Miguel Ibáñez

Phospholipid Membranes Form Specific Nonbilayer Molecular Arrangements That Are Antigenic

Hexagonal phase (HII)-preferring lipids such as phosphatidate, cardiolipin, and phosphatidylserine form nonbilayer molecular arrangements in lipid bilayers. While their presence in biological membranes has not been established, in vitro studies suggest that alterations in membrane properties modify their function. In this study, antiphospholipid monoclonal antibodies were developed against nonbilayer structures. One of the monoclonal antibodies identifies nonplanar surfaces in liposomes and in membranes of cultured cells. These results are the first evidence that natural membranes maintain a fragile balance between bilayer and nonbilayer lipid arrangements. Therefore, these antibodies can be used to evaluate the role of HII-preferring lipids in the modulation of membrane activities. Our studies demonstrated that nonplanar surfaces are highly immunogenic. Although these structures are normally transient, their formation can be stabilized by temperature variations, drugs, antibiotics, apolar peptides, and divalent cations. Our studies demonstrated that abnormal exposure of nonbilayer arrangements may induce autoimmune responses as found in the antiphospholipid syndrome.

Possible prebiotic significance of polyamines in the condensation, protection, encapsulation, and biological properties of DNA.

Some properties of DNA condensed with spermidine have been compared with the properties of DNA condensed with Co3+(NH3)6 to determine whether condensation of DNA with these trivalent cations protects DNA against the action of DNase I and increases transcription and encapsulation of DNA into liposomes. It was shown that DNA condensed with Co3+(NH3)6 was resistant to the action of the endonuclease DNase I such as DNA condensed with spermidine was. However, DNA condensed with Co3+(NH3)6 was significantly less active in transcription with theE. coli RNA polymerase than DNA-spermidine condensed forms. In addition, it was demonstrated that both compacted forms of DNA were more efficiently encapsulated into neutral liposomes; however, negatively, charged liposomes were scarcely formed in the presence of DNA condensed with Co3+(NH3)6. These experiments and the well documented properties of polyamines increasing the resistance to radiations and hydrolysis of nucleic acids, as well as their biological activities, such as replication, transcription, and translation, together with the low concentration of Co3+ in the environment, lead us to propose spermidine as a plausible prebiotic DNA condensing agent rather than Co3+ and the basic proteins proposed by other authors. Then, we consider the possible role and relevance of the polyamine-nucleic acids complexes in the evolution of life.

Transbilayer diffusion of divalent cations into liposomes mediated by lipidic particles of phosphatidate

Liposomes formed from egg-yolk phosphatidylcholine:egg-yolk phosphatidate (molar ratio 2:1) containing pBR322 DNA and DNase I were induced to form, with divalent cations, bilayer/nonbilayer phase transitions of phosphatidate which allowed cation diffusion into liposomes; then cation diffusion was measured by the activation of the hydrolysis of DNase I on DNA. The formation of phosphatidate transitions on liposomes was demonstrated by freeze-fracture and 31P NMR, and a direct correlation between the formation of phosphatidate transitions and the transbilayer diffusion of cations was found: only Ca2+ and Mn2+, which induce phase transitions, were able to penetrate liposomes and triggered the DNase I activity; in addition, Ca2+ at higher concentrations (10 mM) caused fusion of liposomes, whereas Mn2+ did not, suggesting that transitions induced by Mn2+ participated only in the diffusion of this ion; furthermore, Mg2+ neither formed phase transitions nor triggered the enzymatic activity. The liposomes studied represent more dynamic structures that can form phosphatidate structures involved in both (1) the interchange of divalent cations with the surroundings, thereby modulating encapsulated enzymes, and (2) the fusion of lipid vesicles probably implicated in the enrichment of liposomal content in the early Precambian Earth.

Lepromatous leprosy patients produce antibodies that recognise non-bilayer lipid arrangements containing mycolic acids

Non-bilayer phospholipid arrangements are three-dimensional structures that form when anionic phospholipids with an intermediate structure of the tubular hexagonal phase II are present in a bilayer of lipids. Antibodies that recognise these arrangements have been described in patients with antiphospholipid syndrome and/or systemic lupus erythematosus and in those with preeclampsia; these antibodies have also been documented in an experimental murine model of lupus, in which they are associated with immunopathology. Here, we demonstrate the presence of antibodies against non-bilayer phospholipid arrangements containing mycolic acids in the sera of lepromatous leprosy (LL) patients, but not those of healthy volunteers. The presence of antibodies that recognise these non-bilayer lipid arrangements may contribute to the hypergammaglobulinaemia observed in LL patients. We also found IgM and IgG anti-cardiolipin antibodies in 77% of the patients. This positive correlation between the anti-mycolic-non-bilayer arrangements and anti-cardiolipin antibodies suggests that both types of antibodies are produced by a common mechanism, as was demonstrated in the experimental murine model of lupus, in which there was a correlation between the anti-non-bilayer phospholipid arrangements and anti-cardiolipin antibodies. Antibodies to non-bilayer lipid arrangements may represent a previously unrecognised pathogenic mechanism in LL and the detection of these antibodies may be a tool for the early diagnosis of LL patients.

Diffusion of Mn2+ ions into liposomes mediated by phosphatidate and monitored by the activation of an encapsulated enzymatic system

SummaryTransbilayer diffusion of Mn2+ ions occurred in liposomes formed from dipalmitoyl-phosphatidylcholine or egg-yolk phosphatidylcholine and egg-yolk phosphatidate (molar ratio 2∶1) containing DNA and DNase I within their aqueous compartments. Cation diffusion was demonstrated by the hydrolytic activity of DNase I, activated by the Mn2+ ions that diffused into the vesicles, and this was confirmed by light scattering. Phosphatidate, a cone-shaped lipid which has been synthesized under simulated prebiotic conditions, was necessary for cation diffusion across the liposome membranes. Such liposomes represent a simple precellular system that interchanges cations with the surroundings and provides a microenvironment for enzymatic reactions, as evidenced by the hydrolysis of DNA by DNase I inside these closed lipid compartments.

Antibodies to non-bilayer phospholipid arrangements induce a murine autoimmune disease resembling human lupus

Antibodies recognizing non‐bilayer phospholipid arrangements (NPA) in membrane models and in cell membranes in vivo, triggered an autoimmune‐like disease in mice. This exhibited features similar to human lupus and was induced by injecting mice either with the H308 monoclonal antibody specific to NPA, with sera from mice which already had developed the autoimmune disease, or withliposomes treated with the NPA inductors chlorpromazine or procainamide; or with these NPA inductors alone. All these procedures revealed the involvement of antibodies to non‐bilayer phospholipids in inducing this autoimmune‐like disease. Unraveling the mechanisms of these antibodies might contribute to a better understanding of the molecular and immunological basis of autoimmune diseases like lupus and, hopefully, towards the development of better therapeutic strategies.

Liposomes with polyribonucleotides as model of precellular systems

A study of the encapsulation of poly(U) and poly(C) within liposomes made from dipalmitoylphosphatidyl choline (DPPC), from egg yold phosphatidyl choline (PC), and from PC with cholesterol (CHOL) was made. The liposomes were prepared under anoxic conditions following the reverse-phase evaporation method. Determinations showed that 36 to 70% of the available lipids form liposomes and 2 to 5% of the polyribonucleotides can be entrapped by liposomes. The encapsulation of polyribonucleotides has also been measured in the presence of urea, cyanamide and Zn++, condensing agents in prebiotic polymerization reactions. DPPC and PC:CHOL liposomes were formed in the presence of 1.0 M urea, although no PC liposomes were formed. The three types of liposomes were readily formed at 0.01 M urea, but in no case an enhancement of encapsulation efficiency of poly(U) was observed due to the presence of urea. Similar results were obtained with cyanamide. An enhanced encapsulation of poly(U) by the three types of liposomes was observed when Zn++ was in the range of 0.001 to 0.01 M. Poly(U) encapsulation was 15 to 25 times higher when liposomes were prepared from DPPC at 0.01 M Zn++. Similar results were obtained with poly(C). The advantages of DPPC-polyribonucleotide liposomes as precellular systems are discussed.

Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

Molecular organization of the non-bilayer phospholipid arrangements that induce an autoimmune disease resembling human lupus in mice

Abstract Non-bilayer phospholipid arrangements are three-dimensional structures that can form when anionic phospholipids with an intermediate form of the tubular hexagonal phase II (HII), such as phosphatidic acid, phosphatidylserine or cardiolipin, are present in a bilayer of lipids. The drugs chlorpromazine and procainamide, which trigger a lupus-like disease in humans, can induce the formation of non-bilayer phospholipid arrangements, and we have previously shown that liposomes with non-bilayer arrangements induced by these drugs cause an autoimmune disease resembling human lupus in mice. Here we show that liposomes with non-bilayer phospholipid arrangements induced by Mn2+ cause a similar disease in mice. We extensively characterize the physical properties and immunological reactivity of liposomes made of the zwitterionic lipid phosphatidylcholine and a HII-preferring lipid, in the absence or presence of Mn2+, chlorpromazine or procainamide. We use an hapten inhibition assay to define the epitope recognized by sera of mice with the disease, and by a monoclonal antibody that binds specifically to non-bilayer phospholipid arrangements, and we report that phosphorylcholine and glycerolphosphorylcholine, which form part of the polar region of phosphatidylcholine, are the only haptens that block the binding of the tested antibodies to non-bilayer arrangements. We propose a model in which the negatively charged HII-preferring lipids form an inverted micelle by electrostatic interactions with the positive charge of Mn2+, chlorpromazine or procainamide; the inverted micelle is inserted into the bilayer of phosphatidylcholine, whose polar regions are exposed and become targets for antibody production. This model may be relevant in the pathogenesis of human lupus.

Characterization of cationic liposomes having IL-2 expressed on their external surface, and their affinity to cervical cancer cells expressing the IL-2 receptor

Anionic, cationic, and neutral liposomes were constructed to contain IL-2 in order to evaluate their affinity to a cervical cancer cell line (INBL) and to determine whether they can present IL-2 on their external surface. When these liposomes were co-cultured with INBL, the anionic liposomes were the only ones found to be cytotoxic. When neutral and cationic liposomes were constructed in the presence of IL-2, IL-2 was detected only on the surface of cationic liposomes by using a fluorescent anti-IL-2 antibody. By co-culturing INBL with IL-2-containing cationic liposomes, and by using fluorescent anti-IL-2 antibody, we found a strong IL-2 presence on the cell membranes thus suggesting a high affinity of the liposomes to the INBL cells.