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1
Myler PJ, Stuart KD, 2000. Recent developments from the Leishmania genome project. Curr Opin Microbiol 3 :412–416.
-
2
Saxena A, Worthey EA, Yan S, Leland A, Stuart KD, Myler PJ, 2003. Evaluation of differential gene expression in Leishmania major Friedlin procyclics and metacyclics using DNA microarray analysis. Mol Biochem Parasitol 129 :103–114.
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3
Almeida R, Gilmartin BJ, McCann SH, Norrish A, Ivens AC, Lawson D, Levick MP, Smith DF, Dyall SD, Vetrie D, Freeman TC, Coulson RM, Sampaio I, Schneider H, Blackwell JM, 2004. Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome. Mol Biochem Parasitol 136 :87–100.
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4
Drummelsmith J, Brochu V, Girard I, Messier N, Ouellette M, 2003. Proteome mapping of the protozoan parasite leishmania and application to the study of drug targets and resistance mechanisms. Mol Cell Proteomics 2 :146–155.
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5
Mikus J, Steverding D, 2000. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. Parasitol Int 48 :265–269.
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6
Sereno D, Lemesre JL, 1997. Use of an enzymatic micromethod to quantify amastigote stage of Leishmania amazonensis in vitro. Parasitol Res 83 :401–403.
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7
Changsen C, Franzblau SG, Palittapongarnpim P, 2003. Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter. Antimicrob Agents Chemother 47 :3682–3687.
-
8
Dorsky DI, Wells M, Harrington RD, 1996. Detection of HIV-1 infection with a green fluorescent protein reporter system. J Acquir Immune Defic Syndr Hum Retrovirol 13 :308–313.
-
9
Buckner FS, Verlinde CLMJ, La Flamme AC, Van Voorhis WC, 1996. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing β-galactosidase. Antimicrob Agents Chemother 40 :2592–2597.
-
10
Naylor LH, 1999. Reporter gene technology: the future looks bright. Biochem Pharmacol 58 :749–757.
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11
Chan MM, Bulinski JC, Chang KP, Fong D, 2003. A microplate assay for Leishmania amazonensis promastigotes expressing multimeric green fluorescent protein. Parasitol Res 89 :266–271.
-
12
Roy G, Dumas C, Sereno D, Wu Y, Singh AK, Tremblay MJ, Ouellette M, Olivier M, Papadopoulou B, 2000. Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models. Mol Biochem Parasitol 110 :195–206.
-
13
LeBowitz JH, Coburn CM, McMahon-Pratt D, Beverley SM, 1990. Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes. Proc Natl Acad Sci USA 87 :9736–9740.
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14
Sutcliffe JG, 1978. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci USA 75 :3737–3741.
-
15
Moore JT, Davis ST, Dev IK, 1997. The development of beta-lactamase as a highly versatile genetic reporter for eukaryotic cells. Anal Biochem 247 :203–209.
-
16
Neal RA, Allen S, McCoy N, Olliaro P, Croft SL, 1995. The sensitivity of Leishmania species to aminosidine. J Antimicrob Chemother 35 :577–584.
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17
Chang KP, Nacy CA, Pearson RD, 1986. Intracellular parasitism of macrophages in leishmaniasis: in vitro systems and their applications. Methods Enzymol 132 :603–626.
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18
Escobar P, Matu S, Marques C, Croft SL, 2002. Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH(3) (edelfosine) and amphotericin B. Acta Trop 81 :151–157.
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19
Amato VS, Padilha AR, Nicodemo AC, Duarte MI, Valentini M, Uip DE, Boulos M, Neto VA, 2000. Use of itraconazole in the treatment of mucocutaneous leishmaniasis: a pilot study. Int J Infect Dis 4 :153–157.
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20
Dogra J, Saxena VN, 1996. Itraconazole and leishmaniasis: a randomised double-blind trial in cutaneous disease. Int J Parasitol 26 :1413–1415.
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21
Alrajhi AA, Ibrahim EA, De Vol EB, Khairat M, Faris RM, Maguire JH, 2002. Fluconazole for the treatment of cutaneous leishmaniasis caused by Leishmania major.N Engl J Med 346 :891–895.
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COLORIMETRIC ASSAY FOR SCREENING COMPOUNDS AGAINST LEISHMANIA AMASTIGOTES GROWN IN MACROPHAGES
FREDERICK S. BUCKNER
FREDERICK S. BUCKNER
Department of Medicine, University of Washington, Seattle, Washington
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AARON J. WILSON
AARON J. WILSON
Department of Medicine, University of Washington, Seattle, Washington
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An estimated 12 million persons throughout the world suffer from the protozoan disease leishmaniasis. Current treatments have liabilities including poor activity against some forms of leishmaniasis, toxicity, or the need for parenteral administration. Higher throughput methods to screen chemical compounds are needed to facilitate the search for new antileishmania drugs. In the mammalian host, Leishmania parasites exist as amastigotes that replicate within macrophages. Therefore, an in vitro screening assay using intramacrophage amastigotes most closely represents the natural infection. We have transfected strains of Leishmania major and Leishmania amazonensis with the β-lactamase gene, which catalyzes a colorimetric reaction with the substrate nitrocephin. The growth of these β-lactamase–expressing Leishmania within macrophages was quantified in 96-well plates using an optical density plate reader, thus simplifying the methodology for scoring inhibitor assays. This simple and relatively inexpensive colorimetric assay helps improve throughput for screening compounds for antileishmania activity.
Author Notes
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1
Myler PJ, Stuart KD, 2000. Recent developments from the Leishmania genome project. Curr Opin Microbiol 3 :412–416.
-
2
Saxena A, Worthey EA, Yan S, Leland A, Stuart KD, Myler PJ, 2003. Evaluation of differential gene expression in Leishmania major Friedlin procyclics and metacyclics using DNA microarray analysis. Mol Biochem Parasitol 129 :103–114.
-
3
Almeida R, Gilmartin BJ, McCann SH, Norrish A, Ivens AC, Lawson D, Levick MP, Smith DF, Dyall SD, Vetrie D, Freeman TC, Coulson RM, Sampaio I, Schneider H, Blackwell JM, 2004. Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome. Mol Biochem Parasitol 136 :87–100.
-
4
Drummelsmith J, Brochu V, Girard I, Messier N, Ouellette M, 2003. Proteome mapping of the protozoan parasite leishmania and application to the study of drug targets and resistance mechanisms. Mol Cell Proteomics 2 :146–155.
-
5
Mikus J, Steverding D, 2000. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. Parasitol Int 48 :265–269.
-
6
Sereno D, Lemesre JL, 1997. Use of an enzymatic micromethod to quantify amastigote stage of Leishmania amazonensis in vitro. Parasitol Res 83 :401–403.
-
7
Changsen C, Franzblau SG, Palittapongarnpim P, 2003. Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter. Antimicrob Agents Chemother 47 :3682–3687.
-
8
Dorsky DI, Wells M, Harrington RD, 1996. Detection of HIV-1 infection with a green fluorescent protein reporter system. J Acquir Immune Defic Syndr Hum Retrovirol 13 :308–313.
-
9
Buckner FS, Verlinde CLMJ, La Flamme AC, Van Voorhis WC, 1996. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing β-galactosidase. Antimicrob Agents Chemother 40 :2592–2597.
-
10
Naylor LH, 1999. Reporter gene technology: the future looks bright. Biochem Pharmacol 58 :749–757.
-
11
Chan MM, Bulinski JC, Chang KP, Fong D, 2003. A microplate assay for Leishmania amazonensis promastigotes expressing multimeric green fluorescent protein. Parasitol Res 89 :266–271.
-
12
Roy G, Dumas C, Sereno D, Wu Y, Singh AK, Tremblay MJ, Ouellette M, Olivier M, Papadopoulou B, 2000. Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models. Mol Biochem Parasitol 110 :195–206.
-
13
LeBowitz JH, Coburn CM, McMahon-Pratt D, Beverley SM, 1990. Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes. Proc Natl Acad Sci USA 87 :9736–9740.
-
14
Sutcliffe JG, 1978. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci USA 75 :3737–3741.
-
15
Moore JT, Davis ST, Dev IK, 1997. The development of beta-lactamase as a highly versatile genetic reporter for eukaryotic cells. Anal Biochem 247 :203–209.
-
16
Neal RA, Allen S, McCoy N, Olliaro P, Croft SL, 1995. The sensitivity of Leishmania species to aminosidine. J Antimicrob Chemother 35 :577–584.
-
17
Chang KP, Nacy CA, Pearson RD, 1986. Intracellular parasitism of macrophages in leishmaniasis: in vitro systems and their applications. Methods Enzymol 132 :603–626.
-
18
Escobar P, Matu S, Marques C, Croft SL, 2002. Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH(3) (edelfosine) and amphotericin B. Acta Trop 81 :151–157.
-
19
Amato VS, Padilha AR, Nicodemo AC, Duarte MI, Valentini M, Uip DE, Boulos M, Neto VA, 2000. Use of itraconazole in the treatment of mucocutaneous leishmaniasis: a pilot study. Int J Infect Dis 4 :153–157.
-
20
Dogra J, Saxena VN, 1996. Itraconazole and leishmaniasis: a randomised double-blind trial in cutaneous disease. Int J Parasitol 26 :1413–1415.
-
21
Alrajhi AA, Ibrahim EA, De Vol EB, Khairat M, Faris RM, Maguire JH, 2002. Fluconazole for the treatment of cutaneous leishmaniasis caused by Leishmania major.N Engl J Med 346 :891–895.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 33 | 33 | 8 |
| Full Text Views | 352 | 77 | 0 |
| PDF Downloads | 96 | 25 | 0 |