Jose G. Avila

Human islet transplantation from pancreases with prolonged cold ischemia using additional preservation by the two-layer (UW solution/perfluorochemical) cold-storage method

Background. A two-layer (University of Wisconsin solution/perfluorochemical [UW/PFC]) cold-storage method delivers sufficient oxygen to the pancreas during preservation and restores the ischemically damaged pancreas. In this study, we determined whether the additional preservation by the two-layer method could improve islet recovery from human pancreases with prolonged cold storage in UW. Methods. Human pancreases were procured from cadaveric organ donors and preserved by the two-layer method (UW/PFC) for 2.9±0.7 hours (mean±SEM) at 4°C after 11.8±1.5 hours of cold storage in UW (UW/PFC group, n=7), or by cold UW alone for 11.3±0.3 hours (UW group, n=14). The selected pancreases met the criteria of having at least 10 hours of cold storage in UW. All were processed by using a standard protocol of Liberase perfusion with Pefabloc by way of the duct, gentle mechanical dissociation, and Ficoll gradient purification. Transplanted islets were selected with the criteria of the Edmonton protocol (>5,000 islet equivalents [IE]/kg recipient body weight). Results. The islet recovery was significantly increased in the UW/PFC group compared with the UW group (349.2±44.1×103 and 214.0±31.0×103 IE, respectively;P <0.05). This resulted in islet yields of 4.6±1.0×103 IE/g of pancreas in the UW/PFC group compared with 2.0±0.3×103 IE/g of pancreas in the UW group (P <0.05). Five of 7 cases (71%) in the UW/PFC group and 5 of 14 cases (36%) in the UW group were transplanted. The islet grafts in the UW/PFC group improved the ability of glycemic control and decreased exogenous insulin administration in all recipients. Conclusions. Improvements in methods to preserve and recover ischemically damaged human pancreases before islet isolation and transplant could be extremely beneficial to the field of clinical islet transplantation. This preliminary study shows that additional short preservation by the two-layer (UW/PFC) cold-storage method can significantly improve islet recovery and increase opportunities of islet transplantation from human pancreases after prolonged cold ischemia.

Islet xenotransplantation using gal-deficient neonatal donors improves engraftment and function.

Significant deficiencies in understanding of xenospecific immunity have impeded the success of preclinical trials in xenoislet transplantation. Although galactose‐α1,3‐galactose, the gal epitope, has emerged as the principal target of rejection in pig‐to‐primate models of solid organ transplant, the importance of gal‐specific immunity in islet xenotransplant models has yet to be clearly demonstrated. Here, we directly compare the immunogenicity, survival and function of neonatal porcine islets (NPIs) from gal‐expressing wild‐type (WT) or gal‐deficient galactosyl transferase knockout (GTKO) donors. Paired diabetic rhesus macaques were transplanted with either WT (n = 5) or GTKO (n = 5) NPIs. Recipient blood glucose, transaminase and serum xenoantibody levels were used to monitor response to transplant. Four of five GTKO versus one of five WT recipients achieved insulin‐independent normoglycemia; transplantation of WT islets resulted in significantly greater transaminitis. The WT NPIs were more susceptible to antibody and complement binding and destruction in vitro. Our results confirm that gal is an important variable in xenoislet transplantation. The GTKO NPI recipients have improved rates of normoglycemia, likely due to decreased susceptibility of xenografts to innate immunity mediated by complement and preformed xenoantibody. Therefore, the use of GTKO donors is an important step toward improved consistency and interpretability of results in future xenoislet studies.

Influence of pancreas preservation on human islet isolation outcomes: impact of the two-layer method.

Background. Human pancreas preservation for islet transplantation holds additional challenges and considerations compared with whole pancreas transplantation. The purpose of this study was to clarify the limitations of the University of Wisconsin (UW) solution and the potentials of the two-layer method (TLM) for pancreas preservation before human islet isolation. Methods. We retrospectively evaluated human islet isolation records between January 2001 and February 2003. One hundred forty-two human pancreata were procured from cadaveric donors and preserved by means of the UW solution (n=112) or TLM (n=30). Human islet isolations were performed using a standard protocol and assessed by islet recovery and in vitro function of islets. Results. Eight to ten hours of cold ischemia in the UW solution is a critical point for successful islet isolations. It is difficult to recover a sufficient number of viable islets for transplantation from human pancreata with more than 10 hours of cold storage in the UW solution. The overall islet recovery in the TLM group was significantly higher than in the UW group. With 10 to 16 hours of cold storage, the success rates of islet isolations remained at 62% in the TLM group but decreased to 22% in the UW group. Transplanted islets in the TLM group worked well in the recipients. Conclusions. There are time limitations for using the UW solution for pancreas preservation before human islet isolation. The TLM is a potential method to prolong the optimal cold storage time for successful islet isolations.

LFA-1–specific therapy prolongs allograft survival in rhesus macaques

Outcomes in transplantation have been limited by suboptimal long-term graft survival and toxicities associated with current immunosuppressive approaches. T cell costimulation blockade has shown promise as an alternative strategy to avoid the side effects of conventional immunosuppressive therapies, but targeting CD28-mediated costimulation alone has proven insufficient to prevent graft rejection in primates. Donor-specific memory T (TM) cells have been implicated in costimulation blockade-resistant transplant rejection, due to their enhanced effector function and decreased reliance on costimulatory signaling. Thus, we have tested a potential strategy to overcome TM cell-driven rejection by targeting molecules preferentially expressed on these cells, such as the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1). Here, we show that short-term treatment (i.e., induction therapy) with the LFA-1-specific antibody TS-1/22 in combination with either basiliximab (an IL-2Rα-specific mAb) and sirolimus (a mammalian target of rapamycin inhibitor) or belatacept (a high-affinity variant of the CD28 costimulation-blocker CTLA4Ig) prolonged islet allograft survival in nonhuman primates relative to control treatments. Moreover, TS-1/22 masked LFA-1 on TM cells in vivo and inhibited the generation of alloproliferative and cytokine-producing effector T cells that expressed high levels of LFA-1 in vitro. These results support the use of LFA-1-specific induction therapy to neutralize costimulation blockade-resistant populations of T cells and further evaluation of LFA-1-specific therapeutics for use in transplantation.

Experience with a novel efalizumab-based immunosuppressive regimen to facilitate single donor islet cell transplantation

Islet transplantation is an experimental therapy for selected patients with type 1 diabetes (T1DM). It remains limited by immunosuppressive drug toxicity, progressive loss of insulin independence, allosensitization and the need for multiple islet donors. We describe our experience with an efalizumab‐based immunosuppressive regimen as compared to the prevailing standard regimen, the Edmonton protocol. Twelve patients with T1DM received islet transplants: eight were treated with the Edmonton protocol; four were treated with daclizumab induction, a 6‐month course of tacrolimus, and maintenance with efalizumab and mycophenolate mofetil. The primary endpoint was insulin independence after one islet infusion. Only two Edmonton protocol treated patients achieved the primary endpoint; six required islets from multiple donors, and all experienced leukopenia, mouth ulcers, anemia, diarrhea and hypertransaminasemia. Four became allosensitized. All patients treated with the efalizumab‐based regimen achieved insulin independence with normal hemoglobin A1c after a single islet cell infusion and remained insulin independent while on efalizumab. These patients experienced significantly fewer side effects and none became allosensitized. Trial continuation was terminated by withdrawal of efalizumab from the market. These data suggest that this efalizumab‐based regimen prevents islet rejection, is well tolerated, and allows for single donor islet transplantation.

Nondepleting Anti‐CD40‐Based Therapy Prolongs Allograft Survival in Nonhuman Primates

Costimulation blockade of the CD40/CD154 pathway has been effective at preventing allograft rejection in numerous transplantation models. This strategy has largely depended on mAbs directed against CD154, limiting the potential for translation due to its association with thromboembolic events. Though targeting CD40 as an alternative to CD154 has been successful at preventing allograft rejection in preclinical models, there have been no reports on the effects of CD40‐specific agents in human transplant recipients. This delay in clinical translation may in part be explained by the presence of cellular depletion with many CD40‐specific mAbs. As such, the optimal biologic properties of CD40‐directed immunotherapy remain to be determined. In this report, we have characterized 3A8, a human CD40‐specific mAb and evaluated its efficacy in a rhesus macaque model of islet cell transplantation. Despite partially agonistic properties and the inability to block CD40 binding of soluble CD154 (sCD154) in vitro, 3A8‐based therapy markedly prolonged islet allograft survival without depleting B cells. Our results indicate that the allograft‐protective effects of CD40‐directed costimulation blockade do not require sCD154 blockade, complete antagonism or cellular depletion, and serve to support and guide the continued development of CD40‐specific agents for clinical translation.

Functional MR microimaging of pancreatic β-cell activation

The increasing incidence of diabetes and the need to further understand its cellular basis has resulted in the development of new diagnostic and therapeutic techniques. Nonetheless, the quest to noninvasively ascertain β-cell mass and function has not been achieved. Manganese (Mn)-enhanced MRI is presented here as a tool to image β-cell functionality in cell culture and isolated islets. Similar to calcium, extracellular Mn was taken up by glucose-activated β-cells resulting in 200% increase in MRI contrast enhancement, versus nonactivated cells. Similarly, glucose-activated islets showed an increase in MRI contrast up to 45%. Although glucose-stimulated Ca influx was depressed in the presence of 100 μM Mn, no significant effect was seen at lower Mn concentrations. Moreover, islets exposed to Mn showed normal glucose sensitivity and insulin secretion. These results demonstrate a link between image contrast enhancement and β-cell activation in vitro, and provide the basis for future noninvasive in vivo imaging of islet functionality and β-cell mass.

Alternative immunomodulatory strategies for xenotransplantation: CD40/154 pathway-sparing regimens promote xenograft survival.

Immunosuppressive therapies that block the CD40/CD154 costimulatory pathway have proven to be uniquely effective in preclinical xenotransplant models. Given the challenges facing clinical translation of CD40/CD154 pathway blockade, we examined the efficacy and tolerability of CD40/CD154 pathway‐sparing immunomodulatory strategies in a pig‐to‐nonhuman primate islet xenotransplant model. Rhesus macaques were rendered diabetic with streptozocin and given an intraportal infusion of ∼50 000 islet equivalents/kg wild‐type neonatal porcine islets. Base immunosuppression for all recipients included maintenance therapy with belatacept and mycophenolate mofetil plus induction with basiliximab and LFA‐1 blockade. Cohort 1 recipients (n = 3) were treated with the base regimen alone; cohort 2 recipients (n = 5) were additionally treated with tacrolimus induction and cohort 3 recipients (n = 5) were treated with alefacept in place of basiliximab, and more intense LFA‐1 blockade. Three of five recipients in both cohorts 2 and 3 achieved sustained insulin‐independent normoglycemia (median rejection‐free survivals 60 and 111 days, respectively), compared to zero of three recipients in cohort 1. These data show that CD40/CD154 pathway‐sparing regimens can promote xenoislet survival. Further optimization of these strategies is warranted to aid the clinical translation of islet xenotransplantation.

Short-term storage of the ischemically damaged human pancreas by the two-layer method prior to islet isolation.

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata in the canine pancreas transplant model. In this study, we applied a short-term preservation of the TLM to human pancreata after prolonged cold ischemia prior to islet isolation, and investigated the mechanisms of resuscitation of the ischemically damaged human pancreas by the TLM. Human pancreata were procured from cadaveric donors and preserved by the TLM for 3.2 ± 0.5 h after 11.1 ± 0.9 h of cold storage in UW (TLM group), or by cold UW alone for 11.0 ± 0.3 h (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro functional viability of isolated islets were significantly increased in the TLM group compared with the UW group. According to the criteria of the Edmonton protocol, 10/14 cases (71%) in the TLM group were transplanted to patients with type I diabetes mellitus compared with only 5/21 cases (24%) in the UW group. In the metabolic assessment of human pancreata, levels of energetic parameters (ATP, total adenylates, and energy charge) were significantly increased, and malondialdehyde (MDA) levels were significantly decreased after the TLM preservation. There was no observable change in the incidence or degree of mitochondrial injury after the TLM preservation. Additional short-term storage by the TLM resuscitates the ischemically damaged human pancreas by regenerating the energetic status and prevents further damage by oxidative stress, ultimately leading to improvements of islet recovery and in vitro function. Use of the TLM following prolonged storage in UW provides an excellent adjunctive protocol for treating human pancreata for the rigors of the islet isolation process.

Intra‐Ductal Glutamine Administration Reduces Oxidative Injury During Human Pancreatic Islet Isolation

Oxidative stress during islet isolation induces a cascade of events injuring islets and hampering islet engraftment. This study evaluated islet isolation and transplantation outcomes after intra‐ductal glutamine administration. Human pancreata deemed unsuitable for pancreas or islet transplantation were treated with either a 5 mM solution of l‐glutamine (n = 6) or collagenase enzyme alone (n = 6) through the main pancreatic duct. Islet yield, viability, in vitro function; markers of oxidative stress [malondialdehyde (MDA) and Glutathione (GSH)] and apoptosis were assessed. Islet yields were significantly increased in the glutamine group compared to controls (318, 559 ± 25, 800 vs. 165, 582 ± 39, 944 mean ± SEM, p < 0.01). The amount of apoptotic cells per islet was smaller in the glutamine group than the control. The percentage of nude mice rendered normoglycemic with glutamine‐treated islets was higher than the controls (83% n = 10/12 vs. 26% n = 6/23; p < 0.01), and the time to reach normoglycemia was decreased in the glutamine group (1.83 ± 0.4 vs. 7.3 ± 3 days; p < 0.01). Glutamine administration increased GSH levels (7.6 ± 1.7 nmol/mg protein vs. 4.03 ± 0.5 in control, p < 0.05) and reduced lipid‐peroxidation (MDA 2.45 ± 0.7 nmol/mg of protein vs. 6.54 ± 1.7 in control; p < 0.05). We conclude that intra‐ductal administration of glutamine reduces oxidative injury and apoptosis and improves islet yield and islet graft function after transplantation.