(A) HMEEC releases IL-1α by treatment with NTHi lysate. Cell culture medium was collected at 4, 24 and 48 hrs after treatment. Released IL-1α was quantified using ELISA (ND – not detectable). Treatment with NTHi lysate up-regulates IL-1α expression in a time dependent manner in Hela, A549 and HMEEC cells (B). Cells were treated with NTHi lysate for 2, 4 and 8 hours, and real time quantitative PCR was performed after RNA purification and reverse transcription. Inoculation of NTHi lysate into the middle ear results in the up-regulation of IL-1α mRNA in the mouse model (C). Mice were transtympanically inoculated with NTHi lysate and mucosal RNA was extracted after 6, 9, 12 and 24. Control animals received PBS. The RNA samples were reverse transcribed and analyzed by real time quantitative PCR. Values are given as mean ± standard deviation. N = 3. *: p < 0.05, **: p < 0.01.