Protection against toxoplasmosis in mice immunized with different antigens of Toxoplasma gondii incorporated into liposomes.

Different toxoplasma antigens were entrapped within liposomes and evaluated, in this form, for their ability to protect Swiss mice against toxoplasma infection: soluble tachyzoite antigen (L/TAg), tissue cyst (L/CAg), tachyzoite plus tissue cyst (L/TCAg) or purified antigen of tachyzoite (L/pTAg). The protein used in L/pTAg was purified from tachyzoites using a stage-specific monoclonal antibody which reacted at a molecular weight of 32 kD in SDS PAGE and silver stain using reduced condition. To compare the immuno-adjuvant action of liposomes and of Freund's Complete Adjuvant (FCA), another group of mice was immunized with soluble tachyzoite antigen (STAg) emulsified in FCA (FCA/TAg). Control groups were inoculated with (STAg) alone, phosphate-buffered saline (PBS), FCA with PBS (FCA/PBS) and empty liposomes (L/PBS). Mice were inoculated subcutaneously with these antigens six, four and two weeks before a challenge with 80 tissue cysts of the P strain of Toxoplasma gondii orally. All mice immunized with or without adjuvant showed a humoral response, as measured by Elisa. However, no correlation was found between antibody titer and protection against the challenge. All mice immunized with L/pTAg or L/TCAg survived (100), whereas 80% and 90% of mice from groups which received respectively PBS or FCA/PBS and L/PBS died. All mice immunized with antigens entrapped within liposomes (L/TAg, L/CAg, L/TCAg and L/pTAg) showed low numbers of intracerebral cysts.     

In vitro activation of tumoricidal properties in mouse macrophages using the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) incorporated in liposomes

Summary We investigated the ability of free or liposome-incorporated synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) to generate tumoricidal properties in mouse macrophages. As FMLP contains three hydrophobic amino acid residues, it can readily be incorporated into multilamellar vesicles (MLV) consisting of phosphatidylcholine (PC) and phosphatidylserine (PS). The incorporation of FMLP into MLV with a PC:PS ratio of 7:3 mol at FMLP concentrations of up to 10^–4 M did not affect the phagocytosis of liposomes by mouse peritoneal macrophages. Studies with radioactive FMLP revealed that higher levels of FMLP can be delivered to macrophages by liposomes than in the free, nonencapsulated form. Treatment of mouse macrophages with liposome-encapsulated FMLP, but not with free FMLP, generated tumoricidal properties in the macrophages. The mechanism appears to involve an intracellular site since 100-fold concentrations of free FMLP or free N-acetyl-methionyl-leucyl-phenylalanine, the FMLP agonist, failed to competitively inhibit the macrophage's tumoricidal properties generated by liposome-encapsulated FMLP.     

Macrophage activation by PAF incorporated into dipalmitoylphosphatidylcholine-cholesterol liposomes

The phospholipid mediator, platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine), was recently reported to activate macrophage-monocyte cells as well as neutrophils or platelets. PAF was incorporated into dipalmitoylphosphatidylcholine-cholesterol liposomes, and their effect on guinea pig peritoneal macrophages was examined. PAF incorporated into liposomes was found to activate macrophages much more potently than PAF in the free form, whereas the effect of PAF in liposome on platelets was weaker than that of PAF in the free form. A large difference between PAF in liposomes and PAF in the free form was observed in the rate of degradation of PAF during incubation with macrophages. This rapid degradation of PAF in the free form may partially explain the poor activation by PAF of macrophages.     

Identification of phosphoproteins associated with human neutrophil granules following chemotactic peptide stimulation

Regulated exocytosis of neutrophil intracellular storage granules is necessary for neutrophil participation in the inflammatory response. The signal transduction pathways that participate in neutrophil exocytosis are complex and poorly defined. Several protein kinases, including p38 MAPK and the nonreceptor tyrosine kinases, Hck and Fgr, participate in this response. However, the downstream targets of these kinases that regulate exocytosis are unknown. The present study combined a novel inhibitor of neutrophil exocytosis with proteomic techniques to identify phosphopeptides and phosphoproteins from a population of gelatinase and specific granules isolated from unstimulated and fMLF-stimulated neutrophils. To prevent loss of granule-associated phosphoproteins upon exocytosis, neutrophils were pretreated with a TAT-fusion protein containing a SNARE domain from SNAP-23 (TAT-SNAP-23), which inhibited fMLF-stimulated CD66b-containing granule exocytosis by 100±10%. Following TAT-SNAP-23 pretreatment, neutrophils were stimulated with the chemotactic peptide fMLF for 0 min, 1 min, and 2 min. Granules were isolated by gradient centrifugation and subjected to proteolytic digestion with trypsin or chymotrypsin to obtain peptides from the outer surface of the granule. Phosphopeptides were enriched by gallium or TiO2 affinity chromatography, and phosphopeptides and phosphorylation sites were identified by reversed phase high performance liquid chromatography-electrospray ionization-tandem MS. This resulted in the identification of 243 unique phosphopeptides corresponding to 235 proteins, including known regulators of vesicle trafficking. The analysis identified 79 phosphoproteins from resting neutrophils, 81 following 1 min of fMLF stimulation, and 118 following 2 min of stimulation. Bioinformatic analysis identified a potential Src tyrosine kinase motif from a phosphopeptide corresponding to G protein coupled receptor kinase 5 (GRK5). Phosphorylation of GRK5 by Src was confirmed by an in vitro kinase reaction and by precursor ion scanning for phospho-tyrosine specific immonium ions containing Tyr251 and Tyr253. Immunoprecipitation of phosphorylated GRK5 from intact cells was reduced by a Src inhibitor. In conclusion, targets of signal transduction pathways were identified that are candidates to regulate neutrophil granule exocytosis.     

Ion selectivity of the nerve membrane sodium channel incorporated into liposomes

Tetrodotoxin-sensitive sodium channels of lobster nerve membranes were incorporated into soybean liposomes by the freeze-thaw-sonication procedure and their ionic selectivity was studied. Veratridine and grayanotoxin-I were used to activate the sodium channels and the increment of the ionic flux through them was specifically abolished by tetrodotoxin. The drug-sensitive 22Na+, 42K+, 86Rb+ and 137Cs+ influxes were measured. The permeability ratios calculated directly from ion fluxes showed that the channels preferably allow the passage of Na+. No anion influx ([32P]phosphate, [35S]sulfate, 36Cl) sensitive to the drugs was observed. The data reveal that the sodium channels incorporated into liposomes remain cation-selective and discriminate among different cations.     

Lectin interactions with the variant surface glycoprotein from Trypanosoma brucei brucei incorporated into liposomes

The variant specific surface glycoprotein from Trypanosoma brucei brucei is incorporated into lipid vesicles using 8M urea as an unfolding reagent. Pronase treatment of these proteoliposomes removes most of the protein, leaving a glycophospholipopeptide which is the membrane attachment site. We show here that lectins, specific for mannose and galactose are able to recognize oligosaccharide residues on these proteoliposomes, using a straightforward aggregation assay. The relevance of these results obtained with the liposome model system to the accessibility of the surface antigens in living trypanosomes is discussed.     

Binding of choleragen and anti-ganglioside antibodies to gangliosides incorporated into preformed liposomes

Exogenously added gangliosides were taken up and incorporated into liposomes just as they are incorporated into cells. Ganglioside GM1 was rapidly taken up by liposomes containing dimyristoyl- or dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate. When incubated with a wide range of GM1 concentrations for 18 h, the liposomes incorporated about 10% of the added ganglioside. The rate of GM1 uptake by preformed liposomes was both time- and temperature-dependent. The liposomes also incorporated other gangliosides to a similar extent. The GM1 taken up by preformed liposomes was predominantly located on the outer surface of the liposomes and did not appear to be internalized into the inner half of the lipid bilayer. Liposomes containing GM1 added after liposome formation bound as many anti-GM1 antibodies and as much choleragen as liposomes having GM1 added during the formation of the lipid bilayers. Thus, preformed liposomes sensitized by incubation with GM1 are a good model system for studying the interactions of antibodies and toxins with membrane-associated gangliosides.     

Differences in the microtubule organization between normal and cml granulocytes after stimulation with chemotactic peptide

Chemotaxis of polymorphonuclear leukocytes (PMNL) from chronic myeloid leukemia (CML) patients followed in a gradient of a chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (fMLP) is consistently defective in all the phases of the disease. Chemoattractant-induced polymerization of cytoskeletal proteins (actin and tubulin) plays a major role in regulation of cell shape and cellular motility. To study the role of microtubules in defective chemotaxis, we have compared fMLP-induced alterations in organization of microtubules in PMNL from CML patients with those from normal subjects by laser confocal microscopy. Our analysis shows differences in microtubule organization between normal and CML PMNL and suggests that both nucleation of new microtubule and elongation of pre-existing microtubules are essential for PMNL chemotaxis.     

Voltage dependence of liver gap-junction channels reconstituted into liposomes and incorporated into planar bilayers.

The voltage dependence of rat liver gap junctions was investigated using non-denaturing solubilization and reconstitution of gap-junction protein into proteoliposomes in controlled conditions of connexon aggregation. The presence of liver connexin 32 in reconstituted proteoliposomes was checked with specific antibodies. The proteoliposomes were inserted into planar lipid bilayers by fusion. The single-channel conductance was voltage independent, and its magnitude was 700-1900 pS in 1 M NaCl, as expected from other reports, assuming that conductance is linear with ion activity. The channels were open at zero voltage and completely closed above 40 mV in either direction. This steep voltage dependence corresponded to an open/closed-state voltage difference of 19 mV and to 3.5 gating charges moving through the field. When several channels were inserted into the bilayer, a large fraction of the membrane conductance became voltage insensitive. These results show that the isolated channel units are highly voltage dependent and are consistent with the assumption that aggregated connexons interact through links which prevent voltage-sensitive conformational changes.     

Polymorphonuclear leukocyte-platelet interactions: acetylglyceryl ether phosphocholine-induced platelet activation under stimulation with chemotactic peptide

A mixture of rabbit polymorphonuclear leukocytes (PMNs) and platelets at concentrations of 5 X 10(6) PMN and 3.5 X 10(8) platelets/ml Tyrode's solution was stimulated with the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). A micromolar concentration of FMLP elicited an immediate weak aggregation, followed by a strong aggregation with a time lag of about 1 min. Microscopic examination showed that the immediate aggregation was due to PMNs and the delayed one was more complex and involved platelets. The delayed aggregation was dependent upon the concentrations of both the PMNs and FMLP. The delayed aggregation was completely blocked by pretreatment of the PMN-platelet mixture with 8 microM CV-3988, a specific receptor antagonist of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), or by the application of platelets desensitized to AGEPC. The time course of AGEPC production by PMNs was well matched to that of the biphasic aggregation response. Furthermore, nordihydroguaiaretic acid inhibited both the AGEPC production by PMNs and the delayed aggregation in a similar dose-dependent manner. These result demonstrate that AGEPC, newly-generated by PMNs under FMLP-stimulation, is of primary importance in platelet aggregation in a PMN-platelet mixed system.     

Effects of alkyl glycosides incorporated into liposomes prepared from synthetic amphiphiles on their tissue distribution in Ehrlich solid tumor-bearing mice.

A study of the effects of alkyl glycosides incorporated into synthetic liposomes with respect to their stability, their in vivo distribution in Ehrlich solid tumor-bearing mice and their in vitro interaction with liver cells was undertaken. The synthetic liposomes were prepared from N,N-didodecyl-N alpha-[6-(trimethylammonio)hexanoyl]-L-alaninamide bromide (N+C5Ala2C12) and labeled with 99mTc. n-Dodecyl glucoside (DG) and n-dodecyl sucrose (DS) were used as alkyl glycosides. The stability was hardly changed by incorporation of alkyl glycosides into the liposomes in saline and serum. The uptake of DG- and DS-modified N+C5Ala2C12 liposomes decreased in liver and spleen compared with that of unmodified N+C5Ala2C12 liposomes, resulting in an increase in blood and other tissues such as tumor, duodenum and kidney, where the DS-modified N+C5Ala2C12 liposomes had a marked tendency. It was observed with electron micrographs that the size of N+C5Ala2C12 liposomes became small by incorporation of alkyl glycoside. The smaller N+C5Ala2C12 liposomes were found to result in the lower uptake in liver. The interaction of the liposomes with liver cells in vitro indicated that both DG- and DS-modified liposomes had a low affinity for liver cells compared with the unmodified liposomes and the extent of interaction of the DS-modified liposomes was weaker than that of the DG-modified liposomes.     

Preparation and characterization of lyophilized liposomes with incorporated quercetin

Liposomes composed of egg-phosphatidylcholine (EPC) incorporating quercetin (QR) were prepared by the thin-film hydration method (TFHM) and the monophase solution method (MSM). A rapid and slow freeze-drying process was applied for both laboratory and industrial scales. The purpose of this study was to compare the two methods of liposome preparation, and further determine whether the lyophilization process affects the liposome physicochemical characteristics (size, polydispersity index, and ?-potential) and incorporation of quercetin.     

Vertical toxoplasmosis in a murine model. Protection after immunization with antigens of Toxoplasma gondii incorporated into liposomes

Distinct Toxoplasma gondii antigens were entrapped within liposomes and evaluated for their ability to protect Balb/c mice against congenital transmission: soluble tachyzoite antigen (L/STAg), soluble tissue cyst antigen (L/SCAg), soluble tachyzoite plus tissue cyst (L/STCAg) or purified 32kDa antigen of tachyzoite (L/pTAg). Soluble tachyzoite antigen alone in PBS (STAg) or emulsified in Freund's Complete Adjuvant (FCA/STAg) was also evaluated. Dams were inoculated subcutaneously with these antigens 6, 4 and 2 weeks prior to a challenge with four tissue cysts of the P strain of T. gondii orally between 10 and 14 days of pregnancy. Significant diminution differences were observed between the frequency of infected pups born of the dams immunized with the antigens incorporated into liposomes and that of pups born of the dams immunized with antigen emulsified in FCA or non immunized group (p<0.05). There was a significant decrease in the number of pups born dead in the groups L/STAg, L/SCAg and L/pTAg when compared with pups from all other groups (p <0.05). All dams immunized with or without adjuvant showed an antibody response and a proliferation of T-cells. However, no correlation was found between immune response and protection against the challenge.     

Stimulation of superoxide release in neutrophils by 1-oleoyl-2-acetylglycerol incorporated into pH-sensitive liposomes

Incorporation of 1-oleoyl-2-acetylglycerol (OAG) into multilamellar liposomes composed of egg phosphatidylethanolamine (PE) and arachidonic acid (AA) resulted in a significant enhancement of superoxide release by guinea pig neutrophils when compared to free OAG. OAG incorporated into liposomes containing phosphatidylcholine and arachidonic acid were generally less effective than free OAG. The potency of the liposomes correlates well with the ability of the liposomes to undergo lipid mixing at acidic pH. The enhanced effect of liposome-associated OAG could be related to exposure to an acidic environment in the endosomes/lysosomes once liposomes are endocytosed by neutrophils.     

Light-driven proton transport of bacteriorhodopsin incorporated into long-term stable liposomes of a polymerizable sulfolipid

The chromoprotein bacteriorhodopsin from Halobacterium halobium has been incorporated into liposomes made of a fully synthetic, polymerizable lipid. Bacteriorhodopsin is found to be active in these polymer liposomes. The advantage in the use of such polymer systems concerning long-term stability in comparison with liposomes made of natural lipid is demonstrated.     

Temperature dependence of optical properties of chlorophyll a incorporated into phosphatidylcholine liposomes

Temperature-dependent spectral changes of chlorophyll a (Chl a) incorporated into liposomes of two types of phosphatidylcholine are studied. When Chl a incorporated into the liposomes is cooled down to 5°C from the temperature of the gel-to-liquid crystalline phase transition of the lipid, the red shift as well as the increase in half-bandwidth of the red peak of Chl a are only slight. By measuring the difference spectra produced by substracting the absorption spectrum at the phase transition temperature of the lipid from that at lower temperature, it is shown that the component absorbing at longer wavelength (675–685 nm) than the peak of the red maximum (about 670 nm) significantly increases at the expense of the component absorbing at shorter wavelength (657–668 nm). The positions of positive and negative peaks depend on the temperature and the molar ratio of the lipid to Chl a. The absorbance change is most pronounced on cooling below the phase transition temperature of the lipid. The temperature-induced absorbance change is almost completely reversible. The results indicate that the aggregated forms of Chl a in liposomes can be spectrophotometrically detected in the gel phase of the lipid.     

Lectin-receptor interactions in liposomes II. Interaction of wheat germ agglutinin with phospatidylcholine liposomes containing incorporated monosialoganglioside

Bovine brain gangliosides incorporated into phospholipid liposomes provide receptors for wheat germ agglutinin. Purified monosialogangliosides were mixed with egg phosphatidylcholine, and unilamellar liposomes were generated. Addition of wheat germ agglutinin induced the liposomes to fuse, and gel filtration analysis revealed that the lectin was incorporated into the fused liposomes. The fusion process was studied by following the changes in the 190° light scattering. Increasing the proportion of the monosialoganglioside in the liposomes was found to increase both the extent of the lectin-induced liposome fusion and the rate of the reaction; below a threshold of approx. 5 mol %, the process was extremely slow. The increase in light scattering could be prevented by the addition of the hapten inhibitor, N-acetyl-d-glucosamine (1 mM). Addition of the inhibitor, subsequent to the lectin, caused a partial decrease in light scattering due to the dissociation of unfused vesicle aggregates. Electron microscopic examination revealed that the ganglioside-containing liposomes were vesicles, 244±25 Å (S.D.) in diameter. Upon addition of wheat germ agglutinin, the vesicles appeared to fuse to form larger vesicles, corresponding to dimers and trimers of the initial vesicles. Inhibition studies with a variety of monosaccharides indicated that the sialic acid moieties present in the gangliosides acted as the lectin-receptor sites. This was confirmed by the observation that wheat germ agglutinin did not interact with phosphatidylcholine vesicles containing desialyated ganglioside.     

Stability and absorption of liposomes with incorporated insulin in the small intestine

Lecithin-cholesterol liposomes with the incorporated insulin were studied in vitro for their stability to the action of some factors of gastrointestinal tract--hydrochloric acid, gastric juice, pepsin, trypsin, bile. A considerable destructive action of bile on liposomes is established. Modification of the lipid composition of liposomes makes it possible to increase their stability to the bile action. Suction of liposomes from the isolated sites of rat small intestine is studied. Insulin from liposomes is established to be sucked most intensive from the initial parts of small intestine. In this case it penetrates into the blood bed not in a free state but in the composition of liposomes.