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    • br Methods The donor pig herd Auckland Island

    2018-11-07


    Methods The donor pig herd (Auckland Island pigs; Living Cell Technologies, Manukau, New Zealand, and Diatranz Otsuka Ltd, Auckland New Zealand) was maintained in a designated pathogen-free facility and screened for an extensive panel of infectious agents including porcine endogenous retrovirus (PERV) (Garkavenko et al., 2004). Newborn piglets from the donor herd were shipped to a cell processing facility (Auckland, New Zealand). They were anesthetized and bled. The procured pancreata were then brought into a cell processing room and digested under good manufacturing practices (GMP) as described previously (Hillberg et al., 2013). Digested pancreata were cultured using spinner flasks for 3days before encapsulation (Hillberg et al., 2013). After counting islet yield, cultured neonatal porcine islets were encapsulated using alginate-poly-l-ornithine-alginate (APA) (Hillberg et al., 2013). APA-encapsulated neonatal porcine islets were cultured for an additional three weeks. Then APA-encapsulated neonatal porcine islets were shipped to Argentine using RPMI culture media at cold temperature. From August 2011 to July 2012, encapsulated neonatal porcine islets were transplanted into the peritoneal cavity via a laparoscope (Fig. 1) at Hospital Interzonal General de Agudos Eva Peron, San Martin, Provincia de Buenos Aires. The study protocol (ClinicalTrials.gov Identifier NCT01739829) was granted ethics approval in Argentina from the Ministry of Health, Buenos Aires Province and the local institutional ethics committee. All patients provided written consent forms to accept this c-di-AMP treatment. Main inclusion criteria for this study are as follows. There were two groups consisting of patients who received either approximately 5000isletequivalent(IEQ)/body weight kg twice (total approximately 10,000IEQ/kg) (group 1, n=4) or 10,000IEQ/kg twice (total approximately 20,000IEQ/kg) (group 2, n=4). The second transplantation was conducted at approximately 3months after the first transplantation. At the time of second transplantation, abdominal cavity was observed via laparoscopy (Fig. 1B) and samples of encapsulated islets were retrieved for histological analysis. Outcomes of safety were determined from adverse event reports. PERV provirus and PERV genomic RNA were detected using a PCR-based method (Wynyard et al., 2014). PERV antibodies were tested on patient sera (Wynyard et al., 2014). Outcomes of efficacy were determined from HbA1c, daily insulin dose (averaged during 2weeks), and frequency of unaware hypoglycemic events assessed on a regular basis. The regular assessments were conducted 5 times before the first transplantation within approximately 2months, at the time of transplantation and 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 15, 16, 24, and 29months after the first transplantation. To assess the function of transplanted encapsulated neonatal porcine islets, transplant estimated function (TEF) was used (Caumo et al., 2011). TEF was calculated as follows: To assess the efficacy of prevention of unaware hypoglycemia, numbers of unaware hypoglycemic events per 4weeks were assessed using continuous glucose monitoring and diary cards in which any symptoms of hypoglycemia were recorded when patients noticed. For the baseline, unaware hypoglycemic events were counted from week −4 to week −1 before transplantation. To assess the long-term effect, HbA1c, daily insulin dose, TEF and unaware hypoglycemic events were analyzed at >300days and 600days. Oral glucose tolerance tests (OGTTs) were performed in selected patients (patients 2, 5, 6, 7, and 8) whose fasting blood glucose levels were <150mg/dl approximately 15months after the 1st implantation. Both long-action insulin (NPH) and short acting insulin (insulin aspart) were suspended 24h and 5h before OGTT, respectively. Fifty grams of glucose was taken within 10min and blood glucose levels were measured before OGTT, at 30, 60, 90 and 120min.
    Results
    Discussion In terms of safety, there was only one procedure related serious adverse event and it was resolved without residual effects. For the observed period PERV infection was not seen for any of the patients tested (Morozov et al., in press) and is consistent with an earlier study using the same technology (Matsumoto et al., 2014; Wynyard et al., 2014).