Flow cytometric assessment of Leishmania spp metacyclic differentiation: Validation by morphological features and specific markers

Saraiva, Elvira M.; Pinto-da-Silva, Lúcia Helena; Wanderley, João Luiz Mendes; Bonomo, Adriana; Barcinski, Marcello A.; Moreira, M E C

doi:10.1016/j.exppara.2005.01.004

Cited by 45 publications

(51 citation statements)

References 27 publications

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“…As the mean life of AZT in serum is of 1.5 h (Nascimento et al 2004), the inhibitory activity of this drug only on the first 24 h of exposition may be explained due to the drug's phosphorilation by the parasite. Saraiva et al (2005) report that stationary phase promastigotes present body dimensions of 8.68×1.2-1.5 μm which is in accordance to the morphometry of promastigotes from our control groups. Also Ueda-Nakamura et al (2001) reported the morphological characteristics of L. amazonensis promastigotes from the 3rd day of culture which are in accordance to the ones found in our study.…”

Section: Discussionsupporting

confidence: 88%

“…The stationary phase is characterized by a stasis of growth with increase in infectivity. This differentiation process is called metaciclogenesis and is vital to the parasite's survival (Gossage et al 2003;Saraiva et al 2005). Some studies have been conducted to evaluate the in vitro promastigotes morphological alterations induced by drugs as to determine their toxicity and anti-leishmanial activities ).…”

Section: Introductionmentioning

confidence: 99%

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Morphological alterations and growth inhibition of Leishmania (L.)amazonensis promastigotes exposed to zidovudine (AZT)

Araújo

Batista

et al. 2010

Parasitol Res

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Leishmania parasites cause a worldwide public health disease and its treatment is still based on pentavalent antimonials which present financial and toxicologic limitations. Some nucleosidic derivatives have demonstrated anti-leishmanial properties and this study aims to evaluate the in vitro morphologic alterations and growth inhibition of Leishmania (L.) amazonensis promastigotes exposed to zidovudine at several concentrations. The citotoxicity of zidovudine (AZT) to macrophages was determined by an MTT assay. After which the promastigotes were exposed to concentrations of AZT, ranging from 1 to 50 μM. The evaluation of survival and morphometry alterations were performed in two distinct phases of in vitro growth, on the third and sixth days, representing the logarithmic and stationary phases, respectively. Slides with the promastigotes were photographed and analyzed using Image J. A significant reduction of parasite number in the logarithmic phase of in vitro growth was observed when the parasites were submitted to 20, 30, 40, and 50 μM of AZT. Morphometric alterations were observed such as an increase in width of the body, cytoplasmic granulations and vacuolizations. These data indicate the toxicity of AZT which prevents the parasite's multiplication, indicating a promising use of AZT as an anti-leishmania drug.

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Section: Discussionsupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Morphological alterations and growth inhibition of Leishmania (L.)amazonensis promastigotes exposed to zidovudine (AZT)

Araújo

Batista

et al. 2010

Parasitol Res

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“…In view of the morphological differences between the procyclic and metacyclic forms, flow cytometric analysis can be applied for the discrimination of both forms (28). Briefly, nonfluorimetric measurements were performed (Cell Lab Quanta; Beckman Coulter) by suspending 10 l of promastigote culture in 1 ml phosphate-buffered saline and analyzing the light scatter of the mixture.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Sensitivity Testing of Leishmania Clinical Field Isolates: Preconditioning of Promastigotes Enhances Infectivity for Macrophage Host Cells

Luz

Vermeersch

Dujardin

et al. 2009

Antimicrob Agents Chemother

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Diagnostic material from patients with leishmaniasis is generally available as promastigotes, and proper testing for susceptibility to first-line drugs by the intracellular amastigote assay is frequently hampered by the poor infectivity of the promastigotes for the macrophage host cell. Several conditions for optimization of the in vitro metacyclogenesis and cell infectivity of Leishmania donovani, L. guyanensis, and L. braziliensis field strains obtained from patients receiving standard antimony medication were investigated. Triggering logphase promastigotes to become amastigote-like by increasing the temperature or acidifying the culture medium was not successful. Adequate metacyclogenesis and the highest levels of macrophage infection were obtained after 5-day-old late-log-phase promastigote cultures were preconditioned at 25°C to pH 5.4 for 24 h in Schneider's medium prior to infection. The susceptibility assay with primary peritoneal mouse macrophages included pentavalent antimony (Sb V ; sodium stibogluconate), trivalent antimony (Sb III ; potassium antimonyl tartrate), miltefosine, and the experimental drug PX-6518. All strains were sensitive to miltefosine (50% inhibitory concentration [IC 50 ] < 10 M) and PX-6518 (IC 50 < 2 g/ml) but showed distinct susceptibility to Sb V and/or Sb III , depending on whether they were derived from cured, relapse, or nonresponder patients. Within the available set of Leishmania species and strains, simultaneous Sb V -Sb III resistance was clearly associated with treatment failure; however, a larger set of isolates is still needed to judge the predictive value of Sb V -Sb III susceptibility profiling on treatment outcome. In conclusion, the proposed conditioning protocol further contributes toward a more standardized laboratory model for evaluation of the drug sensitivities of field isolates.As the failure of first-line treatment for all clinical forms of leishmaniasis is a growing problem, it is pivotal to monitor the efficacy of standard drugs and map the prevalence of drug resistance in areas where the disease is endemic (7,9,24). Diagnostic field isolates are mostly provided to the laboratory as promastigotes, but it remains an experimental challenge to appropriately adapt them to the amastigote-macrophage model, still considered the "gold standard" for susceptibility evaluation (23, 31). Infection of the macrophage is generally achieved with metacyclic promastigotes, but unfortunately, infection is subject to a high degree of variability, among several other factors affecting the outcome of the sensitivity test (8, 10). These factors strongly suggest the need for the further standardization of susceptibility testing of clinical field isolates.In the normal course of events, infective metacyclic promastigotes are inoculated by the sand fly into the mammalian host, where they rapidly penetrate susceptible cells, undergo intracellular transformation to the amastigote form, and start dividing. The procyclic and metacyclic phases observed during in vitro culture appear to...

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“…Once in the vertebrate host, Leishmania metacyclic promastigotes interact with macrophages through ligand receptor-mediated residues on their surfaces and mannose receptors on the macrophage surface (Mosser et al 1987, Da Silva et al 1989. Metacyclic promastigotes interact with macrophages differently than non-infective forms (Courret et al 1999, Saraiva et al 2005 and they have been shown to impair the capacity of macrophages to present antigens (Courret et al 1999). However, most studies use stationary phase cultures, which contain mostly (80%) non-infective forms, for experimental infections (Scott 1989, da Costa et al 1992, Afonso & Scott 1993, Santiago et al 1999, Ji et al 2003.…”

mentioning

confidence: 99%

Low and high-dose intradermal infection with Leishmania majorand Leishmania amazonensis in C57BL/6 mice

Côrtes

Carneiro

Santos

et al. 2010

Mem. Inst. Oswaldo Cruz

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Flow cytometric assessment of Leishmania spp metacyclic differentiation: Validation by morphological features and specific markers

Cited by 45 publications

References 27 publications

Morphological alterations and growth inhibition of Leishmania (L.)amazonensis promastigotes exposed to zidovudine (AZT)

Morphological alterations and growth inhibition of Leishmania (L.)amazonensis promastigotes exposed to zidovudine (AZT)

In Vitro Sensitivity Testing of Leishmania Clinical Field Isolates: Preconditioning of Promastigotes Enhances Infectivity for Macrophage Host Cells

Low and high-dose intradermal infection with Leishmania majorand Leishmania amazonensis in C57BL/6 mice

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