02% deoxyribonuclease I was added. The solution was pelleted, resuspended in cul ture medium, and brought to a single protein inhibitor cell suspension by repeated pipetting followed by passing through a 105m pore mesh. Cells were cultured at 37 C in humidified 5% CO295% air. Medium was replaced every 57 days. Cultures were used between 12 and 15 days in vitro. At this point they typi cally consisted Inhibitors,Modulators,Libraries of 75% type I astrocytes and 25% micro glia. Separately, to obtain astrocyte enriched cultures, non astrocytes were detached from the flasks by shaking, and removed by changing the medium. We confirmed that astrocyte enriched cultures consisted of 95% astrocytes. The BV 2 murine microglial cell lines were kindly pro vided by Dr. Sung Joong Lee.

The cells were grown and maintained in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum and 1% penicillinstreptomycin at 37 C in a humidified incubator under 5% CO2. Cultures of Mtb and preparation of s Mtb Cultures of Mtb H37Rv were grown to mid log phase in Inhibitors,Modulators,Libraries Middlebrook 7H9 liquid medium supplemented with oleic acidalbumindextrosecatalase Inhibitors,Modulators,Libraries , washed three times in sterile saline, and resuspended in RPMI 1640 medium at the various concentrations. Separate culture suspensions were sonicated for 10 min on ice, in order to obtain non infective cell lysates, as described previously. S Mtb was pooled and applied to an immobilized polymyxin B column. Preparations of the s Mtb used in experi ments were tested for lipopolysaccharide contami nation by a Limulus amebocyte lysate assay and contained less than 50 pgml at the concentrations of the s Mtb used in experiments.

In order to show that the stimulatory capacity Inhibitors,Modulators,Libraries of the s Mtb was not the result of contamination with LPS, experiments were performed with the addition of the specific LPS inhibiting oligopeptide polymyxin B before s Mtb stimulation. Reagents and antibodies LPS and peptidoglycan was purchased from Sigma. BLP, a synthetic bacterial lipopeptide derived from the immunologi cally active N terminus of bacterial lipoproteins, Inhibitors,Modulators,Libraries was pur chased from Invitrogen. NAC, DPI, allopurinol and rotenone were purchased from Calbio chem. Dimethyl sulfoxide was added to cultures at 0. 1% as a sol vent control. Specific inhibitors of p38 MAPK, SB203580, and specific inhibitors of MEK, U0126, and PD98059 were purchased from Calbiochem.

The expression plas mids that encode p47phox WT and DN, and TAT Ser345 peptide were that kindly provided by Dr. J. El Benna. Cells were transfected using LipofectAMINE as indicated by the manufacturer. Specific Abs against ERK12, phospho ERK12, p38, phospho p38 Abs were purchased from Cell signalling Technology. Anti Dectin 1 mAb was from Serotec. Abs to p47phox, and actin were purchased from Santa Cruz Biotechnology. The anti phospho p47phos Ab was used, as previously described. Anti IL 1 mAb and isotype mAb were purchased from R D system.