Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis.

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites. Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection. Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches

Synthesis and antitrypanosomal activities of novel pyridylchalcones

Collaboration with the London School of Hygiene and Tropical Medicine. The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.A library of novel pyridylchalcones were synthesised and screened against Trypanosoma brucei rhodesiense. Eight were shown to have good activity with the most potent 8 having an IC50 value of 0.29 M. Cytotoxicity testing with human KB cells showed a good selectivity profile for this compound with a selectivity index of 47. Little activity was seen when the library was tested against Leishmania donovani. In conclusion, pyridylchalcones are promising leads in the development of novel compounds for the treatment of human African trypanosomiasis (HAT)

Dose-dependent effect and pharmacokinetics of fexinidazole and its metabolites in a mouse model of human African trypanosomiasis.

Human African trypanosomiasis (HAT) is a neglected tropical disease, with a population of 70 million at risk. Current treatment options are limited. In the search for new therapeutics, the repurposing of the broad-spectrum antiprotozoal drug fexinidazole has completed Phase III trials with the anticipation that it will be the first oral treatment for HAT. This study used the recently validated bioluminescence imaging model to assess the dose and rate of kill effect of fexinidazole in infected mice, and the dose-dependent effect of fexinidazole on trypanosome infection. Pharmacokinetics of fexinidazole in plasma and central nervous system (CNS) compartments were similar in both infected and uninfected mice. Drug distribution within the CNS was further examined by microdialysis, showing similar levels in the cortex and hippocampus. However, high variability in drug distribution and exposure was found between mice

Development of an imaging model of a CNS infection with African trypanosomes

The study of late-stage human African trypanosomiasis (HAT) within mouse models is lengthy and complex, with the removal of brain tissue being required to monitor parasitic burden. This results in the inability to track real-time infections within the central nervous system (CNS). With nearly 70 million people at risk of HAT infection in Africa every year, research into new drug therapies which are capable of crossing the blood brain barrier and efficacious towards the parasite are imperative. However, progress has been slow and difficult, partly due to the limitations with the current drug relapse mouse model for African trypanosomiasis (T. b. brucei GVR35), which requires a follow-up time of 180-days. In this study, we report the generation of highly bioluminescent parasites and their use in an in vivo imaging model of late-stage African trypanosomiasis. Bloodstream forms of the chronic model strain GVR35 were transfected with a “red-shifted” luciferase, which produced detectable signal in CNS at 21-days p.i mimicking that of the wild type line. This model enabled the tracking of a single animal through the entire chronic infection, with the detection of parasites occurring earlier than blood film microscopy. The model was further employed to assess the effects of known anti-trypanosomal drugs on bioluminescence, and to demonstrate how the reduction in bioluminescent signal combined with qPCR can determine a dose-dependent effect after treatment. The non-invasive in vivo imaging model will reduce the time and numbers of mice required to assess preclinical efficacy of new anti-trypanosomal drugs. This study shows the development and optimisation of a new, efficient method to evaluate novel anti-trypanosomal drugs in vivo with the added advantage of reducing the current drug relapse model from 180-days to 90-days

A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late-stage human African trypanosomiasis

Objectives To optimize the Trypanosoma brucei brucei GVR35 VSL-2 bioluminescent strain as an innovative drug evaluation model for late-stage human African trypanosomiasis.<p></p> Methods An IVIS® Lumina II imaging system was used to detect bioluminescent T. b. brucei GVR35 parasites in mice to evaluate parasite localization and disease progression. Drug treatment was assessed using qualitative bioluminescence imaging and real-time quantitative PCR (qPCR).<p></p> Results We have shown that drug dose–response can be evaluated using bioluminescence imaging and confirmed quantification of tissue parasite load using qPCR. The model was also able to detect drug relapse earlier than the traditional blood film detection and even in the absence of any detectable peripheral parasites.<p></p> Conclusions We have developed and optimized a new, efficient method to evaluate novel anti-trypanosomal drugs in vivo and reduce the current 180 day drug relapse experiment to a 90 day model. The non-invasive in vivo imaging model reduces the time required to assess preclinical efficacy of new anti-trypanosomal drugs.<p></p&gt

Limit of detection <i>in vivo</i>.

<p>(<b>A</b>) Six sets of three BALB/c mice were inoculated i.p. with either 20, 100, 500, 5000, or 50000 bloodstream form <i>T.b. brucei</i> GVR35 clone VSL2. An additional set of three mice was inoculated with 30000 non-transformed parasites (WT). All of the IVIS Lumina images were acquired using large binning, 5 minute exposures, 15 minutes after infection and 10 minutes after administration of luciferin (150 mg kg⁻¹). It was possible to visualise as few as 100 parasites in the intra-peritoneal space. (<b>B</b>) A dose response curve generated from the <i>in vivo</i> limit of detection data. Mean abdominal bioluminescence was recorded from each group. Linear regression analysis shows a very strong positive correlation between bioluminescence and parasite inoculum (R²>0.99). Dotted line indicates mean bioluminescence plus two standard deviations, from mice infected with wild type parasites.</p

Limit of detection <i>in vitro</i>.

<p>(<b>A</b>) Images of two 96-well microtitre plates containing dilutions of <i>T.b. brucei</i> GVR35 clone VSL2 and a non-transformed control (WT). Each plate was imaged using an IVIS Lumina (Perkin Elmer) with 1 minute exposure and medium binning. Both cell lines were serially diluted from 1×10⁶ to 1×10³ (upper plate) and from 1×10³ to 1×10² parasites ml⁻¹ (cell numbers shown above each plate). 100 VSL2 parasites could be clearly visualised. Note that the imaging software automatically adjusts the heat-map scale to account for the intensity of the well containing the highest number of parasites. (<b>B</b>) <i>In vitro</i> linear regression plots generated from both plates. Each point corresponds to bioluminescence represented by the total flux recorded from a single well. In both cases, linear regression analysis shows a very strong positive correlation between bioluminescence and parasite number (R²>0.99). The graphs show readings from duplicate wells. In the upper graph, duplicate values were extremely close and are not individually distinguishable. In the lower graph, duplicates are shown as red squares and blue triangles, and the dotted line indicates the background in blank wells (green triangles), plus two standard deviations.</p