A New Experimental Model for Assessing Drug Efficacy against Trypanosoma cruzi Infection Based on Highly Sensitive In Vivo Imaging

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, one of the world’s major neglected infections. Although development of improved antiparasitic drugs is considered a priority, there have been no significant treatment advances in the past 40 years. Factors that have limited progress include an incomplete understanding of pathogenesis, tissue tropism, and disease progression. In addition, in vivo models, which allow parasite burdens to be tracked throughout the chronic stage of infection, have been lacking. To address these issues, we have developed a highly sensitive in vivo imaging system based on bioluminescent T. cruzi, which express a red-shifted luciferase that emits light in the tissue-penetrating orange-red region of the spectrum. The exquisite sensitivity of this noninvasive murine model has been exploited to monitor parasite burden in real time throughout the chronic stage, has allowed the identification of the gastrointestinal tract as the major niche of long-term infection, and has demonstrated that chagasic heart disease can develop in the absence of locally persistent parasites. Here, we review the parameters of the imaging system and describe how this experimental model can be incorporated into drug development programs as a valuable tool for assessing efficacy against both acute and chronic T. cruzi infections.

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Limited Ability of Posaconazole To Cure both Acute and Chronic Trypanosoma cruzi Infections Revealed by Highly SensitiveIn VivoImaging

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00520-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4653-4661 ◽

Cited By ~ 84

Author(s):

Amanda Fortes Francisco ◽

Michael D. Lewis ◽

Shiromani Jayawardhana ◽

Martin C. Taylor ◽

Eric Chatelain ◽

...

Keyword(s):

Clinical Trial ◽

Trypanosoma Cruzi ◽

Chagas Disease ◽

Imaging System ◽

Ex Vivo ◽

Parasite Burden ◽

Significant Activity ◽

Content Type ◽

Chronic Stage

ABSTRACTThe antifungal drug posaconazole has shown significant activity againstTrypanosoma cruziin vitroand in experimental murine models. Despite this, in a recent clinical trial it displayed limited curative potential. Drug testing is problematic in experimental Chagas disease because of difficulties in demonstrating sterile cure, particularly during the chronic stage of infection when parasite burden is extremely low and tissue distribution is ill defined. To better assess posaconazole efficacy against acute and chronic Chagas disease, we have exploited a highly sensitive bioluminescence imaging system which generates data with greater accuracy than other methods, including PCR-based approaches. Mice inoculated with bioluminescentT. cruziwere assessed byin vivoandex vivoimaging, with cyclophosphamide-induced immunosuppression used to enhance the detection of relapse. Posaconazole was found to be significantly inferior to benznidazole as a treatment for both acute and chronicT. cruziinfections. Whereas 20 days treatment with benznidazole was 100% successful in achieving sterile cure, posaconazole failed in almost all cases. Treatment of chronic infections with posaconazole did however significantly reduce infection-induced splenomegaly, even in the absence of parasitological cure. The imaging-based screening system also revealed that adipose tissue is a major site of recrudescence in mice treated with posaconazole in the acute, but not the chronic stage of infection. Thisin vivoscreening model for Chagas disease is predictive, reproducible and adaptable to diverse treatment schedules. It should provide greater assurance that drugs are not advanced prematurely into clinical trial.

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Immunity conferred by drug-cured experimental Trypanosoma cruzi infections is long-lasting and cross-strain protective

10.1101/741462 ◽

2019 ◽

Author(s):

Gurdip Singh Mann ◽

Amanda F. Francisco ◽

Shiromani Jayawardhana ◽

Martin C. Taylor ◽

Michael D. Lewis ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Bioluminescence Imaging ◽

Viral Vector ◽

Parasite Burden ◽

Whole Body ◽

Luciferase Reporter ◽

Chronic Stage ◽

Route Of Infection ◽

Highly Sensitive

ABSTRACTBackgroundThe long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole body in vivo imaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge with Trypanosoma cruzi.Methodology/Principal FindingsMice were infected with T. cruzi strains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Mice infected for 14 days or more, prior to the initiation of treatment, were highly protected from challenge with both homologous and heterologous strains (>99% reduction in parasite burden). Sterile protection against homologous challenge was frequently observed. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimal T. cruzi-specific IFN-γ+ T cell response.Conclusions/SignificanceOur findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response.AUTHOR SUMMARYChagas disease, which is caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem throughout Latin America. Attempts to develop a vaccine have been hampered by technical difficulties in monitoring the extremely low parasite burden during the life-long chronic stage of infection. To circumvent these issues, we used highly sensitive bioluminescence imaging to assess the ability of recombinant viral vector vaccines and drug-cured infections to confer protection against experimental challenge in mice. We observed that drug-cured infections were much more effective than subunit vaccines, with many instances of sterile protection. Efficacy was independent of the route of infection or size of the challenge inoculum, and was undiminished after almost a year. In addition, drug-cured infections conferred a high level of cross-strain protection. The highly sensitive imaging procedures enabled us to visualise parasite distribution in mice where sterile protection was not achieved. This suggested that to confer sterile protection, vaccines must prevent the infection of organs/tissues that act as parasite reservoirs during the chronic stage. Once established at these sites, parasites become largely refractory to vaccine-induced elimination.

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Nanotechnology: from In Vivo Imaging System to Controlled Drug Delivery

Nanoscale Research Letters ◽

10.1186/s11671-017-2249-8 ◽

2017 ◽

Vol 12 (1) ◽

Cited By ~ 41

Author(s):

Maria Mir ◽

Saba Ishtiaq ◽

Samreen Rabia ◽

Maryam Khatoon ◽

Ahmad Zeb ◽

...

Keyword(s):

Drug Delivery ◽

In Vivo Imaging ◽

Imaging System ◽

Controlled Drug Delivery ◽

In Vivo Imaging System

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Real-Time In Vivo Bioluminescent Imaging for Evaluating the Efficacy of Antibiotics in a Rat Staphylococcus aureus Endocarditis Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.1.380-387.2005 ◽

2005 ◽

Vol 49 (1) ◽

pp. 380-387 ◽

Cited By ~ 68

Author(s):

Yan Q. Xiong ◽

Julie Willard ◽

Jagath L. Kadurugamuwa ◽

Jun Yu ◽

Kevin P. Francis ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Real Time ◽

Imaging System ◽

Bioluminescent Imaging ◽

Vancomycin Therapy ◽

Treatment Groups ◽

Highly Sensitive ◽

Relevant Animal Model ◽

And Control

ABSTRACT Therapeutic options for invasive Staphylococcus aureus infections have become limited due to rising antimicrobial resistance, making relevant animal model testing of new candidate agents more crucial than ever. In the present studies, a rat model of aortic infective endocarditis (IE) caused by a bioluminescently engineered, biofilm-positive S. aureus strain was used to evaluate real-time antibiotic efficacy directly. This strain was vancomycin and cefazolin susceptible but gentamicin resistant. Bioluminescence was detected and quantified daily in antibiotic-treated and control animals with IE, using a highly sensitive in vivo imaging system (IVIS). Persistent and increasing cardiac bioluminescent signals (BLS) were observed in untreated animals. Three days of vancomycin therapy caused significant reductions in both cardiac BLS (>10-fold versus control) and S. aureus densities in cardiac vegetations (P < 0.005 versus control). However, 3 days after discontinuation of vancomycin therapy, a greater than threefold increase in cardiac BLS was observed, indicating relapsing IE (which was confirmed by quantitative culture). Cefazolin resulted in modest decreases in cardiac BLS and bacterial densities. These microbiologic and cardiac BLS differences during therapy correlated with a longer time-above-MIC for vancomycin (>12 h) than for cefazolin (∼4 h). Gentamicin caused neither a reduction in cardiac S. aureus densities nor a reduction in BLS. There were significant correlations between cardiac BLS and S. aureus densities in vegetations in all treatment groups. These data suggest that bioluminescent imaging provides a substantial advance in the real-time monitoring of the efficacy of therapy of invasive S. aureus infections in live animals.

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In Vivo Analysis of Trypanosoma cruzi Persistence Foci at Single-Cell Resolution

mBio ◽

10.1128/mbio.01242-20 ◽

2020 ◽

Vol 11 (4) ◽

Cited By ~ 2

Author(s):

Alexander I. Ward ◽

Michael D. Lewis ◽

Archie A. Khan ◽

Conor J. McCann ◽

Amanda F. Francisco ◽

...

Keyword(s):

Skeletal Muscle ◽

Immune Response ◽

Smooth Muscle ◽

Trypanosoma Cruzi ◽

Parasite Burden ◽

Circular Muscle ◽

Muscle Layer ◽

Chronic Infections ◽

Content Type ◽

Chronic Stage

ABSTRACT Infections with Trypanosoma cruzi are usually lifelong despite generating a strong adaptive immune response. Identifying the sites of parasite persistence is therefore crucial to understanding how T. cruzi avoids immune-mediated destruction. However, this is a major technical challenge, because the parasite burden during chronic infections is extremely low. Here, we describe an integrated approach involving comprehensive tissue processing, ex vivo imaging, and confocal microscopy, which allowed us to visualize infected host cells in murine tissue with exquisite sensitivity. Using bioluminescence-guided tissue sampling, with a detection level of <20 parasites, we showed that in the colon, smooth muscle myocytes in the circular muscle layer are the most common infected host cell type. Typically, during chronic infections, the entire colon of a mouse contains only a few hundred parasites, often concentrated in a small number of cells each containing >200 parasites, which we term mega-nests. In contrast, during the acute stage, when the total parasite burden is considerably higher and many cells are infected, nests containing >50 parasites are rarely found. In C3H/HeN mice, but not BALB/c mice, we identified skeletal muscle as a major site of persistence during the chronic stage, with most parasites being found in large mega-nests within the muscle fibers. Finally, we report that parasites are also frequently found in the skin during chronic murine infections, often in multiple infection foci. In addition to being a site of parasite persistence, this anatomical reservoir could play an important role in insect-mediated transmission and have implications for drug development. IMPORTANCE Trypanosoma cruzi causes Chagas disease, the most important parasitic infection in Latin America. Major pathologies include severe damage to the heart and digestive tract, although symptoms do not usually appear until decades after infection. Research has been hampered by the complex nature of the disease and technical difficulties in locating the extremely low number of parasites. Here, using highly sensitive imaging technology, we reveal the sites of parasite persistence during chronic-stage infections of experimental mice at single-cell resolution. We show that parasites are frequently located in smooth muscle cells in the circular muscle layer of the colon and that skeletal muscle cells and the skin can also be important reservoirs. This information provides a framework for investigating how the parasite is able to survive as a lifelong infection, despite a vigorous immune response. It also informs drug development strategies by identifying tissue sites that must be accessed to achieve a curative outcome.

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Preclinical orthotopic xenograft model of renal pelvis cancer in which cancer growth could be traced by an in vivo imaging system

International Journal of Urology ◽

10.1111/iju.13829 ◽

2018 ◽

Vol 26 (1) ◽

pp. 138-139 ◽

Cited By ~ 2

Author(s):

Teruki Shimizu ◽

Masatsugu Miyashita ◽

Atsuko Fujihara ◽

Fumiya Hongo ◽

Osamu Ukimura ◽

...

Keyword(s):

Renal Pelvis ◽

In Vivo Imaging ◽

Imaging System ◽

Xenograft Model ◽

Cancer Growth ◽

Orthotopic Xenograft ◽

Orthotopic Xenograft Model ◽

In Vivo Imaging System

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116 INCORPORATION AND DEVELOPMENTAL TOXICITY OF QUANTUM DOT NANOPARTICLES IN AMPHIBIAN LARVAE

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab116 ◽

2017 ◽

Vol 29 (1) ◽

pp. 166 ◽

Cited By ~ 1

Author(s):

A. R. Julien ◽

S. B. Park ◽

C. K. Vance ◽

P. L. Ryan ◽

S. T. Willard ◽

...

Keyword(s):

Quantum Dot ◽

In Vivo Imaging ◽

Half Life ◽

Imaging System ◽

Fluorescence Emission ◽

Total Flux ◽

Intestinal Lumen ◽

Post Exposure ◽

Amphibian Larvae

The use of nanoparticles both commercially and pharmaceutically has increased over the past decade, including fluorescent quantum dot nanoparticles (QD) in biochemical research for in vivo imaging. Previous studies have reported the toxic effects of nanoparticles, but their effects on larval metamorphosis and animal development and growth have not been thoroughly examined. Additionally, the method of uptake of nanoparticles by larval systems is unknown. Amphibian larvae are an ideal model for assessing toxicity because of their sensitivity to environmental contaminants and rapid and easily observable developmental stages. We used Anaxyrus fowleri tadpoles to investigate QD (≤ 25 nm diameter) integration into larvae and possible deleterious effects on their growth and development. Tadpoles (A. fowleri; n = 5/group) were placed in 24-well plates containing 1 mL of distilled water and increasing concentrations of QD (0, 1, and 2 nM) 72 h post-hatch. The fluorescence emission of QD in wells was detected at various time points (1, 2, 24, 48, and 72 h) using the in vivo imaging system (IVIS). A subset of tadpoles was killed (MS-222) and sectioned for histopathology. Remaining tadpoles were monitored throughout development. Fluorescence emission of QD in sectioned tadpoles was visualised using an EVOS Cell Imaging System. Developmental metrics of living tadpoles were recorded until metamorphosis. Fluorescence intensity between controls and dosage groups were analysed by ANOVA-1, followed by Student’s l.s.d. test to evaluate the effects of QD concentration and exposure time. The threshold of significance was P < 0.05. The rate of incorporation of QD into tadpoles was determined using the equation y = C + Ao*2(–x/t1/2), where t1/2 is the half-life of QD remaining in solution. The IVIS imaging revealed a rapid decrease of QD fluorescence (total flux) signals from the aqueous tadpole environment. Decreases in fluorescence occurred within 1 h post-exposure and appeared dose and time dependent, with signal nearly gone within 48 h. Half-life of total flux (time necessary for tadpoles to absorb half of the QD in solution) is 20.75 h (R2 = 0.92) and 2.54 h (R2 = 0.96) for 1 nm and 2 nm QD in solution, respectively. The EVOS imaging revealed integration of QD and localization into tadpole tissues. Fluorescence was exclusively found within the mouth, gills, and sections of the intestinal lumen of exposed tadpoles within the first hour. Dose-dependent increases in fluorescence within tissue were observed at each time-point. No signal was observed in controls. In remaining live tadpoles, QD treated tadpoles were smaller in size [t(34) = 2.35, P = 0.024] than controls. Findings reveal that (1) A. fowleri tadpoles integrate and accumulate nanoparticles, without detectable excretion within 72 h post-exposure, and (2) nanoparticles impede normal tadpole development. Ongoing studies are determining the effects of QD exposure on complete tadpole metamorphosis. The work was supported by USDA-ARS Biophotonics Initiative grant #58–6402–3-018.

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