Mean eVAS responses following treatment with placebo (A), oral morphine immediate release 40 mg (B) and oral tapentadol immediate release 100 mg (C) at 60–90 min after drug intake.

<p>The blue dots are the responses to the test stimulus without conditioning stimulus, the orange dots the responses to the test stimulus with conditioning stimulus. The broken line is the mean temperature of the test stimulus. Values are ± SEM.</p

The setup used to induce Conditioned Pain Modulation (CPM).

<p>The volunteer sits on the bed while a thermal test stimulus is applied to the arm <i>via</i> a 3×3 cm thermode (insert top right). The leg is placed in a bucket filled with cold water (<i>ie</i>. the conditioning stimulus) and the subject scores the heat pain to the arm using an electronic visual analogue scale (insert top middle). The cold water is applied via the Icydip system; the heat pain via Medoc’s pathway system.</p

Consort flow diagram.

<p>The test stimulus temperatures (heat pain) as determined prior to treatment averaged to 47 ± 0.5°C, 46.6 ± 0.5°C and 46.9 ± 0.5°C on placebo, morphine and tapentadol experimental sessions (one-way anova: NS). The target conditioning stimulus temperatures were 7.9 ± 0.5°C, 8.1 ± 0.6°C and 8.3 ± 0.6°C on placebo, morphine and tapentadol experimental sessions (one-way anova: NS).</p

HSV-1 antigens recognized by human TG-derived CD8 T-cells.

<p>Representative data from TG-TCL of 6 TG donors assayed for T-cell reactivity to proteins encoded by individual HSV-1 open reading frames (ORFs). Mean IFN-γ secretion levels, shown as arbitrary OD₄₅₀ values, by TG-TCL exposed in duplicate to Cos-7 cells that co-express the respective donor-specific HLA class I allele and the individual HSV-1 ORFs arrayed in nominal genomic order on the x-axis. The names of the HSV-1 ORFs and corresponding proteins specifically recognized are indicated by an arrow. The HSV-1 ORFs recognized by the TG-TCL of donors TG2 and TG7 are shown in colors of the respective TG donor-specific HLA class I allele. ICP, infected cell polypeptide; VP, virion protein and in case of a viral glycoprotein (e.g. gB, glycoprotein B). Currently, no proteins names are available for HSV-1 ORFs <i>UL6</i> and <i>UL25</i>.</p

Localization and phenotype of T-cells in HSV-1 latently infected human TG.

<p>(A) Representative image of an HSV-1 latently infected TG stained by immunohistochemistry (IHC) for CD3 (red). Inset: magnification of the TG tissue showing a cluster of CD3⁺ cells in panel A. (B; left panel) Double immunofluoresence staining for CD4 (red) and CD8 (green) combined with DNA counterstaining (DAPI; blue nuclei). The white arrows signify autofluorescent cytoplasmatic granules in neurons containing lipofuscin and neuron outlines are marked with white dotted lines. (B; right panel) Consecutive TG tissue sections stained for CD8 (brown) and granzyme B (brown), CD8 (brown) and TIA-1 (brown), and CD3 (red) and CD137 (red). Sections were developed with diaminobenzidine (brown staining pattern) or 3-amino-9-ethylcarbazole (red staining pattern) and counterstained with hematoxylin (blue nuclei). Magnifications were: (A) ×20 and inset ×200, (B; left panel) ×400 and (B; right panel) ×1000. Representative images from 10 HSV-1 latently infected TG donors analyzed.</p

HSV-1 antigens recognized by human TG-derived CD4 T-cells.

<p>(A) Representative data from the TG-TCL of the donors TG2 and TG3 assayed for T-cell reactivity by a proliferation assay to proteins encoded by individual HSV-1 open reading frames (ORFs). Cell lysates of mock- and HSV-1 infected Cos-7 cells were used as negative and positive controls, respectively. Mean [³H]-thymidine incorporation by TG-TCL exposed in duplicate to γ-irradiated donor HLA-DQ/DR-matched allogeneic peripheral blood mononuclear cells (PBMC) pulsed with lysates generated from Cos-7 cells transfected with the individual HSV-1 ORFs arrayed in nominal genomic order on the x-axis. The names of the HSV-1 ORFs and corresponding proteins driving the positive responses are indicated by an arrow. ICP47, infected cell polypeptide 47 and VP16, virus protein 16. (B) Proliferation assay data of the TG-TCL of donor TG2 with γ-irradiated HLA-DQ/DR TG 2-matched allogeneic PBMC pulsed with whole HSV-1 ICP47 protein (gene US12) spanning synthetic peptides (15-meric peptides with 10 amino acid (aa) overlap) as antigen presenting cell (APC). (C; left panel) Proliferation assay data of the TG-TCL of donor TG3 with γ-irradiated HLA-DQ/DR-matched allogeneic PBMC pulsed with the indicated recombinant HSV-1 VP16 protein (gene UL48) fragments as APC. (C; right panel) Proliferation assay data of the TG-TCL of donor TG3 with γ-irradiated HLA-DQ/DR-matched allogeneic PBMC pulsed with HSV-1 VP16 protein fragment (aa151–240) spanning synthetic peptides (13-meric peptides with 8 aa overlap) as APC. Data are presented as mean counts per minute of triplicate experiments. Data presented in (B) and (C) are the means ± standard error of the mean.</p

Comparison of T-cell cytolytic granule and cytokine transcripts to HSV-1 and VZV DNA load, and CD8β transcript levels, in human TG.

<p>(A) Scatter plot showing the mean HSV-1 and VZV genome equivalent copies (gec) per 10⁵ TG cells. (B) Comparison of HSV-1 and VZV DNA load in TG of individual donors. (C) Comparison between relative CD8β transcript levels and HSV-1 and VZV DNA load. (D) Comparison of HSV-1 DNA load with relative transcript levels of perforin, granzyme B (grB), interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α). (E) Comparison of VZV DNA load with relative transcript levels of perforin, grB, IFN-γ and TNF-α. (F) Comparison between relative CD8β transcript levels and perforin, granzyme B, IFN-γ and TNF-α. (G) Comparison of the transcript levels of CD8β, perforin, grB, IFN-γ and TNF-α between paired left and right TG of individual donors. The paired <i>T</i>-test (A), Spearman correlation (B–F) and Wilcoxon matched pairs test (G) were used for statistical analysis. Data of 26 TG specimens analyzed.</p

HSV-1 epitope-specific CD8 T-cells are localized close to sensory neuron cell bodies in the contralateral human TG.

<p>Representative image of the TG tissue of donor TG2 stained with DAPI (blue), anti-CD8 (green) and tetramers (red) that consisted of both the synthetic HSV-1 peptides ICP0_642–651 and ICP8_1096–1105 conjugated to HLA-A*0201. Inserts, lower left and upper right corner, are enlargements of areas containing tetramer-positive CD8 T-cells. The white arrows and arrowheads signify autofluorescent granules containing lipofuscin and tetramer-positive CD8 T-cells, respectively. Neuron outlines are marked with a white dashed line. Magnification was ×400.</p

Phenotype and HLA class I allele restriction of HSV-1 reactive T-cells recovered from human TG.

*<p>TG-derived T-cell lines were incubated with mock– and HSV-1–infected autologous B-cell lines and assayed by flow cytometry for intra-cellular interferon gamma (IFN-γ) expression.</p>#<p>The ratio of CD4 and CD8 T-cells of the respective TG-derived T-cell lines are indicated.</p>$<p>Patient HLA class I allele restricted HSV-1 reactive CD8 T-cell responses were defined using partially HLA class I matched BLCL. The values represent mean net percentages of live/CD3-gated IFN-γ⁺ T-cells (HSV-1 minus mock) of at least 2 separate experiments.</p><p>nd, not done.</p

HSV-1 antigens and epitopes recognized by CD8 T-cells recovered from human TG.

*<p>HSV-1 gene and protein names, and expression kinetic class, are from reference 1 and Genbank NC_001806. The amino acid (aa) location of CD8 T-cell epitopes identified are in parentheses. na, not applicable.</p>#<p>HLA allele by which the indicated proteins and peptides are recognized by the specific CD8 T-cells.</p