Generation of Mature Toxoplasma gondii Bradyzoites in Human Immortalized Myogenic KD3 Cells.

Toxoplasma gondii is a zoonotic protozoan parasite and one of the most successful foodborne pathogens. Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue. Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models. In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited. We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites. Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo-like features, including pepsin resistance, oral infectivity, and antifolate resistance. In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics. In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO2 levels. We also discuss critical aspects of the procedure and suggest improvements. Key features • This protocol describes a scalable human myotube-based in vitro system capable of generating encysted bradyzoites featuring in vivo hallmarks. • Bradyzoite differentiation is facilitated through CO2 depletion but without additional artificial stress factors like alkaline pH. • Functional maturation occurs over four weeks.

The in vivo metabolism of Furazadrol in greyhounds.

Samples of the 'dietary supplement' Furazadrol sourced through the internet have been reported to contain the designer anabolic androgenic steroids [1',2']isoxazolo[4',5':2,3]-5α-androstan-17ß-ol (furazadrol F) and [1',2']isoxazolo[4',3':2,3]-5α-androstan-17ß-ol (isofurazadrol IF). These steroids contain an isoxazole fused to the A-ring and were designed to offer anabolic activity while evading detection, raising concerns over the potential for abuse of this preparation in sports. The metabolism of Furazadrol (F:IF, 10:1) was studied by in vivo methods in greyhounds. Urinary phase II Furazadrol metabolites were detected as glucuronides after a controlled administration. These phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli ß-glucuronidase to afford the corresponding phase I metabolites. Using a library of synthetically derived reference materials, the identities of seven urinary Furazadrol metabolites were confirmed. Major confirmed metabolites were isofurazadrol IF, 4α-hydroxyfurazadrol 4α-HF and 16α-hydroxy oxidised furazadrol 16α-HOF, whereas the minor confirmed metabolites were furazadrol F, 4ß-hydroxyfurazadrol 4ß-HF, 16ß-hydroxyfurazadrol 16ß-HF and 16ß-hydroxy oxidised furazadrol 16ß-HOF. One major hydroxyfurazadrol and two dihydroxyfurazadrol metabolites remained unidentified. Qualitative excretion profiles, limits of detection and extraction recoveries were established for furazadrol F and major confirmed metabolites. These investigations identify the key urinary metabolites of Furazadrol following oral administration, which can be incorporated into routine screening by anti-doping laboratories to aid the regulation of greyhound racing.

Prehospital statin and aspirin use and the prevalence of severe sepsis and acute lung injury/acute respiratory distress syndrome.

OBJECTIVES: To determine whether prehospital statin use is associated with a lower risk of sepsis, acute lung injury/acute respiratory distress syndrome, and mortality in critically ill patients. We also investigated the effect of combined prehospital use of both statins and aspirin. DESIGN: Cross-sectional analysis of a prospective cohort. PATIENTS: A total of 575 critically ill patients admitted to the medical or surgical intensive care unit of an academic tertiary-care hospital. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Of 575 patients, 149 (26%) were on statin therapy before hospitalization. A multivariable analysis including age, gender, current tobacco use, prehospital aspirin use, race, and Acute Physiology and Chronic Health Evaluation II score revealed that patients on statin therapy before hospitalization were less likely to have or develop severe sepsis (odds ratio 0.62, 95% confidence interval 0.40-0.96) or acute lung injury/acute respiratory distress syndrome (odds ratio 0.60, 95% confidence interval 0.36-0.99) during the first four intensive care unit days. In-hospital mortalities for patients with and without prehospital statin use (odds ratio 1.06, 95% confidence interval 0.62-1.83) were similar. Patients who had prehospital use of both statins and aspirin had the lowest rates of severe sepsis, acute lung injury/acute respiratory distress syndrome, and mortality. CONCLUSIONS: Prehospital use of statins may be protective against sepsis and acute lung injury. This effect may be potentiated by prehospital aspirin use.