Ex vivo gene transfer of viral interleukin-10 to BB rat islets: no protection after transplantation to diabetic BB rats

Allogeneic and autoimmune islet destruction limits the success of islet transplantation in autoimmune diabetic patients. This study was designed to investigate whether ex vivo gene transfer of viral interleukin-10 (vIL-10) protects BioBreeding (BB) rat islets from autoimmune destruction after transplantation into diabetic BB recipients. Islets were transduced with adenoviral constructs (Ad) expressing the enhanced green fluorescent protein (eGFP), alpha-1 antitrypsin (AAT) or vIL-10. Transduction efficiency was demonstrated by eGFP-positive cells and vIL-10 production. Islet function was determined in vitro by measuring insulin content and insulin secretion and in vivo by grafting AdvIL-10-transduced islets into syngeneic streptozotocin (SZ)-diabetic, congenic Lewis (LEW.1 W) rats. Finally, gene-modified BB rat islets were grafted into autoimmune diabetic BB rats. Ad-transduction efficiency of islets increased with virus titre and did not interfere with insulin content and insulin secretion. Ad-transduction did not induce Fas on islet cells. AdvIL-10-transduced LEW.1 W rat islets survived permanently in SZ-diabetic LEW.1 W rats. In diabetic BB rats AdvIL-10-transduced BB rat islets were rapidly destroyed. Prolongation of islet culture prior to transplantation improved the survival of gene-modified islets in BB rats. Several genes including those coding for chemokines and other peptides associated with inflammation were down-regulated in islets after prolonged culture, possibly contributing to improved islet graft function in vivo. Islets transduced ex vivo with vIL-10 are principally able to cure SZ-diabetic rats. Autoimmune islet destruction in diabetic BB rats is not prevented by ex vivo vIL-10 gene transfer to grafted islets. Graft survival in autoimmune diabetic rats may be enhanced by improvements in culture conditions prior to transplantation.     

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter >150 μm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter <100 μm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 μm/min in small islets and 2.8 μm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150 μm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.     

Reduction of diffusion barriers in isolated rat islets improves survival,but not insulin secretion or transplantation outcome

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter > 150 μm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter &lt; 100 μm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 μm/min in small islets and 2.8 μm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150μm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets.     

In vivo multimodal imaging of transplanted pancreatic islets

Interest is increasing in the transplantation of pancreatic islets as a means to achieve insulin independence in individuals with type I diabetes. The success of this approach is hampered by the absence of methods to follow the fate of transplanted islets non-invasively. In vivo imaging seems to be the most appropriate technique to achieve this goal in small animals and eventually in humans. Here we describe a protocol for labeling and subsequent imaging of transplanted islets in vivo using magnetic resonance imaging (MRI) and optical imaging. The whole series of experiments can be carried out in roughly 48 h. We believe that our approach can significantly advance the current ability to determine islet distribution, and possibly survival, after transplantation. This information would be essential not only for the long-term monitoring of graft function but also for the design of improved transplantation and immunomodulatory methods.     

Beneficial Effect of Insulin Treatment on Islet Transplantation Outcomes in Akita Mice

Islet transplantation is a promising potential therapy for patients with type 1 diabetes. The outcome of islet transplantation depends on the transplantation of a sufficient amount of β-cell mass. However, the initial loss of islets after transplantation is problematic. We hypothesized the hyperglycemic status of the recipient may negatively affect graft survival. Therefore, in the present study, we evaluated the effect of insulin treatment on islet transplantation involving a suboptimal amount of islets in Akita mice, which is a diabetes model mouse with an Insulin 2 gene missense mutation. Fifty islets were transplanted under the left kidney capsule of the recipient mouse with or without insulin treatment. For insulin treatment, sustained-release insulin implants were implanted subcutaneously into recipient mice 2 weeks before transplantation and maintained for 4 weeks. Islet transplantation without insulin treatment did not reverse hyperglycemia. In contrast, the group that received transplants in combination with insulin treatment exhibited improved fasting blood glucose levels until 18 weeks after transplantation, even after insulin treatment was discontinued. The group that underwent islet transplantation in combination with insulin treatment had better glucose tolerance than the group that did not undergo insulin treatment. Insulin treatment improved graft survival from the acute phase (i.e., 1 day after transplantation) to the chronic phase (i.e., 18 weeks after transplantation). Islet apoptosis increased with increasing glucose concentration in the medium or blood in both the in vitro culture and in vivo transplantation experiments. Expression profile analysis of grafts indicated that genes related to immune response, chemotaxis, and inflammatory response were specifically upregulated when islets were transplanted into mice with hyperglycemia compared to those with normoglycemia. Thus, the results demonstrate that insulin treatment protects islets from the initial rapid loss that is usually observed after transplantation and positively affects the outcome of islet transplantation in Akita mice.     

Long-term cryopreservation of reaggregated pancreatic islets resulting in successful transplantation in rats

Preservation of pancreatic islets for long-term storage of islets used for transplantation or research has long been a goal. Unfortunately, few studies on long-term islet cryopreservation (1 month and longer) have reported positive outcomes in terms of islet yield, survival and function. In general, single cells have been shown to tolerate the cryopreservation procedure better than tissues/multicellular structures like islets. Thus, we optimized a method to cryopreserve single islet cells and, after thawing, reaggregated them into islet spheroids. Cryopreserved (CP) single human islet cells formed spheroids efficiently within 3–5 days after thawing. Approximately 79% of islet cells were recovered following the single-cell cryopreservation protocol. Viability after long-term cryopreservation (4 weeks or more) was significantly higher in the CP islet cell spheroids (97.4 ± 0.4%) compared to CP native islets (14.6 ± 0.4%). Moreover, CP islet cell spheroids had excellent viability even after weeks in culture (88.5 ± 1.6%). Metabolic activity was 4–5 times higher in CP islet cell spheroids than CP native islets at 24 and 48 h after thawing. Diabetic rats transplanted with CP islet cell spheroids were normoglycemic for 10 months, identical to diabetic rats transplanted with fresh islets. However, the animals receiving fresh islets required a higher volume of transplanted tissue to achieve normoglycemia compared to those transplanted with CP islet cell spheroids. By cryopreserving single cells instead of intact islets, we achieved highly viable and functional islets after thawing that required lower tissue volumes to reverse diabetes in rats.     

Small rat islets are superior to large islets in in vitro function and in transplantation outcomes

Barriers to the use of islet transplantation as a practical treatment for diabetes include the limited number of available donor pancreata. This project was designed to determine whether the size of the islet could influence the success rate of islet transplantations in rats. Islets from adult rats were divided into two groups containing small (diameter <125 microm) or large (diameter >150 microm) islets. An average pancreas yielded three times more small islets than large. Smaller islets were approximately 20% more viable, with large islets containing a scattered pattern of necrotic and apoptotic cells or central core cell death. Small islets in culture consumed twice as much oxygen as large islets when normalized for the same islet equivalents. In static incubation, small islets released three times more insulin under basal conditions than did large islets. During exposure to high glucose conditions, the small islets released four times more insulin than the same islet equivalencies of large islets, and five times more insulin was released by the small islets in response to glucose and depolarization with K+. Most importantly, the small islets were far superior to large islets when transplanted into diabetic animals. When marginal islet equivalencies were used for renal subcapsular transplantation, large islets failed to produce euglycemia in any recipient rats, whereas small islets were successful 80% of the time. The results indicate that small islets are superior to large islets in in vitro testing and for transplantation into the kidney capsule of diabetic rats.     

Regulation of RNA metabolism in relation to insulin production and oxidative metabolism in mouse pancreatic islets in vitro

This study was undertaken to investigate the long-term effects of different substrates, in particular glucose, on the regulation of islet RNA metabolism and the relationship of this regulation to the metabolism and insulin production of the islet B-cell. For this purpose collagenase-isolated mouse islets were used either in the fresh state or after culture for 2 or 5 days in RPMI 1640 plus 10% calf serum supplemented with various test compounds. Islets cultured with 16.7 mM glucose contained more RNA than those cultured with 3.3 mM glucose. Culture of islets in glucose at low concentrations inhibited glucose-stimulated RNA synthesis and this inhibitory effect was reversed by prolonged exposure to high glucose concentrations. Culture with 10 mM leucine and 3.3 mM glucose or with 10 mM 2-ketoisocaproate and 3.3 mM glucose increased the total RNA content of islets as compared to that of islets cultured with 3.3 mM glucose alone. Islets cultured with 5 mM theophylline maintained a high RNA content in the presence of 3.3 mM glucose. Theophylline also increased the islet RNA content when added together with 16.7 mM glucose, as compared to 16.7 mM glucose alone. Theophylline probably exerted this effect by decreasing the rate of RNA degradation. Changes in islet RNA metabolism showed a close correlation to changes in islet total protein biosynthesis, whereas islet (pro)insulin biosynthesis and insulin release exhibited different glucose-dependency patterns. The response of islet oxygen uptake to glucose was similar to that of islet RNA and protein biosynthesis. It is concluded that the RNA content of the pancreatic islets is controlled at the levels of both synthesis and degradation. Glucose stimulates the RNA synthesis and inhibits its degradation. Moreover, the results suggest that regulation of RNA synthesis may be mediated through islet metabolic fluxes and the cAMP system.     

Overexpression of thioredoxin in islets transduced by a lentiviral vector prolongs graft survival in autoimmune diabetic NOD mice

   

Pancreatic islet transplantation is considered an appropriate treatment to achieve insulin independence in type I diabetic patients. However, islet isolation and transplantation-induced oxidative stress and autoimmune-mediated destruction are still the major obstacles to the long-term survival of graft islets in this potential therapy. To protect islet grafts from inflammatory damage and prolong their survival, we transduced islets with an antioxidative gene thioredoxin (TRX) using a lentiviral vector before transplantation. We hypothesized that the overexpression of TRX in islets would prolong islet graft survival when transplanted into diabetic non-obese diabetic (NOD) mice.     

Method for isolation of pancreatic blood vessels,their culture and coculture with islets of langerhans

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 μm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.     

Incorporation of Bone Marrow Cells in Pancreatic Pseudoislets Improves Posttransplant Vascularization and Endocrine Function

Failure of revascularization is known to be the major reason for the poor outcome of pancreatic islet transplantation. In this study, we analyzed whether pseudoislets composed of islet cells and bone marrow cells can improve vascularization and function of islet transplants. Pancreatic islets isolated from Syrian golden hamsters were dispersed into single cells for the generation of pseudoislets containing 4×10³ cells. To create bone marrow cell-enriched pseudoislets 2×10³ islet cells were co-cultured with 2×10³ bone marrow cells. Pseudoislets and bone marrow cell-enriched pseudoislets were transplanted syngeneically into skinfold chambers to study graft vascularization by intravital fluorescence microscopy. Native islet transplants served as controls. Bone marrow cell-enriched pseudoislets showed a significantly improved vascularization compared to native islets and pseudoislets. Moreover, bone marrow cell-enriched pseudoislets but not pseudoislets normalized blood glucose levels after transplantation of 1000 islet equivalents under the kidney capsule of streptozotocin-induced diabetic animals, although the bone marrow cell-enriched pseudoislets contained only 50% of islet cells compared to pseudoislets and native islets. Fluorescence microscopy of bone marrow cell-enriched pseudoislets composed of bone marrow cells from GFP-expressing mice showed a distinct fraction of cells expressing both GFP and insulin, indicating a differentiation of bone marrow-derived cells to an insulin-producing cell-type. Thus, enrichment of pseudoislets by bone marrow cells enhances vascularization after transplantation and increases the amount of insulin-producing tissue. Accordingly, bone marrow cell-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation.     

Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices

Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets added.     

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes ^1,2. More recently, advances in human islet transplantation have further strengthened this view ^1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.     

Rat islet isolation yield and function are donor strain dependent

Effective rat islet isolation is pertinent for successful islet transplantation and islet studies in vitro. To determine which rat strain yields the highest number of pure and functional islets, four commonly used rat strains were compared with regard to islet yield, islet purity and islet function. Secretory responses were assessed by stimulation with glucose, and by stimulation with glucose plus 3-isobutyl-1-methylxanthine (IBMX). We show that rat islet function and isolation yield are donor strain dependent. Albino Oxford (AO) rats donated twice as many islets than Wistar, Lewis and Sprague Dawley (SD) rats. Stimulation with glucose plus IBMX resulted in an average five-fold increase of the stimulation index of AO, Lewis, Wistar and SD rats compared to stimulation with glucose only. AO islets had improved secretory responses after a one-week culture period, but required the addition of IBMX to glucose to elicit a distinguished stimulated insulin secretion after 2 days of culture. Islets from SD rats showed inferior results with regard to purity immediately after isolation and with regard to function after short- and after long-time culture. Because Lewis islets possessed the highest secretory response to glucose (without IBMX) immediately after isolation, Lewis rats may be preferred as islet donors for immediate use. The addition of IBMX to glucose for in vitro functional testing is recommended because it elicits high insulin secretory responses of islets regardless of the rat strain. AO rats are preferred for culture experiments since the number of experimental animals is reduced two-fold compared to Lewis, Wistar and SD rats.     

Nonhuman primate models in type 1 diabetes research

The recent success of "steroid-free" immunosuppressive protocols and improvements in islet preparation techniques have proven that pancreatic islet transplantation (PIT) is a valid therapeutic approach for patients with type 1 diabetes. However, there are major obstacles to overcome before PIT can become a routine therapeutic procedure, such as the need for chronic immunosuppression, the loss of functional islet mass after transplantation requiring multiple islet infusion to achieve euglycemia without exogenous administration of insulin, and the shortage of human tissue for transplantation. With reference to the first obstacle, stable islet allograft function without immunosuppressive therapy has been achieved after tolerance was induced in diabetic primates. With reference to the second obstacle, different strategies, including gene transfer of antiapoptotic genes, have been used to protect isolated islets before and after transplantation. With reference to the third obstacle, pigs are an attractive islet source because they breed rapidly, there is a long history of porcine insulin use in humans, and there is the potential for genetic engineering. To accomplish islet transplantation, experimental opportunities must be balanced by complementary characteristics of basic mouse and rat models and preclinical large animal models. Well-designed preclinical studies in primates can provide the quality of information required to translate islet transplant research safely into clinical transplantation.     

Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal

Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet-extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post-transplantation. FPCM was developed by embedding fibroblasts within type-I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over-growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome.     

Glutathione Ethyl Ester Supplementation during Pancreatic Islet Isolation Improves Viability and Transplant Outcomes in a Murine Marginal Islet Mass Model

Background

The success of pancreatic islet transplantation still faces many challenges, mainly related to cell damage during islet isolation and early post-transplant. The increased generation of reactive oxygen species (ROS) during islet isolation and the consumption of antioxidant defenses appear to be an important pathway related to islet damage.

Methodology/Principal Findings

In the present study we evaluated whether supplementation of glutathione-ethyl-ester (GEE) during islet isolation could improve islet viability and transplant outcomes in a murine marginal islet mass model. We also cultured human islets for 24 hours in standard CMRL media with or without GEE supplementation. Supplementation of GEE decreased the content of ROS in isolated islets, leading to a decrease in apoptosis and maintenance of islet viability. A higher percentage of mice transplanted with a marginal mass of GEE treated islets became euglycemic after transplant. The supplementation of 20 mM GEE in cultured human islets significantly reduced the apoptosis rate in comparison to untreated islets.

Conclusions/Significance

GEE supplementation was able to decrease the apoptosis rate and intracellular content of ROS in isolated islets and might be considered a potential intervention to improve islet viability during the isolation process and maintenance in culture before islet transplantation.     

Evaluation of encapsulating and microporous nondegradable hydrogel scaffold designs on islet engraftment in rodent models of diabetes

Islet transplantation is a promising therapeutic option for type 1 diabetes mellitus, yet the current delivery into the hepatic portal vasculature is limited by poor engraftment. Biomaterials have been used as a means to promote engraftment and function at extrahepatic sites, with strategies being categorized as encapsulation or microporous scaffolds that can either isolate or integrate islets with the host tissue, respectively. Although these approaches are typically studied separately using distinct material platforms, herein, we developed nondegradable polyethylene glycol (PEG)‐based hydrogels for islet encapsulation or as microporous scaffolds for islet seeding to compare the initial engraftment and function of islets in syngeneic diabetic mice. Normoglycemia was restored with transplantation of islets within either encapsulating or microporous hydrogels containing 700 islet equivalents (IEQ), with transplantation on microporous hydrogels producing lower blood glucose levels at earlier times. A glucose challenge test at 1 month after transplant indicated that encapsulated islets had a delay in glucose‐stimulated insulin secretion, whereas microporous hydrogels restored normoglycemia in times consistent with native pancreata. Encapsulated islets remained isolated from the host tissue, whereas the microporous scaffolds allowed for revascularization of the islets after transplant. Finally, we compared the inflammatory response after transplantation for the two systems and noted that microporous hydrogels had a substantially increased presence of neutrophils. Collectively, these findings suggest that both encapsulation and microporous PEG scaffold designs allow for stable engraftment of syngeneic islets and the ability to restore normoglycemia, yet the architecture influences islet function and responsiveness after transplantation.