Stacie Jenkins

Cytoprotection of pancreatic islets before and soon after transplantation by gene transfer of the anti-apoptotic Bcl-2 gene.

Isolated pancreatic islet transplantation is a promising alternative to conventional insulin-dependent diabetes treatment but is not yet a practical clinical therapy. In the first few days after pancreatic islet transplantation, substantial donor pancreatic islet dysfunction and apoptosis commonly occur. Islet apoptosis has been documented after extracellular matrix disruption and exposure to proinflammatory cytokines, and during hypoxia before islet revascularization and rejection. These studies show that targeting the apoptosis pathway by adenoviral-mediated gene transfer of the anti-apoptotic Bcl-2 gene exerts a major cytoprotective effect on isolated macaque pancreatic islets. Bcl-2 transfection ex vivo protects these islets from apoptosis induced by disruption of the islet extracellular matrix during pancreatic digestion. Additionally, overexpression of Bcl-2 confers long-term, stable protection and maintenance of functional islet mass after transplantation of macaque islets into diabetic severe combined immunodeficency mice. Notably, genetic modification of pancreatic islets also reduced the islet mass required to achieve stable euglycemia. Ex vivo gene transfer of anti-apoptotic genes has potential as a therapeutic approach to both minimize loss of functional islet mass after transplant and reduce the high donor islet requirement currently needed for successful stable reversal of insulin-dependent diabetes.

Genetically Modified Adenovirus Vector Containing an RGD Peptide in the HI Loop of the Fiber Knob Improves Gene Transfer to Nonhuman Primate Isolated Pancreatic Islets

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islets (PIs) may allow enhanced post‐transplantation survival. The available gene transfer vectors differ greatly in their ability to infect and express genes in different cell types. One limitation associated with the use of viral vectors is related to the virus reliance on the presence of its primary binding site. Tropism of the viral vectors can be altered using retargeting strategies. Results on phage biopanning proved that the RGD motif has in vivo targeting capabilities. This motif interacts especially with cellular integrins of the αVβ3 and αVβ5 types, highly expressed on pancreatic islets. In this report, we have explored the utility of a retargeted adenovirus vector (Ad) containing an RGD motif in the HI loop of the fiber knob in order to improve the infection efficiency to intact isolated nonhuman primate PIs and reduce toxicity after the genetic modification. Nonhuman primate PIs were isolated by a semi‐automated technique. Steptozotocin‐induced diabetic mice with severe combined immunodeficiency disease (SCID) were used as recipients. A recombinant Ad containing a heterologous RGD peptide and expressing luciferase (AdRGDLuc) or green fluorescent protein (AdRGDGFP) were generated in our laboratory. Similar Ads without the RGD peptide were used as a control (AdLuc and AdGFP). Higher transfection efficiency was demonstrated using AdRGDGFP compared with AdGFP (> 80% of the islet cells were infected at 10 particle‐forming units (pfu)/cell using AdRGDGFP vs. 7% after infection with AdGFP). More than 90% of the infected cells were insulin‐producing cells. Significantly higher transgene expression was demonstrated after infection with AdRGDLuc compared with AdLuc at different titers. Analysis of the glucose‐stimulated insulin response demonstrated better performance of PI transfected with AdRGDLuc at low titers (10 pfu/cell in order to achieve > 80% transfection efficiency) compared with AdLuc at high titers. Finally, long‐term euglycemia (> 250 d) was observed in 89% of the animals that received PI infected with AdRGDLuc compared with none of the animals that received PI infected with AdLuc. The present study provides new information about the possibility of tropism modification of Ad vectors to increase the transfection efficiency and transgene expression to isolated PI. Incorporation of the RGD sequence in the HI loop of the fiber knob allows highly efficient transfection efficiency to nonhuman primate insulin‐producing cells and adequate long‐term function of the β‐cell after transplantation.

Elevated T Regulatory Cells in Long-Term Stable Transplant Tolerance in Rhesus Macaques Induced by Anti-CD3 Immunotoxin and Deoxyspergualin

Regulatory T cells (Tregs) are implicated in immune tolerance and are variably dependent on IL-10 for in vivo function. Brief peritransplant treatment of multiple nonhuman primates (NHP) with anti-CD3 immunotoxin and deoxyspergualin has induced stable (5–10 years) rejection-free tolerance to MHC-mismatched allografts, which associated with sustained elevations in serum IL-10. In this study, we demonstrate that resting and activated PBMC from long-term tolerant NHP recipients are biased to secrete high levels of IL-10, compared with normal NHP PBMC. Although IL-10-producing CD4+ Tregs (type 1 regulatory cells (TR1)/IL-10 Tregs) were undetectable (<0.5%) in normal rhesus monkeys, 7.5 ± 1.7% of circulating CD4+ T cells of tolerant rhesus recipients expressed IL-10. In addition to this >15-fold increase in Tr1/IL-10 Tregs, the tolerant monkeys exhibited a nearly 3-fold increase in CD4+CD25+ Tregs, 8.1 ± 3.0% of CD4 T cells vs 2.8 ± 1.4% in normal cohorts (p < 0.02). The frequency of CD4+CD25+IL-10+ cells was elevated 5-fold in tolerant vs normal NHP (1.8 ± 0.9% vs 0.4 ± 0.2%). Rhesus CD4+CD25+ Tregs exhibited a memory phenotype, and expressed high levels of Foxp3 and CTLA-4 compared with CD4+CD25− T cells. Also, NHP CD4+CD25+ Tregs proliferated poorly after activation and suppressed proliferation of CD4+CD25− effector T cells, exhibiting regulatory properties similar to rodent and human CD4+CD25+ Tregs. Of note, depletion of CD4+CD25+ Tregs restored indirect pathway antidonor responses in tolerant NHP. Our study demonstrates an expanded presence of Treg populations in tolerant NHP recipients, suggesting that these adaptations may be involved in maintenance of stable tolerance.

Stable alpha- and beta-islet cell function after tolerance induction to pancreatic islet allografts in diabetic primates.

Pancreatic islet transplantation (PIT) is an attractive alternative for type 1 diabetic patients. PIT is not yet an effective clinical reality due in part to early loss of functional islet mass. In addition, current immunosuppressive drugs have toxic effects on islets and increase the risk of morbidity and mortality. Precise and durable α‐ and β‐cell function is essential for the success of PIT. Therefore, it is important to establish whether PIT can produce adequate long‐term metabolic control, especially in the absence of chronic immunosuppressive therapy (CIT). In the present study, the stability of functional α‐ and β‐cell mass and metabolic function was assessed in streptozotocin (STZ)‐induced diabetic primates following PIT in the absence of CIT. Diabetes was induced in rhesus macaques with STZ, 140 mg/kg. Hyperglycemia was reversed rapidly by PIT coupled with a 14‐day tolerance induction protocol based on F(Ab)2‐IT and DSG (n = 7). Two diabetic animals received the tolerance induction protocol without PIT. Acute rejection was presented in three animals at 70, 353 and 353 days post transplant in the tolerance induction protocol, whereas the controls [F(Ab)2‐IT or DSG alone] showed early 10‐day function but all lost islet function by days 15–70. One recipient [F(Ab)2‐IT or DSG] died euglycemic after a surgical procedure on day 187. At 2 years, three animals studied had a normal FIM evaluated by oral glucose tolerance test, mixed meal test, acute insulin response to glucose, glucose disposal rate, and hyperinsulinemic hypoglycemic clamp. PIT in STZ‐induced diabetic primates resulted in restoration of normal α‐ and β‐cell function. Operational tolerance induction was achieved with only peritransplant administration of F(Ab)2‐IT and DSG sparing the animals from chronic exposure of diabetogenic immunosuppressive drugs. These results offer an exciting new potential for treatment of type 1 diabetes mellitus.

RETRACTED: Tolerance induced by anti-CD3 immunotoxin plus 15-deoxyspergualin associates with donor-specific indirect pathway unresponsiveness☆

Peritransplant treatment with anti-CD3 immunotoxin plus deoxyspergualin induces tolerance to kidney allografts in most rhesus macaque recipients. Tolerant recipients maintain normal function for years without evidence of chronic rejection. Indirect alloantigen presentation is implicated in chronic rejection. Accordingly, we determined if anti-CD3 immunotoxin plus deoxyspergualin induced rejection-free tolerance associates with suppression of anti-donor indirect pathway responses. Tolerant recipients exhibited an early decrease in direct anti-donor responses with recovery to baseline levels by 3 years posttransplantation. In contrast, tolerant monkeys were unresponsive to donor antigens presented by the indirect pathway. Recipients that rejected their allografts retained vigorous direct and indirect anti-donor responses. Therefore, following temporary donor-specific hyporesponsiveness, direct responses recover in tolerant recipients >1.5 years after transplantation. However, tolerant recipients tested at 1.9-4 years posttransplant are specifically unresponsive to donor antigens presented by the indirect pathway. Thus, the rejection-free state of tolerant recipients may depend on mechanisms regulating indirect pathway responsiveness.