Partial hepatectomy Cyclopamine order was performed as described.19 Paraffin-embedded liver sections

(4 μm thick) were used for immunohistochemical staining. Antigen retrieval was achieved by heating the slides in a microwave at high power in 1× citrate buffer for 10 minutes. The tissue sections were blocked in blue blocker for 20 minutes followed by incubation with primary antibody overnight at 4°C. The primary antibody was then linked to biotinylated secondary antibody followed by routine avidin-biotin complex method. Diaminobenzidine was used as the chromogen, which resulted in a brown reaction product. Primary antibodies used were as follows: NRSF (Millipore 07-579; 1:1,000), Oct4 (ab27985; 1:250), KLF4 (SC-20691; 1:500), Nanog (ab80892; find more 1:700), and Myc (ab32072; 1:100). Data are expressed as means ± standard error (SE). Statistical differences were determined by one-way

analysis of variance (ANOVA) followed by Tukey’s honest significant difference test to determine which means were significantly different from each other or from controls using the JMP software (SAS Institute, Cary, NC). The criterion for significance was P < 0.05. We report that primary hepatocytes do express REST, Oct4, cMyc, Klf4, and Nanog in culture (Figs. 1, 2). Their expression is up-regulated with time in culture. However, this up-regulation is further enhanced in cultures incubated with growth factors (Figs. 1, 2). REST and Klf4 message showed a steady increase with time in both the groups peaking at day 10 in culture (Fig. 1A,D). Oct4, cMyc, and Nanog message showed earlier peaks at 4, 2, and 8 days, respectively (Figs. 1B,C,E, 2). Previous studies have shown maximum proliferation of dedifferentiated hepatocytes when plated in culture Casein kinase 1 with GF between days 4 and 10 by bromodeoxyuridine labeling and tritiated thymidine incorporation.15 Expression of REST, Oct4, Nanog,

Myc, and Klf4 protein was maximally induced when there was maximum proliferation in culture (days 4 to 10), suggesting their role in GF-mediated proliferation of hepatocytes in culture. To test if expression of REST and reprogramming factors is dependent on the differentiation status of cells, Matrigel was used to induce hepatocyte differentiation as described. Hepatocytes were incubated as follows: with Matrigel with GF (+MT G+GF), with Matrigel without GF (+MTG−GF), without Matrigel with GF (−MTG+GF), or without Matrigel without GF (−MTG−GF). Matrigel-induced differentiation down-regulated the growth factor induced expression of REST, Klf4, cMyc, and Oct4 message (Fig. 3A-D). The group without Matrigel and with growth factors (−MTG+GF) showed maximum expression of REST, Klf4, and cMyc, whereas the group with Matrigel and without growth factors (+MTG−GF) showed the least expression. The other two groups lay in between (Fig. 3). Albumin (Fig.

1B, left); this indicates that HSCs isolated from a healthy liver have an epithelial phenotype. After cultivation, the expression level of ECAD decreased, whereas the levels of the transdifferentiation markers (NCAD, αSMA, and vimentin) increased. Moreover, NCAD expression was prominent in LX-2 cells (an activated Maraviroc mw HSC cell line), and

there were increases in the levels of αSMA and vimentin (Fig. 1B, right). As expected, MEF cells (a fibroblast cell line) also showed increased expression of NCAD, αSMA, and vimentin. We assessed the expression of TGFβ target genes regulating EMT in HSCs on days 0 and 12 (ECAD expression was distinctly different at these times). The messenger RNA (mRNA) levels of bone morphogenetic protein 7 (Bmp7) and inhibitor of DNA binding 2 (Id2) markedly decreased on day 12, but the levels of Snail, Twist, and PAI-1 increased (Fig. 1C); this suggests that ECAD loss in activated HSCs may promote EMT by inducing TGFβ target genes. Ectopic expression of ECAD prevents cell transformation as well as tumor cell invasion in an adhesion-independent manner.3, 4 Next, the effects of ECAD HIF-1 cancer overexpression on the levels of NCAD, αSMA, and vimentin were examined in primary cultured HSCs that had been

activated (Fig. 1D, left). Forced expression of ECAD repressed the expression levels of NCAD, αSMA, and vimentin in HSCs. Consistently, ECAD overexpression resulted in similar changes in both MEFs and LX-2 cells (Fig. 1D, middle and right). Moreover, forced expression of ECAD increased negative regulators of EMT, Bmp7, and Id2 but reciprocally decreased positive regulators of EMT, Snail, and Twist (Fig. 1E). These results indicate that the expression

of ECAD alters the activation status of HSCs. TGFβ1 stimulates HSC activation and liver fibrosis.6, 13 Therefore, the effect of forced expression of ECAD on the TGFβ1 gene was monitored in subsequent experiments. Overexpression of ECAD elicited a significant decrease in luciferase activity from a TGFβ1 promoter construct in MEF cells, but this was not observed in cells transfected with NCAD (Fig. 2A, left). Instead, TGFβ1 luciferase activity was moderately increased by NCAD overexpression. A decrease in the basal TGFβ1 expression by ECAD was also Selleck MG132 confirmed in LX-2 cells and HSCs (Fig. 2A, middle and right). Real-time PCR analysis verified the ability of ECAD to repress the TGFβ1 gene (Fig. 2B). TGFβ1 promotes the remodeling and deposition of ECM through the activation of downstream target genes such as PAI-1 and MMPs.6, 11, 12 In agreement with the repression of TGFβ1, the basal luciferase activities of PAI-1, MMP2, and MMP9 were all inhibited by ECAD overexpression in both MEFs and LX-2 cells (Fig. 2C). As expected, ECAD expression decreased the transcript levels of PAI-1, MMP2, MMP9, collagen type IA (COL1A), and αSMA (Fig. 2D).

Methods: The medical records of patients with see more active UC who were initially treated with the time-dependent release formulation of mesalamine (4.0 g/day)

and were later switched to the pH-dependent release formulation (3.6 g/day) because of inadequate response to the former were retrospectively analyzed. All patients were treated at the IBD Center, Sapporo Kosei General Hospital from January 2010 to June 2010. The efficacy of the pH-dependent release formulation was evaluated on the basis of the decrease in Lichtiger’s Clinical Activity Index (CAI) score, which was calculated at baseline, 4 weeks and 8 weeks after treatment initiation, respectively. Remission and response were confirmed by a decrease in CAI score of ≤4 and ≥3 points, respectively. Results: Of the 22 patients (mean age, 37.2 years), 11 were females. The mean duration of disease was 9.4 years and the mean baseline CAI score was 7.9. Eight selleck products patients had total colitis, 11 had left-sided colitis, and 3 had proctitis-type colitis. Concomitant treatment with azathioprine and local mesalamine was administered to 9 and 9 subjects, respectively. CAI scores at 4 weeks after treatment initiation significantly decreased to 5.0 (P < 0.001). The response rate at 4 weeks was 54.5%, while the remission rate at 4 weeks and 8 weeks was

45.5% and 68.2%, respectively. Conclusion: The pH-dependent release formulation of mesalamine (3.6 g/day) can prove effective in patients with UC that fails to respond adequately to the MRIP time-dependent

formulation (4.0 g/day). Key Word(s): 1. ulcerative colitis; 2. mesalamine; Presenting Author: SATOSHI MOTOYA Additional Authors: MASAKI YAMASHITA, MASANAO NASUNO, MANABU ISHII, HIROKI TANAKA, AKIMICHI IMAMURA Corresponding Author: HIROKI TANAKA Affiliations: IBD Center, Sapporo Kosei General Hospital Objective: Infliximab (IFX) has been established as a useful treatment option for patients diagnosed with refractory ulcerative colitis (UC). However, the details of IFX treatment in Japanese patients with refractory UC remain unclear. We analyzed the long-term outcomes of IFX treatment for refractory UC and the related prognostic factors in a Japanese cohort. Methods: We retrospectively analyzed the medical records of 77 patients with refractory UC who received IFX treatment at the IBD Center, Sapporo Kosei General Hospital from July 2005 to January 2012. The cumulative colectomy rate following IFX administration was estimated using the Kaplan–Meier method. The background factors that influenced the cumulative colectomy rate were evaluated using multivariate Cox regression analysis. Results: Of the 77 patients (mean age, 36.2 years), 35 were females. The mean duration of disease was 5.7 years, the mean C-reactive protein level was 1.2 mg/dl, and the mean clinical activity index (Lichtiger index) score was 9.5 at baseline.