Expression of the proteins was observed using a fluorescent microscope (Leica Microscopy system Inc., Buffalo Grove, IL, USA). Immunization with recombinant adenovirus.  Six- to 8-week-old female C57BL-6 mice were purchased from Charles River

Laboratories (Wilmington, MA, USA) and maintained under specific pathogen-free conditions at the animal facility buy Alvelestat at Colorado State University. For intranasal immunization (i.n.) of one dose of recombinant adenovirus, 5 × 107 PFU of recombinant adenovirus or AdLac Z was diluted with phosphate-buffered saline (PBS) to a total volume of 20 μl and delivered into the airway of a mouse with a fine pipette tip [12]. Mycobacterium bovis BCG at a dose of 5 × 105 CFU/mouse was diluted in PBS to a total volume of 100 μl and injected subcutaneously. Lymphocyte isolation and in vitro antigen stimulation.  At 4 weeks post-vaccination, mice were humanely euthanized, and the spleens were removed. Single-cell suspensions were prepared as described previously [12] and cultured. Approximately 1 × 106 cells per well were seeded in 96-well plates in RPMI-1640 medium containing 10% foetal calf serum, 100 U/ml penicillin, 100 g/ml streptomycin and 2 mm of l-glutamine at 37 °C in 5% CO2 with or without antigen stimulation (ESAT-6 at 5 μg/ml). ICG-001 The culture supernatants were collected after 24 h

and stored at −80 °C until used. Analysis of ESAT-6-specific T cells by ELISPOT assay.  Isolated splenocytes were seeded at 1 × 106 per well many in a 96-well PVDF microplate (Millipore, Bedford, MA, USA) that was precoated overnight with a mouse IFN-γ capture antibody (R&D Systems, Minneapolis, MN, USA;

1:60 dilution). Cells were incubated for 24 h with or without stimulation by ESAT-6. The plate was then developed by a standardized streptavidin-conjugated alkaline phosphatase and chromogen method (R&D Systems). The number of IFN-γ-releasing cells was determined using a Cellular Technology Ltd Series 5 UV Immunospot Analyzer (Shaker Heights, OH, USA). TNF-α measurement.  The level of TNF-α in the supernatants was measured with a mouse-specific enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems). Stored samples were thawed, and the manufacturer’s protocol was used to assess the concentration. The sensitivity of detection was 2 pg/ml. Low-dose aerosol infection with Mycobacterium tuberculosis.  Animals were infected with live M. tuberculosis H37Rv via the aerosol route in a Glass-Col Airborne inhalation exposure system that delivered approximately 100 CFU M. tuberculosis bacilli per mouse. At 4 weeks post-infection, the protective efficacy was evaluated by plating serial 10-fold dilutions of lung and spleen homogenates on Middlebrook 7H11 agar plates. Plates were then incubated at 37 °C for 21 days, and colonies were counted. Data analysis.