C peptide elisa assay

Rhesus macaques were rendered diabetic chemically through IV administration of streptozocin (STZ, 1250 mg/m² IV; Sigma, St Louis, MO, USA) 3–5 weeks prior to the planned transplantation and diabetes was confirmed by hyperglycemia with concomitant reduction and poor responsiveness of rhesus C-peptide levels (C-peptide ELISA assay; Abcam, Cambridge, MA, USA) to dextrose bolus. On day of transplantation, NPI preparations were washed and suspended in 20–30 ml of CMRL 1066 solution (Corning, Corning, NY, USA) supplemented with 200 units of heparin and etanercept at 3mg/kg (Enbrel; Amgen, Philadelphia, PA, USA). All rhesus macaque recipients underwent mini-laparotomy while under general anesthesia. At least 50,000 IEQs/kg of NPIs were infused through the portal vein via gravity through a 22-gauge catheter. As most piglets were born to parents of heterozygous human CD46 knocked in background, we received a variable number of GKO/heterologous hCD46 piglets each time. Thus, although we prefer to use islets of a single genotype, five out of twelve animals received an islet preparation of mixed genotypes to make up a minimal 50,000 IEQs/kg NPI dosage. All the above procedures were approved by the Duke IACUC.

Rhesus macaques were rendered diabetic through streptozocin induction (STZ, 1250 mg/m² IV; Sigma, St Louis, MO, USA) and diabetes was confirmed by significant reduction and poor responsiveness of rhesus C-peptide levels (C-peptide ELISA assay; Abcam, Cambridge, MA, USA) to dextrose bolus. As NPIs generally take weeks to months to reach maturity, our 7-day extended dual islet transplantation was not designed to achieve normoglycemia in NHP recipients. The use of diabetic recipients was not essential for our study but selected to better simulate the clinical scenario. Additionally, the host immune responses to xenograft in a diabetic animal and a normoglycemic animal may differ.
Five diabetic recipients underwent GKO and GKO/CD46 xenoislet dual transplantation (Table 2) and given an immunosuppressive regimen consisting of Basiliximab at 0.3mg/kg on day of transplant and Post-operative day (POD) 2, Belatacept at 20mg/kg on POD 0 and 4, and Tacrolimus at 0.05mg/kg twice a day starting at POD 1 with a target trough level of 8–12ng/ml. As neonatal porcine islet requires at least 3 weeks to mature in vivo and recipients following NPI infusion do not achieve insulin independence until weeks to months posttransplantation, recipient blood glucose measurements were not indicative of graft function and were taken only to monitor for clinical consequences of hypo- or hyperglycemia.

Rhesus macaques were rendered diabetic through streptozocin induction (STZ, 1250 mg/m² IV; Sigma, St Louis, MO, USA) after baseline IV glucose tolerance testing (IVGTT) data were obtained. Post-diabetes induction IVGTT was performed to confirm significant reduction and poor responsiveness of rhesus C-peptide levels (C-peptide ELISA assay; Abcam, Cambridge, MA, USA) to dextrose bolus. Animals were monitored with twice daily glucose checks and administration of NPH and glargine insulin based on sliding scale to maintain appropriate blood glucose levels. Three diabetic recipients underwent rhesus islet and GTKO xenoislet dual transplantation (Table 1B) and given a robust costimulation blockade based regimen using CTLA-4Ig, anti-CD154, and anti-LFA1 therapy used in our initial GTKO islet xenotransplantation series, (21 (link)) with an experimental endpoint of 7 days after transplant.