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Editor's Notes

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• #20: Development and progression of experimental colitis. Male, 6 month-old C57BL/6 mice (n = 20) were exposed to 2% DSS in drinking water for up to 5 days. Mice were sacrificed at Day 0, 3, 7, and 14 of the experimental period (n = 5 per time point). (A) Daily body weight of mice sacrificed at Day 14; mice sacrificed at earlier time points followed a similar pattern. *P,0.05 compared to initial body weight on Day 0. (B) Disease Activity Index (DAI) was calculated at the time of sacrifice based on weight loss, stool blood, and stool consistency. (C) Colons were excised and measured lengthwise. (D) H&E stained sections were graded for Histologic Severity Score.
• #21: (E) Representative images of colon histology on Day 0, 3, 7, and 14 (original magnification 6 100; arrowhead = mononuclear infiltrates, thick arrow = mucosal ulceration, thin arrow = epithelial hyperplasia). Data represent mean 6 SD, ***P,0.001, *P,0.05 vs. Day 0.
• #22: Adipose tissue inflammation during acute colitis. (A) Colons and adipose tissue [mesenteric (MAT), epididymal (EAT), and subcutaneous (SAT)] from mice sacrificed at Day 0 and Day 7 (n = 4 per timepoint) were analyzed for relative mRNA expression of TNF-a, IL-1b, and IL-6. Target gene expression was normalized to HPRT gene expression; fold change was calculated relative to mean SAT expression at Day 0. Data represent mean 6 SD. ***P,0.001, **P,0.01, *P,0.05 vs. Day 0 of the same tissue; {{{P,0.001, {{P,0.01 vs. EAT and SAT at Day 7; ###P,0.001 vs. MAT at Day 7. (B) H&E stained sections of formalin-fixed MAT, EAT, and SAT from Day 0 and Day 7 of experimental colitis were compared (n = 5 per time point). Representative images are shown (original magnification6400; arrowhead = mononuclear infiltrates; thick arrow = fibrotic connective tissue; thin arrow = adipocytes).
• #24: Time-course of mesenteric adipose tissue cytokine expression. Mice were sacrificed at Day 0, 3, 7, and 14 of experimental colitis (n = 5 per time point). (A) MAT was analyzed for relative mRNA expression of TNF-a, IL-1b, and IL-6. Target gene expression was normalized to HPRT expression; fold change was calculated relative to mean expression at Day 0. Data represent mean 6 SD, ***P,0.001, *P,0.05 vs. Day 0.
• #25: Cytokine expression from MAT adipocytes (Ad), stromal vascular fraction (SVF), and mesenteric lymph nodes (MLN) during acute experimental colitis. (A) The adipocyte-specific hormone, Adiponectin; the preadipocyte marker, Pref-1; and the lymph node marker, MAdCAM-1, were used to confirm successful separation of Ad, SVF, and MLN, respectively. Target gene expression was normalized to HPRT expression; fold change was calculated relative to MLN expression at Day 0 (for Adiponectin and Pref-1) or Ad expression at Day 0 (for MAdCAM-1). (B) TNF-a, IL-1b, and IL-6 expression was compared between Ad, SVF, and MLN at Day 0 and Day 7. Target gene expression was normalized to HPRT expression and adjusted for RNA yield; fold change was calculated relative to Ad expression at Day 0. Data represent mean 6 SD, ***P,0.001, **P,0.01, *P,0.05 vs. Day 0 of the same fraction; {{{P,0.001, {{P,0.01 vs. other fractions at Day 7.
• #31: In vivo expression of interleukin (IL)-17A and IL-17E. IL-17A and IL-17E were detected by both (A) immunoblotting and (B) ELISA in uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of 12 patients with fibrostenosing Crohn’s disease (CD) and from normal gut of 11 healthy control (HC) subjects. (A) Each example shown in the upper panel is representative of experiments performed in 12 patients with CD and 11 HC subjects. Blots were stripped and analyzed for β-actin as an internal loading control. In the lower panel, densitometry of IL-17A and IL-17E expression normalized for β-actin is shown. Results are mean ± SEM. au, Arbitrary units. (B) Results, expressed as pg/100 μg of total protein, are mean ± SEM. *P<0.005 versus Non-strict uninfl and HC tissue samples.
• #32: In vivo expression of interleukin (IL)-17A and IL-17E receptors. (A) IL-17RC (receptor of IL-17A), and (B) IL-17RB (receptor of IL-17E) were detected by immunoblotting in uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of 12 patients with fibrostenosing Crohn’s disease (CD) and from normal gut of 11 healthy control (HC)subjects. Each example shown in the left panels is representative of experiments performed in all patients with CD and all HC subjects. Blots were stripped and analyzed for β-actin as an internal loading control. In the right panels, densitometry of IL-17RC and IL-17RB expression normalized for β-actin is shown. Results are mean ± SEM. au, Arbitrary units.
• #33: Levels of cytokines and pro-fibrogenic mediators in tissue explant organ culture supernatants. Levels of interleukin (IL)-17A, IL-17E, IL-6, and tumor necrosis factor (TNF)-α, expressed as pg/ml, and collagen, expressed as μg/ml, in the supernatants of tissue explants from uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of six patients with fibrostenosing Crohn’s disease (CD) and from normal gut of seven control subjects (HC), cultured for 24 hours in the absence of stimuli, and levels of transforming growth factor (TGF)-β1, expressed as relative units compared with the median expression in control subjects (which was assigned the value 1), in the same cultured tissue explants. Values are mean ± SEM. r.u., Relative units.
• #35: Expression of interleukin (IL)-17A and IL-17E receptors on intestinal myofibroblasts. (A) IL-17RC and (B) IL-17RB were detected by immunoblotting on lysates of myofibroblasts isolated from uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of six patients with fibrostenosing Crohn’s disease (CD) and from normal gut of seven healthy control (HC) subjects. Each example shown in the left panels is representative of experiments performed in all patients with CD and all HC subjects. Blots were stripped and analyzed for β-actin as an internal loading control. In the right panels, densitometry of IL-17RC and IL-17RB expression normalized for β-actin is shown. Results are mean ± SEM. au, Arbitrary units.
• #36: Effect of interleukin (IL)-17A and IL-17E on the production of matrix metalloproteinase (MMP)-3, MMP-12, and tissue inhibitor of metalloproteinase (TIMP)-1 by intestinal myofibroblasts. (A) MMP-3, (B) MMP-12, and (C) (TIMP-1 in culture supernatants of myofibroblasts isolated from uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of six patients with fibrostenosing Crohn’s disease (CD) and from normal gut of six healthy control (HC) subjects, cultured for 24 hours with medium alone or recombinant human (rh) tumor necrosis factor (TNF)-α, or rhIL-17A, or rhIL-17E. Blots are representative of experiments performed in all patients with CD and HC subjects. Lower panels show densitometry of western blots. Values are mean ± SEM. *P<0.05 versus myofibroblasts from the same study group cultured with medium alone. §P<0.05 versus Non-strict uninfl CD and HC myofibroblasts cultured under the same conditions.
• #37: Effect of interleukin (IL)-17A and IL-17E on the production of matrix metalloproteinase (MMP)-3, MMP-12, and tissue inhibitor of metalloproteinase (TIMP)-1 by intestinal myofibroblasts. (A) MMP-3, (B) MMP-12, and (C) (TIMP-1 in culture supernatants of myofibroblasts isolated from uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of six patients with fibrostenosing Crohn’s disease (CD) and from normal gut of six healthy control (HC) subjects, cultured for 24 hours with medium alone or recombinant human (rh) tumor necrosis factor (TNF)-α, or rhIL-17A, or rhIL-17E. Blots are representative of experiments performed in all patients with CD and HC subjects. Lower panels show densitometry of western blots. Values are mean ± SEM. *P<0.05 versus myofibroblasts from the same study group cultured with medium alone. §P<0.05 versus Non-strict uninfl CD and HC myofibroblasts cultured under the same conditions.
• #38: Effect of interleukin (IL)-17A and IL-17E on the production of collagen by intestinal myofibroblasts. Levels of collagen, expressed as μg/ml, in the supernatants of myofibroblasts isolated from uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of six patients with fibrostenosing Crohn’s disease (CD) and from normal gut of six healthy control (HC) subjects, cultured for 24 hours with medium alone or recombinant human (rh)tumor necrosis factor (TNF)-α, or rhIL-17A, or rhIL-17E. Values are mean ± SEM. *P<0.05 versus myofibroblasts from the same study group cultured with medium alone. §P<0.05 versus Non-strict uninfl CD and HC myofibroblasts cultured under the same conditions.
• #39: Wound-healing scratch assay. Effect of recombinant human (rh)IL-17A and rhIL-17E on the migration, assessed by an in vitro wound-healing scratch assay, of myofibroblasts isolated from uninflamed areas of strictured (Strict uninfl) and non-strictured (Non-strict uninfl) gut of six patients with fibrostenosing Crohn’s disease (CD) and from normal gut of seven healthy control (HC) subjects. Myofibroblasts were cultured with rhIL-17A or rhIL-17E or medium alone. Results, expressed as percentage of wound repair, are mean ± SEM. *P<0.05 versus myofibroblasts cultured with medium only at 8, 16, and 24 hours.
• #46: Administration with NOD2-activated hUCB-MSCs enhanced the protective effects against DSS-induced colitis in mice. (A–C) Clinical progression in DSS-induced colitic mice was monitored. Numbers of mice were as follows: naive mice, 10–14; PBS mice, 12–20; fibroblast mice, 6–10; MSC mice, 12–20; MSCώMDP mice, 12–20; and MSC-siNOD2ώMDP mice, 12–29. (A) Mantel–Cox analysis of survival rate, (B) percentage of body weight loss, (C) disease activity index for colitis severity, and (D and E) on day 10, animals were killed for further evaluation. (D) Colon length measurement. (E) Histopathologic analysis of colon. Bar, 500 mm. Six mice per group were used. (F) Enlargement of mesenteric lymph nodes was evaluated. Five mice per group were used. *P < .05, **P < .01, ***P < .001. Results are shown as mean SD.
• #47: NOD2-activated hUCB-MSCs reduce colonic inflammation in mice. (A) DSS-induced colitic mice were injected intraperitoneally with hUCB-MSCs and IL-6, IFN-g, TNF-a, and IL-10 levels in colon were determined on day 5 by enzyme-linked immunosorbent assay. (B) Neutrophil infiltration was determined by colonic MPO activity assay. (C and D) Inflammatory T lymphocyte and phagocyte infiltration were measured by counting cells per microscopic field on immunostained colon sections. (C) CD4ώ cell counts. (D) CD11bώ cell counts. (E) Colonic infiltration of CD4ώCD25ώFoxP3ώ cells were determined by flow cytometry on day 5. *P < .05, **P < .01, ***P < .001. Three to 6 mice per group were used. Results are shown as mean SD.
• #48: NOD2 is crucial for the protective ability of hUCB-MSCs against TNBS-induced colitis. (A–C) Gross and histologic observations in TNBS induced colitic mice were performed. Numbers of mice were as follows: naive mice, 7; EtOH mice, 8; PBS mice, 13; fibroblast mice, 15; MSC mice, 18; MSC ώ MDP mice, 18; and MSC-siNOD2 ώ MDP mice, 13. (A) Survival rate and body weight loss. (B) Measurement of colon length. (C) Histopathologic evaluation of colon sections. Five mice per group were used, Bar, 500 mm. (D) NOD2-deficient hUCB-MSCs without MDP stimulation were injected intraperitoneally into colitic mice. Percentage of survival rate and body weight loss were measured. Numbers of mice were as follows: EtOH mice, 8; PBS mice, 13; MSC-siCTL mice, 18; and MSC-siNOD2 mice, 13. (E) Nine days after colitis induction and hUCB-MSC administration, a second dose of TNBS was inoculated and survival rate was analyzed. Numbers of mice were as follows: EtOH mice, 9; PBS mice, 8; MSC mice, 10; and MSCώMDP mice, 10. Numbers in parentheses represent the percentage of mice that were dead. *P < .05, **P < .01, ***P < .001. Results are shown as mean SD.
• #49: MDP enhanced the immunosuppressive effect of hUCB-MSCs. (A) After treatment with ligands, hUCB-MSCs were co-cultured with hUCB-MNCs and MNC proliferation was determined by the bromodeoxyuridine kit. (B and C) hUCB-MNCs were cultured with the culture media of hUCB-MSCs (UCM). hUCB-MNC proliferation was determined by the bromodeoxyuridine kit. UCM 618 and 620; UCM from 618 and 620 hUCBMSCs. (D) Human Jurkat cells and (E) mouse splenocytes were cultured in the presence of UCM and their proliferation was determined. *P < .05, ***P < .001. Results show 1 representative experiment of at least 3. Results are shown as mean SD.
• #50: MDP-induced PGE2 is responsible for the anti-inflammatory activity of hUCB-MSCs. (A and B) hUCB-MSCs were treated with each indicated ligand. (A) The PGE2 concentration was measured from culture supernatant by enzyme-linked immunosorbent assay. (B) Cellular COX-2 expression was determined by Western blot analysis. (C) PGE2 concentration in UCM was detected. (D) hMNCs and (E) mouse splenocytes were cultured at the presence of various doses of PGE2 and their proliferation was determined by the bromodeoxyuridine kit. (F) hUCB-MSCs were treated with MDP alone or MDP ώ indomethacin and UCM was collected. hUCB-MNCs were cultured in the presence of each UCM and MNC proliferation was determined. *P < .05, **P < .01, ***P < .001. Results show 1 representative experiment of at least 3. Results are shown as mean SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
• #51: MDP enhanced the immunosuppressive effect of hUCB-MSCs through a NOD2-RIP2–dependent pathway. (A) hUCB-MSCs were transfected with siRNAs, and then were treated with MDP. Protein levels of COX-2 were examined by Western blot analysis. (B) UCM was harvested from cells treated with MDP after siRNA transfection. PGE2 concentration was measured in UCM by enzyme-linked immunosorbent assay. (C) siRNA-transfected hUCB-MSCs without MDP stimulation were determined for COX-2 expression on the protein level. (D) hUCB-MSCs were treated with siRNA without MDP stimulation, and PGE2 secretion was detected from culture supernatant using an enzyme-linked immunosorbent assay kit. In addition, PGE2 concentration in UCM was measured. (E) hUCB-MNCs were cultured in UCM and IL-10 production was measured in the culture supernatant. (F) hUCB-MNCs cultured in UCM were analyzed for regulatory T-cell population by flow cytometry. Results are 1 representative experiment of 2 or 3 or the cumulative of 3 independent experiments. *P < .05, **P < .01, ***P < .001. Results are shown as mean SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
• #54: NOD2-mediated PGE2 production and ensuing IL-10 induction are crucial for the attenuation of colitis. (A) siRNA for COX-2 was transfected into hUCB-MSCs. Cells were stimulated with MDP and injected intraperitoneally into DSS-induced colitic mice. Survival rate, disease activity index, and histopathologic score were analyzed. Numbers of mice were as follows: naive mice, 10; PBS mice, 12; MSC-siControl (CTL) mice, 12; MSC-siCOX2ώMDP mice, 12; and MSC-siCTLώMDP mice, 10. (B) COX-2–inhibited hUCB-MSCs were administered into TNBS-induced colitic mice and disease progress was monitored. Numbers of mice were as follows: EtOH mice, 8; PBS mice, 15; MSC-siCTLώMDP mice, 20; and MSCsiCOX2ώMDP mice, 10. (C) PGE2 concentration was measured in both serum and colon of hUCB-MSCs transplanted colitic mice at days 3, 5, and 7. (D) IL-10–neutralizing antibody was injected intraperitoneally daily from day 0 to day 5 into hUCB-MSC–administered colitic mice. Survival rate, body weight loss, and histologic score were evaluated. Numbers of mice were as follows: naive mice, 10; PBS mice, 10; MSCώMDP mice, 10; And MSCώ MDPώIL-10 neutralizing antibody (NA) mice, 10. *P < .05, **P < .01, ***P < .001. Results are shown as mean SD.
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