Figure 2

Lung caspase activity in RSV-infected and uninfected mice. Representative HPLC runs of the Ac-DEVD cleavage reaction by lung specimens from infected (B and D) and uninfected (A and C) mice on days 6 (A-B) and 10 (C-D) after inoculation are shown. Briefly, BALB/c mice were inoculated on day 0 with the RSV-A2 or the mock preparation of HEp-2 culture supernatant. Lung specimens were collected on days 6 and 10 after inoculation and immediately incubated at 37°C in 1.0 mL KH buffer (continuously gassed with 95% O₂: 5% CO₂) with and without 32 μM zVAD-fmk (pan-caspase inhibitor) for 10 min. Ac-DEVD-AMC (37 μM) was then added and the incubations continued for additional 20 min. The tissues were disrupted by vigorous homogenization and the supernatants were separated on HPLC and analyzed for the AMC peak (retention time, R _t , ~4.8 min; the AMC moiety reflects caspase-3 activity). Ac-DEVD-AMC had a R _t of ~2.5 min. The substrate runs were without lung specimen and showed negligible AMC peak areas. Panel E (positive control) shows increased lung tissue caspase activity in a sample from uninfected mouse incubated in vitro in KH buffer (without oxygenation) for 0, 2 and 6 h. The AMC peak area (arbitrary units mg^-1) at 0 h (immediately post tissue collection) was 482,115, at 2 h was 3,417,616 and at 6 h was 6,456,028. Similar results were noted in 10 independent experiments spanning the course of the disease over 15 days.