Harmful Delayed Effects of Exogenous Isolation Enzymes on Isolated Human Islets: Relevance to Clinical Transplantation

Balamurugan, A. N.; He, Jing; Guo, Fei; Stolz, Donna B.; Bertera, Suzanne; Geng, Xuehui; Ge, Xin Janet; Trucco, Massimo; Bottino, Rita

doi:10.1111/j.1600-6143.2005.01078.x

Cited by 54 publications

(36 citation statements)

References 29 publications

(32 reference statements)

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“…Islet perifusion. Glucose-stimulated insulin release was assayed by dynamic perifusion of cultured islets, as described (22). Aliquots of 100 freshly isolated murine islets were cultured in 1.5 ml RPMI-1640 medium (Cellgro Mediatech, Herndon, VA) in the absence or presence of 30 ng/ml sirolimus at 37°C for 24 h, followed by islet perifusion, as described (22).…”

Section: Methodsmentioning

confidence: 99%

“…Glucose-stimulated insulin release was assayed by dynamic perifusion of cultured islets, as described (22). Aliquots of 100 freshly isolated murine islets were cultured in 1.5 ml RPMI-1640 medium (Cellgro Mediatech, Herndon, VA) in the absence or presence of 30 ng/ml sirolimus at 37°C for 24 h, followed by islet perifusion, as described (22). Islets were first equilibrated by perifusing with Kreb KCl s-Ringer bicarbonate buffer (pH 7.4, 2.4 mmol/l CaCl 2 , 120 mmol/l NaCl, 1.2 mmol/l MgSO 4 , 5.4 mmol/l KCl, 1.2 mmol/l KH 2 PO 4 , 20 mmol/l HEPES) supplemented with 2.8 mmol/l glucose for 30 min, followed by perifusion with Krebs-Ringer bicarbonate buffer containing 2.8 or 20 mmol/l glucose at a constant flow rate of 1 ml/min.…”

Section: Methodsmentioning

confidence: 99%

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Sirolimus Is Associated With Reduced Islet Engraftment and Impaired β-Cell Function

et al. 2006

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Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, but only a fraction of transplanted islets can survive and become engrafted, and yet the underlying mechanism remains unclear. In this study, we examined the effect of sirolimus, a key component of the immunosuppressive regimen in clinical islet transplantation, on islet engraftment and function. To distinguish the effect of sirolimus on immune rejection from its effect on islet engraftment, we used a syngeneic model. Diabetic mice were transplanted with 250 islets under the renal capsule, followed by treatment with sirolimus or vehicle for 14 days. Thirty days posttransplantation, islet grafts were retrieved for the determination of insulin content and vascular density. Compared with mocktreated controls, diabetic recipient mice receiving sirolimus exhibited impaired blood glucose profiles and reduced glucose-stimulated insulin secretion, correlating with reduced intragraft insulin content and decreased vascular density. Islets exposed to sirolimus for 24 h in culture displayed significantly diminished glucose-stimulated insulin release, coinciding with decreased pancreas duodenum homeobox-1 and GLUT2 expression in cultured islets. Furthermore, sirolimus-treated diabetic recipient mice, as opposed to mock-treated controls, were associated with dyslipidemia. These data suggest that sirolimus, administered in the early posttransplantation phase, is a confounding factor for reduced islet engraftment and impaired ␤-cell function in transplants. Diabetes 55:2429 -2436, 2006 T he Edmonton protocol for islet transplantation depends on the infusion of ϳ10,000 IE (islet equivalents)/kg body wt, requiring multiple cadaver pancreata per diabetic recipient (1-4). Despite the implantation of such a large quantity of islets, Ͻ30% of transplanted islets can survive the procedure and gain stable engraftment, and yet the mechanism underlying the loss of a vast majority of islet mass in the early posttransplantation phase remains elusive (4,5). Unlike whole-organ transplantation, by which grafts are implanted as vascularized tissue, islets are transplanted as single islets or islet clusters that are considered avascular following collagenase digestion and isolation. Although residual endothelial cells in isolated islets may contribute to islet revascularization (6,7), adequate intraislet blood flow requires the formation of a functional microvascular network that links engrafted islets to surrounding tissues. These data suggest that microvascular perfusion to newly transplanted islets does not resume immediately after transplantation and can take up to 2 weeks before the reestablishment of a functional microvasculature in islet grafts (8,9). This delay in islet revascularization can potentially deprive islets of oxygen and nutrients, resulting in islet cell death, particularly within the core of engrafted islets. There is mounting evidence that impaired islet revascularization is an independent factor that limits the success rate...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Sirolimus Is Associated With Reduced Islet Engraftment and Impaired β-Cell Function

et al. 2006

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“…Insulin secretory characteristics and islet viability Islet insulin secretory ability was assessed in vitro by static or dynamic exposure to Krebs Ringer bicarbonate buffer containing low (2.8 mmol/l) and high (20 mmol/l) glucose [24,27].…”

Section: Methodsmentioning

confidence: 99%

Suitability of human juvenile pancreatic islets for clinical use

et al. 2006

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Aims/hypothesis The limited availability of deceased donor pancreases suitable for pancreas and islet transplantation calls for a broader utilisation of donor tissue for transplantation purposes. Young donors, representing, fortunately, a minor but significant pool of individuals, have been largely under-employed, mainly because of anatomical and functional incompatibilities with potential recipients. For islet transplantation, the isolation of pancreatic islets from young donors rarely occurs, because of technical problems. As a result of the peculiar characteristics of young donor pancreases, the standard isolation procedure does not allow efficient separation of the islets from the surrounding exocrine tissue, and favours the generation of mantled islets. Nonetheless, young donor islets offer high qualitative and clinically appealing characteristics. Subjects and methods We standardised a modified methodology to obtain purified and mantle-free human islets from young donors. This method principally involves efficient delivery of isolation enzyme with reduced mechanical disruption of the pancreas combined with additional filtration steps. Results We were able to obtain purified and mantle-free human islets from donors as young as 6 months of age with good morphological and functional properties. The good qualitative characteristics of the islets, evidenced in vitro, were proven in vivo, as they were qualitatively superior to islets of older donors in transplantation studies. Conclusions/interpretation This study justifies the utilisation of islets derived from young donors for islet transplantation.

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“…Apoptosis takes place during the isolation process when extracellular matrix surrounding islets is disrupted by exogenous isolation enzymes [4]. In addition, nonspecific inflammatory reaction at the site of transplantation could lead to release of proinflammatory cytokines and free radicals, potential inducers of apoptosis [2].…”

Section: Introductionmentioning

confidence: 99%

Dominant negative mutant forms of the cAMP response element binding protein induce apoptosis and decrease the anti-apoptotic action of growth factors in human islets

Sarkar¹,

Gunter

Bouchard

et al. 2007

Diabetologia

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Aims/hypothesis Transplantation of islets is a viable option for the treatment of diabetes. A significant proportion of islets is lost during isolation, storage and after transplantation as a result of apoptosis. cAMP response element binding protein (CREB) is an important cell survival factor. The aim of the present study was to determine whether preservation of CREB function is needed for survival of human islets. Materials and methods To determine the effects of downregulation of CREB activity on beta cell apoptosis in a transplantation setting, adenoviral vectors were used to express two dominant negative mutant forms of CREB in human islets isolated from cadaveric donors. Markers of apoptosis were determined in these transduced islets under basal conditions and following treatment with growth factor. Results Expression of CREB mutants in human islets resulted in significant (p<0.001) activation of caspase-9, a key regulatory enzyme in the mitochondrial pathway of apoptosis, when compared with islets transduced with adenoviral beta galactosidase. Immunocytochemical analysis showed the activation of caspase-9 to be predominantly in beta cells. Other definitive markers of apoptosis such as parallel activation of caspase-3, accumulation of cleaved poly-(ADP-ribose) polymerase and nuclear condensation were also observed. Furthermore, the anti-apoptotic action of growth factors exendin-4 and betacellulin in human islets exposed to cytokines was partially lost when CREB function was impaired. Conclusions/interpretation Our findings suggest that impairment of CREB-mediated transcription could lead to loss of islets by apoptosis with potential implications in islet transplantation as well as in the mechanism of beta cell loss leading to diabetes.

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Harmful Delayed Effects of Exogenous Isolation Enzymes on Isolated Human Islets: Relevance to Clinical Transplantation

Cited by 54 publications

References 29 publications

Sirolimus Is Associated With Reduced Islet Engraftment and Impaired β-Cell Function

Sirolimus Is Associated With Reduced Islet Engraftment and Impaired β-Cell Function

Suitability of human juvenile pancreatic islets for clinical use

Dominant negative mutant forms of the cAMP response element binding protein induce apoptosis and decrease the anti-apoptotic action of growth factors in human islets

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