Updating the modified Thompson test by using whole-body bioluminescence imaging to replace traditional efficacy testing in experimental models of murine malaria.

BACKGROUND: Rodent malaria models are extensively used to predict treatment outcomes in human infections. There is a constant need to improve and refine these models by innovating ways to apply new scientific findings and cutting edge technologies. In addition, and in accordance with the three R's of animal use in research, in vivo studies should be constantly refined to avoid unnecessary pain and distress to the experimental animals by using preemptive euthanasia as soon as the main scientific study objective has been accomplished. METHODS: The new methodology described in this manuscript uses the whole-body bioluminescence signal emitted by transgenic, luciferase-expressing Plasmodium berghei parasites to assess the parasite load predicted parasitaemia (PLPP) in drug and control treated female ICR-CD1 mice infected with 1 × 105 luciferase-expressing P. berghei (ANKA strain) infected erythrocytes. This methodology can replace other time-consuming and expensive methods that are routinely used to measure parasitaemia in infected animals, such as Giemsa-stained thin blood smears and flow cytometry. RESULTS: There is a good correlation between whole-body bioluminescence signal and parasitaemia measured using Giemsa-stained thin blood smears and flow cytometry respectively in donor and study mice in the modified Thompson test. The algebraic formulas which represent these correlations can be successfully used to assess PLPP in donor and study mice. In addition, the new methodology can pinpoint sick animals 2-8 days before they would have been otherwise diagnosed based on behavioural or any other signs of malaria disease. CONCLUSIONS: The new method for predicting parasitaemia in the modified Thompson test is simple, precise, objective, and minimizes false positive results that can lead to the premature removal of animals from study. Furthermore, from the animal welfare perspective of replace, reduce, and refine, this new method facilitates early removal of sick animals from study as soon as the study objective has been achieved, in many cases well before the clinical signs of disease are present.

Engineered Cardiac Microtissue Biomanufacturing Using Human Induced Pluripotent Stem Cell Derived Epicardial Cells.

Epicardial cells are a crucial component in constructing in vitro 3D tissue models of the human heart, contributing to the ECM environment and the resident mesenchymal cell population. Studying the human epicardium and its development from the proepicardial organ is difficult, but induced pluripotent stem cells can provide a source of human epicardial cells for developmental modeling and for biomanufacturing heterotypic cardiac tissues. This study shows that a robust population of epicardial cells (approx. 87.7% WT1+) can be obtained by small molecule modulation of the Wnt signaling pathway. The population maintains WT1 expression and characteristic epithelial morphology over successive passaging, but increases in size and decreases in cell number, suggesting a limit to their expandability in vitro. Further, low passage number epicardial cells formed into more robust 3D microtissues compared to their higher passage counterparts, suggesting that the ideal time frame for use of these epicardial cells for tissue engineering and modeling purposes is early on in their differentiated state. Additionally, the differentiated epicardial cells displayed two distinct morphologic sub populations with a subset of larger, more migratory cells which led expansion of the epicardial cells across various extracellular matrix environments. When incorporated into a mixed 3D co-culture with cardiomyocytes, epicardial cells promoted greater remodeling and migration without impairing cardiomyocyte function. This study provides an important characterization of stem cell-derived epicardial cells, identifying key characteristics that influence their ability to fabricate consistent engineered cardiac tissues.

Lead Optimization of Second-Generation Acridones as Broad-Spectrum Antimalarials.

The global impact of malaria remains staggering despite extensive efforts to eradicate the disease. With increasing drug resistance and the absence of a clinically available vaccine, there is an urgent need for novel, affordable, and safe drugs for prevention and treatment of malaria. Previously, we described a novel antimalarial acridone chemotype that is potent against both blood-stage and liver-stage malaria parasites. Here, we describe an optimization process that has produced a second-generation acridone series with significant improvements in efficacy, metabolic stability, pharmacokinetics, and safety profiles. These findings highlight the therapeutic potential of dual-stage targeting acridones as novel drug candidates for further preclinical development.

Discovery and Structural Optimization of Acridones as Broad-Spectrum Antimalarials.

Malaria remains one of the deadliest diseases in the world today. Novel chemoprophylactic and chemotherapeutic antimalarials are needed to support the renewed eradication agenda. We have discovered a novel antimalarial acridone chemotype with dual-stage activity against both liver-stage and blood-stage malaria. Several lead compounds generated from structural optimization of a large library of novel acridones exhibit efficacy in the following systems: (1) picomolar inhibition of in vitro Plasmodium falciparum blood-stage growth against multidrug-resistant parasites; (2) curative efficacy after oral administration in an erythrocytic Plasmodium yoelii murine malaria model; (3) prevention of in vitro Plasmodium berghei sporozoite-induced development in human hepatocytes; and (4) protection of in vivo P. berghei sporozoite-induced infection in mice. This study offers the first account of liver-stage antimalarial activity in an acridone chemotype. Details of the design, chemistry, structure-activity relationships, safety, metabolic/pharmacokinetic studies, and mechanistic investigation are presented herein.

Use of In Vivo Imaging System Technology in Leishmania major BALB/c Mouse Ear Infection Studies.

Novel treatments for cutaneous leishmaniasis (CL) are needed, due to current lack of effective universal treatments, increasing resistance among the parasite, and toxic effects or impracticality of the current therapeutics. Models of direct infection with high number of Leishmania parasites in the current research of CL involving the BALB/c mouse or Golden Syrian Hamster are considered not suitable for the assessment of antileishmanial drug efficacy because of the lack of disease similarities with humans. The saliva of the sand fly vector is known to affect the host response to infection by the Leishmania parasite. Here, we build upon a previous BALB/c model infected with luciferase-expressing Leishmania major parasites. In the present study, we infect the ear dermis instead of the foot pad or base of the tail, and compare multiple methods of infection, using parasites alone or mixed with either bites from uninfected sand flies (Phlebotomus duboscqi Diptera Psychodidae:Neveu-Lemaire) or salivary gland sonicate from sand flies. Our data show a dose-response of bioluminescent signal (which represents the parasite load at the infection site), dermal lesion development, and Leishmania Donovan Units in liver and spleen. This in vivo L. major ear infection model, once optimized, can be used for assessing the efficacy of drug compounds that have been determined as very effective in the other, high inoculum CL models.

Mimicking lichens: incorporation of yeast strains together with sucrose-secreting cyanobacteria improves survival, growth, ROS removal, and lipid production in a stable mutualistic co-culture production platform.

BACKGROUND: The feasibility of heterotrophic-phototrophic symbioses was tested via pairing of yeast strains Cryptococcus curvatus, Rhodotorula glutinis, or Saccharomyces cerevisiae with a sucrose-secreting cyanobacterium Synechococcus elongatus. RESULTS: The phototroph S. elongatus showed no growth in standard BG-11 medium with yeast extract, but grew well in BG-11 medium alone or supplemented with yeast nitrogen base without amino acids (YNB w/o aa). Among three yeast species, C. curvatus and R. glutinis adapted well to the BG-11 medium supplemented with YNB w/o aa, sucrose, and various concentrations of NaCl needed to maintain sucrose secretion from S. elongatus, while growth of S. cerevisiae was highly dependent on sucrose levels. R. glutinis and C. curvatus grew efficiently and utilized sucrose produced by the partner in co-culture. Co-cultures of S. elongatus and R. glutinis were sustained over 1 month in both batch and in semi-continuous culture, with the final biomass and overall lipid yields in the batch co-culture 40 to 60% higher compared to batch mono-cultures of S. elongatus. The co-cultures showed enhanced levels of palmitoleic and linoleic acids. Furthermore, cyanobacterial growth in co-culture with R. glutinis was significantly superior to axenic growth, as S. elongatus was unable to grow in the absence of the yeast partner when cultivated at lower densities in liquid medium. Accumulated reactive oxygen species was observed to severely inhibit axenic growth of cyanobacteria, which was efficiently alleviated through catalase supply and even more effectively with co-cultures of R. glutinis. CONCLUSIONS: The pairing of a cyanobacterium and eukaryotic heterotroph in the artificial lichen of this study demonstrates the importance of mutual interactions between phototrophs and heterotrophs, e.g., phototrophs provide a carbon source to heterotrophs, and heterotrophs assist phototrophic growth and survival by removing/eliminating oxidative stress. Our results establish a potential stable production platform that combines the metabolic capability of photoautotrophs to capture inorganic carbon with the channeling of the resulting organic carbon directly to a robust heterotroph partner for producing biofuel and other chemical precursors.