A. M. J. Shapiro

Optimal implantation site for pancreatic islet transplantation

Since the first report of successful pancreatic islet transplantation to reverse hyperglycaemia in diabetic rodents, there has been great interest in determining the optimal site for implantation. Although the portal vein remains the most frequently used site clinically, it is not ideal. About half of the islets introduced into the liver die during or shortly after transplantation. Although many patients achieve insulin independence after portal vein infusion of islets, in the long term most resume insulin injections.

High risk of sensitization after failed islet transplantation.

Human Leukocyte Antigen (HLA) antibodies posttransplant have been associated with an increased risk of early graft failure in kidney transplants. Whether this also applies to islet transplantation is not clear. To achieve insulin independence after islet transplants multiple donor infusions may be required. Hence, islet transplant recipients are at risk of sensitization after transplantation. Islet transplant recipients were screened for HLA antibodies posttransplant by flow‐based methods. A total of 98 patients were studied. Twenty‐nine patients (31%) developed de novo donor specific antibodies (DSA) posttransplant. Twenty‐three patients developed DSA while on immunosuppression (IS). Among recipients who have discontinued IS, 10/14 (71%) are broadly sensitized with panel reactive antibody (PRA) ≥50%. The risk of becoming broadly sensitized after transplant was 11/69 (16%) if the recipient was unsensitized prior to transplant. The majority of these antibodies have persisted over time. Appearance of HLA antibodies posttransplant is concerning, and the incidence rises abruptly in subjects weaned completely from IS. This may negatively impact the ability of these individuals to undergo further islet, pancreas or kidney transplantation and should be discussed upfront during evaluation of candidates for islet transplantation.

Portal venous pressure changes after sequential clinical islet transplantation.

Background. Sequential pancreatic islet transplantation via the portal vein has led to insulin independence in patients with type 1 diabetes. Complications associated with the injection of islets into the portal vein have been reported; therefore, in this study we sought to further characterize changes in portal venous pressure associated with islet infusion. Methods. Pre- and posttransplant portal venous pressures were recorded in 50 consecutive transplant procedures in 26 patients receiving highly purified, heparinized allogeneic islet preparations via a radiologically placed portal venous cannula. Doppler ultrasound scans of the portal vein were completed within 24 hr of transplantation. Results. Posttransplant portal vein pressures rose significantly with sequential transplantation (12.4 mm Hg vs. 17.3 mm Hg, P <0.05). Portal pressure change correlated significantly with islet packed cell volume (r =0.66, P <0.001) and also with the number of islets transplanted (r =0.49, P <0.001). Segmental portal vein thrombosis was radiologically detected after two procedures (4%). Conclusion. Multiple sequential islet transplants can be safely performed via the portal vein, provided that care is taken with islet purification and attention is paid to portal venous monitoring.

The role of macrophage migration inhibitory factor on glucose metabolism and diabetes

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in many inflammatory reactions and disorders, and it has become evident that it also affects glucose homeostasis. The protein is produced by pancreatic beta cells and can promote the release of insulin. It also modulates glucose uptake, glycolysis and insulin resistance in insulin target cells such as the adipocyte, myocyte and cardiomyocyte. Possessing both immunological and endocrinological properties, MIF has been associated with the development of type 1 and type 2 diabetes, and it may be important in the setting of islet transplantation. The present review summarises our current knowledge, based on clinical and research data, on the impact of MIF on both physiological and pathological aspects of glucose metabolism.

Portal Vein Thrombosis Is a Potentially Preventable Complication in Clinical Islet Transplantation

Percutaneous transhepatic portal access avoids surgery but is rarely associated with bleeding or portal venous thrombosis (PVT). We herein report our large, single‐center experience of percutaneous islet implantation and evaluate risk factors of PVT and graft function. Prospective data were collected on 268 intraportal islet transplants (122 subjects). A portal venous Doppler ultrasound was obtained on Days 1 and 7 posttransplant. Therapeutic heparinization, complete ablation of the portal catheter tract with Avitene paste and limiting packed cell volume (PCV) to <5 mL completely prevented any portal thrombosis in the most recent 101 islet transplant procedures over the past 5 years. In the previous cumulative experience, partial thrombosis did not affect islet function. Standard liver volume correlated negatively (r =−0.257, p < 0.001) and PCV correlated positively with portal pressure rise (r = 0.463, p < 0.001). Overall, partial portal thrombosis occurred after 10 procedures (overall incidence 3.7%, most recent 101 patient incidence 0%). There were no cases of complete thrombosis and no patient developed sequelae of portal hypertension. In conclusion, portal thrombosis is a preventable complication in clinical islet transplantation, provided therapeutic anticoagulation is maintained and PCV is limited to <5 mL.

Liraglutide, a long-acting human glucagon-like peptide 1 analog, improves glucose homeostasis in marginal mass islet transplantation in mice

The current scarcity of high-quality deceased pancreas donors prevents widespread application of islet transplantation for treatment of labile type 1 diabetes mellitus. Opportunities for the improvement of current techniques include optimization of islet isolation and purification, use of culture with pharmacological insulinotropic agents, strategies to reduce graft rejection and inflammation, and the search for alternative insulin producing tissue. Here, we report our findings on the efficacy of the long-acting human glucagon-like peptide 1 analog, liraglutide, in a mouse model of marginal mass islet transplantation. Liraglutide was administered (200 microg/kg sc twice daily) after a marginal mass syngeneic islet transplant in streptozotocin-induced diabetic BALB/c mice. Time-to-normoglycemia was significantly shorter in liraglutide-treated animals (median 1 vs. 7 d; P = 0.0003), even in recipients receiving sirolimus (median 1 vs. 72.5 d; P < 0.0001). Liraglutide-treated animals also demonstrated improved glucose tolerance as assessed by an ip glucose tolerance test. Liraglutide discontinuation at postoperative d 90 resulted in diminished glucose tolerance during the ip glucose tolerance test, whereas a late-start liraglutide therapy 90 d after transplant resulted in no improvement. These findings suggest that liraglutide therapy mediates early and late insulinotropic effects. In accord with this hypothesis, insulin/terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling fluorescence microscopy showed reduced transplanted beta-cell apoptosis in liraglutide-treated recipients 48 h after transplant. In addition, liraglutide resulted in improved glucose-dependent insulin secretion. Overall, our data show that liraglutide has a beneficial impact on the engraftment and function of syngeneic islet transplants in mice, when administered continuously starting on the day of transplant.

Neonatal porcine islets exhibit natural resistance to hypoxia-induced apoptosis.

Background. Despite the success of the Edmonton protocol for human islet transplantation, an alternate source of islet tissue must be developed if β-cell replacement therapy is to see widespread application. Neonatal porcine islets (NPI) represent one potential source of tissue. When human or rodent islets are transplanted, the majority of cells undergo hypoxia-induce apoptosis soon after the grafts are placed in the recipient. In the present study, we investigated whether NPI were similarly sensitive to hypoxia. Methods. NPI were exposed to hypoxia and hypoxia/reoxygenation using an in vitro hypoxic chamber. Afterwards, viability, frequency of apoptosis, and β-cell function were evaluated. NPI and adult porcine islets were transplanted into chemically diabetic, immunodeficient mice and graft apoptosis was assessed 24 hours and seven days posttransplant. Results. NPI demonstrated a remarkable capacity to resist apoptosis and maintain insulin secretion despite severe stresses such as hypoxia/reoxygenation. One day after transplantation, NPI grafts showed limited apoptosis, confined to rare strongly insulin positive cells. In contrast, adult porcine islet grafts underwent widespread apoptosis. Western blotting revealed that NPI express high levels of at least one potent endogenous antiapoptotic protein (XIAP). Conclusions. The majority of cells within transplanted human islets undergo apoptosis soon after portal infusion. In contrast, NPI have the capacity to resist this early posttransplant apoptosis, with likely reduced antigen release and diminished immune stimulation. NPI appear to contain a population of insulin-low to insulin-negative pre-β-cells, which are resistant to hypoxia-induced apoptosis and still capable of differentiating into mature β-cells.

Anakinra Potentiates the Protective Effects of Etanercept in Transplantation of Marginal Mass Human Islets in Immunodeficient Mice

Anti‐inflammatory agents are used routinely in clinical islet transplantation in an attempt to promote islet engraftment. Infliximab, and more recently etanercept, is being used to neutralize tumor necrosis factor alpha, but this tenet is based on limited preclinical data. One group has promoted the potential of combined etanercept with an IL‐1 receptor antagonist, anakinra in a small clinical study, but without strong preclinical data to justify this approach. We therefore sought to evaluate the impact of combined anakinra and etanercept in a marginal islet mass transplant model using human islets in immunodeficient mice. The combination of anakinra and etanercept led to remarkable improvement in islet engraftment (control 36.4%; anakinra 53.9%; etanercept 45.45%; anakinra and etanercept 87.5% euglycemia, p < 0.05 by log‐rank) compared to single‐drug treated mice or controls. This translated into enhanced metabolic function (area under curve glucose tolerance), improved graft insulin content and marked reduction in beta‐cell specific apoptotis (0.67% anakinra + etanercept vs. 23.5% control, p < 0.001). These results therefore strongly justify the combined short‐term use of anakinra and etanercept in human islet transplantation.

Preliminary Single-Center Canadian Experience of Human Normothermic Ex Vivo Liver Perfusion: Results of a Clinical Trial.

After extensive experimentation, outcomes of a first clinical normothermic machine perfusion (NMP) liver trial in the United Kingdom demonstrated feasibility and clear safety, with improved liver function compared with standard static cold storage (SCS). We present a preliminary single‐center North American experience using identical NMP technology. Ten donor liver grafts were procured, four (40%) from donation after circulatory death (DCD), of which nine were transplanted. One liver did not proceed because of a technical failure with portal cannulation and was discarded. Transplanted NMP grafts were matched 1:3 with transplanted SCS livers. Median NMP was 11.5 h (range 3.3–22.5 h) with one DCD liver perfused for 22.5 h. All transplanted livers functioned, and serum transaminases, bilirubin, international normalized ratio, and lactate levels corrected in NMP recipients similarly to controls. Graft survival at 30 days (primary outcome) was not statistically different between groups on an intent‐to‐treat basis (p = 0.25). Intensive care and hospital stays were significantly more prolonged in the NMP group. This preliminary experience demonstrates feasibility as well as potential technical risks of NMP in a North American setting and highlights a need for larger, randomized studies.

Combined coinhibitory and costimulatory modulation with anti-BTLA and CTLA4Ig facilitates tolerance in murine islet allografts

Complex interactions between positive and negative cosignaling receptors ultimately determine the fate of the immune response. The recently identified coinhibitory receptor, B and T lymphocyte attenuator (BTLA), contributes to regulation of autoimmune and potentially alloimmune responses. We investigated the role of BTLA in a fully major histocompatibility complex‐mismatched mouse islet transplant model. We report that anti‐BTLA mAb (6F7) alone does not accelerate graft rejection. Rather, while CTLA4Ig alone improved allograft survival, the addition of anti‐BTLA mAb to CTLA4Ig led to indefinite (>100 days) allograft survival. Immediately after treatment with anti‐BTLA mAb and CTLA4Ig, islet allografts showed intact islets and insulin production despite a host cellular response, with local accumulation of Foxp3+ cells. We clearly demonstrate that combined therapy with anti‐BTLA mAb and CTLA4Ig mice induced donor‐specific tolerance, since mice accepted a second donor‐specific islet graft without further treatment and rejected third party grafts. CTLA4Ig and anti‐BTLA mAb limited the initial in vivo proliferation of CFSE‐labeled allogeneic lymphocytes, and anti‐BTLA mAb enhanced the proportion of PD‐1 expressing T cells while depleting pathogenic BTLA+ lymphocytes. We conclude that targeting the BTLA pathway in conjunction with CTLA4Ig costimulatory blockade may be a useful strategy for promoting immunological tolerance in murine islet allografts.