We have now examined the role of the two the MAPK pathway and NF?B activation in BCG killing and nitric oxide produc tion. We report that both of those pathways are activated by BCG alone and that opsonization of BCG with SP A leads to enhanced activation of both pathways, contribut ing to elevated intracellular BCG killing. Components and procedures Materials Uracil was purchased from NEN. Fetal bovine serum for culture of rat bone marrow macrophages was purchased from HyClone Lab oratories, all other tissue culture reagents had been from GIBCO BRL. Kinase assay kits, U0126, and antibodies towards phosphorylated and non phos phorylated ERK1 and ERK2 have been obtained from Cell Sig nalling Technologies. All other reagents were bought from Sigma Chemical.

Cells and bacteria Rat bone marrow derived macrophages have been isolated from female Sprague Dawley rats as previously described. Briefly, femurs were removed from rats along with the marrow flushed into 50 ml conical tubes. The cells had been resuspended in DMEM and cultured in DMEM with 10% fetal bovine serum, antibiotics, and 10% L cell conditioned medium for 5 seven days. Docetaxel IC50 Macrophages have been then eliminated from the culture dishes with cold EDTA and plated in 24 or six wells dishes as described for every experiment. Just before infection with BCG, the media was altered to serum and antibiotic absolutely free DMEM. For NF?B experiments, bone marrow macrophages have been pre pared from femurs of transgenic mice expressing a luci ferase gene driven by the HIV one prolonged terminal repeat containing sixB consensus web-sites in its promoter. BCG, Pasteur strain, was obtained in the American Style Culture Collection.

Bacteria had been cultured in Middlebrook Broth supplemented with OADC enrichment, Microcystin-LR structure and 1. five ml aliquots of bacteria at roughly 108 bacteria per ml were stored at 70 C. Colony forming units per ml have been determined by plating serial dilutions in the bacteria onto Middle brook agar plates, and counting colonies after 2 three weeks of development. Purification of SP A SP A was purified from human alveolar proteinosis fluid or Dr. Samuel Hawgood as previously described. Briefly, 1 2 ml of APF in PBS was extracted with 25 ml of one butanol and then dried overnight beneath nitrogen. Dried protein was resuspended in 1 mM HEPES buffer, pH seven. 5, with 0. 15 M NaCl and 20 mM n octyl D glucoside. The pel let was collected by centrifugation at 17,000 ? g as well as system repeated.

The final pellet was resuspended in 5 mM HEPES buffer with one mM EDTA and dia lyzed for 48 hours with buffer adjustments. Soon after dialysis, pol ymyxin B agarose was additional on the SP A and the mixture was rotated for one hour at space temperature. The poly myxin B agarose was eliminated by centrifugation plus the SP A concentration was determined working with the BCA pro tein kit from Pierce. The ultimate SP A preparation was divided into 1 ml aliquots and stored at four C for immedi ate use or twenty C for long lasting storage. The SP A was ana lyzed for purity by SDS Web page and for endotoxin contamination utilizing the Limulus amebocyte lysate assay. Endotoxin levels were rou tinely determined to be under 0. 05 units ml. Infections Frozen stocks of BCG were thawed and vortexed vigor ously by using a glass bead to break up any clumps.

The myco bacteria were collected by centrifugation, then resuspended in PBS. SP A or buffer was added, and also the mixture incubated for 30 minutes at 37 C. The cells in DMEM were then contaminated with the opsonized or buffered mycobacteria for that time periods and in the MOIs as indi cated in each experiment. BCG killing assays To find out the impact of protein tyrosine kinase inhibi tors on BCG killing, a modification from the approach of Chan et al. employing metabolic labelling of viable BCG was utilized as follows, cells were incubated with BCG or SP A BCG for 4 hr at 37 C.