Yasuhiro Fudaba

Biliary Complications after Duct-to-duct Biliary Reconstruction in Living-donor Liver Transplantation: Causes and Treatment

BackgroundIn living-donor liver transplantation (LDLT), biliary complications are recognized as a significant cause of post-transplantation morbidity.MethodsEighty patients who underwent LDLT with duct-to-duct biliary reconstruction at Hiroshima University Hospital were enrolled in this study. The mean follow-up was 24 months (range, 3–72 months). Eighteen patients underwent the basiliximab-based immunosuppressive therapy, and 62 patients underwent non-basiliximab-based immunosuppressive therapy. The development of biliary complications after LDLT was retrospectively analyzed. Biliary complications were initially treated by endoscopic or radiological modalities.ResultsBiliary leakages and strictures occurred in 12 (15%) and 20 (25%) of the 80 patients, respectively. Stepwise multivariate analysis demonstrated bile leakage to be an independent risk factor for the development of biliary stricture (p = 0.001) and basiliximab-based immunosuppressive therapy to be an independent protective factor for postoperative biliary leakage (p = 0.005). The 1-week total doses of steroids were significantly lower in the basiliximab-based immunosuppressive regimes (mean dose: 573mg) than in the non-basiliximab-based ones (mean dose: 1,121mg) (p = 0.01). All patients with biliary leakage were successfully treated with endoscopic or radiological modalities, except one patient who was treated by surgical treatment. Endoscopic or radiological modalities were successful as primary treatment modalities in 12 (60%) of 20 patients with biliary strictures. Lastly, six patients were treated surgically with long-term success, except for one patient with chronic cholangitis who died after 16 months.ConclusionsSteroid-sparing basiliximab-based immunosuppressive therapy reduced the incidence of biliary leakage, and biliary leakage was the independent factor for biliary stricture. The non-surgical and surgical treatments for biliary complications were satisfactory.

Geranylgeranylacetone, a heat shock protein inducer, prevents primary graft nonfunction in rat liver transplantation.

Background. Heat shock proteins (HSPs) are well known as cytoprotective proteins. Geranylgeranylacetone (GGA), a nontoxic anti-ulcer drug, was recently shown to have HSP-inducing capacity. In the present study, the activity of GGA was tested in a rat orthotopic liver transplantation (OLT) model to determine whether the compound has beneficial effects in warm ischemia-reperfusion injury. Methods. Either GGA or a control vehicle was orally administered to donor rats before graft harvest. Harvested livers were subjected to 45-min warm ischemia (37°C) followed by OLT. HSP mRNA expressions and HSP syntheses in the graft livers were evaluated by reverse transcriptase polymerase chain reaction and Western blot analysis, respectively. Results. When the donors were treated with a vehicle, all recipients died of primary nonfunction within 2 days after OLT. In contrast, when the donors were treated with GGA (200 mg/kg per day) for 4 weeks, the 7-day survival rate of recipients was dramatically improved (90%). By giving a high dose of GGA (600 mg/kg per day) for 1 week, a similar improvement in recipient survival was seen (83.3%). GGA administration accumulated mRNA for both HSP72 and HSP90 in the livers even before warm ischemia and facilitated the syntheses of HSP72 and HSP90 after warm ischemia. In addition, GGA pretreatment also significantly reduced the serum levels of tumor necrosis factor-&agr; (TNF-&agr;) after reperfusion. Conclusions. These findings indicate that both the enhanced induction of HSPs and the suppression of a cytotoxic mediator (TNF-&agr;) might be involved in the beneficial effects of GGA on ischemia-reperfusion injury. Thus, oral administration of GGA would be a useful tool for preventing primary nonfunction in liver transplantation.

Rho-associated kinase inhibitor reduces tumor recurrence after liver transplantation in a rat hepatoma model.

Tumor recurrence after liver transplantation still remains a significant problem in patients with hepatocellular carcinoma. The small GTPase Rho/Rho‐associated kinase (ROCK) pathway is involved in the motility and invasiveness of cancer cells. We investigated whether tacrolimus activated the Rho/ROCK signal pathway to promote the invasiveness of rat hepatocellular carcinoma cells. We also investigated whether the ROCK inhibitor Y‐27632 suppressed tumor recurrence after experimental liver transplantation in a rat hepatocellular carcinoma model. Orthotopic liver transplantation was performed in hepatocellular carcinoma cell line McA‐RH7777‐bearing rats. Tacrolimus was administered to liver transplant rats and these rats were divided into two groups: the Y‐27632‐treated (10 mg/kg, for 28 days) group and the Y‐27632‐untreated group. Tacrolimus enhanced the cancer cell migration and stimulated phosphorylation of the myosin light chain (MLC), a downstream effector of Rho/ROCK signaling. Y‐27632 suppressed the cancer cell migration and tacrolimus‐induced MLC phosphorylation. Suppression of tumor recurrence after liver transplantation and significant prolongation of survival were observed in the Y‐27632‐treated rats in comparison with theY‐27632‐untreated rats. Tacrolimus stimulates the Rho/ROCK signal pathway to enhance the invasiveness of hepatocellular carcinoma, and the ROCK inhibitor Y‐27632 can be used as a new antimetastatic agent for the prevention of tumor recurrence after liver transplantation.

Prolongation of liver allograft survival after interleukin-10 gene transduction 24-48 hours before donation.

BACKGROUND Interleukin- (IL) 10 may be a potent regulator for controlling of allograft rejection. A single administration of IL-10 is not effective for controlling graft rejection. Gene transfer is an attractive vehicle for prolonging the expression of short-lived proteins. METHODS Donor or recipient livers were transduced with 1 x 10(10) p.f.u. of replication-deficient adenovirus vectors harboring human IL-10 cDNA (AdCMVhIL-10) via the ileocecal vein before or after rat orthotopic liver transplantation. RESULTS DA allografts given AdCMVhIL-10 24-48 hr before donation survived for more than 56 days in Lewis recipients, although DA allografts given the adenovirus vector 7 days or 6 hr before, and 3 days after transplantation were rejected within 30 days in recipients. Serum levels of human IL-10 in gene-transferred rats were maximum from day 2 to 7. The serum level of human IL-10 then decreased gradually, and human IL-10 was not detected by ELISA 30 days after gene-transduction. In gene-transduced long-term surviving liver allografts, IL-10 was expressed, and the expression of IL-4 was also up-regulated on posttransplant day 3, despite the expression of Th1 cytokines (IL-2 and interferon-gamma), although in rejected liver allografts, IL-2 and interferon-gamma were expressed without expression of IL-4 and IL-10. CONCLUSIONS The prolongation of survival of IL-10 cDNA transferred liver allografts might be due to inhibition of the early phase of alloimmune-response by over expression of IL-10, despite the expression of IL-2 and interferon-gamma.

Prevention of ischemia-reperfusion-induced hepatic microcirculatory disruption by inhibiting stellate cell contraction using rock inhibitor.

Background. We demonstrated that hepatic stellate cells (HSCs) isolated from rat livers exposed to warm ischemia are significantly contractile when compared with HSCs from intact rat livers. This suggests that ischemia-reperfusion (IR)-induced impairment of sinusoidal microcirculation results, at least in part, from contraction of HSCs. Methods. Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) is one of the key regulators of HSCs motility. Therefore we investigated whether Y-27632, a p160ROCK-specific inhibitor, has beneficial effects on warm IR injury in an in vivo rat partial liver IR model and a rat orthotopic liver transplantation model. Results. After reperfusion following 90 min of warm ischemia, livers in untreated control rats had persistent congestion and impaired mitochondrial respiration, as demonstrated by increasing deoxy-hemoglobin and reduced cytochrome oxidase contents in the hepatic tissues using in vivo near-infrared spectroscopy. Serum levels of transaminase and endothelin (ET)-1 in these rats were markedly increased 1 hr after reperfusion. In contrast, when Y-27632 (3–30 mg/kg) was administered orally, hepatic tissue contents of deoxy-hemoglobin and cytochrome oxidase rapidly normalized. In such animals, the elevation of serum transaminase levels, but not that of ET-1 levels, was significantly suppressed. This is consistent with in vitro data demonstrating that Y-27632 causes HSCs to undergo relaxation even in the presence of ET-1. Moreover, in a rat orthotopic liver transplantation model, Y-27632 pretreatment dramatically improved the survival of recipients with liver grafts subjected to 45 min of warm ischemia. Conclusions. Y-27632 attenuates IR-induced hepatic microcirculation disruption by inhibiting contraction of HSCs.

Hepatocyte growth factor prevents chronic allograft dysfunction in liver-transplanted rats.

Background. Hepatocyte growth factor (HGF) is a growth factor with multiple biologic properties, including mitogenic, morphogenic, anti-apoptotic, and antifibrogenic activities. Long-term administration of the deletion variant of HGF (dHGF) might contribute to the prevention of chronic liver allograft dysfunction, which is attributed to immunologic and nonimmunologic reactions. Methods. Low-dose tacrolimus was administered to rat-liver recipients after transplantation. Effects of dHGF on transplanted livers treated with low-dose tacrolimus were investigated. Results. Rats receiving liver transplants treated with only low-dose tacrolimus administration showed chronic allograft dysfunction. Treatment with dHGF prolonged the survival time of rats that received liver allografts and suppressed fibrosis of liver allograft. Treatment with dHGF also suppressed the expression levels of interleukin (IL)-1&bgr;, caspase-1, and transforming growth factor (TGF)-&bgr; mRNAs in liver allografts. Conclusions. The findings indicate that dHGF may prevent chronic liver-allograft dysfunction and thus may become a novel treatment for chronic liver-allograft dysfunction.

Influence of Gastric pH Modifiers on Development of Intestinal Metaplasia Induced by X‐Irradiation in Rats

The influence of gastric pH on intestinal metaplasia was examined in male Crj:CD(SD) rats. At the age of 5 weeks, animals were irradiated with two 10 Gy doses of X‐rays to the gastric region at a 3‐day interval (total 20 Gy), and 6 months after irradiation, received either secretin or histamine in silicon tubes for 2 months or had their bilateral submandibular salivary glands removed. The incidences of intestinal metaplasia in the fundus of animals after administration of secretin or histamine, or removal of the salivary glands were reduced, along with the pH values, as compared with values for rats given X‐rays alone. In both the pyloric and the fundic gland mucosae, the numbers of alkaline phosphatase (ALP)‐positive foci and type B metaplasias (intestinal crypts without Paneth cells) were also significantly decreased (P<0.01). In a second experiment, started six months after irradiation, rats were kept on 1% sodium chloride (NaCl) diet for 6 months. Subsequent removal of salivary glands along with histamine treatment brought about a marked drop in pH and in numbers of ALP‐positive foci after three and five days. The present results thus indicated that development and maintenance of intestinal metaplasia can be influenced by a decrease of pH value.

Near‐Infrared Spectroscopic Analysis of Hemodynamics and Mitochondrial Redox in Right Lobe Grafts in Living‐Donor Liver Transplantation

Near‐infrared spectroscopy (NIRS), which enables non‐destructive evaluation of hemoglobin (Hb) oxygenation and the redox state of cytochromeoxidase (Cyt.aa3) in living tissues, has been employed during surgery to detect possible impairment of hemodynamics and mitochondrial respiration in the anterior segment of a right lobe liver graft in living‐donor liver transplantation (LDLT). Thirty‐six patients undergoing LDLT using a right lobe graft without the middle hepatic vein (MHV) were enrolled in this study. During the course of harvesting and implantation, NIRS measurements were performed on the anterior segments of the liver grafts. In two recipients of liver grafts with Hb residue over 70% in the anterior segment after ex vivo flushing, the MHV tributary was reconstructed, while it was not reconstructed in the other 34 recipients. Of those 34 recipients, 16 recipients of liver graft with 40–70% Hb residue showed transient increase of transaminase levels after LDLT. Of those 16 recipients, six recipients who showed reduction in oxidized Cyt.aa3 in the anterior segment suffered from persistent hyperbilirubinemia after LDLT. In patients showing impairment of mitochondrial redox associated with congestion caused by deprivation of the MHV tributaries, 
 reconstruction of the MHV tributaries might have a beneficial effect.

Comparison of Human T Cell Repertoire Generated in Xenogeneic Porcine and Human Thymus Grafts

Background. Xenogeneic thymus transplantation is an effective approach to achieving T cell tolerance across highly disparate xenogeneic species barriers. We have previously demonstrated that phenotypically normal, specifically tolerant human T cells are generated in porcine thymic grafts. In this study, we assessed the diversity of the human T cell repertoire generated in porcine thymic xenografts. We also examined the ability of porcine thymus grafts to coexist with human thymus grafts. Methods. Fetal swine (SW) or human (HU) thymus with human fetal liver fragments were transplanted under the kidney capsule of 3Gy irradiated NOD/SCID mouse recipients. Thymus tissue was harvested approximately 16 weeks posttransplant for analysis of mixed lymphocyte reactions and spectratyping of human CD4 and CD8 single positive thymocytes. Results. T cell receptor &bgr; genes of human CD4 and CD8 single positive cells developing in HU and SW thymus grafts showed similar, normal CDR3 length distributions. Human T cells developing in SW thymus grafts showed specific unresponsiveness to the major histocompatibility complex of the donor swine in mixed leukocyte reaction assays. In two of three animals receiving SW and HU thymus grafts under opposite kidney capsules, both grafts functioned. In animals with surviving SW grafts, thymocytes from the SW but not the HU grafts showed specific unresponsiveness to the SW donor. Conclusion. Swine thymus grafts support generation of human T cells with a diverse T cell receptor repertoire. Human thymocytes in human thymus grafts are not tolerized by the presence of an additional porcine thymus, but tolerance might be achieved by postthymic encounter with porcine antigens.

Should splenectomy be performed for hepatitis C patients undergoing living‐donor liver transplantation?

Liver cirrhosis associated with hepatitis C virus (HCV) infection has become the most common indication for liver transplantation; however, the recurrence of HCV infection is an almost universal event after liver transplantation. As a result, graft and patient survival are significantly poorer in HCV-positive recipients than in HCV-negative recipients. Although various strategies including a combination therapy of interferon and ribavirin have been used to overcome the recurrence of HCV infection after liver transplantation, the therapeutic outcome is still poor with 10–40% sustained viral response (SVR) because of adverse effects such as cytopenia. In order to overcome this problem, Kishi and colleagues performed splenectomy concurrently with liver transplantation for all HCV-positive liver transplant patients; however, the indication for splenectomy still remains controversial. Therefore, we investigated whether thrombocytopenia acts as an obstacle to the continuation of interferon therapy after living-donor liver transplantation (LDLT); we further clarified the indications for simultaneous splenectomy with LDLT. From January 2000 to August 2006, a total of 29 patients underwent elective LDLT for HCV cirrhosis in Hiroshima University Hospital. Of these, 24 patients who had undergone LDLT by February 2006 and had been followed up for 6 months were enrolled in this study. The mean age of the patients was 52 years (range, 29–69 years). All of the 24 patients were Japanese, and 16 were men. Preoperatively, all patients were sero-positive for the anti-HCV antibody. The HCV genotypes were 1b (n = 23) and 2a (n = 1). The median value of the preoperative HCV-RNA load in the 24 patients was 380 kIU/mL. The survival rate for the HCV-positive patients after LDLT was 71% at 1 year after transplantation. Necroinflammatory activity (A0–A4) and the fibrosis stage (F0–F4) were assessed using the METAVIR scoring system. The presence of recurrent hepatitis was evidenced in 14 of the 20 LDLT patients within 1 year after LDLT. The grades of the necroinflammatory activities observed in these 14 patients within 1 year were as follows: A1 in 10 patients, A2 in two patients and A3 in two patients. Significant fibrosis (F2 or more) was observed in four patients, including two patients who developed fibrosis of F4 within 1 year. Twelve patients were administered antiviral treatment for 6 months to 28 months after LDLT. The treatment protocol consisted of interferon-a2b (3–6 ¥ 10 units, three times/week) plus ribavirin (200–400 mg/ day orally) for at least 48 weeks. From June 2005, the interferon in the treatment regimen was replaced with pegylated interferon-a2b (0.5–1.0 mg/kg/week). Interferon therapy was discontinued in five of the six patients with a platelet count of <100 000/mm within 6 months because of thrombocytopenia (n = 4) and general fatigue with psychosis (n = 1). Therapy was discontinued in one of the six patients with a platelet count of >100 000/mm because of psychosis and general fatigue. The rate of cessation of the antiviral therapy was significantly higher (P = 0.02) in the patients with a platelet count of <100 000/mm prior to the administration of antiviral therapy, as compared with those having a platelet count of >100 000/mm (Fig. 1). Of the 12 patients, two patients responded to the combination therapy, and the serum HCV-RNA test remained negative 6 months after the cessation of the therapy. The other four patients have continued to receive the antiviral therapy for durations ranging from 6 to 18 months (mean 10 months). We next examined the preand post-operative course of the platelet counts in the 20 patients. In four of the nine patients with a preoperative platelet count of <60 000/mm, it exceeded beyond 100 000/mm at 2 months after LDLT. In 10 of the 11 patients with a preoperative platelet count of >60 000/mm, it exceeded 100 000/mm at 2 months after LDLT. The number of patients with a platelet count of >100 000/mm at 2 months after LDLT was significantly lower (P = 0.024) in those patients who had a preoperative platelet count of <60 000/mm, compared with those patients who had a preoperative platelet count of >60 000/mm (Fig. 2). From July 2005, based on the preliminary results, we initiated pre-emptive therapy and performed splenectomy simultaneously with liver transplantation for the patients with a preoperative platelet count of <60 000/mm. Informed consent was obtained from all patients. The platelet count of three patients who had preoperative platelet counts of <60 000/mm and underwent splenectomy with LDLT exceeded 100 000/mm (range 250 000–750 000/mm) 2 months after LDLT. Four patients have pre-emptively received antiviral therapy consisting of pegylated interferon and ribavirin that was initiated approximately 1–2 months after the operation; these patients have continued to receive the pre-emptive interferon therapy for 6–12 months without any post-operative complications, severe bacterial infection or acute rejection. Although the current standard of treatment for recurrent HCV hepatitis after liver transplantation is a combination therapy of interferon and ribavirin, its results are still poor due to a high incidence of adverse effects requiring the use of expensive adjuvant therapy or the discontinuation of the antiviral therapy. The frequency of side-effects necessitating dose reduction or the cessation of antiviral therapy for recurrent HCV hepatitis after liver transplantation has been reported to range from 45% to 85%. Cytopenias and post-operative complications including acute rejection, infections, renal dysfunction and severe debilitation limit the administration of interferon and ribavirin in the very early post-transplant period. Among the common complications of antiviral therapy, we focused on thrombocytopenia. Our results demonstrated that the rate of cessation of antiviral therapy was significantly high in the patients with a platelet count of <100 000/ mm prior to the administration of antiviral therapy. Further, we demonstrated that significantly fewer patients in the group with a preoperative platelet count of <60 000/mm had developed a platelet count of >100 000 mm by 2 months after LDLT, compared with the group with >60 000/mm. These results prompted us to perform splenectomy simultaneously with LDLT in those HCV doi:10.1111/j.1440-1746.2007.04951.x