Islet Transplantation - The Canadian Perspective.

This commentary discusses the evolution of islet transplantation in Canada over the past two decades and discusses the challenge and opportunity for more widespread availability of this therapy in other coun-tries, especially in the US.

Type 1 Diabetes Mellitus: Cellular and Molecular Pathophysiology at A Glance.

Type 1 diabetes mellitus (T1DM) is a disease where destruction of the insulin producing pancreatic beta-cells leads to increased blood sugar levels. Both genetic and environmental factors play a part in the development of T1DM. Currently, numerous loci are specified to be the responsible genetic factors for T1DM; however, the mechanisms of only a few of these genes are known. Although several environmental factors are presumed responsible for progression of T1DM, to date, most of their mechanisms remain undiscovered. After several years of hyperglycemia, late onsets of macrovascular (e.g., cardiovascular) and microvascular (e.g., neurological, ophthalmological, and renal) complications may occur. This review and accompanying figures provides an overview of the etiological factors for T1DM, its pathogenesis at the cellular level, and attributed complications.

Posttransplant Lymphoproliferative Disorder After Clinical Islet Transplantation: Report of the First Two Cases.

We report the first two cases of posttransplant lymphoproliferative disorder (PTLD) in recipients of islet transplants worldwide. First, a 44-year-old recipient of three islet infusions developed PTLD 80 months after his initial transplantation, presenting with abdominal pain and diffuse terminal ileum thickening on imaging. He was treated with surgical excision, reduction of immunosuppression, and rituximab. Seven months later, he developed central nervous system PTLD, presenting with vertigo and diplopia; immunosuppression was discontinued, resulting in graft loss, and he was given high-dose methotrexate and underwent consolidative autologous stem cell transplantation. He remains in remission 37 months after the initial diagnosis. Second, a 58-year-old female recipient of two islet infusions developed PTLD 24 months after initial islet infusion, presenting with pancytopenia secondary to extensive bone marrow involvement. Immunosuppression was discontinued, resulting in graft loss, and she received rituximab and chemotherapy, achieving complete remission. Both patients were monomorphic B cell PTLD subtype by histology and negative for Epstein-Barr virus in tissue or blood. These cases document the first occurrences of this rare complication in islet transplantation, likely secondary to prolonged, intensive immunosuppression, and highlight the varying clinical manifestations of PTLD. Further studies are needed to determine incidence rate and risk factors in islet transplantation.

Sitagliptin plus pantoprazole can restore but not maintain insulin independence after clinical islet transplantation: results of a pilot study.

AIMS: Resuming insulin use due to waning function is common after islet transplantation. Animal studies suggest that gastrointestinal hormones, including gastrin and incretins may increase ß-cell mass. We tested the hypothesis that pantoprazole plus sitagliptin, would restore insulin independence in islet transplant recipients with early graft insufficiency and determined whether this would persist after a 3-month washout. METHODS: Single-centre, uncontrolled, open label study of sitagliptin 100 mg daily plus pantoprazole 40 mg twice daily for 6 months. RESULTS: After 6 months of treatment, two of eight participants (25%) achieved the primary endpoint, defined as HbA1C < 42 mmol/mol (6%), fasting plasma glucose < 7.0 mmol, C-peptide > 0.5 nmol and no insulin use. There was a significant reduction in mean insulin dose, but no change in HbA1C or weight. There were no changes in the acute insulin response to arginine, the mixed meal tolerance test or blinded continuous glucose monitoring. After the washout, no participants met the primary endpoint and HbA1C increased from 45 ± 8 mmol/mol (6.3 ± 0.7%) to 51 ± 6 mmol/mol (6.8 ± 0.6%) (P < 0.05). Two participants had mild-moderate transient gastrointestinal side effects. There were no episodes of hypoglycaemia. CONCLUSIONS: Sitagliptin plus pantoprazole is well tolerated and safe and may restore insulin independence in some islet transplant recipients with early graft insufficiency, but this was not sustained when treatment was withdrawn. A larger, controlled trial is required to confirm the effectiveness of this combination to achieve insulin independence and to confidently exclude any persistent benefit for graft function. (Clinical Trials Registry No.: NCT00768651).

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.

Validation of the BETA-2 Score: An Improved Tool to Estimate Beta Cell Function After Clinical Islet Transplantation Using a Single Fasting Blood Sample.

The beta score, a composite measure of beta cell function after islet transplantation, has limited sensitivity because of its categorical nature and requires a mixed-meal tolerance test (MMTT). We developed a novel score based on a single fasting blood sample. The BETA-2 score used stepwise forward linear regression incorporating glucose (in millimoles per liter), C-peptide (in nanomoles per liter), hemoglobin A1c (as a percentage) and insulin dose (U/kg per day) as continuous variables from the original beta score data set (n = 183 MMTTs). Primary and secondary analyses assessed the score's ability to detect glucose intolerance (90-min MMTT glucose ≥8 mmol/L) and insulin independence, respectively. A validation cohort of islet transplant recipients (n = 114 MMTTs) examined 12 mo after transplantation was used to compare the score's ability to detect these outcomes. The BETA-2 score was expressed as follows (range 0-42): [Formula: see text] A score <20 and ≥15 detected glucose intolerance and insulin independence, respectively, with >82% sensitivity and specificity. The BETA-2 score demonstrated greater discrimination than the beta score for these outcomes (p < 0.05). Using a fasting blood sample, the BETA-2 score estimates graft function as a continuous variable and shows greater discrimination of glucose intolerance and insulin independence after transplantation versus the beta score, allowing frequent assessments of graft function. Studies examining its utility to track long-term graft function are required.

Long-Term Follow-Up of the Edmonton Protocol of Islet Transplantation in the United States.

We report the long-term follow-up of the efficacy and safety of islet transplantation in seven type 1 diabetic subjects from the United States enrolled in the multicenter international Edmonton Protocol who had persistent islet function after completion of the Edmonton Protocol. Subjects were followed up to 12 years with serial testing for sustained islet allograft function as measured by C-peptide. All seven subjects demonstrated continued islet function longer than a decade from the time of first islet transplantation. One subject remained insulin independent without the need for diabetic medications or supplemental transplants. One subject who was insulin-independent for over 8 years experienced graft failure 10.9 years after the first islet transplant. The remaining six subjects demonstrated continued islet function upon trial completion, although three had received a supplemental islet transplant each. At trial completion, five subjects were receiving insulin and two remained insulin independent, although one was treated with liraglutide. The median hemoglobin A1c was 6.3% (45 mmol/mol). All subjects experienced progressive decline in the C-peptide/glucose ratio. No patients experienced severe hypoglycemia, opportunistic infection, or lymphoma. Thus, although the rate and duration of insulin independence was low, the Edmonton Protocol was safe in the long term. Alternative approaches to islet transplantation are under investigation.

Chemical Basis for Qualitative and Quantitative Differences Between ABO Blood Groups and Subgroups: Implications for Organ Transplantation.

Blood group ABH(O) carbohydrate antigens are carried by precursor structures denoted type I-IV chains, creating unique antigen epitopes that may differ in expression between circulating erythrocytes and vascular endothelial cells. Characterization of such differences is invaluable in many clinical settings including transplantation. Monoclonal antibodies were generated and epitope specificities were characterized against chemically synthesized type I-IV ABH and related glycans. Antigen expression was detected on endomyocardial biopsies (n = 50) and spleen (n = 11) by immunohistochemical staining and on erythrocytes by flow cytometry. On vascular endothelial cells of heart and spleen, only type II-based ABH antigens were expressed; type III/IV structures were not detected. Type II-based ABH were expressed on erythrocytes of all blood groups. Group A1 and A2 erythrocytes additionally expressed type III/IV precursors, whereas group B and O erythrocytes did not. Intensity of A/B antigen expression differed among group A1 , A2 , A1 B, A2 B and B erythrocytes. On group A2 erythrocytes, type III H structures were largely un-glycosylated with the terminal "A" sugar α-GalNAc. Together, these studies define qualitative and quantitative differences in ABH antigen expression between erythrocytes and vascular tissues. These expression profiles have important implications that must be considered in clinical settings of ABO-incompatible transplantation when interpreting anti-ABO antibodies measured by hemagglutination assays with reagent erythrocytes.

The combination of anti-NKG2D and CTLA-4 Ig therapy prolongs islet allograft survival in a murine model.

Islet transplantation is an effective means of treating severe type 1 diabetes in patients with life-threatening hypoglycemia. Improvements in glycemic control with correction of HbA1C enhance quality of life irrespective of insulin independence. By antagonizing the Natural Killer Group 2, member D (NKG2D) receptor expression on NK and CD8+ T cells, in combination with blocking CTLA-4 binding sites, we demonstrate a significant delay of graft rejection in islet allotransplant. Anti-NKG2D combined with CTLA-4 Ig (n = 15) results in prolonged allograft survival, with 84.6 ± 10% of the recipients displaying insulin independence compared to controls (n = 10, p < 0.001). The effect of combination therapy on graft survival is superior to treatments alone (CTLA-4 Ig vs. combination p = 0.024, anti-NKG2D vs. combination p < 0.001) indicating an interaction between these pathways. In addition, combination treatment also improves glucose tolerance when compared to controls (n = 10, p = 0.018). Histologically, NKG2D+ cells were significantly decreased within the allograft after 7 days of combination treatment (n = 6, p = 0.029). T cell proliferation was significantly reduced with anti-NKG2D therapy and CD8+ T cell daughter fractions were also significantly decreased with mAb and combination treatment when measured by in vitro mixed lymphocyte reaction (n = 5, p = 0.015, p = 0.005 and p = 0.048). These results demonstrate that inhibition of NKG2D receptors and costimulatory pathways enhance islet allograft survival.

Islet oxygen consumption rate dose predicts insulin independence for first clinical islet allotransplants.

BACKGROUND: Human islet allotransplantation for the treatment of type 1 diabetes is in phase III clinical trials in the U.S. and is the standard of care in several other countries. Current islet product release criteria include viability based on cell membrane integrity stains, glucose-stimulated insulin release, and islet equivalent (IE) dose based on counts. However, only a fraction of patients transplanted with islets that meet or exceed these release criteria become insulin independent following 1 transplant. Measurements of islet oxygen consumption rate (OCR) have been reported as highly predictive of transplant outcome in many models. METHOD: In this article we report on the assessment of clinical islet allograft preparations using OCR dose (or viable IE dose) and current product release assays in a series of 13 first transplant recipients. The predictive capability of each assay was examined and successful graft function was defined as 100% insulin independence within 45 days post-transplant. RESULTS: OCR dose was most predictive of CTO. IE dose was also highly predictive, while glucoses stimulated insulin release and membrane integrity stains were not. CONCLUSION: OCR dose can predict CTO with high specificity and sensitivity and is a useful tool for evaluating islet preparations prior to clinical human islet allotransplantation.

Autologous islet transplantation after total pancreatectomy for renal cell carcinoma metastases.

Pancreatic metastases from renal cell carcinoma (RCC) may have a chronic and highly indolent course, and may be resected for cure after considerable delay following treatment of the primary tumor, in contrast to other more common pancreatic tumors. Surgical resection is the treatment of choice, which may lead to postpancreatectomy diabetes mellitus in the case of extensive resection. We present a 70-year-old patient with multifocal pancreatic metastases from RCC causing obstructive jaundice. A total pancreatectomy was required to excise two distant tumors in the head and tail of the pancreas, together with a segment VI liver resection. An autologous islet transplant (AIT) prepared from the central, uninvolved pancreas was carried out to prevent postpancreatectomy diabetes. The patient was rendered insulin-free and remains so with excellent glycemic control for 1 year of follow-up, and there is no evidence of tumor recurrence. The patient has been treated with adjuvant sunitinib to minimize risk of further recurrence. In conclusion, AIT after pancreatectomy may represent a useful option to treat patients with metastatic RCC. A critical component of this approach was dependent upon elaborate additional testing to exclude contamination of the islet preparation by cancerous cells.

Distinct and opposing roles for the phosphatidylinositol 3-OH kinase catalytic subunits p110α and p110ß in the regulation of insulin secretion from rodent and human beta cells.

AIMS/HYPOTHESIS: Phosphatidylinositol 3-OH kinases (PI3Ks) regulate beta cell mass, gene transcription, and function, although the contribution of the specific isoforms is unknown. As reduced type 1A PI3K signalling is thought to contribute to impaired insulin secretion, we investigated the role of the type 1A PI3K catalytic subunits α and ß (p110α and -ß) in insulin granule recruitment and exocytosis in rodent and human islets. METHODS: The p110α and p110ß subunits were inhibited pharmacologically or by small hairpin (sh)RNA-mediated knockdown, and were directly infused or overexpressed in mouse and human islets, beta cells and INS-1 832/13 cells. Glucose-stimulated insulin secretion (GSIS), single-cell exocytosis, Ca(2+) signalling, plasma membrane granule localisation, and actin density were monitored. RESULTS: Inhibition or knockdown of p110α increased GSIS. This was not due to altered Ca(2+) responses, depolymerisation of cortical actin or increased cortical granule density, but to enhanced Ca(2+)-dependent exocytosis. Intracellular infusion of recombinant PI3Kα (p110α/p85ß) blocked exocytosis. Conversely, knockdown (but not pharmacological inhibition) of p110ß blunted GSIS, reduced cortical granule density and impaired exocytosis. Exocytosis was rescued by direct intracellular infusion of recombinant PI3Kß (p110ß/p85ß) even when p110ß catalytic activity was inhibited. Conversely, both the wild-type p110ß and a catalytically inactive mutant directly facilitated exocytosis. CONCLUSIONS/INTERPRETATION: Type 1A PI3K isoforms have distinct and opposing roles in the acute regulation of insulin secretion. While p110α acts as a negative regulator of beta cell exocytosis and insulin secretion, p110ß is a positive regulator of insulin secretion through a mechanism separate from its catalytic activity.

Targeting cells causing split tolerance allows fully allogeneic islet survival with minimal conditioning in NOD mixed chimeras.

Donor-specific tolerance induced by mixed chimerism is one approach that may eliminate the need for long-term immunosuppressive therapy, while preventing chronic rejection of an islet transplant. However, even in the presence of chimerism it is possible for certain donor tissues or cells to be rejected whereas others from the same donor are accepted (split tolerance). We previously developed a nonmyeloablative protocol that generated mixed chimerism across full major histocompatability complex plus minor mismatches in NOD (nonobese diabetic) mice, however, these chimeras demonstrated split tolerance. In this study, we used radiation chimeras and found that the radiosensitive component of NOD has a greater role in the split tolerance NOD mice develop. We then show that split tolerance is mediated primarily by preexisting NOD lymphocytes and have identified T cells, but not NK cells or B cells, as cells that both resist chimerism induction and mediate split tolerance. Finally, after recognizing the barrier that preexisting T cells impose on the generation of fully tolerant chimeras, the chimerism induction protocol was refined to include nonmyeloablative recipient NOD T cell depletion which generated long-term mixed chimerism across fully allogeneic barriers. Furthermore, these chimeric NOD mice are immunocompetent, diabetes free and accept donor islet allografts.

The voltage-dependent potassium channel subunit Kv2.1 regulates insulin secretion from rodent and human islets independently of its electrical function.

AIMS/HYPOTHESIS: It is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function. METHODS: Wild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1-syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca(2+)-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured. RESULTS: Upregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1-syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca(2+)-responses to glucose, but to reduced SNARE complex formation and Ca(2+)-dependent exocytosis. CONCLUSIONS/INTERPRETATION: Direct interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1-syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel's electrical function.

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.

Human islet distribution program for basic research at a single center.

The Clinical Islet Laboratory at the University of Alberta/Alberta Health Services distributes human islets for basic research when islet preparations fail to meet defined release criteria for transplantation. This report highlights our islet distribution activity for diabetes research over a 3-year period. Shipments of the acinar-enriched fraction for research were not included in this report. In 2010, we distributed 6.3 million islet equivalents (IEQs) of islets through 127 shipments to 8 researchers, locally, nationally, and internationally. The number of preparations for research use was stable over the 3-year period (26, 23, and 29 preparations in 2008, 2009, and 2010, respectively). Islet yield distributed for research per isolation was 201, 212, and 218 × 10(3) IEQs, respectively. The number of basic researchers was stable as well, although there were only 2 researchers before 2007. Recently, each researcher has received fewer islets per shipment (49,820 IEQs in 2010 vs 75,635 IEQs in 2008) but more frequently (21.5 in 2010 vs 11.2 times per year in 2008). This paradigm shift would be desirable for researchers, because in our experience, most require <30,000 IEQs per shipment, and more frequent islet shipments results in a larger sample size for experimentation. After an initial expansion in the number of researchers requesting islets, our islet distribution activity has remained stable over the years in terms of total productivity of islets utilized for research. The current supply-versus-demand ratio in our program appears to be appropriate.

The use of bovine pericardial buttress on linear stapler fails to reduce pancreatic fistula incidence in a porcine pancreatic transection model.

We investigate the effectiveness of buttressing the surgical stapler to reduce postoperative pancreatic fistulae in a porcine model. As a pilot study, pigs (n = 6) underwent laparoscopic distal pancreatectomy using a standard stapler. Daily drain output and lipase were measured postoperative day 5 and 14. In a second study, pancreatic transection was performed to occlude the proximal and distal duct at the pancreatic neck using a standard stapler (n = 6), or stapler with bovine pericardial strip buttress (n = 6). Results. In pilot study, 3/6 animals had drain lipase greater than 3x serum on day 14. In the second series, drain volumes were not significantly different between buttressed and control groups on day 5 (55.3 ± 31.6 and 29.3 ± 14.2 cc, resp.), nor on day 14 (9.5 ± 4.2 cc and 2.5 ± 0.8 cc, resp., P = 0.13). Drain lipase was not statistically significant on day 5 (3,166 ± 1,433 and 6,063 ± 1,872 U/L, resp., P = 0.25) or day 14 (924 ± 541 and 360 ± 250 U/L). By definition, 3/6 developed pancreatic fistula; only one (control) demonstrating a contained collection arising from the staple line. Conclusion. Buttressed stapler failed to protect against pancreatic fistula in this rigorous surgical model.

Late cytomegalovirus transmission and impact of T-depletion in clinical islet transplantation.

The epidemiology of cytomegalovirus infection (CMV) in islet transplantation (IT) is not well defined. This study defines incidence, transmission and clinical sequelae of CMV reactivation or disease in 121 patients receiving 266 islet infusions at a single institution. The donor (D)/recipient (R) serostatus was D+/R- 31.2%, D+/R+ 26.3%, D-/R+ 13.2% and D-/R- 29.3%. CMV prophylaxis with oral ganciclovir/valganciclovir was given in 68%. CMV infection occurred in 14/121 patients (11.6%); six had asymptomatic seroconversion and eight others had positive viremia (six asymptomatic and two with CMV febrile symptoms). Median peak viral loads were 1755 copies/mL (range 625-9 100 000). Risk factors for viremia included lymphocyte depletion (thymoglobulin or alemtuzumab, p < 0.001). Viremia was more common in D+/R+ versus D+/R- (p = 0.12), occurring mostly late after transplant (median 306 days). Presumed transmission from IT occurred in 8/83 of D+/R- procedures (9.6%). Of the two cases of CMV disease, one resulted from islet transmission from a CMV positive donor (D+/R-); the other was due to de novo exogenous infection (D-/R-). Therefore, CMV transmission presents rarely after IT and with low incidence compared to solid organ transplantation, but occurs late posttransplant. The use of lymphocyte depleting therapies is a primary risk factor.

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.