9 research outputs found

    TRAF6ΔDC mice exhibit disruption of microbiotic homeostasis localized to the small intestine.

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    <p><b>(A)</b> Pie charts represent bacterial composition categorized by phylum in the small bowl of control (WT) or TRAF6ΔDC (ΔDC) mice. <b>(B)</b> Heatmap analysis shows relative abundance of taxa as a percentage of total 16S rRNA, organized according to genus or most specific assigned taxon. Color scales reflect proportion contributed by each taxon. Total bacterial genome was isolated from small intestinal contents of control (WT) or TRAF6ΔDC (ΔDC) mice (n = 4, 20 week old littermate and co-housed group). <b>(C)</b> The copy number of total bacterial 16S rRNA bacteria from fecal or small intestine contents of control (WT) and TRAF6ΔDC (ΔDC) was measured by comparing to reference E. coli 16S rRNA plasmids. Some groups were fed by broad-spectrum antibiotic water (Abx) for last 2 weeks before collecting samples. **p < 0.001; n.s., not significant. Data were analyzed by Anova on Prism software (n = 3).</p

    Increased Tregs in GF TRAF6ΔDC lymphoid organs following antibiotic treatment.

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    <p><b>(A)</b> FACS plots gated on CD4<sup>+</sup> T cells show intracellular staining Foxp3 from each indicated organ of germ-free (GF) TRAF6ΔDC bone marrow chimera (ΔDC-BMC) at 8 weeks post-reconstitution. Broad-spectrum antibiotic water was provided to a group of TRAF6ΔDC bone marrow chimeras (ΔDC) for the last 2 weeks. <b>(B)</b> Percentage of Foxp3<sup>+</sup> CD4 T cells were determined from the indicated organs of TRAF6ΔDC bone marrow chimera (ΔDC-BMC) in germ-free (GF) or germ-free under antibiotics (GF-Abx). <b>(C)</b> Representative FACS plots show Foxp3<sup>+</sup> CD4 T cells from each indicated organ of germ-free (GF) B6 mice. Some of the mice were provided with broad-spectrum antibiotic water (GF-Abx) for last 2 weeks. Percentage of Foxp3<sup>+</sup> CD4 T cells were shown in each organs of germ-free B6 mice with (GF-Abx) or without antibiotics (GF). SP, spleen; MLN, mesenteric lymph node; SI, small intestine; COL, colon. **p < 0.01.</p

    Impact of Ad-hHO-1 on H<sub>2</sub>O<sub>2</sub> or hTNF-α and CHX-induced cell death and apoptosis in NPCCs.

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    <p><b>A:</b> Ad-hHO-1 increased cell viability in H<sub>2</sub>O<sub>2</sub>-treated NPCCs. NPCCs were transduced with Ad-hHO-1 for 24 hr before the addition of H<sub>2</sub>O<sub>2</sub> (0, 50, 100, 200, or 400 µM). After incubating the cells with H<sub>2</sub>O<sub>2</sub> for an additional 24 hr, cell viability was measured by CCK-8. <b>B:</b> After treatment with hTNF-α and CHX cell viability was measured by CCK-8. An increased proportion of viable cells were detected in the Ad-hHO-1 group compared with the Ad-GFP control group. NPCCs were infected with Ad-hHO-1 for 24 hr before the addition of hTNF-α (25 ng/ml) and CHX (10 µg/ml). After treatment with hTNF-α and CHX, the NPCCs were incubated for an additional 24 hr. <b>C:</b> FACS analysis of apoptosis in NPCCs after treatment with 400 µM H<sub>2</sub>O<sub>2</sub>. A lower proportion of apoptotic cells were noted in Ad-hHO-1 (50 MOI) infected NPCCs relative to the Ad-GFP control. The NPCCs were treated with H<sub>2</sub>O<sub>2</sub> (400 µM) and FACS analysis was performed using annexin V-PE. Each experiment was repeated three times (data not shown). <b>D:</b> FACS analysis of apoptosis in NPCCs after treatment with hTNF-α and CHX for 24 hr. A smaller proportion of apoptotic cells was detected in Ad-hHO-1 (200 MOI)-infected NPCCs than in the Ad-GFP (200 MOI) group (p<i><0.05)</i>. After treatment with hTNF-α and CHX, apoptosis of cultured NPCCs was measured by FACS using annexin V. The numbers indicate the proportion of apoptotic cells in the NPCCs after 24 hr of incubation with hTNF-α and CHX. <b>E:</b> Lower activity of caspase 3/7 in Ad-hHO-1-infected NPCCs relative to Ad-GFP-infected NPCCs after 24 hr of incubation with H<sub>2</sub>O<sub>2</sub> (400 µM). After Zn(II)PPIX treatment, Ad-hHO-1 increased activity of caspase 3/7. Each experiment was repeated three times. (* p<0.05, ** p<0.01, *** p<0.001, NT: no treatment). <b>F:</b> The activities of caspase 3/7 were measured in Ad-hHO-1-infected NPCCs. The protective effect of Ad-hHO-1 in the NPCCs compared with Ad-GFP after 24 hr of incubation with hTNF-α and CHX. Each experiment was repeated three times. (** p<0.01, *** p<0.001).</p

    Anti-apoptotic effects of fibroblasts from the hHO-1-Tg pig.

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    <p><b>A:</b> Cell viability was measured using the CCK-8. After induction with hTNF-α (25 ng/ml) and CHX (10 µg/ml) for 15 hours, the hHO-1 group had a lower percentage of viable cells. <b>B and C:</b> FACS analysis of hTNF-α and CHX-mediated apoptosis in fibroblasts. Apoptosis was analyzed using annexin V staining. The numbers indicate the proportion of apoptotic cells in the NPCCs after 15 hr incubation with hTNF-α and CHX. <b>D:</b> Activities of caspase 3/7 were measured in fibroblasts. Human HO-1 protected transgenic fibroblasts from apoptosis after 15 hr of incubation with hTNF-α and CHX. After Zn(II)PPIX treatment, Ad-hHO-1 increased activity of caspase 3/7. Each experiment was repeated three times. (* p<0.05, ** p<0.01, *** p<0.001, NT: no treatment).</p

    Anti-oxidant effects of fibroblasts from the hHO-1-Tg pig.

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    <p><b>A:</b> Western blot analysis for hHO-1 and HA in the hHO-1-Tg fibroblasts using anti-HO-1 (1∶200) and anti-HA (1∶4,000) antibodies. The α-tubulin was used as a control. <b>B:</b> Cell viability was higher in the hHO-1-Tg fibroblasts compared to the wild-type. After H<sub>2</sub>O<sub>2</sub> (0, 200, 400 or 800 µM) treatment, cell viability was slightly better in the hHO-1-Tg fibroblast than in the wild-type fibroblasts. Cell viability was measured by CCK-8 after induction with H<sub>2</sub>O<sub>2</sub> for an additional 1 hr. <b>C:</b> Level of ROS was significantly reduced in the hHO-1-Tg fibroblasts in comparison to the wild type fibroblasts. After H<sub>2</sub>O<sub>2</sub> treatment, ROS production was significantly reduced in the hHO-1-Tg fibroblasts compared with the wild-type fibroblasts. Fibroblasts obtained from ear tissues were incubated with 25 µM DCFH-DA for 15 minutes at 37°C, and ROS were measured using FACS. <b>D:</b> Expression of the iNOS gene by real-time PCR in hHO-1-Tg fibroblasts compared with the wild-type fibroblasts. The threshold cycle (Ct) value was defined as the number of PCR cycles at which the fluorescence crossed a fixed threshold above baseline. For relative quantification, the ΔΔCt method was used to measure fold changes of cDNA. (* p<0.05, ** p<0.01, *** p<0.001).</p

    Tissue distribution of hHO-1 protein in hHO-1-Tg pig tissues.

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    <p><b>A:</b> The tissue distribution of the hHO-1 protein was analyzed in various organs by western blot analysis in a 465-day-old male hHO-1 F0 pig. Western blot was performed on heart, kidney, lung, liver, pancreas, spleen, and skin tissue. <b>B:</b> The tissue distribution of the hHO-1 protein was analyzed in various organs by western blot analysis in a 74-day-old male hHO-1 F1 pig. Western blot was performed on heart, kidney, lung, liver, pancreas, spleen, and skin tissue. <b>C:</b> The tissue distribution of the hHO-1 protein was analyzed in various organs by western blot analysis in a 74-day-old male hHO-1 F1 pig. Western blot was performed on heart, kidney, lung, liver, pancreas, spleen, and skin tissue using anti-hHO-1 (1∶1,000) and anti-HA antibodies (1∶4,000). Twenty micrograms of protein were loaded into each lane as indicated by the α-tubulin band. <b>D:</b> Immunohistochemistry of heart, kidney, lung, liver, pancreas, spleen, and skin tissues of hHO-1 F0 pig were performed to analyze expression patterns of hHO-1 and HA. Paraffin sections of various organs were stained with anti-HA antibody (1∶1,000), anti-hHO-1antibody (multiple organs: 1∶100) and control (anti-hHO-1 antibody, anti-HA antibody: data not shown). <b>E:</b> Immunohistochemistry of heart, kidney, lung, liver, pancreas, spleen, and skin tissues of hHO-1 F1 (n = 2) were performed to analyze expression patterns of hHO-1 and HA. Paraffin sections of various organs were stained with anti-HA antibody (1∶1,000), anti-hHO-1antibody (multiple organs: 1∶100) and control (anti-hHO-1 antibody, anti-HA antibody: data not shown).</p
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