You are asked to design experiments to allow you to investigate whether a new drug X2243 affects the ability of bacterium Salmonella Typhimurium to be taken up by peritoneal macrophages and to cause disease in a mouse model of Salmonella infection.
You are given the following pieces of information:
Growth of Salmonella Typhimurium for in oculum preparation
Salmonella Typhimurium strains are routinely grown in Luria–Bertani (LB) medium at 37 °C for 24 h. These cultures routinely contain 5 x 1012 cells/ml.
Salmonella Typhimurium mouse infection model:
BALB/c mice (male, 6-8 weeks old, 20 g) are infected by intraperitoneal injection with 1 x 104 colony forming units (CFU) per mouse.
Infection progression can be followed for 20 days and mice killed when they become severely ill. Day of death is recorded as the day after the mice were killed.
Mice can also be sampled at day 3 to monitor disease progression by measuring bacterial organ burdens in the liver and spleen. Organs are removed aseptically, weighed and homogenised in 0.5 ml sterile water. Dilutions are plated on to LB agar and cells counts performed to determine bacterial cells/g organ.
Uptake of fluorescently labelled bacteria by peritoneal macrophages: peritoneal macrophages can be sampled after three days of infection by lavage of the peritoneal cavity of killed mice with 10 ml PBS (inject 10 ml PBS into the peritoneal cavity of killed mice, gently massage the peritoneal cavity, then remove the PBS into a sterile container using a syringe and needle). The obtained cells are centrifuged at 4000 rpm, washed in PBS, and resuspended in 1 ml and analysed by flow cytometry.
Fluorescent labelling of Salmonella Typhimurium cells
To label Salmonella cells with FITC, 1 x1010 cells were suspended in 1 ml of 0.1 M sodium bicarbonate buffer and FITC added to give a final concentration of 500 µg/ml. Cells were incubated in the dark with end-over-end rotation for 30 min at room temperature, then washed 4 X in PBS to remove unbound dye and the cells resuspended in 1 ml PBS (wrap tube in foil).
Flow cytometry analysis of macrophages
1 x 107 peritoneal cells in 1 ml PBS were incubated with 1 µg PE-labelled anti-F4/80+ antibody per 100 µl reaction at 4 °C for 30 minutes. Cells were pelleted, washed and resuspended in 1 ml PBS prior to analysis by flow cytometry.
Flow cytometry was used to gate on PE-labelled macrophages, then used to determine which macrophages contained FITC-labelled Salmonella cells.
Drug X2243
Drug X2243 is provided in a solution of 50 mg/ml in 10% DMSO. It should be administered by intraperitoneal injection. Mice will receive one dose of drug at 24 hours post-infection.
Note: All mouse intraperitoneal injections should be 100 µl volumes.
Considering the information given, answer the questions below to design an experiment to test the effects of drug X2243 on Salmonella Typhimurium uptake by mouse peritoneal macrophages and its effect on disease progression in a mouse model of Salmonella Typhimurium infection. Please show calculations where appropriate.
Q1. What volume of overnight culture is required to carry out FITC labelling of Salmonella cells? (5%)
Q2. Your FITC stock solution is 10 mg/ml. What volume of FITC stock solution do you need to add to get the required concentration in the labelling reaction? (5%)
Q3. Once you have labelled your cells, what dilution is required to obtain the cell concentration required for intraperitoneal infection of mice? (5%)
Q4. Assume that you want to treat mice with X2243 at 10 mg/kg drug. What dilution will you need to make to obtain the correct drug concentration for treating mice? (10%)
Q5. What will you dilute your drug with? (5%)
Q6. For our mouse experiments, what is the most suitable control for X2243 drug treatment? (5%)
Q7. In previous research publications, researchers have used 16 mice per treatment group for survival studies and 8 mice per group for three-day organ and macrophage experiments.
Considering the power curves shown below which were constructed from data from previous experiments, do you agree with these group sizes?
How many mice per group are required for survival experiments (where you are looking for a difference of a least two days in mean survival time) where you want to be able to detect a two day difference in mean survival time? (15%)
How many mice per group are required for three-day experiments (remember that liver and spleen burdens can be determined from the same mice) where you want to be able to detect a one log difference in organ burden counts (note that organ burdens are expressed as log10 CFU/g)? (15%)
Justify your answers for the number of mice per group chosen.
Power curve for detecting a two day difference in mean survival time. Power curves are plotted for mouse group sizes of 4, 8, 12, 16 and 20.
Power curve for detecting a one log difference in spleen bacterial organ burdens (measured as log10 CFU/g). Power curves are plotted for mouse group sizes of 4, 8, 12, 16 and 20.
Power curve for detecting a one log difference in liver bacterial organ burdens (measured as log10 CFU/g). Power curves are plotted for mouse group sizes of 4, 8, 12, 16 and 20.
Q8. To label macrophages for flow cytometry, you have a stock of PE-labelled anti-F4/80+ antibody at 10 mg/ml. What volume of stock antibody do you need to add to label 1 x 107 peritoneal cells? (5%)
Q9. Create a plan for the experiment required to test whether drug X2243:
affects survival of mice infected with Salmonella Typhimurium
affects bacterial organ burdens
affects uptake of bacteria by peritoneal macrophages.
Your plan should include the number of mice required for each part of the experiment and what procedures would be carried out on different days (you can include tables, flow charts or Gantt charts to illustrate your answers). (30%)