Influence of charge ratio of liposome/DNA complexes on their size after extrusion and transfection efficiency.

Background: Physicochemical characteristics of liposome/DNA complexes influence transfection efficiency and affect each other in a very intricate way. The result of this is discrepancies in conclusions drawn about the individual influence of each one.

Methods: Aiming to elucidate the influence of liposome/DNA charge ratio and size on transfection efficiency and on each other, we used liposome/DNA complexes with charge ratio (+/-) in the range of 1-50 and extruded through membranes of 400, 200, and 100 nm.

In vivo and ex vivo EPR detection of spin-labelled ovalbumin in mice.

In this study, spin-labelled ovalbumin (SL-OVA), free or entrapped in liposomes, was administered to mice subcutaneously (s.c.) or intravenously (i.

Comparative study of structurally related peptidoglycan monomer and muramyl dipeptide on humoral IgG immune response to ovalbumin in mouse.

Structurally related peptidoglycan monomer (PGM) and muramyl dipeptide (MDP) differ in several aspects of biological activity but have in common immunostimulating properties. Comparative study of the effects of these adjuvants on humoral IgG immune response specific for protein antigen ovalbumin (OVA) was carried out in two inbred mouse strains, CBA and NIH/OlaHsd, and their ability to modulate the bias of immune response towards Th1/Th2 was evaluated. MDP had better adjuvant activity at some points than PGM, whereas both adjuvants stimulated Th2-biased immune response specific for OVA.

Liposome fusogenicity and entrapment efficiency of antigen determine the Th1/Th2 bias of antigen-specific immune response.

Liposomes, either alone or in combination with additional immunostimulatory molecules, could be used for the delivery of antigens as vaccine adjuvants. The aim of this study was to investigate the influence of (a) composition (fusogenicity and charge) of large multilamellar liposomes, (b) antigen entrapment efficiency into cationic liposomes and (c) addition of immunostimulatory peptidoglycan monomer (PGM) into liposomal formulations on intensity and direction of antigen-specific humoral immune response. Ovalbumin (OVA) was used as a model antigen and liposomal formulations were tested in a well defined experimental mice model.

Novel mannosyl derivatives of peptidoglycan monomer: Synthesis and biological evaluation of immunomodulatory properties.

The aim of this work was to prepare mannosyl derivatives of peptidoglycan monomer (PGM, beta-d-GlcNAc-(1-->4)-d-MurNAc-l-Ala-d-isoGln-mesoDAP(epsilonNH(2))-d-Ala-d-Ala) in order to study the effects of mannosylation on adjuvant (immunostimulating) activity. Novel Man-OCH(2)CH(CH(3))CO-PGM isomers were substrates for N-acetylmuramyl-l-alanine amidase, like the parent PGM molecule. Adjuvant activity of Man-OCH(2)CH(CH(3))CO-PGM was tested in the mouse model using ovalbumin as an antigen.

Entrapment of ovalbumin into liposomes--factors affecting entrapment efficiency, liposome size, and zeta potential.

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB + 0.15 M NaCl).

The role of antibodies specific for toxic sPLA2s and haemorrhagins in neutralizing potential of antisera raised against Vipera ammodytes ammodytes venom.

The contribution of antibodies directed against the two main toxic groups of proteins in the Vipera ammodytes ammodytes venom, haemorrhagic metalloproteinases (H) and neurotoxic sPLA2s (Atxs), to the overall protective efficacy of the whole venom antisera was investigated. Using ELISA assays we established a high correlation between the protective efficacy of the whole venom antisera in mice and their anti-Atxs antibody content. As the haemorrhage is the prevailing toxic effect of the venom in human, the lack of correlation also with anti-H IgG content exposed that the mouse model might not be optimal to evaluate the neutralizing potential of the venom-specific antisera for human therapy.

Immunomodulatory activity of novel adjuvant formulations based on Montanide ISA oil-based adjuvants and peptidoglycan monomer.

The aim of the study was to directly compare the potential of Montanide ISA720 and ISA206 oil-based adjuvant formulations on the induction of Th1/Th2-type of immune response, and to compare their effect to Complete Freund's adjuvant (CFA), a well known Th1 inducer. IgG isotype profiles (IgG1/IgG2a ratios) and specific cytokine secretion (IFN-gamma and IL-4) as specific markers of Th1/Th2-type of immune response were monitored in experimentally immunised mice using ovalbumin (OVA) as an antigen. Specifically, we wanted to evaluate whether the incorporation of immunostimulating peptidoglycan monomer (PGM) into two oil-based adjuvants (ISA720(PGM) and ISA206(PGM)) influences their capability on Th1/Th2-type of immune response switching.

Comparison of mouse and rabbit model for the assessment of strong PGM-containing oil-based adjuvants.

Peptidoglycan monomer (PGM) is an adjuvant active molecule with potential for use in human and veterinary vaccine. PGM's action is short-lived in mammals hence its effects might be limited. Novel PGM-containing oil-based formulations have been developed recently by incorporation of PGM into Montanide ISA720 and ISA206 adjuvants with the aim to prolong and improve immunostimulating activities of PGM.

Effectiveness of novel PGM-containing incomplete Seppic adjuvants in rabbits.

Peptidoglycan monomer (PGM) is adjuvant active molecule in experimental mice, although its adjuvanticity is much lower in comparison to potent adjuvants. The novel adjuvant formulations were developed by incorporation of PGM into Montanide ISA 206 and Montanide ISA 720 adjuvants, with the aim to enhance its adjuvanticity by protecting it from the fast degradation and metabolic clearance. Adjuvanticity of the novel adjuvant formulations was tested in rabbits for induction of protein-specific antibodies.

Determination of DNA entrapment into liposomes using short monolithic columns.

A high-performance liquid chromatography (HPLC) method for the determination of DNA entrapment efficiency in liposomes has been developed. Plasmid DNA was encapsulated into positively charged liposomes. Non-entrapped DNA was separated by ultracentrifugation from liposomes and supernatant was chromatographed on Convective Interaction Media (CIM) DEAE disk.

Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies.

Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes.

Effect of liposomal formulations and immunostimulating peptidoglycan monomer (PGM) on the immune reaction to ovalbumin in mice.

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDpm(epsilonNH2)-D-Ala-D-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator.

Purification and characterization of l,(l/d)-aminopeptidase from Guinea pig serum.

Mammalian sera contain enzymes that catalyze the hydrolytic degradation of peptidoglycans and molecules of related structure and are relevant for the metabolism of peptidoglycans. We now report on a novel L,(L/D)-aminopeptidase found in human and mammalian sera. The enzyme hydrolyses the pentapeptide L-Ala-D-iso-Gln-meso-DAP(omegaNH(2))-D-Ala-D-Ala yielding the free L-alanine and the respective tetrapeptide (K(M) 18 mM).

Immunogenicity of peptides of measles virus origin and influence of adjuvants.

Epitope-based peptide antigens have been under development for protection against measles virus. The immunogenicity of five peptides composed of the same B cell epitope (BCE) (H236-250 of the measles virus hemagglutinin), and different T cell epitopes of measles virus fusion protein (F421-435, F256-270, F288-302) and nucleoprotein (NP335-345) was studied in mice (subcutaneous immunisation). The adjuvant effects of peptidoglycan monomer (PGM), Montanide ISA 720 and 206 were also investigated.

Entrapment of peptidoglycans and adamantyltripeptides into liposomes: an HPLC assay for determination of encapsulation efficiency.

The encapsulation of different immunomodulating peptides, the peptidoglycan monomer, its semisynthetic derivatives (Adamant-1-yl)-acetyl-peptidoglycan monomer and Boc-Tyr-peptidoglycan monomer, respectively, and of two diastereoisomers of adamantyltripeptides into the large negatively charged multilamellar liposomes was investigated. The reproducible quantitative method using HPLC was established for the determination of the entrapped compounds. It was shown that the tested compounds could be efficiently incorporated into liposomes using either the film or modified film method.

Modified glycopeptides related to cell wall peptidoglycan: conformational studies by NMR and molecular modelling.

Polymeric peptidoglycans of bacterial cell walls, and smaller glycopeptides derived from them, exhibit versatile biological activities including immunomodulating properties. Peptidoglycan monomer (PGM) was isolated from Brevibacterium divaricatum and novel lipophilic derivatives of PGM bearing either (adamantyl-1-yl)-acetyl or Boc-Tyr substituents (Ad-PGM and BocTyr-PGM respectively) have recently been synthesized. We have obtained full assignments of the 1H and 13C spectra, using 2D NMR techniques, for all three compounds in DMSO solutions.

A spin labelling study of immunomodulating peptidoglycan monomer and adamantyltripeptides entrapped into liposomes.

The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes.

Adjuvant activity of peptidoglycan monomer and its metabolic products.

Peptidoglycan monomer (PGM) is a natural compound of bacterial origin. It is a non-toxic, non-pyrogenic, water-soluble immunostimulator potentiating humoral immune response to ovalbumin (OVA) in mice. It is fast degraded and its metabolic products-the pentapeptide (PP) and the disaccharide (DS)-are excreted from the mammalian organism upon parenteral administration.

Influence of adjuvant-active peptidoglycan monomer on specific T cell responses in mice.

Peptidoglycan monomer (PGM) originating from Brevibacterium divaricatum is a non-toxic, non-pyrogenic, water-soluble immunostimulator. It potentiates humoral immune response to ovalbumin (OVA) in mice upregulating both immunoglobulin (IgG) 1 and IgG2a antibody subclasses. This study concerns the influence of PGM on T cell activation and cytokine networks in response to OVA.

Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides.

The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound.