[Show abstract][Hide abstract] ABSTRACT: The shipment of human islets (IE) from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment).
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Islet transplantation is a recognized treatment option for select patients with type I diabetes mellitus. However, islet infusions from multiple donors are often required to achieve insulin independence. Ideally, insulin independence would be achieved routinely with only a single donor. Identification of factors associated with insulin independence after single-donor islet transplantation may help to select recipient-donor combinations with the highest probability of success.
[Show abstract][Hide abstract] ABSTRACT: The optimal endocrine transplant that should follow a successful kidney transplant in patients with type 1 diabetes is currently open to debate. Ideally, a combined kidney and pancreas transplant is carried out simultaneously from an optimal donor as there is clear immunological benefit. However, when a living donor kidney transplant occurs initially, either a pancreas after kidney or islet after kidney transplant may be considered. A pancreas after kidney transplant carries more surgical risk but provides more robust endocrine reserve compared to the alternative option of islet after kidney transplantation. Furthermore, islet transplantation is not universally available, requires specialized manufacturing facilities, and the costs associated with islet manufacture may not be reimbursable by healthcare. From a patient’s perspective, however, islet after kidney transplantation is a highly attractive option to avoid further surgical risk and recovery. This paper discusses the merits and demerits of these alternative transplant options, and debates both competitive aspects and complementarity. We discuss current outcomes and ongoing clinical trials.
[Show abstract][Hide abstract] ABSTRACT: Single nucleotide polymorphisms (SNPs) within the ADCY5 gene, encoding adenylate cyclase 5, are associated with elevated fasting glucose and increased type 2 diabetes (T2D) risk. Despite this, the mechanisms underlying the effects of these polymorphic variants at the level of pancreatic beta cells remain unclear. Here, we show firstly that ADCY5 mRNA expression in islets is lowered by the possession of risk alleles at rs11708067. Next, we demonstrate that ADCY5 is indispensable for coupling glucose, but not GLP-1, to insulin secretion in human islets. Assessed by in situ imaging of recombinant probes, ADCY5 silencing impaired glucose-induced cAMP increases and blocked glucose metabolism towards ATP at concentrations of the sugar >8 mM. However, calcium transient generation and functional connectivity between individual human beta cells were sharply inhibited at all glucose concentrations tested, implying additional, metabolism-independent roles for ADCY5. In contrast, calcium rises were unaffected in ADCY5-depleted islets exposed to GLP-1. Alterations in beta cell ADCY5 expression and impaired glucose signalling thus provide a likely route through which ADCY5 gene polymorphisms influence fasting glucose levels and T2D risk, while exerting more minor effects on incretin action.
[Show abstract][Hide abstract] ABSTRACT: Islet transplantation is today an accepted modality for treating selected patients with frequent hypoglycemic events or severe glycemic lability. Despite tremendous progress in islet isolation, culture, and preservation, clinical use is still restricted to a limited subset, and lifelong immunosuppression is required. Issues surrounding limited islet revascularization and immune destruction remain. One of the major challenges is to prevent alloreactivity and recurrence of autoimmunity against β-cells. These two hurdles can be effectively reduced by immunosuppressive therapy combining induction and maintenance treatments. The introduction of highly potent and selective biologic agents has significantly reduced the frequency of acute rejection and has prolonged graft survival, while minimizing the complications of this therapeutic scheme. This review will address the most important biological agents used in islet transplantation. We provide a historical perspective of their introduction into clinical practice and their role in current clinical protocols, aiming at improved engraftment efficiency, increased long-term survival, and better overall results of clinical islet transplantation.
Current Diabetes Reports 08/2013; · 3.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
American Journal of Transplantation 07/2013; · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Pancreas dissociation is a critical initial component of the islet isolation procedure and introduces high variability based on factors including the enzyme type, specificity and potency. Product refinement and alterations to the application strategies have improved isolation outcomes over time; however, islet utilization from donor organs remains low. In this study we evaluate a low endotoxin-high activity grade neutral protease in clinical islet isolation. Materials and Methods: The use of a non-collagenolytic enzyme, either thermolysin or high active neutral protease, was randomized in clinical islet isolations to evaluate efficacy. Additionally a retrospective comparison to neutral protease NB was conducted. Results: The thermolysin group had lower trapped islet population and increased purity and post-culture islet mass in comparison to high active grade neutral protease. Comparison of neutral protease NB GMP grade to high active neutral protease displayed no measurable difference in islet mass or viability and transplantation outcomes at 1 mo post-transplant were favorable for both groups. Conclusions: High activity neutral protease can generate clinical grade islets and may prove beneficial to islet function and viability based on a reduced endotoxin load but dosing of neutral protease requires ongoing optimization.
[Show abstract][Hide abstract] ABSTRACT: Cellular homeostasis requires intrinsic sensing mechanisms to temper function in the face of prolonged activity. In the pancreatic β-cell, glucose is likely a physiological trigger that activates an adaptive response to stimulation, thereby maintaining cellular homeostasis. Immediate early genes (IEGs) are activated as a first line of defense in cellular homeostasis and are largely responsible for transmitting an environmental cue to a cellular response. Here we examine the regulation and function of the novel β-cell IEG, neuronal PAS domain protein 4 (Npas4). Using MIN6 cells, mouse and human islets, as well as in vivo infusions, we demonstrate that Npas4 is expressed within pancreatic islets and is upregulated by β-cell depolarizing agents. Npas4 tempers β-cell function through a direct inhibitory interaction with the insulin promoter and by blocking the potentiating effects of GLP-1 without significantly reducing glucose-stimulated secretion. Finally, Npas4 expression is induced by classical endoplasmic reticulum (ER) stressors and can prevent thapsigargin- and palmitate-induced dysfunction and cell death. These results suggest that Npas4 is a key activity-dependent regulator that improves β-cell efficiency in the face of stress. We posit that Npas4 could be a novel therapeutic target in type 2 diabetes that could both reduce ER stress and cell death and maintain basal cell function.
[Show abstract][Hide abstract] ABSTRACT: 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 β (p110α and -β) in insulin granule recruitment and exocytosis in rodent and human islets. METHODS: The p110α and p110β 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/p85β) blocked exocytosis. Conversely, knockdown (but not pharmacological inhibition) of p110β blunted GSIS, reduced cortical granule density and impaired exocytosis. Exocytosis was rescued by direct intracellular infusion of recombinant PI3Kβ (p110β/p85β) even when p110β catalytic activity was inhibited. Conversely, both the wild-type p110β 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, p110β is a positive regulator of insulin secretion through a mechanism separate from its catalytic activity.
[Show abstract][Hide abstract] ABSTRACT: Our understanding of adult human β-cells is advancing, but we know little about the function and plasticity of β-cells from infants. We therefore characterized islets and single islet cells from human infants after isolation and culture. Although islet morphology in pancreas biopsies was similar to that in adults, infant islets after isolation and 24-48 hours of culture had less insulin staining, content, and secretion. The cultured infant islets expressed pancreatic and duodenal homeobox 1 and several (Glut1, Cav1.3, Kir6.2) but not all (syntaxin 1A and synaptosomal-associated protein 25) markers of functional islets, suggesting a loss of secretory phenotype in culture. The activity of key ion channels was maintained in isolated infant β-cells, whereas exocytosis was much lower than in adults. We examined whether a functional exocytotic phenotype could be reestablished under conditions thought to promote β-cell differentiation. After a 24- to 28-day expansion and maturation protocol, we found preservation of endocrine markers and hormone expression, an increased proportion of insulin-positive cells, elevated expression of syntaxin 1A and synaptosomal-associated protein 25, and restoration of exocytosis to levels comparable with that in adult β-cells. Thus, human infant islets are prone to loss of their exocytotic phenotype in culture but amenable to experimental approaches aimed at promoting expansion and functional maturation. Control of exocytotic protein expression may be an important mechanism underlying the plasticity of the secretory machinery, an increased understanding of which may lead to improved regenerative approaches to treat diabetes.
[Show abstract][Hide abstract] ABSTRACT: Abstract Background: Several published studies have analyzed microbial contamination rates of islet products, ranging from 0% to 16%. However, few studies make reference to potential clinical consequences for transplant recipients and possible impact on islet survival. Materials and Methods: The current study defines rates of microbiological contamination of islet products under current good manufacturing practice conditions in 164 patients receiving 343 transplants at a single institution. Results: Nineteen (5.5%) islet preparations showed positive microbial growth with a majority (79.4%) due to Gram-positive organisms. The most frequently identified microorganism was coagulase-negative Staphylococcus (nine of 19 [47.3%]), followed by polymicrobial organisms (eight of 19 [42.1%]). No patient developed signs of clinical infection, and there were no hepatic abscesses evident on imaging by ultrasound or magnetic resonance imaging (none of 19 [0%]), despite the use of potent T-depletional induction. Finally, we could not demonstrate any negative impact of microbiological contamination on long-term islet graft survival. Conclusions: Microbiological contamination of the final islet preparation appears to have little or no effect on patients or on islet survival when appropriate antibiotics are given. However, preparation sterility should be guaranteed at all cost in order maximize patient safety and avoid potential complications in immunosuppressed patients.
[Show abstract][Hide abstract] ABSTRACT: Hepatic steatosis is one complication patients may experience following clinical islet transplantation (CIT), yet the cause and consequences of this are poorly understood. The purpose of this case-control study was to examine the relationship between hepatic steatosis, metabolic parameters and graft function in an Albertan cohort of CIT recipients. Hepatic steatosis was detected by magnetic resonance imaging (MRI) in n = 10 cases age-matched with n=10 MRI-negative controls. Progression/regression of steatosis was determined by ultrasound (US) in cases. Hepatic steatosis first appeared 2.8 ± 2.2 (mean ± SD) years post-CIT, and lasted approximately 4.6 ± 2.0 years. In five cases steatosis resolved, with recurrence in two cases during the follow-up period (8.5 ± 3.2 years). No evidence of CIT causing deleterious effects on long-term liver function or graft outcome was observed.