Stimulation of secretory antibodies against Bordetella pertussis antigens in the lungs of mice after oral or intranasal administration of liposome-incorporated cell-surface antigens.

Lipopolysaccharide (LPS) and outer membrane protein (OMP) preparations of Bordetella pertussis were incorporated into multilamellar liposomes composed of soya bean-derived phospholipids which were then used for oral and intranasal immunization of mice. Specific antibody responses of animals immunized by either route were measured in lung washes. A specific IgA response to LPS was detected after immunization with the OMP-containing liposomes but not with the LPS-containing liposomes, indicating adjuvant activity of the proteins. The OMP-containing liposomes were significantly more effective in inducing immune responses than the OMP preparation alone. Responses were highest when mice were given a booster 30 days after primary immunization. Maximum responses occurred 20 days after the booster but specific antibody was still detected 75 days after the secondary immunization. These results suggest that this liposome antigen delivery system has potential in stimulating secretory antibody responses which may be helpful in protecting against infection from B. pertussis.

Antibody responses in the serum and respiratory tract of mice following oral vaccination with liposomes coated with filamentous hemagglutinin and pertussis toxoid.

Mice were orally vaccinated with liposomes coated with filamentous hemagglutinin (FHA) and detoxified pertussis toxin (PT) of Bordetella pertussis. FHA- and PT-specific immunoglobulin G (IgG) was detected in serum, and both IgG and IgA were detected in lung washes following the immunization. Antibody responses in mice immunized with liposomes coated with FHA and PT were significantly higher than those in mice immunized with free FHA and PT, which demonstrated the adjuvanticity of the liposome carrier. The results indicate the potential usefulness of this approach for eliciting immune responses against FHA and PT (and perhaps other pertussis antigens) in humans and its possible utility in large-scale vaccination to protect against both B. pertussis infection and disease.

Treatment of Brucella canis and Brucella abortus in vitro and in vivo by stable plurilamellar vesicle-encapsulated aminoglycosides.

Stable plurilamellar vesicles (SPLVs) entrapping aminoglycosides were used to treat infections due to Brucella species (Brucella canis and Brucella abortus). SPLV-entrapped antibiotics effectively eliminated internalized B. canis in cultures of resident murine peritoneal macrophages and internalized B. abortus in cultures of resident guinea pig peritoneal macrophages. In vivo studies demonstrated that SPLV-entrapped aminoglycosides administered to B. canis-infected mice and B. abortus-infected guinea pigs effectively eliminated bacteria from infected organs. The dosage schedule used involved two intraperitoneal administrations of SPLV-entrapped aminoglycosides at three-day intervals. The results demonstrate the superiority of SPLV-entrapped aminoglycosides to free aminoglycosides in effecting elimination of facultative intracellular bacteria in vitro and in vivo. The use of SPLVs as a drug carrier has broad application to treatment of infections due to other organisms.

Enhanced intraphagocytic killing of Brucella abortus in bovine mononuclear cells by liposomes-containing gentamicin.

In vitro intraphagocytic killing of Brucella abortus in bovine mononuclear leukocytes was enhanced by cationic, anionic, and neutral multilamellar liposomes-containing gentamicin. Free gentamicin not entrapped in liposomes. and liposomes without antibiotic did not enhance intraphagocytic killing of B. abortus in bovine phagocytes. In vivo killing of B. abortus in guinea pigs was also enhanced by liposomes-containing gentamicin when compared to free gentamicin. Liposomes-containing alpha tocopherol acetate failed to enhance in vivo killing of B. abortus.

Plasma membrane-mediated leakage of liposomes induced by interaction with murine thymocytic leukemia cells.

The interaction of liposomes with BW 5147 murine thymocytic leukemia cells was studied using fluorescent probes (entrapped carboxyfluorescein and fluorescent phosphatidylethanolamine) in conjunction with a Ficoll-Paque discontinous gradient system for rapid separation of liposomes from cells. Reversible liposomal binding to discrete sites on the BW cell surface was found to represent the major form of interaction; uptake of intact liposomal contents by a process such as liposome-BW cell membrane fusion was found to apparently represent a minor pathway of interaction (2%). Liposomal lysis was found to be associated with the process of liposomal binding (perhaps as a result of the binding itself). Lysis was followed by release of the entrapped carboxyfluorescein into the media and its subsequent uptake by the cells. This lysis was shown to be dependent upon discrete membrane-associated sites that have some of the properties of proteins. The results of these studies suggest that liposomal binding to the cells, subsequent lysis of the liposomes and cellular uptake of their contents should be seriously considered in all studies of liposome-cell interactions as an alternate mode of interaction to the four modes (fusion, endocytosis, adsorption and lipid exchange) previously emphasized in the literature.

An ELISA test to detect antibody to Bordetella bronchiseptica.

An enzyme-linked immunosorbent assay (ELISA) was developed to measure antibody to Bordetella bronchiseptica in dogs. The ELISA test was more rapid and sensitive and required 50 to 150 times less antigen than the amount of antigen required for the conventional tube agglutination test. A survey of 50 canine serum samples using ELISA suggested that 8% of all sera had titers greater than 1:64, 56% had titers of 1:8 to 1:64, and 36% had titers of less than 1:8. The mean titer of survey sera was 1:46 and the median titer was 1:16. Serum antibody responses in dogs inoculated with a commercially available bacterin were compared with responses in dogs inoculated with experimental endotoxin depleted bacterin.

Interactions of multilamellar phospholipid vesicles with bovine lymphocytes: effects of alpha-tocopherol on lymphocyte blastogenesis.

Multilamellar lipid vesicles (MLV) composed of egg yolk lecithin (EYL) suppressed the response of bovine peripheral blood lymphocytes (BPBL) to phytohemagglutinin (PHA). EYL contains 18:1 and 18:2 as the major unsaturated phospholipids. Dioleoyllecithin (DOL; cis 9) MLV did not suppress BPBL blastogenesis. Dilinoeyllecithin (DLL; cis, cis 9, 12) MLV suppressed BPBL blastogenesis. The suppressive effect could be reversed by increasing the MLV DML concentration. The addition of alpha-tocopherol (alpha-T) at 10 mole% into MLV containing DLL reversed blastogenic suppression of BPBL. MLV composed of mixed saturated phospholipids (dimyristoyllecithin and dipalmitoyllecithin) and alpha-T enhanced the BPBL blastogenic response to PHA. BPBL incubated with varying PHA concentrations (11.82-375 mg/ml) and a constant concentration (2 mumoles/ml) of MLV composed of EYL remained suppressed either when PHA and MLV were added simultaneously or when MLV were incubated for 1 hr prior to the addition of PHA. This suggests that alpha-T may act as an immunomodulator in the blastogenic response to PHA. Results suggest that alpha-T reversion of EYL suppression of BPBL blastogenesis may be due to interactions of alpha-T with unsaturated acyl chains in EYL phospholipids.

Introduction of alpha-tocopherol into bovine lymphocyte surfaces by lipid vesicles: effects of phospholipid composition on alpha-tocopherol transfer.

Liposomes transferred alpha-tocopherol to bovine peripheral blood lymphocytes (BPBL) at 39 degrees C. Phospholipid ([14C] DMPC) transferred into BPBL at rates similar to [3H] alpha-tocopherol but with less efficiency than [3H] alpha-tocopherol from liposomes containing both labels. Liposomes containing unsaturated phospholipids DOPC and DLPC reduced but did not stop the rates of transfer of alpha-T to BPBL when compared to liposomes composed of DMPC. The presence of [14C] cholesterol in liposomes with [3H] alpha-tocopherol does not restrict but possibly enhances the alpha-tocopherol transfer into BPBL.

Effects of mitogen and liposome phospholipid concentration on the blastogenic response of bovine lymphocytes to phytohemagglutinin.

Lymphocytes incubated with liposomes prior to the addition of phytohemagglutinin (PHA) exhibited a time dependent suppression of blastogenesis which was reversible for phosphatidylcholine (PC):cholesterol (Chol) alpha-tocopherol (alpha-T) (1:0.5:0.5), and PC: alpha-T (1:1) liposomes but not for PC and PC:Chol (1:1) liposomes. Incubation of PHA with lymphocytes prior to the addition of liposomes caused a time dependent, reversible suppression of blastogenesis for PC: alpha-T:Chol (1:0.5:0.5), PC: alpha-T (1:1) and PC:Chol (1:1) liposomes but not reversible for PC liposomes. The effects of phospholipid concentration on the blastogenic response to PHA exhibited a concentration dependent suppression from 1 to 2 mumoles phospholipid/ml which was reversible, to varying degrees, at a concentration of 4 mumoles phospholipid/ml.

Liposome carrier vehicle for triiodothryonine.

1. Liposomes (multilamellar vesicles, MLV) were prepared which entrapped triiodothyronine (T3). The MLV were composed of dimyristoylphosphatidylcholine, cholesterol and dicetylphosphate as DMPC alone, DMPC:Chol (7.2 molar ratio) and DMPC:Chol:DCP (7:2:1 molar ratio). 2. The optimal T3 entrapment was within MLV composed of DMPC: Chol:DCP. The entrapped concentrations of T3 was 63.1%. 3. The MLV composed of DMPC:Chol:DCP did not leak T3 from the MLV into buffered saline but did leak T3 into serum (42.5% after 4 h). 4. The MLV composed of DMPC:Chol:DCP exhibited a distribution of T3 between membrane associated and membrane entrapped of 42.58%.

Effects of enrichment of phosphatidylcholine liposomes with cholesterol or alpha-tocopherol on the response of lymphocytes to phytohemagglutinin.

Studies were performed to determine the ability of alpha-tocopherol and cholesterol to influence the effect of phosphatidylcholine (PC) liposomes on the blastogenic response of bovine peripheral blood lymphocytes (BPBL) to phytohemagglutinin (PHA). BPBL were cultured with liposomes having a molar ratio of cholesterol to PC (C/P) ranging from 0 to 2.0, a molar ratio of alpha-tocopherol to PC (E/P) of 1.0 and a molar ratio of cholesterol + alpha-tocopherol to PC [(C + E)/P] of 2.0 and 4.0 PC liposomes significantly suppressed BPBL blastogenic response to PHA. Cholesterol-rich (C/P greater than or equal to 1.0) liposomes, alpha-tocopherol-rich (E/P = 1.0) liposomes and liposomes rich in cholesterol and alpha-tocopherol [(C + E)/P greater than or equal to 2.0] were able to completely reverse PC liposome suppression of BPBL. There was no molar ratio [C/P, E/P or (C + E)/P] that was able to enhance the blastogenic response of BPBL above the response obtained with PHA alone. The results suggest that the augmentation of PC liposomes rich in cholesterol, alpha-tocopherol and cholesterol with alpha-tocopherol (C/P and E/P greater than or equal to 1.0) was equally capable of restoring normal responses in BPBL but did not enhance or suppress the response to PHA.

Serum concentrations of digoxin entrapped in liposomes after intravenous administration in dogs.

1. Digoxin was associated into phosphotidylcholine liposomes at concentrations of 28-33 mol% in Hank's Buffer, pH 7.4 at 28 degrees C. 2. Digoxin-liposomes (digoxin concentration 0.022 mg/kg per dog per day) administered intravenously in five adult male dogs attained therapeutic serum concentrations (0.7-3.0 ng/ml) beginning with day 1 of administration. 3. Digoxin serum concentrations obtained by intravenous digoxin-liposomes compared favorably with normal oral digoxin administration (0.022 mg/kg per dog per day) in all 5 dogs monitoring serum digoxin levels for 7 days showed no significant (P < 0.05) differences in mean serum digoxin concentrations +/- s.e.m. on 6 of 7 days of treatments.

Effect of phenobarbital on serum digoxin concentration during administration of liposome-entrapped digoxin: an examination of possible drug interactions.

The effect of concomitant administration in normal dogs of phenobarbital with either oral free digoxin, oral liposome-entrapped digoxin were examined. Phenobarbital induced a transient rise in serum digoxin concentrations during concomitant administration of oral free digoxin. Phenobarbital did not affect serum concentrations of either oral or intravenous liposome-entrapped digoxin. The results suggest that liposome-entrapped digoxin may be protected against transient fluctuations in serum concentrations during concomitant administration of phenobarbital. The protective liposome encapsulation of digoxin may prevent digoxin toxicosis in dogs induced by elevations in serum digoxin concentrations during concomitant administration of phenobarbital.

Liposome-cell interactions. A rapid assay for cells in suspension culture.

A method has been developed for the rapid separation of cells in suspension from non-cell associated lipid vesicles in various assays for vesicle-cell interation. Separation is achieved on a discontinuous Ficoll-Paque gradient. Cells and free vesicles are totally separated, as evidenced by both radiolabelled vesicles, and vesicles containing the fluorescent dye 6-carboxyfluorescein. The main advantages of this method are the rapidity, efficacy, and gentleness of the separation. Viability of the cells remains consistently high (greater than 96%) throughout the separation. Since this method involves a one-step centrifugation, it precludes the necessity for repeated washings of cells which have been incubated with lipid vesicles.