Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: We used double IHC analysis of MCP-1 or MMP-1 or MMP-9 and CD68 in paraffin-embedded diseased lung from six TB patients who underwent surgery to remove damaged tissue. We are showing representative data. A: negative control (irrelevant antibodies isotype control); B: MCP-1 (blue) and CD68 (red); C: MMP-1 (blue) and CD68 (light red); D: MMP-9 (blue) and CD68 (light red). Images were acquired at 200X total magnification. IHC analysis shows granulomas with CD68-positive cells (macrophages) expressing copious amounts of MCP-1, MMP-1, and MMP-9.

Article Snippet: For IHC, we used heat-induced epitope retrieval in citrate buffer (Thermo/Fisher Scientific Inc., Waltham, MA), rabbit anti-human CCL2/MCP-1 polyclonal antibody LS-C112304 (LifeSpan Bioscience, Inc), rabbit anti-human MMP-1/Collagenase-1 Ab-6 (Thermo/Fisher Scientific, Inc), rabbit anti-human MMP-9 ab13458 (Millipore), rabbit anti-human PAR-1 ab13398 (abcam), mouse anti-human CD68 KP1 (abcam), and the MultiVision Polymer Detection System/anti-mouse-HRP (red) + anti-rabbit-AP (blue) detection system (Lab Vision Products, Thermo Scientific).

Techniques: Negative Control, Expressing

Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: Values are shown as medians (horizontal lines), the 25th and 75th percentiles (boxes), and ranges (whiskers). Legends in the x-axis mean: 1 = two-locus genotype A/- 1G/-; 2 = two-locus genotype A/- 2G/2G; 3 = two-locus genotype GG 1G/-; 4 = two-locus genotype GG 2G/2G ). Section A: Distribution of serum MCP-1 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the serum levels of MCP-1 across genotypes (ANOVA F = 16.31; p = 0.0001). Section B: Distribution of plasma MMP-1 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the plasma levels of MMP-1 across genotypes (ANOVA F = 5.76; p = 0.001). Section C: Distribution of plasma MMP-9 values for tuberculosis patients stratified according to the relevant two-locus genotypes. We observed a significant difference in the plasma levels of MMP-9 across genotypes (ANOVA F = 8.25; p = 0.0001). Comparison of means and standard deviations by genotypes are shown at the right of the whiskers and box figures. The p-values are based on the Bonferroni least significant difference test.

Article Snippet: For IHC, we used heat-induced epitope retrieval in citrate buffer (Thermo/Fisher Scientific Inc., Waltham, MA), rabbit anti-human CCL2/MCP-1 polyclonal antibody LS-C112304 (LifeSpan Bioscience, Inc), rabbit anti-human MMP-1/Collagenase-1 Ab-6 (Thermo/Fisher Scientific, Inc), rabbit anti-human MMP-9 ab13458 (Millipore), rabbit anti-human PAR-1 ab13398 (abcam), mouse anti-human CD68 KP1 (abcam), and the MultiVision Polymer Detection System/anti-mouse-HRP (red) + anti-rabbit-AP (blue) detection system (Lab Vision Products, Thermo Scientific).

Techniques:

Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: We measured the relative changes in MCP-1, MMP-1 , MMP-9, and TIMP gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls. Panel 1 , the effect of 10 μM concentration of CCR2 RS504393 inhibiting compound was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl serum-free RPMI. CCR2 RS504393 inhibiting compound was diluted with DMSO and dispensed in 5 μl volume to produce a final culture concentration of 0.01% DMSO. We also added 5 μl of DMSO to control cultures. The results presented are from six independent experiments showing the mean and standard deviations. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p < 0.01) when testing MCP-1, MMP-1 and MMP-9. We show corrected p-values obtained from student t-tests. Notably, levels of specific mRNAs from non-stimulated cells were not significantly different than those obtained from cultures that proceeded with the CCR2 inhibitor alone. Panel 2 , the effect of 1 μM concentration of MMP-1 inhibitor 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl serum-free RPMI. 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide was diluted in incomplete RPMI. The results presented are from six independent experiments showing the mean and standard deviations from the mean. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p < 0.01) when testing MCP-1, MMP-1 and MMP-9. We show corrected p-values obtained from student t-tests. Of note, levels of specific mRNAs from non-stimulated cells were not significantly different from those obtained from cultures that proceeded with the 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide inhibitor alone. The inhibitors’ concentrations were selected from dose response-experiments.

Article Snippet: For IHC, we used heat-induced epitope retrieval in citrate buffer (Thermo/Fisher Scientific Inc., Waltham, MA), rabbit anti-human CCL2/MCP-1 polyclonal antibody LS-C112304 (LifeSpan Bioscience, Inc), rabbit anti-human MMP-1/Collagenase-1 Ab-6 (Thermo/Fisher Scientific, Inc), rabbit anti-human MMP-9 ab13458 (Millipore), rabbit anti-human PAR-1 ab13398 (abcam), mouse anti-human CD68 KP1 (abcam), and the MultiVision Polymer Detection System/anti-mouse-HRP (red) + anti-rabbit-AP (blue) detection system (Lab Vision Products, Thermo Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, In Vitro, Sonication

Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: Panel 1, we measured the relative changes in MCP-1, MMP-1 , MMP-9, and TIMP gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls. The effect of 50 nM concentration of SCH79797 PAR-1 inhibiting compound was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl incomplete RPMI. PAR-1 SCH79797 inhibiting compound was diluted with DMSO and dispensed in 5 μl volume to produce a final culture concentration of 0.01% DMSO. We also added 5 μl of DMSO to control cultures. The results presented are from six independent experiments showing the mean and standard deviations. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p < 0.001) when testing MCP-1, MMP-1 and MMP-9. We show corrected p-values obtained from student t-tests. Of note, levels of specific mRNAs from non-stimulated cells were not significantly different from those obtained from cultures that proceeded with the PAR-1 inhibitor alone. Panel 2 , the effect of 1 nM exogenous human purified MMP-1 on THP-1 cells exposed to 5 μg/ml sonicated H37Rv M. tuberculosis was assessed. We also assessed the effect of 1 nM exogenous human purified MMP-1 on THP-1 cells exposed to 5 μg/ml sonicated H37Rv M. tuberculosis in the presence or absence of 50 nM concentration of SCH79797 PAR-1 inhibiting compound. We measured the relative changes in MCP-1, MMP-1 , and MMP-9 gene expression by real-time PCR as explained above. Cultures proceeded in 500 μl of incomplete RPMI. The results presented are from three independent experiments showing the mean and standard deviations. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p < 0.001) when testing MCP-1, MMP-1 and MMP-9. We show corrected p-values obtained from student t-tests. The inhibitor’s concentration was selected from dose response-experiments.

Article Snippet: For IHC, we used heat-induced epitope retrieval in citrate buffer (Thermo/Fisher Scientific Inc., Waltham, MA), rabbit anti-human CCL2/MCP-1 polyclonal antibody LS-C112304 (LifeSpan Bioscience, Inc), rabbit anti-human MMP-1/Collagenase-1 Ab-6 (Thermo/Fisher Scientific, Inc), rabbit anti-human MMP-9 ab13458 (Millipore), rabbit anti-human PAR-1 ab13398 (abcam), mouse anti-human CD68 KP1 (abcam), and the MultiVision Polymer Detection System/anti-mouse-HRP (red) + anti-rabbit-AP (blue) detection system (Lab Vision Products, Thermo Scientific).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, In Vitro, Sonication, Purification

Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: Differentially express genes in H37Rv M. tuberculosis infected THP-1 cells and its regulation by PAR-1 inhibitor SCH79797

Article Snippet: For IHC, we used heat-induced epitope retrieval in citrate buffer (Thermo/Fisher Scientific Inc., Waltham, MA), rabbit anti-human CCL2/MCP-1 polyclonal antibody LS-C112304 (LifeSpan Bioscience, Inc), rabbit anti-human MMP-1/Collagenase-1 Ab-6 (Thermo/Fisher Scientific, Inc), rabbit anti-human MMP-9 ab13458 (Millipore), rabbit anti-human PAR-1 ab13398 (abcam), mouse anti-human CD68 KP1 (abcam), and the MultiVision Polymer Detection System/anti-mouse-HRP (red) + anti-rabbit-AP (blue) detection system (Lab Vision Products, Thermo Scientific).

Techniques: Infection

Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: PAR-1 inhibitor treatment of infected THP-1 cells down-regulates MCP-1, MMP-1 and MMP-9 protein secretion, but does not modify M. tuberculosis intra-cellular proliferation

Article Snippet: For IHC, we used heat-induced epitope retrieval in citrate buffer (Thermo/Fisher Scientific Inc., Waltham, MA), rabbit anti-human CCL2/MCP-1 polyclonal antibody LS-C112304 (LifeSpan Bioscience, Inc), rabbit anti-human MMP-1/Collagenase-1 Ab-6 (Thermo/Fisher Scientific, Inc), rabbit anti-human MMP-9 ab13458 (Millipore), rabbit anti-human PAR-1 ab13398 (abcam), mouse anti-human CD68 KP1 (abcam), and the MultiVision Polymer Detection System/anti-mouse-HRP (red) + anti-rabbit-AP (blue) detection system (Lab Vision Products, Thermo Scientific).

Techniques: Infection