Assessment of peripheral blood mononuclear cell proliferation by [2-3H]adenine uptake in the woodchuck model.

The levels of incorporated exogenous [3H]thymidine of peripheral blood mononuclear cells (PBMC) of the woodchuck were low after stimulation with mitogens concanavalin A (ConA), phytohemagglutinin (PHA), and pokeweed mitogen (PWM) when compared with other cell systems. The use of EDTA as an anticoagulant for blood sampling and AIM-V medium for culturing of PBMC improved the [3H]thymidine uptake of PBMC. A pronounced uptake is observed after use of [3H]adenine instead of [3H]thymidine for PBMC proliferation measurement. One likely explanation for the difference in [3H]adenine versus [3H]thymidine uptake is that the alternative pathway for thymidine monophosphate synthesis is important: the conversion of uridine to uridine monophosphate and, thereafter, to thymidine monophosphate. The optimal conditions for mitogen-induced proliferation of PBMC of the woodchuck were 2 micrograms/ml ConA and PHA at day 4 and 0.14 micrograms of PWM/ml at day 5. No consistent differences of [3H]adenine uptake were observed between PBMC from four woodchuck hepatitis virus-infected woodchucks and five uninfected animals.