Human Pancreatic Islets Transfected to Produce an Inhibitor of TNF are Protected against Destruction by Human Leukocytes

Abstract

The objective of this study was to determine whether transfection of human islets with an adenovirus construct encoding an inhibitor of tumor necrosis factor (TNFi) was effective at limiting damage to beta cells induced by human peripheral blood leukocytes (huPBL). Human islets transfected with TNFi or control islets were transplanted under the kidney capsule of NOD-scid mice. After a 15-day engraftment period, half of the mice received injections of activated huPBL and half received buffer injections. Islet graft function was assessed by two different methods, both of which use a species-specific radioimmunoassay to determine human insulin. In some mice, insulin production following intraperitoneal glucose injection was determined in serum. In other mice, total graft insulin content was determined by acid ethanol extraction. Histochemical stains were performed on some kidneys at the termination of the experiment to evaluate graft presence, transgene expression, and huPBL infiltration. In huPBL injected mice, graft performance was maintained in mice whose grafts were transfected with TNFi but declined substantially in control groups with sham transfected or beta-galactosidase transfected islet grafts. Similar results were obtained using either glucose-stimulated insulin release or graft insulin content as a measure of graft survival. There was no significant difference in graft function between control groups receiving buffer injections, regardless of whether the islets had been transfected. Human leukocytes were found in all huPBL groups regardless of islet transfection status. We conclude that transfection of human islets with an adenovirus encoding TNFi protects beta cells from destruction induced by human leukocytes. The local production of TNFi does not prevent graft infiltration by leukocytes, only the destruction of grafts by the infiltrating leukocytes. These results raise the possibility that local expression of an inhibitor of the proinflammatory cytokine TNF-alpha may also prevent graft failure in clinical islet transplantation.

Adenovirus-based vascular endothelial growth factor gene delivery to human pancreatic islets

“…42 Furthermore, reduction of glucose concentration back to basal level has resulted in reduction in insulin response to basal level in all cases, indicating that the islets do remain dynamically responsive to glucose concentration. Our results for the in vitro islet function test are in good agreement with the work of Dobson et al,26 who demonstrated that the transfection of human islets with the adenovirus did not affect graft function in NOD-SCID mice recipients, as human insulin secretion from the transfected and non-transfected grafts did not differ. This is also consistent with the results reported by others showing that transfection of islets with adenoviral vectors does not inhibit islet graft function in allograft or xenograft models.…”

“…26 These mice lack mature T and B lymphocytes and thus cannot mount adaptive immune responses to reject xenografts of human islets. In this model, islet viability is assessed by measuring human Cpeptide and insulin levels in response to glucose challenge.…”

Activation of c-Jun NH2-terminal kinase (JNK) pathway during islet transplantation and prevention of islet graft loss by intraportal injection of JNK inhibitor

“…The low frequency of islet grafting is dependent on poor islet recovery from donors [4] and early islet loss during the first hours after grafting [5,6], termed primary graft nonfunction. Islet transplantation exposes cells to a variety of stressful stimuli, notably proinflammatory cytokines that encourage beta cell death and lead to early graft failure [7][8][9]. The reduction in islet mass immediately after transplantation implicates beta cell death by apoptosis and the prerecruitment of intracellular death-signalling pathways [10][11][12].…”

Biological and Biomaterial Approaches for Improved Islet Transplantation

“…Generalized immunosuppression using steroids (Corbett et al, 1993) Ex vivo gene therapy using IL-1 receptor antagonist (Gysemans et al, 2003) TNF-␣ Administration of soluble TNF-␣ receptor (Farney et al, 1993) Adv transfection of inhibitor of TNF-␣ (TNFi) (Dobson et al, 2000) …”