Self-assembling peptide nanofiber as a novel culture system for isolated porcine hepatocytes

Freshly isolated porcine hepatocytes are a very attractive cell source in the cell-based therapies to treat liver failure because of unlimited availability. However, due to the loss of hepatocyte functions in vitro, there is a need to develop a functional culture system to keep the cells metabolically active. Here we compared the effect of a self-assembling peptide nanofiber (SAPNF) as an extracellular matrix (ECM) with collagen type I on hepatocyte metabolic and secretion activities following hepatocyte isolation. Isolated porcine hepatocytes were cultured in SAPNF and collagen type I. Morphological assessment at different time points was performed by using SEM and phase contrast microscope. Metabolic and secretion activities were comparatively performed in the groups, by means of ammonia, lidocaine, and diazepam as well as albumin. Hepatocytes cultured on SAPNF revealed a three-dimensional spheroidal formation, thus maintaining cell differentiation status during 2 weeks of culture. On the other hand, hepatocytes in collagen revealed a spread shape, and by day 14 no hepatocyte-like cells were observed, but cells with long shape were present, thus revealing a degree of dedifferentiation in collagen culture. Hepatocytes in SAPNF were capable of drug-metabolizing activities and albumin secretion in higher ratio than those cultured on collagen. The present work clearly demonstrates the usefulness of SAPNF for maintaining differentiated functions of porcine hepatocytes in culture.     

A revised scoring system utilizing serum alphafetoprotein levels to expand candidates for living donor transplantation in hepatocellular carcinoma

BACKGROUND: The development of living donor liver transplantation has stimulated discussion about the expansion of tumor burden limits for patients with hepatocellular carcinoma (HCC). Although serum alphafetoprotein (AFP) level is an important predictor of tumor recurrence, it is not included in the existing selection criteria for HCC in transplantation. METHODS: We performed a retrospective study of 63 consecutive adults with HCC diagnosed preoperatively who received living donor liver transplantation from February 1999 to September 2005 and survived over 1 month. The authors devised new scoring criteria that included tumor size, tumor number, and pretransplant AFP level as prognostic factors. The score of each parameter was classified from 1 to 4 points (tumor size, < or =3, 3.1 to 5, 5.1 to 6.5, >6.5 cm; tumor number, 1, 2 or 3, 4 or 5, or > or =6 nodules; and AFP, < or =20, 20.1 to 200, 200.1 to 1000, >1000 ng/mL, respectively). We defined that 3 to 6 points and 7 to 12 points were "transplantable" and "nontransplantable," respectively. The usefulness of the devised criteria was then investigated as a method of selecting candidates with HCC for transplantation. RESULTS: The candidates' overall 3-year survival rate and recurrence-free survival rate were 67% and 70% after transplantation, respectively. Based on pretransplant imaging, 37 (59%), 41 (65%), and 44 (70%) of the 63 patients met the Milan criteria, University of Californica, San Francisco (UCSF) criteria, and the new scoring criteria. Their 3-year survival rates were 80%, 78%, and 79%, respectively. Moreover, based on posttransplant data, the scoring criteria correlated with the risk of death and HCC recurrence (Milan criteria, P = .005 and .001; UCSF criteria, P = .013 and .001 for death and recurrence; scoring criteria, P < .001 for both). CONCLUSIONS: The newly devised scoring criteria could expand usefully current selection criteria for transplantation without detrimentally affecting outcome in the living donor transplantation setting for HCC.     

A novel scheme for graft allocation in non-heart beating donor renal transplantation

Patients waiting more than 3 years for a renal transplant were ranked according to our novel Bristol and Region Allocation by Non-heart beating Donor Score (BRANDS). One kidney from 40 non-heart beating donors was allocated to the highest BRANDS long-waiter and the other kidney allocated according to the UK National Allocation Scheme (NAS). The scheme reduced the number of patients waiting more than 3 years by 20%. Despite longer dialysis time, greater sensitization and more human leukocyte antigen mismatches, BRANDS patients had equivalent 3-year graft survival (BRANDS 91%, NAS 97%, P=0.264) and patient survival (BRANDS 94%, NAS 92%, P=0.99). Results were similar to 242 synchronous recipients from heart-beating donors. Renal function was significantly lower in BRANDS recipients (40 vs. 62 mL/min/1.73 m2, P<0.0001). Transplanting long-waiting patients with kidneys from non-heart beating donors has reduced waiting times without compromising early outcomes. It is unclear if equivalent survival will be sustained in the long term.     

Microvascular transplantation of epiphyseal plates: studies utilizing allograft donor material

Compromised function of an epiphyseal plate caused by trauma, tumor, infection, or congenital malformation can result in significant musculoskeletal deformity. Techniques used to correct or minimize the extent of these deformities include autogenous or allogeneic cancellous bone grafts, nonvascularized cortical allografts, vascularized bone and composite tissue transfers, and distraction osteogenesis. These solutions are not ideal for children because they do not adequately address the actively growing nature of the extremity. Microvascular techniques have enabled the experimental transplantation of vascularized epiphyseal plates with high levels of postoperative viability and subsequent growth and offer a potential advantage over conventional treatments.     

A model utilizing adult murine stem cells for creation of personalized islets for transplantation

Clinical islet cell transplantation has demonstrated great promise for diabetes treatment. Two major obstacles are the organ donor shortage and the immunoresponse. The purpose of this study was to create a model using the patient's own adult stem cell sources, possibly in combination with non-self cells, such as pancreatic, hepatic, or embryonic stem cells, to create "personalized" islets. We hypothesize that the reconstructed islets have the normal capability to produce insulin and glucagon with reduced immunoresponses after transplantation. Stem cells are a proliferating population of master cells that have the ability for self-renewal and multilineage differentiation. The recently developed photolithograph-based, biologic, microelectromechanic system (BioMEMS) technique supplies a useful tool for biomedical applications. Our lab has developed a novel method that integrates the adult stem cell and BioMEMS to reconstruct personalized islets. We selected islet-derived progenitor cells (IPC) for repairing and reconstructing STZ-diabetic islets. A6(+)/PYY(+) or A6(+)/ngn3(+) cells were selected to manipulate the neoislets. After 3 to 4 weeks in culture, the reconstructed cells formed islet-like clusters containing insulin or glucagon producing cells. The pilot results showed the ability of these reconstructed islets to correct hyperglycemia when transplanted into a STZ-diabetic isograft mouse model. Although several technical problems remain with the mouse model, namely, the difficulty to collect enough islets from a single mouse because of animal size, the mouse isograft model is suitable for personalized islet development.     

A new source of hepatocytes for transplantation

INTRODUCTION: The most effective treatment for acute or chronic liver failure is orthotopic liver transplantation. Worldwide there is a shortage of organs for transplantation. This shortage has called for research into new treatments for management of patients with liver failure. One such treatment is hepatocyte transplantation. During liver resections considerable amounts of normal liver are unavoidably resected. We aim to harvest these hepatocytes and to filter the tumor cells from them to provide a source for transplantation. MATERIALS AND METHODS: After liver resection, the largest vessel at the resected liver edge was identified and cannulated. Seglen's two-stage technique of perfusing the liver with EDTA and collagenase was performed to harvest the hepatocytes. Ep-CAM Ags are consistently present on the surface of epithelial cells and in particular in colorectal cancer cells. Therefore, MOC31 antibodies (selective Abs for Ep-CAM) attached to magnetic beads were used to target the tumor cells. These tumor cells are selectively removed using a magnet. CEA staining was then used to ensure the hepatocyte collection was tumor cell free. Five million hepatocytes were rosetted with one million HT29 CRC cells to assess the immunomagnetic filtration technique. RESULTS: The hepatocyte harvesting resulted in 864,000 viable hepatocytes to be harvested per gram of liver. Histochemical staining using CEA demonstrated 75% of the HT29 cells in the hepatocyte collection were removed after one use of magnetic beads. CONCLUSION: We have demonstrated the successful initial stages of harvesting tumor-free hepatocytes from liver resected for malignancy.     

Duct-to-duct biliary reconstruction in living donor liver transplantation utilizing right lobe graft

OBJECTIVE: To assess the feasibility and safety of duct-to-duct biliary anastomosis for living donor liver transplantation (LDLT) utilizing the right lobe. SUMMARY BACKGROUND DATA: Biliary tract complications remain one of the most serious problems after liver transplantation. Roux-en-Y hepaticojejunostomy has been a standard procedure for biliary reconstruction in LDLT with a partial hepatic graft. However, end-to-end choledochocholedochostomy is the technique of choice for biliary reconstruction and yields a more physiologic bilioenteric continuity than can be achieved with Roux-en-Y hepaticojejunostomy. The authors performed right lobe LDLT with end-to-end duct-to-duct biliary anastomosis, and this study assessed retrospectively the relation between the manner of reconstruction and complications. METHODS: Between July 1999 and December 2000, 51 patients (11-67 years of age) underwent 52 right lobe LDLTs with duct-to-duct biliary reconstruction and remained alive more than 1 month after their transplantation. Interrupted biliary anastomosis was performed for 24 transplants and the continuous procedure was used for 28. A biliary tube was inserted downward into the common bile ducts through the recipient's cystic duct in 16 transplants (cystic drainage), or a biliary stent tube was pushed upward into the anastomosis through the cystic duct in four transplants (cystic stent), or upward into the anastomosis through the wall of the common bile duct in 31 transplants (external stent). RESULTS: Biliary anastomotic procedures consisted of 34 single end-to-end anastomoses, 11 double end-to-end anastomoses, and 7 single anastomoses for double hepatic ducts. Overall, 5 patients developed leakage (9.6%) and 12 patients suffered stricture (23.0%). For biliary anastomosis with interrupted suture, the incidence of stricture was significantly higher in the cystic drainage group (53.3%, 8/15) than in the stent group consisting of cystic stent and external stent (0%, 0/8). While the respective incidences of leakage and stricture were 20% and 53.3% for intermittent suture with a cystic drainage tube (n = 15), they were 7.7% and 15.4% for a continuous suture with an external stent (n = 26). There was a significant difference in the incidence of stricture. CONCLUSIONS: Duct-to-duct reconstruction with continuous suture combined with an external stent represents a useful technique for LDLT utilizing the right lobe, but biliary complications remain significant.     

大鼠供肝冷保存时间延长损伤移植肝肝细胞和肝窦内皮细胞

目的 探讨供肝冷保存时间与肝移植后肝细胞和肝窦内皮细胞（SEC）损伤的关系。方法 选取健康雄性SD大鼠作为供、受者，建立原位肝移植（OLT）模型。随机分为3组，冷保存1h组（H=48）：供肝获取后，置于4C的冷保存液中保存1h，再行OLT。冷保存12h组（n=48）：供肝获取后，置于4℃的UW液中保存12h，再行OLT。对照组（H=6）：大鼠只打开腹腔，不进行移植。前2组分别于术后1、6、12、24、48、72、96和168h采取血液及组织标本，对照组仅在开腹时取血液及组织标本，检测各组、各时点血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）及透明质酸（HA）的水平；观察移植肝的病理形态学变化，透射电镜观察其超微结构改变；原位末端脱氧核糖核酸转移酶标记法（TUNEL）检测移植肝细胞的凋亡情况；观察术后168h时的大鼠存活率。结果 冷保存1h和冷保存12h组肝移植后各时点血清ALT、AST及HA均较对照组明显升高（P〈0．05），并且冷保存12h组又明显高于冷保存1h组（P〈0．05）。冷保存12h组术后24h移植肝组织出现片状坏死，而冷保存1h组病理学改变不明显。冷保存12h组肝窦内皮细胞凋亡指数（AI）明显高于冷保存1h组（F=63．58，P〈0．01），两组大鼠移植肝组织均于术后6h出现凋亡高峰，且肝窦内皮细胞的凋亡指数明显高于肝细胞。冷保存1h组和冷保存12h组大鼠肝移植后168h时的存活率分别为100％和50％，两组比较，差异有统计学意义（F=6．39，P〈0．05）。结论 肝移植后肝细胞和肝窦内皮细胞的损伤程度与冷保存时间密切相关。肝窦内皮细胞对冷保存及再灌注损伤的敏感性高于肝细胞，其损伤方式以细胞凋亡为主。     

The cytokinetic behavior of donor hepatocytes after syngenic hepatocyte transplantation into the spleen

The cytokinetic behavior of isolated hepatocytes transplanted into the spleen of syngenic normal Wistar rats was studied. Hepatocyte transplantation (HTX) was performed by the intrasplenic injection of 10(7) isolated hepatocytes. The proliferation index (PI) of intrasplenic donor hepatocytes was assessed by immunocytochemical visualization of DNA-synthesizing cells after pulse-labeling with bromodeoxyuridine (BrdU), a thymidine analogue. A method for determination of intrasplenic liver mass based on tissue glutamate dehydrogenase content was developed. The spontaneous PI of donor hepatocytes at 12 and at 20 weeks post-HTX amounted to around 3%. A significant increase of intrasplenic liver mass was demonstrated between the 12th and 20th week post-HTX (from 8.1 +/- 0.8% to 10.8 +/- 0.8% of spleen weight, P less than 0.05). After partial hepatectomy (PH) at 12 weeks post-HTX, the PI of liver cells in the spleen showed a transient increase up to about 10%, which rapidly declined to the "spontaneous" level of 3%. However, PH did not cause an additional increase in intrasplenic liver mass. This study shows that continuous mitotic activity of intrasplenic hepatocytes results in an actual increase of liver mass in spleen. Although a short-lived increase of proliferative activity of ectopically grafted hepatocytes was shown to occur after PH in the HTX-treated rat, this procedure did not result in an additional increase of intrasplenic liver tissue.     

A donor risk index for graft loss in pediatric living donor kidney transplantation

Pediatric kidney transplant candidates often have multiple potential living donors (LDs); no evidence‐based tool exists to compare potential LDs, or to decide between marginal LDs and deceased donor (DD) kidney transplantation (KT). We developed a pediatric living kidney donor profile index (P‐LKDPI) on the same scale as the DD KDPI by using Cox regression to model the risk of all‐cause graft loss as a function of living donor characteristics and DD KDPI. HLA‐B mismatch (adjusted hazard ratio [aHR] per mismatch = _1.041.27_1.55), HLA‐DR mismatch (aHR per mismatch = _1.021.23_1.49), ABO incompatibility (aHR = _1.203.26_8.81), donor systolic blood pressure (aHR per 10 mm Hg = _1.011.07_1.18), and donor estimated GFR (eGFR; aHR per 10 mL/min/1.73 m² = _0.880.94_0.99) were associated with graft loss after LDKT. Median (interquartile range [IQR]) P‐LKDPI was ?25 (?56 to 12). 68% of donors had P‐LKDPI <0 (less risk than any DD kidney) and 25% of donors had P‐LKDPI >14 (more risk than median DD kidney among pediatric KT recipients during the study period). Strata of LDKT recipients of kidneys with higher P‐LKDPI had a higher cumulative incidence of graft loss (39% at 10 years for P‐LDKPI ≥20, 28% for 20> P‐LKDPI ≥?20, 23% for ?20 > P‐LKDPI ≥?60, 19% for P‐LKDPI <?60 [log rank P < .001]). The P‐LKDPI can aid in organ selection for pediatric KT recipients by allowing comparison of potential LD and DD kidneys.     

应用MRCP术前评估活体肝移植供者胆管系统

Objective To determine the clinical value of MRCP for peroperative evaluation of donor biliary system in living donor liver transplantation (LDLT). Methods A total of 60 living donors for the LDLT were enrolled in this study. Of the 60 donors with a mean age of 32.2 (19-60), 50were male and 10 female. MRCP was performed before and cholangiography was done during the right lobectomy in these donors. The results of MRCP were compared with those of cholangiography to determine the value of MRCP for typing the biliary system in the donors. Results The preoperative MRCP showed that 40 donors were of type Ⅰ biliary tract, 12 of type Ⅱ , 5 of type Ⅲ and 3 of other types. The intraoperative cholangiography showed that the accordance rate of MRCP was 97.4%,91% and 89% for type Ⅰ , type Ⅱ and other types, respectively. The overall rate of accuracy of MRCP was 95% (57/60). Conlusion MRCP can show types of biliary tract in living donors for liver transplantation to provide evidence for plan of surgery.     

应用MRCP术前评估活体肝移植供者胆管系统

Objective To determine the clinical value of MRCP for peroperative evaluation of donor biliary system in living donor liver transplantation (LDLT). Methods A total of 60 living donors for the LDLT were enrolled in this study. Of the 60 donors with a mean age of 32.2 (19-60), 50were male and 10 female. MRCP was performed before and cholangiography was done during the right lobectomy in these donors. The results of MRCP were compared with those of cholangiography to determine the value of MRCP for typing the biliary system in the donors. Results The preoperative MRCP showed that 40 donors were of type Ⅰ biliary tract, 12 of type Ⅱ , 5 of type Ⅲ and 3 of other types. The intraoperative cholangiography showed that the accordance rate of MRCP was 97.4%,91% and 89% for type Ⅰ , type Ⅱ and other types, respectively. The overall rate of accuracy of MRCP was 95% (57/60). Conlusion MRCP can show types of biliary tract in living donors for liver transplantation to provide evidence for plan of surgery.     

应用MRCP术前评估活体肝移植供者胆管系统

目的 评价应用MRCP术前评估活体肝移植供者胆管系统的临床意义.方法 60例活体肝移植供者(男50例,女10例,年龄19～60岁,平均32.3岁),于右半肝切取术前行MRCP检查,术中经胆囊管行胆道造影.回顾性分析这些活体肝移植供者术前MRCP和术中胆道造影结果,二者进行比较,对比术中胆道造影结果评价MRCP在活体肝移植供者胆管分型方面的准确性.结果 根据术前MRCP结果,所有60例活体肝移植供者均可以判断出肝内胆管分型情况,1型胆管40例,2型胆管12例,3型胆管5例,其他3例.术中胆道造影显示1型胆管39例,MRCP正确诊断38例,准确率为97.4%;术中胆道造影显示2型胆管12例,MRCP正确诊断出11例,准确率为91%;术中胆道造影显示其他胆管类型为9例,MRCP正确诊断出8例,准确率为89%;MPCP总体准确率为95%(57/60).结论 MRCP可以在术前较为准确地显示活体肝移植供者胆管分型,为临床制定手术方案提供依据.     

A role for natural killer cells in the rapid death of cultured donor myoblasts after transplantation

BACKGROUND: The rapid and massive death of cultured donor myoblasts after injection in vivo is a major problem for clinical myoblast transfer therapy (MTT). This study shows blood-borne factors are responsible and that ablating the host natural killer (NK) cell response greatly enhances the survival of such donor myoblasts. METHODS: Cultured male donor myoblasts were injected into muscles of female host mice and surviving donor male DNA (myoblasts) quantified using a Y-chromosome specific (Y1) probe. Survival of donor myoblasts transfected with m144, a murine major histocompatibility complex (MHC) class I homologue that protects against NK attack, was quantified. In addition, donor myoblast survival was investigated in host mice following initial (before MTT) and sustained (repeatedly for 3 weeks after MTT) depletion of host NK1.1+ and CD4+/CD8+ cells using specific monoclonal antibodies (either alone or in combination) for up to 3 weeks after MTT, as well as in beige (deficient in NK activity) and in perforin-deficient mdx host mice. RESULTS: A major role for blood-borne factors (especially cells) was confirmed by MTT experiments in irradiated and perfused host mice. Substantially enhanced myoblast survival was seen with donor myoblasts modified by transfection with the m144 molecule or following antibody depletion of host NK1.1+ cells and in beige host mice. Other studies support some role for CD8+ but not CD4+ cells. CONCLUSIONS: These combined data support a central role for host NK cells in the rapid initial death of donor myoblasts. The demonstrated role of NK cells provides strategies to enhance the efficacy of clinical myoblast transplantation.     

Impact of donor immune cells in pancreatic islet transplantation

Autoimmune-mediated cytotoxicity may cause pancreatic islet transplant failure, leading to recurrent diabetes. Protection of islet grafts depends on immunosuppressive control, which may also prevent autoimmune recurrence of diabetes. In this study, we compared the survival of syngeneic islet transplants using different strains of donor mice. We observed extended functional survival in the islet grafts from donors lacking the genetic background and potential of autoimmunity. Without immunosuppression, the islet grafts of NOR and immune-deficient NOD. Scid donors functioned up to 3 weeks in syngeneic islet transplants compared to 3-day survivals with the grafts from NOD donors. T-cell proliferation and activation markers, CD44 and CD69, were upregulated in NOD donors, suggesting that T-cell activation had occurred prior to pancreas procurement. Systemic delivery of a recombinant adenoassociated viral vector (AAV) encoding the viral (vIL-10) IL-10 gene (AAV vIL-10) in NOD recipients protected syngeneic islets from autoimmune destruction. Alternatively, pretreatment of NOD donor mice with AAV vIL-10 prolonged islet graft survival in untreated NOD recipients. Both studies indicate the effectiveness of vIL-10 gene therapy in autoimmune regulation. These results suggest that a donor factor may exist in autoimmune-prone donors. Therefore, autoimmune recurrence of diabetes may result from donor immune cells transferred during islet transplantation. The AAV vIL-10 gene therapy suppressed previously activated donor T cells and protected the grafted islets from autoimmune-mediated destruction.     

Roux-en-Y bleeding after living donor liver transplantation: a novel technique for surgical treatment

Upper gastrointestinal bleeding (GIB) is one of the most common gastroenterologic complications following liver transplantation. The aim of this study is to define the prevalence of GIB due to Roux- en Y (R-Y) enteral anastomoses after living donor liver transplantation (LDLT) and recommend an anastomotic technique for easy surgical intervention. Ninety-five patients underwent 96 LDLT from June 1999 through January 2003. R-Y biliary reconstruction was employed in 43 patients. Anastomoses were end-to-side (ES) in the first 25 patients and side-to-side (SS) type in the last 18 patients. GIB occurred in 13 patients (30%). The R-Y anastomotic line was shown to be the bleeding site in 10 patients. Anastomoses were in ES fashion in 7 of 10 patients (70%). In other words 28% of ES and 17% of SS anastomoses displayed a bleeding episode after LDLT. Four patients required surgical intervention (Three ES, one SS), namely an operative rate of 9%. The type of the jejunojejunostomy, the UNOS or Child-Pugh scores, the presence of preexisting portal hypertension, the duration of portal vein clamping, the GRWR of patients, revealed no statistical significant difference between bleeding and non- bleeding patients. Although statistical analyses did not reveal any significant difference (P =.47), GIB was higher among patients with an ES type of anastomoses. As a result we recommend a jejunojejunostomy in SS fashion on the antimesenteric borders of the jejunal segments with a 3-4 cm blind intestinal segment. The surgical procedure for R-Y bleeding may then be performed without disrupting the jejunojejunostomy.