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1.
Antimicrob Agents Chemother ; 54(12): 5028-41, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20855728

RESUMO

The papain-like cysteine cathepsins expressed by Leishmania play a key role in the life cycle of these parasites, turning them into attractive targets for the development of new drugs. We previously demonstrated that two compounds of a series of peptidomimetic aziridine-2,3-dicarboxylate [Azi(OBn)(2)]-based inhibitors, Boc-(S)-Leu-(R)-Pro-(S,S)-Azi(OBn)(2) (compound 13b) and Boc-(R)-Leu-(S)-Pro-(S,S)-Azi(OBn)(2) (compound 13e), reduced the growth and viability of Leishmania major and the infection rate of macrophages while not showing cytotoxicity against host cells. In the present study, we characterized the mode of action of inhibitors 13b and 13e in L. major. Both compounds targeted leishmanial cathepsin B-like cysteine cathepsin cysteine proteinase C, as shown by fluorescence proteinase activity assays and active-site labeling with biotin-tagged inhibitors. Furthermore, compounds 13b and 13e were potent inducers of cell death in promastigotes, characterized by cell shrinkage, reduction of mitochondrial transmembrane potential, and increased DNA fragmentation. Transmission electron microscopic studies revealed the enrichment of undigested debris in lysosome-like organelles participating in micro- and macroautophagy-like processes. The release of digestive enzymes into the cytoplasm after rupture of membranes of lysosome-like vacuoles resulted in the significant digestion of intracellular compartments. However, the plasma membrane integrity of compound-treated promastigotes was maintained for several hours. Taken together, our results suggest that the induction of cell death in Leishmania by cysteine cathepsin inhibitors 13b and 13e is different from mammalian apoptosis and is caused by incomplete digestion in autophagy-related lysosome-like vacuoles.


Assuntos
Autofagia , Aziridinas/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Leishmania major/efeitos dos fármacos , Lisossomos/metabolismo , Vacúolos/efeitos dos fármacos , Animais , Leishmania major/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Vacúolos/ultraestrutura
2.
Vaccine ; 28(36): 5785-93, 2010 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-20615489

RESUMO

Upon loading with parasite antigen and adoptive transfer, dendritic cells (DC) are able to confer protection against the protozoan parasite Leishmania major. In the present study, we investigated whether viable DC are required for inducing protection. We provide evidence that L. major antigen-loaded DC that had been fixed with paraformaldehyde or exposed to UV irradiation, and even disrupted cells, are able to serve as an effective vaccine. Furthermore, we demonstrate the potential of DC-derived exosomes to mediate protective immunity against cutaneous leishmaniasis. The route of antigen presentation to recipient T cells involves uptake of intravenously injected DC fragments into late endosomal compartments of splenic DC in the recipient. In vitro studies showed that DC fragments induce T-cell proliferation and interleukin 12 secretion by splenocytes. Together, these findings suggest that the development of a cell-free vaccine for immunoprophylaxis against leishmaniasis and other infectious diseases is feasible.


Assuntos
Antígenos de Protozoários/imunologia , Células Dendríticas/imunologia , Exossomos/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/imunologia , Transferência Adotiva , Animais , Apresentação de Antígeno , Células Cultivadas , Células Dendríticas/parasitologia , Feminino , Interleucina-12/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologia , Linfócitos T/imunologia
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