A simple and reproducible method to obtain large numbers of axenic amastigotes of different Leishmania species

Abstract

This work describes a simple method to yield large amounts of Leishmania amastigote-like forms in axenic cultures using promastigotes as the starting population. The method described induced extracellular amastigote transformation of Leishmania amazonensis (97%), Leishmania braziliensis (98%) and Leishmania chagasi (90%). The rounded parasites obtained in axenic cultures were morphologically similar, even at the ultrastructural level, to intracellular amastigotes. Moreover, the axenic amastigotes remained viable as measured by their ability to revert back to promastigotes and to infect BALB/c mice. L. amazonensis and L. braziliensis promastigotes and axenic amastigotes differed in terms of their Western blot profiles. A 46 kDa protein was recognized by specific antibodies only in axenic and lesion-derived L. amazonensis amastigotes and not in promastigotes.

Enhancement of Experimental Cutaneous Leishmaniasis by Leishmania Molecules Is Dependent on Interleukin-4, Serine Protease/Esterase Activity, and Parasite and Host Genetic Backgrounds

“…Despite the fact that axenic amastigotes are not completely identical to lesion-derived amastigotes in terms of protein makeup (34) and mRNA expression pattern (28), their extract promoted the Leishmania infection to a greater degree than the promastigote extract.…”

“…Promastigotes, derived from tissue amastigotes, were cultured at 23°C in Schneider's medium (Sigma Chemical Co., St. Louis, MO), pH 7.2, supplemented with 50 g/ml of gentamicin and 10% heat-inactivated fetal bovine serum (FBS; Gibco Laboratories, Grand Island, NY) for L. amazonensis or 20% FBS for L. braziliensis. L. amazonensis and L. braziliensis axenic amastigotes were obtained by the differentiation of promastigotes in axenic cultures, as described elsewhere (34). The amastigotes were washed three times in ice-cold sterile saline, resuspended in saline, and lysed by exposure to ultrasound (10 1-min, 300-W pulses, with 30-s intervals in between, on ice; Sonifier cell disruptor; Branson Sonic Power Company, Danbury, CT).…”

LeishmaniaInfection Impairs β1-Integrin Function and Chemokine Receptor Expression in Mononuclear Phagocytes

“…Leishmania braziliensis (MHOM/BR/3456) and L. amazonensis (Leila strain, MHOM/BR88/ BA-125) were grown in Schneider's insect medium (Sigma) containing 10% (L. amazonensis) or 20% (L. braziliensis) FBS at 24°C (47). Parasites were washed three times in HBSS, suspended in complete RPMI, and incubated with the phagocytes.…”

Chemical constituents and leishmanicidal activity from leaves of Kielmeyera variabilis

“…Solutions at 200 μg/mL in DMSO (1% in water) were performed for each fraction or compound and then ten microliters of the solution were added to each well of 96-well plates. The plates were incubated at 32 ºC for 72 h, and then the cells viability was determined using the MTT (methyl thiazolyl tetrazolium) assay (Teixeira et al, 2002). The results were expressed as percent inhibition in relation to the controls without drug.…”