17 research outputs found
HBD-1 production by patient unburned skin tissues in cultures supplemented with CD31<sup>+</sup> IMC culture fluids.
<p>Patient #16∼#18 CD31<sup>+</sup> IMC (1×10<sup>6</sup> cells/ml) were individually treated with or without glycyrrhizin (100 µg/ml) for 12 hours. After washing with media, these cells were cultured for an additional 36 hours. Culture fluids harvested from the cultures supplemented with or without glycyrrhizin were added (5∼20%, v/v) to cultures of patient unburned skin tissues (0.2×0.2 cm), and cultured for 48 hours. Culture fluids harvested were assayed for HBD-1 by ELISA. *<i>P</i><0.05; **<i>P</i><0.01 vs. cultures without glycyrrhizin.</p
Culture fluids of lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>+</sup> cells are inhibitory on HBD-1 production by NHEK. A.
<p>Inhibition of HBD production in NHEK cultures supplemented with culture fluids of lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>+</sup> cells. Culture fluids were harvested 48 hours after cultivation of lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>+</sup> cells (1×10<sup>6</sup> cells/ml) derived from burn patients #8, #9, #13, #14 and #15 (open circles). The culture fluids of lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>−</sup> cells of healthy donors #1∼#5 (filled circles) were utilized as a control. Five to 40% (v/v) of these culture fluids were added to cultures of NHEK (1×10<sup>5</sup> cells/ml). Culture fluids harvested from NHEK cultures were assayed for HBD-1 by ELISA. * <i>P</i><0.01 vs control. <b>B–D.</b> Production of IL-10, IL-13 and CCL2 by lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>+</sup> cells. Lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>+</sup> cells (1×10<sup>5</sup> cells/ml, open circles) isolated from peripheral blood of burn patients #8, #9, #13, #14 and #15 were cultured for 12 to 48 hours. As controls, peripheral blood lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>−</sup> cells of healthy donors #1∼#5 (filled circles) were cultured under the same conditions. Supernatants obtained were assayed for IL-10 (<b>B</b>), IL-13 (<b>C</b>) and CCL2 (<b>D</b>) by ELISA. * <i>P</i><0.05, ** <i>P</i><0.01 vs lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>−</sup> cells.</p
HBD-1 production and mRNA expression by NHEK cultured with peripheral blood lineage<sup>−</sup>CD34<sup>+</sup> cells that were isolated from both severely burned patients and healthy donors. A.
<p>HBD-1 production. Lineage<sup>−</sup>CD34<sup>+</sup> cells (1×10<sup>5</sup> cells/ml, upper chamber), isolated from 5 healthy donors (#1∼#5) and 20 burn patients (#1∼#20), were transwell-cultured with NHEK (1×10<sup>5</sup> cells/ml, lower chamber) for 36 hours. After removal of the upper chamber, NHEK in the lower chamber were cultured for an additional 36 hours. Culture fluids obtained were assayed for HBD-1 by ELISA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082926#pone-0082926-g001" target="_blank">Fig. 1A-1</a> shows independent experiments performed using blood specimens from 5 healthy donors and 20 burn patients, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082926#pone-0082926-g001" target="_blank">Fig. 1A-2</a> shows mean ± SEM of the results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082926#pone-0082926-g001" target="_blank">Fig. 1A-1</a>. * <i>P</i><0.001 vs control. <b>B and C.</b> HBD-1 mRNA expression. Lineage<sup>−</sup>CD34<sup>+</sup> cells (1×10<sup>5</sup> cells/ml, upper chamber), isolated from peripheral blood of 5 healthy donors (#1∼#5, <b>B</b>) and 5 severely burn patients (#1, #16, #17, #18, #19, <b>C</b>), were transwell-cultured with NHEK (1×10<sup>5</sup> cells/ml, lower chamber) for 36 hours. After removal of the upper chamber, NHEK in the lower chamber were analyzed for HBD-1 mRNA by RT-PCR.</p
Effect of glycyrrhizin on the resistance of chimeras against sepsis stemming from <i>P. aeruginosa</i> i.d. infections.
<p><b>A.</b> Survival of patient chimera substitutes i.d. infected with 20 LD<sub>50</sub> of <i>P. aeruginosa</i>. The chimeras exposed to the pathogen were treated i.p. with 10 mg/kg (open circles) and 3 mg/kg (open squires) of glycyrrhizin or saline (0.2 ml/mouse, control, open triangles). Each infection experiment was performed by a group of 2–3 mice, and it was repeated 3 times. The results obtained were combined and displayed in the figure. <b>B.</b> Growth of pathogen in kidneys of patient chimeras i.d. infected with 20 LD<sub>50</sub> of <i>P. aeruginosa</i>. Patient chimeras created with unburned skin tissues from patients #8∼#10 and their CD31<sup>+</sup> IMC were exposed to the pathogen, and treated with glycyrrhizin (10 mg/kg) or saline (0.2 ml/mouse, control). Two days after infection, the growth of <i>P. aeruginosa</i> in kidneys of these chimeras was measured by a standard colony counting method. A white bar shows the results obtained from a group of mice grafted with skin alone. Fig. 3B-1 shows 3 independent experiments performed using skin and blood specimens from 3 patients, and Fig. 3B-2 shows mean ± SEM of the results shown in Fig. 3B-1. **<i>P</i><0.01 vs saline-treated control.</p
Importance of skin antimicrobial peptides on the resistance of various chimeras to <i>P. aeruginosa</i> i.d. infections.
<p><b>A-1.</b> γ-Irradiated NOD-SCID IL-2rγ<sup>−/−</sup> mice treated with (mouse B) or without anti-murine AMP rabbit IgG (mouse A) were i.d. infected with 20 LD<sub>50</sub> of <i>P. aeruginosa</i>. <b>B-1.</b> Chimera A (mouse B grafted with NHEK) and chimera B (chimera A inoculated with experimentally induced CD31<sup>+</sup> IMC, patient chimera substitute) were i.d. infected with 20 LD<sub>50</sub> of <i>P. aeruginosa</i> at the skin surrounding the grafted site tissues. <b>C-1.</b> Chimera A (mouse B grafted with NHEK) and chimera C (chimera A inoculated with lineage<sup>–</sup>CD31<sup>−</sup> cells) were i.d. infected with <i>P. aeruginosa</i> in the same fashion. Then, their survivals were observed for a week after the infection. In addition, the antimicrobial peptide production in the grafted site tissues of all groups of chimeras was tested. One day after the IgG treatment (<b>A-2</b>) or 2 days after grafting (<b>B-2, B-2</b>), 5 biopsies of skin tissues were obtained from the grafting sites. Tissues obtained were homogenized together, and the amounts of HBD-1 and MBD-1 in the homogenates were measured by ELISA. Each infection experiment was performed by a group of 2–3 mice, and it was repeated 3 times. The results obtained were combined and displayed in the figure.</p
Effect of glycyrrhizin on the expression of HBD-1 mRNA and HBD-1 production by patient unburned skin tissues transwell-cultured with the same patient CD31<sup>+</sup> IMC.
<p><b>A.</b> mRNA expression. Patient #11 unburned skin tissues (0.2×0.2 cm, lower chamber) were transwell-cultured with their CD31<sup>+</sup> IMC (1×10<sup>5</sup> cells/ml, upper chamber) for 48 hours in the presence or absence of 1 to 100 µg/ml of glycyrrhizin. Skin tissues harvested were analyzed for HBD-1 mRNA by RT-PCR. Data are representative of 4 individual experiments using unburned skin tissues and CD31<sup>+</sup> IMC obtained from patients #12∼#15. <b>B.</b> HBD-1 production. Transwell-cultures between unburned skin tissues from patients #11∼#15 and their CD31<sup>+</sup> IMC were respectively performed in the presence of 100 µg/ml of glycyrrhizin. Culture fluids harvested 48 hours after cultivation were assayed for HBD-1 by ELISA.</p
CCL2 and IL-10 produced by burn patient lineage<sup>−</sup>CD34<sup>+</sup> CD31<sup>+</sup> cells are responsible for their suppressor cell activities on HBD-1 production by NHEK. A.
<p>Effect of mAbs against IL-10, CCL2 and IL-13 on the suppressor activities of lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>+</sup> cell-culture fluids on HBD production by NHEK. NHEK (1×10<sup>5</sup> cells/ml) were cultured with fresh media supplemented with 20% (v/v) of the culture fluids that were previously treated with mAb for IL-10, IL-13 and CCL2 (2.5 µg/ml each) or a combination of these mAbs. Forty-eight hours after cultivation, culture fluids harvested were assayed for HBD-1. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082926#pone-0082926-g004" target="_blank">Figure 4A</a> displays one of the representative results shown in independent experiments using culture fluids of 5 different lineage<sup>−</sup>CD34<sup>+</sup>CD31<sup>+</sup> cell preparations derived from burn patients #8, #9, #13, #14 and #15. <b>B.</b> Effect of rIL-10 and rCCL2 on HBD-1 production by NHEK. NHEK (1×10<sup>5</sup> cells/ml) were treated with rIL-10 (1 ng/ml), rCCL2 (10 ng/ml) or a combination of rIL-10 and rCCL2. Culture fluids obtained 24 hours after treatment were assayed for HBD-1. * <i>P</i><0.01, ** <i>P</i><0.001 vs NHEK cultured with media.</p
HBD-1 production in patient chimeras treated with glycyrrhizin.
<p><b>A.</b> Patient chimeras created with burn patient #1∼#3 unburned skin tissues and their CD31<sup>+</sup> IMC were treated i.p. with glycyrrhizin (10 mg/kg) 6 and 24 hours after the IMC inoculation. As a control, the chimeras were treated with saline (0.2 ml/mouse). Twenty-four hours after the final glycyrrhizin treatment, 5 skin biopsies were obtained from grafted site tissues, homogenized together, and assayed for HBD-1 by ELISA. A white bar shows the results obtained from a group of mice grafted with skin alone. Fig. 2A-1 shows 3 independent experiments performed using skin and blood specimens from 3 patients, and Fig. 2A-2 shows mean ± SEM of the results shown in Fig. 2A-1. **<i>P</i><0.01 vs saline-treated control. <b>B.</b> The recovery of HBD-1 production in patient chimeras treated with glycyrrhizin. The chimeras created with unburned skin tissues from patients #4∼#7 and their CD31<sup>+</sup> IMC were treated twice with 1 to 10 mg/kg of glycyrrhizin. As controls, the chimeras were treated with saline (0.2 ml/mouse). Twenty-four hours after the treatment, 5 skin biopsies were obtained from grafted site tissues, homogenized together, and assayed for HBD-1 by ELISA. A white bar shows the results obtained from a group of mice grafted with skin alone (mean ± SEM of the 4 independent experiments). *<i>P</i><0.05; **<i>P</i><0.01 vs saline-treated control.</p
The tyrosine-sorting motif of the vacuolar sorting receptor VSR4 from <i>Arabidopsis thaliana</i>, which is involved in the interaction between VSR4 and AP1M2, μ1-adaptin type 2 of clathrin adaptor complex 1 subunits, participates in the post-Golgi sorting of VSR4
<p>μ1-Adaptin of adaptor protein (AP) 1 complex, AP1M, is generally accepted to load cargo proteins into clathrin-coated vesicles (CCVs) at the <i>trans</i>-Golgi network through its binding to cargo-recognition sequences (CRSs). Plant vacuolar-sorting receptors (VSRs) function in sorting vacuolar proteins, which are reportedly mediated by CCV. We herein investigated the involvement of CRSs of <i>Arabidopsis thaliana</i> VSR4 in the sorting of VSR4. The results obtained showed the increased localization of VSR4 at the plasma membrane or vacuoles by mutations in CRSs including the tyrosine-sorting motif YMPL or acidic dileucine-like motif EIRAIM, respectively. Interaction analysis using the bimolecular fluorescence complementation (BiFC) system, V10-BiFC, which we developed, indicated an interaction between VSR4 and AP1M2, AP1M type 2, which was attenuated by a YMPL mutation, but not influenced by an EIRAIM mutation. These results demonstrated the significance of the recognition of YMPL in VSR4 by AP1M2 for the post-Golgi sorting of VSR4.</p> <p>Clathrin AP1M2-recognizable tyrosine motif for plant VSR4 sorting.</p
Development of PET Imaging to Visualize Activated Macrophages Accumulated in the Transplanted iPSc-Derived Cardiac Myocytes of Allogeneic Origin for Detecting the Immune Rejection of Allogeneic Cell Transplants in Mice
<div><p>Allogeneic transplantation (Tx) of induced pluripotent stem cells (iPSCs) is a promising tissue regeneration therapy. However, this inevitably induces macrophage-mediated immune response against the graft, limiting its therapeutic efficacy. Monitoring the magnitude of the immune response using imaging tools would be useful for prolonging graft survival and increasing the therapy longevity. Minimally invasive quantitative detection of activated macrophages by medical imaging technologies such as positron emission tomography (PET) imaging targets translocator protein (TSPO), which is highly expressed on mitochondrial membrane, especially in activated macrophage. <i>N</i>,<i>N</i>-diethyl-2-[4-(2-fluoroethoxy) phenyl]-5,7-dimethylpyrazolo[1,5-<i>a</i>]pyrimidine-3-acetamide (DPA-714) is known as a TSPO ligand used in clinical settings. We herein hypothesized that immune rejection of the transplanted iPSC-derived cardiomyocytes (iPSC-CMs) of allogeneic origin may be quantitated using <sup>18</sup>F-DPA-714-PET imaging study. iPSC-CM cell-sheets of C57BL/6 mice origin were transplanted on the surface of the left ventricle (LV) of C57BL/6 mice as a syngeneic cell-transplant model (syngeneic Tx group), or Balb/c mice as an allogeneic model (allogeneic Tx group). <sup>18</sup>F-DPA-714-PET was used to determine the uptake ratio, calculated as the maximum standardized uptake value in the anterior and septal wall of the LV. The uptake ratio was significantly higher in the allogeneic Tx group than in the syngeneic group or the sham group at days 7 and day 10 after the cell transplantation. In addition, the immunochemistry showed significant presence of CD68 and CD3-positive cells at day 7 and 10 in the transplanted graft of the allogeneic Tx group. The expression of TSPO, <i>CD68</i>, <i>IL-1</i> beta, and <i>MCP-1</i> was significantly higher in the allogeneic Tx group than in the syngeneic Tx and the sham groups at day 7. The <sup>18</sup>F-DPA-714-PET imaging study enabled quantitative visualization of the macrophages-mediated immune rejection of the allogeneic iPSC-cardiac. This imaging tool may enable the understanding and monitoring host-immune response of the host, allogeneic cell transplantation therapy.</p></div