Primer sequence for qRT-PCR analysis of gene transcripts.

<p>Primer sequence for qRT-PCR analysis of gene transcripts.</p

Mycobacterium Tuberculosis-Specific TNF-α Is a Potential Biomarker for the Rapid Diagnosis of Active Tuberculosis Disease in Chinese Population

<div><p>Interferon-gamma release assays (IGRAs) have proven to be useful to accurately detect <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) infection, but they cannot reliably discriminate between active tuberculosis (TB) and latent tuberculosis infection (LTBI). This study aims to test whether <i>Mtb</i>-specific tumor necrosis factor-alpha (TNF-α) could be used as a new tool for the rapid diagnosis of active TB disease. The secretion of TNF-α by <i>Mtb</i>-specific antigen-stimulated peripheral blood mononuclear cells (PBMCs) of sixty seven participants was investigated in the study. Our results showed that the total measurement of TNF-α secretion by <i>Mtb</i>-specific antigen-stimulated PBMCs is not a good biomarker for active TB diagnosis. However, we found that calculation of <i>Mtb</i>-specific TNF-α not only distinguish between active and latent TB infection, but also can differentiate active TB from non-TB patients. Using the cutoff value of 136.9 pg/ml for <i>Mtb</i>-specific TNF-α, we were able to differentiate active TB from LTBI. Sensitivity and specificity were 72% and 90.91%. These data suggest that <i>Mtb</i>-specific TNF-α could be a potential biomarker for the diagnosis of active TB disease.</p></div

The cytotoxicity of ESAT-6 to PHA-stimulated PBMCs of three non-TB patients.

<p>Note. PHA: phytohemagglutinin; TB: tuberculosis; AFS: acid fast staining.</p

Additional file 2: of Characteristics of diarrheagenic Escherichia coli among children under 5 years of age with acute diarrhea: a hospital based study

Raw data of MIC. (XLSX 24 kb

Additional file 1: of Characteristics of diarrheagenic Escherichia coli among children under 5 years of age with acute diarrhea: a hospital based study

Clinical information of DEC infected children. (XLSX 27 kb

The secretions of TNF-α and <i>Mtb</i>-specific TNF-α by ESAT-6 or CFP-10-stimulated PBMCs.

<p>PBMCs obtained from active TB patients (n = 25), LTBI individuals (n = 22), healthy control subjects (n = 10) and non-TB patients (n = 10) were stimulated with ESAT-6 or CFP-10. PBMCs stimulated with medium alone were used as a background control. (A) After 16–20 h of incubation, the supernatant was collected and tested for concentrations of secreted TNF-α by ELISA. (B) <i>Mtb</i>-specific TNF-α was calculated by subtracting background TNF-α secreted by medium-stimulated PBMCs from TNF-α secreted by ESAT-6 or CFP-10-stimulated PBMCs. Median values for each group of participants are represented by a horizontal bar. *<i>p</i> < 0.05, **<i>p</i> < 0.001.</p

Statistical analysis of ROC curve for <i>Mtb</i>-specific TNF-α to distinguish between active TB and LTBI.

<p>Note. TB: tuberculosis; LTBI: latent tuberculosis infection; AUC: area under the curve.</p

Blockade of Tim-3 pathway increased IFN-γ production and decreased apoptosis of NK cells in vitro.

<p>Spleen cells harvested from septic mice at 24 h after LPS injection were stimulated with LPS (1 µg/ml) in the presence of anti-Tim-3 Ab (1 µg/ml), Tim-3 Fc protein (5 µg/ml) or control IgG for 24 h. The bar graphs showed the percentages of (A) IFN-γ⁺ NK cells and (B) CD107a⁺ NK cells between the groups. (C) The apoptosis of NK cells (gated on CD3^–NKp46⁺ cells) was analyzed by Annexin V/PI double staining. The percentage of Annexin V⁺PI^– cells (representative of apoptosis cells) was compared between the groups. (D) The expression of surface galectin-9 on peritoneal macrophages (F4/80⁺ cells), neutrophils (CD11b⁺ cells), DCs (CD11c⁺ cells), CD4⁺ T cells, CD8⁺ T cells and NK cells from normal and septic mice at 24 h after LPS injection were analyzed. (E) The MFI of galectin-9 on F4/80⁺ and CD11b⁺ cell populations was shown in the bar graphs. Data are mean ± SEM of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

The percentages and absolute numbers of Tim-3⁺ NK and IFN-γ⁺ NK cells.

<p>Data are mean ± SEM of at least three independent experiments. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 compared with septic mice at 0 h after LPS injection; ^##<i>p</i><0.01, ^###<i>p</i><0.001 compared with septic mice at 12 h after LPS injection.</p><p>The percentages and absolute numbers of Tim-3⁺ NK and IFN-γ⁺ NK cells.</p

Tim-3 expression is inversely associated with NK cell activity.

<p>(A) The expression of Tim-3 and IFN-γ was analyzed in NK cells from septic mice at 24 h after LPS injection. The percentage of IFN-γ⁺ cells between Tim-3⁺ and Tim-3⁻ NK cell subsets was shown in the graphs. (B) Representative flow cytometry histograms of CD69 expression on NK cells from septic mice at 0, 12 and 24 h after LPS injection were shown. (C) The MFI of CD69 on NK cells was shown in the bar graphs. (D) The percentage of CD69⁺ NK cells between Tim-3⁺ and Tim-3⁻ NK cell subsets from septic mice at 24 h after LPS injection was shown. Data are mean ± SEM of at least three independent experiments. **<i>p</i><0.01, ***<i>p</i><0.001.</p