Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species

Amidine derivatives and Leishmania amazonensis: an evaluation of the effect of nitric oxide (NO) production on the parasite-macrophage interaction

Submitted by Sandra Infurna ([email protected]) on 2019-12-17T11:08:40Z No. of bitstreams: 1 RM_Temporal_etal_IOC_2005.pdf: 408055 bytes, checksum: c93f1871cc8d38d92b4333b26a76198b (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-12-17T11:27:26Z (GMT) No. of bitstreams: 1 RM_Temporal_etal_IOC_2005.pdf: 408055 bytes, checksum: c93f1871cc8d38d92b4333b26a76198b (MD5)Made available in DSpace on 2019-12-17T11:27:26Z (GMT). No. of bitstreams: 1 RM_Temporal_etal_IOC_2005.pdf: 408055 bytes, checksum: c93f1871cc8d38d92b4333b26a76198b (MD5) Previous issue date: 2005Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ, Brasil.Universidade Federal Rural do Rio de Janeiro. Departamento de Química. Seropédica, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ, Brasil.Previous work has demonstrated that N-N'-diphenyl-R-benzamidine was highly effective against Leishmania amazonensis promastigotes/axenic amastigotes and Trypanosoma evansi trypomastigotes and the compound with a methoxy substituent, was the most effective derivative in the parasite-macrophage interaction. Comparative analysis of the nitric oxide (NO) released from the culture infection's supernatant showed the amidine to be less effective than pentamidine Isethionate as a reference drug. Additionally, in order to verify if the methoxylated derivative interferes with NO production by L. amazonensis, the effect of the amidine on the constitutive nitric oxide synthase (cNOS) purified from parasites, was examined, but demonstrated less activity in comparison with the reference drug. This data contributes to studies concerning the metabolic targets present in Leishmania parasites for leishmanicidal drugs

Effect of mesoionic 4-phenyl-5-(cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivative salts on the activities of the nitric oxide synthase and arginase of Leishmania amazonensis

Submitted by Sandra Infurna ([email protected]) on 2019-04-11T10:37:23Z No. of bitstreams: 1 LeonorL_Leon_etal_IOC_2008.pdf: 442059 bytes, checksum: 3a58dab3266d52431ed8e7c5b3d66307 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-04-11T10:45:58Z (GMT) No. of bitstreams: 1 LeonorL_Leon_etal_IOC_2008.pdf: 442059 bytes, checksum: 3a58dab3266d52431ed8e7c5b3d66307 (MD5)Made available in DSpace on 2019-04-11T10:45:58Z (GMT). No. of bitstreams: 1 LeonorL_Leon_etal_IOC_2008.pdf: 442059 bytes, checksum: 3a58dab3266d52431ed8e7c5b3d66307 (MD5) Previous issue date: 2008Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ. Brasil.Universidade Federal Rural do Rio de Janeiro. Departamento de Química. Seropédica, RJ, Brasil.Universidade Federal Rural do Rio de Janeiro. Departamento de Química. Seropédica, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Imunologia. Rio de Janeiro, RJ. Brasil.L-arginine is involved in the production of both nitric oxide (NO), mediated by nitric oxide synthase (NOS) and L-ornithine, by arginase activity. It is generally accepted that NO regulation occurs mainly at the transcriptional level of NOS. In a previous work we purported that there is evidence that Leishmania sp. can produce NO from L-arginine. An arginase activity in its gene sequence has also been reported in Leishmania parasites. In a search for intracellular targets as potential antileishmanicidal agents, such as the L-arginine metabolism, we used 1,3,4-thiadiazolium mesoionic compounds, that have been demonstrated to be cytotoxic to the Leishmania amazonensis, when compared to Pentamidine isethionate as a reference drug. Parasites were assayed in absence/presence of 4'- and 3'-methoxy mesoionic derivatives in order to verify the effect on NO production and arginase activity in L. amazonensis. The results indicated that the drugs reduce from 70 to 90% of the NO production by the parasite and act on a soluble nitric oxide synthase purified from L. amazonensis promastigotes and axenic amastigotes