Hollie Burrell-Saward

Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-specific parasite dynamics and heart disease in the absence of locally persistent infection

Chronic Trypanosoma cruzi infections lead to cardiomyopathy in 20–30% of cases. A causal link between cardiac infection and pathology has been difficult to establish because of a lack of robust methods to detect scarce, focally distributed parasites within tissues. We developed a highly sensitive bioluminescence imaging system based on T. cruzi expressing a novel luciferase that emits tissue‐penetrating orange‐red light. This enabled long‐term serial evaluation of parasite burdens in individual mice with an in vivo limit of detection of significantly less than 1000 parasites. Parasite distributions during chronic infections were highly focal and spatiotemporally dynamic, but did not localize to the heart. End‐point ex vivo bioluminescence imaging allowed tissue‐specific quantification of parasite loads with minimal sampling bias. During chronic infections, the gastro‐intestinal tract, specifically the colon and stomach, was the only site where T. cruzi infection was consistently observed. Quantitative PCR‐inferred parasite loads correlated with ex vivo bioluminescence and confirmed the gut as the parasite reservoir. Chronically infected mice developed myocarditis and cardiac fibrosis, despite the absence of locally persistent parasites. These data identify the gut as a permissive niche for long‐term T. cruzi infection and show that canonical features of Chagas disease can occur without continual myocardium‐specific infection.

Highly Sensitive In Vivo Imaging of Trypanosoma brucei Expressing “Red-Shifted” Luciferase

Background Human African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei species complex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by the limitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS. Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in a number of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However, lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spread use of in vivo imaging to investigate infections with T. brucei and other trypanosomatids. Methodology/Principal findings Here, we report a system that allows the detection of fewer than 100 bioluminescent T. brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase (PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene, flanked by an upstream 5′-variant surface glycoprotein untranslated region (UTR) and a downstream 3′-tubulin UTR, into a T. brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was not associated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time, and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with a yellow firefly luciferase reporter. Conclusions/Significance The optimised bioluminescent reporter line described here will significantly enhance the application of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivity provides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. It should be ideally suited to drug evaluation programmes.

Antimalarial Mannoxanes: Hybrid Antimalarial Drugs with Outstanding Oral Activity Profiles and A Potential Dual Mechanism of Action

Antimalarial Mannoxanes: Hybrid Antimalarial Drugs with Outstanding Oral Activity Profiles and A Potential Dual Mechanism of Action James Chadwick, Richard K. Amewu, Francesc Marti, Fatima Bousejra-El Garah, Raman Sharma, Neil G. Berry, Paul A. Stocks, Hollie Burrell-Saward, Sergio Wittlin, e] Matthias Rottmann, e] Reto Brun, e] Donatella Taramelli, Silvia Parapini, Stephen A. Ward, and Paul M. O’Neill*

Second generation analogues of RKA182: synthetic tetraoxanes with outstanding in vitro and in vivo antimalarial activities

A series of polar dispiro-1,2,4,5-tetraoxanes have been designed and synthesized by parallel synthesis. From this series, endoperoxides with activity as low as 0.2 nM have been obtained and representatives of this group have excellent oral activities in the P. bergheiANKA mouse model of malaria.

Synthesis and evaluation of the antimalarial, anticancer, and caspase 3 activities of tetraoxane dimers

The synthesis of a range of mono spiro and dispiro 1,2,4,5-tetraoxane dimers is described. Selected molecules were examined in in vitro assays to determine their antimalarial and anticancer potential. Our studies reveal that several molecules possess potent nanomolar antimalarial and single digit micromolar antiproliferative IC(50)s versus colon (HT29-AK and leukemia (HL60) cell lines.

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. 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). 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. 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.

Antileishmanial Activity, Uptake, and Biodistribution of an Amphotericin B and Poly(α-Glutamic Acid) Complex

ABSTRACT A noncovalent, water-soluble complex of amphotericin B (AMB) and poly(α-glutamic acid) (PGA), with AMB loadings ranging from 25 to 55% (wt/wt) using PGA with a molecular weight range of 50,000 to 70,000, was prepared as a potential new treatment for visceral leishmaniasis (VL). The AMB-PGA complex was shown to be as active as Fungizone (AMB deoxycholate) against intracellular Leishmania donovani amastigotes in differentiated THP-1 cells. The in vitro uptake of the AMB-PGA complex by differentiated THP-1 cells was similar to that of Fungizone and higher than that of AmBisome (liposomal AMB). The AMB-PGA complex also displayed a dose-response profile similar to that of AmBisome in vivo in BALB/c mice against L. donovani, with 50% effective doses (ED50s) of 0.24 ± 0.03 mg/kg of body weight for the AMB-PGA complex and 0.24 ± 0.06 mg/kg for AmBisome. A biodistribution study with mice indicated that the AMB-PGA complex cleared more rapidly from plasma than AmBisome, with a comparable low level of distribution to the kidneys.

Decoding the network of Trypanosoma brucei proteins that determines sensitivity to apolipoprotein-L1

In contrast to Trypanosoma brucei gambiense and T. b. rhodesiense (the causative agents of human African trypanosomiasis), T. b. brucei is lysed by apolipoprotein-L1 (apoL1)-containing human serum trypanolytic factors (TLF), rendering it non-infectious to humans. While the mechanisms of TLF1 uptake, apoL1 membrane integration, and T. b. gambiense and T. b. rhodesiense apoL1-resistance have been extensively characterised, our understanding of the range of factors that drive apoL1 action in T. b. brucei is limited. Selecting our bloodstream-form T. b. brucei RNAi library with recombinant apoL1 identified an array of factors that supports the trypanocidal action of apoL1, including six putative ubiquitin modifiers and several proteins putatively involved in membrane trafficking; we also identified the known apoL1 sensitivity determinants, TbKIFC1 and the V-ATPase. Most prominent amongst the novel apoL1 sensitivity determinants was a putative ubiquitin ligase. Intriguingly, while loss of this ubiquitin ligase reduces parasite sensitivity to apoL1, its loss enhances parasite sensitivity to TLF1-dominated normal human serum, indicating that free and TLF1-bound apoL1 have contrasting modes-of-action. Indeed, loss of the known human serum sensitivity determinants, p67 (lysosomal associated membrane protein) and the cathepsin-L regulator, ‘inhibitor of cysteine peptidase’, had no effect on sensitivity to free apoL1. Our findings highlight a complex network of proteins that influences apoL1 action, with implications for our understanding of the anti-trypanosomal action of human serum.

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.

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.