Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation.

Clinical islet transplantation achieves insulin independence in selected patients, yet current methods for extracting islets from their surrounding pancreatic matrix are suboptimal. The islet basement membrane (BM) influences islet function and survival and is a critical marker of islet integrity following rodent islet isolation. No studies have investigated the impact of islet isolation on BM integrity in human islets, which have a unique duplex structure. To address this, samples were taken from 27 clinical human islet isolations (donor age 41-59, BMI 26-38, cold ischemic time < 10 h). Collagen IV, pan-laminin, perlecan and laminin-α5 in the islet BM were significantly digested by enzyme treatment. In isolated islets, laminin-α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Collagen IV and pan-laminin were present in the disorganized BM of isolated islets, yet a significant reduction in pan-laminin was seen during the initial 24 h culture period. Islet cytotoxicity increased during culture. Therefore, the human islet BM is substantially disrupted during the islet isolation procedure. Islet function and survival may be compromised as a consequence of an incomplete islet BM, which has implications for islet survival and transplanted graft longevity.

Contemporary management and outcomes for infants born with oesophageal atresia.

BACKGROUND: Reports on the management and outcome of rare conditions, such as oesophageal atresia, are frequently limited to case series reporting single-centre experience over many years. The aim of this study was to identify all infants born with oesophageal atresia in the UK and Ireland to describe current clinical practice and outcomes. METHODS: This was a prospective multicentre cohort study of all infants born with oesophageal atresia and/or tracheo-oesophageal fistula in 2008-2009 in the UK and Ireland to record current clinical management and early outcomes. RESULTS: A total of 151 infants admitted to 28 paediatric surgical units were identified. Some aspects of perioperative management were universal, including oesophageal decompression, operative technique and the use of transanastomotic tubes. However, there were a number of areas where clinical practice varied considerably, including the routine use of perioperative chest drains, postoperative contrast studies and antireflux medication, with each of these being employed in 30-50 per cent of patients. There was a trend towards routine postoperative ventilation. CONCLUSION: The prospective methodology used in this study can help identify practices that all surgeons employ and also those that few surgeons use. Areas of clinical equipoise can be recognized and avenues for further research identified.

Regulation of glucagon secretion by glucose: paracrine, intrinsic or both?

Glucagon secretion is regulated by glucose but the mechanisms involved remain hotly debated. Both intrinsic (within the α-cell itself) and paracrine (mediated by factors released ß- and/or δ-cells) have been postulated. Glucagon secretion is maximally suppressed by glucose concentrations that do not affect insulin and somatostatin secretion, a finding that highlights the significance of intrinsic regulation of glucagon secretion. Experiments on islets from mice lacking functional ATP-sensitive potassium channels (K(ATP)-channels) indicate that these channels are critical to the α-cell's capacity to sense changes in extracellular glucose. Here, we review recent data on the intrinsic and paracrine regulation of glucagon secretion in human pancreatic islets. We propose that glucose-induced closure of the K(ATP)-channels, via membrane depolarization, culminates in reduced electrical activity and glucagon secretion by voltage-dependent inactivation of the ion channels involved in action potential firing. We further demonstrate that glucagon secretion measured in islets isolated from donors with type-2 diabetes is reduced at low glucose and that glucose stimulates rather than inhibits secretion in these islets. We finally discuss the relative significance of paracrine and intrinsic regulation in the fed and fasted states and propose a unifying model for the regulation of glucagon secretion that incorporates both modes of control.

The long lifespan and low turnover of human islet beta cells estimated by mathematical modelling of lipofuscin accumulation.

AIMS/HYPOTHESIS: Defects in pancreatic beta cell turnover are implicated in the pathogenesis of type 2 diabetes by genetic markers for diabetes. Decreased beta cell neogenesis could contribute to diabetes. The longevity and turnover of human beta cells is unknown; in rodents <1 year old, a half-life of 30 days is estimated. Intracellular lipofuscin body (LB) accumulation is a hallmark of ageing in neurons. To estimate the lifespan of human beta cells, we measured beta cell LB accumulation in individuals aged 1-81 years. METHODS: LB content was determined by electron microscopical morphometry in sections of beta cells from human (non-diabetic, n = 45; type 2 diabetic, n = 10) and non-human primates (n = 10; 5-30 years) and from 15 mice aged 10-99 weeks. Total cellular LB content was estimated by three-dimensional (3D) mathematical modelling. RESULTS: LB area proportion was significantly correlated with age in human and non-human primates. The proportion of human LB-positive beta cells was significantly related to age, with no apparent differences in type 2 diabetes or obesity. LB content was low in human insulinomas (n = 5) and alpha cells and in mouse beta cells (LB content in mouse <10% human). Using 3D electron microscopy and 3D mathematical modelling, the LB-positive human beta cells (representing aged cells) increased from >or=90% (<10 years) to >or=97% (>20 years) and remained constant thereafter. CONCLUSIONS/INTERPRETATION: Human beta cells, unlike those of young rodents, are long-lived. LB proportions in type 2 diabetes and obesity suggest that little adaptive change occurs in the adult human beta cell population, which is largely established by age 20 years.

Islet Transplantation in the UK.

This commentary outlines the development and current status of the UK Islet Transplant Programme in the UK. The author makes the case that it is now time for similar fully funded beta-cell programmes to be made available in many other countries as well.

Effect of Etanercept Concentration on Human Islet Integrity.

BACKGROUND: Etanercept is widely used as an antiinflammatory drug to improve engraftment after intraportal islet transplantation. In contrast to other immunosuppressive agents, very little is known about detrimental effects of etanercept on islets. The aim of this pilot study was to define the toxic range of etanercept. METHODS: Human islets isolated from 8 donors were cultured for 4-5 days at 37°C in culture medium supplemented with etanercept at concentrations from 2.5 to 40 µg/mL, corresponding to potential in vivo levels within the portal vein. After culture, islet equivalent (IEQ) yield, fragmentation index (islet number/IEQ), purity, viability, and stimulated insulin release (2 vs 20 mmol/L) were assessed and normalized to islets before culture. RESULTS: Yield (73 ± 8%) and viability (91 ± 4%) were highest with 5 µg/mL etanercept. Islet loss was evident when etanercept was ≥10 µg/mL (55 ± 7%; P < .05 vs control). Fragmentation (154 ± 34%; P < .05) was markedly increased and viability (81 ± 4%, P < .05) markedly decreased with etanercept >10 µg/mL. The accumulation of cell debris at concentrations ≥20 µg/mL resulted in a significant reduction of islet purity (84 ± 3%; P < .05). Etanercept did not interfere with stimulated insulin secretion at concentrations ≤10 µg/mL. The maximum stimulation index was noted at 2.5 µg/mL (1.8 ± 0.1). CONCLUSIONS: Etanercept is tolerated by isolated human islets at concentrations <10 µg/mL. Our data suggest that the tight range between benefit and toxicity should be considered for dosage and administration of etanercept.

Hypoxia-Induced Damage in Human Islets Is Reduced With the Use of Mesenchymal Stem Cell-Preconditioned Medium.

BACKGROUND: Mesenchymal stem cells (MSCs) are protective for islets when cotransplanted in a hypoxic environment. However, the risk of neoplasia is increased when MSCs are transplanted into immunosuppressed patients. This initial study aimed to investigate whether the production of protective factors from MSC can be stimulated by different culture conditions to benefit human islets cultured in hypoxia. METHODS: MSC were isolated from human adipose tissue and cultured for 2 days in supplemented Minimum Essential Media α (MEMα) and 21% (21%-MEMα) or 1% oxygen (1%-MEMα). Native MEMα served as control. After MSC harvesting, cell-depleted media were frozen at -20°C until use for human islet culture in 2% oxygen for 72-96 hours before islet characterization. Data were normalized to control islets cultured in native MEMα and 2% oxygen (mean ± SEM). RESULTS: After culture in 21%- or 1%-MEMα, islet recovery increased to 117 ± 12% (NS) and 138 ± 12% (P < .05), respectively. Viability did not change after culture in native MEMα (59 ± 2%), 21%-MEMα (59 ± 3%), or 1%-MEMα (61 ± 3%). Compared with control samples, the glucose stimulation index was increased after culture in 21%-MEMα (P < .05) or 1%-MEMα (P < .05). Overall survival was higher in 1%-MEMα (143 ± 14%) than in 21%-MEMα (119 ± 14%; NS) or native MEMα (P < .05). CONCLUSIONS: This study demonstrates that MSC-preconditioned MEMα increases survival and in vitro function of hypoxic human islets. These findings indicate that hypoxic MSCs seem to produce factors that improve survival of islets suffering from hypoxia.

The importance of pancreatic embryonic epithelium for mesenchyme-to-epithelial transition during islet development.

Stem or progenitor cells are a promising potential alternative source of pancreatic islets for transplantation in the treatment of juvenile-onset diabetes. However, to derive islets from such cells, it is important to elucidate the mechanisms of normal pancreatic development. Previous work in our laboratory has shown that, contrary to previous thinking, pancreatic mesenchyme when combined with pancreatic epithelium can contribute cells to islets. However, the signals and role of individual tissues involved in this mesenchyme-to-epithelial transition (MET) have yet to be elucidated. The aim of this study was to investigate whether MET can occur in the absence of pancreatic epithelium. Chick and quail eggs were incubated for 4 days and the dorsal pancreatic buds and stomach rudiments were microdissected. Mesenchyme and epithelium of the organ rudiments were separated after collagenase treatment. Separated pancreatic mesenchyme were cultured alone and in combination with stomach (nonpancreatic). After 7 days of culture, the specimens were analysed using immunohistochemistry for quail-specific nucleolar antigen (QCPN), insulin, and islet precursor cell marker (ISL-1). Pancreatic mesenchyme when cultured in the absence of epithelium did not differentiate into islets, but differentiated into fibroblast-like cells. When pancreatic mesenchyme were cultured in combination with stomach epithelium, there was no evidence of mesenchymally derived islets. We have demonstrated that pancreatic mesenchyme require pancreatic epithelium to differentiate into islet cells. These findings further increase our understanding of normal pancreatic islet development and may help to elucidate the molecular mechanisms of MET in islet development.

Comparison of the collagen VI content within the islet-exocrine interface of the head, body, and tail regions of the human pancreas.

Efficient islet isolation depends on the use of collagenase to digest the extracellular matrix within the islet-exocrine interface, the molecular structure of which is poorly understood. Recently it has been reported that transplantable yields of islets can be isolated from the tail segment of the pancreas alone. This study aimed to quantify and compare the amount of collagenase-resistant collagen VI within the islet-exocrine interface of the head, body, and tail of the human pancreas. Human adult pancreata (n = 5) were retrieved from heart-beating donors (age range, 40-62 years; cold ischemia times <10 hours). Tissue blocks from the head, body, and tail region of each pancreas were fixed in formalin and processed for immuno-labelling of collagen VI, which was quantified in the islet-exocrine interface using a Zeiss KS-400 image analysis system. Data were expressed as area of collagen at the interface relative to the islet area. Statistical analysis was done using paired t test. The mean islet areas in the head, body, and tail regions were not significantly different. Collagen VI was uniformly present within the islet-exocrine interface of all regions of the pancreas and was 0.326 +/- 0.064, 0.324 +/- 0.060, and 0.334 +/- 0.052 microm(2)/islet area (P = .441) in the head, body, and tail, respectively. The content of collagen VI within the islet-exocrine interface was uniform throughout all parts of the adult pancreas. Targeting this collagen subtype with novel collagenase blends may result in consistently improved islet yields and enable a wider number of available donor pancreata to be used.

Perfluorocarbon emulsions prevent hypoxia of pancreatic ß-cells.

As oxygen carriers, perfluorocarbon emulsions might be useful to decrease hypoxia of pancreatic islets before transplantation. However, their hydrophobicity prevents their homogenisation in culture medium. To increase the surface of contact between islets and Perfluorooctyl bromide (PFOB), and consequently oxygen delivery, we tested effect of a PFOB emulsion in culture medium on ß-cell lines and rat pancreatic islets. RINm5F ß-cell line or pancreatic rat islets were incubated for 3 days in the presence of PFOB emulsion in media (3.5% w/v). Preoxygenation of the medium was performed before culture. Cell viability was assessed by apoptotic markers (Bax and Bcl-2) and by staining (fluoresceine diacetate and propidium iodide). ß-Cell functionality was determined by insulin release during a glucose stimulation test and. Hypoxia markers, HIF-1α and VEGF, were studied at days 1 and 3 using RT-PCR, Western blotting, and ELISA. PFOB emulsions preserved viability and functionality of RINm5F cells with a decrease of HIF-1α and VEGF expression. Islets viability was preserved during 3 days of culture. Secretion of VEGF was higher in untreated control (0.09 ± 0.041 µg VEGF/mg total protein) than in PFOB emulsion incubated islets (0.02 ± 0.19 µg VEGF/mg total protein, n = 4, p < 0.05) at day 1. At day 3, VEGF secretion was increased as compared to day 1 in control (0.23 ± 0.04 µg VEGF/mg total protein) but it was imbalance by the presence of PFOB emulsion (0.09 ± 0.03 µg VEGF/mg total protein, n = 5, p < 0.05). While insulin secretion was maintained in response to a glucose stimulation test until day 3 when islets were incubated in the presence of PFOB emulsion preoxygenated (0.81 ± 0.16 at day 1 vs. 0.75 ± 0.24 at day 3), the ability to secrete insulin in the presence of high glucose concentration was lost in islets controls (0.51 ± 0.18 at day 1 vs. 0.21 ± 0.13 at day 3). Atmospheric oxygen delivery by PFOB emulsion might be sufficient to decrease islets hypoxia. However, to improve islets functionality, overoxygenation is needed. Finally, maintenance of islet viability and functionality for several days after isolation could improve the outcome of islets transplantation.

Vascular endothelial growth factor as a survival factor for human islets: effect of immunosuppressive drugs.

AIMS/HYPOTHESIS: Rapamycin, part of the immunosuppressive regimen of the Edmonton protocol, has been shown to inhibit vascular endothelial growth factor (VEGF) production and VEGF-mediated survival signalling in tumour cell lines. This study investigates the survival-promoting activities of VEGF in human islets and the effects of rapamycin on islet viability. MATERIALS AND METHODS: Levels of VEGF and its receptors in isolated human islets and whole pancreas was determined by western blotting and immunostaining. Islet viability following VEGF or immunosuppressive drug treatment was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Islet VEGF release was measured by ELISA. Mouse islets infected with an adenovirus expressing the gene for VEGF were transplanted syngeneically into streptozotocin-induced diabetic mice, with blood glucose levels measured three times per week. RESULTS: Isolated human islets produced multiple isoforms of VEGF and VEGF receptors 1, 2 and 3 and the coreceptor neuropilin 1. Exogenous VEGF (10 ng/ml) prevented human islet death induced by serum starvation, which suggests that VEGF can act as a survival factor for human islets. Transplantation of mouse islets infected with a VEGF-expressing adenovirus in a syngeneic model, improved glycaemic control at day 1 post-transplantation (p < 0.05). Rapamycin at 10 and 100 ng/ml significantly reduced islet VEGF release (by 37 +/- 4% and 43 +/- 6%, respectively; p < 0.05) and at 100 ng/ml reduced islet viability (by 36 +/- 9%) and insulin release (by 47 +/- 7%, all vs vehicle-treated controls; p < 0.05). Tacrolimus had no effect on islet VEGF release or viability. CONCLUSIONS/INTERPRETATION: Our data suggest that rapamycin may have deleterious effects on islet survival post-transplantation, both through a direct effect on islet viability and indirectly through blockade of VEGF-mediated revascularisation.

Endoscopic balloon dilatation of esophageal strictures in infants and children: 17 years' experience and a literature review.

PURPOSE: Whereas endoscopic balloon dilatation (EBD) of benign esophageal strictures is an established mode of therapy in adults, this has not been accepted universally in the pediatric population. The aim of this study is to report the safety, efficacy, and long-term results of EBD for children in the authors' center. METHODS: Between 1986 and 2002, a total of 77 children (median age, 1.8 years; range, 2 months to 20 years) were treated by EBD for various causes: 2 had achalasia, and 75 had esophageal strictures (postesophageal atresia repair, 63; reflux esophagitis, 7; postfundoplication, 2; caustic injury, 3). Dilatations were performed using flexible endoscopy and fluoroscopic screening under general anesthesia. RESULTS: A total of 260 dilatations were carried out with the mean number of EBD per patient being 3.4 (range, 1 to 19). A mean period of 5 months (maximum, 28 months) for each patient was required. Four complications of esophageal perforations (1.5%) were observed, but only one required surgical repair because of persistent leakage. The remaining patients have undergone long-term follow-up (median follow-up, 6.6 years), and all are asymptomatic. CONCLUSIONS: This large series has shown that EBD can provide a safe and effective mean of relieving esophageal strictures with good long-term results.