We’ve examined the function of the two the MAPK pathway and NF?B activation in BCG killing and nitric oxide produc tion. We report that the two of these pathways are activated by BCG alone and that opsonization of BCG with SP A contributes to enhanced activation of both pathways, contribut ing to enhanced intracellular BCG killing. Materials and approaches Products Uracil was bought from NEN. Fetal bovine serum for culture of rat bone marrow macrophages was obtained 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 had been obtained from Cell Sig nalling Technologies. All other reagents were obtained 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 and also 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 five seven days. Macrophages were then removed from your culture dishes with cold EDTA and plated in 24 or six wells dishes as described for each experiment. Prior to infection with BCG, the media was transformed to serum and antibiotic no cost DMEM. For NF?B experiments, bone marrow macrophages were pre pared from femurs of transgenic mice expressing a luci ferase gene driven from the HIV 1 prolonged terminal repeat containing sixB consensus web-sites in its promoter. BCG, Pasteur strain, was obtained through the American Form Culture Assortment.

Bacteria had been cultured in Middlebrook Broth supplemented with OADC enrichment, why and one. five ml aliquots of bacteria at approximately 108 bacteria per ml had been stored at 70 C. Colony forming units per ml had been established by plating serial dilutions with the bacteria onto Middle brook agar plates, and counting colonies immediately after two 3 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 after which dried overnight under nitrogen. Dried protein was resuspended in one mM HEPES buffer, pH seven. five, with 0. 15 M NaCl and 20 mM n octyl D glucoside. The pel allow was collected by centrifugation at 17,000 ? g plus the approach repeated.

The last pellet was resuspended in 5 mM HEPES buffer with 1 mM EDTA and dia lyzed for 48 hours with buffer changes. Right after dialysis, pol ymyxin B agarose was added for the SP A plus the mixture was rotated for one hour at area temperature. The poly myxin B agarose was eliminated by centrifugation along with the SP A concentration was determined applying the BCA pro tein kit from Pierce. The final SP A preparation was divided into 1 ml aliquots and stored at 4 C for immedi ate use or twenty C for long run storage. The SP A was ana lyzed for purity by SDS Webpage and for endotoxin contamination utilizing the Limulus amebocyte lysate assay. Endotoxin levels had been rou tinely established for being less than 0. 05 units ml. Infections Frozen stocks of BCG had been thawed and vortexed vigor ously with a glass bead to break up any clumps.

The myco bacteria were collected by centrifugation, after which resuspended in PBS. SP A or buffer was extra, along with the mixture incubated for 30 minutes at 37 C. The cells in DMEM were then infected with the opsonized or buffered mycobacteria for the time intervals and on the MOIs as indi cated in just about every experiment. BCG killing assays To determine the result of protein tyrosine kinase inhibi tors on BCG killing, a modification on the process of Chan et al. using metabolic labelling of viable BCG was used as follows, cells have been incubated with BCG or SP A BCG for 4 hr at 37 C.