Davide Mineo

Long-term insulin independence and improvement in insulin secretion after supplemental islet infusion under exenatide and etanercept.

Background. Progressive graft dysfunction (GDF) and loss of insulin independence (II) have been invariably observed in islet transplant recipients under the “Edmonton protocol.” To reestablish II, we performed supplemental islet infusions (SI) in recipients of allogeneic islet transplant alone, displaying GDF. To improve the engraftment and long-term graft function of SI, exenatide (EXN) and etanercept treatment at islet infusion, and long-term EXN treatment were tested in a non-randomized pilot clinical trial. Methods. Patients with GDF received SI under Edmonton-like immunosuppression with daclizumab induction, either without interventions (SI-control; n=5) or with EXN and etanercept treatment (SI-EXN; n=4). Clinical and metabolic profiles were assessed during 18-month follow-up. Results. Long-term II (18 months) was observed in 100% of SI-EXN and in 20% of SI-control (P=0.04). SI-EXN subjects demonstrated restoration of function better than that seen after initial islet infusions. Comparison of SI-EXN and SI-control groups demonstrated better responses in SI-EXN subjects at 3 months post-SI. During the 18 months of follow-up, function was sustained in the SI-EXN subjects better than in SI-controls. Acute effects of EXN during mixed meal tolerance test and intravenous glucose tolerance test results in improved first and second phase insulin release in response to intravenous glucose tolerance test and suppressed postprandial hyperglucagonemia after mixed meal tolerance test. Conclusion. These results suggest that the combination of EXN and etanercept improve engraftment and long-term islet survival and function in subjects undergoing SI. This data, however, must be interpreted with some caution because of small sample size, lack of randomization, and sequential comparison with historical controls.

Allosensitization of islet allograft recipients.

Background. The immune monitoring of islet transplant recipients includes the assessment of panel reactive antibodies (PRA). A negative association of PRA+ with allogeneic solid organ graft survival has been recognized, but scattered data is available for islet transplantation. Methods. We performed a retrospective analysis of PRA status in 66 patients with type 1 diabetes mellitus recipient of islet allografts between 1985 and 2006. Results. Pretransplant PRA+ was observed in 10 subjects in the old trials and associated with kidney transplantation and/or pregnancies. Thirteen subjects displayed PRA+ at follow-up, eight of whom were de novo. Overall, PRA+ did not correlate with islet graft outcome: long-term graft survival was observed in the presence of basal or persistent PRA+ and graft dysfunction occurred also in the absence of PRA+. Loss of graft function was associated with PRA+ after lowering of immunosuppression or after infection episodes. Loss of C-peptide did not affect kidney graft function even in simultaneous islet-kidney transplant recipients. Mostly, PRA remained negative under adequate immunosuppression. Patients whose immunosuppression was discontinued invariably developed PRA+. Conclusions. Monitoring of PRA under immunosuppression may have little clinical value under adequate immunosuppression in islet transplant recipients. The implications of allosensitization after discontinuation of immunosuppression need to be evaluated to define the real clinical impact in this patient population.

Islet transplantation with alemtuzumab induction and calcineurin-free maintenance immunosuppression results in improved short- and long-term outcomes.

Background. Only a minority of islet transplant recipients maintain insulin independence at 5 years under the Edmonton protocol of immunosuppression. New immunosuppressive strategies are required to improve long-term outcomes. Materials and Methods. Three subjects with unstable type 1 diabetes mellitus underwent islet transplantation with alemtuzumab induction and sirolimus-tacrolimus maintenance for 3 months and then sirolimus-mycophenolic acid maintenance thereafter. Follow-up was more than 2 years. Comparison was with 16 historical subjects transplanted under the Miami version of the Edmonton protocol. Results. Insulin independence was achieved in 2 of 3 alemtuzumab and 14 of 16 historical subjects. Those who did not achieve insulin independence only received a single islet infusion. Insulin-independence rates remained unchanged in the alemtuzumab group, but decreased from 14 of 16 (88%) to 6 of 16 (38%) in the historical group over 2 years. Insulin requirements increased in the historical group while remaining stable in the alemtuzumab group. Comparison of functional measures at 3 months suggested better engraftment with alemtuzumab (P=NS). Further comparison of alemtuzumab versus historical groups, up to 24 months, demonstrated significantly better: Mixed meal stimulation index (24 months, 1.0±0.08 [n=3] vs. 0.5±0.06 pmol/mL [n=6], P<0.01), mixed meal peak C-peptide (24 months, 5.0±0.5 [n=3] vs. 3.1±0.3 nmol/mL [n=6], P<0.05), HbA1c (24 months, 5.4±0.15 [n=3] vs. 6.3±0.12 pmol/mL [n=10], P<0.01). Administration of alemtuzumab was well tolerated. There was no increased incidence of infections in alemtuzumab subjects despite profound, prolonged lymphocyte depletion. Conclusions. Islet transplantation with alemtuzumab induction was well tolerated and resulted in improved short- and long-term outcomes. Further investigation is underway for validation.

Combined islet and hematopoietic stem cell allotransplantation: a clinical pilot trial to induce chimerism and graft tolerance.

To prevent graft rejection and avoid immunosuppression‐related side‐effects, we attempted to induce recipient chimerism and graft tolerance in islet transplantation by donor CD34+hematopoietic stem cell (HSC) infusion. Six patients with brittle type 1 Diabetes Mellitus received a single‐donor allogeneic islet transplant (8611 ± 2113 IEQ/kg) followed by high doses of donor HSC (4.3 ± 1.9 × 106 HSC/kg), at days 5 and 11 posttransplant, without ablative conditioning. An ‘Edmonton‐like’ immunosuppression was administered, with a single dose of anti‐TNFα antibody (Infliximab) added to induction. Immunosuppression was weaned per protocol starting 12 months posttransplant. After transplantation, glucose control significantly improved, with 3 recipients achieving insulin‐independence for a short time (24 ± 23 days). No severe hypoglycemia or protocol‐related adverse events occurred. Graft function was maximal at 3 months then declined. Two recipients rejected within 6 months due to low immunosuppressive trough levels, whereas 4 completed 1‐year follow‐up with functioning grafts. Graft failure occurred within 4 months from weaning (478 ± 25 days posttransplant). Peripheral chimerism, as donor leukocytes, was maximal at 1‐month (5.92 ± 0.48%), highly reduced at 1‐year (0.20 ± 0.08%), and was undetectable at graft failure. CD25+T‐lymphocytes significantly decreased at 3 months, but partially recovered thereafter. Combined islet and HSC allotransplantation using an ‘Edmonton‐like’ immunosuppression, without ablative conditioning, did not lead to stable chimerism and graft tolerance.

Long-term metabolic and hormonal effects of exenatide on islet transplant recipients with allograft dysfunction.

The initial success of islet transplantation (ITx) is followed by graft dysfunction (GDF) and insulin reintroduction. Exenatide, a GLP-1 agonist, increases insulin and decreases glucagon secretion and has potential for β-cell regeneration. To improve functional islet mass, exenatide treatment was given to ITx recipients with GDF. The objective of this study was to assess metabolic and hormonal effects of exenatide in GDF. In this prospective, single-arm, nonrandomized study, 11 type 1 diabetes recipients of ITx with GDF had HbA1c, weight, insulin requirements, and 5-h mixed meal tolerance test (MMTT; with/without exenatide given before test) at baseline, 3, 6, and 12 months after initiating exenatide treatment. Baseline MMTT showed postprandial hyperglycemia and hyperglucagonemia. Daily exenatide treatment resulted in improved glucose, increased amylin/insulin ratio, and decreased proinsulin/insulin ratio as assessed by MMTT. Glucagon responses remained unchanged. Exenatide administration 1 h before MMTT showed decreased glucagon and glucose at 0 min and attenuation in their postprandial rise. Time-to-peak glucose was delayed, followed by insulin, proinsulin, amylin, and C-peptide, indicating glucose-driven insulin secretion. Five subjects completed 12-month follow-up. Glucose and glucagon suppression responses after MMTT with exenatide were no longer observed. Retrospective 3-month analysis of these subjects revealed higher and sustained glucagon levels that did not suppress as profoundly with exenatide administration, associated with higher glucose levels and increased C-peptide responses. In conclusion, Exenatide suppresses the abnormal postprandial hyperglucagonemia and hyperglycemia observed in GDF. Changes in amylin and proinsulin secretion may reflect more efficient insulin processing. Different degrees of responsiveness to exenatide were identified. These may help guide the clinical management of ITx recipients.

Minimization and withdrawal of steroids in pancreas and islet transplantation

For reducing the corticosteroid (CS)‐related side‐effects, especially cardiovascular events, CS‐sparing protocols have become increasingly common in pancreas transplantation (PT). Lympho‐depleting induction antibodies, such as rabbit anti‐thymocyte globulin (rATG) or alemtuzumab, have been widely used in successful trials. The results of various CS‐sparing protocols combining calcineurin inhibitors (CNI) and mycophenolate or sirolimus, have been mixed for rejection and survival rates. Most of the studies were uncontrolled trials of low‐risk patients, therefore the grade of evidence is limited. Large‐scale prospective studies with long‐term follow up are necessary to assess risks and benefits of CS‐sparing regimens in PT before recommending such strategies as standard practice. Islet allo‐transplantation for patients with brittle type 1 diabetes mellitus, less invasive and safer procedure than PT, has been attempted since late 1980s, but diabetogenic immunosuppressants at maintenance, mainly CS and high‐dose CNI, prevented satisfactory results (10% insulin‐independence at 1‐year post‐transplant). Since 2000, CS‐free and CNI‐reducing protocols, including more potent induction [daclizumab, OKT3γ1(ala‐ala) anti‐CD3 antibody, rATG] and maintenance (sirolimus, mycophenolate) agents, have significantly improved short‐term outcomes whereas long‐term are still inadequate (from 80% to 20% insulin‐independence from 1‐ to 5‐year post‐transplant). Main limitations are allo‐ and autoimmunity, immunosuppression‐related islet and systemic toxicity and transplant site unsuitability, which tolerogenic protocols and biotechnological solutions may solve.

Immune profiling by multiple gene expression analysis in patients at-risk and with type 1 diabetes.

There is a need for biomarkers to monitor the development and progression of type 1 DM. We analyzed mRNA expression levels for granzyme B, perforin, fas ligand, TNF-α, IFN-γ, Foxp3, IL-10, TGF-β, IL-4, IL-6, IL-17, Activation-induced cytidine deaminase (AID) and Immunoglobulin G gamma chain (IgG) genes in peripheral blood of at-risk, new-onset and long-term type 1 DM , and healthy controls. The majority of the genes were suppressed in long-term type 1 DM compared to controls and new-onset patients. IFN-γ, IL-4 and IL-10 mRNA levels were significantly higher in new-onset compared to at-risk and long-term groups. There was decreased mRNA expression for AID and IgG and up-regulation of IFN-γ with age in controls. Data suggest an overall depressed immunity in long-term type 1 DM. Increased gene expression levels for IFN-γ, IL-4 and IL-10 in new-onset patients from at-risk patients might be used as potential markers for progression of the disease.

Chimerism and liver transplant tolerance

Chimerism and tolerance in a recipient of a deceased-donor liver transplant. Alexander SI, Smith N, Hu M, Verran D, Shun A, Dorney S, Smith A, Webster B, Shaw PJ, Lammi A, Stormon MO. Complete hematopoietic chimerism and tolerance of a liver allograft from a deceased male donor developed in a 9-year-old girl, with no evidence of graft-versus-host disease 17 months after transplantation. The tolerance was preceded by a period of severe hemolysis, reflecting partial chimerism that was refractory to standard therapies. The hemolysis resolved after the gradual withdrawal of all immunosuppressive therapy. [Abstract reproduced by permission of N Engl J Med 2008;358:369–374]

Islet and Pancreas Transplantation

Islet allotransplantation for patients with brittle type 1 diabetes mellitus (T1DM) is a minimally invasive and relatively safe procedure that can induce sustained, normalized glucose control and restore C-peptide secretion, with reduction of hypoglycemic episodes, stabilization or delay of chronic complications, and better quality of life. Current immunosuppressive protocols have significantly improved short-term outcomes, whereas long-term results are still inadequate (from 80% to 10% insulin-independence from 1 to 5 years post-transplant). Principal limitations include: imperfections in the islet isolation process, autoand alloimmunity, allosensitization, immunosuppression-related toxicity, and unsuitability of the intrahepatic implantation site. More efficient isolation methods, safer and more efficient immunosuppressive agents in tolerogenic strategies, and alternative transplant site(s) may resolve these limitations in the near future. Simultaneous pancreas– kidney (SPK) transplantation is the optimal treatment for patients with T1DM with end-stage renal disease. Restoration of normoglycemia after pancreas transplant, as well as of renal function after kidney transplant, results in significant improvement of neuropathy, retinopathy, and nephropathy. Novel immunosuppressive therapies, improvements in surgical techniques, and better understanding of postoperative recipient care have improved results of SPK transplants consistently over the past decade. Future directions include optimization of immunosuppression, allowing freedom from insulin injection therapy while maintaining normoglycemia, and avoidance of chronic transplant glomerulopathy, with durable normalization of kidney function, thus improving quality of life as well as extending patient survival. 2.1 Type 1 Diabetes Mellitus Type 1 diabetes mellitus (T1DM) is a cell-specific autoimmune disease triggered by environmental factors (e.g., viral infections, toxins, diet nutrients or antigens) in genetically predisposed individuals [e.g., human leukocyte antigen (HLA) D. Mineo (B) Diabetes Research Institute, University of Miami, Miami, FL, USA e-mail: dmineo@med.miami.edu 41 S. Efrat (ed.), Stem Cell Therapy for Diabetes, Stem Cell Biology and Regenerative Medicine, DOI 10.1007/978-1-60761-366-4_2, C