Leishmania major and L. donovani: A method for rapid purification of amastigotes

Glaser, Theresa A.; Wells, Steven J.; Spithill, Terry W.; Pettitt, J. M.; Humphris, Danielle C.; Mukkada, Antony J.

doi:10.1016/0014-4894(90)90039-f

Cited by 58 publications

(28 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In mouse lesion purified amastigotes, mouse caspase-7 was detected, and is likely to be responsible for the DEVDase activity observed. Its presence does not seem to be due to mouse protein contamination, which has been observed previously, 52 as neither mouse caspase-3 nor actin was detected in the purified amastigotes. Interestingly, in mammals it was proposed that active caspase-7 is translocated to the mitochondria and the microsomes following Fas stimulation.…”

Section: Discussionsupporting

confidence: 65%

Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

2002

View full text Add to dashboard Cite

Unicellular organisms, such as the protozoan parasite Leishmania, can be stimulated to show some morphological and biochemical features characteristic of mammalian apoptosis. This study demonstrates that under a variety of stress conditions such as serum deprivation, heat shock and nitric oxide, cell death can be induced leading to genomic DNA fragmentation into oligonucleosomes. DNA fragmentation was observed, without induction, in the infectious stages of the parasite, and correlated with the presence of internucleosomal nuclease activity, visualisation of 45 to 59 kDa nucleases and detection of TUNEL-positive nuclei. DNA fragmentation was not dependent on active effector downstream caspases nor on the lysosomal cathepsin L-like enyzmes CPA and CPB. These data are consistent with the presence of a caspase-independent cell death mechanism in Leishmania, induced by stress and differentiation that differs significantly from metazoa.

show abstract

Section: Discussionsupporting

confidence: 65%

Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

2002

View full text Add to dashboard Cite

show abstract

“…Cultures were initiated at 5 ϫ 10 5 parasites per ml and grown at 24°C with shaking. L. donovani amastigotes used in subsequent experiments were freshly isolated from the spleens of Golden hamsters (24). Axenic amastigote cell line LdBOB (derived from MHOM/S.D./62/1S-CL2D) was maintained in culture as described previously (25).…”

Section: Methodsmentioning

confidence: 99%

Dual Action of Antimonial Drugs on Thiol Redox Metabolism in the Human Pathogen Leishmania donovani

Wyllie

Cunningham

Fairlamb

2004

Journal of Biological Chemistry

272

245

View full text Add to dashboard Cite

Despite extensive use of antimonial compounds in the treatment of leishmaniasis, their mode of action remains uncertain. Here we show that trivalent antimony (Sb III ) interferes with trypanothione metabolism in drug-sensitive Leishmania parasites by two inherently distinct mechanisms. First, Sb III decreases thiol buffering capacity by inducing rapid efflux of intracellular trypanothione and glutathione in approximately equimolar amounts. Second, Sb III inhibits trypanothione reductase in intact cells resulting in accumulation of the disulfide forms of trypanothione and glutathione. These two mechanisms combine to profoundly compromise the thiol redox potential in both amastigote and promastigote stages of the life cycle. Furthermore, we demonstrate that sodium stibogluconate, a pentavalent antimonial used clinically for the treatment for leishmaniasis, induces similar effects on thiol redox metabolism in axenically cultured amastigotes. These observations suggest ways in which current antimony therapies could be improved, overcoming the growing problem of antimony resistance.Leishmania parasites cause a wide spectrum of human and animal infections ranging from life-threatening visceral disease to disfiguring mucosal and cutaneous forms of the disease. These "neglected" diseases are a significant cause of morbidity and mortality in 88 countries in the Americas, Africa, Asia, and Southern Europe; millions of people are at risk, and 400,000 new cases are reported annually (1). Leishmania spp. are obligate intracellular parasites of the vertebrate reticuloendothelial system, where they multiply as amastigotes in macrophage phagolysosomes; transmission is by blood-sucking sandflies, in which they proliferate as extracellular promastigotes. Treatment of the leishmaniases is far from ideal, and pentavalent antimonial (Sb V ) 1 preparations such as sodium stibogluconate (Pentostam) have been the front-line drugs for more than half a century. However, the clinical value of antimony therapy is now threatened because of the emergence of drug resistance (2) and co-infection with human immunodeficiency virus (3). Sb V is generally regarded as a pro-drug that first has to be activated by conversion to the trivalent form (Sb III ) (4). However, the site of reduction (host macrophage, amastigote, or both) and the mechanism of reduction (enzymatic or nonenzymatic) remain unclear (4 -8). The mode of action of these drugs is poorly understood. Sb III reversibly inhibits trypanothione reductase in vitro (9), but this has not been demonstrated in the intact parasite. However, inhibition of this unique and essential enzyme (10 -12) is of particular interest, because trypanothione (N 1 ,N 8Ϫ bis(glutathionyl) spermidine (T[SH] 2 )) is a key intermediate in the regulation of thiol redox homeostasis, as well as in defense against chemical (13,14) and oxidative stress (15, 16).Paradoxically, more is known about the mechanism of resistance to Sb III than its mode of action (17). A considerable body of evidence implicates trypanothione a...

show abstract

“…After 6 days of incubation the U937 cells were harvested. Amastigote purification was done as described [10] by homogenizalion of lhe cells, followed by differential filtration through polycarbonate filters of decreasing pore size (Isopore. Millipore.…”

Section: Antigensmentioning

confidence: 99%

Dichotomy of the human T cell response to Leishmania antigens. II. Absent or Th2-like response to gp63 and Thl-like response to lipophosphoglycan- associated protein in cells from cured visceral leishmaniasis patients

Kurtzhals¹,

Hey²,

Jardim

et al. 1994

Clinical and Experimental Immunology

View full text Add to dashboard Cite

SUMMARY The T cell response to different Leishmania donovani aniigens was investigated using peripheral blood mononuciear cells (PBMC) from Kenyans cured of visceral leishmaniasis and non‐exposed Danes. Crude promastigote and amastigote antigens both induced proliferation and interferon‐gamma (IFN‐γ) production in PBMC from cured patients, while cells from non‐exposed donors gave weak responses. A similar pattern was indticed by lipophosphoglycan‐associated protein (LPGAP). By contrast, the major surface protease of Leishmania. gp63, induced only a weak proliferative response without IFN‐γ production in five of 17 samples from cured patients. Four of the five responding cultures produced IL‐4, i.e. the response to this antigen was of the Th2 type. Furthermore, sera from acutely ill visceral leishmaniasis patients contained high levels of IgG antibodies to gp63. The Th2‐tike response to gp63 in patients cured of visceral leishmaniasis differs from the Thl‐like response to the same antigen observed in patients cured of cutaneous leishmaniasis.

show abstract

Leishmania major and L. donovani: A method for rapid purification of amastigotes

Cited by 58 publications

References 6 publications

Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

Dual Action of Antimonial Drugs on Thiol Redox Metabolism in the Human Pathogen Leishmania donovani

Dichotomy of the human T cell response to Leishmania antigens. II. Absent or Th2-like response to gp63 and Thl-like response to lipophosphoglycan- associated protein in cells from cured visceral leishmaniasis patients

Contact Info

Product

Resources

About