Extended survival versus accelerated rejection of nonhuman primate islet allografts: Effect of mesenchymal stem cell source and timing.

Mesenchymal stem cells (MSC) have been shown to be immunomodulatory, tissue regenerative, and graft promoting; however, several questions remain with regard to ideal MSC source and timing of administration. In this study, we utilized a rigorous preclinical model of allogeneic islet cell transplantation, incorporating reduced immune suppression and near to complete mismatch of major histocompatibility antigens between the diabetic cynomolgus monkey recipient and the islet donor, to evaluate both the graft promoting impact of MSC source, that is, derived from the islet recipient, the islet donor or an unrelated third party as well as the impact of timing. Co-transplant of MSC and islets on post-operative day 0, followed by additional IV MSC infusions in the first posttransplant month, resulted in prolongation of rejection free and overall islet survival and superior metabolic control for animals treated with recipient as compared to donor or third-party MSC. Immunological analyses demonstrated that infusion of MSC from either source did not prevent alloantibody formation to the islet or MSC donor; however, treatment with recipient MSC resulted in significant downregulation of memory T cells, decreased anti-donor T cell proliferation, and a trend toward increased Tregulatory:Tconventional ratios.

Transplantation of PEGylated islets enhances therapeutic efficacy in a diabetic nonhuman primate model.

Islet cell transplantation can lead to insulin independence, reduced hypoglycemia, and amelioration of diabetes complications in patients with type 1 diabetes. The systemic delivery of anti-inflammatory agents, while considered crucial to limit the early loss of islets associated with intrahepatic infusion, increases the burden of immunosuppression. In an effort to decrease the pharmaceutical load to the patient, we modified the pancreatic islet surface with long-chain poly(ethylene glycol) (PEG) to mitigate detrimental host-implant interactions. The effect of PEGylation on islet engraftment and long-term survival was examined in a robust nonhuman primate model via three paired transplants of dosages 4300, 8300, and 10 000 islet equivalents per kg body weight. A reduced immunosuppressive regimen of anti-thymocyte globulin induction plus tacrolimus in the first posttransplant month followed by maintenance with sirolimus monotherapy was employed. To limit transplant variability, two of the three pairs were closely MHC-matched recipients and received MHC-disparate PEGylated or untreated islets isolated from the same donors. Recipients of PEGylated islets exhibited significantly improved early c-peptide levels, reduced exogenous insulin requirements, and superior glycemic control, as compared to recipients of untreated islets. These results indicate that this simple islet modification procedure may improve islet engraftment and survival in the setting of reduced immunosuppression.

Bioengineering the Endocrine Pancreas: Intraomental Islet Transplantation Within a Biologic Resorbable Scaffold.

Transplantation of pancreatic islets is a therapeutic option to preserve or restore ß-cell function. Our study was aimed at developing a clinically applicable protocol for extrahepatic transplantation of pancreatic islets. The potency of islets implanted onto the omentum, using an in situ-generated adherent, resorbable plasma-thrombin biologic scaffold, was evaluated in diabetic rat and nonhuman primate (NHP) models. Intraomental islet engraftment in the biologic scaffold was confirmed by achievement of improved metabolic function and preservation of islet cytoarchitecture, with reconstitution of rich intrainsular vascular networks in both species. Long-term nonfasting normoglycemia and adequate glucose clearance (tolerance tests) were achieved in both intrahepatic and intraomental sites in rats. Intraomental graft recipients displayed lower levels of serum biomarkers of islet distress (e.g., acute serum insulin) and inflammation (e.g., leptin and α2-macroglobulin). Importantly, low-purity (30:70% endocrine:exocrine) syngeneic rat islet preparations displayed function equivalent to that of pure (>95% endocrine) preparations after intraomental biologic scaffold implantation. Moreover, the biologic scaffold sustained allogeneic islet engraftment in immunosuppressed recipients. Collectively, our feasibility/efficacy data, along with the simplicity of the procedure and the safety of the biologic scaffold components, represented sufficient preclinical testing to proceed to a pilot phase I/II clinical trial.

Choice of immunosuppression influences cytomegalovirus DNAemia in cynomolgus monkey (Macaca fascicularis) islet allograft recipients.

This retrospective study reviews the results of our experience with the occurrence of CMV DNAemia in islet cell transplanted cynomolgus monkeys subjected to different immunosuppressive protocols, including induction treatment with thymoglobulin (TMG), with a combination of thymoglobulin and fludarabine (FLUD), with cyclophosphamide, or with daclizumab. CMV DNA in the peripheral blood (CMV DNAemia) of 47 monkeys was quantified by real-time PCR on a weekly to biweekly basis. As compared to other immunosuppressive regimens, and in association with greater decreases in WBC, lymphocyte, CD3+CD4+, and CD3+CD8+ lymphocyte counts, frequent CMV DNAemia occurred earlier (within the first month posttransplant), and was of greater severity and duration in recipients of TMG ± FLUD. Treatment of recipients with alternative induction agents that resulted in less dramatic reductions in WBC and lymphocyte counts, however, resulted in occurrence of CMV DNAemia after postoperative day 60. The frequency, average intensity, duration, and area under the curve (AUC) for CMV DNAemia in animals receiving TMG ± FLUD were 75-100%, 4.02 ± 1.75 copies/ng DNA, 23.0 ± 5.3 days, and 367.0 ± 121.1 days × copies/ng DNA, respectively; corresponding values in animals receiving other treatments (0-44%, 0.19 ± 0.10 copies/ng DNA, 0.5 ± 0.3 days, and 75.4 ± 40.2 days × copies/ng DNA, respectively) were significantly different. The value of WBC, T and B cells at the nadir of cell depletion greatly affects the occurrence of CMV DNAemia. No animals developed CMV DNAemia within the next 3 weeks when the lowest value of WBC, lymphocyte, CD3+, CD3+CD4+, CD3+CD8+, or CD20+ cells was above 4500, 1800, 300, 200, 150, or 300 cells/µl, respectively. Oral valganciclovir prophylaxis did not completely prevent the appearance of CMV DNAemia.

Mesenchymal stem cells enhance allogeneic islet engraftment in nonhuman primates.

OBJECTIVE: To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation. RESEARCH DESIGN AND METHODS: Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype, gene expression, differentiation potential, and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels, reduction of exogenous insulin requirement (EIR), C-peptide levels, changes in peripheral blood T regulatory cells, and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection. RESULTS: MSC phenotype and a normal karyotype were observed through passage 11. IL-6, IL-10, vascular endothelial growth factor, TGF-ß, hepatocyte growth factor, and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8), as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood. CONCLUSIONS: MSCs may provide an important approach for enhancement of islet engraftment, thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore, MSCs may serve as a new, safe, and effective antirejection therapy.

Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.

BACKGROUND: Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model. METHODS: Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 microg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10-25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. RESULTS: Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. CONCLUSIONS: These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.

Omental anatomy of non-human primates.

The anatomy and physiology of the omentum provide optimum reconstructive characteristics and the omentum may be used as a free or pedicled autograft, but also as the receptor site for engraftment of glandular islets. Our purpose was the study of the omental anatomy of non-human primate (NHP), in order to determine an experimental model for pancreatic islets transplantation. Seventeen cadavers NHP (age range 4 years to 23 years) were utilised in this anatomical study. Both cynomolgus monkeys (macaca fascicularis) and baboons (papio hamadryas) were analysed. The animals were without known medical or anatomical abnormalities. We studied the morphology of the omentum, with an emphasis on arterial vascularisation. The omental anatomy of the NHP is very similar to that of humans. The main difference lies in the shape of the lesser sac, which has a complete caudal recess. The arterial vascularisation has a double origin. Based on the anastomosis between them and on the vascular density, the NHP omentum can be divided into four vascular areas. Our results demonstrate that one or two long pedicled flaps can be constructed from the omentum.

Selective venous sampling in recurrent and persistent hyperparathyroidism: indication, technique, and results.

Between 1992 and 2002, 542 patients underwent a surgical treatment for hyperparathyroidism in our department. Twenty-three selective venous sampling procedures (SVS) were performed because of the failure of the other methods of diagnosis. These patients have recurrent or persistent hyperparathyroidism. Noninvasive methods of topographical diagnosis have failed or they have given contradictory results. In our experience, the surgeon needs a precise localization of the pathological glands in these difficult cases. In our series of SVS, specificity was 85.7% and sensitivity was 94.7%. Our results show that a high postoperative gradient of parathyroid hormone in the internal thoracic veins indicates an ectopic pathological gland in the thymus. A high gradient in a vertebral vein indicates a pathological superior parathyroid gland, usually in a retro-esophageal position.

Islet allograft survival in nonhuman primates immunosuppressed with basiliximab, RAD, and FTY720.

OBJECTIVE: In a preclinical, nonhuman primate islet allotransplant model, the authors evaluated a novel immunosuppressive combination of basiliximab for induction and of RAD and FTY720 for maintenance. METHODS: Five ABO-compatible and mixed lymphocyte reactivity-mismatched streptozotocin-induced diabetic juvenile cynomolgus monkeys underwent transplantation intraportally with 48-hr cultured 10,000 islet equivalents per kilogram. Induction immunosuppression was with intravenous basiliximab (10 mg on postoperative days 0 and 4). Maintenance immunosuppression was with RAD (everolimus) (0.075 mg/kg per day administered subcutaneously) and FTY720 (0.3 mg/kg per day administered orally), both administered on day -2 through day 180 posttransplant. RESULTS: All five recipients tolerated their transplants and immunosuppressive therapy well, without adverse events or infectious complications. Insulin requirements pretransplant were 2.6 to 4.0 U/kg per day. All recipients became normoglycemic and insulin-independent posttransplant. Posttransplant serum C-peptide levels averaged 2.7 ng/mL (range, 0.6-6.2 ng/mL). Morning blood glucose levels ranged from less than 100 mg/dL to 150 mg/dL. Posttransplant acute C-peptide response to intravenous arginine averaged 1.3 ng/mL (range, 0.23-2.72 ng/mL). In one recipient with subtherapeutic RAD blood levels on day 7 posttransplant, exogenous insulin was resumed 100 days posttransplant; basal C-peptide levels remained positive in this recipient and averaged 2.6 ng/mL. The other four recipients remained insulin-independent for more than 6 months. CONCLUSIONS: This study provides preliminary evidence of the safety and efficacy of corticosteroid- and calcineurin inhibitor-free immunosuppression in a relevant preclinical transplant model. These findings provide a strong rationale for evaluating this nondiabetogenic regimen in a clinical trial of islet transplants in type 1 diabetic recipients.