Growth and survival of YH<i>Δtpx in</i> the lungs and spleen of BALBc mice.

<p>Mice were infected with 3 × 10⁵ bacteria. At different time points the infected mice were sacrificed and the numbers of bacteria in the lung (A) or spleen (B) were measured. The results for each time point are the means and SDs of four mice in each experimental group. The experiments have been reproductively repeated twice with similar CFU counts in lungs and spleens. Statistical significance was determined by Student's <i>t</i> test (***, P<0.0001). C. Changes in spleen gross anatomy after infection with the WT, the mutant and the complemented strains. Spleens were collected at 2 and 3 weeks after infection. D. Lung histology of mice infected with the WT, the mutant and the complemented strains. Histopathological examination was performed using three mice in each group. Three sections from each mouse were examined. The images shown are representative of lung sections from three animals in each experimental group. Enlarged images of the boxed regions on the left panel (magnification, 4×) are shown on the right panel (magnification, 10×).</p

Confirmation of the <i>M. tuberculosis tpx</i> gene deletion.

<p>A. Southern blotting analysis of DNA from the WT and the YHΔ<i>tpx.</i> which was digested with EcoRV and PvuI and hybridized with a probe synthesized to make the complemented construct. B. PCR amplification of DNA from the WT and the YHΔ<i>tpx.</i> 1. WT, 2. <i>tpx</i> mutant. The primers used were designed in the coding region of the <i>tpx</i> gene. 3. WT, 4. <i>tpx</i> mutant. The primers used were for the amplification of complemented construct. M, molecular weight marker (Invitrogen). The experiments were repeated twice, with identical results.</p

Inactivation of <i>tpx</i> gene renders the mutant more susceptible to oxidative and nitrosative stresses.

<p>Survival of YH<i>Δtpx</i> compared with WT and the complemented strains in response to H₂O₂ at 5 and 10 mM (A), DETA/NO at 1.25, 2.5 and 5 mM (B), paraquat at 10 and 20 mM (C) and GSNO at 5 and 10 mM (D). The data shown is a representative of three independent experiments. Data are represented as mean±SD of triplicate tests. The CFU counts in the Δ<i>tpx</i> are significantly lower using a t-test than in WT after exposure to both H₂O₂ and NO for 24 hours (p<0.0001 all concentrations for both stress conditions).</p

Biochemical analyses of peroxidase activities in <i>M. tuberculosis</i> WT, <i>tpx</i> mutant and the complemented strain.

<p>Presence of hydrogen peroxide was measured by xylenol orange assay. The open bar, initial H₂O₂ at 1 mM. The solid bar, initial H₂O₂ at 0.5 mM. Control, H₂O₂ only. The data was repeated twice with similar results.</p

Survival of SCID mice (<i>n</i> = 6 per group) infected intravenously with <i>M. tuberculosis</i> H37Rv WT, YHΔ<i>tpx</i> and the complemented strains.

<p>Deletion of <i>tpx</i> gene led to no death of the mice over 60 days after infection.</p

Growth and survival of <i>M. tuberculosis</i> Δ<i>tpx</i> in resting and IFN-γ-activated macrophages.

<p>A. Infection in resting bone marrow derived macrophages from BALB/c mice. B. Infection in IFNγ activated bone marrow derived macrophages from BALB/c mice. These results are the means and standard deviation derived from one representative of three independent experiments. C. Infection in resting bone marrow derived macrophages from C57BL/6 mice. D. Infection in IFNγ activated bone marrow derived macrophages from C57BL/6 mice. E. Infection in resting bone marrow derived macrophages from iNOS KO mice. F. Infection in IFNγ activated bone marrow derived macrophages from iNOS KO mice. The results are the means and SDs derived from triplicate wells. The experiments have been reproductively repeated once. Solid square: WT <i>M. tuberculosis</i> H37Rv. Open square: YHΔ<i>tpx.</i> Solid triangle, YH<i>tpx</i>Comp.</p

Number of individuals among test (periodontitis) and control (healthy) groups according to HSP60 levels being undetectable, less or greater than 1000 ng/ml.

<p>Asterisk refers to statistically significant differences (P<0.05) between groups within each category as computed with Chi-square test.</p

Case control study

*<p>Differences were computed with parametric (Independent t-test) or non parametric (Mann-Whitney U-Test) dependent upon the data frequency distribution. Categorical variables were analyzed by Chi-square testing.</p>**<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001198#pone-0001198-g002" target="_blank">Figure 2</a>.</p

Differences in plasma concentrations between patients suffering from periodontitis (N = 80) and age-matched unaffected controls (N = 48).

<p>Boxes refer to the 25^th (bottom) and 75^th (up) percentiles and the median is the large horizontal line, fences refer to the 10^th (lower) and 90^th (upper) percentiles respectively. Open circles represent outliers whereas asterisks stand for extreme observations with the subject number. Statistical differences are computed with Mann-Whitney U-test.</p

Mean values (standard errors) and mean changes (standard errors) (compared to baseline) of HSP10 (A–B), BiP (C–D) and HSP60 (E–F) before, 24 hrs after and 6 months after periodontal therapy.

<p>Positive differences indicate a relative increase in plasma concentrations of various markers compared to the pre-treatment baseline. Subjects who received intensive periodontal therapy (open circles, N = 40) showed greater plasma concentrations of HSP10 at each time visit when compared to control therapy group subjects (filled circles, N = 40). No other changes were observed. Asterisks refer to statistically significant difference (P<0.05) between groups as computed with analysis of covariance.</p