Antonio Gangemi

Islet Transplantation for Brittle Type 1 Diabetes: The UIC Protocol

This prospective phase 1/2 trial investigated the safety and reproducibility of allogeneic islet transplantation (Tx) in type I diabetic (T1DM) patients and tested a strategy to achieve insulin‐independence with lower islet mass. Ten C‐peptide negative T1DM subjects with hypoglycemic unawareness received 1–3 intraportal allogeneic islet Tx and were followed for 15 months. Four subjects (Group 1) received the Edmonton immunosuppression regimen (daclizumab, sirolimus, tacrolimus). Six subjects (Group 2) received the University of Illinois protocol (etanercept, exenatide and the Edmonton regimen). All subjects became insulin‐ independent. Group 1 received a mean total number of islets (EIN) of 1460 080 ± 418 330 in 2 (n = 2) or 3 (n = 2) Tx, whereas Group 2 became insulin‐ independent after 1 Tx (537 495 ± 190 968 EIN, p = 0.028). All Group 1 subjects remained insulin free through the follow‐up. Two Group 2 subjects resumed insulin: one after immunosuppression reduction during an infectious complication, the other with exenatide intolerance. HbA1c reached normal range in both groups (6.5 ± 0.6 at baseline to 5.6 ± 0.5 after 2–3 Tx in Group 1 vs. 7.8 ± 1.1 to 5.8 ± 0.3 after 1 Tx in Group 2). HYPO scores markedly decreased in both groups. Combined treatment of etanercept and exenatide improves islet graft function and facilitates achievement of insulin‐independence with less islets.

Successful rescue of refractory, severe antibody mediated rejection with splenectomy

Antibody-mediated rejection (AMR) commonly occurs after transplantation of ABO-incompatible and sensitized renal transplant. Treatment regimens commonly include a combination of plasmapheresis (PL) and intravenous immunoglobulin (IVIG). However, some cases of AMR remain refractory to treatment. We report a case series of four patients with AMR refractory to standard therapy (ST) who resolved after splenectomy. Four living donor kidney transplant recipients were diagnosed with AMR. Two patients were ABO incompatible, one was cross-match positive and one had no obvious predisposing factors. After failure of therapy with corticosteroids, PL, IVIG, Thymoglobulin, and Rituximab (three patients) or Campath (one patient), AMR was treated with laparoscopic splenectomy. After an average of 11 days of ST, laparoscopic splenectomy was performed for rescue. The urinary output improved immediately in all patients, serum creatinine levels decreased within 48 hr, and ABO titers fell in the ABO-incompatible patient and the cross-match became negative in the two sensitized patients. Splenectomy may play a role in the treatment of AMR refractory to ST.

Encapsulation of Human Islets in Novel Inhomogeneous Alginate-Ca2+/Ba2+ Microbeads : In Vitro and In Vivo Function

Microencapsulation may allow for immunosuppression-free islet transplantation. Herein we investigated whether human islets can be shipped safely to a remote encapsulation core facility and maintain in vitro and in vivo functionality. In non-encapsulated islets before and encapsulated islets after shipment, viability was 88.3±2.5 and 87.5±2.7% (n=6, p=0.30). Stimulation index after static glucose incubation was 5.4±0.5 and 6.3±0.4 (n=6, p=0.18), respectively. After intraperitoneal transplantation, long-term normoglycemia was consistently achieved with 3,000, 5,000, and 10,000 IEQ encapsulated human islets. When transplanting 1,000 IEQ, mice returned to hyperglycemia after 30-55 (n=4/7) and 160 days (n=3/7). Transplanted mice showed human oral glucose tolerance with lower glucose levels than non-diabetic control mice. Capsules retrieved after transplantation were intact, with only minimal overgrowth. This study shows that human islets maintained the viability and in vitro function after encapsulation and the inhomogeneous alginate-Ca2+/Ba2+ microbeads allow for long-term in vivo human islet graft function, despite long-distance shipment.

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.

Lessons learned in pediatric small bowel and liver transplantation from living-related donors.

Background. Children are the primary candidates for intestinal transplant with more than 70% requiring a combined liver-bowel transplant. We report our single-center experience with living donor intestinal transplantation (LDITx) and combined living donor intestinal and liver transplant (CLDILTx) in pediatric patients. Patients and Methods. Between October 2002 and June 2006, 13 living donor intestinal grafts were transplanted in 10 recipients. In five cases CLDILTx was performed. The intestinal grafts consisted of a 150-cm segment of ileum, whereas the liver transplant was completed using standard left lateral grafts. Results. No complications occurred in any donors. In CLDILTx recipients, the patient survival at 1 and 2 years was 100%, the liver graft survival 100%, and the bowel graft survival 80%; the patient who lost the initial intestinal graft was successfully retransplanted. In LDITx recipients, the patient and graft survival at 1 and 3 years were 60% and 50%, respectively. Two isolated LDITx recipients, both 6 months of age and low body weight (mean, 6 vs. 9 kg) died within 4 months posttransplant. One LDITx recipient developed chronic rejection 3.5 years after the original transplant and died after retransplant. All patients who are alive with functioning grafts are currently on full enteral feeding without need for any intravenous supplementation, except for a recipient of CLDILTx, currently on total parenteral nutrition for late fistula. Conclusions. The early outcomes of intestinal transplantation from living donors are promising, particularly for candidates in need of CLDLITx. In this subgroup, the elimination of the high mortality on the cadaver waiting list (∼30%) represents a substantial advantage.

Adult living-donor liver transplantation with ABO-incompatible grafts

Background. Liver transplantation using ABO-incompatible grafts is rarely performed because the reported outcome is poorer than with compatible grafts. We report our positive experience with adult-to-adult living-donor liver transplant (LDLT) using ABO-incompatible grafts. Methods. The immunosuppressive protocol consisted of plasmapheresis/intravenous immunoglobulin infusion before LDLT followed by thymoglobulin induction and splenectomy, maintenance with tacrolimus/cyclosporine (FK/CSA), mycophenolate mofetil, and a rapid steroid taper. Plasmapheresis was planned for up to 3 months after LDLT aiming at maintaining the anti-ABO titers level below 1:16. Liver biopsies were routinely stained for humoral rejection with complement 4d (C4d) and for biliary damage with cytokeratin 7. Results. Between January 2003 and September 2004, five patients, mean age 59 years, received an ABO-incompatible LDLT. Patient and graft survival was 80% at mean follow-up of 43 months (range, 34–54) for the four surviving patients. One patient died 4 months after LDLT. Humoral rejection occurred in one patient whereas acute cellular rejection was diagnosed in four patients. Conclusions. ABO-incompatible LDLT can be performed with patient and graft survival similar to compatible LDLT. Minimization of immunosuppression is possible, and chronic biliary damage is not the norm. Better tools than complement 4d staining must be researched to diagnose the features of immunologic damage to the graft. If these results will be confirmed in a greater number of patients, ABO-incompatible LDLT may be proposed when ABO-compatible donors are not available or when the ABO-incompatible donor is the better candidate.

Use of low dose tacrolimus, mycophenolate mofetil and maintenance IL-2 receptor blockade in an islet transplant recipient.

Abstract:  The utilization of dual maintenance therapy with tacrolimus and sirolimus (the Edmonton protocol) has been widely adopted as standard immunosuppression for islet cell transplantation. This immunosuppression regimen has numerous toxicities including renal dysfunction, anemia, and recurrent aphthous ulcers. We present a case of a 63‐yr‐old Caucasian female who received an isolated islet transplant. Over the first six months post‐transplant, the patient developed severe anemia, intractable aphthous ulcers, and renal dysfunction. Islet transplant function was excellent and the patient is insulin‐independent since the end of the second month post‐transplant. However, because of the above toxicities, a decision was made to change her immunosuppression regimen eight months post‐transplant to low dose tacrolimus, mycophenolate mofetil, and a monthly maintenance infusion of daclizumab. Since then, her aphthous ulcers have disappeared, renal function has improved, and islet cell function remains stable.

Increased albumin concentration reduces apoptosis and improves functionality of human islets.

Providing sufficient islet mass is important for successful islet transplantation. Apoptosis plays a major role in post-isolation islet cell death, and prevention of apoptosis could improve transplant outcomes. The purpose of this study was to determine whether increased concentration of human albumin (HA) in pre-transplantation culture of human islets would reduce apoptosis. Human islets were cultured in CMRL with 1.5 or 5% of HA for 24 h and apoptosis was evaluated indirectly by measuring caspase 3 activity and tetramethylrhodamine-ethyl-ester (TMRE) in dissociated islets. Islet function and viability were evaluated. Islets cultured in higher albumin concentration presented with lower caspase 3 activity (43.9 ± 3.9 vs. 67.4 ± 11.1, p = 0.011), and had increased insulin secretory capacity (Stimulation index 3.76 ± 0.91 vs 1.23 ± 0.21, p = 0.023). We conclude that an increase in albumin concentration can prevent apoptosis in isolated human islets. These findings may have implications for islet transplant outcomes.

Excellent outcome using an alternative technique for arterial reconstruction in living-related liver transplant: sphenoid anastomosis

Thrombosis of the hepatic artery (HAT) after living-related liver transplantation (LRLT) is a significant cause of graft loss. Microsurgical methods can decrease the incidence of HAT between 0% and 2% [1]. We use alternative technique, which does not require an operating microscope. From February 2002 to October 2005, 35 adult LRLTs and four pediatric LRLTs were performed for end-stage liver disease. Twenty recipients were male and 15 female, mean age 51 years (range: 18–74). The four pediatric patients, all younger than 6 months and below 10 kg of weight, underwent combined living donor liver/small bowel transplant for short gut syndrome and Total Parenteral Nutrition induced end-stage liver disease. The size of each pediatric hepatic artery was between 2 and 2.5 mm. The recipient operation has been described in detail elsewhere [2,3]. The arterial anastomosis is performed with the aid of 4· surgical loupes. The arteries are rinsed with heparin. Longitudinal arteriotomies in the recipient and donor vessels are performed. The arteriotomy in the donor artery is performed in the posterior wall, with the length of incision approximately equal to the diameter of the artery. A mirror arteriotomy is performed in the anterior wall of the recipient artery (Fig. 1). The cross-section of each vessel resembles a wedge, hence the term sphenoid. The anastomosis is performed with running 7/0 Prolene. The first stitch is placed from the apex of the graft arteriotomy to the middle of the posterior wall of the recipient artery (Fig. 1). The back wall is sutured first from the ‘inside’ with perfect visualization of the intima. The suture is run in both directions beginning with a ‘parachute’ technique until the corners of the arteriotomy on each side are reached. No corner stitches must be placed as the two ends of the running posterior wall suture can be placed on slight tension and open the field for the suturing of the anterior wall (Fig. 2). Then, a second 7/0 Prolene is placed from the apex of the recipient arteriotomy to the mid portion of the anterior wall of the artery of the graft. The suture is run in both directions to join the suture run on the opposite wall. Arterial flow is reestablished before the sutures are tied to allow further expansion at the anastomosis site and avoid a ‘pursestring’ effect. Thirty-five donors underwent right liver and four left lateral hepatectomy. In the adult recipients, the arterial anastomosis was constructed 14 times with the Right Hepatic artery of the recipient; 14 times with the Proper Hepatic artery; three times each with a Replaced Hepatic artery and a patch of the Gastroduodenal-Common Hepatic artery and in one re-transplant with an arterial conduit from the aorta. In four cases, an interposition venous or arterial conduit was used. In the pediatric recipients, the anastomsosis was invariably performed using the Left Hepatic artery of the graft and the Proper Figure 1 Schematic presentation of how the anastomosis is started. The suture is placed between the apex of the graft vessel’s arteriotomy and the hepatic artery of the graft.