Individual patient's data on hkH37Rv stimulated release of IFN-γ, IL-2, IL-10, ratios of IFN-gamma/IL-10, IL-2/IL-10 and ELISpot responses to ESAT-6 and hkH37Rv.

<p>Individual patient's data on hkH37Rv stimulated release of IFN-γ, IL-2, IL-10, ratios of IFN-gamma/IL-10, IL-2/IL-10 and ELISpot responses to ESAT-6 and hkH37Rv.</p

IFN-gamma ELISpot response of peripheral blood mononuclear cells (PBMC) stimulated with a range of <i>Mycobacterium tuberculosis</i> antigens, expressed as IFN-gamma spot forming cells per million (SFC/10⁶) PBMC.

<p>TB-IRIS patients with multi-drug resistant (MDR) TB are shown in black full circles, rifampicin mono-resistant (RM) cases are shown as a cross, and fully sensitive (FS) TB cases are shown as open circles.</p

The ratio of secreted cytokines measured by luminex analysis in the supernatants of peripheral blood mononuclear cells (PBMC) stimulated with heat killed H37Rv (MOI = 1∶1) for 24 hours.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046481#s3" target="_blank">Results</a> from TB-IRIS patients with multi-drug resistant (MDR) TB are shown in black full circles (n = 7), rifampicin mono-resistant (RM) cases are shown as a cross (n = 3), and fully sensitive (FS) TB cases are shown as open circles (n = 10).</p

Multiplex cytokine analysis (corrected for multiple comparisons).

(1)<p>p value comparing FS unstimulated with FS stimulated.</p>(2)<p>p value comparing MDR unstimulated with MDR stimulated.</p>(3)<p>p value comparing FS stimulated with MDR stimulated.</p><p>N/A: comparison not applicable due to very low values.</p

IFN-gamma response of PBMC to peptide pools and recombinant proteins in the overnight ELISpot assay (n = 29).

<p>“PP” indicates the peptide pools and “r” indicates recombinant proteins of ESAT-6 and CFP-10. No significant difference was found between the peptide pool and recombinant protein for either ESAT-6 or CFP-10 (Wilcoxon matched pairs test). Lines indicate median response.</p

Recombinant ESAT-6 protein profiles on SELDI-TOF mass spectrometry.

<p>(A) Recombinant rESAT-6 protein. (B) Recombinant ESAT-6 stimulated whole blood at Day 0 showing a peak at the predicted molecular weight of 11.7 kDa. (C) Recombinant ESAT-6 stimulated whole blood after 7 days of incubation showing that rESAT-6 cannot be detected, while a new peak appears at 10.8 kDa.</p

Detection of Calgranulin A (S100A8) by Western blot: Supernatant of whole blood stimulated with (A) rESAT-6 and (B) rCFP-10, was probed with Calgranulin A polycolonal antibody at Day 0 and after 7 days of incubation.

<p>The data indicates the presence of Calgranulin A after 7 days of culture, but not on Day 0.</p

Detection of recombinant proteins by Western blot: Supernatant of whole blood stimulated with (A) rESAT-6 and (B) rCFP-10 at Day 0 and after 7 days of incubation, in 2 donors, showing the presence of both recombinant proteins after 7 days of incubation, including positive (recombinant proteins alone) and negative (supernatant of unstimulated whole blood) controls.

<p>Detection of recombinant proteins by Western blot: Supernatant of whole blood stimulated with (A) rESAT-6 and (B) rCFP-10 at Day 0 and after 7 days of incubation, in 2 donors, showing the presence of both recombinant proteins after 7 days of incubation, including positive (recombinant proteins alone) and negative (supernatant of unstimulated whole blood) controls.</p

Detection of His-Tagged recombinant proteins by Western blot: in the supernatant of whole blood stimulated with rESAT-6 and rCFP-10 at Day 0 and after 7 days of incubation compared to a positive control, showing that the His-tag is detectable at both Day 0 and Day 7 of culture.

<p>Detection of His-Tagged recombinant proteins by Western blot: in the supernatant of whole blood stimulated with rESAT-6 and rCFP-10 at Day 0 and after 7 days of incubation compared to a positive control, showing that the His-tag is detectable at both Day 0 and Day 7 of culture.</p

QFT-IT results, representing the Antigen-Nil IU/ml INF-gamma values (n = 29, Panel A).

<p>The solid line represents the median. <b>IFN-gamma response to peptide pools (covering ESAT-6, CFP-10, and Rv2654) and recombinant ESAT-6 and CFP-10 in the 7 day WBA (n = 29, Panel B).</b> “PP” indicates peptide pool and “r” indicates the recombinant proteins. Recombinant proteins induced significantly higher responses than the peptide pools (p<0.0001 for both ESAT-6 and CFP-10). IFN-gamma concentrations are shown as pg/ml. Lines indicate median response.</p