Takahito Yagi

Twist expression promotes migration and invasion in hepatocellular carcinoma.

BackgroundTwist, a transcription factor of the basic helix-loop-helix class, is reported to regulate cancer metastasis. It is known to induce epithelial-mesenchymal transition (EMT). In this study, we evaluated the expression of twist and its effect on cell migration in hepatocellular carcinoma (HCC).MethodsWe examined twist expression using immunohistochemistry in 20 tissue samples of hepatocellular carcinoma, and assessed twist expression in HCC cell lines by RT-PCR and Western blot analysis. Ectopic twist expression was created by introducing a twist construct in the twist-negative HCC cell lines. Endogenous twist expression was blocked by twist siRNA in the twist-positive HCC cell lines. We studied EMT related markers, E-cadherin, Vimentin, and N-cadherin by Western blot analysis. Cell proliferation was measured by MTT assay, and cell migration was measured by in vitro wound healing assay. We used immunofluorescent vinculin staining to visualize focal adhesion.ResultsWe detected strong and intermediate twist expression in 7 of 20 tumor samples, and no significant twist expression was found in the tumor-free resection margins. In addition, we detected twist expression in HLE, HLF, and SK-Hep1 cells, but not in PLC/RPF/5, HepG2, and Huh7 cells. Ectopic twist-expressing cells demonstrated enhanced cell motility, but twist expression did not affect cell proliferation. Twist expression induced epithelial-mesenchymal transition together with related morphologic changes. Focal adhesion contact was reduced significantly in ectopic twist-expressing cells. Twist-siRNA-treated HLE, HLF, and SK-Hep1 cells demonstrated a reduction in cell migration by 50, 40 and 18%, respectively.ConclusionTwist induces migratory effect on hepatocellular carcinoma by causing epithelial-mesenchymal transition.

Elevation of serum interleukin-18 levels and activation of kupffer cells in biliary atresia

BACKGROUND/PURPOSE Interleukin-18 (IL-18)/interferon-gamma-inducing factor (IGIF) is a novel proinflammatory cytokine that can induce interferon gamma (IFN-gamma). In addition, IL-18 enhances intracellular adhesion molecule-1 (ICAM-1) expression as well as Fas ligand (FasL) expression, and induces apoptosis in hepatic injury. The aim of this study was to clarify the potential role of IL-18 in the pathogenesis of the progressive inflammation and fibrosis in biliary atresia (BA). METHODS Six children with BA before hepatic portoenterostomy (HPE), 13 with BA including 7 without jaundice and 6 with persistent jaundice after HPE, and 16 healthy controls were examined. Blood samples were obtained preoperatively from 6 patients, after HPE from 13, and after liver transplantation from 4. The IL-18 level was determined by an enzyme-linked immunosorbent assay (ELISA). Immunohistochemically, liver specimens from BA patients were studied using a monoclonal antibody to macrophage-associated antigen (CD68). RESULTS IL-18 levels were elevated in the patients before HPE compared with those of the controls (349+/-54 pg/mL v. 138+/-13 pg/mL, P<.0001). After HPE, extremely high concentrations of IL-18 were observed in patients with persistent jaundice (532+/-95 pg/mL, P<.0001), and the IL-18 levels were significantly high even in the patients without jaundice (249+/-29 pg/mL, P<0.005). The high IL-18 level lasted for a long time even in the patients without jaundice after HPE. In contrast, the IL-18 levels immediately decreased after liver transplantation. Immunohistochemically, the number of CD68-positive Kupffer cells was significantly higher, and the size was larger in the livers of the patients than in the controls. The proliferation of CD68-positive cells was much more conspicuous in the liver specimens obtained during liver transplantation than in those at the time of HPE. CONCLUSIONS Our findings showed elevation of serum IL-18 levels and activation of Kupffer cells in BA. IL-18 released from activated Kupffer cells might play an important role in the pathophysiology of the progressive inflammation and fibrosis in BA. Furthermore, IL-18 level may be related to the prognosis in patients with BA.

Effects of prophylactic splenic artery modulation on portal overperfusion and liver regeneration in small-for-size graft.

Background. The small-for-size (SFS) syndrome is caused by excessive portal inflow into a small-sized liver graft. Various approaches for portal decompression have been used, but details of their impact on liver regeneration in SFS graft remain unclear. We examined the effect of prophylactic splenic artery modulation (SAM). Methods. We conducted a retrospective cohort study. The study group was 39 consecutive adult-to-adult living liver transplantation recipients, with a graft-to-recipient body weight ratio of less than 0.8. Patients were assigned into the non-SAM group (n=18, without any portal inflow attenuation) or SAM group (n=21, preoperative embolization in 15 patients and intraoperative ligation in 6 patients). Hepatic hemodynamics, graft function, liver regeneration, and outcome were evaluated. Results. In the SAM group, the excessive portal flow was significantly reduced (P<0.01) and the effect of embolization on portal decompression was equivalent to that of ligation. In the acute postoperative phase, serum transaminases, interleukin-6, and tumor necrosis factor-&agr;, were lower in the SAM group than in non-SAM group. In both groups, a negative correlation was observed between graft-to-recipient body weight ratio and liver regeneration rate at 2 weeks after living donor liver transplantation. Splenic artery modulation was advantageous for liver regeneration, and significantly improved clinical features, hyperbilirubinemia, and prolonged ascites. Small-for-size syndrome occurred in five patients of the non-SAM group, and only one of SAM group (P=0.038). Conclusion. In SFS graft with severe portal hypertension, prophylactic splenic embolization/ligation seems to relieve portal overperfusion injury and contributes in improvement of posttransplantation prognosis through liver regeneration.

Serum interferon-gamma-inducing factor/IL-18 levels in primary biliary cirrhosis

Primary biliary cirrhosis is an autoimmune disease of the liver in which T helper 1 cytokines predominate over those of T helper 2 in the pathogenesis. Interleukin‐18 (IL‐18), for which the gene was recently cloned, is a novel T helper 1 cytokine, which augments interferon‐gamma production. We designed this study to clarify the role of IL‐18 in primary biliary cirrhosis and to examine whether serum IL‐18 level can be a prognostic indicator for the disease. Serum IL‐18 levels were measured using an enzyme linked immuno sorbent assay with mouse monoclonal antibodies. Twenty‐two healthy volunteers, 31 patients with primary biliary cirrhosis (Scheuers stage I, 13; II, 10; and IV, 8), 20 patients with autoimmune hepatitis, 11 patients with virus‐related liver cirrhosis and six patients with obstructive jaundice were enrolled. Significant differences of serum IL‐18 levels were observed between patients with Scheuers stage IV and those with stage I, or II, virus‐related liver cirrhosis and obstructive jaundice (P < 0·05). The IL‐18 levels in primary biliary cirrhosis increased according to the disease progression, and fell promptly after living‐related liver transplantation. Moreover, serum IL‐18 levels in primary biliary cirrhosis were correlated with serum bilirubin concentrations and the Risk scores of the Mayo Clinic prognostic model for the disease. The IL‐18 levels observed in patients with autoimmune hepatitis were also elevated, and correlated with the activity of the disease. These results indicate that serum interleukin‐18 levels reflect the severity of primary biliary cirrhosis, the activity of autoimmune hepatitis, and may be an additive prognostic indicator in primary biliary cirrhosis.

Long-term outcomes of endoscopic management for biliary strictures after living donor liver transplantation with duct-to-duct reconstruction

Biliary strictures after living donor liver transplantation (LDLT) with duct‐to‐duct (D‐D) reconstruction are associated with postoperative morbidity and mortality. The aims of this study were to evaluate the long‐term outcomes of endoscopic deployment of plastic stents, and to investigate factors associated with the stent deployment failure. Between April 2001 and May 2007, 96 patients received LDLT with D‐D reconstruction at Okayama University Hospital. Among them, 41 patients (43%) had anastomotic biliary strictures, and all were referred first for endoscopic retrograde cholangiography (ERC). When deployment was unsuccessful, a percutaneous transhepatic procedure was employed. Successful stent deployment was achieved in 35 out of total 41 patients (85%) by both procedures. Among the 35 patients, 28 had their stents removed as a result of strictures resolution. Eight patients underwent ERC and repeated stent deployment as a result of recurrence of the strictures. Finally, 21 out of 41 (51%) patients with biliary stricture were completely treated by endoscopic therapy during the observation period (median 873 days: range 77–2060). By multivariate analysis, biliary leakage was associated with stent deployment failure. Endoscopic deployment of plastic stents is a first‐line therapy for patients with biliary stricture after LDLT.

Restored expression of the tumor suppressor gene RUNX3 reduces cancer stem cells in hepatocellular carcinoma by suppressing Jagged1-Notch signaling

Runt-related transcription factor 3 (RUNX3) is a candidate tumor suppressor gene that is downregulated in various cancers. In the present study, we analyzed the regulatory function of RUNX3 on Jagged-1 (JAG1) expression and cancer stem cell (CSC) signaling in hepatocellular carcinoma (HCC). Eleven HCC cell lines and 30 human HCC tissues were used. RUNX3 and JAG1 expression levels were analyzed by immunoblotting and immunohistochemistry. Ectopic RUNX3 expression was induced by introducing RUNX3 cDNA into the RUNX3-negative HCC cell line Hep3B and Huh7 cells. Furthermore endogenous RUNX3 expression was knocked down by RUNX3 siRNA in SK-Hep-1 cells. In order to analyze JAG1 transcriptional regulation, we conducted reporter assays, chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSAs). Tumorigenicity was analyzed using a SCID mouse liver injection model. An inverse correlation was observed between RUNX3 expression and JAG1 expression in most HCC cell lines and tissues. Restoring RUNX3 expression decreased the expression of JAG1 in Hep3B and Huh7 cells, whereas JAG1 expression was upregulated in RUNX3 siRNA-treated SK-Hep-1 cells. Reporter assays, ChIP assays and EMSAs revealed that RUNX3 directly bound to the transcriptional regulatory region of JAG1 and suppressed JAG1 transcription. Moreover, RUNX3 restoration downregulated CSCs by suppressing JAG1-mediated Notch signaling. The tumorigenic capacity of RUNX3-expressing Hep3B cells was lower compared to that of control Hep3B cells. RUNX3 expression suppressed JAG1 expression and resulted in downregulation of tumorigenesis by suppression of JAG1-mediated CSCs.

Risk factors for acute renal injury in living donor liver transplantation: evaluation of the RIFLE criteria.

Acute renal injury (ARI) is a serious complication after liver transplantation. This study investigated the usefulness of the RIFLE criteria in living donor liver transplantation (LDLT) and the prognostic impact of ARI after LDLT. We analyzed 200 consecutive adult LDLT patients, categorized as risk (R), injury (I), or failure (F), according to the RIFLE criteria. ARI occurred in 60.5% of patients: R‐class, 23.5%; I‐class, 21%; and F‐class, 16%. Four patients in Group‐A (normal renal function and R‐class) and 26 patients in Group‐B (severe ARI: I‐ and F‐class) required renal replacement therapy (P < 0.001). Mild ARI did not affect postoperative prognosis regarding hospital mortality rate in Group A (3.2%), which was superior to that in Group B (15.8%; P = 0.0015). Fourteen patients in Group B developed chronic kidney disease (KDIGO stage 3/4). The 1‐, 5‐ and 10‐year survival rates were 96.7%, 90.6%, and 88.1% for Group A and 71.1%, 65.9%, and 59.3% for Group B, respectively (P < 0.0001). Multivariate analysis revealed risk factors for severe ARI as MELD ≥20 [odds ratio (OR) 2.9], small‐for‐size graft (GW/RBW <0.7%; OR 3.1), blood loss/body weight >55 ml/kg (OR 3.7), overexposure to calcineurin inhibitor (OR 2.5), and preoperative diabetes mellitus (OR 3.2). The RIFLE criteria offer a useful predictive tool after LDLT. Severe ARI, defined beyond class‐I, could have negative prognostic impact in the acute and late postoperative phases. Perioperative treatment strategies should be designed and balanced based on the risk factors for the further improvement of transplant prognosis.

Short-term high-dose followed by long-term low-dose hepatitis B immunoglobulin and lamivudine therapy prevented recurrent hepatitis B after liver transplantation.

Hepatitis B immunoglobulin (HBIg) and lamivudine combination has been accepted as the best way to control hepatitis B recurrence after liver transplantation. However, the optimal dose of HBIg and the target titer of hepatitis B surface antibody (HBsAb) remain unclear. We report our satisfactory experience with high-dose HBIg in the early period followed by low-dose HBIg with lamivudine. Subjects comprised five patients with fulminant hepatitis (FH) and 18 patients with liver cirrhosis (LC) who underwent liver transplantation. HBIg at a dosage of 200 IU/kg per day was administered for one week postoperatively. Thereafter, HBIg was administered only for HBsAb titer <100 IU/L. After six months, HBIg was withdrawn in FH and administered in LC only for HBsAb titer <10 IU/L. Lamivudine was administered to two FH and all LC cases. Although two patients with LC showed transient hepatitis B surface antigen (HBsAg) recurrence, all patients remained HBsAg-negative at the final follow-up date. This method allows reliable and cost-effective control of hepatitis B recurrence.

Preoperative proximal splenic artery embolization: a safe and efficacious portal decompression technique that improves the outcome of live donor liver transplantation.

Terminal liver cirrhosis is associated with marked severe portal hypertension, which increases the risk of intraoperative hemorrhage and graft hyper‐perfusion, especially, in small‐for‐size graft. In cases with developed collateral vessels, we often face difficulties in perihepatic dissection with blood stanching against bleeding during recipient hepatectomy. For aseptic preoperative portal decompression, we established the proximal splenic artery embolization (PSAE) technique. Sixty adult living donor liver transplantation recipients with viral/alcoholic hepatic failure were divided into two groups; PSAE group (n = 30) and non‐PSAE (n = 30). In the PSAE group, the splenic artery was embolized proximal to the splenic hilum 12–18 h before surgery. PSAE enabled shortening of operating time, reduced blood loss, led to less need for transfusion, and significantly reduced the post‐transplant portal venous velocity and ascites. PSAE was not associated with complications, e.g., splenic infarction, abscess, or portal thrombosis. Six of the non‐PSAE patients required additional surgical intervention to resolve postoperative hemorrhage and three patients required secondary PSAE for arterial‐steal‐syndrome. The hospital mortality rate of PSAE patients (3.3%) was significantly better than that of the PSAE group (13.3%, P < 0.05). Preoperative noninvasive PSAE makes more efficient use of portal decompression; thus, it can potentially contribute to improvement of outcome.

Bacterial Lipopolysaccharide Induces Transforming Growth Factor β and Hepatocyte Growth Factor through Tolllike Receptor 2 in Cultured Human Colon Cancer Cells

This study examined, in human cancer cell lines, the pattern of cytokine production stimulated by lipopolysaccharide (LPS), a major component of the outer surface of Gram-negative bacteria, and characterized the expression pattern of CD14, cell surface LPS receptor antigen, and toll-like receptors (TLRs), which appear to be key regulators of the innate immune response system. Two colon cancer cell lines (DLD and LoVo), a hepatocellular carcinoma cell line and a myelomonocytic cell line were incubated with LPS for 0–72 h, and transforming growth factor (TGF) β1 and β2, hepatocyte growth factor (HGF) and interleukins 6, 8 and 15 were assayed. The only changes induced by incubation with LPS were significant increases in TGFβ1 production at 12 h, and in HGF production at 72 h, in LPS-stimulated DLD cells, and significant increases in TGFβ2 production after 12 h and in HGF after 72 h in LoVo cells. Using reverse transcriptase-polymerase chain reaction analysis, expression of CD14 and TLR-2 mRNA was detected in DLD and LoVo cells, and expression of TLR-4 mRNA was detected in PLC/PRF/5 and KG-1 cells. These results suggest that LPS induces TGFβ and HGF production mediated by CD14/TLR-2 in cultured human colon cancer cell lines.