Antibodies reactive with liposomal phospholipids are produced during experimental Trypanosoma rhodesiense infections in rabbits

Antibodies against phospholipids appeared spontaneously during the course of experimental Trypanosoma rhodesiense infections in rabbits. These antibodies were observed in rabbits infected either with a lethal strain or with a strain newly discovered to give a spontaneous self-cure. Serum antibodies reacting with liposomes containing dimyristoyl phosphatidylcholine (DMPC), phosphatidylinositol (Pl), phosphatidylinositol phosphate (PlP), or cardiolipin were detected at 3 to 4 wk by complement-mediated release of trapped marker from liposomes. Antibodies were also detected against a trypanosomal lipid fraction (TrF2) that contained Pl as a major constituent. The antibody activities against DMPC, Pl, or TrF2 all reacted (or cross-reacted) with DMPC, and were removed from the serum by adsorbing with liposomes containing DMPC as the only phospholipid. Phosphocholine inhibited the antibodies reactive with liposomes containing either DMPC or DMPC and Pl as phospholipids. Antibodies against PlP, however, reacted only with liposomes containing PlP and were not removed by adsorbing with liposomes lacking PlP. We conclude that anti-phospholipid antibodies appear during the course of trypanosomal infections that either undergo apparent self-cure or are lethal, and at least two anti-phospholipid antibody specificities can be detected.     

Simple preparation and characterization of cationic liposomes associated with a monoclonal antibody against glioma-associated antigen (immunoliposomes)

Abstract

In this study we prepared and characterized monoclonal antibody associated cationic liposomes (immunoliposomes) to be used as a vehicle for human gene therapy of malignant glioma. This association method is especially amenable to mass production. The immunoliposomes consist of N-(a-trimethylammonio-acetyl)-didodecyl-D-glutamate chloride (TMAG), dilauroyl phosphatidylcholine (DLPC), and dioleoyl phosphatidyl- ethanolamine (DOPE) in a molar ratio of 1:2:2 as TMAG:DLPC:DOPE. Their preparation required only the addition of a solution containing plasmid DNA and a monoclonal antibody against glioma-associated antigen to a lipid film of the above three lipids. The association of antibody on the surface of immunoliposomes was confirmed by an immunochemical procedure. Liposome-mediated LacZ gene transfection of human glioma cells resulted in p-galactosidase activity about 2- to 3-fold higher when immunoliposomes were used as compared to control liposomes that were not associated to antibody. Also, the production of human (3-interferon (HuIFN-P) into the medium was 2- to 7-fold higher when HuIFN-P gene was transfected. Based on the present results, the immunoliposomes associating a monoclonal antibody against glioma-associated antigen may become effective carriers for gene transfer to human glioma cells.     

Antioxidant mechanism of Mn(II) in phospholipid peroxidation.

Antioxidant action of Mn2+ on radical-mediated lipid peroxidation without added iron in microsomal lipid liposomes and on iron-supported lipid peroxidation in phospholipid liposomes or in microsomes was investigated. High concentrations of Mn2+ above 50 microM inhibited 2,2'-azobis (2-amidinopropane) (ABAP)-supported lipid peroxidation without added iron at the early stage, while upon prolonged incubation, malondialdehyde production was rather enhanced as compared with the control in the absence of Mn2+. However, in a lipid-soluble radical initiator, 2,2'-azobis (2,4-dimethyl-valeronitrile) (AMVN)-supported lipid peroxidation of methyl linoleate in methanol Mn2+ apparently did not scavenge lipid radicals and lipid peroxyl radicals, contrary to a previous report. At concentrations lower than 5 microM, Mn2+ competitively inhibited Fe(2+)-pyrophosphate-supported lipid peroxidation in liposomes consisting of phosphatidylcholine with arachidonic acid at the beta-position and phosphatidylserine dipalmitoyl, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in the presence of iron complex in microsomes. Iron reduction responsible for lipid peroxidation in microsomes was not influenced by Mn2+.     

Effect of lipid composition upon fusion of liposomes with Sendai virus membranes

How the lipid composition of liposomes determines their ability to fuse with Sendai virus membranes was tested. Liposomes were made of compositions designed to test postulated mechanisms of membrane fusion that require specific lipids. Fusion does not require the presence of lipids that can form micelles such as gangliosides or lipids that can undergo lamellar to hexagonal phase transitions such as phosphatidylethanolamine (PE), nor is a phosphatidylinositol (PI) to phosphatidic acid (PA) conversion required, since fusion occurs with liposomes containing phosphatidylcholine (PC) and any one of many different negatively charged lipids such as gangliosides, phosphatidylserine (PS), phosphatidylglycerol, dicetyl phosphate, PI, or PA. A negatively charged lipid is required since fusion does not occur with neutral liposomes containing PC and a neutral lipid such as globoside, sphingomyelin, or PE. Fusion of Sendai virus membranes with liposomes that contain PC and PS does not require Ca2+, so an anhydrous complex with Ca2+ or a Ca2+-induced lateral phase separation is not required although the possibility remains that viral binding causes a lateral phase separation. Sendai virus membranes can fuse with liposomes containing only PS, so a packing defect between domains of two different lipids is not required. The concentration of PS required for fusion to occur is approximately 10-fold higher than that required for ganglioside GD1a, which has been shown to act as a Sendai virus receptor. When cholesterol is added as a third lipid to liposomes containing PC and GD1a, the amount of fusion decreases if the GD1a concentration is low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of concanavalin A to free inositol and liposomes containing phosphatidylinositol

We previously reported that concanavalin A could bind specifically to liposomes containing phospholipids and lacking glycoconjugates (Biochem. Biophys. Res. Comm. 74, 208, 1977). In the present study we show that the binding of concanavalin A to the liposomes was greatly increased (up to 5 fold) by the presence of phosphatidylinositol in the liposomes. Furthermore, the binding of concanavalin A to phosphatidylinositol-liposomes was specific and could be inhibited by either alpha-methyl mannoside or by myo-inositol. We also found that concanavalin A-induced lymphocyte mitogenesis could be inhibited either by alpha-methyl mannoside or by myo-inositol. Simultaneous addition of both inhibitors to concanavalin A and liposomes showed that inhibition was non-competitive: alpha-methyl mannoside was more inhibitory to liposomes lacking phosphatidylinositol, and myo-inositol was more inhibitory to liposomes containing phosphatidylinositol. This suggests that the binding site for inositol might be different than that for mannose. Equilibrium dialysis and Scatchard plots revealed 4 binding sites each for inositol and mannose at neutral pH. The binding constants of concanavalin A were 0.13 X 10(4) and 0.25 X 10(4) liters/mole respectively for inositol and mannose. We conclude that concanavalin A binds specifically to the inositol portion of phosphatidylinositol.     

Influence of glycolipids on immune reactions of phospholipid antigens in liposomes

Complement-dependent immune damage to liposomes mediated by a murine monoclonal antibody to the liposomal bilayer was completely inhibited by ceramide tetrasaccharide (globoside) at an 8% concentration in the liposomes. Lower concentrations of globoside, or higher concentrations of ceramide tri-, di-, or monohexoside, were not inhibitory. At a globoside concentration of 5.8%, inhibition of the monoclonal antibody activity was reduced and inhibition was related to antibody concentration; but at 2% globoside, suppression of antibody activity was not observed at all. Analysis of space-filling models revealed that at 8% globoside the distance between adjacent tetrasaccharides of globoside approached the dimensions of the antigen-binding end of a 7S immunoglobulin molecule. We therefore propose that globoside, and perhaps other glycolipids, can exert steric hindrance to the binding of extracellular proteins to nonglycolipid constituents of the lipid bilayer. We conclude that microheterogeneity among polar groups of glycolipids may be a novel mechanism for allowing selective access of proteins to phospholipids on the lipid bilayer.     

Influence of glycolipids on immune reactions of phospholipid antigens in liposomes

Complement-dependent immune damage to liposomes mediated by a murine monoclonal antibody to the liposomal bilayer was completely inhibited by ceramide tetrasaccharide (globoside) at an 8% concentration in the liposomes. Lower concentrations of globoside, or higher concentrations of ceramide tri-, di-, or monohexoside, were not inhibitory. At a globoside concentration of 5.8%, inhibition of the monoclonal antibody activity was reduced and inhibition was related to antibody concentration; but at 2% globoside, suppression of antibody activity was not observed at all. Analysis of space-filling models revealed that at 8% globoside the distance between adjacent tetrasaccharides of globoside approached the dimensions of the antigen-binding end of a 7S immunoglobulin molecule. We therefore propose that globoside, and perhaps other glycolipids, can exert steric hindrance to the binding of extracellular proteins to nonglycolipid constituents of the lipid bilayer. We conclude that microheterogeneity among polar groups of glycolipids may be a novel mechanism for allowing selective access of proteins to phospholipids on the lipid bilayer.     

Hydrolysis of phosphatidylcholine liposomes by phospholipases A2. Effects of the local anesthetic dibucaine.

(1) Dibucaine evokes a downward shift in the phase transition temperature of saturated phosphatidylcholines, while it also affects the pretransition. (2) The binding of dibucaine to phosphatidylcholine liposomes increases sharply when the lipid is transformed from the gel phase to the liquid-crystalline phase. (3) The activity of Naja naja phospholipase A2 towards dimyristoyl phosphatidylcholine liposomes is either stimulated or inhibited by dibucaine, depending on whether the substrate is in the gel or the liquid-crystalline state, respectively, whereas the activity of pancreatic phospholipase A2 is inhibited by the anesthetic irrespective of the physical state of the substrate. This observation is further substantiated by the results of studies on liposomes prepared from mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine. (4) The uptake of dibucaine by positively charged liposomes composed of phosphatidylcholine and stearylamine is considerably reduced in comparison with pure phosphatidylcholine liposomes. This decrease is paralleled by a reduction of the inhibitory and stimulatory effects of dibucaine on the hydrolysis of such liposomes by pancreatic and Naja naja phospholipase, respectively. (5) The inhibitory action of dibucaine towards the pancreatic phospholipase is lowered by increasing CaCl2 concentrations. This reduction is accompanied by a decreased uptake of anesthetic by the liposomes.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

Antibodies induced by liposomal protein exhibit dual binding to protein and lipid epitopes

Natural polyreactive antibodies can accommodate chemically unrelated epitopes, such as lipids and proteins, in a single antigen binding site. Because liposomes containing lipid A as an adjuvant can induce antibodies directed against specific lipids, we immunized mice with liposomes containing lipid A together with a protein or peptide antigen to determine whether monoclonal antibodies generated after immunization would be specifically directed both to the liposomal lipid (either cholesterol or galactosylceramide) and also to the accompanying liposomal protein or peptide. Monoclonal antibodies were obtained that bound, by ELISA, to cholesterol and to recombinant gp140 envelope protein from HIV-1, or to galactosylceramide and to an HIV-1 envelope peptide. Surface plasmon resonance studies with the former antibody showed that the liposomal cholesterol and liposomal gp140 each contributed to the overall binding energy of the antibody to liposomes containing cholesterol and protein.     

Interaction between sphingomyelin and a cytolysin from the sea anemone Stoichactis helianthus

The cytolytic toxin from the sea anemone Stoichactis helianthus was inhibited up to 90–95% by suspensions of sphingomyelin but not by phosphatidylcholine or other membrane lipids. When the toxin was incubated with spingomyelin and the mixture fractionated either by isoelectric focusing or Sephadex gel filtration, the residual hemolytic units migrated together with the lipid and not as free toxin. Incubation with phosphatidylcholine, however, did not shift the toxin peak in either type of column.A toxin-ferritin conjugate retaining hemolytic activity was observed by negative staining to bind to liposomers prepared with sphingomyelin but not with liposomes containing phosphatidylcholine. The results provide evidence that the membrane binding site of the toxin is sphingomyelin.     

Liposomal Drug with Carnosine and Lipoic Acid: Preparation,Antioxidant and Antiplatelet Properties

The conditions for producing phosphatidylcholine liposomes containing lipoic acid and carnosine together were determined. The obtained liposomes are 180–250-nm spherical particles with an efficiency of lipoic acid inclusion of 50–70% (for carnosine, 17–33%). Based on the model of the oxidation of phosphatidylcholine by hydrogen peroxide, an antioxidant effect of carnosine, lipoic acid or lipoic acid with carnosine together was demonstrated; it consisted in inhibition of lipid peroxidation process, which was manifested in a decrease in the formation of lipid peroxidation products that react with thiobarbituric acid. It was established that lipoic acid (5 mM) and carnosine (0.1–10 mM) in liposomes exhibit an antioxidant effect. At the same time, it was demonstrated that the content of the appropriate lipid peroxidation products in liposomes with antioxidants (lipoic acid + carnosine) was 15 times lower than in control liposomes (without antioxidants). The effect of the obtained liposomal drugs on the platelet aggregation induced by arachidonic acid was evaluated. It was found that the liposomal drug containing lipoic acid (1.5 mM) and carnosine (2.1 mM) inhibited platelet aggregation by 50–55% relative to the control (platelets and arachidonic acid), while liposomes without antioxidants and water-soluble forms of carnosine and lipoic acid had almost no effect on platelet aggregation caused by arachidonic acid.

     

Specific recognition of malondialdehyde and malondialdehyde acetaldehyde adducts on oxidized LDL and apoptotic cells by complement anaphylatoxin C3a

Oxidatively modified low-density lipoproteins (Ox-LDL) and complement anaphylatoxins C3a and C5a are colocalized in atherosclerotic lesions. Anaphylatoxin C3a also binds and breaks bacterial lipid membranes and phosphatidylcholine liposomes. The role of oxidized lipid adducts in C3a binding to Ox-LDL and apoptotic cells was investigated. Recombinant human C3a bound specifically to low-density lipoprotein and bovine serum albumin modified with malondialdehyde (MDA) and malondialdehyde acetaldehyde (MAA) in chemiluminescence immunoassays. No binding was observed to native proteins, LDL oxidized with copper ions (CuOx-LDL), or phosphocholine. C3a binding to MAA-LDL was inhibited by two monoclonal antibodies specific for MAA-LDL. On agarose gel electrophoresis, C3a comigrated with MDA-LDL and MAA-LDL, but not with native LDL or CuOx-LDL. C3a bound to apoptotic cells in flow cytometry. C3a opsonized MAA-LDL and was taken up by J774A.1 macrophages in immunofluorescence analysis. Complement-activated human serum samples (n=30) showed increased C3a binding to MAA-LDL (P<0.001) and MDA-LDL (P<0.001) compared to nonactivated samples. The amount of C3a bound to MAA-LDL was associated with total complement activity, C3a desArg concentration, and IgG antibody levels to MAA-LDL. Proteins containing MDA adducts or MAA adducts may bind C3a in vivo and contribute to inflammatory processes involving activation of the complement system in atherosclerosis.     

Liposome-mediated delivery of antibody to a Drosophila cell line

Large, unilamellar vesicles composed of equimolar amounts of acidic phosopholipids and phosphatidylethanolamine were able to deliver fluorescent dye [5(6)-carboxyfluorescein] or a monoclonal antibody directed against intermediate-filament proteins to a Drosophila cell line (Kc cells). Millimolar Ca2+ or protamine sulfate in microgram quantities triggered rapid, synchronous delivery of either solute. Delivery required a specific lipid composition: liposomes composed of 1:1 mole ratios of phosphatidylethanolamine:phosphatidylserine were able to deliver their contents, but not if phosphatidylcholine was substituted for phosphatidylethanolamine. Light microscopic observation of Kc cells incubated with free dye or antibody alone showed very little uptake, a result indicating that encapsulation within liposomes is a prerequisite for substantial delivery. Moreover, the stability of adhering vesicles in the absence of calcium or protamine sulfate, the lipid specificity, and the rapid onset of intracellular fluorescence after triggering suggest that vesicle-cell fusion is the predominant mode of solute uptake. Fusion of liposomes with the cell membrane was confirmed by freeze-fracture electron microscopy, which showed liposome vesicles first adhering to cell surfaces, then undergoing fusion when calcium or protamine sulfate was added.     

Membrane damage of liposomes by the mushroom toxin phallolysin

We have investigated the membrane-damaging effect of phallolysin on liposomes varying in phospholipid composition, net charge and physical constitution. Liposomes were prepared from lipids extracted from bovine or human erythrocyte ghosts. The liposomes composed of bovine lipids (the intact cell showing little sensitivity to phallolysin) were found comparably sensitive to those prepared from lipids of human red cells (these cells being of high sensitivity). In addition, artificial mixtures of lipids were used for the preparation of liposomes, consisting of (a) negatively charged phospholipids such as dicetyl phosphate or phosphatidylserine, (b) cholesterol, and (c) either sphingomyelin (as the major component of erythrocytes from ruminants) or phosphatidylcholine (as the major component of erythrocytes from non-ruminants). Again, we found only little difference in the susceptibilities of sphingomyelin- and phosphatidylcholine-containing liposomes. On the other hand, the susceptibility depended on the presence of phospholipids with negative net charges. Omittance of phosphatidylcholine or dicetyl phosphate, or replacement by the positively charged stearylamine, decreased the susceptibility by a factor of more than 20. Finally, we prepared liposomes from dicetyl phosphate, cholesterol and phosphatidylcholine in two physical states: large unilamellar and smaller multilamellar liposomes. The unilamellar liposomes were about 10-times more sensitive to phallolysin. We conclude: (1) Phallolysin damages phospholipid-membranes in the absence of receptor proteins, but high concentrations of the toxin are required. (2) Membrane damage takes place with liposomes containing phosphatidylcholine as well as those containing sphingomyelin. (3) Phallolysin damages only liposomes containing phospholipids with a negative net charge.     

Liposome-mediated transfer of integral membrane glycoproteins into the plasma membrane of cultured cells

Cultured mouse 3T3 cells treated with phosphatidylserine or phosphatidylserine/phosphatidylcholine (3: 7 mole ratio) liposomes containing ortho- and paramyxovirus envelope glycoproteins become susceptible to killing by virus-specific cytotoxic T lymphocytes indicating that the liposome-derived glycoproteins have been inserted into the cellular plasma membrane. Cells incubated with liposomes of similar lipid composition containing viral antigens plus a dinitrophenylated lipid hapten were killed by both virus- and hapten-specific T lymphocytes indicating that both protein and lipid components are inserted into the plasma membrane. We consider that assimilation of liposome-derived antigens into the plasma membrane results from fusion of liposomes with the plasma membrane. Cells incubated with phosphatidylcholine liposomes containing lipid haptens and viral glycoproteins were not killed by cytotoxic lymphocytes indicating that liposomes of this composition do not fuse with the plasma membrane. Liposome-derived paramyxovirus glycoproteins inserted into the plasma membrane retain their functional activity as shown by their ability to induce cell fusion. These experiments demonstrate the feasibility of using liposomes as carriers for introducing integral membrane (glyco)proteins into the plasma membrane of cultured cells and establish a new approach for studying the role of individual (glyco)proteins in the expression of specific cell surface properties.     

Monoclonal antibodies to phosphatidylinositol phosphate neutralize human immunodeficiency virus type 1: role of phosphate-binding subsites

Both a murine monoclonal antibody to phosphatidylinositol phosphate (PIP) and a human monoclonal antibody (4E10) that is known to have broadly neutralizing capabilities against primary isolates of human immunodeficiency virus type 1 (HIV-1) bound to PIP, as determined by enzyme-linked immunosorbent assay. Each of the antibodies had antigen subsite binding specificities in aqueous medium for small phosphate-containing molecules and for inositol. The anti-PIP monoclonal antibody inhibited infection by two HIV-1 primary isolates in neutralization assays employing primary human peripheral blood mononuclear cells. The data suggest that PIP or related lipids having free phosphates could serve as targets for the neutralization of HIV-1.     

The physical state of membrane lipids modulates the activation of the first component of complement.

Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

Incorporation of the purified Epstein Barr virus/C3d receptor (CR2) into liposomes and demonstration of its dual ligand binding functions

The 145-kDa molecule that has been identified as the C3d receptor CR2 was isolated from lysates of Raji cells by affinity chromatography by using the monoclonal antibody (MoAb)HB-5. The purified protein was incorporated into 14C-phosphatidylcholine liposomes by deoxycholate dialysis followed by flotation on discontinuous sucrose gradients. Incorporation of the receptor was verified by testing the gradient fractions for CR2 by an enzyme-linked immunosorbent assay. Liposomes were shown to be unilamellar vesicles ranging in diameter from 25 to 100 nm by electron microscopy. The external orientation of CR2 in the membranes was demonstrated by immunoelectron microscopy. The functional activities of liposomes containing CR2 and liposomes without protein were compared. CR2 liposomes bound to EC3d, but not to E, and this binding was inhibited by the anti-CR2 MoAb OKB7 and by a MoAb specific for C3d. Control liposomes failed to bind to either E or EC3d. The ability of CR2 to function as a receptor for Epstein Barr virus (EBV) was tested in two ways. First, CR2 liposomes bound to B95-8, a cell line expressing EBV membrane antigens, but not to B95-8 cells treated with the viral DNA polymerase inhibitor phosphonoformic acid. Second, liposomes containing CR2 were shown by ultracentrifugal analyses to bind directly to purified EBV, and this binding was also inhibited by OKB7. Control liposomes did not bind to B95-8 cells or to EBV. These findings show that CR2 purified from detergent extracts of Raji cells can be reconstituted into lipid membranes with maintenance of its dual functions as a receptor for C3d and EBV.