Marginal Islet Mass Research Articles

Diannexin Decreases Inflammatory Cell Infiltration Into the Islet Graft, Reduces β-Cell Apoptosis, and Improves Early Graft Function

A major unmet challenge is to reduce the islet mass needed for insulin independence in type 1 diabetic recipients after islet transplantation. The recombinant homodimer of human annexin V, diannexin, has completed a Phase II Clinical Trial in Kidney Transplantation (NCT00615966). We developed a marginal islet mass transplantation model (10-12 islets per gram of recipient body weight) and investigated whether diannexin prevents β-cell apoptosis and improves islet graft function. Diannexin was administered to islet cell donors shortly before pancreas harvest, added to isolation reagents, and infused into recipients at the time of transplantation and repeated daily until day 4. In the syngeneic marginal islet mass transplantation model, the median time needed to achieve normoglycemia was reduced from 17.0 days among untreated controls to 3.5 days among diannexin-treated recipients (P=0.004). Histologic analysis of islet grafts harvested on day 3 posttransplantation revealed decreased macrophage (44.7%±9.8% vs. 19.2%±3.2%, P=0.007) and T-cell infiltration (25.9%±5.5% vs. 9.1%±1.1%, P=0.004), and a lower rate of islet cell apoptosis (20.5%±2.8% vs. 7.6%±2.3%, P=0.01) with diannexin treatment. Expression profiling of the islet grafts showed significantly lower levels of mRNA for the proapoptotic molecule Bid, but higher levels of interleukin-6, interferon-γ, and immunosuppressive cytokine interleukin-10. Our findings demonstrate that diannexin improves the early function of marginal mass islet grafts, and its effects are associated with reductions in inflammatory cell infiltration and β-cell death by apoptosis after islet transplantation.

Mesenchymal Stem Cells Prevent Acute Rejection and Prolong Graft Function in Pancreatic Islet Transplantation

Pancreatic islet transplantation is a promising cell-based therapy for type 1 diabetes (insulin-dependent diabetes mellitus), a disease triggered by the immune response against autoantigens of beta-cells. However, the recurrence of immune response after transplantation and the diabetogenic and growth-stunting side effects of immunosuppressants are major challenges to the application of islet transplantation. Mesenchymal stem cells (MSCs) have recently been reported to modulate the immune response in allogeneic transplantation. The ability of MSCs, either syngeneic or allogeneic to recipients, to prevent acute rejection and improve glycemic control was investigated in rats with diabetes given a marginal mass of pancreatic islets through the portal vein. Reduced glucose levels and low-grade rejections were observed up to 15 days after transplantation upon triple-dose administration of MSCs, indicating that MSCs prolong graft function by preventing acute rejection. The efficacy of MSCs was associated with a reduction of pro-inflammatory cytokines and was independent of the administration route. Efficacy was similar for MSCs whether syngeneic or allogeneic to recipients and comparable to that of immunosuppressive therapy. The results show that MSCs modulate the immune response through a down-regulation of pro-inflammatory cytokines, suggesting that MSCs may prevent acute rejection and improve graft function in portal vein pancreatic islet transplantation.

Prediction of Marginal Mass Required for Successful Islet Transplantation

ABSTRACTIslet quality assessment methods for predicting diabetes reversal (DR) following transplantation are needed. We investigated two islet parameters, oxygen consumption rate (OCR) and OCR per DNA content, to predict transplantation outcome and explored the impact of islet quality on marginal islet mass for DR. Outcomes in immunosuppressed diabetic mice were evaluated by transplanting mixtures of healthy and purposely damaged rat islets for systematic variation of OCR/DNA over a wide range. The probability of DR increased with increasing transplanted OCR and OCR/DNA. On coordinates of OCR versus OCR/DNA, data fell into regions in which DR occurred in all, some, or none of the animals with a sharp threshold of around 150-nmol/min mg DNA. A model incorporating both parameters predicted transplantation outcome with sensitivity and specificity of 93%% and 94%%, respectively. Marginal mass was not constant, depended on OCR/DNA, and increased from 2,800 to over 100,000 islet equivalents/kg body weight as OCR/DNA decreased. We conclude that measurements of OCR and OCR/DNA are useful for predicting transplantation outcome in this model system, and OCR/DNA can be used to estimate the marginal mass required for reversing diabetes. Because human clinical islet preparations in a previous study had OCR/DNA

Comparison of Four Pancreatic Islet Implantation Sites

Although the liver is the most common site for pancreatic islet transplantation, it is not optimal. We compared kidney, liver, muscle, and omentum as transplantation sites with regard to operative feasibility, and the efficiency of implantation and glycemic control. Islets from C57BL/6 mice were transplanted into diabetic syngeneic recipients. The mean operative time and mortality were measured to assess feasibility. To assess implantation efficiency, the marginal mass required to cure diabetes and the mean time taken to achieve normoglycemia were measured. A glucose tolerance test was performed to assess glycemic control efficiency. The data are listed in the order of the kidney, liver, muscle, and omentum, respectively. The mean mortality rate was 6.7, 20.0, 7.1, and 12.5%; the mean operative time was 10.2, 27.4, 11.2, and 19.8 min; the marginal islet mass was 100, 600, 600, and 200 islet equivalence units and the mean time to reach euglycemia was 3.0, 15.1, 26.6, and 13.9 days. The glucose kinetics of omental pouch islets was the most similar to controls. Thus, a strategic approach is required for deciding on the best transplantation recipient sites after considering donor sources and islet volume. Alternatives can be chosen based on safety or efficacy.

Porcine Marginal Mass Islet Autografts Resist Metabolic Failure Over Time and Are Enhanced by Early Treatment with Liraglutide

Although insulin independence is maintained in most islet recipients at 1 yr after transplant, extended follow-up has revealed that many patients will eventually require insulin therapy. Previous studies have shown that islet autografts are prone to chronic failure in large animals and humans, suggesting that nonimmunological events contribute to islet graft functional decay. Early intervention with therapies that promote graft stability should provide a measurable benefit over time. In this study, the efficacy of the long-acting glucagon-like peptide-1 analog liraglutide was explored in a porcine marginal mass islet autograft transplant model. Incubation with liraglutide enhanced porcine islet survival and function after prolonged culture. Most vehicle-treated (83%) and liraglutide-treated (80%) animals became insulin independent after islet autotransplantation. Although liraglutide therapy did not improve insulin independence rates or blood glucose levels after transplant, a significant increase in insulin secretion and acute-phase insulin response was observed in treated animals. Surprisingly, no evidence for deterioration of graft function was observed in any of the transplanted animals over more than 18 months of follow-up despite significant weight gain; in fact, an enhanced response to glucose developed over time even in control animals. Histological analysis showed that intraportally transplanted islets remained highly insulin positive, retained alpha-cells, and did not form amyloid deposits. This study demonstrates that marginal mass porcine islet autografts have stable long-term function, even in the presence of an increasing metabolic demand. These results are discrepant with previous large animal studies and suggest that porcine islets may be resistant to metabolic failure.

The Use of an Approved Biodegradable Polymer Scaffold as a Solid Support System for Improvement of Islet Engraftment

The aim of this study was to investigate whether the use of a medically approved biodegradable scaffold as a solid support system would enhance graft survival following transplantation into the omental pouch in a preclinical large animal model. Six beagle dogs underwent total pancreatectomy followed by islet autotransplantation into the omental pouch. Four dogs received islets seeded in a biodegradable polymer scaffold and two received free islets without a scaffold. All four animals that received islets in the scaffold became normoglycemic without exogeneous insulin injection. One dog, transplanted with the largest number of islets, maintained a normal metabolic state until the graft was removed at 5 months posttransplant. In two out of the three that received a marginal islet mass, insulin independence was sustained up to 2 months. In contrast, two dogs transplanted with a similar marginal mass without the scaffold never became normoglycemic. Histological examination of the grafts in the scaffold showed numerous well-granulated, insulin-containing cells as well as glucagon-positive cells. These results indicate that biodegradable scaffolds may enhance survival and function of islet grafts. Manipulation of the microenvironment of transplanted islets may constitute the basis for new approaches to enhance islet engraftment.

The Role of Macrophage Migration Inhibitory Factor in Mouse Islet Transplantation

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many tissues including pancreatic beta-cells. This study investigates the impact of MIF on islet transplantation using MIF knock-out (MIFko) mice. Early islet function, assessed with a syngeneic marginal islet mass transplant model, was enhanced when using MIFko islets (P<0.05 compared with wild-type [WT] controls). This result was supported by increased in vitro resistance of MIFko islets to apoptosis (terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling assay), and by improved glucose metabolism (lower blood glucose levels, reduced glucose areas under curve and higher insulin release during intraperitoneal glucose challenges, and in vitro in the absence of MIF, P<0.01). The beneficial impact of MIFko islets was insufficient to delay allogeneic islet rejection. However, the rejection of WT islet allografts was marginally delayed in MIFko recipients by 6 days when compared with WT recipient (P<0.05). This effect is supported by the lower activity of MIF-deficient macrophages, assessed in vitro and in vivo by cotransplantation of islet/macrophages. Leukocyte infiltration of the graft and donor-specific lymphocyte activity (mixed lymphocyte reaction, interferon gamma ELISPOT) were similar in both groups. These data indicate that targeting MIF has the potential to improve early function after syngeneic islet transplantation, but has only a marginal impact on allogeneic rejection.

A fibrin gel carrier system for islet transplantation into kidney subcapsule

Islet transplantation is a promising therapeutic option for type 1 diabetes, and actively performed in the clinic as well as in the animal experiments. For the rodent experiments, islet transplantation into kidney subcapsule is widely used to assess islet quality, however, it is often difficult to do using a polyethylene tubing and fine needle because of inherent dead volume of needle and stickiness of the tubing to islets. This problem makes it difficult to interpret the physiological response to different islet doses. Here, we developed a simple fibrin gel carrier system for islet transplantation into kidney subcapsule and utilized it to determine the marginal islet mass sufficient for correction of hyperglycemia in diabetic nude mice.

Hepatocyte Growth Factor Enhances Engraftment and Function of Nonhuman Primate Islets

OBJECTIVE—Adenoviral delivery of hepatocyte growth factor (HGF) to rodent islets improves islet graft survival and function, markedly reducing the number of islets required to achieve glucose control. Here, we asked whether these prior observations in rodent models extend to nonhuman primate (NHP) islets.RESEARCH DESIGN AND METHODS—NHP islets were transduced with murine (Ad.mHGF) or human (Ad.hHGF) adenoviral HGF (Ad.HGF) at low multiplicity of infection and studied in vitro. To study the function of Ad.HGF-transduced NHP islets in vivo, a renal subcapsular marginal mass islet transplant model was developed in streptozotocin-induced diabetic NOD-SCID mice.RESULTS—Baseline glucose values were 454.7 ± 11.3 mg/dl (n = 7). Transplant of 500 NHP islet equivalents (IE) had only a marginal effect on blood glucose (369.1 ± 9.7 mg/dl, n = 5). In striking contrast, 500 NHP IE transduced with Ad.mHGF promptly and continuously corrected blood glucose (142.0 ± 6.2 mg/dl, n = 7) for the 6-week duration of the experiment. Unilateral nephrectomy resulted in an immediate return of glucose to baseline diabetic levels. Interestingly, adenoviral DNA, as well as mouse HGF (mHGF) mRNA derived from the adenovirus, were present for 42 days posttransplantation. Surprisingly, transplant of 500 IE with Ad.hHGF, as compared with Ad.mHGF, resulted in only marginal correction of blood glucose, suggesting that human HGF is less efficient than mHGF in this system.CONCLUSIONS—These studies demonstrate that mHGF markedly improves islet transplant outcomes in the highest preclinical species examined to date. HGF has promise as an agent that can improve islet mass and function in transplant models and likely in other models of types 1 and 2 diabetes.

Hepatocyte Growth Factor Enhances Engraftment and Function of Nonhuman Primate Islets

OBJECTIVE—Adenoviral delivery of hepatocyte growth factor (HGF) to rodent islets improves islet graft survival and function, markedly reducing the number of islets required to achieve glucose control. Here, we asked whether these prior observations in rodent models extend to nonhuman primate (NHP) islets. RESEARCH DESIGN AND METHODS—NHP islets were transduced with murine (Ad.mHGF) or human (Ad.hHGF) adenoviral HGF (Ad.HGF) at low multiplicity of infection and studied in vitro. To study the function of Ad.HGF-transduced NHP islets in vivo, a renal subcapsular marginal mass islet transplant model was developed in streptozotocin-induced diabetic NOD-SCID mice. RESULTS—Baseline glucose values were 454.7 ± 11.3 mg/dl (n = 7). Transplant of 500 NHP islet equivalents (IE) had only a marginal effect on blood glucose (369.1 ± 9.7 mg/dl, n = 5). In striking contrast, 500 NHP IE transduced with Ad.mHGF promptly and continuously corrected blood glucose (142.0 ± 6.2 mg/dl, n = 7) for the 6-week duration of the experiment. Unilateral nephrectomy resulted in an immediate return of glucose to baseline diabetic levels. Interestingly, adenoviral DNA, as well as mouse HGF (mHGF) mRNA derived from the adenovirus, were present for 42 days posttransplantation. Surprisingly, transplant of 500 IE with Ad.hHGF, as compared with Ad.mHGF, resulted in only marginal correction of blood glucose, suggesting that human HGF is less efficient than mHGF in this system. CONCLUSIONS—These studies demonstrate that mHGF markedly improves islet transplant outcomes in the highest preclinical species examined to date. HGF has promise as an agent that can improve islet mass and function in transplant models and likely in other models of types 1 and 2 diabetes.

Liraglutide, a Long-Acting Human Glucagon-Like Peptide 1 Analog, Improves Glucose Homeostasis in Marginal Mass Islet Transplantation in Mice

The current scarcity of high-quality deceased pancreas donors prevents widespread application of islet transplantation for treatment of labile type 1 diabetes mellitus. Opportunities for the improvement of current techniques include optimization of islet isolation and purification, use of culture with pharmacological insulinotropic agents, strategies to reduce graft rejection and inflammation, and the search for alternative insulin producing tissue. Here, we report our findings on the efficacy of the long-acting human glucagon-like peptide 1 analog, liraglutide, in a mouse model of marginal mass islet transplantation. Liraglutide was administered (200 microg/kg sc twice daily) after a marginal mass syngeneic islet transplant in streptozotocin-induced diabetic BALB/c mice. Time-to-normoglycemia was significantly shorter in liraglutide-treated animals (median 1 vs. 7 d; P = 0.0003), even in recipients receiving sirolimus (median 1 vs. 72.5 d; P < 0.0001). Liraglutide-treated animals also demonstrated improved glucose tolerance as assessed by an ip glucose tolerance test. Liraglutide discontinuation at postoperative d 90 resulted in diminished glucose tolerance during the ip glucose tolerance test, whereas a late-start liraglutide therapy 90 d after transplant resulted in no improvement. These findings suggest that liraglutide therapy mediates early and late insulinotropic effects. In accord with this hypothesis, insulin/terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling fluorescence microscopy showed reduced transplanted beta-cell apoptosis in liraglutide-treated recipients 48 h after transplant. In addition, liraglutide resulted in improved glucose-dependent insulin secretion. Overall, our data show that liraglutide has a beneficial impact on the engraftment and function of syngeneic islet transplants in mice, when administered continuously starting on the day of transplant.

The Caspase Selective Inhibitor EP1013 Augments Human Islet Graft Function and Longevity in Marginal Mass Islet Transplantation in Mice

Clinical islet transplantation can provide insulin independence in patients with type 1 diabetes, but chronic graft failure has been observed. This has been attributed in part to loss of >or=60% of the transplanted islets in the peritransplant period, resulting in a marginal implant mass. Strategies designed to maximize survival of the initial islet mass are likely to have major impact in enhancing long-term clinical outcomes. EP1013 (N-benzyloxycabonyl-Val Asp-fluoromethyl ketone [zVD-FMK]), is a broad-spectrum caspase selective inhibitor with no observed toxicity in rodents. The therapeutic benefit of EP1013 was examined in a syngeneic rodent islet transplant model using deceased donor human islets to determine whether the amount of tissue required to restore euglycemia in diabetic animals could be reduced. EP1013 (combined pretransplant islet culture for 2 h and in vivo treatment for days 0-5 posttransplant) significantly improved marginal islet mass function following syngeneic islet transplantation in mice, even at lower doses, compared with previous studies using the pan-caspase inhibitor N-benzyloxycabonyl-Val Ala-Asp-fluoromethyl ketone (zVAD-FMK). EP1013 supplementation in vitro improved human islet yields following prolonged culture and reversed diabetes following implantation of a marginal human islet mass (80-90% reduction) into mice. Our data suggest that EP1013 therapy will markedly reduce the islet mass required in clinical islet transplantation, improving insulin independence rates following single-donor infusion.

Caspase Inhibitor Therapy Enhances Marginal Mass Islet Graft Survival and Preserves Long-Term Function in Islet Transplantation

Islet transplantation can provide insulin independence in patients with type 1 diabetes, but islets derived from two or more donors are often required. A significant fraction of the functional islet mass is lost to apoptosis in the immediate posttransplant period. The caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-FMK) has been used therapeutically to prevent apoptosis in experimental animal models of ischemic injury, autoimmunity, and degenerative disease. In the current study, zVAD-FMK therapy was examined in a syngeneic islet transplant model to determine whether caspase inhibition could improve survival of transplanted islets. zVAD-FMK therapy significantly improved marginal islet mass function in renal subcapsular transplantation, where 90% of zVAD-FMK-treated mice became euglycemic with 250 islets, versus 27% of the control animals (P < 0.001). The benefit of zVAD-FMK therapy was further demonstrated after intraportal transplantation, where 75% of zVAD-FMK-treated animals established euglycemia with only 500 islets, and all of the controls remained severely diabetic (P < 0.001). zVAD-FMK pretreatment of isolated islets in the absence of systemic therapy resulted in no significant benefit compared with controls. Long-term follow-up of transplanted animals beyond 1 year posttransplant using glucose tolerance tests confirmed that a short course of zVAD-FMK therapy could prevent metabolic dysfunction of islet grafts over time. In addition, short-term zVAD-FMK treatment significantly reduced posttransplant apoptosis in islet grafts and resulted in preservation of graft insulin reserve over time. Our data suggest that caspase inhibitor therapy will reduce the islet mass required in clinical islet transplantation, perhaps to a level that would routinely allow for insulin independence after single-donor infusion.

Prolonged Survival of Allogeneic Islets in Cynomolgus Monkeys After Short-Term Anti-CD154-Based Therapy: Nonimmunologic Graft Failure?

Conventional drug therapy and several anti-CD154 mAb-based regimens were tested in the nonhuman primate (NHP) islet allograft model and found to be inadequate because islets were lost to rejection. Short-term therapy with an optimized donor-specific transfusion (DST) + rapamycin (RPM) + anti-CD154 mAb regimen enables immunosuppression drug-free islet allograft function for months following cessation of therapy in the NHP islet allograft model. After a substantial period of drug-free graft function, these allografts slowly and progressively lost function. Pathologic studies failed to identify islet allograft rejection as a destructive islet invasive lymphocytic infiltration of the allograft was not detected. To evaluate the mechanism, immunologic versus nonimmunologic, of the late islet allograft loss in hosts receiving the optimized therapeutic regimen, we performed experiments with islet autografts and studied islet function in NHPs with partial pancreatectomy. The results in both experiments utilizing autologous islet allografts and partially pancreatectomized hosts reinforce the view that the presence of a marginal islet mass leads to slowly progressive nonimmunological islet loss. Long-term clinically successful islet cell transplantation cannot be realized in the absence of parallel improvements in tolerizing regimens and in the preparation of adequate numbers of islets.

Overexpression of Glutathione Peroxidase With Two Isoforms of Superoxide Dismutase Protects Mouse Islets From Oxidative Injury and Improves Islet Graft Function

Primary nonfunction of transplanted islets results in part from their sensitivity to reactive oxygen species (ROS) generated during the isolation and transplantation process. Our aim was to examine whether coexpression of antioxidant enzymes to detoxify multiple ROS increased the resistance of mouse islets to oxidative stress and improved the initial function of islet grafts. Islets from transgenic mice expressing combinations of human copper/zinc superoxide dismutase (SOD), extracellular SOD, and cellular glutathione peroxidase (Gpx-1) were subjected to oxidative stress in vitro. Relative viability after hypoxanthine/xanthine oxidase treatment was as follows: extracellular SOD + Gpx-1 + Cu/Zn SOD > extracellular SOD + Gpx-1 > extracellular SOD > wild type. Expression of all three enzymes was the only combination protective against hypoxia/reoxygenation. Islets from transgenic or control wild-type mice were then transplanted into streptozotocin-induced diabetic recipients in a syngeneic marginal islet mass model, and blood glucose levels were monitored for 7 days. In contrast to single- and double-transgenic grafts, triple-transgenic grafts significantly improved control of blood glucose compared with wild type. Our results indicate that coexpression of antioxidant enzymes has a complementary beneficial effect and may be a useful approach to reduce primary nonfunction of islet grafts.

Effect of donor treatment with heparinoids on graft function after intraportal transplantation of a marginal islet mass: An experimental study

Heparinoids interact with factors that are involved in ischemia-reperfusion injury and thus may prevent organ injury. We therefore studied the effects on subsequent intraportal islet transplantation of systemic administration of unfractionated and N-desulphated heparin to donors prior to pancreatectomy. Donor rats were given an intravenous injection of either heparin (1.3 mg/kg or 13.3 mg/kg; 200 U/kg or 2000 U/kg, respectively) or N-desulphated heparin (50 mg/kg; ∼5 U/kg) at 5 to 10 minutes prior to pancreas procurement. Five hundred freshly isolated islets were injected intraportally into syngeneic male Lewis recipients that had developed streptozotocin-induced diabetes. Blood glucose and body weight were monitored for 5 weeks thereafter. Rats transplanted with islets from donors given high dose heparin showed a fall in blood glucose from 25.1 ± 1.4 to 11.0 ± 2.7 mmol/L ( P < .01) with 60% of animals euglycemic within the first week. In contrast, the controls, did not show a fall in glucose levels at 1 week and none had become euglycaemic. Normalization of glycemia was slower in recipients of islets from animals treated with the lower dose of heparin. Results were intermediate with islets from donors given N-desulphated heparin. Nevertheless, all heparinoids used in this study caused more than a doubling of the number of animals achieving normoglycemia by 3 to 4 weeks. We hypothesize that pretreatment of the donor with heparin protects islet integrity during procurement and isolation and hence accelerates islet engraftment and remodelling. Since the effect was seen with N-desulphated heparin, which has negligible anticoagulant properties, we believe the mechanism to be independent of the anticoagulant activity.

Hepatocyte growth factor gene therapy for islet transplantation

Recent clinical studies have documented that human islet transplantation has the potential to replace pancreatic endocrine function in patients with type 1 diabetes. These studies have also highlighted an enormous shortage of human islets that impedes the use of islet transplantation in clinical practice on a larger scale. To address this problem, one potential approach is to use islet growth factors to increase beta cell replication, to improve beta cell function and to enhance beta cell survival. In that context, transgenic mice overexpressing hepatocyte growth factor (HGF) in the pancreatic beta cell display increased beta cell proliferation, function and survival. More importantly, HGF-overexpressing transgenic mouse islets markedly improve transplant performance in severe combined immunodeficiency (SCID) mice and reduce the number of islets required for successful islet transplantation. Recently, adenoviral-mediated gene transfer of HGF into normal rodent islets has confirmed the beneficial effects of HGF in improving islet transplant outcomes in two marginal mass islet transplant models in rodents: islet transplant under the kidney capsule in SCID mice; and portal islet allograft transplantation in rats treated with the Edmonton immunosuppressive regimen. These studies suggest that ex vivo HGF gene therapy has the potential to reduce the number of human islets required for successful islet transplantation.

Adenovirus Transduction Induces Expression of Multiple Chemokines and Chemokine Receptors in Murine β Cells and Pancreatic Islets

Adenoviral vectors are highly efficient for transferring genes to islets. However, the inflammatory and immune responses stimulated by adenovirus may be detrimental to islet survival. Given the role of chemokines and their receptors in inflammation, we analyzed their expression in isolated murine islets, in a murine β cell line and in syngeneic islet grafts after adenovirus transduction (AdRSVLacZ). AdRSVLacZ transduction enhanced and induced the expression of a variety of chemokines. Transduced syngeneic transplanted islets showed significantly enhanced expression of multiple chemokines and receptors, including monocyte chemoattractant protein-1 (MCP-1), CC chemokine receptor 2 (CCR2) and regulated upon activation, normal T cell expressed and secreted (RANTES), compared with untransduced islet grafts. AdRSVLacZ-transduced islet grafts had significant mononuclear infiltrates, and in situ hybridization demonstrated intragraft expression of MCP-1, CCR2 and RANTES. Although adenovirus transduction did not impair in vitro insulin secretion, diabetes was reversed in only one of six recipients of a marginal mass of AdRSVLacZ-transduced islets, compared with six of six control recipients. In conclusion, multiple chemokines and chemokine receptors are expressed by murine islets constitutively and in response to adenovirus transduction. Adenovirus transduction impairs engraftment of marginal mass of transplanted islets. This is not because of direct vector toxicity of islet secretory capacity, but may be related to host innate immunity in response to adenovirus vector.

Sirolimus and islet transplants

IMMUNOSUPPRESSION is a critical determinant of the success of islet transplants. Tailoring immunosuppression to the specific setting of marginal mass islet cell transplants in autoimmune type 1 diabetic recipients would likely increase the safety, success rate, and applicability of such transplants. Tailored immunosuppression would ideally inhibit antigen-independent innate immune responses during the engraftment of transplanted islets, control alloimmune responses, and thwart autoimmune responses without diabetogenic and nephrotoxic side effects. Rejectionfree protocols need to be administered until markers of imminent islet rejection are validated or until robust function of a dysfunctional graft is restored. Sirolimus is a macrocyclic lactone with potent immunosuppressive properties. Its mechanism of action involves the blockade of IL-2 and other T-cell growth factor-mediated signal transduction and the inhibition of cell cycle progression. Sirolimus is conceivably the single most important contribution to the immunosuppressive armamentarium in islet transplantation. The immunosuppressive synergism between sirolimus and cyclosporin A (CsA) markedly reduces the incidence of solid organ and islet allograft rejection; it also enables steroids to be avoided and CsA doses to be profoundly reduced. In vitro studies suggested competitive inhibition between sirolimus and tacrolimus. Synergism between those two drugs was demonstrated in rat transplant models. The synergism between sirolimus (and other mammalian targets of rapamycin antagonists) and sphingosine 1-phosphate receptor agonists enables both steroids and calcineurin inhibitors to be avoided, thereby protecting allografts from rejection as well as from diabetogenic and nephrotoxic side effects. The synergism between sirolimus and selective blockers of costimulatory pathways helps induce immunologic tolerance and may, in the future, facilitate minimization or discontinuation of maintenance immunosuppression. The distinctive profile of sirolimus suggests an important role for it as an immunosuppressive and tolerogenic agent for diabetic recipients of islet transplants. Studies of the tolerogenic potential of sirolimus have been performed predominantly in cardiac and islet transplant models in mice. Sirolimus was used at a dose of 0.2 mg/kg/d intraperitoneally (IP) daily for the first 3 days, then every other day until the advent of hyperglycemia. It markedly prolonged the survival of islet allografts across a complete major histocompatibility barrier in interleukin (IL)-2 / and IL-4 / double knockout mice. Thus, sirolimus blocks T-cell growth factor signals beyond IL-2 and IL-4, especially IL-7 and IL-15; these rapamycin-sensitive T-cell growth factor signals are critical in the execution of islet allograft rejection. Combined costimulatory blockade (anti-CD154 monoclonal antibody and CTLA4-Ig) and sirolimus monotherapy 0.2 mg/kg/d IP on days 0, 1, 2, then every other day through day 14 posttransplant both facilitated indefinite survival of cardiac allografts in the Balb/c to C3H/He strain combination. Adding CsA, but not sirolimus, to treatment with anti-CD154 monoclonal antibody and CTLA4-Ig blocked the capacity of costimulatory blockade to produce permanent engraftment. Subsequently, islet transplant experiments were conducted in IL-2-deficient mice treated with sirolimus, and cardiac allograft transplant experiments in Bcl-XL transgenic mice treated with costimulatory blockade. Those experiments assessed the importance of alloreactive T-cell pool size, and more specifically activated-induced cell death of alloreactive T cells (in peripheral tolerance induction). They confirmed the hypothesis that sirolimus blocks the proliferative component of IL-2 signaling but does not inhibit priming of activation-induced cell death. They also demonstrated that deletion of activated T cells, through activation-induced cell death or growth factor withdrawal, is necessary to achieve peripheral tolerance across major histocompatibility barriers. The efficacy of sirolimus in preventing spontaneous and recurrent autoimmune diabetes has been studied in NOD mice, a clinically relevant model of autoimmune type 1 diabetes. Sirolimus was administered per os (PO) 3 times per week from 8 to 10 weeks of age; it prevented onset of autoimmune diabetes in 90% of NOD mice at a dose of 0.6

Adenovirus-mediated Hepatocyte Growth Factor Expression in Mouse Islets Improves Pancreatic Islet Transplant Performance and Reduces Beta Cell Death

Hepatocyte growth factor (HGF) increases beta cell proliferation and function in rat insulin promoter (RIP)-targeted transgenic mice. RIP-HGF mouse islets also function superiorly to normal islets in a transplant setting. Here, we aimed to determine whether viral gene transfer of the HGF gene into mouse islets ex vivo could enhance the performance of normal islets in a streptozotocin-diabetic severe combined immunodeficient mouse marginal islet mass model in which 300 uninfected or adenovirus (Adv) LacZ-transduced islet equivalents were insufficient to correct hyperglycemia. In dramatic contrast, 300 AdvHGF-transduced islet equivalents promptly (day 1) and significantly (p < 0.01) decreased random non-fasting blood glucose levels, from 351 +/- 20 mg/dl to an average of 191 +/- 7 mg/dl over 8 weeks. At day 1 post-transplant, beta cell death was significantly (p < 0.05) decreased, and the total insulin content was significantly (p < 0.05) increased in AdvHGF-transduced islets containing grafts. This anti-beta cell death action of HGF was independently confirmed in RIP-HGF mice and in INS-1 cells, both treated with streptozotocin. Activation of the phosphatidylinositol 3-kinase/Akt intracellular-signaling pathway appeared to be involved in this beta cell protective effect of HGF in vitro. In summary, adenoviral delivery of HGF to murine islets ex vivo improves islet transplant survival and blood glucose control in a subcapsular renal graft model in immuno-incompetent diabetic mice.