H Yoshino

Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume.

The naked plasmid DNA transfer method of rapid injection with large volume has been useful for gene therapy in experimental study. However, only small animals like rodents have usually been reported on. In this study, the authors attempted to transfect naked plasmid DNA to the porcine liver by modified hydrodynamic method. We decided to transfer plasmid DNA to a part of the liver using the angio-catheter to reduce the liver damage. To discern the condition of injection, naked plasmid DNA-encoding green fluorescent protein (GFP) was transferred for use as a marker gene. The GFP gene expression was markedly observed in gene-transferred pig livers. In large animals, not only the naked gene quantity, the solution volume containing the plasmid DNA and the injection speed, but also the additional treatments of the portal vein and the hepatic artery preparation were crucial. We found that the following injection condition were needed: plasmid DNA, 3 mg; the solution volume, 150 ml and the injection speed, 5 ml/s. The portal vein and the hepatic artery were clamped during gene delivery and the blood flow of the portal vein was flushed out using normal saline. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig) gene was used to test for secretory protein. CTLA4-Ig gene was injected with a large volume of solution via the hepatic vein to the left outer lobe of the liver selectively. CTLA4-Ig was detected in the pig blood at a maximum serum level of 161.7 ng/ml 1 day after gene transfer, and the CTLA4-Ig was detected for several weeks. Our new technique of inserting a catheter into only a selected portion of the liver reduced liver toxicity and increased gene transfer efficiency. This is the first report of successful gene transfer, using a hydrodynamic method, to the segmental liver in pigs, and achieved more than enough secretory protein for the clinically therapeutic level in pigs.

Molecular investigation of hepatitis E virus infection in domestic and miniature pigs used for medical experiments

Abstract:  Background:  Hepatitis E virus (HEV) infection is highly prevalent among domestic pigs in Japan. It has been reported that pig handlers such as farmers and veterinarians are at increased risk of contracting HEV infection. Pigs are regarded as the most acceptable candidate animals for xenotransplantation and, recently, they are being used as experimental animals.

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

Living-Donor Liver Transplantation in 126 Patients with Biliary Atresia: Single-Center Experience

OBJECTIVES To describe our experience with 126 consecutive living-donor liver transplantation (LDLT) procedures performed because of biliary atresia and to evaluate the optimal timing of the operation. PATIENTS AND METHODS Between May 2001 and January 2010,126 patients with biliary atresia underwent 130 LDLT procedures. Mean (SD) patient age was 3.3 (4.2) years, and body weight was 13.8 (10.7) kg. Donors included 64 fathers, 63 mothers, and 3 other individuals. The left lateral segment was the most commonly used graft (75%). Patients were divided into 3 groups according to body weight: group 1, less than 8 kg (n = 40); group 2,8 to 20 kg (n = 63); and group 3, more than 20 kg (n = 23). Medical records were reviewed retrospectively. Follow up was 4.5 (2.7) years. RESULTS All group 3 donors underwent left lobectomy, and all group 1 donors underwent left lateral segmentectomy. No donors required a second operation or died. Comparison of the 3 groups demonstrated that recipient Pediatric End-Stage Liver Disease score in group 1 was highest, operative blood loss in group 2 was lowest (78 mL/kg), and operative time in group 3 was longest (1201 minutes). Hepatic artery complications occurred more frequently in group 1 (17.9%), and biliary stenosis (43.5%) and gastrointestinal perforation (8.7%) occurred more frequently in group 3. The overall patient survival rates at 1, 5, and 9 years was 98%, 97%, and 97%, respectively. Five-year patient survival rate in groups 1,2, and 3 were 92.5%, 100%, and 95.7%, respectively. Gastrointestinal perforation (n = 2) was the primary cause of death. CONCLUSIONS Living-donor liver transplantation is an effective treatment of biliary atresia, with good long-term outcome. It seems that the most suitable time to perform LDLT to treat biliary atresia is when the patient weighs 8 to 20 kg.

Gastrointestinal bleeding after living-related liver transplantation

We examined the prevalence of gastrointestinal bleeding in patients undergoing living-related liver transplantation (LRLT). Seventy-seven patients (37 male and 40 female) underwent LRLT at the University of Tokyo Hospital from January 1996 through December 1999. Forty-nine patients were children or adolescents and 28 patients were adults. The mean follow-up period was 21.3 ± 12.8 months. Nine of the 77 recipients had gastrointestinal bleeding after transplantation. The incidence of posttransplant bleeding was significantly higher in adult recipients than in pediatric recipients (25% vs 4%, P < 0.05). The ratio of graft volume to standard liver volume was significantly lower in adult recipients than in pediatric recipients (41 ± 6% vs 86 ± 26%, P < 0.05). Portal vein thrombosis (PVT) developed after LRLT in 8 patients. Variceal bleeding subsequently occurred in all 4 adult patients with PVT but in only 1 of 4 pediatric patients. Small-for-size grafts may cause transient portal hypertension, which increases the risk of gastrointestinal bleeding.

Immunosuppressive effect of chenodeoxycholic acid on natural killer cell activity in patients with biliary atresia and hepatitis C virus-related liver cirrhosis.

Patients with severe liver diseases, such as liver cirrhosis and biliary atresia, have low natural killer (NK) cell activity. The relations between NK activity and measures of liver function, including serum levels of total bilirubin, total bile acids, bile acid components, aspartate aminotransferase, and alanine aminotransferase, and platelet count were examined in patients with biliary atresia (6 boys and 6 girls; mean age, 4.8 ± 5.7 years) and patients with liver cirrhosis due to hepatitis C virus infection (10 men and 2 women; mean age, 54.3 ± 13.8 years). Univariate analysis showed that platelet count was positively correlated with NK activity in patients with biliary atresia (r = 0.611, P < 0.05). Serum levels of free chenodeoxycholic acid were negatively correlated with NK activity both in patients with biliary atresia (r = −0.647, P < 0.05) and in patients with hepatitis C virus-related liver cirrhosis (r = −0.876, P < 0.01). None of the other free bile acids or conjugated bile acids or other indicators of liver function were correlated with NK activity. Multiple stepwise regression analysis showed that only levels of free chenodeoxycholic acid were independently correlated with NK activity. All patients with biliary atresia underwent liver transplantation from living related donors. NK activity had increased significantly two months after transplantation (from 24.1 ± 20.2% to 49.2 ± 12.5%, P < 0.01). In contrast, levels of free chenodeoxycholic acid in transplant recipients had decreased significantly two months after transplantation (from 1.22 ± 1.16 to 0.26 ± 0.21 μmol/l, P < 0.05). In conclusion, in patients with biliary atresia or liver cirrhosis, NK activity in peripheral blood decreases, mostly because of free chenodeoxycholic acid.

The influence of living donor liver transplantation on families with or without siblings

Abstract:  With child living donor liver transplantation (LDLT), two people from one family, a child patient and one parent and one parent are forced to be hospitalized, so the family faces very servere problems. This is especially severe in cases when pediatric patients have siblings. Our research investigated the influence of LDLT upon families with siblings as compared to those without, especially the impact of surgery and hospitalization on family relationships. Families with siblings had more stress about child care and they tended to be more anxious about child development. The siblings had a lot of stress and mental problems. Therefore, the families of LDLT are seen to need special supports, especially those who had siblings.