HIV Aspartyl Peptidase Inhibitors Interfere with Cellular Proliferation, Ultrastructure and Macrophage Infection of Leishmania amazonensis

Submitted by Sandra Infurna ([email protected]) on 2019-01-08T13:43:09Z No. of bitstreams: 1 Ellenf_Altoe_etal_IOC_2009.pdf: 1452755 bytes, checksum: 77127a59920cef6bca71296107f6ec63 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-01-08T13:51:34Z (GMT) No. of bitstreams: 1 Ellenf_Altoe_etal_IOC_2009.pdf: 1452755 bytes, checksum: 77127a59920cef6bca71296107f6ec63 (MD5)Made available in DSpace on 2019-01-08T13:51:34Z (GMT). No. of bitstreams: 1 Ellenf_Altoe_etal_IOC_2009.pdf: 1452755 bytes, checksum: 77127a59920cef6bca71296107f6ec63 (MD5) Previous issue date: 2009Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Microbiologia Prof. Paulo de Góes. Departamento de Microbiologia Geral,. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Microbiologia Prof. Paulo de Góes. Departamento de Microbiologia Geral,. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Microbiologia Prof. Paulo de Góes. Departamento de Microbiologia Geral,. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis

Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis

Submitted by Sandra Infurna ([email protected]) on 2020-03-20T18:40:39Z No. of bitstreams: 1 VitorEVidal_ClaudiaLevy_etal_IOC_2019.pdf: 2297099 bytes, checksum: cb1aca4fd57f8498a88ea0341a2b9d0b (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2020-03-20T18:52:50Z (GMT) No. of bitstreams: 1 VitorEVidal_ClaudiaLevy_etal_IOC_2019.pdf: 2297099 bytes, checksum: cb1aca4fd57f8498a88ea0341a2b9d0b (MD5)Made available in DSpace on 2020-03-20T18:52:50Z (GMT). No. of bitstreams: 1 VitorEVidal_ClaudiaLevy_etal_IOC_2019.pdf: 2297099 bytes, checksum: cb1aca4fd57f8498a88ea0341a2b9d0b (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Estudos Integrados em Protozoologia. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Estudos Integrados em Protozoologia. Rio de Janeiro, RJ, Brasil..Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular de Parasitas e Vetores. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Laboratório de Imunofarmacologia Parasitária. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Laboratório de Estudos Avançados de Microrganismos Emergentes e Resistentes. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Estudos Integrados em Protozoologia. Rio de Janeiro, RJ, Brasil.Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome

Effect of HIV PIs on the growth rate of <i>Leishmania amazonensis.</i>

<p>The growth pattern of <i>L. amazonensis</i> was followed for parasites cultivated at 26°C in the absence (control) or presence of lopinavir, nelfinavir, amprenavir, saquinavir and indinavir at 50 µM (HIV PIs). Subsequently, lopinavir and nelfinavir were assayed in concentrations ranging from 15 to 50 µM. Amprenavir, saquinavir and indinavir were tested in concentrations ranging from 50 to 500 µM. The PIs were added to the cultures at 0 hour and viable cells were counted daily by trypan blue exclusion and motility. In all experiments, the data from DMSO represents the concentration present in the highest dose of each compound. Data shown are the mean±standard error (S.E.) of three independent experiments performed in triplicate.</p

Effect of the pre-treatment of promastigotes with HIV PIs on the <i>Leishmania amazonensis</i>–macrophage interaction.

<p>The promastigotes of <i>L. amazonensis</i> were treated or not with nelfinavir, amprenavir or lopinavir (as indicated) for 1 hour prior to macrophage–parasite interactions, then the cells were washed with PBS. Parasites maintained their viability under this experimental condition (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004918#s2" target="_blank">Methods</a> section). Macrophages were then infected with promastigote forms for 1 hour at 37°C, and macrophage monolayers were washed with PBS to remove unbound parasites. The association index was determined after 1 hour of infection by light microscopy, counting at least 200 cells in each of duplicated coverslips. Each bar represents the mean±standard error of at least three independent experiments.</p

Effect of HIV PIs on peptidase expression by <i>L. amazonensis.</i>

<p>Differential peptidase expression observed in <i>L. amazonensis</i> promastigote forms grown in the absence (control) or in the presence of nelfinavir, amprenavir and lopianvir at 100 µM. Gelatin-SDS-PAGE: The gel strips, containing the equivalent to 5×10⁶ cells of parasite extract obtained after 4 hours of incubation with each inhibitor, were incubated at 37°C for 48 hours in 50 mM sodium phosphate buffer (pH 5.5) supplemented with 2 mM DTT. Numbers on the left indicate relative molecular mass of the peptidases, expressed in kilodaltons (kDa). Immunoblotting: anti-gp63 or anti-cpb antibodies were employed to detect gp63- and cpb-like molecules, respectively, in the whole cellular extract from <i>L. amazonensis</i> grown in the absence (control) or in the presence of nelfinavir, lopianvir and amprenavir for 4 hours at 26°C; Anti-tubulin monoclonal antibody was used as a control for sample loading in the gels. The graphics represent the densitometric measurements of the proteolytic halos observed in the gelatin-SDS-PAGE. The values represent mean±standard deviation of three independent measurements. Similar densitometric analyses were performed using the reactive polypeptides detected in the immunobloting assays (data not shown). Symbol denotes systems treated with PIs that had densitometric units significantly different from the control (<i>P</i><0.05; Student's t test).</p

Toxicity of HIV PIs in mouse peritoneal macrophages and susceptibility of intracellular parasites to HIV PIs in macrophages.

<p>Initially, the macrophages (1×10⁵ cells) were incubated in a 96-well plate for 24 hours in the absence or in the presence of different concentrations (as indicated) of the following PIs: nelfinavir, amprenavir and lopinavir. After this period, the viability of the macrophage cells was determined spectrophotometrically at 490 nm by means of MTT assay. The dotted line separates the graphic in two portions: minor than and equal or major than 95% of macrophage viability. Control represents untreated macrophages (A). After that, we studied the susceptibility of intracellular parasites to HIV PIs in macrophages. Macrophages were infected with promastigotes of <i>L. amazonensis</i> for 1 hour at 37°C, followed by exhaustive washing in PBS and then the interaction was incubated for additional 24 hours in the absence (control), or in the presence of nelfinavir, amprenavir or lopinavir as indicated. The concentrations employed correspond to those in which more than 95% of the macrophage cells were viable as demonstrated in (A). Finally, the association index was determined by light microscopy, counting at least 200 cells in each of duplicated coverslips. Control system is shown as 100%. Each bar represents the mean±standard error of at least three independent experiments (B).</p