Journal: Scientific Reports

Article Title: Infection with Mycobacterium tuberculosis induces the Warburg effect in mouse lungs

doi: 10.1038/srep18176

Figure Lengend Snippet: Confocal imaging of HK3, LDHA and ATP6a3 isoform of V-ATPase expression in macrophages and CD3 + cells in mouse lungs ( A–F ). Immunohistochemical staining, confocal imaging, and 3D reconstruction and analysis were carried out using 20 μm lung tissue sections obtained from uninfected control and M. tuberculosis -infected C57BL/6 mice at day 30 post-infection (D30). Tissue sections were stained for nuclei (DAPI, blue); HK3, LDHA, or ATP6a3 isoform of V-ATPase (green); M. tuberculosis (red), CD3 + cells (T lymphocyte, white); and IBA-1 (macrophage, cyan). Bar = 150 μm. Quantitative analysis of HK3, LDHA and ATP6a3 expression by confocal imaging in macrophages and CD3 + cells in lungs of infected and uninfected mice ( G–I ). The expression levels of each target protein under both conditions were obtained by measuring the positive pixels and their intensities from same number of cells of the specific regions of interests (ROIs). Three sections per lung region were examined for each animal and 4 animals per condition were analyzed. Data shown are means of fold change ± SDs of the measurements from four mice at each time point relative to background expressions from each condition. *** indicates p ≤ 0.005.

Article Snippet: Lung tissue sections were analyzed by five-color immunohistochemical staining with antibodies for nuclei: DAPI; M. tuberculosis : biotin antibody: Genetex, Irvine, CA; hexokinase 3 (HK3): Biorbyt, Cambridge, UK; subunit isoform a3 of V0 domain of V-type H + ATPase (ATP6a3): Santa Cruz, Dallas, TX; lactate dehydrogenase A (LDHA), or HIF-1α: Abcam, Cambridge, MA; macrophage: ionized calcium-binding adapter molecule 1 (IBA-1), a marker exclusively for tissue macrophages, Abcam; and CD3: a marker for lymphocytes, Abcam.

Techniques: Imaging, Expressing, Immunohistochemical staining, Staining, Control, Infection

Journal: Biology

Article Title: Quantitative Rapid Test for Detection and Monitoring of Active Pulmonary Tuberculosis in Nonhuman Primates

doi: 10.3390/biology10121260

Figure Lengend Snippet: Distinctive increase in serum SAA1, IP-10 and IL-6 after MTB infection. SAA1, IP-10 and IL-6 levels were measured by UCP-LFA in serum from MTB Erdman (500 CFU, n = 53; 15 CFU, n = 12; 1-7 CFU, n = 10) infected rhesus macaques with or without BCG pre-vaccination ( n = 75, ). Endpoints varied between 6 weeks and 52 weeks after infection. Results are displayed as the Ratio value (R) between Test (T) and Flow-Control (FC) signal based on relative fluorescence units (RFUs; excitation at 980nm and emission at 550 nm) measured at the respective lines. ( a , b ) Analysis of three biomarker levels pre and postinfection in MTB Erdman infected rhesus macaques ( n = 75). ( c , d ) Analysis in untreated, infected control animals from high- (500 CFU, n = 24) and low-dose (15 CFU, n = 12) MTB infection studies ( n = 36). ( a , c ) Significant differences between biomarker levels ( y -axis) pre and postinfection were determined by Wilcoxon matched-pairs signed rank tests. ( b , d ) The ability to distinguish pre from postinfection states in the same animals was evaluated by ROC curve analysis, including Area Under the Curve (AUC) measurements (ROC-AUC). AUC: area under the curve; p -values: **** p < 0.0001.

Article Snippet: Animals were experimentally infected with MTB strain Erdman K01 (obtained through BEI Resources) by endobronchial instillation.

Techniques: Infection, Control, Fluorescence, Biomarker Discovery

Journal: Biology

Article Title: Quantitative Rapid Test for Detection and Monitoring of Active Pulmonary Tuberculosis in Nonhuman Primates

doi: 10.3390/biology10121260

Figure Lengend Snippet: SAA1, IP-10, and IL-6 serum levels increased after MTB infection. SAA1, IP-10 and IL-6 levels were measured by UCP-LFA in serum from high-dose (500 CFU, n = 24, red dots; left panels) or low-dose (15 CFU, n = 12, green triangles; right panels) MTB Erdman infected rhesus macaques ( n = 36) at different timepoints ( x -axis) pre and postinfection. Cutoff values for positivity (blue dashed lines) were calculated by applying the Youden’s index as described in b. ( a – c ) The levels of SAA1, IP-10, and IL-6 increase after MTB infection. Differences between high and low doses at 3/4, 6, and 12 weeks postinfection were determined by Mann–Whitney U tests indicated in the right panel. p -values: * p < 0.05, *** p < 0.001.

Article Snippet: Animals were experimentally infected with MTB strain Erdman K01 (obtained through BEI Resources) by endobronchial instillation.

Techniques: Infection, MANN-WHITNEY

Journal: Biology

Article Title: Quantitative Rapid Test for Detection and Monitoring of Active Pulmonary Tuberculosis in Nonhuman Primates

doi: 10.3390/biology10121260

Figure Lengend Snippet: Correlation between SAA1, IP-10, and IL-6 levels and total/ lung pathology score at the endpoint. ( a ) SAA1, ( b ) IP-10, and ( c ) IL-6 levels ( x -axis) were measured by UCP-LFA in serum from MTB Erdman infected rhesus macaques ( n = 75, see ). Tuberculosis pathology (PA; y -axis) scoring methods are described in Materials and Methods (2.2), and PA is expressed as arbitrary units (AU). R 2 is the square of the Spearman correlation coefficient. ( d ) Values above the cutoff per biomarker (described in b) were considered positive. 3BM ( x -axis) was generated using the sum of all positive tests results from the individual markers. ( a – d ) SAA1, IP-10, IL-6, and 3BM showed a good correlation with total- and lung pathology scores at the endpoint.

Article Snippet: Animals were experimentally infected with MTB strain Erdman K01 (obtained through BEI Resources) by endobronchial instillation.

Techniques: Infection, Biomarker Discovery, Generated

Journal: Biology

Article Title: Quantitative Rapid Test for Detection and Monitoring of Active Pulmonary Tuberculosis in Nonhuman Primates

doi: 10.3390/biology10121260

Figure Lengend Snippet: SAA1, IP-10, and IL-6 levels in two rhesus genotypes with/without BCG vaccination after MTB infection. SAA1, IP-10, and IL-6 levels were measured by UCP-LFA in serum from high-dose (500 CFU) MTB Erdman infected Chinese-type (CHN, n = 18) and Indian-type (IND, n = 12) rhesus macaques. 12 macaques were unvaccinated (CHN, n = 6, red dots; IND, n = 6, green squares); 18 were vaccinated with BCG 17 weeks before infectious challenge with MTB, of which 12 by intradermal injection (BCG i.d.; CHN, n = 6, yellow rhombus, and IND, n = 6, purple hexagons) and 6 by pulmonary mucosal administration (BCG muc; CHN, n = 6, blue triangles)) ( a – c ) Kaplan–Meier curves were plotted, showing the proportion of positive NHP over time for the Chinese vs. Indian rhesus macaques with/without BCG vaccination.

Article Snippet: Animals were experimentally infected with MTB strain Erdman K01 (obtained through BEI Resources) by endobronchial instillation.

Techniques: Infection, Injection

Journal: Biology

Article Title: Quantitative Rapid Test for Detection and Monitoring of Active Pulmonary Tuberculosis in Nonhuman Primates

doi: 10.3390/biology10121260

Figure Lengend Snippet: SAA1, IP-10, and IL-6 levels in serum increased after infection and are associated with the disease severity. SAA1, IP-10, and IL-6 levels ( y -axis) were measured by UCP-LFA in serum from high-dose (500 CFU) MTB Erdman infected rhesus macaques ( n = 23), of which 16 animals developed progressive disease and reached a premature humane endpoint (HE), while the endpoint by protocol was fixed at 50 weeks after challenge (EE). The median values of each group are indicated by horizontal lines. Cut-off for positivity (blue dashed lines) as described in b. Each coloured square (BCG-vaccinated, n = 12) or dot (unvaccinated, n = 11) represents an individual NHP. ( a – c ) SAA1, IP-10, and IL-6 postinfection levels were significantly increased in the humane endpoint (HE) group only. SAA1, IP-10 and IL-6 levels in NHPs with the HE in week 3, 6 ( n = 16) and week 12 ( n = 13) were higher than the EE ( n = 7). Significant differences between each timepoint versus week 0 were determined by Wilcoxon matched-pairs signed rank tests. Since three macaques reached a HE before week 12 in the HE group, only 13 data points are available for week 12. Differences between HE versus EE at per time point were determined by Mann-Whitney U tests indicated in the right panel (EE). p -values: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Animals were experimentally infected with MTB strain Erdman K01 (obtained through BEI Resources) by endobronchial instillation.

Techniques: Infection, MANN-WHITNEY

Journal: Biology

Article Title: Quantitative Rapid Test for Detection and Monitoring of Active Pulmonary Tuberculosis in Nonhuman Primates

doi: 10.3390/biology10121260

Figure Lengend Snippet: Correlation between SAA1, IP-10, and IL-6 levels in serum and disease severity. SAA1, IP-10, and IL-6 levels were measured by UCP-LFA in serum from high-dose (500 CFU) MTB Erdman infected rhesus macaques ( n = 23). (Since three rhesus macaques reached a humane endpoint before week 12, only 20 instead of 23 measurements are depicted at week12). The animals were sorted according to the protein levels at each time point. The top 33% of the value is defined as high responders, the middle 33% is defined as medium responders, and the last 33% is defined as low responders. All animals with undetectable protein levels are defined as low responders. Kaplan–Meier curves were plotted of individual endpoints for the high vs. the medium vs. the low responders after MTB infection. High responders after MTB infection arrive at HE earlier than medium or low responders.

Article Snippet: Animals were experimentally infected with MTB strain Erdman K01 (obtained through BEI Resources) by endobronchial instillation.

Techniques: Infection

Journal: Biology

Article Title: Quantitative Rapid Test for Detection and Monitoring of Active Pulmonary Tuberculosis in Nonhuman Primates

doi: 10.3390/biology10121260

Figure Lengend Snippet: SAA1, IP-10, and IL-6 decreased to the baseline levels upon antibiotic treatment. SAA1, IP-10, and IL-6 levels ( y -axis) were measured by UCP-LFA in serum from low-dose (15 CFU) MTB Erdman infected rhesus macaques ( n = 18), six of them were treated by rifampin (RIF) and isoniazid (INH) from week four postinfection for eight weeks until endpoint at week 12 twelve The median values of each group are indicated by horizontal lines. Red dots indicate untreated macaques; yellow squares indicate RIF + INH treated macaques. Cutoff (blue dashed lines) applied was described in b. ( a – c ) The SAA1, IP-10, and IL-6 levels were decreased after RIF + INH treatment. Significant differences between each timepoint versus week 0 were determined by Wilcoxon matched-pairs signed rank tests; differences between untreated versus RIF + INH treated at per time point were determined by Mann-Whitney U tests indicated in the right panel (RIF + INH). p -values: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Animals were experimentally infected with MTB strain Erdman K01 (obtained through BEI Resources) by endobronchial instillation.

Techniques: Infection, MANN-WHITNEY