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1.
Parasitol Res ; 106(6): 1327-37, 2010 May.
Article in English | MEDLINE | ID: mdl-20237802

ABSTRACT

The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported. Mouse immune sera depleted complement-induced damage in epimastigotes characterized by morphological changes and death. The purpose of this work was to study the mechanism of death in epimastigotes exposed to decomplemented mouse immune serum. Epimastigotes were maintained in RPMI medium. Immune sera were prepared in mice by immunization with whole crude epimastigote extracts. Viable epimastigotes were incubated with decomplemented normal or immune sera at 37 degrees C. By electron microscopy, agglutinated parasites showed characteristic patterns of membrane fusion between two or more parasites; this fusion also produced interdigitation of the subpellicular microtubules. Apoptosis was determined by flow cytometry using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V assays. Nuclear features were examined by 4'-,6-diamidino-2'-phenylindole diHCI cytochemistry that demonstrated apoptotic nuclear condensation. Caspase activity was also measured. TUNEL results showed that parasites incubated with decomplemented immune sera took up 26% of specific fluorescence as compared to 1.3% in parasites incubated with decomplemented normal sera. The Annexin-V-Fluos staining kit revealed that epimastigotes incubated with decomplemented immune sera exposed phosphatidylserine on the external leaflet of the plasma membrane. The incubation of parasites with immune sera showed caspase 3 activity. We conclude that specific antibodies are able to induce agglutination and apoptosis in epimastigotes, although the pathway is not elucidated.


Subject(s)
Antibodies, Protozoan/immunology , Apoptosis , Complement System Proteins/immunology , Trypanosoma cruzi/immunology , Animals , Annexin A5/analysis , Caspase 3/analysis , Female , In Situ Nick-End Labeling , Mice , Microbial Viability , Microscopy, Electron , Trypanosoma cruzi/chemistry , Trypanosoma cruzi/ultrastructure
2.
Exp Parasitol ; 123(4): 362-8, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19735657

ABSTRACT

This study describes the first days of Taenia crassiceps infection in BALB/c substrains, BALB/cAnN and BALB/cJ, using two stocks of the same strains which were kept in different animal facilities, conventional and pathogen-free conditions, respectively. This study shows that parasite growth restriction shown by conventional BALB/cJ mice changed to parasite growth permissiveness when pathogen-free BALB/cJ mice were used. In addition, the higher number of macrophages, NK cells and intraperitoneal level of IFN-gamma found in the conventional restrictive BALB/cJ substrain vanished when the permissiveness to the parasite growth increased. No differences were found in DNA sequences of parasites collected before and after the change in the permissiveness to parasite growth which favors the possibility that the observed modifications could be due to changes in the murine strains and/or their maintenance conditions.


Subject(s)
Cysticercosis/immunology , Cysticercus/growth & development , Animals , Cysticercosis/parasitology , Cysticercus/genetics , Cysticercus/immunology , DNA, Helminth/chemistry , DNA, Intergenic/chemistry , Female , Flow Cytometry , Interferon-gamma/metabolism , Mice , Mice, Inbred BALB C , Peritoneum/cytology , Peritoneum/immunology , Polymorphism, Genetic , Specific Pathogen-Free Organisms
3.
Exp Parasitol ; 121(3): 199-207, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19041644

ABSTRACT

Macrophages (Mphi) and dendritic cells (DC) are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a method employed by multiple pathogens to ensure their survival in the infected cell. Leishmania has been shown to protect Mphi and neutrophils from both natural and induced apoptosis. As shown in this study, apoptosis in monocyte-derived dendritic cells (moDC) induced by treatment with camptothecin was downregulated by coincubation with L. mexicana, as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low molecular weight DNA fragments, and annexin V binding to phosphatidylserine. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in moDC. The capacity of L. mexicana to delay apoptosis induction in the infected moDC may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/physiology , Camptothecin/pharmacology , Dendritic Cells/parasitology , Leishmania mexicana/physiology , Animals , Apoptosis/drug effects , Azure Stains , Caspase 3/metabolism , Cell Survival , Cells, Cultured , DNA Fragmentation , Dendritic Cells/cytology , Dendritic Cells/drug effects , Down-Regulation , Flow Cytometry , Humans , In Situ Nick-End Labeling , Monocytes/cytology
4.
PLoS Negl Trop Dis ; 1(1): e1, 2007 Oct 31.
Article in English | MEDLINE | ID: mdl-17989778

ABSTRACT

BACKGROUND: Chagas disease affects around 18 million people in the American continent. Unfortunately, there is no satisfactory treatment for the disease. The drugs currently used are not specific and exert serious toxic effects. Thus, there is an urgent need for drugs that are effective. Looking for molecules to eliminate the parasite, we have targeted a central enzyme of the glycolytic pathway: triosephosphate isomerase (TIM). The homodimeric enzyme is catalytically active only as a dimer. Because there are significant differences in the interface of the enzymes from the parasite and humans, we searched for small molecules that specifically disrupt contact between the two subunits of the enzyme from Trypanosoma cruzi but not those of TIM from Homo sapiens (HTIM), and tested if they kill the parasite. METHODOLOGY/PRINCIPAL FINDINGS: Dithiodianiline (DTDA) at nanomolar concentrations completely inactivates recombinant TIM of T. cruzi (TcTIM). It also inactivated HTIM, but at concentrations around 400 times higher. DTDA was also tested on four TcTIM mutants with each of its four cysteines replaced with either valine or alanine. The sensitivity of the mutants to DTDA was markedly similar to that of the wild type. The crystal structure of the TcTIM soaked in DTDA at 2.15 A resolution, and the data on the mutants showed that inactivation resulted from alterations of the dimer interface. DTDA also prevented the growth of Escherichia coli cells transformed with TcTIM, had no effect on normal E. coli, and also killed T. cruzi epimastigotes in culture. CONCLUSIONS/SIGNIFICANCE: By targeting on the dimer interface of oligomeric enzymes from parasites, it is possible to discover small molecules that selectively thwart the life of the parasite. Also, the conformational changes that DTDA induces in the dimer interface of the trypanosomal enzyme are unique and identify a region of the interface that could be targeted for drug discovery.


Subject(s)
Triose-Phosphate Isomerase/metabolism , Trypanosoma cruzi/drug effects , Aniline Compounds/pharmacology , Animals , Chagas Disease/drug therapy , Chagas Disease/epidemiology , Cysteine/analysis , Dimerization , Escherichia coli/enzymology , Escherichia coli/genetics , Humans , Incidence , Kinetics , Models, Molecular , Protein Conformation , Recombinant Proteins/drug effects , Sequence Deletion , Triose-Phosphate Isomerase/chemistry , Triose-Phosphate Isomerase/genetics , Trypanosoma cruzi/enzymology , Trypanosoma cruzi/growth & development , X-Ray Diffraction
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