Relapse and survival following ingenol mebutate treatment of B16 tumours grown in C57BL/6 mice treated with anakinra.

<p>(A) Relapse rates after C57BL/6 mice bearing B16 tumours were treated with ingenol mebuate or placebo, and received daily injections of PBS or anakinra, days 1–7. (n = 9–12 mice per group). Mice were scored positive when a tumour was clearly visible (≥1–2 mm in diameter). Statistics compared + anakinra with + PBS in ingenol mebutate treated groups using the log-rank (Mantel-Cox) test. (B) Survival of the mice described in A; mice were euthanized when tumours reached 100 mm². Statistics as in A.</p

Neutrophil recruitment and apoptosis.

<p>(A) Neutrophil recruitment to treatments sites (left bar chart) and to within ≈200 μm of tumour (right bar chart). Treatment sites; day 2 post initiation of ingenol mebutate treatment, treatment sites were excised and processed for immunohistochemistry and stained with the neutrophil marker, anti-Ly6G. Slides were scanned and analysed by Aperio Pixel count software for brown staining (default settings) excluding areas containing tumour (as melanosomes provide a false positive signal). Two sections per mouse, 6 mice per group. Statistics by Kolmogorov-Smirnov tests (differences in variance between groups was >4). Within ≈200 μm of tumour; in sections where the tumour mass could be readily identified (by the presence of black melanosomes), brown staining surrounding the tumour (within ≈200 μm) was quantitated as above. One section per mouse, 4–5 mice per group. Statistics by Mann Whitney U test (non-parametric data distribution and differences in variance <4). (B) Images illustrating the reduced density of anti-Ly6G staining neutrophils (brown stain) within ≈200 μm of the tumour mass in anakinra versus PBS treated mice. The tumours are delineated by white lines and identified by the presence of black melanosomes. Sections are oriented with the skin (not shown) at the top, with the tumours located in the dermis. (C) ApoTag staining of the sections described in A. Six mice per group, 2/3 sections per mouse, statistics by 2 way ANOVA (parametric data distribution and differences in variance <4, drug and mouse as fixed factors, 2/3 sections per mouse as dependent variables). (Examples of the staining are shown in Figure F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153975#pone.0153975.s001" target="_blank">S1 File</a>).</p

Relapse and survival following ingenol mebutate treatment of B16 tumours grown in MyD88^-/- and C57BL/6 mice.

<p>(A) Relapse rates following (i) ingenol mebutate treatment of B16 tumours grown in MyD88^-/- mice (n = 25), (ii) ingenol mebutate treatment of B16 tumours grown in C57BL/6 mice (n = 21), (iii) placebo treatment of B16 tumours grown in MyD88^-/- mice (n = 18) and (iv) placebo treatment of B16 tumours grown in C57BL/6 mice (n = 19). Mice were scored positive when a tumour was clearly visible (≥1–2 mm in diameter). Data from two independent experiments. Ingenol mebutate treatment groups were significantly different p = 0.021, log-rank (Mantel-Cox) test. (B) Survival rates of the same mice described in A; mice were euthanized when tumours reached 100 mm². Ingenol mebutate treatment groups were significantly different p = 0.018, log-rank (Mantel-Cox) test.</p

IL-1α and IL-1β protein levels after ingenol mebutate treatment.

<p>(A, B) C57BL/6 and MyD88^-/- mice with B16 tumours were treated topically with ingenol mebutate day 0 and 1, treatment sites were excised and IL-1α and IL-1β levels were measured in extracts using BD BD™ Cytometric Bead Array (n = 6 mice per group and time point). Statistics by Kolmogorov-Smirnov tests (differences in variance >4). (C) Cultured adult human keratinocytes were treated with the indicated concentration of ingenol mebutate for 16 hours and the supernatants analysed by Western using an anti-IL-1α antibody. Arrow indicates the position of the 18 kDa bioactive form of IL-1α.</p

Expression analysis of G Protein-Coupled Receptors in mouse macrophages-1

Of 91 murine cell types and tissues. Data points show normalised values and similar cell types are grouped according to bar colour; blue indicates primary macrophage cell types, purple indicates bone-related cell types, red indicates other immune cell types, green indicates stem cell populations, orange indicates whole tissue samples, yellow indicates neuronal and retinal cell types and pink indicates cell lines. Additional file gives details of the 91 cell types and tissues profiled.<p><b>Copyright information:</b></p><p>Taken from "Expression analysis of G Protein-Coupled Receptors in mouse macrophages"</p><p>http://www.immunome-research.com/content/4/1/5</p><p>Immunome Research 2008;4():5-5.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2394514.</p><p></p

Expression analysis of G Protein-Coupled Receptors in mouse macrophages-2

over a timecourse of 0, 2, 6, and 24 h, and 0, 1 and 7 h, respectively. Data points show gene expression relative to untreated control for each cell population (0 h).<p><b>Copyright information:</b></p><p>Taken from "Expression analysis of G Protein-Coupled Receptors in mouse macrophages"</p><p>http://www.immunome-research.com/content/4/1/5</p><p>Immunome Research 2008;4():5-5.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2394514.</p><p></p

Expression analysis of G Protein-Coupled Receptors in mouse macrophages-0

Of 91 murine cell types and tissues. Data points show normalised values and similar cell types are grouped according to bar colour; blue indicates primary macrophage cell types, purple indicates bone-related cell types, red indicates other immune cell types, green indicates stem cell populations, orange indicates whole tissue samples, yellow indicates neuronal and retinal cell types and pink indicates cell lines. Additional file gives details of the 91 cell types and tissues profiled.<p><b>Copyright information:</b></p><p>Taken from "Expression analysis of G Protein-Coupled Receptors in mouse macrophages"</p><p>http://www.immunome-research.com/content/4/1/5</p><p>Immunome Research 2008;4():5-5.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2394514.</p><p></p

Slfn4 over-expression caused splenomegaly.

<p>(A) Macroscopic appearance of spleens from <i>Slfn4</i> over-expressing mice (right) and from MacBlue littermate controls (left). (B) Organ weights are expressed as percentage of body weight. Data are combined from four independent experiments and are displayed as mean + SEM.</p

Macrophage-specific expression of the UAS-<i>Slfn4</i>-V5 transgene.

<p>(A) Elements of the UAS-<i>Slfn4</i>-V5 transgene include six UAS, a kozak sequence, and the open reading frame of <i>Slfn4</i> followed by a V5-tag. Upon crossing of the UAS-<i>Slfn4</i>-V5 mouse with the MacBlue mouse, the offspring (MacBlue/UAS-<i>Slfn4</i>-V5 mice) contain the GAL4-expressing module, the GAL4-reporting module, and the UAS-<i>Slfn4</i>-V5 transgene. GAL4/VP16 protein binding to both the UAS induces the expression of ECFP and <i>Slfn4</i>-V5 specifically in cells of the myeloid lineage. (B and C) RNA from bone marrow (BM) or BMM from the offspring of four UAS-<i>Slfn4</i>-V5 founder lines (F1–F4) was extracted and cDNA was prepared. <i>Slfn4</i> mRNA levels relative to <i>hprt</i> were determined by quantitative real-time PCR. Data are combined from at least two independent experiments (mean + range) and are presented as expression relative to BM controls to enable normalization across different the transgenic lines (B), or as expression relative to <i>hprt</i> (no normalization across the transgenic lines) (C).</p

Induction of <i>Slfn</i> expression by LPS in BMM, and in the CIA mouse model.

<p>(A) BMM were stimulated with LPS over a time course (0h no treatment control, 2h, 4h, 8h, 24h), RNA was extracted and reverse transcribed, and the expression of <i>Slfn1</i>, <i>Slfn2</i>, <i>Slfn4</i>, <i>Slfn5</i>, <i>Slfn8</i>, and <i>Slfn9</i> was determined using quantitative real-time PCR. Data (mean + SEM) are combined from 3 independent experiments. * <i>P</i>&lt;0.05 compared to control; ** <i>P</i>&lt;0.01 compared to control; *** <i>P</i>&lt;0.001 compared to control. (B) RNA from whole joints (disease affected, score 3; or unaffected, score 0) from mice with CIA was used to synthesise cDNA. mRNA expression of <i>Slfn1</i>, <i>Slfn2</i>, <i>Slfn4</i>, <i>Slfn5</i>, <i>Slfn8</i>, <i>Slfn9</i>, <i>csf1r</i>, and <i>Emr1</i> was determined using real-time PCR. The data, displayed as fold induction relative to control (score 0), are combined from two independent experiments (mean + range).</p