Calculated CD73/CD39 content per mg heart tissue under basal conditions and after I/R.

<p>The number of cell surface molecules was calculated by multiplying the absolute cell count/mg heart tissue with the percentage of CD73⁺/CD39⁺ cells and with the number of CD73/CD39 molecules on those cells. <b>A</b>: Calculated CD73 content per mg heart tissue. <b>B</b>: Calculated CD39 content per mg heart tissue. Values are means ± SD of n = 5 experiments. * P<0.05; n.d. = not detectable.</p

Abundance of CD73 and CD39 on leukocytes in blood and heart under basal conditions.

<p><b>A</b>: CD73⁺ and CD39⁺ cells per leukocyte population in cardiac tissue. <b>B</b>: CD73/CD39 surface density on CD73⁺/CD39⁺ cells per leukocyte population in cardiac tissue. <b>C</b>: CD73⁺ and CD39⁺ cells per leukocyte population in blood. <b>D</b>: CD73/CD39 surface density on CD73⁺/CD39⁺ cells per leukocyte population in blood. Only positive gated CD73 or CD39 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034730#pone-0034730-g003" target="_blank">Fig. 3A, 3C</a>) were considered for the calculation of antigen density. Values are means ± SD of n = 5 experiments. n.d. = not detectable.</p

Leukocyte subpopulations in myocardial tissue under basal conditions.

<p><b>A:</b> Gating strategy used to identify different leukocyte subpopulations by multiparametric flow cytometry. CD45⁺ cells were gated and DAPI-staining was used to exclude dead and apoptotic immune cells. Living CD45⁺ cells were then divided in subleukocyte populations using a panel of cell-specific fluorochrome-labeld antibodies. Lymphocytes were gated into CD45R(B220)⁺ cells (B-cells) and CD3⁺ cells (T-cells). T-cells were subdivided in CD4⁺ cells (T-helper cells) and CD8⁺ cells (cytotoxic T-cells). Myeloid cells were characterized as CD11b⁺ cells and further subdivided in CD11c⁺ cells (APCs) and Ly6g⁺ cells (granulocytes). <b>B:</b> Leukocyte subpopulations in the unstressed heart. Values are means ± SD of n = 5 experiments.</p

Comparison of cardiac CD73⁺ leukocytes and coronary endothelial cells under basal conditions and after I/R.

<p>Values are calculated from data given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034730#pone-0034730-t001" target="_blank">Table 1</a>. Values are means ± SEM of n = 5 experiments.</p

Expression of CD73 and CD39 on cardiac cells and circulating blood cells under basal conditions (n = 4–5).

*<p>Analysed by fluorescence microscopy.</p

Changes of total leukocytes, CD73⁺/CD39⁺ cells and CD73/CD39 surface density after I/R.

<p><b>A</b>: Representative flow cytometry plots of digested hearts under basal conditions compared to 3 days after I/R. <b>B</b>: Representative flow cytometry histograms of CD73 expression (red) under basal conditions compared to 3 days after I/R. Grey histograms = fluorescence minus one control (FMO). <b>C</b>: Representative flow cytometry histograms of CD39 expression (blue). Grey histograms = fluorescence minus one control (FMO). <b>D</b>: Increase of leukocyte populations within myocardial tissue after I/R. * P<0.05 for cells/mg heart tissue under basal conditions vs. I/R. CTC = cytotoxic t-cells, THC = T-helper cells, Treg = regulatory t-cells, BC = B-cells, NKC = NK cells, Gr = Granulocytes, Mo = Monocytes, APC = Antigen-presenting cells. <b>E</b>: Changes of CD73⁺/CD39⁺ cells per leukocyte population after I/R. <b>F</b>: Changes of CD73/CD39 surface density on CD73⁺/CD39⁺ leukocytes after I/R. Analysis was done 3 days after I/R. Values are means ± SD of n = 5 experiments. n.d. = not detectable.</p

Analysis of the different cell fractions present within the unstressed heart.

<p>Fluorescence microscopy of intact murine cardiomyocytes (<b>A</b>) and non-cardiomyocytes (<b>B</b>) extracted from the murine heart. Red = CD45⁺ cells (fluorescence microscopy). Blue = nuclear stain with DAPI. <b>C</b>: Representative flow cytometry plot of non-cardiomyocytes. Black = CD45⁺ cells (lower right), light grey = endothelial cells (CD31⁺, upper left) and dark grey = CD31⁻ CD45⁻ (lower left). <b>D</b>: Representative Ter-119/CD45 plot of non-cardiomyocytes to derive the ratio of erythrocytes to leukocytes in myocardial tissue. Assuming a ratio of erythrocytes/leukocytes in peripheral blood to be 1000∶1, contamination of blood derived leukocytes was calculated to be <0.2% (n = 5).</p

Purinergic Signaling on Leukocytes Infiltrating the LPS-Injured Lung

<div><p>Extracellular nucleotides and nucleosides have been implicated as important signaling molecules in the pathogenesis of acute lung injury (ALI). While adenosine is known to inhibit T cell activation, little information is available as to ATP and NAD degrading enzymes, the expression of ATP and adenosine receptors/transporters in different T cell subsets. ALI was induced by challenging mice with intra-tracheal instillation of 60 µl (3 µg/g) LPS. After 3 d and 7 d blood, lung tissue and bronchoalveolar lavage was collected and immune cells were analyzed using flow cytometry. The transcriptional phenotype of T helper cells, cytotoxic and regulatory T cells sorted by FACS was assessed by measuring the expression profile of 28 genes related to purinergic signaling using TaqMan Array Micro Fluidic Cards. Catabolism of ATP, NAD and cAMP by activated CD4⁺ T cells was evaluated by HPLC. CD73 was found to be highly abundant on lymphoid cells with little abundance on myeloid cells, while the opposite was true for CD39. After ALI, the abundance of CD39 and CD73 significantly increased on all T cell subsets derived from lung tissue and bronchoalveolar space. Expression analysis in T cell subsets of the lung revealed ATP (<i>Cd39</i>, <i>Cd73</i>) and NAD (<i>Cd38</i>, <i>Cd157</i>, <i>Cd296</i>, <i>Pc-1</i>) degrading enzymes. However, only transcription of <i>Cd38</i>, <i>Cd39</i>, <i>Cd73</i>, <i>Ent1</i> and <i>A2a receptor</i> was significantly upregulated after ALI in T helper cells. CD4⁺ T cells from injured lung rapidly metabolized extracellular ATP to AMP and adenosine but not NAD or cAMP. These findings show that lung T cells – the dominant cell fraction in the later phase of ALI – exhibit a unique expression pattern of purinergic signaling molecules. Adenosine is formed by T cells at an enhanced rate from ATP but not from NAD and together with upregulated A2a receptor is likely to modulate the healing process after acute lung injury.</p></div

"Imposition et liberté d’exercice du culte », note sous 8 décisions CEDH (depuis CEDH, 29 sept. 2010, n° 8916/05, Les Témoins de Jéhovah c. France) et une Cass. (Com, 15 janv. 2013, n° 12-11.642, Assoc. L’Arche de Marie) in chronique de jurisprudence fiscale

<p>(A+B) The percentage of cells expressing CD73 was high within the T cell subsets but low within the myeloid cell, B cell and NK cell populations. The percentage of CD73 expressing cells tended to be increased in T helper cells after ALI. (C+D) As assessed by means of the MFI CD73 was highly expressed on the different T cell subsets and showed a comparatively low expression on myeloid cells, B cells and NK cells. After LPS installation particular T helper cells, NKC and M&M showed an increased abundance of CD73. Data are mean ± SD (n = 5 mice per group). Statistical significance was assessed by one-way ANOVA with Dunnett's post hoc test. *P<0.05, **P<0.01, ***P<0.0001. Under unstressed conditions CD73 staining of regulatory T cells (IS) and T helper cells (BAL) was detected in only n = 2, thus statistical significance was not assessed. ALI  =  acute lung injury, AM  =  alveolar macrophages, APC  =  antigen-presenting cells, BAL  =  bronchoalveolar lavage, BC  =  B cells, CTC  =  cytotoxic T cells, Gr  =  granulocytes, IS  =  interstitial lung tissue, MFI  =  mean fluorescence intensity, M&M  =  monocytes and macrophages, n.d.  =  not detected, NKC  =  natural killer cells, SD  =  standard deviation, THC  =  T helper cells, Treg  =  regulatory T cells.</p

Gene expression of adenosine and ATP receptors in the T cell subsets isolated from the lung under basal conditions and 7d after LPS exposure determined by quantitative <i>real-time</i> PCR.

<p>(A) In the unstressed lung, T cells expressed predominantly the <i>A2a receptor</i>. The <i>P2x4</i> and <i>P2y6 receptors</i> were moderately expressed while <i>P2x5/7</i> and <i>P2y2 receptors</i> displayed a very low expression level. Gene expression was normalized to beta-actin and relative expression levels are depicted. (B) LPS administration significantly induced the <i>A2a receptor</i> expression in T helper cells 7 d post instillation. Since expression of some target mRNAs was below the detection limit in at least one of the conditions, fold changes (control vs. 7 d) were not calculated but the relative expression levels were depicted. Data are mean ± SD (n = 4 mice per group). Expression levels under basal conditions were compared to that in the injured lung 7 d post induction of ALI and statistical significance was then assessed by Mann-Whitney U test. *P<0.05, **P<0.01, ***P<0.0001. ALI  =  acute lung injury, ATP  =  adenosine triphosphate, AU  =  arbitrary units, LPS  =  lipopolysaccharide, n.d.  =  not detected, SD  =  standard deviation.</p