involved, 4, .75% of lung involved. (ii) primary lesions: 0-no primary lesions present, 1, a single primary lesion, 2, two or more multi-focal primary lesions, 3, two or more multifocal to coalescing primary lesions, 4, multiple coalescing and extensive primary lesions. (iii) secondary lesions: 0, no secondary lesions present, 1, ,25% of lung involved, 2, 25–50% of lung involved, 3, 50–75% of lung involved, 4, .75% of lung involved. Necrosis (iv), mineralization (v) and fibrosis (vi) were scored based on severity as follows: 0, none, 1, minimal, 2, mild, 3, moderate, 4, marked. Subcategory scores were added for the final total score for each organ (Range: 0–24). Immunohistochemical scoring For immunohistochemical scoring, the total lung area was divided into primary lesions (lesions with a central core of necrosis) and primary lesion free (PLF) lungs. Within these regions the overall immunohistochemical scoring was determined based on extent of staining (0, none; 1, ,25%; 2, 25–50%; 3, 50–75%; 4, .75%) and staining intensity (0, none: 1, mild; 2, moderate; 3, marked; 4, extensive). The resultant total overall scores were converted to a four-point scale. Isolation of guinea pig alveolar macrophages Guinea pig alveolar macrophages were collected from naive, non-infected guinea pigs by repeated infusion and aspiration of 10 ml of 16 Hank’s balanced salt solution (MP Biomedicals, Solon, OH) three times. Cells were pelleted by centrifugation and washed twice with 16 PBS and the cell numbers and viability determined using a hemocytometer in combination with Trypan blue dye exclusion (Gibco Life Technologies, Grand Island, NY). The numbers of viable macrophages were adjusted to 26105 cells/ ml in a serum free media (Aim-V; Gibco, Grand Island, NY) containing bovine serum albumin, L-glutamine, streptomycin sulfate, and gentamicin sulfate. In vitro culture of guinea pig alveolar macrophages Bronchoalveolar lavage cells (26105/well) were allowed to adhere for 1 hour to each well of a 24 well flat-bottom plate (Becton Dickinson Labware, Franklin Lakes, NJ). Media and non- adherent cells were removed and the monolayers washed with 16 PBS. Representative wells were stained with Diff-Quik staining (Dade Behring, Newark, DE) and the staining revealed over 95% cells to have the morphologic characteristics of alveolar macro- phages. Adherent alveolar macrophages that were treated with either non-oxidized low density lipoprotein or copper-oxidized low density lipoprotein (Biomedical Technologies, Stoughton, MA) at a dose of 25 mg/ml then infected or mock-infected with M. tuberculosis H37Rv (MOI 5:1). At 4 hrs, 24 hrs, 3 days and 7 days after the infection, one set of wells were analyzed for cell viability using CellTitre 96 Acqueous solution cell proliferation assay (Promega, Madison, WI) following manufacturers protocol and from another set of wells, the adherent macrophages were washed and gently scraped from the surface and resuspended 16 PBS. The macrophages were then homogenized, plated on nutrient 7H11 agar plates following serial dilution and M. tuberculosis numbers determine after three weeks of culture at 37uC and data expressed as colony-forming units. Different set of the primary alveolar macrophages from the same experiment but grown in eight well chamber glass slides were fixed with 10% methanol. Cells were permeabilized with 0.1% Triton X-100 and endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. Cells were stained with diluted Rhodamine B (Sigma, St Louis, MO) for 30 minutes (for M. tuberculosis) and de-stained twice with an acid alcohol solution. Oxidized LDL in the Guinea Pig Tuberculosis Model Slides were then blocked using Tyramide Signal Amplification (TSA kit; Invitrogen, Carlsbad, CA) blocking Solution. Primary polyclonal rabbit anti-OxLDL (Calbiochem, San Diego, CA) antibody was added to the slides and the slides were incubated overnight at 4uC. After washing, slides were incubated at room temperature with the TSA goat anti-rabbit IgG HRP for 1 hour. TSA Alexa Fluor 488 tyramide and 0.0015% hydrogen peroxide is added for detection of antibody complex. Slides were counter- stained with diluted Hoechst in 16 PBS. Slides were mounted using ProLong Gold (Invitrogen, CA). Images were taken on a Nikon Eclipe 80i with DAPI, FITC and TRITC cubes and composite images produced with Adobe Photoshop 7.0. Statistical analysis The statistical differences of lesion or immunostaining scores among different time points were analyzed using non-parametric Kruskal-Wallis one-way analysis of variance. The specific statistical differences between two individual group scores were analyzed using the Mann-Whitney t test. The statistical differences between serum OxLDL levels were analyzed using two-way ANOVA and non-paired student t test. Statistical analysis of correlation was performed using Pearson’s correlation method and coefficient of correlation for the pairs of groups discussed in the results. All of the statistical tests were done using the Prism software (version 4.03, Graph Pad, La Jolla, CA). Results Lung lesion burden during M. tuberculosis infection The histopathologic changes within the lungs of M. tuberculosis- infected guinea pigs were quantified using a scoring system that correlates disease progression with lesion burden or immunohis- tochemical scores. Figure 1 shows data the median lung lesion scores (n = 5) on days 5, 15, 20, 30 and 60 after infection in both saline-vaccinated and BCG-vaccinated guinea pigs. The number and extent of the lung lesions progressed rapidly from 5 to 60 days and lesion burden at days 30 and 60 of infection were statistically significant from day 5 values. Lesions were less severe and disease progression was delayed in guinea pigs vaccinated with BCG prior to virulent challenge. Serum OxLDL levels Serum OxLDL levels were measured by ELISA in uninfected guinea pigs and in BCG or saline vaccinated animals on days 15, 30 and 60 of infection. Serum OxLDL levels increased significantly in the mock-vaccinated animals on days 30 and 60 (p,0.05) compared to the BCG-vaccinated animals (Figure 2). The increase in the serum OxLDL levels correlated with the progression of disease expressed by lesion scores (r = 0.96). The highest serum OxLDL levels were observed in three of five animals on day 60 of the infection. Despite the elevation in serum OxLDL levels on day 60, there was no significant correlation between bacterial burden in lungs, spleen and lymph node or the extent of histopathologic lesion severity. However, the progressive increase in mean OxLDL levels and median lung lesion scores correlated on days 30 and 60. BCG vaccination abrogated the OxLDL increase in M. tuberculosis infected animals even out to 60 days of infection. OxLDL expression in lungs Oxidized LDL, formed during oxidative stress conditions, is taken up and accumulates within the cytoplasm of macrophages and other cell types at the site of chronic inflammation [25]. To determine whether lung accumulation of OxLDL was diffuse or PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e34148