Invariant natural killer T cells infiltrate intestinal allografts undergoing acute cellular rejection.

Immunological responses in human intestinal allografts are poorly understood and accurate diagnosis of acute cellular rejection remains difficult. Here, human intestinal allografts were analyzed by multi-color quantitative fluorescent immunohistochemical morphometry in order to monitor the clinical course of rejection. Morphometry gave two-dimensional plots based on size and circularity, and identified phenotypes of individual cells infiltrating the allograft by fluorescent staining. Using this method, invariant TCRVα24(+) NKT (iNKT) cells were observed in the intestinal allograft during rejection. Because these were not identified in the normal donor intestine before surgery, this finding was considered to be a signature of acute cellular rejection of the intestinal allograft. Infiltrating iNKT cells released IL-4 and IL-5, Th2-related cytokines that antagonize the Th1 responses that induce acute cellular rejection. Histological observation suggested eosinophilic enteritis in the mucosa with elevation of IL-4 and IL-5. In conclusion, iNKT cells were recruited to the intestine; however, because higher levels of IL-4 and IL-5 may contribute to eosinophilic enteritis, timely steroid administration is recommended for allograft injury due to enteritis, as well as acute cellular rejection.

Thrombotic microangiopathy-like disorder after living-donor liver transplantation: a single-center experience in Japan.

AIM: To investigate thrombotic microangiopathy (TMA) in liver transplantion, because TMA is an infrequent but life-threatening complication in the transplantation field. METHODS: A total of 206 patients who underwent living-donor liver transplantation (LDLT) were evaluated, and the TMA-like disorder (TMALD) occurred in seven recipients. RESULTS: These TMALD recipients showed poor outcomes in comparison with other 199 recipients. Although two TMALD recipients successfully recovered, the other five recipients finally died despite intensive treatments including repeated plasma exchange (PE) and re-transplantation. Histopathological analysis of liver biopsies after LDLT revealed obvious differences according to the outcomes. Qualitative analysis of antibodies against a disintegrin-like domain and metalloproteinase with thrombospondin type 1 motifs (ADAMTS-13) were negative in all patients. The fragmentation of red cells, the microhemorrhagic macules and the platelet counts were early markers for the suspicion of TMALD after LDLT. Although the absolute values of von Willebrand factor (vWF) and ADAMTS-13 did not necessarily reflect TMALD, the vWF/ADAMTS-13 ratio had a clear diagnostic value in all cases. The establishment of adequate treatments for TMALD, such as PE for ADAMTS-13 replenishment or treatments against inhibitory antibodies, must be decided according to each case. CONCLUSION: The optimal induction of adequate therapies based on early recognition of TMALD by the reliable markers may confer a large advantage for TMALD after LDLT.

Living-donor liver transplantation for progressive familial intrahepatic cholestasis.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) results in liver cirrhosis during the disease course, although the etiology includes unknown mechanisms. Some PFIC patients require liver transplantation (LT). METHODS: In this study, 11 patients with PFIC type 1 (PFIC1) and 3 patients with PFIC type 2 (PFIC2) who underwent living-donor LT (LDLT) were evaluated. RESULTS: Digestive symptoms after LDLT were confirmed in 10 PFIC1 recipients (90.9%); 8 PFIC1 recipients showed steatosis after LDLT (72.7%), which began during the early postoperative period (71.5±55.1 days). Seven of the eight steatosis-positive PFIC1 recipients (87.5%) showed a steatosis degree of ≥80%, which was complicated with steatohepatitis and resulted in fibrosis. Cirrhotic findings persisted in six PFIC1 recipients even after LDLT (54.5%), and three PFIC1 recipients finally died. The survival rates of the PFIC1 recipients at 5, 10, and 15 years were 90.9%, 72.7%, and 54.5%, respectively. In contrast, the PFIC2 recipients showed good courses and outcomes without any steatosis after LDLT. CONCLUSIONS: The clinical courses and outcomes after LDLT are still not sufficient in PFIC1 recipients owing to steatosis/steatohepatitis and subsequent fibrosis, in contrast to PFIC2 recipients. PFIC2 is good indication for LDLT. PFIC1 patients require LT during the disease course; therefore, we suggest that the therapeutic strategies for PFIC1 patients, including the timing of LDLT, under the donor limitation should be reconsidered. The establishment of more advanced treatments for PFIC1 patients is required to improve the long-term prognosis of these patients.

Progressive familial intrahepatic cholestasis: a single-center experience of living-donor liver transplantation during two decades in Japan.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) results in liver cirrhosis. Therefore, some PFIC patients require liver transplantation (LT). Although three types of PFIC have been identified, their etiologies include unknown mechanisms. PATIENTS: A total of 717 recipients who underwent living-donor LT (LDLT) at <20 yr old were enrolled in this study. Among these recipients, 14 PFIC recipients comprising 11 PFIC type 1 (PFIC1) and three PFIC type 2 (PFIC2) were evaluated. RESULTS: Three of 11 PFIC1 recipients died, while all three PFIC2 recipients survived. Eight of 11 PFIC1 recipients showed steatosis after LDLT. Among the eight steatosis-positive PFIC1 recipients, seven showed severe steatosis and seven were complicated with steatohepatitis. Nine of 11 PFIC1 recipients showed fibrosis after LDLT, and eight of the nine fibrosis-positive PFIC1 recipients showed severe fibrosis. In contrast to the PFIC1 recipients, the PFIC2 recipients did not show any steatosis or fibrosis after LDLT. CONCLUSIONS: The clinical courses and outcomes of PFIC1 recipients after LDLT are still not sufficient owing to steatosis/fibrosis, unlike the case for PFIC2 recipients. As PFIC1 patients will require LT during the long-term progression of the disease, further strategy improvements are required for PFIC1 patients.

Prediction of pancreatic tissue densities by an analytical test gradient system before purification maximizes human islet recovery for islet autotransplantation/allotransplantation.

BACKGROUND: Using standard density gradient (SDG) ranges for human islet purification frequently results in islet loss and transplantation of lower islet mass. Measuring the densities of islet and acinar tissue beforehand to customize the gradient range for the actual COBE 2991 cell processor (COBE) purification is likely to maximize the recovery of islets. We developed an analytical test gradient system (ATGS) for predicting pancreatic tissue densities before COBE purification to minimize islet loss during purification. METHODS: Human islets were isolated from deceased donor (n=30) and chronic pancreatitis pancreata (n=30). Pancreatic tissue densities were measured before purification by the ATGS, and the density gradient range for islet purification in a COBE was customized based on density profiles determined by the ATGS. The efficiency of custom density gradients (CDGs) to recover high islet yield was compared with predefined SDGs. RESULTS: Pancreatic tissue densities from autografts were significantly higher than in allograft preparations. In allograft purifications, a higher proportion of islets were recovered using ATGS-guided CDGs (85.9%±18.0%) compared with the SDG method (69.2%±27.0%; P=0.048). Acinar contamination at 60%, 70%, and 80% cumulative islet yield for allografts was significantly lower in the CDG group. In autograft purifications, more islets were recovered with CDGs (81.9%±28.0%) than SDGs (55.8%±22.8%; P=0.03). CDGs effectively reduced islet loss by minimizing islet sedimentation in the COBE bag. CONCLUSIONS: Using ATGS-guided CDGs maximizes the islet recovery for successful transplantations by reducing acinar contamination in allograft preparations and by reducing sedimentation of islets in the COBE bag in autograft preparations.

Pediatric orthotopic living-donor liver transplantation cures pulmonary hypertension caused by Abernethy malformation type Ib.

A 3.3-yr-old boy was diagnosed with PH caused by a PSS of Abernethy malformation type Ib. After control of PH, he underwent OLDLT at 4.9 yr. His PV flowed directly into the confluence of the CCLMHV and the IVC. To shorten the anhepatic phase, hepatic arterial flow was partially maintained. Removal of the native liver began simultaneously with the graft harvest. The proximal PV was cut at the optimal point for reconstruction. The distal PV was cut at the concrescence of the PV and the CCLMHV. After IVC-plasty, the LHV of the graft was attached with an anterior patch by venous grafting and was then anastomosed to the IVC. Although the mPAP temporarily increased above the mean arterial pressure, mPAP was stable during OLDLT. FNH and steatosis were confirmed histopathologically. In summary, pediatric OLDLT was performed successfully in PH caused by PSS.

Liver transplantation for congenital biliary dilatation: a single-center experience.

BACKGROUND: Congenital biliary dilatation is a rare disease. Although the possibility of refractory cholangitis and/or the frequency of malignant tumors legitimize hepatobiliary surgery, repeated cholangitis and biliary obstruction result in secondary liver cirrhosis even after polysurgery. There are no definitive guidelines on liver transplantation for congenital biliary dilatation. PATIENTS: A total of 1,101 liver transplantation recipients were enrolled in this study. Eleven patients with congenital biliary dilatation including 5 patients with Caroli's disease were retrospectively analyzed in detail. RESULTS: Nine of 11 patients underwent initial operations before liver transplantation while 2 patients with Caroli's disease received liver transplantation as initial surgery, with good outcomes. All patients had intractable symptoms caused by liver cirrhosis, and growth delay was considerable in patients aged <20 years. Histopathological analysis of the native liver revealed hepatic fibrosis (≥F2). One patient with ABO incompatibility died. One patient with Caroli's disease accompanied with intrahepatic carcinoma survives 11.8 years after liver transplantation without any recurrences. CONCLUSIONS: Patients with congenital biliary dilatation with refractory symptoms and complications secondary to liver failure are appropriate candidates for liver transplantation. We suggest that liver transplantation is an effective therapeutic option for patients with congenital biliary dilatation with due consideration to many accompanying factors, such as clinical course, growth delay, image findings and histopathological analysis.

Comprehensive and innovative techniques for liver transplantation in rats: a surgical guide.

AIM: To investigate our learning curves of orthotopic liver transplantation (OLT) in rats and the most important factor for successful surgery. METHODS: We describe the surgical procedures for our rat OLT model, and determined the operator learning curves. The various factors that contributed to successful surgery were determined. The most important surgical factors were evaluated between successful and unsuccessful surgeries. RESULTS: Learning curve data indicated that 50 cases were required for operator training to start a study. Operative time, blood loss, warm ischemic time, anhepatic phase, unstable systemic hemodynamic state, and body temperature after surgery significantly affected surgery success by univariate analysis, while the anhepatic phase was the most critical factor for success by multivariate analysis. CONCLUSION: OLT in rats is the only liver transplantation model that provides clinically relevant and reliable results. Shortened anhepatic phase is key to success in this model.

Analysis of donor- and isolation-related variables from non-heart-beating donors (NHBDs) using the Kyoto islet isolation method.

Recently, we demonstrated that islet transplantation from non-heart-beating donors (NHBDs) using the Kyoto islet isolation method (KIIM) successfully reversed patients' diabetes state. In this study, we evaluated the effects of donor- and isolation-related variables on islet isolation results from NHBDs by KIIM. Twenty-one islet preparations from the pancreata of NHBDs were isolated by KIIM. Islet preparations that met transplantation criteria and achieved improved patient diabetes control after transplantation were defined as successful isolations. Potential risk factors deemed to affect islet isolation results, such as age, gender, body mass index, hospital stay, donors' blood biochemical tests, a modified pancreata procurement method, and isolation and purification procedure-related variables, were analyzed. Seventeen out of 21 islet isolations (81%) were successful isolations. Postpurification islet yield was 447,639 +/- 39,902 islet equivalents (IE) in the successful isolation group and 108,007 +/- 31,532 IE in the failure group. Donor age was significantly younger in the success group (41.9 +/- 4.0 years old in the success group vs. 57.5 +/- 2.2 years old in the failure group, p = 0.003). Chronic pancreatitis significantly decreased islet yields (p = 0.006). Phase I time was significantly shorter (p = 0.010) and undigested tissue volume was significantly smaller (p = 0.020) in the success group. Purity was in positive correlation to postpurification islet yield, while donor age was in reverse correlation to postpurification islet yield. KIIM enables us to perform islet transplantation from NHBDs; however, the decision to use pancreata from older donors or those with chronic pancreatitis requires careful consideration.

Living donor islet transplantation, the alternative approach to overcome the obstacles limiting transplant.

We performed the world's first successful living donor islet transplantation for unstable diabetes. A total of 408,114 islet equivalents were isolated from half a living pancreas and transplanted immediately to the recipient who was a 27-year-old female. The donor was a 56-year-old female in good health, mother of the recipient. The islets functioned immediately, and the recipient was weaned completely from insulin on the 22nd posttransplant day, and has maintained excellent glycemic control since. The donor was discharged on the 18th postoperative day with normal oral glucose tolerance test and without complications. Living donor islet transplantation could cure one insulin-dependent diabetes mellitus patients with a single donor. There are some advantages in the living donor islet transplantation: (a) living donor can alleviate the issue of donor shortage; (b) highly potent islets can be isolated from a living donor; and (c) the recipient can be treated with immunosuppressant and controlled blood glucose level tightly prior to the transplantation. These are important factors in overcoming the obstacles limiting islet transplantation. We believe that the living donor islet transplantation may become an additional option in treating insulin-dependent diabetes.

A new mouse model for intraportal islet transplantation with limited hepatic lobe as a graft site.

Intraportal site is the standard for grafting in clinical islet transplantation. In the mouse model, the whole liver has been used as the grafting site to mimic clinical islet transplantation. However, this model lacks the potency to directly assess the contribution of the islet graft to diabetes control. Only demonstrating the immediate recurrence of diabetes in a surviving recipient after the removal of the islet graft can validate this assessment. In this study, we develop a mouse model of intraportal islet transplantation equipped with the potency of this assessment by injecting islets selectively into the right hepatic lobe under temporal clamp of the left portal vein. The mouse of this model survives after the right hepatectomy by which the islet graft is removed. This model can be applied to investigate both the specific graft-recipient interaction in the liver and the islet graft contribution to the control of diabetes.

Procurement of the human pancreas for pancreatic islet transplantation from marginal cadaver donors.

BACKGROUND: Recent advances in pancreatic islet transplantation (PIT) have contributed significantly to the treatment of patients with type 1 diabetes. The specific aim of this study was to develop an effective technique for the procurement of pancreas for PIT from nonheart-beating-donor (NHBDs). METHODS: Between January 2004 and August 2004, eight human pancreata were procured and processed for isolation of islets at a cell processing center. After confirmation of brain death status, a double balloon catheter was inserted to prevent warm ischemic damage to the donor pancreas by using an in situ regional organ cooling system that was originally developed for procurement of kidneys. The catheter position of the cooling system was modified specifically for the pancreas and kidney. Furthermore, we worked in cooperation with a kidney procurement team to protect the pancreas during kidney procurement. RESULTS: Warm ischemic time could be controlled with the modified in situ regional cooling system at 3.0 +/- 0.8 min (mean +/- SE). The operations for procurement of the kidneys and pancreata lasted 45.6 +/- 3.6 min and 10.6 +/- 1.8 min, respectively. Islet yield per isolation was 444,426 +/- 35,172 IE (islet equivalent). All eight cases met the criteria for PIT based on the Edmonton protocol. CONCLUSION: We developed a novel procurement technique in cooperation with our kidney procurement team. This protocol for the procurement of pancreas and kidney from a NHBD enabled us to transplant islets into a type 1 diabetic patient and kidney into a renal failure patient.

Successful islet transplantation from nonheartbeating donor pancreata using modified Ricordi islet isolation method.

BACKGROUND: Current success of islet transplantation has led to donor shortage and the need for marginal donor utilization to alleviate this shortage. The goal of this study was to improve the efficacy of islet transplantation using nonheartbeating donors (NHBDs). METHODS: First, we used porcine pancreata for the implementation of several strategies and applied to human pancreata. These strategies included ductal injection with trypsin inhibitor for protection of pancreatic ducts, ET-Kyoto solution for pancreas preservation, and Iodixanol for islet purification. RESULTS: These strategies significantly improved both porcine and human islet isolation efficacy. Average 399,469+/-36,411 IE human islets were obtained from NHBDs (n=13). All islet preparations met transplantation criteria and 11 out of 13 cases (85%) were transplanted into six type 1 diabetic patients for the first time in Japan. All islets started to secrete insulin and all patients showed better blood glucose control without hypoglycemic loss of consciousness. The average HbA1c levels of the six recipients significantly improved from 7.5+/-0.4% at transplant to 5.1+/-0.2% currently (P<0.0003). The average insulin amounts of the six recipients significantly reduced from 49.2+/-3.3 units at transplant to 11+/-4.4 units (P<0.0005) and five out of six patients reduced to less than half dose. The first patient is now insulin free, the first such case in Japan. CONCLUSION: This demonstrates that our current protocol makes it feasible to use NHBDs for islet transplant into type 1 diabetic patients efficiently.

A single transplantation of the islets can produce glycemic stability and reduction of basal insulin requirement.

We investigated glycemic stability and insulin requirement 1 month after a single transplantation of the islets from non-heart-beating donors or a living donor. Overall blood glucose levels decreased immediately after transplantation. The M-value and mean amplitude of glycemic excursions (MAGE) decreased significantly from 53.0 (range, 8.9-91.0) to 4.2 (0.6-8.8, P<0.05) and from 8.5 mM (4.8-11.7) to 3.3 mM (2.0-4.5, P<0.05), respectively. The values after transplantation were lower than the first quartile of 102 type 2 diabetic control patients. The estimated HbA1c level decreased significantly from 7.9% (5.7-10.9) to 5.4% (4.7-5.9, P<0.05). The supplement of basal insulin decreased 43% from 0.31 units/kg/day (0.16-0.37) to 0.18 units/kg/day (0-0.22, P<0.05), while that of stimulated insulin did not decrease significantly, from 0.28 units/kg/day (0.13-0.51) to 0.21 units/kg/day (0-0.41). Thus, only one islet transplantation can be sufficient to attain metabolic stability, probably by effective supply of basal insulin secretion, sufficient to avoid life-threatening severe hypoglycemia and prevent or delay the progress of secondary complications of diabetes by decreasing the HbA1c level.

Pancreatic islet transplantation for treating diabetes.

Pancreatic islet transplantation is one of the options for treating diabetes and has been shown to improve the quality of life of severe diabetic patients. Since the Edmonton protocol was announced, islet transplantation have advanced considerably, including islet after kidney transplantation, utilisation of non-heart-beating donors, single-donor islet transplantation and living-donor islet transplantation. These advances were based on revised immunosuppression protocols, improved pancreas procurement and islet isolation methods, and enhanced islet engraftment. Further improvements are necessary to make islet transplantation a routine clinical treatment. To synergise efforts towards a cure for type 1 diabetes, a Diabetes Research Institute (DRI) Federation is currently being established to include leading diabetes research centres worldwide, including DRIs in Miami, Edmonton and Kyoto among others.

Temporal decline in sirolimus elimination immediately after pancreatic islet transplantation.

Pancreatic islet transplantation is a curable treatment for type 1 diabetes and has been put into practice in various countries. In this study, we analyzed the pharmacokinetic characteristics of sirolimus and tacrolimus in six Japanese patients with pancreatic islet transplants immediately after surgery, and monitored efficacy and toxicity. The patients were treated with immunosuppressive therapy based on the Edmonton protocol, that is, sirolimus and low-dose tacrolimus. Pharmacokinetic analyses were performed using the nonlinear mixed-effects modeling program NONMEM. Large inter- and intra-individual variability was observed in the pharmacokinetics of sirolimus and tacrolimus. A model with increased apparent clearance in the postoperative period explained well the intra-individual variability in the pharmacokinetics of both drugs. The most frequent drug-induced toxicity was a decrease in the white blood cell count, and two of six patients required the administration of granulocyte colony-stimulating factor. Clinical laboratory tests immediately before the transplantation and cytochrome P450 3A5 genotype were not related to the high blood concentrations of sirolimus after the loading dose. From these results, the apparent clearance of sirolimus and tacrolimus might temporally decline immediately after pancreatic islet transplantation. A high trough concentration of sirolimus might increase the risk of hematological toxicy, and adjustment of the dosage for immunosuppressive treatment will be necessary in Japanese patients.

Follow-up study of the first successful living donor islet transplantation.

BACKGROUND: Islet transplantation has become an option for the treatment of insulin-dependent diabetes mellitus and is usually performed using brain-dead heartbeating donors. However, we have very limited number of such donors in Japan; therefore, it is not allowed to perform islet transplantation with brain-dead donors. In order to perform islet transplantation in Japan, we need to seek new donor resources. METHODS: We performed the first successful living-donor islet transplantation. In this case, the recipient had brittle diabetes with hypoglycemic unawareness. The donor was deemed qualified after undergoing both metabolic and preoperative assessments. Distal pancreatectomy was performed using open laparotomy and more than 400,000 islets were isolated and transplanted immediately. RESULTS: The recipient has been insulin independent posttransplant with positive C-peptide for more than one year. She no longer suffers from hypoglycemic unawareness and displayed a substantial improvement in hemoglobulin (Hb) A1C. The donor's clinical course was uneventful, which allowed her to return to her job within one month. She maintained normal fasting C-peptide and HbA1C levels during follow-up period. CONCLUSION: In our first case of living donor islet transplantation, both the donor and the recipient have been maintaining excellent glycemic control with no untreatable complications for more than one year.

Cell permeable peptide of JNK inhibitor prevents islet apoptosis immediately after isolation and improves islet graft function.

Although application of the Edmonton protocol has markedly improved outcomes for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has proven to remain low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Pancreas preservation with the two-layer method (TLM) has proven to improve transplant results by protecting isolated islets against apoptosis through the mitochondrial pathway. However, pancreas storage with TLM cannot protect against activation of c-Jun NH2-terminal kinase (JNK) in isolated islets. This study investigated whether delivery of a JNK inhibitory peptide (JNKI) via the protein transduction system can prevent apoptosis of islet cells immediately after isolation. For efficient delivery of the (JNKI into isolated islets, we synthesized JNKI as a C-terminal fusion peptide with the 11-arginine protein transduction domain (11R-JNKI). 11R efficiently delivered the JNKI into isolated islets and 11R-JNKI prevented islet apoptosis immediately after isolation and improved islet graft function. These findings suggest that peptide drugs could be useful for the prevention of the impairment of islet cells and lead to improvement in the outcomes for pancreatic islet transplantation.

Insulin independence after living-donor distal pancreatectomy and islet allotransplantation.

Rising demand for islet transplantation will lead to severe donor shortage in the near future, especially in countries where cadaveric organ donation is scarce. We undertook a successful transplantation of living-donor islets for unstable diabetes. The recipient was a 27-year-old woman who had had brittle, insulin-dependent diabetes mellitus for 12 years. The donor, who was a healthy 56-year-old woman and mother of the recipient, underwent a distal pancreatectomy. After isolation, 408 114 islet equivalents were transplanted immediately. The transplants functioned immediately and the recipient became insulin-independent 22 days after the operation. The donor had no complications and both women showed healthy glucose tolerance. Transplantation of living-donor islets from the distal pancreas can be sufficient to reverse brittle diabetes.

Impact of right lobe with middle hepatic vein graft in living-donor liver transplantation.

Technical improvements in adult-to-adult living-donor liver transplantation (LDLT) have led to the use of right-lobe grafts to overcome the problems encountered with 'small-for-size grafts'. The major controversy remains that the venous drainage from anterior segment substantially depends on tributaries of the middle hepatic vein (MHV), and deprivation of such tributaries may critically influence the postoperative graft function. Right-lobe grafts with MHV could resolve the potential problem of congestion in anterior segment. From December 2000 to January 2004, we performed 217 right-lobe LDLTs for adult patients. Of these, 40 patients received a right lobe with MHV graft (18.4%). The overall cumulative 3-year graft survival rate of a right lobe with (n = 40) and without MHV (n = 177) was 86.2% and 74.8% (p = NS). The proximal side of the MHV and the drainage vein of segment IV to the MHV (the left medial superior vein) were preserved in 24 patients. All of them needed venous interposition graft for anastomosis. All patients had a patent right hepatic vein (RHV) and MHV anastomosis during the follow-up period. We adopted the right lobe with MHV graft in 40 LDLT cases. Vein graft is essential for safe MHV anastomosis in cases which preserve proximal side of the MHV.