Publications (28) View all
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Article: Low-Dose Rapamycin Unmasks the Protective Potential of Targeting Intragraft NF-κB for Islet Transplants.
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ABSTRACT: Islet-grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by NF-κB. Thus NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H2(d)) to C57/BL6 (H2(b)) mouse islet allografts by genetically-engineering islets to express the NF-κB super-repressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early-immediate pro-inflammatory response; with the most highly up-regulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10 and Ccl2; the cytokines Tnfα and Il6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50-95 % in islets and MIN6 β-cells in vitro, by inhibiting NF-;κB-dependent gene transcription. Histological and RTqPCR analysis at post-operative day (POD) 10 revealed that IκBα transduced islet allografts exhibited improved islet architecture and strong insulin-labelling with decreased Ccl2 and Il6 mRNA levels compared to GFP-transduced control grafts. Despite these protective effects, NF-κB blocked islet allografts were promptly rejected in our MHC mismatched mouse model. However, IκBα expressing grafts did harbour localized 'pockets' of Foxp3⁺ mononuclear cells not evident in control grafts. This result suggested to us that the effect of NF-κB blockade might synergize with regulatory Tcell sparing Rapamycin. Indeed, combining intragraft IκBα expression with low-dose Rapamycin increased the mean survival time of islet allografts from 20 to 81 days; with20 % of grafts surviving for greater than 100 days. In conclusion, Rapamycin unmasks the protective potential of intragraft NF-κB blockade which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.Cell Transplantation 10/2012; · 5.13 Impact Factor -
Article: A PRE-EXISTENT HYPOXIC GENE SIGNATURE PREDICTS IMPAIRED ISLET GRAFT FUNCTION AND GLUCOSE HOMEOSTASIS.
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ABSTRACT: We examined whether hypoxic exposure prior to the event of transplantation would have a positive or negative effect upon later islet graft function. Mouse islets, exposed to hypoxic culture were transplanted into syngeneic recipients. Islet graft function, beta cell physiology, as well as molecular changes were examined. Expression of hypoxia-response genes in human islets pre- and post-transplant was examined by microarray. Hypoxia-pre-exposed murine islet grafts provided poor glycemic control in their syngeneic recipients, marked by persistent hyperglycemia and pronounced glucose intolerance with failed first and second phase glucose-stimulated insulin secretion in vivo. Mechanistically, hypoxic pre-exposure stabilized HIF-1α with a concomitant increase in hypoxic-response genes including LDHA; a molecular gene set which would favor glycolysis and lactate production and impair glucose sensing. Indeed, static incubation studies showed hypoxia-exposed islets exhibited dysregulated glucose responsiveness with elevated basal insulin secretion. Isolated human islets, prior to transplantation, express a characteristic hypoxiaresponse gene expression signature, including high levels of LDHA, which is maintained post transplant. Hypoxic pre-exposure of an islet graft drives a HIF-dependent switch to glycolysis with subsequent poor glycemic control and loss of GSIS. Early intervention to reverse or prevent these hypoxia-induced metabolic gene changes may improve clinical islet transplantation.Cell Transplantation 10/2012; · 5.13 Impact Factor -
Article: HUMAN ISLETS EXPRESS A MARKED PRO-INFLAMMATORY MOLECULAR SIGNATURE PRIOR TO TRANSPLANTATION.
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ABSTRACT: : In the context of islet transplantation, experimental models show that induction of islet intrinsic NF-κB-dependent pro-inflammatory genes can contribute to islet graft rejection. Isolation of human islets triggers activation of the NF-κB and mitogen-activated kinase (MAPK) stress response pathways. However, the down stream NF-κB-target genes induced in human islets during the isolation process are poorly described. Therefore in this study, using microarray, bioinformatic, and RTqPCR approaches, we determined the pattern of genes expressed by a set of 14 human islet preparations. We found that isolated human islets express a panel of genes reminiscent of cells undergoing a marked NF- κB-dependent pro-inflammatory response. Expressed genes included; matrix metallopeptidase 1 (MMP1) and Fibronectin 1 (FN1), factors involved in tissue remodelling, adhesion and cell migration; inflammatory cytokines IL-1 β and IL-8; genes regulating cell survival including A20 and ATF3; and notably high expression of a set of chemokines that would favour neutrophil and monocyte recruitment including CXCL2, CCL2, CXCL12, CXCL1, CXCL6, CCL28. Of note, the inflammatory profile of isolated human islets was maintained after transplantation into RAG-/- recipients. Thus human islets can provide a reservoir of NF- κBdependent inflammatory factors that have the potential to contribute to the anti-islet-graft immune response. To test this hypothesis we extracted rodent islets under optimal conditions, forced activation of NF- κB, and transplanted them into allogenic recipients. These NF- κB activated islets not only expressed the same chemokine profile observed in human islets, but also struggled to maintain normoglycemia post transplantation. Further, NF- κB activated islets were rejected with a faster tempo as compared to non-NF- κB-activated rodent islets. Thus isolated human islets can make cell autonomous contributions to the ensuing allograft response by elaborating inflammatory factors that contribute to their own demise. These data highlight the potential importance of islet intrinsic pro-inflammatory responses as targets for therapeutic intervention.Cell Transplantation 03/2012; · 5.13 Impact Factor -
Article: Pre-clinical model of composite foetal pig pancreas fragment/renal xenotransplantation to treat renal failure and diabetes.
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ABSTRACT: Development of a limitless source of β cells for xenotransplantation into patients suffering type 1 diabetes and renal failure that can control their diabetes and provide normal renal function in one procedure would be a major achievement. For the islet tissue to survive transplantation, as an islet-kidney composite graft this would have significant advantages. It would simplify the surgical procedure; remove the complications caused by the exocrine pancreas whilst reversing diabetes and uraemia. It was our hypothesis that a composite foetal porcine pancreas fragment (FPPF)/renal graft could achieve these objectives in a large pre-clinical animal model as a means to establish whether this would be feasible before moving to the clinic. Inbred 'Westran' pig FPPF were transplanted under the kidney capsule of syngeneic Westran pig recipients without immunosuppression. Following maturation of the FPPF under the renal subcapsular space of this recipient, this kidney bearing the composite FPPF piggyback graft was removed and transplanted into another nephrectomized and pancreatectomized recipient to demonstrate function. Under the kidney capsule of the first transplant group (n = 6), the FPPF-transplanted tissue developed and matured to form islet cell nests. These composite FPPF/renal grafts were then successfully removed and transplanted into the second functional assessment recipient group. This second group of six composite FPPF/renal-grafted pigs had normal renal function for more than 44 days and normal glucose homoeostasis without exogenous insulin as assessed by normal glucose tolerance tests, K values and normal glucagon secretion. Histological analysis showed despite the ischaemic insult during the composite kidney transplant procedure, there was appropriate development of islet-like structures up to and beyond 224 days after the original transplantation under the kidney capsule. This study shows that the use of composite FPPF/renal grafts can cure both diabetes and renal failure with a single-transplant procedure. Using such composite grafts for xenotransplantation would simplify the surgical procedure and protect the islet graft from the immediate innate immune response.Xenotransplantation 11/2011; 18(6):390-9. · 2.33 Impact Factor -
Article: Interleukin-21 is critically required in autoimmune and allogeneic responses to islet tissue in murine models.
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ABSTRACT: Type 1 diabetes is an incurable chronic autoimmune disease. Although transplantation of pancreatic islets may serve as a surrogate source of insulin, recipients are subjected to a life of immunosuppression. Interleukin (IL)-21 is necessary for type 1 diabetes in NOD mice. We examined the efficacy of an IL-21-targeted therapy on prevention of diabetes in NOD mice, in combination with syngeneic islet transplantation. In addition, we assessed the role of IL-21 responsiveness in islet allograft rejection in mouse animal models. NOD mice were treated with IL-21R/Fc, an IL-21-neutralizing chimeric protein. This procedure was combined with syngeneic islet transplantation to treat diabetic NOD mice. Survival of allogeneic islet grafts in IL-21R-deficient mice was also assessed. Evidence is provided that IL-21 is continually required by the autoimmune infiltrate, such that insulitis was reduced and reversed and diabetes inhibited by neutralization of IL-21 at a late preclinical stage. Recovery from autoimmune diabetes was achieved by combining neutralization of IL-21 with islet transplantation. Furthermore, IL-21-responsiveness by CD8+ T-cells was sufficient to mediate islet allograft rejection. Neutralization of IL-21 in NOD mice can inhibit diabetes, and when paired with islet transplantation, this therapeutic approach restored normoglycemia. The influence of IL-21 on a graft-mounted immune response was robust, since the absence of IL-21 signaling prevented islet allograft rejection. These findings suggest that therapeutic manipulation of IL-21 may serve as a suitable treatment for patients with type 1 diabetes.Diabetes 03/2011; 60(3):867-75. · 8.29 Impact Factor
