9 research outputs found

    Assay specificity and possible additional analyses.

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    <p>(A) ActCasp3 staining (left) and side scatter signal (right) in the DP gate with and without A2 tetramer stimulation for 24 h in the presence or absence of caspase inhibitor Z-VAD (OMe)-FMK. (B) Simultaneous stimulation of PD670-labeled and unlabeled cells in the same well with K<sup>b</sup>-Q7 tetramer for 24 h. ActCasp3 staining in the PD670 positive and negative DP gate with and without stimulation with tetramer for 24 h. (C) PD670 signal (X axis) in the DP gate with and without stimulation with A2, Q7, G4, and E1 tetramers for 24 h. (D) Vα2 (left) and CD69 (middle) staining in the DP gate with and without stimulation with A2, Q7, G4, and E1 tetramers for 24 h. Tetramer-PE signal (right) in ActCasp3<sup>+</sup> and ActCasp3<sup>−</sup> DP gate compared with that of non-treated B6 DP thymocytes. (E) Vα2 (left), CD69 (middle) and tetramer-PE (right) signals in the DP gate with and without stimulation with K<sup>b</sup>-A2 tetramer for 24 h in the presence or absence of caspase inhibitor Z-VAD (OMe)-FMK.</p

    Alternative gating strategies.

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    <p>(A) ActCasp3 (Y axis) and Vα2 staining (X axis) in the DP gate with and without stimulation with anti-CD3/28 beads or E1 and A2 H-2K<sup>b</sup> tetramers for 24 h. (B) ActCasp3 (Y axis) and side scatter signal (X axis) in the DP gate with and without stimulation with anti-CD3/28 beads or E1 and A2 tetramers for 24 h. (C) ActCasp3 (Y axis) and forward scatter signal (X axis) in the DP gate with and without stimulation with anti-CD3/28 beads or E1 and A2 tetramers for 24 h.</p

    Detection of dual Vα expressing cells in subsets gated based on Vα2-positive or Vα3.2-positive populations.

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    <p>Cells positive for subunits indicated above each plot were analyzed for the expression of the other subunit. Gates were set based on total lymphocyte population (gray lines). Top panel represents detection of dual Vα expressing cells by surface staining, bottom panel by intracellular staining. Results are representative of six mice per genotype.</p

    Development of triple-transgenic cells in hosts lacking MHC class I and II yields TCR<sup>int</sup>, single α-chain thymocytes.

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    <p>(A) Bone marrow from B6 mice was used to reconstitute irradiated CD45.1 (top) or MHC<sup>o/o</sup> (bottom) hosts. After eight weeks, thymocytes were harvested and analyzed for expression of either α-chain in TCR<sup>lo</sup>, TCR<sup>int</sup>, and TCR<sup>hi</sup>, Vβ5-positive gates. (B) Bone marrow from triple-transgenic mice was used to reconstitute irradiated CD45.1 (top) or MHC<sup>o/o</sup> (bottom) hosts. After eight weeks, thymocytes were harvested and analyzed for expression of either α-chain in TCR<sup>lo</sup>, TCR<sup>int</sup>, and TCR<sup>hi</sup>, Vβ5-positive gates. FACS plots representative of 7-9 mice per group in three independent experiments.</p

    Vβ5 transgene skews expression of Vα2 from CD4 to CD8 subset, but Vα3.2 skews to CD8s with or without Vβ5 transgene.

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    <p>(A) LN cells from B6 mice (WT), <i>Tcra</i><sup>−/−</sup> mice transgenic for Vα2 and Vα3.2 miniloci, with or without transgenic rearranged Vβ5, were gated for Vα2 or Vα3.2 expression to show the assortment of cells to CD4 or CD8 subpopulations. FACS plots representative of >10 mice per genotype. (B) shows the CD8∶CD4 ratio of peripheral T cells from mouse strains expressing all the potential combinations of transgenes used here: Vα2 and Vα3.2 miniloci individually or together, and rearranged Vβ5 transgene. Data represent 8–17 mice per genotype.</p

    Vα3.2-expressing T cells are more frequent than Vα2-expressing cells in dual α-chain minilocus mice.

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    <p>(A) Vα2, Vα3.2 and Vβ5 expression in lymph node T cells from B6 mice (WT), <i>Tcra</i><sup>−/−</sup> mice transgenic for Vα2 or Vα3.2 miniloci, <i>Tcra</i><sup>−/−</sup> mice transgenic for both Vα2 and Vα3.2 miniloci, and <i>Tcra</i><sup>−/−</sup> mice transgenic for Vα2 and Vα3.2 miniloci plus the rearranged Vβ5 gene. FACS plots representative of >10 mice per genotype. (B) Ratio of Vα3: Vα2-bearing T cells in CD4 and CD8 peripheral T cell subsets in the mice strains described in (A). Data represent >10 mice per genotype.</p

    Triple-transgenic thymocytes exhibit efficient allelic exclusion of TCR Vα chains.

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    <p>(A) Double-negative (DN) and CD4 and CD8 single positive (SP) thymocytes from B6 (top), Vα2 Vα3.2 (middle) and Vα2 Vα3.2 Vβ5 (bottom) mice were analyzed for surface expression of Vα2, Vα3.2 and Vβ5 in TCR<sup>int</sup> and TCR<sup>hi</sup> gate. (B) DN and CD4 and CD8 SP thymocytes from B6 (top), Vα2 Vα3.2 (middle) and Vα2 Vα3.2 Vβ5 (bottom) mice were analyzed for intracellular expression of Vα2, Vα3.2 and Vβ5 in TCR<sup>int</sup> and TCR<sup>hi</sup> gate. FACS plots representative of >10 mice per genotype and are derived from the same dataset as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114320#pone-0114320-g004" target="_blank">Fig. 4</a>.</p

    data_sheet_1_Neutrophils and Activated Macrophages Control Mucosal Immunity by Proteolytic Cleavage of Antileukoproteinase.docx

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    <p>Antileukoproteinase or secretory leukocyte peptidase inhibitor is a small protein which protects the mucosal linings against excessive proteolysis, inflammation, and microbial infection. We discovered that gelatinase B or matrix metalloproteinase (MMP)-9, a secreted zinc-dependent endopeptidase typically found at sites of inflammation, destroys antileukoproteinase by cleavages within both of its two functional domains: the anti-microbial N-terminal and the anti-proteolytic C-terminal domains. Cleaved antileukoproteinase possessed a significantly lower ability to bind lipopolysaccharides (LPS) and a reduced capacity to inhibit neutrophil elastase (NE) activity. Whereas intact antileukoproteinase repressed proinflammatory transcript [prostaglandin-endoperoxide synthase 2 (PTGS2) and IL6] synthesis and protein secretion [e.g., of MMP-9] in human CD14<sup>+</sup> blood monocytes stimulated with LPS, this effect was reduced or lost for cleaved antileukoproteinase. We demonstrated the in vivo presence of antileukoproteinase cleavage fragments in lower airway secretions of non-cystic fibrosis bronchiectasis patients with considerable levels of neutrophils and, hence, elastase and MMP-9 activity. As a comparison, other MMPs (MMP-2, MMP-7, and MMP-8) and serine proteases (NE, cathepsin G, and proteinase 3) were also able to cleave antileukoproteinase with similar or reduced efficiency. In conclusion, in specific mucosal pathologies, such as bronchiectasis, neutrophils, and macrophage subsets control local immune reactions by proteolytic regulation, here described as the balance between MMPs (in particular MMP-9), serine proteases and local tissue inhibitors.</p
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