Development of an Alamar Blue (TM) Viability Assay in 384-Well Format for High Throughput Whole Cell Screening of Trypanosoma brucei brucei Bloodstream Form Strain 427

Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Queensland, Australia.
The American journal of tropical medicine and hygiene (Impact Factor: 2.7). 10/2009; 81(4):665-74. DOI: 10.4269/ajtmh.2009.09-0015
Source: PubMed


There is an urgent need for new compounds for the drug development pipeline for treatment of patients with African sleeping sickness. One approach for identifying such compounds is by high throughput screening (HTS) of compound collections. For time and cost considerations, there is a need for the development of an assay that uses at least 384-well formats. To our knowledge, there are currently no viability assays for whole cell screening of trypanosomes in the 384-well plate format. We have developed and optimized an Alamar Blue viability assay in a 384-well format for Trypanosoma brucei brucei bloodstream form strain 427 (BS427). The assay had a Z' > 0.5 and tolerated a final dimethyl-sulfoxide concentration of 0.42%. Drug sensitivity was compared with those reported from previously developed 96-well methods and was found to be comparable. The sensitivity and cost benefit of the Alamar Blue assay make it an excellent candidate for HTS application.

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    • "When added to cells, the PrestoBlue TM reagent – containing a non-fluorescent, cell-permeant compound – is modified by the reducing environment of the viable cells, becoming highly fluorescent, which can be detected using fluorescence or absorbance measurements . PrestoBlue TM reagent is more sensitive than AlamarBlue ® , which is a redox indicator of enzyme activity widely used in whole organism screening [18] and is extensively used in screening tests of viability and cytotoxicity [18] [22] [12] [29] [1] [27]. PrestoBlue TM was directly added to the cells in the culture medium at a final concentration of 10%. "
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    ABSTRACT: During therapeutic interventions, blood concentrations of intravenously applied drugs are higher, and their onset of pharmacological action is faster than with other routes of drug administration. However, acute drug therapy often produces nephrotoxic side effects, as commonly seen after treatment with Ketorolac or Gentamicin leading to questions about their use, especially for patients at risk for acute renal failure. Omega-6(n-6) and omega-3(n-3) polyunsaturated fatty acids (PUFA) affect eicosanoid metabolism, which plays a role in the regulation of inflammation. Eicosanoids derived from n-6 FA have proinflammatory and immunoactive functions, whereas eicosanoids derived from n-3 PUFA have anti-inflammatory and cytoprotective properties. We hypothesized that providing such injectable drugs with nephrotoxic potential in combination with n3-PUFAs from the outset, might afford rapid cytoprotection of renal cells, given the recent evidence that intravenously administered n3-PUFAs are rapidly incorporated into cell membranes. We used intraglomerular mesangial cells (MES13) that are sensitive to treatment with Ketorolac or Gentamicin instead of proximal tubular cells which do not respond to Ketorolac. We found a significant inhibition of Ketorolac (0.25, 0.5, 1 mM) or Gentamicin (2.5, 5 mM) induced cytotoxicity after pretreatment of MES13 cells with 0.01% of 20%w/v LipOmega-3 Emulsion 9/1, containing 90:10 wt/wt mixture of fish oil derived triglycerides to medium chain triglycerides.
    Toxicology Reports 10/2014; 1. DOI:10.1016/j.toxrep.2014.10.011
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    • "The traditional route to identify such therapeutic agents has been to search for compounds that will target genes or pathways necessary for cell viability. Indeed, whole-cell based high-throughput screening for trypanostatic and trypanocidal agents is well established (Hesse et al., 1995; Mackey et al., 2006; Sykes and Avery, 2009; Jones et al., 2010; De Rycker et al., 2012; Sykes et al., 2012; Vodnala et al., 2013) and multiple classes of compounds with potential for development into new therapeutics are being pursued (Mackey et al., 2006; Jones et al., 2010; Sykes et al., 2012; Vodnala et al., 2013). However, phenotypic screens for genes or pathways involved in the establishment, maintenance or transmission of infection could provide an alternative or complementary route to the identification of novel therapeutics with anti-virulence or transmission-blocking potential. "
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    ABSTRACT: African trypanosomes are sustained in the bloodstream of their mammalian hosts by their extreme capacity for antigenic variation. However, for life cycle progression, trypanosomes also must generate transmission stages called stumpy forms that are pre-adapted to survive when taken up during the bloodmeal of the disease vector, tsetse flies. These stumpy forms are rather different to the proliferative slender forms that maintain the bloodstream parasitaemia. Firstly, they are non proliferative and morphologically distinct, secondly, they show particular sensitivity to environmental cues that signal entry to the tsetse fly and, thirdly, they are relatively robust such that they survive the changes in temperature, pH and proteolytic environment encountered within the tsetse midgut. These characteristics require regulated changes in gene expression to pre-adapt the parasite and the use of environmental sensing mechanisms, both of which allow the rapid initiation of differentiation to tsetse midgut procyclic forms upon transmission. Interestingly, the generation of stumpy forms is also regulated and periodic in the mammalian blood, this being governed by a density-sensing mechanism whereby a parasite-derived signal drives cell cycle arrest and cellular development both to optimize transmission and to prevent uncontrolled parasite multiplication overwhelming the host. In this review we detail recent developments in our understanding of the molecular mechanisms that underpin the production of stumpy forms in the mammalian bloodstream and their signal perception pathways both in the mammalian bloodstream and upon entry into the tsetse fly. These discoveries are discussed in the context of conserved eukaryotic signaling and differentiation mechanisms. Further, their potential to act as targets for therapeutic strategies that disrupt parasite development either in the mammalian bloodstream or upon their transmission to tsetse flies is also discussed.
    Frontiers in Cellular and Infection Microbiology 11/2013; 3:78. DOI:10.3389/fcimb.2013.00078 · 3.72 Impact Factor
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    • "Sleeping sickness or known as Human African Trypamosomiasis (HAT) is a disease that has affects 36 countries of sub-Saharan Africa, annually reported as 17,500 new cases (Sykes and Avery 2009). This disease was considered to be the first or second greatest cause of mortality, compared to HIV/AIDS, in those communities (WHO 2006). "
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    Phytomedicine: international journal of phytotherapy and phytopharmacology 10/2013; 21(3). DOI:10.1016/j.phymed.2013.09.011 · 3.13 Impact Factor
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