RESUMO
These studies are focused on antagonizing organophosphorous (OP) intoxications by a new conceptual approach using recombinant enzymes encapsulated within sterically stabilized liposomes to enhance diisopropylfluorophosphate (DFP) degradation. The OP hydrolyzing enzyme, organophosphorous acid anhydrolase (OPAA), encapsulated within the liposomes, was employed either alone or in combination with pralidoxime (2-PAM) and/or atropine. The recombinant OPAA enzyme, from the ALTEROMONAS: strain JD6, has high substrate specificity toward a wide range of OP compounds, e.g., DFP, soman, and sarin. The rate of DFP hydrolysis by liposomes containing OPAA (SL)* was measured by determining the changes in fluoride-ion concentration using a fluoride ion-selective electrode. This enzyme carrier system serves as a biodegradable protective environment for the OP-metabolizing enzyme (OPAA), resulting in an enhanced antidotal protection against the lethal effects of DFP. Free OPAA alone showed some antidotal protection; however, the protection with 2-PAM and/or atropine was greatly enhanced when combined with (SL)*.
Assuntos
Inibidores da Colinesterase/toxicidade , Esterases/farmacologia , Isoflurofato/antagonistas & inibidores , Isoflurofato/toxicidade , Lipossomos , Animais , Arildialquilfosfatase , Portadores de Fármacos , Isoflurofato/metabolismo , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sarina/metabolismo , Soman/metabolismo , Especificidade por SubstratoRESUMO
This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.
Assuntos
Antídotos/administração & dosagem , Esterases/administração & dosagem , Intoxicação por Organofosfatos , Animais , Arildialquilfosfatase , Atropina/farmacologia , Hidrólise , Isoflurofato/farmacocinética , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Compostos de Pralidoxima/farmacologia , Proteínas Recombinantes/administração & dosagemRESUMO
Plasmodium circumsporozoite (CS) protein-induced antibody and T-cell responses are considered to be important in protective immunity. Since the key repeat determinant of the CS protein may actually restrict the recognition of other potential T- and B-cell sites, a modified Plasmodium falciparum CS protein lacking the central repeat region, RLF, was expressed in Escherichia coli. On purification, RLF was encapsulated into liposomes [L(RLF)] and used for the in vivo induction of cytolytic T lymphocytes (CTL) and antibodies. Immunization of B10.Br (H-2k) mice with L(RLF), but not with RLF, induced CD8+ CTL specific for the P. falciparum CS protein CTL epitope, amino acid residues 368-390. Anti-L(RLF) serum reacted with antigens on intact sporozoites and inhibited sporozoite invasion of hepatoma cells. Antibody specificity studies in New Zealand White rabbits revealed new B-cell sites localized in amino acid residues 84-94, 91-99, 97-106 and 367-375. Although the mechanisms by which liposomes enhance cellular and humoral immune responses remain unknown, liposome-formulated vaccines have been well tolerated in humans; hence, their use in vaccines, when efficacy depends on antibody and CTL responses, may be broadly applicable.
Assuntos
Ativação Linfocitária/efeitos dos fármacos , Vacinas Antimaláricas/administração & dosagem , Plasmodium falciparum/imunologia , Proteínas de Protozoários/administração & dosagem , Linfócitos T Citotóxicos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Formação de Anticorpos/efeitos dos fármacos , Sequência de Bases , Antígenos CD8/imunologia , Portadores de Fármacos , Epitopos/imunologia , Feminino , Imunização , Lipossomos , Ativação Linfocitária/imunologia , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/farmacologia , Camundongos , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/farmacologia , Coelhos , Sequências Repetitivas de Ácido Nucleico , Linfócitos T Citotóxicos/imunologiaRESUMO
This investigation effort is focused on increasing organophosphate (OP) degradation by phosphotriesterase to antagonize OP intoxication. For these studies, sterically stabilized liposomes encapsulating recombinant phosphotriesterase were employed. This enzyme was obtained from Flavobacterium sp. and was expressed in Escherichia coli. It has a broad substrate specificity, which includes parathion, paraoxon, soman, sarin, diisopropylfluorophosphate, and other organophosphorous compounds. Paraoxon is rapidly hydrolyzed by phosphotriesterase to the less toxic 4-nitrophenol and diethylphosphate. This enzyme was isolated and purified over 1600-fold and subsequently encapsulated within sterically stabilized liposomes (SL). The properties of this encapsulated phosphotriesterase were investigated. When these liposomes containing phosphotriesterase were incubated with paraoxon, it readily degraded the paraoxon. Hydrolysis of paraoxon did not occur when these sterically stabilized liposomes contained no phosphotriesterase. These sterically stabilized liposomes (SL) containing phosphotriesterases (SL)* were employed as a carrier model to antagonize the toxic effects of paraoxon by hydrolyzing it to the less toxic 4-nitrophenol and diethylphosphate. This enzyme-SL complex (SL)* was administered intravenously to mice either alone or in combination with pralidoxime (2-PAM) and/or atropine intraperitoneally. These results indicate that this carrier model system provides a striking enhanced protective effects against the lethal effects of paraoxon. Moreover when these carrier liposomes were administered with 2-PAM and/or atropine, a dramatic enhanced protection was observed.
Assuntos
Esterases/administração & dosagem , Inseticidas/intoxicação , Paraoxon/intoxicação , Animais , Arildialquilfosfatase , Portadores de Fármacos , Ponto Isoelétrico , Lipossomos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Paraoxon/antagonistas & inibidores , Compostos de Pralidoxima/farmacologia , Proteínas Recombinantes/administração & dosagemRESUMO
Seventeen malaria-naive volunteers received a recombinant Plasmodium falciparum vaccine (RLF) containing the carboxy- and the amino-terminal of the circumsporozoite protein (CSP) antigen without the central tetrapeptide repeats. The vaccine was formulated in liposomes with either a low or high dose of 3-deacylated monophosphoryl lipid A (MPL) and administered with alum by intramuscular injection. Both formulations were well tolerated and immunogenic. MPL increased sporozoite antibody titers measured by ELISA, Western blot, and immunofluorescence assay. One high-dose MPL vaccine formulation recipient developed a CSP-specific cytotoxic T lymphocyte response. After homologous sporozoite challenge, immunized volunteers developed patent malaria. There was no correlation between prepatent period and antibody titers to the amino- or carboxy-terminal. The absence of delay in patency argues against inclusion of the amino-terminal in future vaccines. A significant cytotoxic T lymphocyte response may have been suppressed by the inclusion of alum as an adjuvant.