The immunogenicity of p24 protein from HIV-1 virus is strongly supported and modulated by coupling with liposomes and mannan.

Anti-viral and anti-tumor vaccines aim to induce cytotoxic CD8+ T cells (CTL) and antibodies. Conserved protein antigens, such as p24 from human immunodeficiency virus, represent promising component for elicitation CTLs, nevertheless with suboptimal immunogenicity, if formulated as recombinant protein. To enhance immunogenicity and CTL response, recombinant proteins may be targeted to dendritic cells (DC) for cross presentation on MHCI, where mannose receptor and/or other lectin receptors could play an important role. Here, we constructed liposomal carrier-based vaccine composed of recombinant p24 antigen bound by metallochelating linkage onto surface of nanoliposomes with surface mannans coupled by aminooxy ligation. Generated mannosylated proteonanoliposomes were analyzed by dynamic light scattering, isothermal titration, and electron microscopy. Using murine DC line MutuDC and murine bone marrow derived DC (BMDC) we evaluated their immunogenicity and immunomodulatory activity. We show that p24 mannosylated proteonanoliposomes activate DC for enhanced MHCI, MHCII and CD40, CD80, and CD86 surface expression both on MutuDC and BMDC. p24 mannosylated liposomes were internalized by MutuDC with p24 intracellular localization within 1 to 3 h. The combination of metallochelating and aminooxy ligation could be used simultaneously to generate nanoliposomal adjuvanted recombinant protein-based vaccines versatile for combination of recombinant antigens relevant for antibody and CTL elicitation.

Alpha-tocopheryl succinate induces apoptosis by targeting ubiquinone-binding sites in mitochondrial respiratory complex II.

Alpha-tocopheryl succinate (alpha-TOS) is a selective inducer of apoptosis in cancer cells, which involves the accumulation of reactive oxygen species (ROS). The molecular target of alpha-TOS has not been identified. Here, we show that alpha-TOS inhibits succinate dehydrogenase (SDH) activity of complex II (CII) by interacting with the proximal and distal ubiquinone (UbQ)-binding site (Q(P) and Q(D), respectively). This is based on biochemical analyses and molecular modelling, revealing similar or stronger interaction energy of alpha-TOS compared to that of UbQ for the Q(P) and Q(D) sites, respectively. CybL-mutant cells with dysfunctional CII failed to accumulate ROS and underwent apoptosis in the presence of alpha-TOS. Similar resistance was observed when CybL was knocked down with siRNA. Reconstitution of functional CII rendered CybL-mutant cells susceptible to alpha-TOS. We propose that alpha-TOS displaces UbQ in CII causing electrons generated by SDH to recombine with molecular oxygen to yield ROS. Our data highlight CII, a known tumour suppressor, as a novel target for cancer therapy.

Stimulation of nonspecific immunity, haemopoiesis and protection of mice against radiation injury by 1-adamantylamide-L-alanyl-D-isoglutamine incorporated in liposomes.

1-Adamantylamide-L-alanyl-D-isoglutamine (adamantylamide dipeptide (AdDP)) belongs to a group of desmuramyl muramyl peptide derivatives which are able to protect an organism from some viral infections. Encapsulation of AdDP to egg phosphatidyl choline liposomes and the targeting of this drug to lymphatic node macrophages via subcutaneous (s.c.) administration proved to be the efficient way to protect mice against irradiation when administered s.c., 24 h prior to lethal gamma-irradiation (long-term survival rate in the range of 40% compared with 0% in saline or free drug control). Parameters characteristic for the recovery of haemopoiesis in the bone marrow (number of granulocyte-macrophage haemopoietic progenitor cells, granulocyte-macrophage colony forming cells (GM-CFC)) were significantly improved in comparison with the controls and free drug on day 10 after 6.5 Gy irradiation. The haemopoietic effect was observed in the broad application time window (72 h before and 48 h after irradiation). Very high radioprotective effect of s.c. administered liposomal AdDP (L-AdDP) can be explained (together with induction of haemopoiesis) by the effective and long-lasting activation of nonspecific immunity, which withholds the onset of septicemia in early days after irradiation. Induction of nonspecific immunity was proven in Candida albicans infectious model. L-AdDP significantly increased both the survival time and score (about 40% survival compared with 0% in controls and free drug). In conclusion, L-AdDP could be therapeutically beneficial to moderate the haemopoietic damage (undesirable effect of radiotherapy or chemotherapy) and induce the non-specific immunity to support the antimicrobial treatment of immunocompromised patients.

Linkup of a fast protein liquid chromatography system with a stirred thermostated cell for sterile preparation of liposomes by the proliposome-liposome method: application to encapsulation of antibiotics, synthetic peptide immunomodulators, and a photosensitizer.

The proliposome-liposome method is based on the conversion of the initial proliposome preparation into a liposome dispersion by dilution with the aqueous phase. This technique is characterized by an extremely high entrapment efficiency and is suitable for the encapsulation of a wide range of drugs with different water and alcohol solubility. A description of a home-made stirred thermostated cell and its linkup with an FPLC system for a rapid and automated preparation of multilamellar liposomes under strictly controlled conditions (temperature, dilution rate, and schedule) is presented. The highly reproducible procedure yields multilamellar liposomes with a high encapsulation efficiency for various drugs. Carboxyfluorescein, as a model hydrophilic compound, was entrapped with an efficiency of 81 +/- 2%. The antibiotics neomycin and gentamycin were entrapped with efficiencies of 65 and 69%, respectively. Synthetic immunomodulators adamantylamide dipeptide, muramyl dipeptide, and beta-D-GlcNAc-norMurNAc-L-Abu-D-isoGln were entrapped with efficiencies of 87, 62, and 85%, respectively. The photosensitizer mesotetra-(parasulfophenyl)-porphin was entrapped with an efficiency of 65%. The cell has been designed for laboratory-scale preparation of liposomes (300-1000 mg of phospholipid per run) in a procedure taking less than 90 min. The method can be readily scaled up and linked with secondary processing methods, such as pressure extrusion through polycarbonate filters.

Stimulation of haemopoiesis and protection of mice against radiation injury by synthetic analogues of muramyldipeptide incorporated in liposomes.

Protection from undesirable effects of radiotherapy or chemotherapy, primarily from myelosuppression, remains still a crucial problem to be studied. Attention has been therefore paid to various immunomodulatory agents that through the monocyte/macrophage system induced production of cytokines, which can induce and operate restoration of haemopoiesis and thus act radioprotectively. Some synthetic analogues of MDP free of undesirable side-effects, were synthesized in the Czech Republic. Lipophilic beta-D-GlcNstearoyl-(1- > 4)-norMurNAc-L-Abu-D-isoGln (DDD-St) was designed to be easily entrapped into liposomes and this liposomal DDD-St protected efficiently mice against irradiation, when administered i.p., i.v. or s.c. 24 h prior to lethal irradiation (survival rate in the range of 30-80% compared with 0% in control). Especially the subcutaneous application of liposomal DDD-St was very efficient. The parameters characteristic of recovery of haemopoiesis in bone marrow on day 10 after 6.5 Gy irradiation were significantly improved in comparison with the controls. Very high radioprotective effect of s.c. administered liposomal DDD-St can be explained (together with induction of haemopoiesis) by an effective and long-lasting activation of nonspecific immunity, which is able to withhold an onset of septicemia in early days after irradiation. In conclusion, the liposomal DDD-St should be therapeutically beneficial in moderating the haemopoietic damage, which is an undesirable effect of radiotherapy or chemotherapy.

Comparison of mouse and chick embryo liver and hepatoma cell lines as model systems used for enzymological estimation of toxicity potentials of organic pollutants.

Cytochromes P450-dependent monooxygenase activities were determined and compared in mouse liver microsomes and in hepatoma cell homogenates after exposure to prototype inducers of individual P450 enzymes. In vivo inductions of levels of mouse hepatic monooxygenase activities have been found as effective biochemical markers of toxicity potentials of a series of classes of xenobiotics (CYP1A induction for toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, coplanar polychlorinated biphenyls, polycyclic aromatic hydrocarbons and related pollutants; CYP2E induction for dialkylnitrosamines and organic solvents, e.g. acetone and ethanol; CYP2B and CYP3A induction for phenobarbital- and dexamethasone-type of xenobiotics). A specific induction of CYP1A-dependent O-dealkylase activities by TCDD was found in Hepa-1 and Hep G2 cell cultures, but no in vitro induction of other P450 enzymes was found after the treatment with phenobarbital, acetone or dexamethasone. Therefore, mouse liver is a suitable in vivo system for the testing of inducing effects of xenobiotics on all relevant P450 forms, while hepatoma cell cultures are usable only for the bioassay of TCDD-like toxicity.

Adjuvant effect of liposomes and adamantylamide dipeptide on antigenicity of entrapped synthetic peptide derived from HIV-1 transmembrane region glycoprotein gp41.

The synthetic peptide antigen (Ag) (the primary structure Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile- Cys-Thr derived from the envelope glycoprotein gp41 of the human immunodeficiency virus type 1 (HIV-1) and exerting specificity with all HIV-1-positive sera available in the Czech Republic (and also in a panel of 10,000 sera from WHO)) was conjugated with bovine serum albumin (BSA) and encapsulated into liposomes. Adjuvant activities of liposomes with various lipid compositions were compared with Freund's complete adjuvant (FCA) and with aluminium hydroxide (AL). The immune response to BSA-Ag liposomes with coentrapped adamantylamide dipeptide (AdDP) was comparable with that of FCA in terms of longevity and levels of specific antibodies in mouse sera.