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• W1960949488 abstract "The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti-CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only two of five demonstrating function beyond 5 months. The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti-CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only two of five demonstrating function beyond 5 months. Xenotransplantation (xenoTx) of porcine islets is poised to become a therapeutic alternative to pancreas and islet allotransplantation (alloTx) for patients with Type 1 diabetes (1Ekser B Ezzelarab M Hara H et al.Clinical xenotransplantation: The next medical revolution?.Lancet. 2012; 379: 672-683Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 2van der Windt DJ Bottino R Kumar G et al.Clinical islet xenotransplantation: How close are we?.Diabetes. 2012; 61: 3046-3055Crossref PubMed Scopus (99) Google Scholar, 3Cooper DKC Bottino R Satyananda V Wijkstrom M Trucco M Toward clinical islet xenotransplantation—Are revisions to the IXA guidelines warranted?.Xenotransplantation. 2013; 2: 68-74Crossref Scopus (14) Google Scholar, 4Dufrane D Gianello P Pig islet xenotransplantation in human: Structural and physiological compatibility for human clinical application.Transplant Rev. 2012; 26: 183-188Crossref Scopus (41) Google Scholar, 5Elliott RB Towards xenotransplantation of pig islets in the clinic.Curr Opin Organ Transplant. 2011; 16: 195-200Crossref PubMed Scopus (56) Google Scholar). A significant advance has been the ability to produce pigs with specific genetic modifications (6Phelps CJ Koike C Vaught TD et al.Production of α1,3-galactosyltransferase-deficient pigs.Science. 2003; 299: 411-414Crossref PubMed Scopus (907) Google Scholar, 7Phelps C Ball S Vaught T et al.Production and characterization of transgenic pigs expressing porcine CTLA4-Ig.Xenotransplantation. 2009; 16: 477-485Crossref PubMed Scopus (107) Google Scholar, 8Phelps C Vaught T Ball S et al.Multi-transgenic pigs designed for xenoislet transplants.Xenotransplantation. 2009; 16 (Abstract #IXA-O-7.3): 374Crossref Scopus (109) Google Scholar, 9Yazaki S Iwamoto M Onishi A et al.Production of cloned pigs expressing human thrombomodulin in endothelial cells.Xenotransplantation. 2012; 19: 82-91Crossref PubMed Scopus (24) Google Scholar, 10Klymiuk N van Buerck L Bähr A et al.Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice.Diabetes. 2012; 61: 1527-1537Crossref PubMed Scopus (101) Google Scholar, 11Ayares D Vaught T Ball S et al.Islet-specific expression of TFPI, CD39, and CTLA4Ig in transgenic pigs designed for xenoislet transplantation.Xenotransplantation. 2011; 18 (Abstract #120): 269Google Scholar, 12Ayares D Phelps C Vaught T et al.Multi-transgenic pigs for vascularized pig organ xenografts.Xenotransplantation. 2011; 18 (Abstract #119): 269Google Scholar, 13Ayares D Multi-transgenic pigs for xenotransplantation.Reprod Fertil Dev. 2012; 25: 320Crossref Google Scholar), which may prove useful in overcoming the metabolic and immunological barriers between species, and ultimately contribute to reduce the islet mass as well as the intensity of immunosuppressive therapy necessary to sustain islet graft survival. As also seen in the human islet alloTx setting, intraportal infusion of pig islets in monkeys results in an immediate loss of islets, along with the release of insulin and C-peptide, making it more difficult to achieve and maintain normoglycemia (14Korsgren O Nilsson B Berne C et al.Current status of clinical islet transplantation.Transplantation. 2005; 79: 1289-1293Crossref PubMed Scopus (115) Google Scholar). Some of the mechanisms involved in early islet loss have been characterized as the instant blood-mediated inflammatory reaction (IBMIR) (15Bennet W Sundberg B Groth CG et al.Incompatibility between human blood and isolated islets of Langerhans: A finding with implications for clinical intraportal islet transplantation?.Diabetes. 1999; 48: 1907-1914Crossref PubMed Scopus (366) Google Scholar, 16Nilsson B Ekdahl KN Lorsgren O Control of instant blood-mediated inflammatory reaction to improve islets of Langerhans engraftment.Curr Opin Organ Transplant. 2011; 6: 620-626Crossref Scopus (141) Google Scholar, 17van der Windt DJ Bottino R Casu A Campanile N Cooper DK Rapid loss of intraportally transplanted islets: An overview of pathophysiology and preventive strategies.Xenotransplantation. 2007; 14: 288-297Crossref PubMed Scopus (136) Google Scholar). Previously, our group achieved insulin-independence in diabetic, immunosuppressed cynomolgus monkeys following the transplantation (Tx) of islets from pigs expressing a single human complement-regulatory protein (hCD46) (18van der Windt DJ Bottino R Casu A et al.Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets.Am J Transplant. 2009; 9: 2716-2726Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). However, while hCD46 was associated with successful engraftment when compared with WT pig islets, a reduction of early islet loss was not observed, suggesting that other modulatory transgenes would be beneficial to islet survival. Several studies have indicated that tissue factor, complement and coagulation activation, antibody binding and inflammation contribute to primary nonfunction (15Bennet W Sundberg B Groth CG et al.Incompatibility between human blood and isolated islets of Langerhans: A finding with implications for clinical intraportal islet transplantation?.Diabetes. 1999; 48: 1907-1914Crossref PubMed Scopus (366) Google Scholar,19van der Windt DJ Marigliano M He J et al.Early islet damage after direct exposure of pig islets to blood: Has humoral immunity been underestimated?.Cell Transplant. 2012; 21: 1791-1802Crossref PubMed Scopus (43) Google Scholar, 20Beuneu C Vosters O Ling Z et al.N-Acetylcysteine derivative inhibits procoagulant activity of human islet cells.Diabetologia. 2007; 50: 343-347Crossref PubMed Scopus (20) Google Scholar, 21Johansson H Lukinius A Moberg L et al.Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation.Diabetes. 2005; 54: 1755-1762Crossref PubMed Scopus (263) Google Scholar). To reduce such effects, new genetically engineered (GE) pigs have been generated on a background of α1,3-galactosyltransferase gene-knockout and ubiquitous expression of hCD46 (GTKO/hCD46 pigs). Specific transgenes were selected to target relevant mechanisms. Human tissue factor pathway inhibitor (hTFPI) was aimed at inhibition of coagulation and inflammation associated with IBMIR (22Berman DM Cabrera O Kenyon NM et al.Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.Transplantation. 2007; 84: 308-315Crossref PubMed Scopus (64) Google Scholar,23Lin CC Ezzelarab M Hara H et al.Atorvastatin or transgenic expression of TFPI inhibits coagulation initiated by anti-nonGal IgG binding to porcine aortic endothelial cell.J Thromb Haemost. 2010; 8: 2001-2010Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Human CD39, through its ATPase activity, has been shown to decrease platelet activation and prevent clotting in transgenic mouse models (24Dwyer KM Robson SC Nandurkar HH et al.Thromboregulatory manifestations in human CD39 transgenic mice and the implications for thrombotic disease and transplantation.J Clin Invest. 2004; 113: 1440-1446Crossref PubMed Scopus (0) Google Scholar,25Dwyer KM Mysore TB Crikis S et al.The transgenic expression of human CD39 on murine islets inhibits clotting of human blood.Transplantation. 2006; 82: 428-432Crossref PubMed Scopus (54) Google Scholar). In addition, the porcine CTLA4-Ig transgene was incorporated with the goal of inhibiting the cellular immune response (26Phelps CJ Ball SF Vaught TD et al.Production and characterization of transgenic pigs expressing porcine CTLA4-Ig.Xenotransplantation. 2009; 16: 477-485Crossref PubMed Scopus (107) Google Scholar,27Koshika T Phelps C Fang J et al.Relative efficiency of porcine and human cytotoxic T-lymphocyte antigen 4 immunoglobulin in inhibiting human CD4+ T-cell responses co-stimulated by porcine and human B7 molecules.Immunology. 2011; 134: 386-397Crossref Scopus (23) Google Scholar). Upon reaching sufficient adult size and age, the pigs’ pancreases were harvested for isolation of the islets, which were infused into diabetic, immunosuppressed cynomolgus monkeys. We report the effects of these novel GE pig islets on early islet loss and Tx outcome in the first five experiments. Despite significant limitations, this study provides insights into the use of GE pigs in islet xenoTx. Six female pigs aged 16–36 months, weighing 350–400 lbs (159–181 kg) (Revivicor, Blacksburg, VA) were sources of islets (Table 1). The production of these GE pigs and their glucose metabolism are detailed in Wijkstrom et al (28Wijkstrom M, Iwase H, Ekser B, et al. Glucose metabolism in pigs expressing human genes under an insulin promoter. Submitted.Google Scholar) (Supplementary Methods).Table 1:Characteristics of islet-source pigsPig no.Genetic manipulation13-GE = GTKO/CD46/CD39; 4-GE = GTKO/CD46/TFPI/CTL4-Ig; 5-GE = GTKO/CD46/TFPI/CTL4-Ig/CD39.Pig weight (kg)Islet yield (IEQ)Islet yield per gram pancreas (IEQ/g)Viability (%)Purity (%)Stimulation index high/low g-high + theoph/low gP388-034-GE2All four of the 4-GE and 5-GE pigs are clones of each other with respect to the 4-GE background. The 4-GE cell line 548A.3 was used to clone P384-02 and P388-03. This same 4-GE cell line was subsequently transfected with the ins-CD39 vector, to generate the two 5-GE cloned animals, P388-01 and P388-2. While P388-03 was part of the cell pool transfected with ins-CD39, genotype analysis showed that this animal was a “no-take” with respect to integration of the CD39 transgene, and thus only 4-GE.∼160338 083135691852.0–8.1P384-024-GE2All four of the 4-GE and 5-GE pigs are clones of each other with respect to the 4-GE background. The 4-GE cell line 548A.3 was used to clone P384-02 and P388-03. This same 4-GE cell line was subsequently transfected with the ins-CD39 vector, to generate the two 5-GE cloned animals, P388-01 and P388-2. While P388-03 was part of the cell pool transfected with ins-CD39, genotype analysis showed that this animal was a “no-take” with respect to integration of the CD39 transgene, and thus only 4-GE.∼160399 083223794752.3–6.3P388-25-GE2All four of the 4-GE and 5-GE pigs are clones of each other with respect to the 4-GE background. The 4-GE cell line 548A.3 was used to clone P384-02 and P388-03. This same 4-GE cell line was subsequently transfected with the ins-CD39 vector, to generate the two 5-GE cloned animals, P388-01 and P388-2. While P388-03 was part of the cell pool transfected with ins-CD39, genotype analysis showed that this animal was a “no-take” with respect to integration of the CD39 transgene, and thus only 4-GE.∼160331 846186994753.1–5.3P462-043-GE2All four of the 4-GE and 5-GE pigs are clones of each other with respect to the 4-GE background. The 4-GE cell line 548A.3 was used to clone P384-02 and P388-03. This same 4-GE cell line was subsequently transfected with the ins-CD39 vector, to generate the two 5-GE cloned animals, P388-01 and P388-2. While P388-03 was part of the cell pool transfected with ins-CD39, genotype analysis showed that this animal was a “no-take” with respect to integration of the CD39 transgene, and thus only 4-GE.∼160325 4201574953Islets from P462-04 and P474-07 were pooled and the combined prep was tested.803Islets from P462-04 and P474-07 were pooled and the combined prep was tested.2.6–6.93Islets from P462-04 and P474-07 were pooled and the combined prep was tested.P474-073-GE2All four of the 4-GE and 5-GE pigs are clones of each other with respect to the 4-GE background. The 4-GE cell line 548A.3 was used to clone P384-02 and P388-03. This same 4-GE cell line was subsequently transfected with the ins-CD39 vector, to generate the two 5-GE cloned animals, P388-01 and P388-2. While P388-03 was part of the cell pool transfected with ins-CD39, genotype analysis showed that this animal was a “no-take” with respect to integration of the CD39 transgene, and thus only 4-GE.∼160300 7501012953Islets from P462-04 and P474-07 were pooled and the combined prep was tested.803Islets from P462-04 and P474-07 were pooled and the combined prep was tested.2.6–6.93Islets from P462-04 and P474-07 were pooled and the combined prep was tested.P388-015-GE2All four of the 4-GE and 5-GE pigs are clones of each other with respect to the 4-GE background. The 4-GE cell line 548A.3 was used to clone P384-02 and P388-03. This same 4-GE cell line was subsequently transfected with the ins-CD39 vector, to generate the two 5-GE cloned animals, P388-01 and P388-2. While P388-03 was part of the cell pool transfected with ins-CD39, genotype analysis showed that this animal was a “no-take” with respect to integration of the CD39 transgene, and thus only 4-GE.∼180149 606120290752.2–10.8g, glucose; IEQ, islet equivalents; theoph, theophylline.1 3-GE = GTKO/CD46/CD39; 4-GE = GTKO/CD46/TFPI/CTL4-Ig; 5-GE = GTKO/CD46/TFPI/CTL4-Ig/CD39.2 All four of the 4-GE and 5-GE pigs are clones of each other with respect to the 4-GE background. The 4-GE cell line 548A.3 was used to clone P384-02 and P388-03. This same 4-GE cell line was subsequently transfected with the ins-CD39 vector, to generate the two 5-GE cloned animals, P388-01 and P388-2. While P388-03 was part of the cell pool transfected with ins-CD39, genotype analysis showed that this animal was a “no-take” with respect to integration of the CD39 transgene, and thus only 4-GE.3 Islets from P462-04 and P474-07 were pooled and the combined prep was tested. Open table in a new tab g, glucose; IEQ, islet equivalents; theoph, theophylline. Five male cynomolgus monkeys (Macaca fascicularis; Three Springs Scientific, Perkasie, PA, and Alpha Genesis, Yemassee, SC) aged 2.5–4.0 years, weighing 2.5–4.1 kg, were islet recipients (Table 2). One monkey received islets from two GTKO/CD46/hCD39 (3-GE) cloned pigs (P462-04 and P474-07).Table 2:Islet mass transplanted (IEQ/kg), number of recipient CD3+ T cells on day of transplantation, mean mycophenolate mofetil (MMF) trough levels after transplantation and graft survivalRecipient monkey no.Monkey weight at time of Tx (kg)Pig islet donor no.Islet mass transplanted (IEQ/kg)Number of CD3+ T cells on day of Tx (cells/μL)Mean (± SEM) MMF trough levels (µg/mL)Graft survival (days)M2-113.3P388-03100 0004910.59 ± 0.150M3-112.8P384-02100 0003401.25 ± 0.17365M1-114.1P388-275 0004412.78 ± 0.53160M14-122.5P462-04; P474-07100 0001831.63 ± 0.405M12-122.8P388-0150 000911.86 ± 0.473IEQ, islet equivalents; Tx, transplantation. Open table in a new tab IEQ, islet equivalents; Tx, transplantation. All procedures that impacted the care of animals were in compliance with guidelines in the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 2011), and approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Pig pancreases were excised as nonsurvival procedures, as described (29Bottino R Balamurugan AN Smetanka C et al.Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies.Xenotransplantation. 2007; 14: 74-82Crossref PubMed Scopus (68) Google Scholar,30Echeverri GJ McGrath K Bottino R et al.Endoscopic gastric submucosal transplantation of islets (ENDO-STI): Technique and initial results in diabetic pigs.Am J Transplant. 2009; 9: 2485-2496Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Pancreas was transported within 60 min to the laboratory for immediate islet isolation, purification, and culture. Islets were counted as islet equivalents (IEQ) (29Bottino R Balamurugan AN Smetanka C et al.Isolation outcome and functional characteristics of young and adult pig pancreatic islets for transplantation studies.Xenotransplantation. 2007; 14: 74-82Crossref PubMed Scopus (68) Google Scholar). CIzyme™ Collagenase MA and BP Protease were used (VitaCyte, Indianapolis, IN) following the manufacturer’s guidelines. Viability was determined by double fluorescent calcein-AM/propidium iodide staining, a method validated for human islets (31Lorenzo A Razzaboni B Weir GC Yankner BA Pancreatic islet cell toxicity of amylin associated with type-2 diabetes mellitus.Nature. 1994; 368: 756-760Crossref PubMed Scopus (731) Google Scholar) (Supplementary Methods). Islet preparations were stained with dithizone and the percent of dithizone-positive aggregates (at least 50) over whole tissue was used to express purity (32Bottino R Balamurugan AN Bertera S Pietropaolo M Trucco M Piganelli JD Preservation of human islet cell functional mass by anti-oxidative action of a novel SOD mimic compound.Diabetes. 2002; 51: 2561-2567Crossref PubMed Scopus (135) Google Scholar). For qualitative analysis, islets were subjected to dynamic secretagogue challenges (21Johansson H Lukinius A Moberg L et al.Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation.Diabetes. 2005; 54: 1755-1762Crossref PubMed Scopus (263) Google Scholar) (Supplementary Methods). Table 1 summarizes islet graft characteristics. To facilitate blood withdrawal and intravenous (i.v.) drug administration, catheters were inserted into the carotid artery and jugular vein and connected through a tether and jacket system to the exterior of the animal cage (33Cooper DKC Ye Y Niekrasz M Heart transplantation in primates.in: Cramer DV Podesta L Makowka L Handbook of animal models in transplantation research. CRC Press, Boca Raton, FL1994: 173-200Google Scholar). The vascular lines were removed 5–6 weeks later (approximately 2 weeks after islet Tx) to minimize the risk of infection. Additional blood draws were obtained from the femoral vein, after ketamine sedation (Ketaset, Fort Dodge, IA; 10 mg/kg). Two to three weeks before islet Tx, diabetes was induced by i.v. streptozotocin (Zanosar, Sicor Pharmaceuticals, Irvine, CA; 125 mg/kg, never exceeding 1500 mg/m2 to avoid nephrotoxicity). Blood glucose levels were measured using Precision Xtra (Becton Dickinson, Franklin Lakes, NJ). Monkeys were considered diabetic if the following conditions were met: (i) hyperglycemia (>350 mg/dL) on at least two occasions, (ii) baseline primate C-peptide reduced by >75% (34Jonasson O Jones CW Bauman A John E Manaligod J Tso MO The pathophysiology of experimental insulin-deficient diabetes in the monkey. Implications for pancreatic transplantation.Ann Surg. 1985; 201: 27-39PubMed Google Scholar), (iii) no increase in C-peptide after intravenous glucose tolerance test and arginine stimulation test (35Casu A Bottino R Balamurugan AN et al.Metabolic aspects of pig-to-monkey (Macaca fascicularis) islet transplantation: Implications for translation into clinical practice.Diabetologia. 2008; 51: 120-129Crossref PubMed Scopus (79) Google Scholar) and (iv) need for exogenous insulin to prevent ketoacidosis. All monkeys met these criteria. Monkey C-peptide was measured by radioimmunoassay (RIA; Millipore, Billerica, MA) using anti-human antibodies, and concentrations were confirmed with Immulite System (UPMC Presbyterian Hospital Laboratory). Serum porcine C-peptide was measured by radioimmunoassay (RIA, Millipore) using antibodies that do not cross-react with monkey C-peptide. The successful induction of diabetes was confirmed by histological immunostaining of pancreatic tissue at necropsy. All islet preparations were cultured overnight prior to Tx, with the exception of islets from pig P462-04 that were cultured for 1 week and mixed with a second islet batch (P474-07) cultured overnight (Table 2). Culture was in CMRL-1066 (Life Technologies, Carlsbad, CA) supplemented with 10% heat-inactivated porcine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 2 mmol/L L-glutamine (all from Life Technologies) at 24°C in 5% CO2. Prior to Tx, islets were resuspended in fresh 20 mL CMRL-1066 medium with the addition of low molecular weight dextran sulfate (4.5 mg/kg of recipient; Sigma–Aldrich, St. Louis, MO) (36Goto M Johansson H Maeda A Elgue G Korsgren O Nilsson B Low molecular weight dextran sulfate prevents the instant blood-mediated inflammatory reaction induced by adult porcine islets.Transplantation. 2004; 77: 741-747Crossref PubMed Scopus (95) Google Scholar). The insulin content of the transplant medium was negligible (<0.5 U). The islets were infused intraportally by gravity over 5–10 min. Immediately before islet infusion, anti-inflammatory and anticoagulant treatment was administered (Table 3). Activated clotting time (ACT) was monitored (I-Stat; Abbott, Princeton, NJ) and anticoagulants discontinued if the ACT >190 s (37Rood PP Bottino R Belamurugan AN et al.Reduction of early graft loss after intraportal porcine islet transplantation in monkeys.Transplantation. 2007; 82: 202-210Crossref Scopus (71) Google Scholar). Postoperative treatment consisted of prophylactic cefazolin (10 mg/kg intramuscularly [i.m.] × 2 daily) and buprenorphine (0.03 mg/kg i.m. × 2 daily) for 3 days. Monkeys were allowed to eat on the evening of surgery.Table 3:Treatment aimed at reducing the IBMIR, immunosuppressive regimen and supportive therapyTreatmentDose and administrationRouteProstacyclin (Flolan; GlaxoSmithKline, Philadelphia, PA)20 ng/kg/minDay of Tx30 min prior to Tx, 3 h infusioni.v.Methylprednisolone (SoluMedrol; Pfizer, New York, NY)10 mg/kgDay of Txi.v.Dextran sulfate5 mg/kgDay of Txi.v.2 mg/kg/h; Target: ACT 150–190 sDay of Tx6 h infusion. Discontinued if ACT > 190 si.v.Antithymocyte globulin (ATG; Thymogolbulin; Genzyme, Cambridge, MA)25 mg/5–25 mg; Target: <500 CD3+ cells/µLDays −3, −1Over 7 hi.v.Mycophenolate mofetil (MMF; Cellcept; Roche, Nutley, NJ)100 mg/kg/day; Target trough level: 3–5 µg/mLFrom day −7p.o.Anti-CD154 mAb (NIH NHP Reagent Resource, Boston, MA)25 mg/kgDays −1, 0, 3, 7, 11 and 15 and every other week subsequentlyi.v.Aspirin81 mg/dayFrom days −7 to +7p.o.Ganciclovir (Cytovene; Roche, Welwyn Garden City, UK)5 mg/kg/dayFrom day of Tx until removal of vascular linesi.v.Valganciclovir (Valcyte; Genentech, San Francisco, CA)15 mg/kg x2 dailyAfter line removalp.o.Famotidine (APP Pharmaceuticals, Schaumburg, IL, and Baxter Healthcare, Deerfield, IL)0.25 mg/kg/dayFrom day of Tx until removal of vascular linesi.v.1 mg/kg/dayAfter line removalp.o.ACT, activated clotting time; IBMIR, instant blood-mediated inflammatory reaction; i.v., intravenous; p.o., by mouth; Tx, transplantation. Open table in a new tab ACT, activated clotting time; IBMIR, instant blood-mediated inflammatory reaction; i.v., intravenous; p.o., by mouth; Tx, transplantation. Prostacyclin, methylprednisolone, dextran sulfate and aspirin were administered for their anticoagulant and/or anti-inflammatory effects; antithymocyte globulin (ATG) for induction and mycophenolate mofetil (MMF) and anti-CD154mAb for maintenance immunosuppression (Table 3). Ganciclovir and valganciclovir were administered to prevent cytomegalovirus reactivation, and famotidine to prevent peptic ulceration. Serum porcine C-peptide was measured 1, 2 and 24 h post-Tx, and expressed as ng/mL per 10 000 IEQ/kg to take into account differences in islet mass infused. Blood glucose was measured at least every 2 h during the first day and every 4 h on day 1. During the first 24 h post-Tx, the blood glucose target was 100–150 mg/dL. If the blood glucose rose >150 mg/dL, insulin was administered. If it fell <100 mg/dL, dextrose was infused i.v. The total amount of dextrose (expressed as grams/24 h) needed to maintain glycemia within the target range and to prevent hypoglycemia as a result of insulin leakage was calculated (17van der Windt DJ Bottino R Casu A Campanile N Cooper DK Rapid loss of intraportally transplanted islets: An overview of pathophysiology and preventive strategies.Xenotransplantation. 2007; 14: 288-297Crossref PubMed Scopus (136) Google Scholar). Quantification of serum human IL-6 (Quantikine ELISA; R&D Systems, Minneapolis MN) and SC5b-9 (complement complex) Plus Enzyme Immunoassays (Quidel, San Diego, CA) was carried out. Monkeys were followed for 6–12 months or until graft failure. Graft function was defined by detectable fasting porcine C-peptide with no need for exogenous insulin to maintain blood glucose levels <200 mg/dL or with exogenous insulin dose <50% of pre-Tx requirement. Fasting blood glucose levels were measured daily and C-peptide weekly. The livers and pancreases of all recipients were examined at necropsy. Additionally, specimens from the 3-4-5 GE donor pancreases prior to islet isolation and from transgenic hCD46 pigs used as donors in our previous study (18van der Windt DJ Bottino R Casu A et al.Long-term controlled normoglycemia in diabetic non-human primates after transplantation with hCD46 transgenic porcine islets.Am J Transplant. 2009; 9: 2716-2726Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar) were examined. Tissue sections were fixed in both 10% buffered formalin and 4% paraformaldehyde. Formalin-paraffin-embedded sections were stained with hematoxylin/eosin, using standard procedures. Paraformaldehyde-fixed tissues were used for immunofluorescence analysis. Primary and secondary antibodies are listed in Table 4. Nuclear staining was done with TO-Pro-3 iodide (Molecular Probes, Eugene, OR).Table 4:Antibodies for immunocytochemistry and immunofluorescenceAntibodyDilutionSupplierImmunocytochemistryRabbit anti-proinsulin1:100Scytek Laboratories (Logan, UT)Rabbit anti-glucagon1:50Zymed (San Francisco, CA)ImmunofluorescencePrimaryMouse anti-human CD461:100Thermo Fisher Scientific (Fremont, CA)Mouse anti-human CD31:20BD Pharmingen (San Jose, CA)Rabbit anti-human C4d1:20EMELCA Bio-science (Bergen op Zoom, the Netherlands)Goat anti-human lgG1:1000Kirkegaard & Perry (Gaithersburg, MD)Rabbit or mouse anti-insulin1:100Santa Cruz Biotechnology (Santa Cruz, CA)Goat anti-glucagon1:50Santa Cruz BiotechnologyRabbit anti-human TFPI1:10American Diagnostic (Hauppauge, NY)Mouse anti-CD1521:10Serotec (Raleigh, NC)SecondaryGoat anti-mouse Cy31:500Jackson ImmunoResearch (West Grove, PA)Goat anti-rabbit Cy31:500Jackson ImmunoResearchDonkey anti-goat Cy31:500Jackson ImmunoResearchGoat anti-rabbit Alexa 4881:500Molecular Probes (Eugene, OR)Goat anti-mouse Alexa 4881:500Molecular ProbesTFPI, tissue factor pathway inhibitor. Open table in a new tab TFPI, tissue factor pathway inhibitor. Images were captured by a Photometrics Cool SNAP digital camera (Roper Scientific, Tucson, AZ) and Nikon C1 confocal system at 40× objective lens (Nikon Instruments, Melville, NY), and analyzed by MetaMorph imaging analysis software (Molecular Devices, Downington, PA). Photographs were taken through a Nikon Eclipse E800 microscope (Nikon Instruments). Cultured porcine pancreatic cells enriched in islets (50–60% islets/whole tissue) were dissociated into single-cell suspensions by gentle agitation in 0.25% trypsin-EDTA (Invitrogen, Carlsbad, CA). The suspension was filtered through a 100 µm nylon cell strainer (Becton Dickinson) to" @default.
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• W1960949488 date "2014-10-01" @default.
• W1960949488 modified "2023-10-13" @default.
• W1960949488 title "Pig-to-Monkey Islet Xenotransplantation Using Multi-Transgenic Pigs" @default.
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