Humoral immune response against epidermal growth factor encapsulated in dehydration rehydration vesicles of different phospholipid composition.

We investigated the influence of dehydration-rehydration vesicles (DRV) phospholipid composition and the addition of other components on human recombinant epidermal growth factor (hrEGF) encapsulation efficiency and its release from liposomes. Encapsulation of EGF into DRV composed of phosphatidylcholine with different unsaturation levels was around 20-35%. The best result was obtained with dipalmitoyl phosphatidylcholine: cholesterol (DPPC:Ch) liposomes (35%) corresponding to the lowest hrEGF release during one month of storage. Even with this phospholipid composition, modification of the DRV procedure by including an extrusion step did not improve hrEGF encapsulation efficiency, rendering less stable particles. The inclusion of recombinant P64k from Neisseria meningitidis (rP64k), as such or conjugated to hrEGF, decreased the encapsulation efficiency of the latter protein into DRV or freeze and thaw multilamellar vesicles (FATMLV). The hrEGF release from liposomes could be related to the interaction between this polypeptide and the bilayer, as evidenced by increased carboxyfluorescein release from hrEGF-DRV; less susceptibility to fluorescence quenching by acrylamide in the presence of liposomes; and a measurable decrease of phospholipid phase transition Delta enthalpy (DeltaH). DRV comprising saturated phospholipids (DPPC:Ch or distearoyl phosphatidylcholine [DSPC]:Ch) and containing the conjugate EGF-P64k induced a more efficient immune response against hrEGF than unsaturated phospholipid and alum in terms of total IgG, IgG(2a), and IgG(2b) subclasses and the ability of antibody to inhibit the interaction of the EGF receptor with hrEGF.

Changes in the surface potential of Lactobacillus acidophilus under freeze-thawing stress.

The zeta potential of Lactobacillus acidophilus CRL 640, a measure of the net distribution of electrical charges on the bacterial surface, is a function of the glucose concentration in the growing media. With 2% glucose, cells in the stationary phase showed a zeta potential of -45 +/- 2 mV. With these cells, the zeta potential after freezing and thawing decreased to -32 +/- 2 mV and there was a decrease in viability. The changes in the surface potential correlated with damage to the cell surface as shown by electron microscopy. Freeze-thawed cells incubated in a rich medium recovered a zeta potential of -38 +/- 2 mV without cell growth. L. acidophilus CRL 640 showed the same value of surface potential as control cells when they were frozen and thawed in 2 M glycerol.

Influence of growth temperature on cryotolerance and lipid composition of Lactobacillus acidophilus.

In order to correlate the lipid composition of the membrane of Lactobacillus acidophilus CRL 640 with the freeze-thaw behaviour of the cultures grown at different temperatures, fatty acid methyl esters (FAMEs) from extracts grown at 25, 30, 37 and 40 degrees C were obtained and compared. Cultures grown at 25 degrees C (M25) exhibited more resistance to the freeze-thaw process probably because of an increase in C18:2 and C16:0 fatty acids. This culture also exhibited a lesser amount of phospholipids as shown by the sugar: phosphorus ratio. In all cases, the presence of the uncommon 10-hydroxyoctadecanoic acid was determined. From the extracts of the M25 and M37 cultures, diacylphosphatidylglycerol, cardiolipin, diglycosyldiglycerides, triglycosyldiglycerides and neutral lipids were isolated and identified. The structural elucidation was carried out by FAMEs and sugar analysis and by mass spectrometry using fast atom bombardment ionization. The changes in lipid composition due to different growth temperatures could be indicative of the resistance of the bacteria to freeze-thaw processes.

Permeability and stability properties of membranes formed by lipids extracted from Lactobacillus acidophilus grown at different temperatures.

Lactobacillus acidophilus CRL 640 grown at 25 and 37 degrees C showed a high content of cardiolipin, phosphatidylglycerol, and glycolipids. Cultures grown at 25 degrees C showed a twofold increase in glycolipids in relation to phospholipids, a twofold increase in the C16:0 and a fourfold increase in the C18:2 fatty acids. In contrast, the C19-cyc and the 10-hydroxy acid (C18:0-10 OH) species showed a noticeable decrease. Extracts of total lipids of bacteria grown at 25 and 37 degrees C dispersed in water yielded particles having a high negative surface potential as measured by electrophoretic mobility. Vesicles prepared by extrusion of these dispersions through polycarbonate membranes of 100-nm pore diameter showed high trapping of carboxyfluorescein (CF), which remained unchanged for at least 20 h. The fluorescence anisotropy measured with diphenylhexatriene (DPH) and the generalized polarization of Laurdan were significantly lower in vesicles prepared with lipids containing the highest glycolipid ratio, in comparison to those of bacteria grown at 37 degrees C. No phase transition was detected between 5 and 50 degrees C as measured with both probes. In accordance with these results, no significant release of the trapped CF in this range of temperature was detected. Bile salts and NaCl promoted an increase in the fluorescence, which is interpreted as a change in the permeability properties of the membrane. This effect was lower with KCl, while CaCl2 did not cause any change. The greater permeability change was observed in vesicles with a low glycolipid/phospholipid ratio. NaCl did not affect the packing of the interface as measured with Laurdan, in contrast to CaCl2. The action of Ca+2 may be ascribed to the binding to the negatively charged lipids, such as phosphatidyl glycerol and cardiolipin. It is concluded that the higher glycolipid/phospholipid ratio and the fatty acids C18:2 and C16:0 enhance the lipid membrane stability and decrease the organization in the interfacial and hydrocarbon zones. These results are congruent with the behavior of entire bacteria subject to osmotic and freeze/thaw stresses.