regulatory cells may account, at least in part, for the extreme difficulty of treating the infections they cause [7] and the lack of correlation between their susceptibility to drugs under axenic conditions and in vivo. At present the only available vaccine against tuberculosis, M. bovis Bacillus Calmette-Gue´rin (BCG), has proven unreliable in being able to protect against pulmonary tuberculosis in adults [41]. It has been proposed by some epidemiological studies that BCG protects the host in countries where low levels of environmental NTM exposure occurs compared to lack of BCG protection in developing countries where high levels of environmental NTM exposure occurs [42]. We propose that the widely varying degrees of BCG efficacy present in different studies may be due to environmental NTM exposure and pre-sensitization of individuals to regulatory T cell expansion resulting in cross reactivity during M. tuberculosis [24] disease. Further research is required to link the virulence and molecular mechanisms underlying the increased pathogenicity of BRA100 M. massiliense epidemic isolates, whether related to changes in surface composition under the selective pressure of GTA disinfection or otherwise, which are presently being investigated in our laboratories. Materials and Methods Bacterial Cultures CIP 108297 (the M. massiliense type strain) [2] and the BRA100 isolate, CRM-0019 [13], were routinely grown at 30uC in liquid Middlebrook 7H9-OADC medium (Difco) supplemented with 0.05% Tween 80 or on solid Middlebrook 7H11-OADC medium. GTA-susceptibility testing The susceptibility of M. massiliense to glutaraldehyde in suspension tests was determined as described by Griffiths et al. [43]. Mice Specific-pathogen-free female GKO mice, from 6 to 8 weeks old, were purchased from the Jackson Laboratories, Bar Harbor, Maine. Mice were maintained in the Biosafety Level III animal laboratory at Colorado State University, and were given sterile water, mouse chow, bedding, and enrichment for the duration of the experiments. The specific pathogen-free nature of the mouse colonies was demonstrated by testing sentinel animals. All experimental protocols were approved by the Animal Care and Usage Committee of Colorado State University. The CSU animal assurance welfare number is A3572-01. Experimental infection of BMDM and bacterial enumeration Macrophages were infected with mycobacteria, and 2 hours later, the monolayers were washed to remove extracellular bacilli. The numbers of intracellular mycobacteria were measured by plating. Briefly, monolayers were washed at each time point to remove extracellular bacilli and 1 ml double-distilled H2O containing 0.05% Tween 80 was added to monolayers and incubated for 10 min to lyse macrophages. After passing through a 26-gauge needle five times, the lysates were serially diluted and plated onto Middlebrook 7H11 agar plates. Monolayers that weren’t lysed were replenished with fresh medium. Cellular viability assays BMDM cell death was assayed by trypan blue exclusion and determined by flow cytometry using BDTM Viability Counting Increased Virulence in M. massiliense Infection Beads, as described by the manufacturer (BD PharMingen, San Jose, CA USA 95131) cell viability-staining methods [32]. Experimental infections in mice Mice were challenged with reference strain Mycobacterium massiliense CIP 108297 and a clinical epidemic BRA100 strain, Mycobacterium massiliense CRM-0019, using a Glas-Col (Terre Haute, Inc.) aerosol generator calibrated to deliver either a HDA of ,1,000 bacilli per animal. At days 1, 30 and 60 following infection, bacterial loads in the lungs and spleen, lung histology, and mononuclear and lymphocytic cellular expressions were determined. Bacterial counts were determined by plating serial dilutions of homogenates of lungs on nutrient 7H11 agar and counting colony-forming units after 5-10 days incubation at 30uC. A total of five animals were infected for each time point. Histological analysis The accessory lobe of the lung from each mouse was fixed with 10% formalin in phosphate buffered saline (PBS). Tissue sections were stained using haematoxylin and eosin and acid-fast stains as previously reported [20,23]. Lung cell digestion Briefly, single cell suspensions were prepared as described previously [20]. Lungs and spleens were aseptically removed, teased apart and treated with a solution of deoxyribonuclease IV (DNAse) (Sigma Chemical, 30 mg/ml) and collagenase XI (Sigma Chemical, 0.7 mg/ml) for 45 min at 37uC. To obtain a single-cell suspension, the organs were gently passed through cell strainers (Becton Dickinson, Lincoln Park, NJ). The remaining erythrocytes were lysed with Gey’s solution (0.15 M NH4Cl, 10 mM KHCO3) and the cells were washed with Dulbecco’s modified Eagle’s minimal essential medium. Cell suspensions from each individual mouse were incubated with monoclonal antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll-a protein (PerCP), or allophycocyanin (APC) at 4uC for 30 minutes in the dark as described previously [20]. Total cell numbers were determined by flow cytometry using BDTM Liquid Counting Beads, as described by the manufacturer (BD PharMin- gen, San Jose, CA USA 95131). All analyses were performed with an acquisition of at least 100,000 total events. Intracytoplasmic cytokine staining Cells were first stained for cell surface markers as indicated above and thereafter the same cell suspensions were prepared for intracellular staining as described before [20,23]. For flow cytometry analysis, single-cell suspensions of lung from each mice were re-suspended in PBS (Sigma-Aldrich) containing 0.1% of Sodium Azide (PBS+Na/Az). Cells were incubated in the dark for 25 min at 37uC with pre-determined optimal titrations of specific antibody (directly conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridin-cholorophyll-protein (PerCP), allo- phycocyanin (APC), Pacific Blue, Alexa 700); or after biotin antibody incubations washed and incubated for 25 minutes more with streptavidin Qdot800 (Invitrogen), followed by two washes in PBS containing 4% sodium azide. Measurement of intracellular cytokines was conducted by pre-incubating lung cells with monensin (3 mM) (Golgi Stop, BD PharMingen), anti-CD3 and anti-CD28 (both at 0.2 mg/106 cells) for 4 h at 37uC, 5% CO2. The cells were then surface stained, incubated for 30 minutes at 37uC, washed then fixed and permeabilized with Perm Fix/Perm Wash (BD Pharmingen). Finally, the cells were stained with intracellular Foxp3 (FJK-16s), IL-12 (clone REM-11) TNFa (clone PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24726