Mikiko Ueda

Yeast cell-surface display—applications of molecular display

In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed. Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology. This mini-review describes molecular display using yeast and its various applications.

Analyses of Peripheral Blood Mononuclear Cells in Operational Tolerance After Pediatric Living Donor Liver Transplantation

Operational tolerance (graft acceptance in an immunosuppression (IS)‐free environment) after living‐donor liver transplantation (LDLT) could occur by our elective protocol in some patients. There is, nevertheless, no reliable parameter to monitor patients who may discontinue IS without a risk of rejection. To identify such parameters, we systemically phenotyped peripheral blood mononuclear cells from operationally tolerant patients. An increase was observed in the frequency of CD4+CD25high+ cells, B cells and Vδ1/Vδ2 γδT‐cells ratio in operationally tolerant patients (Gr‐tol; n = 12), compared with those from age‐matched volunteers (Gr‐vol; n = 24) or patients on IS (Gr‐IS; n = 19). The frequency of NK cells was decreased in Gr‐tol, compared with those in Gr‐IS or Gr‐vol. The frequency of NKT cells was decreased after LDLT, compared with that in Gr‐vol. Although the contribution of those subsets to the tolerant state remains elusive, the results may provide important clues for reliable indicators of tolerance after LDLT.

Anatomical variations and surgical strategies in right lobe living donor liver transplantation: lessons from 120 cases.

Background. Anatomical variations in right liver lobe are common. However, clinical implications and surgical management of these variations in living donor liver transplantation have not been analyzed systematically. Methods. Surgical anatomy of vascular and biliary structures in 120 right lobe grafts were reevaluated by reviewing the results of preoperative (computerized tomography and Doppler ultrasonography) and intraoperative (cholangiography) imaging as well as surgical findings. The data were analyzed in relation to surgical management of anatomical variations. Results. The incidence of variants leading to multiple portal vein anastomoses was 7.5%. The incidence of dual right hepatic veins was 0.8%; 30% of the grafts had significant accessory hepatic veins (>5 mm) and 13.9% of these were multiple. All of them were successfully reconstructed with technical modifications including venoplasty and venous grafts, except for two cases with multiple intraparenchymal portal vein branches to the anterior segment. The incidence of dual hepatic arteries was 1.7%, but only one of them was reconstructed without negative sequelae. The incidence of variants potentially leading to multiple bile duct anastomoses was 35.0%, and eventually 39.2% of the grafts had multiple orifices. With a variety of techniques including ductoplasty, hepaticohepaticostomy, and biliary stent, total incidence of leakage and stenosis was 10.8% and 9.2%, respectively. Although ductoplasty, internal stent or no stenting, seemed to be associated with increased risk of complications, anatomical variants, multiple bile ducts, and duct-to-duct reconstruction did not bear a significant risk. Conclusions. Anatomical variations of vascular and biliary structures in right lobe grafts are common. However, most can be managed safely with technical modifications. Only cases with intraparenchymal origin of the anterior portal vein(s) may form a relative contraindication, especially when combined with similar biliary variants. Otherwise, intraoperative assessment of biliary anatomy was enough for successful management. Detailed and precise assessment of vascular and biliary anatomy is vital for appropriate surgical management.

Living-donor liver transplantation for hepatocellular carcinoma.

In cadaveric liver transplantation, the Milan criteria have been accepted as the selection criteria for hepatocellular carcinoma (HCC) patients in considering organ allocation. However, the situation is different in living-donor liver transplantation (LDLT), in which the donor has a strong preference for altruism. The authors describe herein their experience with LDLT for HCC patients using their patient selection criteria. From February 1999 to March 2002, right lobe LDLT was performed in 56 patients with HCC. The authors’ exclusion criteria included only those with extrahepatic metastasis or vascular invasion detected during preoperative evaluation. Thirty patients (54%) were in tumor, node, metastases stage IVa and 25 patients (45%) did not meet the Milan criteria at the time of LDLT. The follow-up period was 1 to 39 months (median, 11 months). The overall survival rates at 1 and 3 years were 73% and 55%, respectively, and the latter was significantly lower than that of adult right lobe LDLT without HCC (71% at 3 years). Fourteen patients died because of postoperative complications without tumor recurrence. Thirty-six patients survived without recurrence and six patients had recurrence. Among the six patients with recurrence, four had survived for 11 to 36 months after LDLT. In the analysis of patients who survived longer than 3 months after transplantation, 19 of 20 within the Milan criteria survived without recurrence. However, 15 of 20 patients beyond the criteria also survived without recurrence for 3 to 33 months (median, 12 months) and three of five patients with recurrence were alive for 11 to 36 months (median, 20 months). Histopathologic grading and microscopic portal venous invasion had significant negative impact on tumor recurrence. LDLT was an effective treatment for uncontrollable hepatocellular carcinoma. Because many patients who did not meet the Milan criteria survived without tumor recurrence after transplantation, different patient selection criteria are necessary in LDLT to save those with advanced HCC.

Expansion of selection criteria for patients with hepatocellular carcinoma in living donor liver transplantation

In the present study, the results of living donor liver transplantation (LDLT) for 125 hepatocellular carcinoma (HCC) patients were analyzed to determine optimal criteria exceeding the Milan criteria (MC) but still with predictably good outcomes. On the basis of pretransplant imaging studies, 70 patients met the MC, and 55 patients did not. Patients who exceeded the MC but presented with ≤10 tumors all ≤5 cm in diameter (n = 30) displayed 5‐year recurrence rates (7.3%) similar to those of patients within the MC (9.7%, P = 0.8787). According to the results of multivariate analysis of risk factors for recurrence among preoperative tumor variables, we have defined the new criteria, namely ≤10 tumors all ≤5 cm in diameter and protein induced by vitamin K absence or antagonist‐II (PIVKA‐II) ≤400 mAU/mL. The 78 patients who met the new criteria showed significantly lower 5‐year recurrence rates (4.9%) than the 40 patients who exceeded them (60.5%, P < 0.0001). Similarly, 5‐year survival rates significantly differed between these groups (86.7% versus 34.4%, respectively; P < 0.0001). In conclusion, selection criteria for patients with HCC undergoing LDLT may be safely extended to ≤10 tumors all ≤5 cm in diameter and PIVKA‐II ≤400 mAU/mL with acceptable outcomes. Liver Transpl 13: 1637–1644, 2007.

Safety of the donor in living-related liver transplantation-an analysis of 100 parental donors

The safety and lack of undue operative stress on the donor are documented from an analysis of 100 parental donors, whose children (3 months to 17 years old), received LRLTx at our institution between June 1992 and May 1994. Survival rate of recipients was 86%. No primary nonfunctioning liver was observed. The donors were 56 mothers and 44 fathers. Their ages ranged from 19 to 51 years and their weight ranged from 44 to 80 kg. They received partial liver resections to harvest the grafts. With regard to the liver graft, the left lobe was used in 24 cases (group L) and the left lateral segment was used in 75 cases (group S). The right lobe was used in one case. In the two groups, blood losses were 242 +/- 5 (S) and 312 +/- 14 ml (L); operation times were 6.22 +/- 0.11 (S) and 7.15 +/- 0.21 hr (L), respectively; in both groups, the postoperative hospital stay was 11 days (S, L). No significant differences between the two groups were observed in peripheral RBC and WBC count or serum AST. An increase in total bilirubin was not observed. In the exceptional case using the right lobe, blood loss of 2300 ml necessitated a blood transfusion of 1000 ml, and the total bilirubin increased up to 4.0 mg/dl on the third postoperative day, which prolonged the postoperative hospital stay to 17 days. These results conclusively suggest that safety is guaranteed when the left lobe or the left lateral segment is used as the liver graft for LRLTx.

End-to-side portocaval shunting for a small-for-size graft in living donor liver transplantation

In the development of adult‐to‐adult living donor liver transplantation (LDLT), the small‐for‐size graft has been associated with poor clinical outcome. Persistent portal hypertension or portal venous overperfusion are considered to be causative factors, and partial diversion of portal flow to systemic circulation may be effective for avoiding injuries that occur in the small‐for‐size (SFS) graft. Recently, we constructed an end‐to‐side portocaval shunting using 1 of the portal branches and anastomosed the other branch with the portal vein of the graft in 2 cases of LDLT recipients transplanted with a SFS graft. With the suppression of portal hypertension, as well as sufficient portal flow to the graft, the recipients recovered successfully with favorable graft function. This new and simple technique may be able to be used as a feasible and effective method to attenuate the SFS syndrome. (Liver Transpl 2004;10:807–810.)

Acute humoral rejection and C4d immunostaining in ABO blood type‐incompatible liver transplantation

Complement C4d deposition in graft capillaries has been reported to be associated with antibody‐mediated rejection in kidney and other solid organ transplantation. The correlation of C4d deposits and humoral rejection in liver transplants, however, is not well understood. We investigated the C4d immunostaining pattern in 34 patients whose liver biopsy was taken within the first 3 postoperative weeks for suspected acute rejection after ABO blood type‐incompatible liver transplantation. The staining pattern was classified as positive (portal stromal staining), indeterminate (endothelial staining only), and negative (no staining). Positive C4d immunostaining was seen in 17 (50%) patients and was significantly associated with high (×64 or more) postoperative antidonor A/B antibody (immunoglobulin M (IgM)) titers (88 vs. 35%, P = 0.002) and poorer overall survival rate (41 vs. 88%, P = 0.007). Ten of 11 (91%) cases with histological acute humoral rejection (periportal edema and necrosis (PEN) or portal hemorrhagic edema) were positive for C4d, all of which showed high postoperative antibody titers. The other histologies associated with C4d positivity was purulent cholangitis (n = 4), coagulative hepatocyte necrosis (n = 1), acute cellular rejection (n = 1), and hepatocanalicular cholestasis (n = 1). Full clinical recovery was observed in only 6 of 17 (35%) C4d‐positive patients, and tended to be associated with a lower rejection activity index (RAI). In conclusion, our study indicates that C4d deposits in the portal stroma can be a hallmark of acute humoral rejection in ABO‐incompatible liver transplantation, and allograft damage can be reversible in a minority of cases. Liver Transpl 12:457–464, 2006.

Long-term outcomes of 600 living donor liver transplants for pediatric patients at a single center.

This report concerns the long‐term outcome of living donor liver transplantation (LDLT) for pediatric patients at a single center. Between June 1990 and December 2003, a total of 600 LDLTs, including 568 primary transplantations and 32 retransplantations, were performed for pediatric patients, who were immunosuppressed with FK506 and low‐dose corticosteroids. Patient survival at 1, 5, and 10 years were 84.6%, 82.4%, and 77.2%, respectively, and the corresponding findings for graft survivals were 84.1%, 80.9%, and 74.5%. Multivariate analysis demonstrated that fulminant hepatic failure (FHF), a graft vs. body weight (GBWR) ratio of <0.8, and ABO‐incompatible transplants were independently associated with both patient and graft survival. The retransplantation rate was 6%, and 55 patients (9.7%) have been completely weaned off immunosuppressants. Long‐term patient and graft survival after pediatric LDLT for a large cohort of children at our hospital were found to be as good as those for cadaveric liver transplantation, although this series includes 13% liver transplantations with ABO‐incompatible donors, which are obviously inferior in patient and graft survival. To obtain better outcomes for patients with FHF and for patients with ABO‐incompatible transplants, immunosuppressive therapy needs to be improved. Liver Transpl 12:1326‐1336, 2006.

Living-donor liver transplantation with monosegments.

Background. Living-donor liver transplantation is now an established technique to treat children with end-stage liver disease. Implantation of left-lateral segment grafts can be a problem in small infants because of a large-for-size graft. We report 10 cases of transplantation using monosegment grafts from living donors. Method. Of 506 children transplanted between June 1990 and June 2002, 10 patients (median age 196 days, median weight 5.9 kg) received monosegment living-donor liver transplants. The indication for using this technique was infants with an estimated graft-to-recipient weight ratio of over 4.0%. Results. Graft and patient survival was 80.0%. There were no differences in donor operation time and blood loss between monosegmentectomy and left-lateral segmentectomy (n=281). Monosegmental transplantation had a high incidence of vascular complications (20.0%). Conclusion. Monosegmental living- donor liver transplantation is a feasible option with satisfactory graft survival in small babies with liver failure.