James Shapiro

Long-term outcome of living related liver transplantation for patients with intrapulmonary shunting and strategy for complications

BACKGROUND In 320 living related liver transplantation performed between June 1990 and September 1997, there were 21 living related liver transplantation for patients with intrapulmonary shunting, manifested by digital clubbing, cyanosis, and dyspnea. We report the long-term outcome for more 6 months and our strategy to overcome complications in these recipients. PATIENTS A total of 21 patients (age range 2-33 years, 19 children and 2 adults, 6 males and 15 females) were classified into three grades according to shunt ratio calculated by TcMAA pulmonary scintigraphy; 5 in mild group (shunt ratio: less than 20%), 6 in moderated group (20%-40%), and 10 in severe group (more than 40%). The original underlying liver disease was biliary atresia in all patients. RESULTS Spearmens correlation coefficient rank test revealed that shunt ratio correlated significantly with PaO2 in room air (P=0.0001), PaO2 in 100% oxygen (P=0.0004), hematocrit (P=0.0276), and period of dyspnea before transplantation (P=0.023). COMPLICATIONS Wound infection occurred in 80, 66, and 80%, and bile leakage in 20, 0, 40% in mild, moderate, and severe group, respectively. Patients who had portal vein thrombosis, and intracranial complication were classified as severe group and the incidence was 20 and 20%, respectively. The patient actuarial one year survival was 80, 66.7, and 48%, in mild, moderate, and severe group, respectively, although there was no significant difference. All patients who survived improved hepatopulmonary syndrome and the length of period required for the resolution was significantly correlated to the preoperative shunt ratio (P=0.023). COMMENTS Patients with severe shunting are susceptible to wound infection and bile leak. The trend of higher incidence of portal thrombosis and intracranial complications in the severe group was closely related high hematocrit. Secure surgical technique to reduce bile leak and delayed primary wound closure to reduce wound infection were found to be effective. Anticoagulant therapy by infusing heparin through the portal vein followed by coumadin could prevent fatal portal vein thrombosis without counter risk of fatal cerebral hemorrhage.

The Standardization of Pancreatic Donors for Islet Isolations

Background. Islet transplantation has proven to be a successful treatment for type 1 diabetes mellitus. The aim of this study was to establish an algorithm by which the combination of the donor quality and pancreas quality was given a numerical score from 0 to 100 for use in determining the quality of a pancreas for islet isolation. Methods. We retrospectively analyzed 326 pancreases and the outcomes of their respective isolations. Specific donor variables and physical characteristics were identified and weighted according to their influence on the success of the isolation. For each variable, ranges and point weightings were established based on our laboratory experience and literature review. Results. Analysis of the data showed a strong association of the donor point with isolation outcome. Pancreases with lower donor point scores had lower transplant success rates, whereas higher donor point scores in turn produced higher transplant rates. Conclusions. This scoring system has proven to be useful in guiding the decision process as to whether to accept a particular pancreas for a favorable isolation outcome.

In vitro and in vivo improvement of islet survival following treatment with nerve growth factor.

Background. Nerve growth factor (NGF) has been reported to play an important regulatory role in pancreatic beta-cell function. However, the usefulness of NGF in a transplantation setting is unknown. Methods. A marginal number of islet cells (260 islet equivalents/recipient) cultured for 24 hr with NGF (500 ng/ml) was syngeneically transplanted under the kidney capsule of streptozotocin-induced diabetic Balb/c mice. Fluorescence microscopy was used to evaluate islet viability. Islet function was evaluated in vitro and in vivo by static assay and glucose tolerance test, respectively. Results. In vitro, improved viability and survival were found in murine islets cultured in serum-free medium for 96 hr with 500 ng/ml NGF (P<0.05). NGF-treated islets had more insulin secretion than islets cultured without NGF in response to 2.8 mmol/L glucose (P<0.05), and 20 mmol/L glucose conditions. In vivo, 67% of recipients with a submarginal number of islets cultured in NGF attained normoglycemia for more than 120 days, whereas transplanted islets without NGF treatment survived a maximum of 13 days in control mice. At posttransplant day 4, recipients of NGF-cultured islets showed significant improvement of glucose tolerance. On immunohistochemistry, the kidney capsules containing NGF-cultured islets displayed higher insulin content, and more dilated neoplastic microvessels than control renal capsules. The number of apoptotic cells using TUNEL staining decreased by nearly 50% in NGF-cultured islet grafts in comparison to control islet grafts. Conclusions. The above data suggest potential advantages of NGF for islet survival following transplantation. This neurotrophic approach may prove beneficial in human islet transplantation.

Recognition and Killing of Human and Murine Pancreatic b Cells by the NK Receptor NKp46

Type 1 diabetes is an incurable disease that is currently treated by insulin injections or in rare cases by islet transplantation. We have recently shown that NKp46, a major killer receptor expressed by NK cells, recognizes an unknown ligand expressed by β cells and that in the absence of NKp46, or when its activity is blocked, diabetes development is inhibited. In this study, we investigate whether NKp46 is involved in the killing of human β cells that are intended to be used for transplantation, and we also thoroughly characterize the interaction between NKp46 and its human and mouse β cell ligands. We show that human β cells express an unknown ligand for NKp46 and are killed in an NKp46-dependent manner. We further demonstrate that the expression of the NKp46 ligand is detected on human β cells already at the embryonic stage and that it appears on murine β cells only following birth. Because the NKp46 ligand is detected on healthy β cells, we wondered why type 1 diabetes does not develop in all individuals and show that NK cells are absent from the vicinity of islets of healthy mice and are detected in situ in proximity with β cells in NOD mice. We also investigate the molecular mechanisms controlling NKp46 interactions with its β cell ligand and demonstrate that the recognition is confined to the membrane proximal domain and stalk region of NKp46 and that two glycosylated residues of NKp46, Thr125 and Asn216, are critical for this recognition.

Intra‐Ductal Glutamine Administration Reduces Oxidative Injury During Human Pancreatic Islet Isolation

Oxidative stress during islet isolation induces a cascade of events injuring islets and hampering islet engraftment. This study evaluated islet isolation and transplantation outcomes after intra‐ductal glutamine administration. Human pancreata deemed unsuitable for pancreas or islet transplantation were treated with either a 5 mM solution of l‐glutamine (n = 6) or collagenase enzyme alone (n = 6) through the main pancreatic duct. Islet yield, viability, in vitro function; markers of oxidative stress [malondialdehyde (MDA) and Glutathione (GSH)] and apoptosis were assessed. Islet yields were significantly increased in the glutamine group compared to controls (318, 559 ± 25, 800 vs. 165, 582 ± 39, 944 mean ± SEM, p < 0.01). The amount of apoptotic cells per islet was smaller in the glutamine group than the control. The percentage of nude mice rendered normoglycemic with glutamine‐treated islets was higher than the controls (83% n = 10/12 vs. 26% n = 6/23; p < 0.01), and the time to reach normoglycemia was decreased in the glutamine group (1.83 ± 0.4 vs. 7.3 ± 3 days; p < 0.01). Glutamine administration increased GSH levels (7.6 ± 1.7 nmol/mg protein vs. 4.03 ± 0.5 in control, p < 0.05) and reduced lipid‐peroxidation (MDA 2.45 ± 0.7 nmol/mg of protein vs. 6.54 ± 1.7 in control; p < 0.05). We conclude that intra‐ductal administration of glutamine reduces oxidative injury and apoptosis and improves islet yield and islet graft function after transplantation.

Improved outcomes in islet isolation and transplantation by the use of a novel hemoglobin-based O2 carrier.

During isolation, islets are exposed to warm ischemia. In this study, intraductal administration of oxygenated polymerized, stroma‐free hemoglobin‐pyridoxalated (Poly SFH‐P) was performed to improve O2 delivery. Rat pancreata subjected to 30‐min warm ischemia were perfused intraductally with collagenase in oxygenated Poly SFH‐P/RPMI or RPMI (control). PO2 was increased by Poly SFH‐P (381.7 ± 35.3 mmHg vs. 202.3 ± 28.2, p = 0.01) and pH maintained within physiological range (7.4–7.2 vs. 7.1–6.6, p = 0.009). Islet viability (77%± 4.6 vs. 63%± 4.7, p = 0.04) was improved and apoptosis lower with Poly SFH‐P (caspase‐3: 34,714 ± 2167 vs. 45,985 ± 1382, respectively, p = 0.01). Poly SFH‐P improved islet responsiveness to glucose as determined by increased intracellular Ca2+ levels and improved insulin secretion (SI 5.4 ± 0.1 vs. 3.1 ± 0.2, p = 0.03). Mitochondrial integrity was improved in Poly SFH‐P‐treated islets, which showed higher percentage change in membrane potential after glucose stimulation (14.7%± 1.8 vs. 9.8 ± 1.4, respectively, p < 0.05). O2 delivery by Poly SFH‐P did not increase oxidative stress (GSH 7.1 ± 2.9 nm/mg protein for Poly SFH‐P vs. 6.8 ± 2.4 control, p = 0.9) or oxidative injury (MDA 1.8 ± 0.9 nmol/mg protein vs. 6.2 ± 2.4, p = 0.19). Time to reach normoglycemia in transplanted diabetic nude mice was shorter (1.8 ± 0.4 vs. 7 ± 2.5 days, p = 0.02), and glucose tolerance improved in the Poly SFH‐P group (AUC 8106 ± 590 vs. 10,863 ± 946, p = 0.03). Oxygenated Poly SFH‐P improves islet isolation and transplantation outcomes by preserving mitochondrial integrity.

Surgical aspects of human islet isolation

Islet transplantation is a safe and effective procedure; however, it depends on the critical step of isolating high quality of islets from whole pancreas. Human islet isolation requires considerable experience and expertise, and is frequently seen as ‘an art rather than a science’. However, without scientific knowledge of pancreatic anatomy, real experience can not be gained. In particular, an understanding ductal anatomy is important to perform human islet isolation. This review is based on clinical experience of more than 900 human islet isolations performed over 10 years and aims to highlight pancreatic anatomy and surgical techniques in islet processing.

Multicentric outcome analysis of sirolimus‐based immunosuppression in 252 liver transplant recipients

The use of sirolimus (SRL) in orthotopic liver transplantation (OLT) has been controversial after experimental data suggested an increased risk of hepatic artery thrombosis (HAT). To assess the safety and efficacy of SRL as de novo immunosuppression in OLT recipients. Outcomes of 252 OLT patients who received SRL were compared with outcomes of 291 OLT recipients who received calcineurin inhibitor in a retrospective study. Primary outcomes of this study were: patient‐ and graft survivals, vascular, biliary, wound complications and rejection rates. Secondary outcomes were: postoperative infection rate, bone marrow and renal function and changes of lipid levels. Patient‐ and graft survivals, rejection and infection rates were similar. In the SRL group, HAT occurred in 1.2%, biliary complications in 19.4%, and incisional hernias in 9.1%. In the control group the incidence of HAT was 5.8% (P = 0.004), biliary complications 18.5% (P = NS) and incisional hernias 7.2% (P = NS). Patients on SRL experienced significantly higher levels of serum triglycerides but fewer acute cellular rejections. Bone marrow and renal functions were similar in both the groups. Our findings would suggest that SRL is safe and effective for very selected OLT recipients. Randomized controlled trials are necessary to confirm our results.

Portal vein thrombosis complicating islet transplantation in a recipient with the Factor V Leiden mutation.

A 31-year-old Native American/ white female with Type 1 diabetes mellitus of 26 years duration enrolled in the international multicenter trial of the Immune Tolerance Network (NS01) to replicate the Edmonton Protocol for islet transplantation. She used oral contraceptives. She had undergone two prior islet transplant procedures complicated by sirolimus induced mucositis, diarrhea, and anemia, treated with replacement of sirolimus with mycophenolate mofetil. She received a third transplant of 314,039 islets (4,906 islet equivalents per kg) with a purity of 30% in 9 ml of packed-cell volume in two aliquots through a 5-Fr cholangiogram catheter in the right branch of the portal vein. Each aliquot contained heparin, 35 u/kg of patient body weight. After one hour, she developed abdominal pain, hypotension, and anemia. Enoxaparin was withheld. An abdominal ultrasound on postprocedure day 1 revealed a perihepatic fluid collection consistent with blood and patent hepatic vessels without evidence of thrombosis. She received 4 units of packed red blood cells over 48 hours, and her hemoglobin stabilized. An abdominal ultrasound on postprocedure day 7 demonstrated a nonocclusive thrombus in the right portal vein, 30 mm in length (Fig. 1). She was anticoagulated with heparin and then warfarin. Serial ultrasounds revealed resolution of the thrombosis by 12 weeks after the event. A thrombophilia evaluation for antithrombin III, protein C and S, anticardiolipin antibodies, homocysteine, prothrombin gene mutation, and Factor V Leiden (FVL) revealed heterozygosity for the FVL mutation, and negative or normal otherwise. Subsequent genetic analysis of her mother revealed heterozygosity for FVL. Portal vein thrombosis after islet allotransplantation has been reported previ

Clinical Results After Islet Transplantation

After the discovery of insulin,1it was believed that diabetes was cured; however, the chronic complications of diabetes soon became evident. For the patient with Type 1 diabetes, the fine control of hyperglycemia to prevent retinopathy, nephropathy, neuropathy, or vascular disease has to be offset against the risk of hypoglycemia. The Diabetes Control and Complications Trial of the National Institute of Diabetes and Digestive and Kidney Diseases has unequivocally confirmed that hyperglycemia is associated with progressive complications, yet at the same time intensive insulin therapy has been associated with a three-fold increased risk of hypoglycemia.2These observations provide the stimulus for the development of alternative means of therapy. Although insulin analogues will undoubtedly facilitate this, to date they have not been associated with euglycemia. Pancreas transplantation alone has been associated with normoglycemia, but it is a major surgical procedure that is associated with significant morbidity, some mortality, and a one-year graft survival rate of about 86%.3,4Islet transplantation, on the other hand, is a simple procedure, but it has been associated with poor long-term success; at best, 10% of recipients have insulin independence at one year.5Last year we published preliminary results that confirmed that islet transplantation can be successful in establishing insulin independence,6and now we provide an update of these results as fully detailed in our recent report.7 Patients with Type 1 diabetes were selected for these studies. All had confirmed C-peptide absence and were selected on the basis of labile glucose values, as confirmed by a mean amplitude of glycemic excursion8of greater than 11.1 mmol/L, or by recurrent hypoglycemia, particularly if it is associated with decreased patient-awareness of hypoglycemia. In addition to labile glucose values, patients typically demonstrated disruption of their daily lives by diabetes. All patients must have …