Lipid mediators in resolving skin blisters in humans.

<p>Blister content was collected in 3% sodium citrate 24 h and 72 h after application of cantharidin with cells separated from oedema by centrifugation. Red blood cells were lysed and the remaining leukocytes counted by haemocytometer while oedema volume recorded and analysed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry for eicosanoid levels (n = 17).</p

Total leukocyte and oedema profile in resolving skin blisters in humans.

<p>Blister content was collected in 3% sodium citrate 24 h and 72 h after application of cantharidin with cells separated from oedema by centrifugation. Red blood cells were lysed and the remaining leukocytes counted by haemocytometer while oedema volume recorded. Temporal differences from 24 to 72 h for total cells, leukocyte concentration and oedema volume are shown (n = 17).</p

Cytokine profiles in resolving skin blisters in humans.

<p>Blister content was collected in 3% sodium citrate 24 h and 72 h after application of cantharidin with cells separated from oedema by centrifugation. Red blood cells were lysed and the remaining leukocytes counted by haemocytometer while oedema volume recorded and processed by assay for cytokine concentration (A). Correlations were made between cytokine concentration and PMN infiltration at 24 h and 72 h (B) (n = 17).</p

Leukocyte subtype profiles in resolving skin blisters in humans.

<p>Blister content was collected in 3% sodium citrate 24 h and 72 h after application of cantharidin with cells separated from oedema by centrifugation. Red blood cells were lysed and the remaining leukocytes counted by haemocytometer. Leukocytes were incubated with fluorescent antibodies, processed for flow cytometry, and gated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089375#pone-0089375-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089375#pone-0089375-g003" target="_blank">3</a>. Temporal changes in cell populations from 24 h to 72 h are shown (n = 17).</p

Characterisation of inflammatory cell infiltrates into skin blisters at 72(resolution) – I.

<p>Representative dot plots for flow cytometric gating are shown for healthy male volunteers (n = 17). Blister contents were collected from the remaining blister 72 h after application of cantharidin in 3% sodium citrate, with cells separated from oedema by centrifugation. Leukocytes were enumerated by haemocytometer and oedema volume recorded. Leukocytes were incubated with antibodies and processed by flow cytometry. Gating strategies firstly identified CD3⁺ T cells, CD19⁺ B cells and CD56⁺CD16^+/− NK cells. The remaining lymphocyte-deplete population was gated into HLA-DR⁺ and HLA-DR⁻ cells. Arrows indicate gating strategy.</p

Characterisation of peripheral blood leukocytes from healthy volunteers- II.

<p>HLA-DR⁺ and HLA-DR⁻ cells identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089375#pone-0089375-g001" target="_blank">Figure 1</a> were further analysed. HLA-DR⁺ cells were characterised into CD14^hi/CD16⁻ , CD14^hi/CD16⁺, CD14^lo/CD16⁺ monocytes and CD14⁻/CD16⁻ dendritic cells. HLA-DR⁻ cells comprised of typical PMNs (CD16^hi, CD66⁺) and a CD16^lo population. On extended characterisation, HLA-DR⁻/CD16^lo cells were identified as Siglec-8⁺ eosinophils and the HLA-DR⁺/CD14⁻/CD16⁻ dendritic cells comprised of CD141⁺ and CD11c⁺ dendritic cell subpopulations. Arrows indicate gating strategy.</p

Characterisation of inflammatory cell infiltrates into skin blisters at 24(onset) – II.

<p>HLA-DR⁺ and HLA-DR⁻ cells identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089375#pone-0089375-g003" target="_blank">Figure 3</a> were further analysed. HLA-DR⁺ cells were characterised into CD14⁺/CD16^lo monocytes/macrophages and HLA-DR⁺/CD14⁻/CD16⁻ dendritic cells. HLA-DR⁻ cells comprised of typical PMNs (CD16^hi, CD66⁺) and a CD16^lo population. On extended characterisation, HLA-DR⁻/CD16^lo were identified as a mixture of Siglec-8⁺ eosinophils and Annexin V/7AAD⁺ apoptotic/dead PMNs. The HLA-DR⁺/CD14⁻/CD16⁻ dendritic cells comprised of CD141⁺ and CD11c⁺ dendritic cell subpopulations. Arrows indicate gating strategy.</p

Correlations in cell profiles from onset to resolution.

<p>A wide range of PMN numbers infiltrate skin blisters early in the response (onset), and an immune response induces appropriate resolution. Correlation between change in PMN numbers (72 h minus 24 h) and total PMN numbers at onset are expressed as either total cells or as percentages.</p

Distribution of luciferin-dependent bioluminescence in cardiovascular tissue from <i>Cox2</i>^{<i>fLuc/+</i>} mice.

<p>(a) Quantification of basal expression from the aortic tree, vena cava, chambers of the heart and, for comparison, brain from <i>Cox2</i>^{<i>fLuc/+</i>} mice and (b) and representative images of bioluminescence. Arteries, veins and chambers of the heart were essentially devoid of expression from the <i>Cox2</i> gene, in comparison with the brain as a reference tissue. The only exception to this was weak, but detectable, expression in the region of the aortic arch. n=3.</p

Distribution of luciferin-dependent bioluminescence in tissues from <i>Cox2</i>^{<i>fLuc/+</i>} mice.

<p>(a) Basal expression from organs of the <i>Cox2</i>^{<i>fLuc/+</i>} mice was visualized by bioluminescent imaging of tissues dissected from <i>Cox2</i>^{<i>fLuc/+</i>} reporter mice after injection of D-luciferin in vivo (125mg/kg i.p.). (b) Imaging data are expressed as maximum luminescent emission from each tissue. Basal <i>Cox2</i> gene driven luciferase expression was present in many tissues including the vas deferens, brain, intestine, and thymus but was notably low to absent in the aorta (highlighted with red circles). Sub-division of the (c) brain, (d) intestine, (e) kidney and (f) stomach revealed regional expression patterns within each tissue. n=5.</p