Correction of diabetes mellitus by transplanting minimal mass of syngeneic islets into vascularized small intestinal segment.

Transplantation of mature islets into portal vein has been most effective thus far, although attrition of transplanted islets constitutes a major limitation, and alternative approaches are required. We analyzed the mechanisms by which islets engrafted, vascularized and functioned over the long term in the small intestinal submucosa. To determine engraftment, survival and function, 350 syngenic islets were transplanted into either intestinal segments or portal vein of diabetic rats. Islet reorganization, vascularization and function were analyzed by histological analysis, RT-PCR analysis as well as glycemic control over up to 1 year. Transplantation of syngeneic islets in marginal numbers successfully restored normoglycemia in diabetic rats. Transplantation of semi-pure islet preparation did not impair their engraftment, vascularization and function. Islets were morphologically intact and expressed insulin as well as glucagon over the year. Expression of angiogenic genes permitted revascularization of transplanted islets. We identified the expression of transcription factors required for maintenance of beta cells. These studies demonstrated that marginal mass of transplanted islets was sufficient to restore euglycemia in streptozotocin-treated rats. These superior results were obtained despite use of an impure preparation of islets in animals with small intestinal segment. Our findings will help advance new horizons for cell therapy in patients with diabetes.

Feasibility of localized immunosuppression: 1. Exploratory studies with glucocorticoids in a biohybrid device designed for cell transplantation.

Emerging biotechnologies, such as the use of biohybrid devices for cellular therapies, are showing increasing therapeutic promise for the treatment of various diseases, including type 1 diabetes mellitus. The functionality of such devices could be greatly enhanced if successful localized immunosuppression regimens could be established, since they would eliminate the many otherwise unavoidable side effects of currently used systemic immunosuppressive therapies. The existence of local immune privilege at some specialized tissues, such as the eye, CNS, or pregnant uterus, supports the feasibility of localized immunomodulation, and such an approach is particularly well-suited for cell transplant therapies where all transplanted tissue is localized within a device. Following the success of syngeneic transplantation in a subcutaneous prevascularized device as a bioartificial pancreas in a rodent model, we now report the first results of exploratory in vivo islet allograft studies in rats using locally delivered glucocorticoids (dexamethasone phosphate and the soft steroid loteprednol etabonate). Following in vitro assessments, in silico drug distribution models were used to establish tentative therapeutic dose ranges. Sustained local delivery was achieved via implantable osmotic mini-pumps through a central sprinkler, as well as with a sustained-delivery formulation for loteprednol etabonate using poly(D,L-lactic) acid (PLA) microspheres. Doses delivered locally were approximately hundred-fold smaller than those typically used in systemic treatments. While several solubility, stability, and implantation problems still remain to be addressed, both compounds showed promise in their ability to prolong graft survival after tapering of systemic immunosuppression, compared to control groups.

Effects of systemic immunosuppression on islet engraftment and function into a subcutaneous biocompatible device.

The aim of this study was to explore the effect of sirolimus (Sir) and tacrolimus (Tac) on islets implanted into a subcutaneous (SC), prevascularized device in syngeneic rats. Animals received a 40-day treatment with Tac and Sir (alone or in combination) starting either on day 0 or 40 days after islet transplantation. Controls received no treatment. A 40-day washout period was performed after immunosuppression (IS). Glycemia and intravenous glucose tolerance tests (IVGTT) were assessed at follow-up. In the control group, 75% of recipients achieved stable normoglycemia after islet transplantation, while none reversed diabetes with any IS regimen started on day 0. Graft dysfunction was irreversible after IS withdrawal. Glucose clearance (IVGTT) was significantly impaired among Tac-treated compared with control groups (P < .05 with IS; P < .01 after washout). Among animals with established grafts, islet dysfunction which occurred under IS treatment persisted after washout in animals treated with Tac and Sir plus Tac. When compared with controls, glucose clearance was significantly impaired in the Tac and Tac plus Sir groups before and after IS (P < .01, Tac; P < 0.01, Tac plus Sir). Sir and Tac showed profound deleterious effects on islet cell engraftment and function, which may hinder the success of implantation into biohybrid devices. Nondiabetogenic IS protocols must be developed for clinical application of islet transplantation into biohybrid devices.

Ischemic preconditioning improves islet recovery after pancreas cold preservation.

Increasing evidence supports the beneficial effects of ischemic preconditioning (IPC) of organs on subsequent ischemia. The aim of this study was to assess the effects of IPC of the pancreas on islet cell recovery after cold preservation using a rat model. The pancreas was deprived of perfusion (celiac artery and superior mesenteric artery occlusion) for 10 minutes followed by 10 minutes of reperfusion. Islet isolation was performed after 18 hours of cold ischemia. Glands undergoing IPC yielded significantly greater numbers of islets than controls. Following overnight culture, a significantly greater proportion of islets was recovered from IPC-treated pancreata. Microarray genomic analysis of pancreatic tissue revealed a significant differential expression of approximately 600 unique mRNA strands within IPC pancreata compared to only <100 unique mRNA strands within non-IPC pancreata (>2-fold change; P < .05). Proteomic analysis revealed significant differential expression of at least 5 proteins >1.5-fold change; P < .05) within the IPC vs control group. Our data indicated that IPC of the pancreas prior to cold preservation was associated with improved islet cell recovery after cold ischemia. IPC of the pancreas may represent a viable therapeutic intervention to increase islet transplantation success from a single donor and to maximize organ utilization.

c-Jun N-terminal kinase 1 is deleterious to the function and survival of murine pancreatic islets.

AIMS/HYPOTHESIS: Inhibition of c-jun N-terminal kinase (JNK) favours pancreatic islet function and survival. Since two JNK isoforms are present in the pancreas (JNK1 and JNK2), we addressed their specific roles in experimental islet transplantation. METHODS: C57BL/6J (wild-type [WT]), Jnk1 (also known as Mapk8)(-/-) and Jnk2 (also known as Mapk9)(-/-) mice were used as donor/recipients in a syngeneic islet transplantation model. Islet cell composition, function, viability, production of cytokines and of vascular endothelial growth factor (VEGF) were also studied in vitro. RESULTS: Jnk1 ( -/- ) islets secreted more insulin in response to glucose and were more resistant to cytokine-induced cell death compared with WT and Jnk2 (-/-) islets (p < 0.01). Cytokines reduced VEGF production in WT and Jnk2 (-/-) but not Jnk1 ( -/- ) islets; VEGF blockade restored Jnk1 ( -/- ) islet susceptibility to cytokine-induced cell death. Transplantation of Jnk1 ( -/- ) or WT islets into WT recipients made diabetic had similar outcomes. However, Jnk1 ( -/- ) recipients of WT islets had shorter time to diabetes reversal (17 vs 55 days in WT, p = 0.033), while none of the Jnk2 (-/-) recipients had diabetes reversal (0% vs 71% in WT, p = 0.0003). Co-culture of WT islets with macrophages from each strain revealed a discordant cytokine production. CONCLUSIONS/INTERPRETATION: We have shown a deleterious effect of JNK2 deficiency on islet graft outcome, most likely related to JNK1 activation, suggesting that specific JNK1 blockade may be superior to general JNK inhibition, particularly when administered to transplant recipients.

Human alpha 1-antitrypsin therapy induces fatal anaphylaxis in non-obese diabetic mice.

Previous studies have shown that human alpha-1 antitrypsin (hAAT) gene delivery prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. Furthermore, hAAT protein administration prolongs acceptance of islet allografts. Therefore, we evaluated the use of purified hAAT protein therapy to prevent T1D in NOD mice. Female NOD, non-obese resistant (NOR), Balb/c and C57BL/6 mice were injected intraperitoneally with vehicle alone or vehicle containing hAAT, human albumin or mouse albumin (or mg/injection/mouse; 2x/week). Preparations of clinical-grade hAAT included API(R), Aralast, Prolastin and Zemaira. Surprisingly, hAAT administration was associated with a high rate of fatal anaphylaxis. In studies seeking T1D prevention at 4 weeks of age, 100% mice died after six injections of hAAT. When administrated at 8-10 weeks of age, most (80-100%) NOD mice died following the fourth injection of hAAT, while 0% of Balb/c and C57BL/6 mice and 10% of NOR mice died. Interestingly, repeated injections of human albumin, but not mouse albumin, also induced sudden death in NOD mice. Antibodies to hAAT were induced 2-3 weeks after hAAT administration and death was prevented by treatment with anti-platelet-activating factor along with anti-histamine. In studies of disease reversal in NOD mice, using the four pharmaceutical grade formulations of hAAT, anaphylactic deaths were observed with all hAAT preparations. The propensity for fatal anaphylaxis following antigenic administration appears to be NOD- but not hAAT-specific. The susceptibility of NOD mice to hypersensitivity provides a significant limitation for testing of hAAT. Development of strategies to avoid this unwanted response is required to use this promising therapeutic agent for T1D.

beta-Cell specific cytoprotection by prolactin on human islets.

INTRODUCTION: Many cytoprotective agents have been reported to improve islet isolation and transplantation outcomes. However, several of these agents improve all cell subsets within an islet preparation; selection of non-beta-cell components (eg, acinar cells) may have a negative effect on beta-cell function and survival. In this study, we examined the effect of prolactin (PRL) supplementation in the culture medium to determine whether it exerted beta-cell-selective cytoprotection on islet viability and function. MATERIALS AND METHODS: Human islets were precultured with or without recombinant human PRL (500 microg/L) for 48 hours. The fractional viability and cellular composition of non-beta-cell and beta-cell-specific components were assessed using FACS and Laser Scanning Cytometry (LSC). Islet potency was assessed in vivo by transplantation into chemically induced diabetic immunodeficient mice. RESULTS: The relative viable beta-cell mass and the relative islet beta-cell content in the PRL group were 28% higher (P = .018) and 19% higher (P = .029) than the control group, respectively. All transplanted mice achieved normoglycemia in both groups, indicating that PRL treatment did not alter islet function. CONCLUSION: PRL treatment improved beta-cell-specific viability and survival of human islets in vitro. The development of novel beta-cell-specific cytoprotective strategies may be of assistance in improving islet transplantation.

Rapamycin impairs beta-cell proliferation in vivo.

During pregnancy a high rate of beta-cell proliferation occurs, making of this a useful model for the study of islet cell expansion in vivo. We used the murine pregnancy model to assess the effect of Rapamycin treatment on islet cell proliferation in vivo. Rapamycin is routinely used for the prevention of graft rejection in transplanted patients, including islet transplant recipients. As expected, pregnancy led to increased beta-cell proliferation, islet yield and skewing in size distribution after isolation and pancreatic insulin content, when compared to non-pregnant females. Rapamycin treatment resulted in reduced beta cell proliferation in pregnant mice, while minimal effects of Rapamycin treatment were observed on islet function both in vivo and in vitro. Rapamycin treatment of islets resulted in reduced phosphorylation of p70s6k, a downstream effector molecule of mTOR and increased ERK1/2 phosphorylation. In conclusion, beta-cell replication is reduced under Rapamycin treatment in vivo, suggesting that this mechanism may be operational and impair beta-cell renewal in transplanted patients.

Prolonged islet allograft survival by alpha-1 antitrypsin: the role of humoral immunity.

Immunomodulatory properties have been recognized for human alpha-1 antitrypsin (hAAT). However, production of anti-hAAT antibodies in mice may inactivate the protein. In this study, we evaluated the effects of chronic hAAT administration on allogeneic islet graft survival. Chemically diabetic mice lacking an efficient humoral response due to the targeted disruption of the Ig mu-chain (muMT mice) or wild-type (WT) C57BL/6 mice received DBA/2 mouse islets under the kidney capsule. hAAT (Prolastin or Aralast) was given intraperitoneally on day 0 and every 3 days thereafter. Control animals received no treatment. hAAT administration in WT mice resulted in prolongation of islet allograft survival in a dose-dependent fashion in both hAAT-treated groups. Lack of Ig response (muMT mice) per se conferred a beneficial effect on graft survival that worsened in the Prolastin-treated groups but improved in the Aralast-treated group. Our data indicate that systemic administration of hAAT results in prolongation of islet allograft survival. Absence of mature B cells and Ig mu-chain resulted in improved graft survival, pointing to a role for B cells in the rejection process in this model. Treatment with Prolastin worsened graft survival in muMT mice, whereas Aralast did improve it, suggesting a different efficacy and possible actions of the two drug formulations.

Alpha-1 antitrypsin treatment of spontaneously diabetic nonobese diabetic mice receiving islet allografts.

Alpha-1 antitrypsin (AAT) is a serine protease inhibitor able to prevent diabetes onset in nonobese diabetic (NOD) mice and to prolong islet allograft survival in a nonautoimmune murine model. In this study, we explored the effect of chronic administration of human AAT (hAAT) on allogeneic (C57BL/6) islet graft survival in spontaneously diabetic female NOD mice. Mice received intraperitoneal treatment with saline, Prolastin (1 or 2 mg/mouse) or Aralast (2 mg/mouse) on days -1, 0, 3, 6, and 9. Saline-treated mice rejected the grafts 10.0 +/- 2.5 days after transplantation (n = 9). Prolastin 1 mg (n = 9) and 2 mg (n = 3) resulted in rejection on 8.7 +/- 1.4 (not significant) and 13.0 +/- 4.3 days (P < .03), respectively. Aralast-treated mice showed prolongation of graft survival (13 +/- 5.9 days; n = 5; P < .03). Notably, repeated administrations of either hAAT formulation led to sudden death of a proportion of treated animals. Collectively, our preliminary data indicate that prolongation of islet allograft survival in the stringent autoimmune diabetic NOD mouse model can be achieved with hAAT monotherapy. The death of a proportion of treated animals may be consequent to immunization to hAAT and lethal hypersensitivity. Interestingly, this phenomenon was not observed in a non-autoimmune mouse strain (C57BL/6) despite extended hAAT treatment (>100 days).

Soft corticosteroids for local immunosuppression: exploring the possibility for the use of loteprednol etabonate for islet transplantation.

Transplantation of pancreatic islets into subcutaneous, neovascularized devices is one of the possibilities explored as part of our search for a cure of diabetes. We have recently reported that syngeneic transplantation in a subcutaneous prevascularized device can restore euglycemia and sustain long-term function in rats and that explanted grafts showed preserved islets and intense vascular networks. Because all of the transplanted tissue is localized within the device, if such a bioartificial pancreas approach is used, localized immunosuppression might provide sufficient protection against rejection to achieve long-term function, while also avoiding the serious systemic side effects and the susceptibility for opportunistic infections that are commonly associated with systemic immunosuppressive therapies as only much smaller and localized doses are needed. Soft steroids are obvious candidates because soft drugs are specifically designed to produce targeted local activity, but no systemic side effects due to prompt metabolic (preferably extrahepatic, e.g., hydrolytic) inactivation. However, local concentrations that are effective for immunosuppression, but non-toxic to insulin-producing beta-cells have to be found, and nontrivial difficulties related to long-term local deliverability have to be addressed. Here, we report preliminary results obtained using in vitro studies with human islets used to establish a tentative therapeutic concentration range together with fully scaled three-dimensional finite element method (FEM)-based Comsol multiphysics computational models that were used to explore various possibilities to achieve and maintain these concentration levels within the device.

Prolonged islet graft survival in NOD mice by blockade of the CD40-CD154 pathway of T-cell costimulation.

Allorejection and recurrence of autoimmunity are the major barriers to transplantation of islets of Langerhans for the cure of type 1 diabetes in humans. CD40-CD154 (CD40 ligand) interaction blockade by the use of anti-CD154 monoclonal antibody (mAb) has shown efficacy in preventing allorejection in several models of organ and cell transplantation. Here we report the beneficial effect of the chronic administration of a hamster anti-murine CD154 mAb, MR1, in prolonging islet graft survival in NOD mice. We explored the transplantation of C57BL/6 islets into spontaneously diabetic NOD mice, a combination in which both allogeneic and autoimmune components are implicated in graft loss. Recipients were treated either with an irrelevant control antibody or with MR1. MR1 administration was effective in prolonging allograft survival, but did not provide permanent protection from diabetes recurrence. The autoimmune component of graft loss was studied in spontaneously diabetic NOD mice that received syngeneic islets from young male NOD mice. In this combination, a less dramatic yet substantial delay in diabetes recurrence was observed in the MR1-treated recipients when compared with the control group. Finally, the allogeneic component was explored by transplanting C57BL/6 islets into chemically induced diabetic male NOD mice. In this setting, long-term graft survival (>100 days) was achieved in MR1-treated mice, whereas control recipients rejected their grafts within 25 days. In conclusion, chronic blockade of CD154 results in permanent protection from allorejection and significantly delays recurrence of diabetes in NOD mice.

Proteins linked to a protein transduction domain efficiently transduce pancreatic islets.

The resounding success of a new immunosuppressive regimen known as the Edmonton protocol demonstrates that islet cell transplantation is becoming a therapeutic reality for diabetes. However, under the Edmonton protocol, a single donor does not provide enough islets to attain the insulin independence of a transplant recipient. This limitation is mainly caused by islet apoptosis triggered during isolation. In this study, we describe a highly efficient system of transiently transferring anti-apoptotic proteins into pancreatic islets, thus opening an exciting new therapeutic opportunity to improve the viability of transplantable islets. We fused beta-galactosidase to the 11-amino acid residues that constitute the protein transduction domain (PTD) of the HIV/TAT protein and transduced pancreatic islets ex vivo with this fusion protein in a dose-dependent manner with >80% efficiency. We observed that transduction of the anti-apoptotic proteins Bcl-X(L) and PEA-15 fused to TAT/PTD prevented apoptosis induced by tumor necrosis factor-alpha in a pancreatic beta-cell line, indicating that TAT/PTD anti-apoptotic proteins retained their biological activity. Finally, we demonstrated that TAT-fusion proteins did not affect the insulin secretion capability of islets, as determined by glucose static incubation and by reversion of hyperglycemia in diabetic immunodeficient mice.

Heme oxygenase-1 induction in islet cells results in protection from apoptosis and improved in vivo function after transplantation.

Transplantation of islets of Langerhans represents a viable therapeutic approach for the treatment of type 1 diabetes. Unfortunately, transplanted islets are susceptible to allogeneic recognition and rejection, recurrence of autoimmunity, and destruction by local inflammation at the site of implantation. The last of these phenomena might not only result in functional impairment and death of islet cells but could also contribute to amplifying the subsequent specific immune response. Induction of islet cell protection against inflammation could therefore be postulated to be a powerful means to improve overall graft fate. Heme oxygenase-1 (HO-1) has been described as an inducible protein capable of cytoprotection via radical scavenging and apoptosis prevention. The purpose of the present study was to analyze whether HO-1 upregulation in a beta-cell line and in freshly isolated murine islets could result in protection from apoptosis and improve in vivo functional performance. HO-1 upregulation was induced reproducibly with protoporphyrins and was correlated with protection from apoptosis induced in vitro with proinflammatory cytokines or Fas engagement. Furthermore, in vivo HO-1 upregulation resulted in improved islet function in a model of marginal mass islet transplantation in rodents. Strategies aimed at inducing HO-1 upregulation might result in improved success in islet transplantation.

Patterns of engraftment in different strains of immunodeficient mice reconstituted with human peripheral blood lymphocytes.

BACKGROUND: Models of immunodeficient mice reconstituted with a competent human immune system would represent an invaluable tool for the study of transplantation immunobiology allergy, autoimmunity, and infectious diseases. Severe combined immune deficiency (scid) mice can be successfully reconstituted with human peripheral blood lymphocytes (PBLs), but rates and levels of engraftment are poor. New strains of mice with diverse immunodeficiencies have been recently characterized or developed, which might prove to be advantageous for in vivo studies of human immune reactivity. METHODS: We have compared rates and patterns of human PBL engraftment in four available immunodeficient murine strains; scid-beige, nonobese diabetic (NOD)-scid, NOD-scid-beta2 m- and rag-. T- and B-lymphocyte engraftment, phenotype of engrafted cells, and occurrence of graft-versus-host disease (GVHD) were studied and compared. RESULTS: Successful engraftment of human PBL was readily obtained in the majority of scid-beige, NOD-scid, and NOD-scid-beta2 m- with a single i.p. administration of human PBLs, whereas it was seldom achieved in rag- animals. Human Ig levels were accordingly remarkably low in rag- recipients but, interestingly also in NOD-scid-beta2 m- mice. Engraftment was readily observed not only in peripheral blood but also in spleen and bone marrow of successfully reconstituted animals. Phenotypic analysis of engrafted human cells showed preserved CD4/CD8 ratios and a clear skewing toward an activated phenotype. GVHD was invariably observed in successfully reconstituted animals. CONCLUSIONS: Our data indicate that a high rate of reconstitution with human lymphocytes can be achieved in scid-beige, NOD-scid, and NOD-scid-beta2 m- mice. Human Ig are produced at high levels, except in NOD-scid-beta2 m-, including xenoreactive natural antibodies. Scid-beige and NOD-scid appear therefore better suited than NOD-scid-beta2 m- or rag- for analysis of human immunoreactivity in vivo. An important caveat is the invariable occurrence of GVHD that precludes long-term studies in this model system.

Endotoxin-mediated delayed islet graft function is associated with increased intra-islet cytokine production and islet cell apoptosis.

BACKGROUND: Primary nonfunction resulting in immediate graft loss is responsible for the failure of a large number of islet transplants. Evidence is accumulating to single out endotoxin contamination of the various reagents needed for islet isolation as a major cause of early graft loss. METHODS: Islets isolated with endotoxin-containing (400 endotoxin units/ml) collagenase type V and "endotoxin-free" (3.1 endotoxin units/ml) Liberase were compared. Graft function was assessed using a syngeneic murine model of marginal islet mass transplantation. Pro-inflammatory cytokine production by islets was measured by ELISA in culture supernatants, and quantitative reverse transcriptase-PCR. Islet cell apoptosis was measured using the annexin assay. RESULTS: Graft function was significantly delayed when islets were isolated with endotoxin-containing collagenase. Addition of endotoxin to the Liberase solution similarly delayed graft function. After 18 hr in culture, collagenase-isolated islets released higher amounts of proinflammatory cytokines compared with Liberase-isolated islets (interleukin-6: 2,185+/-1,174 pg/ml vs. 520+/-201 pg/ml; tumor necrosis factor-alpha: 304+/-298 pg/ml vs. 0; IL-1beta: 12.5 pg/ml+/-12.5 vs. 0). This observation correlated with higher cytokine mRNA expression in collagenase-isolated islets. The percentage of apoptotic islet cells immediately after isolation was 17.2%+/-9.4 in collagenase-isolated islets and 7.1%+/-2.1 in Liberase-isolated islets. CONCLUSIONS: We propose that endotoxin contamination is the primum movens of a chain of events that involves intra-islet cytokine production and release and islet cell apoptosis, and endotoxin contamination can ultimately lead to primary nonfunction in vivo. This emphasizes the fact that using endotoxin-free reagents during isolation is a key factor for successful islet transplantation.

Early assessment of apoptosis in isolated islets of Langerhans.

BACKGROUND: There is substantial evidence to link early graft loss after islet transplantation to isolation-induced islet cell apoptosis. Measurement of caspase 3 activity and detection of the lost cell membrane asymmetry, revealed by annexin V binding, are newly available assays that allow the analysis of early events of apoptosis. METHODS: In this study, we compared these tests with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and analysis of DNA fragmentation after gel electrophoresis in freshly isolated islets obtained from rats, before and after treatment with interleukin-1 beta, interferon gamma, and tumor necrosis factor a, cytokines known to induce islet cell damage. RESULTS: A measurable level of apoptosis was observed the day after isolation when caspase 3 activity and annexin V binding were used as assays, although no substantial DNA fragmentation was detected with TUNEL assay and DNA gel electrophoresis. Baseline caspase 3 activity was 0.8+/-0.3 U/100 islet equivalents and it increased to 1.4+/-0.45 U/100 islet equivalents 3 hr after cytokine stimulation (P<0.05 vs. unstimulated islets). The baseline level of apoptosis, as detected by annexin V binding, was 21.1%+/-5.8%, and it increased to 27.5%+/-8.1% 6 hr after addition of the cytokine cocktail (P<0.01 vs. unstimulated islets). An increase in the number of TUNEL-positive nuclei was detected 24 hr after stimulation and peaked at 48 hr. DNA laddering was also evident 24 hr after cytokine treatment. CONCLUSION: These data suggest that measurement of caspase 3 activity and annexin V binding analysis might represent reliable markers of early events of islet cell apoptosis.

Absence of CSF-1-dependent macrophages does not improve function of transplanted islets of Langerhans.

A role of macrophage-mediated inflammatory events in early islet graft loss is increasingly acknowledged. Osteopetrotic mice (op/op) have a complete absence of CSF-1, and thus of most tissue macrophages. We have investigated whether the absence of CSF-1-dependent macrophages in the graft itself or at the transplant site could decrease the delay to function of a syngeneic marginal islet mass. Islets transplanted into op/op or control recipients reversed diabetes in 59 days vs. 10 days (p = 0.28, NS). Islets isolated from op/op or control mice reversed diabetes in 11 days vs. 10 days. IL-1 and TNF-alpha release by cultured islets was markedly decreased for op/op islets compared with control islets (IL-1: 0 vs. 4.2 pg/ml, p = 0.07; TNF-alpha: 67 vs. 311 pg/ml, p = 0.002). In contrast, IL-6 release by op/op islets was significantly increased (11.1 vs. 4.3 ng/ml, p = 0.006). CSF-1-dependent tissue macrophages may not be critical in the inflammatory insult to islet transplants. Alternate patterns of intraislet release of deleterious proinflammatory cytokines may exist.

Islet cell transplantation: in vivo and in vitro functional assessment of nonhuman primate pancreatic islets.

Transplantation of pancreatic islets of Langerhans as a therapeutic approach for treatment of type I diabetes offers an alternative to subcutaneous insulin injections. Normalization of blood glucose levels by transplanted islets may prevent the development of diabetes-related complications. Problems related to rejection, recurrence of autoimmunity, and local inflammation upon transplantation of islets into the liver need to be solved before the implementation of islet cell transplantation can be viewed as a justifiable procedure in a large cohort of patients. Islet cell isolation has been quite successful in small animals, but the translation of this approach to nonhuman primates has been less rewarding. One of the main problems encountered in nonhuman primate models is the difficulty of isolating an adequate number of functional islets for transplantation. The aim of the present study was to develop a method for isolating a sufficient number of viable islets from nonhuman primates to allow for reversal of diabetes. By implementing minor modifications in the automated method for human islet isolation we were able to obtain viable, functional islets that responded normally to glucose stimulation in vitro. These islets were also able to reverse diabetes in immunocompromised nude mice, rendered diabetic by streptozotocin. This method of islet cell isolation has enabled us to proceed with protocols of allogeneic islet cell transplantation in preclinical, nonhuman primate models.

In Vivo Assessment of Human Islet Potency.

Transplantation of insulin-producing cells into immunodeficient mice represents an important tool for the assessment of viability and function of cellular products intended to restore beta cell function in the clinical and research applications. The in vivo bioassay allows for the assessment of the ability of the cellular product to restore and maintain euglycemia in vivo after transplantation into chemically-induced diabetic recipients. Herein we describe the protocol utilized for the in vivo potency assessment of human islet cell products. Modifications of the protocol enable the use of the bioassay for the potency assessment of human or animal insulin-producing cells from adult, neonatal or fetal sources or generated from stem cells.