Cells were infected with a multiplicity of infection of 2-10 bacteria per cell and incubated for 3 hours at 37°C in 5% CO2. After incubation, monolayers were thoroughly washed with phosphate-buffered saline to remove extracellular bacteria and fresh medium was added. To evaluate the bacterial growth, supernatants were aspirated and monolayers were lysed with 0.5% Nonidet P40 (Roche Diagnostics, Mannheim, Germany) at 3 hours and days 1, 4, and 7 after infection. Serial 10-fold dilutions

of cellular lysates were plated on Middlebrook 7 H11 plates and incubated for 3 weeks at 37°C in 5% CO2, and colonies were counted. Intracellular growth was expressed as the growth rate, which is the slope of the function of log10 CFU values throughout the infection period (3 hours and days 1, 4, and 7). Three #Selleckchem GSK1210151A randurls[1|1|,|CHEM1|]# or more independent experiments were performed for each assayed strain. Cytokine analysis Culture PND-1186 nmr supernatants from control and infected THP-1 cells were harvested after 3 hours and on days 1, 4, and 7, frozen

at -70°C, and assayed using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (BD Biosciences, Lincoln Park, NJ) to measure levels of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10). Statistical analysis Three independent experiments were performed per strain. The means and standard errors were determined for each measurement in both intracellular growth and cytokine production. One-way analysis of variance with repetitive measures was used to determine P values, which were adjusted using the Bonferroni method. All the comparisons were carried out using the program SPSS 17.0. Acknowledgements This study was partially funded by the Fondo de Investigaciones Sanitarias (FIS060882; FIS061467; FIS06/90490; 06/90357), Junta de Andalucía (0453/06, 151/05), and the Instituto de Salud Carlos III (CIBER Enfermedades Respiratorias CB06/06/0058 and

the Spanish Network for the Research in Infectious Diseases [REIPI RD06/0008]). N.A.R. received a grant from the Consejería de Educación de la Comunidad de Madrid and the European Social Fund (3334/2004). We are grateful to Joaquin Navarro from the Immunology Ribonucleotide reductase Department in Gregorio Marañón Hospital for assessing us with the cytokine assays and to the INDAL-TB group in Almería for the recruitment of cases and compilation of clinical data. We are grateful to Thomas O’Boyle for editing and proofreading the final version of the manuscript. References 1. WHO: Global tuberculosis control: surveillance, planning, financing. WHO report 2008. WHO/HTM/TB/2008.393.Geneva. 2008. 2. Frieden TR, Sterling TR, Munsiff SS, Watt CJ, Dye C: Tuberculosis. Lancet 2003,362(9387):887–899.PubMedCrossRef 3.