ABSTRACT
The toxic effects of miltefosine on the epithelial cells of the gastrointestinal tract and its hemolytic action on erythrocytes have limited its use as an antileishmanial agent. As part of our search for new strategies to overcome the side effects of miltefosine during the treatment of leishmaniasis, we have developed stable miltefosine-loaded lipid nanoparticles in an attempt to reduce the toxic effects of the drug. We have evaluated lipid nanoparticles containing varying amounts of miltefosine and cholesterol, prepared by sonication, in terms of their physicochemical properties, preliminary stability, hemolytic potential toward erythrocytes, and cytotoxicity to macrophages and to promastigote and amastigote forms of Leishmania (L.) chagasi. Miltefosine loading into lipid nanoparticles was 100% for low drug concentrations (7.0 to 20.0mg/mL). Particle size decreased from 143nm (control) to between 43 and 69nm. From fluorescence studies, it was observed that the presence of miltefosine and cholesterol (below 103µM) promoted ordering effects in the phospholipid region of the nanoparticles. The formulation containing 15mg/mL miltefosine was stable for at least six months at 4°C and in simulated gastrointestinal fluids, and did not promote epithelial gastrointestinal irritability in Balb/C mice. When loaded into lipid nanoparticles, the hemolytic potential of miltefosine and its cytotoxicity to macrophages diminished, while its antiparasitic activity remained unaltered. The results suggested that miltefosine-loaded lipid nanoparticles may be promising for the treatment of leishmaniasis and might be suitable for oral and parenteral use.
Subject(s)
Drug Carriers/chemistry , Nanoparticles/chemistry , Phosphorylcholine/analogs & derivatives , Animals , Antiprotozoal Agents/administration & dosage , Cell Death/drug effects , Cells, Cultured , Drug Stability , Erythrocytes/drug effects , Gastrointestinal Diseases/drug therapy , Gastrointestinal Diseases/pathology , Hemolysis/drug effects , Humans , Lipids/chemistry , Macrophages/drug effects , Mice , Mice, Inbred BALB C , Particle Size , Phosphorylcholine/administration & dosage , RAW 264.7 CellsABSTRACT
This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.
Subject(s)
Chagas Disease/parasitology , Protozoan Proteins/genetics , Trypanosoma cruzi/growth & development , Trypanosoma cruzi/genetics , Amino Acid Sequence , Gene Expression Regulation, Developmental , Humans , Life Cycle Stages , Molecular Sequence Data , Protozoan Proteins/metabolism , Sequence Alignment , Trypanosoma cruzi/isolation & purification , Trypanosoma cruzi/metabolismABSTRACT
The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.
Subject(s)
Chromosome Mapping , Cysteine Endopeptidases/genetics , DNA, Protozoan/genetics , Expressed Sequence Tags , Leishmania/genetics , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Leishmania/enzymology , Molecular Sequence DataABSTRACT
The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32 percent are not related to any other sequences in database and 68 percent presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.
Subject(s)
Animals , Chromosome Mapping , Cysteine Endopeptidases/genetics , DNA, Protozoan/genetics , Expressed Sequence Tags , Leishmania/genetics , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , Leishmania/enzymology , Molecular Sequence DataABSTRACT
We report the molecular characterization of a novel reiterated family of transcribed oligo(A)-terminated, interspersed DNA elements in the genome of Trypanosoma cruzi. Steady-state level of transcripts of this sequence family appeared to be developmentally regulated, since only in the replicative forms the parasite showed expression of related sequences with a major band around 3 kb. The presence of frame shifts or premature stop codons predicts that transcripts are not translated. The sequence family also contains truncated forms of retrotransposons elements that may become potential hot spots for retroelement insertion. Sequences homologous to this family are interspersed at many chromosomes including the subtelomeric regions
Subject(s)
Animals , DNA, Protozoan , Genome, Protozoan , Interspersed Repetitive Sequences , Trypanosoma cruziSubject(s)
Male , Female , Humans , Chagas Disease/genetics , Trypanosoma cruzi/genetics , Trypanosoma cruzi/immunologyABSTRACT
No Trypanosoma cruzi, agente etiológico da doença de Chagas, a maioria das proteínas antigênicas descritas até hoje possuem domínios repetitivos. Em trabalhos anteriores nosso grupo isolou um clone recombinante genômico, chamado H49, que codifica repetições de 68 aminoácidos presentes em um antígeno de alto peso molecular (Cotrim et al., 1990). O antígeno H49 é uma proteína estrutural associada ao citoesqueleto do parasita, localizando-se na região de interação do flagelo com o corpo celular (Cotrim et al., 1990 e 1995). Levin e col. (1989) isolaram um clone de cDNA, denominado JL8, codificando repetições de 14 aminoácidos presentes em uma proteína de 200 kb. O antígeno JL8 tem localização citoplasmática e sua função ainda não foi determinada (Lafaille et al., 1989, Levin et al., 1989). Os antígenos H49 e JL8 contém epitopos imunodominantes reconhecidos por anticorpos presentes na maioria dos soros de pacientes chagásicos crônicos (Franco da Silveira, 1992). O objetivo do nosso trabalho foi anlisar a organização dos genes h49 e jL8, e construir um mapa físico da região cromosômica que contém os loci h49 e jL8. Isto foi feito por análises de "Southern blot" contendo megafragmentos genômicos obtidos por digestões com enzimas de restrição que raramente tem sítios de reconhecimento. Utilizamos também o isolamento de clones YAC com sobreposição ("overlapping") constituindo um "cotig"dos cromosomas. Análises de restrição do DNA genômico e sequencialmento de clones genômicos e de cDNA indicaram que a maioria da proteína H49 é constituída de repetições de 68 aminoácidos, dispostas em tandem. O tamanho do gene foi estimado ser de aproximadamente 8 kb, baseando-se no tamanho do transcrito e da proteína nativa (300 kDa). A conservação dos nucleotídeos entre as repetições indicam que a homogeneização seletiva de sequência, as dá provalvemente através de conservação gênica mantendo conservada a sequência de aminoácidos. As formas alélicas do gene h49 foram mapeadas em dois fragmentos genômicos de cerca de 20 kb gerado por digestão com a enzima de restrição EcoRI. Em algumas cepas um alelo adicional foi localizado em um fragmento Eco RI de 9,7 kb. Baseando-se no tamanho do gene h49 (8 kb) nós assumimos que o fragmento genômico de 9,7 kb deve conter uma cópia do gene h49 enquanto o fragmento de 20 kb conteria duas cópias. Em várias cepas de T. cruzi, os genes h49 e jL8 foram mapeados em duas bandas cromosômicas de 2,6 Mb. ... (au)
