Kinetics of cytokines production between <i>Maf-</i> and <i>Mtb</i>-infected groups following treatment.

<p>The kinetics of cytokine expression showing either strong or weak evidence of difference between lineage groups following overnight incubation with Medium alone (I) or ESAT-6/CFP-10 (II). Line plots show the predicted mean and its 95% confidence interval (95% CI) of log-2 transformed cytokines concentration in <i>Maf-</i> and <i>Mtb</i>-infected groups at 0, 2 and 6 months of treatment. Wald test through contrasts analysis following a random-intercept model adjusted for age, sex and ethnicity was used to assess interaction between lineage group and time point on cytokine production. The legend shows <i>Maf</i>-infected group (dashed lines) and <i>Mtb</i>-infected group (solid lines) respectively. P-values of the interactions are shown.</p

Ingenuity network of direct relationship among genes and cytokines differentially expressed between <i>Maf-</i> and <i>Mtb</i>-infected patients.

<p>Ingenuity network showing 13 of the 23 pro-inflammatory cytokines and genes that were differentially expressed between <i>Maf-</i> and <i>Mtb-</i> infected patients, centred on NF-κ complex. Genes or cytokines that were higher in <i>Mtb-</i> compared to <i>Maf</i>- infected patients are depicted in red, those that were lower in green.</p

Demographic, clinical and haematology characteristics of <i>M</i>. <i>africanum</i> and <i>M</i>. <i>tuberculosis</i> patients.

<p>Demographic, clinical and haematology characteristics of <i>M</i>. <i>africanum</i> and <i>M</i>. <i>tuberculosis</i> patients.</p

Kinetics of gene expression in <i>Maf-</i> and <i>Mtb</i>-infected groups following treatment.

<p>The expression kinetics of genes that showed significant differences between <i>Maf-</i> and <i>Mtb</i>-infected groups following overnight incubation with Medium alone (A), ESAT-6/CFP-10 (B) and live <i>Maf</i> (C). Line plots show the predicted mean and its 95% CI of log2 transformed gene expression levels in <i>Maf-</i> and <i>Mtb</i>-infected groups at 0, 2 and 6 months of treatment. Wald test through contrasts analysis following a random-intercept model adjusted for age, sex and ethnicity was used to assess interaction between lineage group and time point on gene expression. The legend shows <i>Maf-infected</i> group (dashed lines) and <i>Mtb-infected</i> group (solid lines) respectively. P-values of the interactions are shown.</p

Gene expression profiles differ between <i>Maf-</i> and <i>Mtb</i>-infected patients before and after treatment.

<p>dcRT-MLPA was performed on RNA extracted from whole blood incubated overnight with medium only (A), ESAT-6/CFP-10 (B) and live <i>Maf</i> (C). Median gene expression levels (peak areas normalized to GAPDH and log2 transformed) of the indicated genes are shown in box-and-whisker plots. Equal number of samples were analysed at 0 and 6 months of treatment in each group of <i>Maf</i>-infected (n = 20; white boxes) and <i>Mtb</i>-infected (n = 31; grey boxes) patients, respectively. Log-2 transformed gene expression data were compared between the groups using contrast analysis following a random-intercept model adjusted for age, sex and ethnicity, and Sidak multiple comparison correction. Wald test was used to assess the significance at each time point. P-values of significant differences are shown.</p

Differential cytokine production between <i>Maf-</i> and <i>Mtb</i>-infected patients before and after treatment.

<p>Whole blood incubated overnight with medium only (I), revealed differences in the concentrations of IL12p70 (A), PDGF-ββ (B), MIP-1α (C), IL-15 (D) and IL-8 (E) between <i>Mtb-</i> and <i>Maf</i>-infected patients at 6 month of treatment. (II) Only ESAT-6/CFP-10 stimulation induced significant differences in cytokine production between <i>Mtb-</i> and <i>Maf</i>-infected patients above the background level of IFN-γ (A), IL-2 (B), IL-1ra (C), TNF-α (D), GM-CSF (E) and Rantes (F). Dot plots show log-2 transformed cytokine concentrations measured with Bio-Plex assay. Horizontal bars indicate median cytokine concentration by lineage groups, <i>Maf</i>-infected patients (closed circles, n = 26 and 20) and <i>Mtb</i>-infected patients (open circles, n = 49 and 31) respectively at 0 and 6 months of TB treatment. Log-2 transformed cytokine concentrations were compared between lineage groups using a random-intercept model adjusted for age, sex and ethnicity, and Sidak multiple comparison correction. Contrasts analysis was used after estimation to compute the difference in each cytokine concentration between lineage groups and used Wald test for assessing the significance at each time point. P-values of the differences are shown.</p

UL49.5 is responsible for TAP-inhibition in virus-infected natural host cells.

<p>Bovine cells (MDBK), porcine cells (PK15), and equine cells (E.derm) were infected with wild type BHV-1, PRV, or EHV-1, respectively, or with the corresponding UL49.5-negative recombinant viruses. In all experiments, mock-treated (uninfected) cells from the relevant host species were used as a control. Peptide transport was assessed at 5 hrs post-infection in the presence and absence of ATP (black and open bars, respectively). The data are expressed as percentage of translocation, relative to the translocation observed in control cells (defined as 100%).</p

UL49.5 of PRV and EHV-1 do not interfere with peptide binding to TAP.

<p>To evaluate peptide binding to the TAP complex, microsomal membranes from MJS TAP1-GFP cells (control; ▪), or MJS TAP1-GFP cells expressing UL49.5 of PRV (▴) or EHV-1 (▾) were incubated with increasing concentrations of the radiolabeled peptide (RR[¹²⁵I]YQKSTEL). Unspecific binding was determined in the presence of 200-fold excess of ICP47 (data not shown). The amount of specifically bound peptide per amount of microsomal protein is plotted against the peptide concentration. K_d values for control MJS: 277±58 nM, for MJS UL49.5^PRV: 351±42 nM and for MJS UL49.5^EHV-1: 236±71 nM.</p

UL49.5 homologs arrest TAP in a translocation-incompetent state.

<p>The lateral mobility of the TAP complex was analyzed in MJS TAP1-GFP cells using confocal microscopy and FRAP. A circular spot in the ER was bleached and recovery of fluorescence was monitored. The half-time for recovery was determined and used to calculate the diffusion coefficient D. Where indicated, ATP was depleted (-ATP) or saturating amounts of substrate peptides (long side chain peptides, l.s.c.p.) were micro-injected into the cells.</p

UL49.5 of EHV-1 and EHV-4 block ATP binding to human and equine TAP.

<p>(A, B, C) MJS cells expressing the UL49.5 proteins or the HCMV-derived TAP-inhibitor US6 and (D) E.derm cells expressing EHV-4 UL49.5 were lysed using digitonin or NP40 as indicated. Post nuclear lysates were incubated with ATP-agarose. The pellet (P) contains the ATP-binding proteins. The supernatant (S) contains proteins incapable of binding ATP. ATP-bound (ATP-agarose beads; pellet) and unbound (soluble; supernatant) fractions were separated by centrifugation and analyzed using SDS-PAGE and immunoblotting (IB) with antibodies against the proteins indicated.</p