The bacterial lysate (Lysate) was stored at −80 °C, and 5 μg mL−1 lysate was used in all the experiments. His-tag-fused (6 ×) pneumolysin was expressed in and purified from an Escherichia coli strain, and residual lipopolysaccharide was removed by passage over End-X resin as described previously (Srivastava et al., 2005). All media described below were supplemented with 10% fetal bovine serum (GIBCO), penicillin (100 U mL−1) and streptomycin (0.1 mg mL−1). A549 (human alveolar epithelial) and BEAS-2B (human bronchial epithelial) cells were maintained in RPMI-1640 (Hyclone). HeLa (human cervix epithelial) cells were maintained

in MEM (Hyclone). HM3 (human colon epithelial) cells were maintained in DME H-21 (UCSF Cell Culture Facility). Cells were cultured at 37 °C in a humidified Small molecule library PR-171 in vitro 5% CO2 air-jacketed incubator. Total RNA was isolated using TRIzol® Reagent following Invitrogen’s instruction. SYBR Green PCR Master Mix (Applied Biosystems) was used for Q-PCR. Synthesis of cDNA from total RNA was performed using TaqMan Reverse Transcription Reagents (Applied Biosystems). The primer sequence information for human IL-1β, TNF-α and cox2 was as follows: IL-1β primers, 5′-AAACAGATGAAGTGCTCCTTCCAGG-3′ and 5′-TGGAGAACACCACTTGTTGCTCCA-3′; TNF-α primers, 5′-CAGAGGGAAGAGTTCCCCAG-3′

and 5′-CCTTGGTCTGGTAGGAGACG-3′; cox2 primers, 5′-GAATCATTCACCAGGCAAATTG-3′ and 5′-TCTGTACTGCGGGTGGAACA-3′. Reactions were amplified and quantified

using a 7500 Real-Time PCR System and the manufacturer’s software (Applied Biosystems). Relative quantities of IL-1β, TNF-α and cox2 mRNA were calculated using the comparative CT method PtdIns(3,4)P2 and normalized by human GAPDH (5′-CCCTCCAAAATCAAGTGG-3′ and 5′-CCATCCACAGTCTTCTGG-3′) for the amount of RNA used in each reaction. The culture supernatants were collected and used to determine the levels of secreted TNF-α using a Human TNF-α Immunoassay (R&D systems). Supernatants were filtered through 0.22-μm filters and used to quantify the TNF-α according to the manufacturer’s instructions. The minimum detectable dose of TNF-α was 0.5 pg mL−1 as reported by the manufacturer of the ELISA kit. The experiments were repeated three times. Epithelial cells act as the first line of host defense against microorganisms by producing a range of molecules for clearance. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes. Because IL-1β and TNF-α have been identified as prominent proinflammatory cytokines, we examined cytokine expression in response to clinical isolates of S. pneumoniae in human epithelial cells. We used real-time Q-PCR to quantify the level of mRNA expressions in human epithelial HeLa cells following incubation with clinical isolates of S. pneumoniae strains D39, 6B, 19F and 23F. As shown in Fig.