2-Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate.

2-Methoxyestradiol, once considered an inacitve end-metabolite of estradiol, has recently emerged as a very promising agent for cancer treatment. It is orally active in a wide range of tumor models, and inhibits tumor growth at doses showing no clinical signs of toxicity. 2ME2 targets both the tumor cell and endothelial cell compartments by inducing apoptosis in rapidly proliferating cells and inhibiting blood vessel formation at several stages in the angiogenic cascade. Moreover, the ability of 2ME2 to inhibit metastatic spread in several models adds to its therapeutic value for cancer treatment at various stages of the disease. Though the mechanism of action is still undefined, several potential molecular targets and pathways of activation have been suggested.

Interaction of anti-cholesterol antibodies with human lipoproteins.

Inoculation of mice with cholesterol-rich liposomes containing the adjuvant monophosphoryl lipid A results in the production of antiserum containing IgM Ab to cholesterol. The specificity of the Ab was to cholesterol and structurally similar sterols containing a 3 beta-hydroxyl group. Anti-cholesterol binding activity was significantly diminished if the 3 beta-hydroxyl was altered by either epimerization, substitution, oxidation, or esterification. A similar specificity for 3 beta-hydroxy-sterols was observed for an anti-cholesterol IgM mAb. Both hyperimmune serum and the mAb reacted with intact human very-low-/intermediate-density lipoprotein (VLDL/IDL) and low-density lipoproteins (LDL), but not high-density lipoproteins (HDL), in an ELISA, but could react with total lipid extracts containing cholesterol that were prepared from all three lipoprotein classes. Functionally, immune serum or the mAb aggregated and induced a fusion-like reaction with VLDL/IDL and LDL at low temperatures: these aggregates result in spherical structures visible with light microscopy. Similarly, binding of anti-cholesterol A to small cholesterol-rich liposomes resulted in the appearance of vesicular structures with approximately 20- to 200-fold increased diameters. These data demonstrate that the anti-cholesterol Ab recognize unesterified cholesterol in VLDL/IDL and LDL; high-density lipoprotein cholesterol in the intact lipoprotein, however, appears to be protected from reaction with these Ab.

Role of atheroma liposomes and malondialdehyde-modified low-density lipoproteins in complement activation.

We investigated the ability of atheroma-associated liposomes and malondialdehyde (MDA)-modified low-density lipoproteins (MDA-LDL) to activate complement. Complement activation markers C3a, Bb, C4d and SC5b-9 were measured in both normal and complement-deficient sera. We found that MDA-LDL was able to generate C3a and SC5b-9, predominantly by the alternative pathway. High-density lipoproteins modified with MDA were also capable of C3a generation although to a lesser degree. The presence of atheroma-associated liposomes did not result in detectable levels of complement activation markers. We conclude that MDA-modified lipoproteins may represent a possible source for complement activation within atherosclerotic lesions.

Immunization with cholesterol-rich liposomes induces anti-cholesterol antibodies and reduces diet-induced hypercholesterolemia and plaque formation.

Immunization of rabbits with a protein-free formulation consisting of liposomes containing 71% cholesterol and lipid A as an adjuvant induced anticholesterol antibodies that caused complement-dependent lysis of liposomes lacking lipid A. The antibodies, immunoglobulin G (IgG) and immunoglobulin M (IgM), also recognized nonoxidized crystalline cholesterol as an antigen by enzyme-linked immunosorbent assay (ELISA). The effects of immunization against cholesterol on elevations in serum cholesterol and development of atherosclerosis were examined in rabbits fed a diet containing 0.5% to 1.0% cholesterol. Although the mean serum cholesterol level, mainly in the form of very-low-density lipoprotein cholesterol, rose as much as 60-fold in the nonimmunized rabbits, the elevation was significantly less--as much as 35% lower--in the immunized rabbits. Elevation of serum cholesterol was accompanied by an apparent drop in the level of antibodies on initiating the diet, followed by a rebound on stopping the diet, thus suggesting that the antibodies were adsorbed to cholesterol that was present in circulating lipoproteins. When lipoprotein fractions--composed of either very-low-density and intermediate-density lipoproteins derived from cholesterol-fed nonimmunized rabbits or human low-density lipoproteins--were tested as capture antigens by solid-phase ELISA, reactivity was observed with IgG and IgM antibodies present in the serum of immunized rabbits. Immunization also resulted in a marked decrease in the risk of developing atherosclerosis. Analysis of aortic atherosclerosis by quantitative histologic examination and fatty streaks by automated morphometric probability-of-occurrence mapping showed diminished atherosclerosis in most areas of the aorta in vaccine recipients. It is proposed that immunization with liposomes containing 71% cholesterol and lipid A can reduce diet-induced hypercholesterolemia and atherosclerosis.

Analysis of anticholesterol antibodies using hydrophobic membranes.

An analytical immunoblotting procedure and a serological enzyme-linked immunosorbent assay (ELISA) for the characterization of antibodies to cholesterol are described. Hydrophobic membranes consisting of polyvinylidene fluoride (PVDF) are used to immobilize cholesterol for immunodetection by anti-sterol antibodies. To determine whether antibodies to cholesterol were induced after immunization with liposomal cholesterol, we separated total lipid extracts of very-low density lipoproteins by thin layer chromatography (TLC) on silica gel plates and transferred the separated lipid classes to PVDF membranes using isopropanol to facilitate passive diffusion. Lipid transfer was confirmed by exposure of membranes to iodine vapors or by staining of cholesterol with filipin complex. Serum from immunized mice reacted with cholesterol, whereas pre-immune serum or serum from mice injected with control liposomes did not bind. To determine the amount of anti-cholesterol activity in serum, we coated microtiter plates consisting of PVDF membrane wells with cholesterol. The PVDF membrane-based ELISA was found to be more reproducible and four-fold more sensitive than the conventional ELISA on polystyrene plates. These techniques may be useful in the analysis of anti-sterol antibodies and antibodies to other hydrophobic antigens.

ATP specifically bound as a hapten to a monoclonal anti-phospholipid antibody retains phosphate donor activity.

We have previously reported that each of four monoclonal antibodies to a phospholipid, phosphatidylinositol phosphate (PIP), has a phosphate binding subsite in the antigen binding site that can bind ATP (Molec. Immunol. 21, 863-868, 1984). We have now observed that antibody-bound ATP has the ability to donate a phosphate group in the phosphorylation reaction of glucose to glucose-6-phosphate catalyzed by hexokinase. The phosphorylation reaction proceeds equally efficiently when ATP is provided as free (nonbound) ATP or as antibody-bound ATP. We conclude that an anti-phospholipid antibody can serve as a carrier of a functionally active nucleotide.

Antibodies to cholesterol, cholesterol conjugates and liposomes: implications for atherosclerosis and autoimmunity.

Polyclonal and monoclonal antibodies to cholesterol are readily induced by injecting cholesterol-loaded liposomes containing lipid A as an adjuvant. Analysis of the literature reveals that conjugates of cholesterol, and conjugates of analogues of cholesterol, with heterologous proteins or lipids have been used as antigens in various studies since 1925, and this has led to successful development of immunoassays for steroid hormones. It is concluded that cholesterol is a highly immunogenic molecule. The ability of monoclonal antibodies to cholesterol to react with liposomes containing cholesterol to cause complement-dependent immune damage to the liposomes is strongly influenced by the lipid composition of the liposomes, the amount of cholesterol in the liposomes, and the reaction temperature. The antibodies also react with crystalline cholesterol in a solid-phase ELISA and, depending on the particular monoclonal antibody, immune reactivity may or may not be observed with cholesterol esters, cholesterol analogues, or steroid hormones. Analysis by ELISA has revealed that virtually all normal human sera contain varying levels of naturally occurring IgG and IgM autoantibodies to cholesterol. Naturally occurring autoantibodies to cholesterol are also observed in pigs, but not in guinea pigs. Possible implications of these investigations for theories of immune mechanisms that may have beneficial or detrimental roles in processes of aging, atherosclerosis, and vascular diseases are discussed.

Antibodies to liposomal phosphatidylcholine and phosphatidylsulfocholine.

Antibodies against dimyristoyl phosphatidylsulfocholine or dimyristoyl phosphatidylcholine were raised in rabbits after injection of liposomes containing phosphatidylsulfocholine or phosphatidylcholine, cholesterol, and lipid A. The antibody activities were assayed by complement-dependent immune damage to liposomes and by a solid-phase, enzyme-linked immunosorbent assay using purified dimyristoyl phosphatidylcholine or dimyristoyl phosphatidylsulfocholine as antigen. Each antiserum raised against phosphatidylsulfocholine reacted with liposomes containing phosphatidylcholine, and each antiserum raised against phosphatidylcholine reacted with liposomes containing phosphatidylsulfocholine. However, adsorption of dimyristoyl phosphatidylsulfocholine antiserum with liposomes containing dimyristoyl phosphatidylcholine removed all activity against dimyristoyl phosphatidylcholine, but did not eliminate antibody activity against dimyristoyl phosphatidylsulfocholine. These results indicate that the antiserum against phosphatidylsulfocholine contained mixed populations of antibodies. Polyclonal antisera that have been appropriately adsorbed can therefore be obtained with a high degree of specificity for phosphatidylsulfocholine and such antisera can distinguish between phosphatidylsulfocholine and phosphatidylcholine.

Immunogenicity of liposomal malaria sporozoite antigen in monkeys: adjuvant effects of aluminium hydroxide and non-pyrogenic liposomal lipid A.

The immunogenicity of a recombinant protein (R32tet32) containing sequences from the tetrapeptide repeat region of the circumsporozoite protein of Plasmodium falciparum was enhanced by encapsulation in liposomes containing lipid A and adsorption of the liposomes with alum. The toxicities and efficacies of preparations containing different types and doses of lipid A were assessed by studying pyrogenicity in rabbits and adjuvanticity in monkeys. In each case liposomal lipid A was 25-fold to 200-fold less pyrogenic than free lipid A. Monophosphoryl lipid A, whether free or in liposomes, was the least pyrogenic of the three lipid A preparations tested. High antibody levels were obtained after immunization of rhesus monkeys with a formulation consisting of alum-adsorbed liposomes in which the liposomes contained R32tet32 and a strongly pyrogenic dose of native lipid A. Excellent antibody levels were also observed in monkeys immunized with a combination of R32tet32 encapsulated in alum-adsorbed liposomes containing non-pyrogenic doses of monophosphoryl lipid A and alum. The adjuvant effect was related to the dose of the lipid A in the liposomes, and the adjuvant effect was still strongly expressed despite suppression of the pyrogenic effect of lipid A. Antibody levels were considerably lower in monkeys immunized with liposomes lacking lipid A. It was concluded that a non-pyrogenic formulation of alum-adsorbed liposomes, in which the liposomes contained both lipid A and an encapsulated synthetic sporozoite antigen, shows considerable promise for inducing high titres of antibodies to sporozoites.

Anaphylactoid reactions mediated by autoantibodies to cholesterol in miniature pigs.

Antoantibodies to cholesterol were detected and purified from normal (nonimmunized) pig serum. The antibodies were assayed by ELISA with crystalline cholesterol as an Ag and by C-dependent damage to cholesterol-laden liposomes. Intravenous injection of liposomes containing cholesterol into anesthetized animals caused decreased hemolytic complement titers, and induced a reaction consisting of transient neutropenia, thrombocytopenia, respiratory distress, cyanosis, pulmonary and systemic hypertension, and decreased cardiac output. Plasma levels of thromboxane B2 and 6-keto-prostaglandin F1 alpha increased 1300 and 200%, respectively, and leukocyte and platelet counts decreased by 36 and 38%, respectively. Injection of cholesterol-free liposomes did not induce the reaction. These results show that naturally occurring autoantibodies to cholesterol can initiate C activation and can be associated with anaphylactoid reaction to exogenously administered cholesterol in pigs.

Inhibition of interferon gamma-induced macrophage microbicidal activity against Leishmania major by liposomes: inhibition is dependent upon composition of phospholipid headgroups and fatty acids.

Multilamellar liposomes of phosphatidylcholine and phosphatidylserine at a 7:3 molar ratio significantly inhibited activation of murine resident peritoneal macrophages by recombinant murine interferon-gamma for cytotoxicity against amastigotes of the protozoan parasite Leishmania major; other macrophage effector functions, such as particle phagocytosis or tumoricidal activity, were unaffected. This inhibition was not due to direct toxic effects of liposomes against parasite or macrophage, was fully reversible, and was directed at one or more early events in macrophage-LK interactions which ultimately induce microbicidal activity. Liposomes containing some natural phospholipids (phosphatidylserine, phosphatidylethanolamine, phosphatidic acid or diphosphatidyl glycerol), but not phosphatidylcholine, phosphatidylglycerol, or several synthetic saturated phospholipids, prevented the induction of macrophage microbicidal activity. Inhibition by liposomes of various composition was not related to the efficiency with which these vesicles were ingested by macrophages. Inhibitory activity was directly influenced by changes in the phospholipid head group, as well as by the number of unsaturated bonds in phospholipid fatty acids: for a given phospholipid in liposomes, inhibition was directly related to the number of unsaturated bonds among the fatty acids. These data support a role for phospholipids in postbinding regulation of macrophage activation and add to our understanding of how liposome delivery systems can be designed to avoid potential microbicidal suppressive effects.

Cross-reactions of nucleic acids with monoclonal antibodies to phosphatidylinositol phosphate and cholesterol.

Four monoclonal IgM antibodies to phosphatidylinositol phosphate (PIP), four antibodies to cholesterol and one antibody to liposomes containing phosphatidylcholine, cholesterol and dicetyl phosphate were tested for reactivity with denatured DNA. Three of four antibodies to PIP cross-reacted strongly with denatured DNA. The other antibodies did react with denatured DNA but only very weakly. The binding to DNA was competed by synthetic polynucleotides. In competitive assays, one of the anti-PIP antibodies was particularly reactive with poly(dT) and another with poly(I) and poly(dG). Binding of an anti-cholesterol antibody to ssDNA was also inhibited by poly(I) and poly(dG). Two of the anti-PIP antibodies were also reactive with mononucleotides, and all four bound inositol hexaphosphate. High concns of nucleosides did not compete for binding, indicating that phosphate is involved in the binding site. Phospholipids, particularly those containing inositol phosphate, also competed for binding to DNA, but to varying extents, indicating a variable overlap in the antibody binding site for DNA and phospholipid determinants. These antibodies, induced by immunization with liposomes, showed cross-reactivity characteristics often found with certain types of autoantibodies, but they did not bear the H130 idiotype, which was identified on IgM anti-DNA autoantibodies from MRL-lpr/lpr mice.

Antibodies to cholesterol.

Cholesterol-dependent complement activation has been proposed as a factor that might influence the pathogenesis of atherosclerosis. Although antibodies to cholesterol conjugates have been reported, cholesterol is widely regarded as a poorly immunogenic substance. Monoclonal IgM complement-fixing antibodies to cholesterol were obtained in the present study after immunizing mice with liposomes containing high amounts of cholesterol (71 mol % relative to phosphatidylcholine) and lipid A as an adjuvant. Clones were selected for the ability of secreted antibodies to react with liposomes containing 71% cholesterol but not with liposomes containing 43% cholesterol. The antibodies also reacted with crystalline cholesterol in a solid-phase enzyme-linked immunosorbent assay. Binding of monoclonal antibodies to the surface of crystalline cholesterol was demonstrated by electron microscopy by utilizing a second antibody (anti-IgM) labeled with colloidal gold. The immunization period required to induce monoclonal antibodies was very short (3 days) and a high fraction of the hybrid cells (at least 70%) were secreting detectable antibodies to cholesterol. The results demonstrate that cholesterol can be a highly immunogenic molecule and that complement-fixing antibodies to cholesterol can be readily obtained.

Antibodies to phospholipids and liposomes: binding of antibodies to cells.

Binding of two monoclonal anti-liposome antibodies to the surface of cultured murine peritoneal macrophages was investigated by indirect immunofluorescence and enzyme-linked immunosorbent assay. Neither antibody bound to cultures of freshly explanted, nonadherent macrophages, but immunoreactivity was observed following cell adherence to tissue culture plastic. Fluorescent microscopic evaluation revealed heterogeneity in staining patterns of the antibodies on adherent cells. Binding both to viable and fixed adherent macrophages was observed even after a 10,000-fold dilution of antibody. Treatment of adherent macrophage cultures with trypsin increased antibody binding. Further treatment of trypsinized-macrophages with alkaline phosphatase or neuraminidase did not affect antibody binding, but phospholipase D and, to a greater extent, phospholipase C resulted in a marked decrease in cellular binding. The data indicate that antibodies produced against liposomes appear to bind to surface phospholipids of macrophages, but binding can be influenced by the physiological state of the macrophage and overlying cell surface proteins.

Inhibition of lymphokine-induced macrophage microbicidal activity against Leishmania major by liposomes: characterization of the physicochemical requirements for liposome inhibition.

Resident peritoneal macrophages from untreated mice develop potent microbicidal activity against amastigotes of Leishmania major after in vitro treatment with lymphokine (LK) from mitogen-stimulated spleen cells. LK-induced macrophage microbicidal activity was completely and selectively abrogated by treatment with phosphatidylcholine-phosphatidylserine (PC/PS) liposomes. Other macrophage effector functions (phagocytosis, tumoricidal activity) were unaffected, as was cytotoxicity by macrophages activated in vivo or by LK in vitro before liposome treatment. Activation factors in LK were not adsorbed or destroyed by liposomes. Liposome-induced inhibition was unaffected by indomethacin and was fully reversible: macrophages washed free of liposomes developed strong microbicidal activity with subsequent LK treatment. Changes in liposomal lipid composition markedly altered suppressive effects, but inhibition was not dependent on liposome size, cholesterol content, charge, or number of lamellae. Liposomes composed of PC alone or in combination with any of five different phospholipids were not suppressive. In contrast, inhibition was directly dependent on PS concentration within PC/PS liposomes. Phosphoserine was not inhibitory nor was dimyristoyl PS (synthetic saturated PS). However, the lysophospholipid metabolite of PS, lysoPS, was strongly suppressive. These studies suggest that the reversible and selective inhibition of LK-induced macrophage microbicidal activity by PC/PS liposomes is mediated by PS and its lysoPS metabolite.

Enhanced efficacy of liposome-encapsulated ribavirin against Rift Valley fever virus infection in mice.

Administration of liposome-encapsulated ribavirin to mice led to ribavirin concentrations in the liver, the primary site of Rift Valley fever virus proliferation, that were fivefold greater than those attained with the same doses of free ribavirin. Liposomal ribavirin given at a dose of either 25 or 50 mg of drug per kg of body weight protected mice against a rapidly lethal high-titer challenge with Rift Valley fever virus, whereas similar doses of free drug or empty liposomes had no detectable benefit. Hence, tissue targeting of ribavirin with liposomes substantially increased the therapeutic index by increasing the efficacy of the treatment. By using liposomes as drug carriers, a nontoxic, low-dose regimen of ribavirin had a therapeutic effect that was comparable to that achieved with higher but potentially more toxic doses of free ribavirin.

Macrophage activation for microbicidal activity against Leishmania major: inhibition of lymphokine activation by phosphatidylcholine-phosphatidylserine liposomes.

Resident peritoneal macrophages from untreated mice develop microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica (current nomenclature = Leishmania major) after in vitro exposure to LK from antigen-stimulated leukocyte culture fluids. This LK-induced macrophage microbicidal activity was completely abrogated by addition of 7:3 phosphatidylcholine: phosphatidylserine liposomes. Liposome inhibition was not due to direct toxic effects against the parasite or macrophage effector cell; factors in LK that induce macrophage microbicidal activity were not adsorbed or destroyed by liposome treatment. Other phagocytic particles, such as latex beads, had no effect on microbicidal activity. Moreover, liposome inhibition of activated macrophage effector function was relatively selective: LK-induced macrophage tumoricidal activity was not affected by liposome treatment. Liposome inhibition was dependent upon liposome dose (5 nmoles/culture) and time of addition of leishmania-infected, LK-treated macrophage cultures. Addition of liposomes through the initial 8 hr of culture completely inhibited LK-induced macrophage microbicidal activity; liposomes added after 16 hr had no effect. Similarly, microbicidal activity by macrophages activated in vivo by BCG or Corynebacterium parvum was not affected by liposome treatment. Liposome treatment also did not affect the increased resistance to infection induced in macrophages by LK. These data suggest that liposomes interfere with one or more early events in the induction of activated macrophages (macrophage-LK interaction) and not with the cytotoxic mechanism itself (parasite-macrophage interaction). These studies add to the growing body of data that implicate cell lipid in regulatory events controlling macrophage effector function.

Differential inhibition of macrophage microbicidal activity by liposomes.

In vitro culture of murine resident peritoneal macrophages with lymphokine (LK)-rich leukocyte culture fluids induces enhanced microbicidal activity against amastigotes of the protozoan parasite Leishmania tropica. Macrophages infected with Leishmania and treated with LKs after infection acquire the capacity to kill the intracellular parasite within 72 h. When compared with control macrophage cultures treated with medium lacking LKs, 80 to 90% fewer macrophages treated with LKs contained amastigotes. In experiments designed to test liposome delivery of LKs to infected macrophages, addition of multilamellar liposomes composed of phosphatidylcholine and phosphatidylserine (molar ratio, 7:3) completely abrogated LK-induced microbicidal activity. Liposomes containing only phosphatidylcholine were not inhibitory. Inhibition of LK activity by the liposomes occurred regardless of whether the liposomes contained LKs. Liposomal inhibition of activated macrophage effector activity was limited to intracellular killing; LK-induced macrophage extracellular cytolysis (i.e., tumor cytotoxicity) was not affected by liposome treatment. These data indicate that elucidation of the effects of liposome composition on acquired host defense mechanisms may be useful for the design of drug delivery systems that allow expression or augmentation of immunologically induced mechanisms for the intracellular destruction of infectious agents.