Additional file 1: of Are the public getting the message about antimicrobial resistance?

Antibiotic questionnaire

Human TCRs and CDR3s sequenced from healthy volunteers and HIV-infected patients, before and after 14 weeks of therapy

<p>We have employed an error-correcting, high-throughput transcript sequencing protocol to profile impact of HIV infection upon the T-cell receptor repertoire.</p> <p>We have sequences both alpha and beta chain TCR repertoires from 2.5ml of peripheral blood for 16 antiretroviral (ART)-naive HIV patients (de-identified reference numbers prefixed 'P0'). Two samples per patient were processed: one immediately before starting therapy ('v1') and another shortly after starting ('v2', after an average 14 weeks). We additionally sequenced the repertoires of ten healthy volunteers ('HV'), taking two blood samples three months apart for four of those donors (HV01 to HV04) in order to compare TCR dynamics over a comparable time frame. One of these healthy donors (HV01) and a further healthy donor (HVD1) also gave a larger blood sample, which was separated into CD4+ and CD8+ T-cell populations by FACS, and their TCR repertoires were seqeunced.</p> <p>Having amplified and sequenced the TCR repertoires of these 100 samples, we identified their VJ recombinations using a modified version of Decombinator (called vDCR), TCR analysis software designed in our lab. We then made use of random barcode sequences introduced before amplification to error- and frequency-correct our Decombinator assignations (DCRs), before translating them and extracting their complementarity determining region 3 (CDR3) sequences.</p> <p>Here we present the results of these analyses. The .dcrcdr3 files in this fileset consist of a unique DCR assignation per line, the CDR3 sequence it encodes, and the frequency with which that TCR appeared in the data following error-correction. Each line follows the format:</p> <p>'V, J, Vdel, Jdel, insert: CDR3, freq'</p> <p>(V = V gene, J = J gene, Vdel = number of deletions from V, Jdel = number of deletions from J, insert = string of nucleotides from the end of the deleted V to the start of the deleted J, CDR3 = translation CDR3 sequence, from the second conserved cysteine residue in the V to the conserved phenylalanine of the FGXG motif of the J, freq = error-corrected frequency of that assignation.)</p> <p>The raw sequence data fastq files from which these TCRs were extracted is available in the Sequence Read Archive (SRA) under the Study accession number SRP045430. The AccessionKey.xls spreadsheet cross-references all filenames with their appropriate SRA individual accession numbers.</p> <p>The Python scripts which were used to generate this data from that raw fastq data are also available on figshare (see links below).</p

Bioinformatic and modular analysis of the TST signature in HIV negative patients with active TB.

<p>(a) Representative example of histological leukocytic infiltrate associated with a clinically positive TST. (b) Frequency distribution of median fold increases in TST transcript abundance in HIV-1 seronegative patients with active TB (n = 16) compared to site of saline injections (n = 8). TFBS enrichment analysis for this gene list is shown indicating the number of genes and statistical enrichment (Z score) associated with selected transcription factors (c). Associations with top twelve most statistically enriched REACTOME functional pathways are represented in a network plot (d), in which the edges indicate associations between genes (blue nodes) and named pathways (red nodes), and the node size is proportional to the number of associations. (e-g) Module scores represent log ratio of geometric mean of gene expression for each module within TST compared to saline biopsies (data points represent median ±IQR).</p

Summary of demographic, clinical and laboratory data of study groups.

<p>Summary of demographic, clinical and laboratory data of study groups.</p

Exaggerated Th2 responses in HIV-1 infected patients with unmasking TB-IRIS.

<p>In patients with active TB, the TST signature in HIV-1 negative patients with clinically positive TSTs (n = 16) and HIV-1 infected patients with unmasking IRIS (n = 3), relative to the mean of saline controls (n = 8), are compared by (a) Venn diagram, and (b) scatter plot of median gene expression showing correlation (r²) and covariance (slope), highlighting the genes that show statistically significantly increased expression in red. (c) For genes that show increased expression in HIV-1 infected TB-IRIS patients, associations with statistically enriched functional pathways is represented in a network plot in which the edges indicate associations between genes (blue nodes) and named pathways (red nodes), and the node size is proportional to the number of associations. Relative enrichment of IL4/13 stimulated gene expression modules in (d) TST (median ±IQR), and (e) peripheral blood transcriptomes (data points for individual patients and median for each group) in the groups indicated (*p<0.05 Mann-Whitney test).</p

Increased immunostaining for IRF4 in HIV-1 infected patients with unmasking TB-IRIS.

<p>(a) Immunostaining of IRF4 in TST biopsies from three separate patients from each of the study groups indicated (white arrows indicate areas of positive IRF4 staining associated with inflammatory infiltrates). (b) Selected inflammatory cell infiltrates in TB-IRIS cases (indicated by red squares) are shown at higher magnification. Black scale bar = 400 μM and white scale bar = 80 μM. </p

Preserved type 1 IFN responses uncoupled from other immune responses in HIV-1/TB co-infected patients with clinically negative TSTs.

<p>(a) In patients with active TB, the TST signature in HIV-1 negative patients with clinically positive TSTs (n = 16) and HIV-1 infected patients with clinically anergic TST (n = 14) are compared in a Venn diagram. For genes that show increased expression in this group of HIV-1 infected patients relative to the mean of saline controls (n = 8), (b) TFBS enrichment analysis is shown indicating the number of genes and statistical enrichment (Z score) associated for selected TFs, and associations with twelve most statistically enriched REACTOME functional pathways is represented in a network plot (c), in which the edges indicate associations between genes (blue nodes) and named pathways (red nodes), and the node size is proportional to the number of associations. Ratios of the median signal for stimulus specific type 1:2 IFN modules are shown within the (d) TST and (e) blood transcriptomes for different groups of patients with active TB as indicated, showing data points for individual patients and median for each group (*p<0.05 Mann-Whitney test).</p