Bhardwaj. other bacteria (10) and the attenuated mutants of infection (26). In addition, the intracellular nature of and its ability to survive and multiply in the intracellular environment highlight the need to generate CMI responses to combat infection. During host TAK-700 (Orteronel) immune responses to infection, dendritic cells (DC) play a key role in the generation of adaptive immune responses (2). DC are the most potent stimulators of na?ve T-cell responses and reside in most tissues and organs (16, 23) at major portals of microbial entry (24). Here, they perform a sentinel-like function, continuously sampling their external environment (24) for foreign antigens. Under physiological conditions, DC exist in an immature state primed for antigen uptake and processing. Pathogen recognition (via receptors such as the toll-like receptor family), together with the production of proinflammatory signals, initiates cell maturation, which transforms the DC into efficient T-cell stimulators (16). During maturation, DC up-regulate their expression of chemokine receptors (25), as well as molecules essential for the activation of T cells, such as major histocompatibility complex (MHC) molecules and the costimulatory molecules CD80 and CD86 (2). The DC then leave the peripheral tissues and migrate to the draining lymph nodes (16), final maturation occurring under the control of specific T cells through the interaction of CD40 and CD40L (14). In this study, we employed DC as a delivery vector to generate CMI responses to in an attempt to develop an effective vaccination regimen for protection against infection. MATERIALS AND TAK-700 (Orteronel) METHODS Experimental animals. BALB/c mice were obtained from Charles River Ltd. and maintained under specific-pathogen-free conditions with free access to food and water. All procedures carried out were EXT1 in accordance with the requirements of the Animal (Scientific Procedures) Act of 1986. Isolation and culture of dendritic cells from murine bone marrow. A method was derived from established methods (19, 23, 27, 29), described briefly as follows. Bone marrow was extracted from murine rear tibiae and fibulas and cultured at a concentration of 2 106 cells ml?1 in six-well tissue culture plates. Standard culture medium was comprised of RPMI 1640 (Sigma, United Kingdom) supplemented with 10% heat-inactivated fetal bovine serum (Sigma, United Kingdom), 1% penicillin-streptomycin-glutamine (Sigma, United Kingdom), and 50 M 2-mercaptoethanol. The culture medium was supplemented with 20 ng ml?1 granulocyte-macrophage colony-stimulating factor (R&D Systems) and 10 ng ml?1 tumor necrosis factor alpha (R&D Systems). Cells were cultured for 96 h at 37C in the presence of 5% CO2 in a fully humidified atmosphere, after which time they were removed from the culture plates by gentle scraping. After being washed, the cell suspension was layered onto 13.7% (wt/vol) metrizamide (Sigma, United Kingdom) and the DC were purified using centrifugation (23a). Flow cytometry. Cells were counted, washed, and then resuspended in phosphate-buffered saline (PBS) supplemented with 2.5% heat-inactivated fetal bovine serum at a concentration of 106 cells per 100 l. The appropriate fluorochrome-labeled antibodies at the correct concentrations for each fluorochrome were added (all antibodies were obtained from Pharmingen, BD Biosciences, United Kingdom) and the cells were incubated at 4C for 30 min. Next, 4% paraformaldehyde was then added to prevent any further binding. Samples were then fixed at 4C overnight before analysis on a Becton Dickinson FACScan flow cytometer using Cell Quest Pro analysis software. Growth of NCTC 4845 was grown in nutrient broth in a static culture for 18 h at 37C. Viable counts were obtained following use of a culture by culturing aliquots at 37C overnight on nutrient agar TAK-700 (Orteronel) plates. Heat inactivation of bacteria. Bacterial cells were harvested by centrifugation and washed three times in PBS before resuspending them in 1/10 the original volume of PBS. The bacterial cell suspension was then incubated in a water bath at 80C for 3 h with occasional shaking. After inactivation, the suspension was checked TAK-700 (Orteronel) for viability by.