LDL and ~160% in HDL. Addition of LPP markedly enhanced alpha-tocopherol transfer leading to 350-400% enrichment in LDL or HDL at 4 h. Higher (~10 fold) enrichment was achieved after 20 h incubation with LPP. Facilitation of alpha-tocopherol transfer was (i) more than 50% higher with human vs pig LPP (despite similar PLTP phospholipid transfer activity), (ii) reduced by specific CETP activity inhibition, (iii) not fully suppressed by heat inactivation, and (iv) not restored by purified PLTP. In conclusion, alpha-tocopherol content in LDL and HDL can be markedly raised by rapid transfer from an alpha-tocopherol-rich emulsion. Our results indicate that alpha-tocopherol mass transfer between emulsion particles and lipoproteins is mediated by more than one single mechanism and that this transfer may be facilitated not only by PLTP but likely also by other plasma proteins such as CETP.

The inflammatory effect of unmethylated CpG DNA sequences represents a major obstacle to the use of cationic lipids for in vivo gene therapy. Although the mechanism of CpG-induced inflammatory response is rather well understood nowadays, few solutions have been designed to circumvent this effect in gene therapy experiments. Our previous work has shown that a refractory state towards inflammation can be elicited by preinjecting cationic liposomes. Here, we present evidence that diC14-amidine liposomes confer new anti-inflammatory properties to phospholipids from low-density lipoprotein (LDL) and even to synthetic phospholipids for which such an observation has not been reported so far. Whereas oxidation of LDL lipids was a prerequisite for any anti-inflammatory activity, lipid oxidation is no longer required in our experiments, suggesting that cationic lipids transport phospholipids through a different route and affect different pathways. This opens up new possibilities for manipulating inflammatory responses in gene therapy protocols but also in a general manner in immunological experiments.

Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated with such transport has been relatively tedious, even though significant progress has been made in the characterization of the intracellular trafficking of lipid/DNA complexes. Surprisingly little is known about the effects of these delivery vectors on cell functioning. In this report, we show that both cationic lipids and cationic lipid/DNA complexes mobilize the intracellular calcium. Removal of extracellular calcium did not significantly abolish this effect and preincubating cells with thapsigargin led to a decrease in [Ca2+]i, indicating that calcium was released mainly from internal calcium stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122, blocked the [Ca2+]i rise, suggesting an inositol dependent mechanism.

The interaction of mice submandibular gland cells with LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), a cationic peptide with immunomodulatory properties, was investigated. LL-37 at a concentration that did not affect the integrity of the cells increased the uptake of calcium and activated a calcium-insensitive phospholipase A(2) (PLA(2)). The small release of ATP induced by LL-37 could not account for this stimulation because apyrase did not significantly block the response to LL-37. The divalent cation magnesium inhibited the response to LL-37, but this inhibition was probably nonspecific because it also inhibited the in vitro bacteriostatic effect of the peptide. The increase of calcium uptake by LL-37 was not affected by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a rather specific inhibitor of P2X(7) receptors in mice. LL-37 also increased [Ca(2+)](i) in cells from mice invalidated for these receptors. LL-37 had no effect on the response to carbachol. It inhibited the increase of [Ca(2+)](i) and the activation of phospholipase D by ATP. It potentiated the activation of the PLA(2) by the nucleotide. Finally, LL-37 increased the fluidity of the plasma membrane of submandibular gland cells. In conclusion, our results suggest that LL-37 is an autocrine regulator of submandibular gland cells. It does not stimulate mouse P2X(7) receptors but modulates their responses.

The present study evaluated the prophylactic potential of ProDer p 1, the recombinant precursor form of the major mite allergen Der p 1, combined with the cationic lipid diC14-amidine in a murine model of house dust mite allergy. Naive mice vaccinated with the amidine/allergen complex developed a Th1-biased immune response characterized by the absence of specific IgE, the production of specific IgG2a, and the presence of IFN-gamma in splenocyte cultures. In contrast, ProDer p 1 adjuvanted with alum induced typical strictly Th2-biased allergic responses with strong IgG1 and IgE titers and IL-5 secretion. Removal of negatively charged sialic acids in ProDer p 1 or increasing the ionic strength reduced the binding of ProDer p 1 to the cationic liposomes and resulted in a decrease of the allergen immunogenicity, suggesting that complexation is required for triggering an optimal immune response. Finally, prophylactic vaccination with ProDer p 1-diC14-amidine reduced drastically the production of specific IgE and airway eosinophilia following subsequent immunization with Der p 1-alum and challenge with aerosolized house dust mite extracts. In conclusion, recombinant ProDer p 1 complexed with diC14-amidine could represent an efficient prophylactic vaccine against house dust mite allergy., Evaluation Studies, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Interaction of cationic lipid/DNA complex with the plasma is a limiting step for the cationic lipid-mediated intravenous gene transfer and expression process. Most of the plasma components that interact with the complex and inhibit its transfection efficiency are still unknown. In the present work, human plasma proteins and lipoproteins that bind to a cationic lipid/DNA complex were isolated on a sucrose density gradient and identified by 2-D gel electrophoresis. Protein binding did not result in complex dissociation or DNA degradation. The effects of several complex-binding plasma components on the transfection efficiency were studied using lung endothelial cells cultured in vitro. Lipoprotein particles caused a drastic loss of the transfection efficiency of the complex. Surprisingly, fibrinogen was found to activate the transfection process. The roles of these complex-binding plasma components on the complex uptake efficiency were quantitatively assessed using radiolabeled plasmid DNA and qualitatively evaluated using fluorescence microscopy. A good correlation was found between the effects of the complex-binding plasma components on the transfection and on cell uptake efficiencies. In contrast to what was generally believed, our data suggest that disruption of the complex does not occur when it is in contact with the plasma and therefore could not be responsible for the loss of transfection activity. Instead, coating of complexes with plasma components seems to be responsible for reduced uptake by cells, which in turn results in reduced transfection.

In this report, we show that intravenous (i.v.) injection into mice of a complex made of the cationic lipid diC14-amidine and the luciferase reporter plasmid (pCMV-luc) results in efficient gene expression in several organs but elicits an inflammatory response characterized by a release of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) into the serum of treated animals. A single preinjection of free diC14-amidine liposomes improves the i.v. transfection efficiency of the diC14-amidine/protamine/pCMV-luc complex as much as 40 times. This improvement is correlated with the ability of free liposomes to inhibit TNF-alpha but not IFN-gamma production resulting from complex injection. TNF-alpha-rich serum obtained from mice injected with diC14-amidine/protamine/pCMV-luc complex inhibits luciferase expression in transfected mouse lung endothelial (MLE) cells cultured in vitro, whereas IFN-gamma has no effect. This inhibitory effect can be partly abolished by treating the mouse serum with a specific anti-TNF-alpha antibody. These data point out that cationic lipids are potent inhibitors of the inflammatory response to the CpG motifs in plasmid DNA. This property is shown to enhance the in vivo transfection efficiency.

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection.

DiC14-amidine is a cationic lipid which induces pro-inflammatory cytokine secretion in immune cells upon interaction with the Toll-like receptor 4 (TLR4)/Myeloid Differentiation factor-2 (MD-2) membrane bound-complex, the natural sensor of bacterial lipopolysaccharides (LPS) [1-3].The aim of the present work is to characterize the interaction between diC14-amidine and the TLR4/MD2 receptor complex. Taking advantage of the species-dependent activity of TLR4 agonists [4], we compared the TLR4 agonist activity of diC14-amidine in four different species in order to map domains in TLR4 and MD2 that are important for diC14-amidine TLR4-agonist activity. We demonstrate that, while LPS is an agonist in all species, diC14-amidine is a full agonist for human, mouse and cat receptors, but a poor agonist for horse. Using chimeric constructs made from human and horse TLR4 and single mutants, we identify two regions in the human TLR4 that modulates the agonist activity of diC14-amidine. Interestingly, these regions in TLR4 are different from the previously identified bacterial lipopolysaccharides binding domains [5]. How ligand binding to two different regions of the same receptor affects the cellular pathways is currently under investigation.[1] Tanaka, T. et al. (2008) Eur.J.Immunol. 38: 1351-1357.[2] Lonez, C. Et al. (2008) Prog Lipid Res. 47:340-7.[3] Lonez, C; et al. (2012) Adv Drug Deliv Rev. 64:1749-58.[4] Walsh, C. et al. (2008) J.Immunol. 181: 1245-1254.[5] Park, B. S. et al. (2009) Nature 458: 1191-1195.

Cationic liposomes are used as vectors for gene delivery both in vitro and in vivo. Comprehension of both DNA/liposome interactions on a molecular level and a description of structural modifications involved, are prerequisites to an optimization of the transfection protocol and, thus, successful application in therapy. Formation and stability of a DNA/cationic liposome complex were investigated here at different DNA:lipid molar ratios (ρ). Isothermal titration calorimetry (ITC) of cationic liposomes with plasmid DNA was used to characterize the DNA-lipid interaction. Two processes were shown to be involved in the complex formation. A fast exothermic process was attributed to the electrostatic binding of DNA to the liposome surface. A subsequent slower endothermic reaction is likely to be caused by the fusion of the two components and their rearrangement into a new structure. Fluorescence and differential scanning calorimetry confirmed this interpretation. A kinetic model analyzes the ITC profile in terms of DNA/cationic liposome interactions.

Human immunodeficiency virus (HIV) glycoprotein (gp) 160 processing by host cell proteinases is an essential step for viral fusion and infectivity. We have identified a rat liver subcellular fraction which specifically processes gp160 into gp120 and gp41. Using equilibration of microsomes in sucrose gradients, the gp160 cleavage activity was associated with particles equilibrating at low densities, well-separated from the endoplasmic reticulum, cis-Golgi network, Golgi stacks, lysosomes and plasma membrane. Its density distribution was compatible with light secretory vesicles derived from the trans-Golgi network (TGN) or to endosomes, but association with endosomes was not supported by free flow electrophoresis. Although furin and pro-protein convertase (PC) 7/LPC have been proposed as the major gp160 processing convertases, the rat liver microsomal gp160 processing activity was essentially resolved from furin and only partially overlapped PC7/LPC. These data suggest that proteinase(s) other than furin and PC7/LPC, presumably located in TGN-derived vesicles, may participate in the gp160 processing into gp120 and gp41.

Use of mannan-coated N-t-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine (diC14-amidine) as an adjuvant for a DNA vaccine encoding glycoprotein 160 of human immunodeficiency virus type-1 (HIV-1) enhanced the antigen-specific immune responses. The role of interferon-gamma (IFN-gamma) and interleukin-12 in the mechanism of adjuvant action was also evaluated. Coating of diC14-amidine with mannan significantly augmented the HIV-specific delayed-type hypersensitivity reaction induced by the immunogenic DNA. HIV-1-specific cytotoxic T lymphocyte activity was also markedly enhanced by the mannan-diC14-amidine cocktail. An immunomodulatory effect of this cocktail was inhibited by treatment with anti-IFN-gamma monoclonal antibody in vivo, which suggests that IFN-gamma plays an important role in inducing cell-mediated immunity by the DNA vaccine containing this adjuvant. The results of both antigen-specific immunoglobulin isotype analysis and cytokine measurement showed that the immunogenic DNA incorporated into mannan-coated diC14-amidine elicits Th1-biased immune responses.

We describe here a new cationic amphiphile, N-t-butyl-N'-tetradecyl-3-tetradecylaminopropionamidine (diC14-amidine), which interacts with plasmid DNA and generates hydrophobic stable complexes resistant against DNase I. In partition experiments between two non-miscible phases, DNA was transferred into an organic phase upon complex formation with diC14-amidine-containing vesicles. Finally, vesicles made of a diC14-amidine and phosphatidylethanolamine (PE) (1:1, mol:mol) mixture or pure diC14-amidine were efficient in mediating transfection of adherent (CHO) and suspension (K562) cell lines, using the chloramphenicol acetyltransferase (CAT) gene as reporter.

Intracellular proteolytic processing of human immunodeficiency virus envelope glycoprotein precursor (gp160) is an essential step for virus infectivity. Northern blot analysis provided evidence that furin and PC1, but not PC2, are expressed in the CD4+ human lymphoblastoid H9 cell line, suggesting the possible participation of these convertases in human immunodeficiency virus (HIV) gp160 proteolytic processing. Purified PC1 and furin cleaved specifically in vitro gp160 into gp120 (HIV-I SU) and gp41 (HIV-I TM). NH2-terminal sequence analysis of the produced gp41 (HIV-I TM) demonstrated that the cleavage occurred within the sequence Arg-Glu-Lys-Arg decreases Ala-Val-Gly-Ile, which is identical to the bond cleaved in vivo. Transition state analog peptides were designed and tested in vitro for their ability to inhibit the PC1- or furin-mediated gp160 cleavage. The best inhibitor was decanoyl-Arg-Lys-Arg-Arg-psi [CH2NH]-Phe-Leu-Gly-Phe-NH2.

For several retroviruses, the N-terminal hydrophobic sequence of the viral envelope glycoprotein has been shown to play a crucial role in the interaction between the virus and the host cell membrane. We report here on the interaction of a synthetic 16 residues peptide corresponding to the gp41 NH2-terminal sequence of Human Immunodeficiency Virus with the phospholipid bilayer. Fluorescence energy transfer measurements show that this peptide can induce lipid mixing of large unilamellar vesicles (LUV) of various compositions at pH 7.4 and 37 degrees C. LUV undergo fusion, provided they contained phosphatidylethanolamine (PE) in their lipid composition. To provide insight into the mechanism of the fusion event, the peptide secondary structure and orientation in the lipid bilayer were determined using Fourier Transform Infrared Spectroscopy (FTIR). The peptide adopts mainly a beta-sheet conformation in the absence of lipids. After interaction with LUV the beta-sheet is partly converted into alpha-helix. The orientation of the peptide with respect to the lipid acyl chains depends on the presence of PE in the lipid bilayer. The peptide is inserted into the lipid bilayer with the helix axis oriented parallel to the lipid acyl chains in the fused vesicles, whereas it is adsorbed parallel to the lipid/water interface in the aggregated vesicles. The role of the two kinds of orientation during the fusion event is discussed.

Cationic liposomes have been used over the past decades with gene therapy or vaccination trials in mind, considering them primarily as the smart Trojan horse allowing to go trough the cell walls. Fusion-promoting lipids such as DOPE were thus added with some success to try to enhance the destabilizing properties of some cationic lipids, while others were intrinsically destabilizing. It is difficult to conceive that lipids that destabilize membranes would act innocently on the cell physiology. After all, lipids are not only the backbone of membranes, they also act as facilitators of membrane functions such as endocytosis, budding, curving; they regulate protein membrane activity and can even serve as signal transmitters (bioactive lipids). Rather than being considered as responsible for harmful side-effects that should be minimized, cationic lipids could be considered as a potential immunostimulating or pharmacological agents. We illustrate these aspects with diC14-amidine, which forms liposomes with a bilayer at the edge of instability. DiC14-amidine fuses easily with cell membranes, modifies cell signaling and activates immune responses through destabilization and/or activation of specific membrane receptors, like TLR4 [1,2]. Like it was shown for natural lipids, this demonstrates that cationic lipids are not only membrane destabilizing agents but might affect cell membrane components function.1. Lonez C, Vandenbranden M, Ruysschaert JM. Prog Lipid Res. 2008. 47(5):340-7.2. Tanaka T et al. Eur J Immunol. 2008. 38(5):1351-7.

It has been recently demonstrated that diC14-amidine, a cationic lipid, activate cytokine secretion (TNF-alpha, IL-12, IFN-gamma, IP-10) in dendritic cells through a Toll-like Receptor-4-dependent mechanism [Tanaka et al., 2008]. This receptor is involved in the recognition of the bacterial endotoxins (lipopolysaccharides, LPS) and activation of innate immune system. DiC14-amidine could activate this cascade by different mechanism [Lonez et al., 2008].Our first hypothesis is that diC14-amidine interacts with the co-receptor of TLR4, MD-2. DiC14-amidine has 2 acyl chains that mimic LPS acyl chains. Molecular dynamics simulations of the insertion of diC14-amidine in the MD-2 cavity revealed that two amidine molecules do occupy a volume identical to that of one tetra-acylated lipid A molecule, an antagonist of TLR4, in the cavity. A non-exclusive alternative could consist in a modification of the membrane environment of TLR4, upon insertion of cationic lipids in the membrane bilayer. The ability of diC14-amidine liposomes to fuse with cell membranes was demonstrated by confocal microscopy or (FRET) Fluorescence Resonance Energy Transfer measurements. Fusion (lipid mixing) with the cell membrane would be a way to insert cationic lipid in the lipid bilayer of the plasma membrane and to modify the lipid-protein interactions involved in the function of membrane proteins. Biophysical studies (AFM, X-ray scattering) revealed that amidine molecules adopts an interdigitated structure and does not require additional lipids (PE) to be fusogenic. This raises also the question of the role of interdigitated structures in the fusion (lipid mixing) mechanism.ReferencesLonez C, Vandenbranden M, Ruysschaert JM. Prog Lipid Res. 2008. 47(5):340-7.Tanaka T et al. Eur J Immunol. 2008. 38(5):1351-7.

Cationic lipids have been extensively used as carriers of biologically active molecules (nucleic acids, peptides and proteins) into cells. Recent data provided evidence that cationic lipids are not just inert transporters but do activate specific cellular cascades. This review illustrates these activating properties with a few examples. Cell activation raises the question of which receptors are involved. Some cationic lipids seem to satisfy specific structural requirements of Toll-like receptors (TLR4) as they activate TLR4-dependent pathways. However, cationic lipids display a large structural diversity and it is likely that they are also recognized by receptors with a broader specificity. Alternatives are proposed and discussed to explain this broad specificity.

Background Recombinant hypoallergens that display reduced allergenicity but retain T-cell reactivity represent promising candidates to improve the safety and efficacy of allergen-specific vaccines or immunotherapy. Objective The current study reports the immunologic characterization of a hypoallergenic variant of the major mite allergen Der p 1. Methods The recombinant proform of Der p 1 (ProDer p 1) was expressed in Escherichia coli (ProDer p 1 coli ), purified and characterized at the level of its secondary structure, and IgE and T-cell reactivities. Moreover, the prophylactic potential of ProDer p 1 coli vaccinations was evaluated in a murine Der p 1 sensitization model. Results After purification and refolding, ProDer p 1 coli remained aggregated with a higher β-sheet content and altered Der p 1 conformational epitopes compared with the correctly folded monomeric ProDer p 1 produced in Chinese hamster ovary cells. Both ProDer p 1 forms were able to retain the Der p 1–specific T-cell reactivity but direct ELISA, competitive inhibition, and rat basophil leukemia assays clearly showed that ProDer p 1 coli displays a very weak IgE reactivity. Mice vaccinations with aggregated ProDer p 1 adjuvanted with alum induced a T H 1-biased immune response that prevented the subsequent allergic response after Der p 1 sensitization and airway challenge with aerosolized mite extracts. Furthermore, ProDer p 1 coli treatment inhibited the development of airway eosinophilia and airway hyperresponsiveness to inhaled methacholine. Conclusion Aggregated forms of Der p 1 could represent hypoallergens suitable for the prevention of mite allergy.

DiC14-amidine cationic liposomes were recently shown to promote Th1 responses when mixed with allergen. To further define the mode of action of diC14-amidine as potential vaccine adjuvant, we characterized its effects on mouse and human myeloid dendritic cells (DC). First, we observed that, as compared with two other cationic liposomes, only diC14-amidine liposomes induced the production of IL-12p40 and TNF-alpha by mouse bone marrow-derived DC. DiC14-amidine liposomes also activated human DC, as shown by synthesis of IL-12p40 and TNF-alpha, accumulation of IL-6, IFN-beta and CXCL10 mRNA, and up-regulation of membrane expression of CD80 and CD86. DC stimulation by diC14-amidine liposomes was associated with activation of NF-kappaB, ERK1/2, JNK and p38 MAP kinases. Finally, we demonstrated in mouse and human cells that diC14-amidine liposomes use Toll-like receptor 4 to elicit both MyD88-dependent and Toll/IL-1R-containing adaptor inducing interferon IFN-beta (TRIF)-dependent responses.

The complete amino-acid sequence of viral fusion proteins has been analyzed by the Eisenberg procedure. The region surrounding the cleavage site contains a highly hydrophilic region immediately followed by a membrane-like region. Since the effective cleavage between these two domains seems required to expose the fusogenic domain (located at the N-terminal sequence of the transmembrane like region) which is assumed to interact with the lipid membrane of the host cell, we have focused our analysis on the conformation and mode of insertion of this membrane-like domain in a lipid monolayer. It was inserted as an alpha-helical structure into a dipalmitoylphosphatidylcholine (DPPC) monolayer and its orientation at the lipid/water interface was determined using a theoretical analysis procedure allowing the assembly of membrane components. For each viral protein sequence these N-terminal helical segments oriented obliquely with respect to the lipid/water interface. This rather unusual orientation is envisaged as a prerequisite to membrane destabilization and fusogenic activity.

Cationic lipids are widely used as vectors to deliver DNA into mammalian cells in vitro and in vivo. However, cationic lipid/DNA lipoplexes induce an inflammatory response, characterized by pro-inflammatory cytokine secretion, which severely limits their use. The main goal of this work is to identify the organs and the cell type involved in TNF-alpha secretion after lipoplex injection. We determined the kinetics of distribution of the cationic lipid/DNA complex in blood, lung, liver and spleen and quantified the TNF-alpha amount in organ homogenates and in the serum at different points of times. Increase in TNF-alpha production was only observed in the spleen and no significant increase of TNF-alpha production could be observed in the other organs. Fractionation of spleen cells revealed that macrophages were mainly responsible for TNF-alpha secretion. This observation was verified in vivo by using macrophage-removing agents. In conclusion, we show here that the TNF-alpha secreted in the serum after intravenous injection of lipoplexes comes mainly from the splenic macrophages.

International audience; We report DNA immunisation experiments in cattle using plasmid constructs that encoded glycoprotein E2 from bovine viral diarrhoea virus (BVDV)-1 (E2.1) and BVDV-2 (E2.2). The coding sequences were optimised for efficient expression in mammalian cells. A modified leader peptide sequence from protein gD of BoHV1 was inserted upstream of the E2 coding sequences for efficient membrane export of the proteins. Recombinant E2 were efficiently expressed in COS7 cells and they presented the native viral epitopes as judged by differential recognition by antisera from cattle infected with BVDV-1 or BVDV-2. Inoculation of pooled plasmid DNA in young cattle elicited antibodies capable of neutralising viral strains representing the major circulating BVDV genotypes.

It has been shown that a preinjection of diC14-amidine cationic liposomes decreased TNF-alpha secretion induced by lipoplexes intravenous injection. We showed here that free cationic liposomes inhibit CpG sequences- or lipopolysaccharides-induced TNF-alpha secretion by macrophages. Surprisingly, this effect was strictly dependent on serum. Free cationic liposomes alone did not reveal any anti-inflammatory activity. Low-density lipoproteins and triglyceride-rich lipoproteins were identified as the serum components that confer to the liposomes an anti-inflammatory activity. Lipid fractions of these lipoproteins were able to reproduce the effect of the total lipoproteins and could inhibit, in association with diC14-amidine liposomes, the CpG-induced TNF-alpha secretion. Serum components confer to cationic liposomes new properties that can be used to modulate the inflammatory response directed against CpG sequences and lipopolysaccharides.

It has been previously shown that transfection activity of cationic liposome/DNA lipoplexes delivered systemically is drastically inhibited by lipoproteins (Tandia, B. M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J. M., and Elouahabi, A. (2003) Mol Ther. 8, 264-273). In this work, we have compared the binding/uptake and transfection activities of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and diC14-amidine (3-tetradecylamino-N-tert-butyl-N'-tetra-decylpropionamidine)-containing lipoplexes in the presence or absence of purified low density lipoproteins and high density lipoprotein. Binding/uptake of both lipoplexes by the mouse lung endothelial cell line was inhibited to a similar extent in the presence of lipoproteins. In contrast, transfection activity of diC14-amidine-containing lipoplexes was almost completely inhibited (approximately by 95%), whereas approximately 40% transfection activity of DOTAP-containing lipoplexes was preserved in the presence of lipoproteins. Interestingly, the ability of lipoproteins to inhibit the transfection efficiency of lipoplexes was well correlated with their ability to undergo lipid mixing with the cationic lipid bilayer as revealed by fluorescence resonance energy transfer assay. Incubation of lipoplexes with increased doses of lipoproteins resulted in enhanced lipid mixing and reduced transfection activity of the lipoplexes in mouse lung endothelial cells. The role of lipid mixing in transfection was further demonstrated using lipid-mixing inhibitor, lyso-phosphatidylcholine, or activator (dioleoylphosphatidylethanolamine). Incorporation of Lyso-PC into diC14-amidine-containing lipoplexes completely abolished their capacity to undergo lipid mixing with lipoproteins and allowed them to reach a high transfection efficiency in the presence of lipoproteins. On the other hand, the incorporation of dioleoylphosphatidylethanolamine into DOTAP/DNA lipoplex activated lipid mixing with the lipoproteins and was shown to be detrimental toward the transfection activity of these lipoplexes. Taken together, these results indicate that fusion of lipoplexes with lipoproteins is a limiting factor for in vivo transfection.

Background Modified allergens with reduced IgE-binding activity represent an elegant approach to circumvent the risk of anaphylactic reactions in allergen-specific immunotherapy. Objective The current work investigated the effect of heat denaturation on the allergenic properties of recombinant ProDer p 1, a precursor form of the major house dust mite allergen Der p 1. Methods The IgE reactivity was estimated by direct and competition ELISA. The immunogenicity of heat-denatured ProDer p 1 was evaluated in naive and Der p 1-allergic mice. Results Heat denaturation in reducing conditions drastically reduced the in vitro ProDer p 1 IgE reactivity toward human allergic sera. In naive mice, heat-denatured ProDer p 1 generated mixed T H 1-T H 2 responses characterized by the absence of specific IgE with concomitant rise in specific IgG2a titers and the presence of IL-5 and IFN-γ in splenocyte cultures. In contrast, natural Der p 1 or native ProDer p 1 induced typical strict T H 2-biased allergic responses with strong IgG1 and IgE titers, whereas spleen cells exhibited only high IL-5 secretion. Moreover, native or heat-denatured ProDer p 1 vaccinations prevented airway eosinophil infiltrations after challenge. Although native or heat-treated ProDer p 1 adjuvanted with SBAS1b induced mixed T H 1-T H 2 responses in allergic mice, heat-denatured ProDer p 1, compared with native ProDer p 1, proved to be more effective in redirecting the T H 2-allergic response toward T H 1. Conclusion Taken together, our results suggest that variants of Der p 1 with reduced IgE-binding reactivity could represent hypoallergenic molecules suitable for allergen-specific immunotherapy.

Glucocorticoids (GC) are widely used anti-inflammatory agents known to suppress T cell activation by interfering with the TCR activation cascade. The attenuation of early TCR signaling events by these compounds has been recently attributed to a selective displacement of key signaling proteins from membrane lipid rafts. In this study, we demonstrate that GC displace the acyl-bound adaptor proteins linker for activation of T cells and phosphoprotein associated with glycosphingolipid-enriched microdomains from lipid rafts of murine T cell hybridomas, possibly by inhibiting their palmitoylation status. Analysis of the lipid content of the membrane rafts revealed that GC treatment led to a significant decrease in palmitic acid content. Moreover, we found an overall decrease in the proportion of raft-associated saturated fatty acids. These changes were consistent with a decrease in fluorescence anisotropy of isolated lipid rafts, indicating an increase in their fluidity. These findings identify the mechanisms underlying the complex inhibitory effects of glucocorticoids on early TCR signaling and suggest that some of the inhibitory properties of GC on T cell responses may be related to their ability to affect the membrane lipid composition and the palmitoylation status of important signaling molecules.

Publisher Summary This chapter describes cationic liposome–mediated gene transfer that has become a widely used tool for in vitro transfection of eukaryotic cells and as a promising and safe alternative for in vivo gene therapy applications. It involves the formation of a cationic lipid–DNA complex that interacts efficiently with the cell surface, leading to the entry and expression of the exogenous DNA. The use of cationic lipids as DNA delivery systems for gene therapy is limited by the relatively low efficiency of the gene transfer process compared with viral vectors. Improvement of the in vitro and in vivo cationic lipid–mediated gene transfection efficiency can be attempted by use of trial and error approaches consisting of synthesizing and testing a large number of cationic lipid derivatives. Alternatively, a deeper understanding of the intermediate steps and mechanisms involved in the gene transfer process mediated by cationic lipids could allow the design of new rational strategies to improve the transfection efficiency. Two key steps are involved in the cationic lipid–mediated gene transfer process: (1) interaction between the cationic liposomes and DNA leading to the formation of a complex and (2) interaction between the complex and the target cells leading to DNA entry and expression of the transgene.

The cationic lipid-mediated gene transfer process involves sequential steps: internalization of the cationic lipid-DNA complexes inside the cells via an endocytosis-like mechanism, escape from endosomes, dissociation of the complex, and finally entry of free DNA into the nucleus. However, cationic lipid-DNA complex dissociation in the cytoplasm and the ability of the subsequently released DNA to enter the nucleus have not yet been demonstrated. In this report we showed, using confocal laser scanning analysis, that microinjection of a double fluorescent-labeled cationic lipid-pCMV-LacZ plasmid complex into the cytoplasm of HeLa cells results in efficient complex dissociation. However, the released DNA did not enter the nucleus, and no significant transfection could be detected. In contrast, nuclear microinjection of the cationic lipid-pCMV-LacZ plasmid complex resulted in efficient complex dissociation and transfection of all the cells. Taken together, the data suggest that intracellular dissociation of the cationic lipid-DNA complex is not a limiting step for transfection as previously thought.

We present experimental evidence that a complex made of a double long chain cationic amphiphile and recombinant mRNA facilitates the entry and expression of genetic material into cells. Combining the properties of the self replicating recombinant mRNA driven by the Semliki Forest Virus (SFV) replicon and the transfection potentialities of a new cationic amphiphile (N-t-butyl-N′-tetradecyl-3-tetradecylaminopropionamidine) yields a highly efficient mRNA transfection system conferring up to 100% infectivity. The preparation and characterization of the long chain amidine cationic amphiphile-mRNA complex as well as the influence of the diC14-amidine/RNA ratio on the infective activity are described.

Human immunodeficiency virus (HIV) membrane has been reconstituted from the recombinant envelope glycoprotein precursor (gp160) by a detergent dialysis technique. Electron microscopy shows that gp160-virosomes are spherical vesicles with a mean diameter identical to that of viral particles. Enzyme-linked immunosorbent assay and immunogold labeling demonstrate efficient association of gp160 with lipid vesicles and proteolysis treatment reveals an asymmetric insertion with about 90% of glycoproteins having their gp120-moiety pointing outside. Glycoproteins are organized as dimers and tetramers and gp160 retains its ability to specifically bind CD4 receptor after reconstitution into virosome.

Peptides of 12, 16 and 24 amino acids length corresponding to the NH2 terminal sequence of SIV gp32 were synthesized. Fluorescence energy transfer studies have shown that those peptides can induce lipid mixing of SUV (Small Unilamellar Vesicles) of various compositions at pH 7.4 and 37 degrees C. LUV (Large Unilamellar Vesicles) were shown to undergo fusion, provided they contained PE in their lipid composition. This work is an attempt to determine how the fusogenic activity depends on the structure of the peptide inserted into a lipidic environment. The peptides secondary structure and orientation in the lipid bilayer were determined using Fourier Transform infrared spectroscopy (FTIR). They adopt mainly a beta-sheet conformation in the absence of lipids. After interaction with DOPC SUV, the beta-sheet is partly converted into alpha-helix oriented obliquely with respect to the membrane interface. We bring here evidence that this oblique orientation is a prerequisite to the fusion process.

A general procedure allowing the acido-basic properties of lipophilic compounds to be defined is described. Drugs are spread at the air-water interface and the surface potential is recorded at different pH values of the aqueous subphase. Comparison of this experimental parameter and the Gouy-Chapman theory prediction allows the calculation of the acid (or basic) dissociation constants and the surface charge density.

We recently presented evidence (Vandenbranden, M., De Coen, J.L., Jeener, R., Kanarek, L. and Ruysschaert, J.M. (1981) Mol. Immunol. 8, 621-631) for the existence of two conformational rabbit serum IgG immunoglobulin isomers. In the present report, their capacity to interact with lipid is investigated in model membranes. (1) One isomer, IgG(H), behaves like several intrinsic membrane proteins: it induces a large surface pressure increase when injected under a lipid monolayer in the close packed state and increases by 20-fold the conductance of a planar bilayer. The other isomer, IgG(S) doesn't interact significantly with the lipids. (2) IgG(H) marked a preference for monolayers made of lipids with a negatively charged polar headgroup and bearing unsaturations in their acyl chains. Penetration is stronger with lipid monolayer in the fluid state than in the rigid state. (3) As previously shown (Vanderbranden, M., De Coen, J.L., Jeener, R., Kanarek, L. and Ruysschaert, J.M. (1918) Mol. Immunol. 18, 621-631), circular dichroism spectra and antigen precipitation tests don't allow to detect any difference in the overall secondary conformation and antigen recognition properties of the two isomers. (4) Papain cleavage of the hinge region suppresses the hydrophobic properties of IgG towards lipid monolayers. (5) The hypothesis of a binding of the hinge region with the lipid bilayer is discussed.

A pre-sequence of 25 amino acids is required for import of yeast cytochrome oxidase subunit IV into mitochondria. Structure and orientation of the 25 amino acids synthesized peptide (p25) in a lipid bilayer were investigated by infrared attenuated total reflection spectroscopy. This method allowed to overcome the difficulties related to the optical turbidity due to the light scattering on membrane fragments which prevents the use of circular dichroism. We demonstrate here that incubation of the peptide with DOPC (dioleoylphosphatidylcholine) and DOPC-CL (dioleoylphosphatidylcholine - cardiolipin) liposomes is accompanied by an increase in alpha-helical content as compared to beta structure. Polarisation measurements indicate that the amphipathic helical segment is inserted parallel to the lipid acyl chains in cardiolipin containing liposomes.

Summary Fluorescence polarization and differential scanning calorimetry measurements made on dipalmitoyl phosphatidyl choline-stearylamine mixed liposomes show striking changes in the biophysical properties of these vesicles. The liposomes formed have transition temperature largely shifted to higher temperature and evolve to larger structure. These results strongly suggest that the alteration of liposomal structures due to the incorporation of lipid soluble compound like drugs requires physico-chemical studies before any screening with liposomes as pharmacological capsules.

We have measured the rate of hydrolysis of liposomes made of DL-α-dipalmitoylphosphatidylcholine (DPPC) and L-α-dimyristoylphosphatidylcholine by a soluble fraction of highly purified lysosomes isolated from rat liver. Phospholipids are hydrolyzed into lysophospho-lipids and fatty acids at a rate which is maximal near the temperature characteristic of the gel to liquid crystalline phase transition of the lipid bilayer. This strong influence of the physical properties of the substrate on the enzyme activity suggests a structural analogy between the lysosomal phospholipases of the A type (EC 3.1.1.32 and EC 3.1.1.4) and the pancreatic phospholipase A2.