Cultivation and characterization of stable Leishmania guyanensis complex axenic amastigotes derived from infected U937 cells.

Abstract

Abstract. The study of the differential regulation of several genes, in both Leishmania parasite life cycle forms, has been simplified by the development of in vitro axenic amastigote culture. Different reports have described extracellular amastigote production and maintenance from several Leishmania spp. A general approach to induce amastigote-like transformation includes progressive pH and temperature changes. Production of axenic amastigotes in continuous cultures using amastigotes recovered from macrophages is described in this report. Leishmania (Viannia) panamensis (M/HOM/PA/71/LS94) and Leishmania (V). guyanensis (M/HOM/BR/75/M4147) intracellular amastigotes were recovered from the human macrophage-like U937 cell line previously infected with promastigotes. The parasites were immediately adapted for growth and kept as axenic amastigotes at 34ЊC and acidic pH. These organisms were able to infect macrophage cell lines, maintain amastigote morphologic features, and express stage-specific transcripts. The relevance of axenic amastigotes in characterizing virulence factors in American leishmaniasis is discussed.

Cultivation of Clinically Significant Hemoflagellates

“…Variation exists between Leishmania spp. on susceptibility to heat shock-induced transformation (37,81,104). Heat shocked leishmanias were more likely to be abnormal promastigotes rather than bona fide amastigotes, in contrast to the real-life situation, in which insect-borne promastigotes are injected into the mammalian host at about 37°C, penetrating a macrophage, and proliferating (80).…”

“…Puentes et al (81) isolated amastigotes of L. guyanensis and L. panamensis from infected U937 human macrophage-like cells in RPMI 1640 medium with 20% fetal calf serum at a pH of 5.0, establishing them in culture using the same medium at pH 5.5 and 34°C in 5% CO 2 . Amastigote growth over a period of 9 to 10 days produced populations of 4 ϫ 10 7 to 5 ϫ 10 7 cells/ml, with doubling times of 20 to 22 h. Among the criteria used for amastigote characterization were morphological fea- tures, surface antigens, thymidine incorporation, cysteine proteinase production, amastigote-specific gene markers, and, finally, infectivity for U937 cells (81).…”

“…Amastigote growth over a period of 9 to 10 days produced populations of 4 ϫ 10 7 to 5 ϫ 10 7 cells/ml, with doubling times of 20 to 22 h. Among the criteria used for amastigote characterization were morphological fea- tures, surface antigens, thymidine incorporation, cysteine proteinase production, amastigote-specific gene markers, and, finally, infectivity for U937 cells (81). Whole-tissue-based cultivation.…”

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

“…The Leishmania in vitro culture contributes considerably to our knowledge of the parasite's biology 24 . In its development cycle, Leishmania presents under two different aspects, the flagellar one, named promastigote, that survives extracellularly and is found in the intestinal tract of the vector (Phlebotominae) and the intracellular one, known as amastigote, present in the macrophages, in the bone-marrow and in cells of the endoplasmic reticle of the mammal host 24 .…”

“…In its development cycle, Leishmania presents under two different aspects, the flagellar one, named promastigote, that survives extracellularly and is found in the intestinal tract of the vector (Phlebotominae) and the intracellular one, known as amastigote, present in the macrophages, in the bone-marrow and in cells of the endoplasmic reticle of the mammal host 24 . The amastigotes have been described in cell cultures ever since this technique began to be used 14,25,30 .…”