No transmission of porcine endogenous retrovirus after transplantation of adult porcine islets into diabetic nude mice and immunosuppressed rats

BACKGROUND: The aim of this study was to investigate whether transmission of porcine endogenous retrovirus (PERV) occurs in a model of diabetes reversal by the xenotransplantation of adult porcine islets (APIs) into immunoincompetent diabetic rodents. METHODS: Black-6 nu/nu mice and Lewis rats were immunosuppressed with cyclosporin A (CsA) and FTY 720, and rendered diabetic with streptozotocin. Purified APIs were transplanted into the renal subcapsular space; 5,000 islet equivalents (IEQs) were used in the nude mice (n = 4) and 40,000 IEQs in the rats (n = 4). The nude mice were sacrificed at 75 days after transplantation. In order to confirm chronic xenograft function, the graft-bearing kidney was removed prior to sacrifice. The rats were followed until xenograft rejection, at which time they were sacrificed. Immediately after sacrifice, tissue samples (liver, spleen, and small intestine) were taken for analysis. Quantitative polymerase chain reaction (PCR) was used to assess evidence of PERV transmission, and porcine cell chimerism. RESULTS: All animals became normoglycemic within 48 h of transplantation. The nude mice remained normoglycemic during the 75-day study period, with removal of the graft-bearing kidney resulting in prompt hyperglycemia. The rats remained normoglycemic until xenograft rejection, which occurred at 66 +/- 28 days. Despite the evidence of porcine cell microchimerism in recipients, real-time PCR detected no evidence of PERV transmission in any of the tissue specimens tested. CONCLUSIONS: There was no evidence of PERV transmission following transplantation of pig islets into diabetic nude mice and immunosuppressed rats.     

Delayed functional maturation of neonatal porcine islets in recipients under strict glycemic control

BACKGROUND: The aim of this study was to compare the functional maturation of neonatal porcine islet (NPI) grafts exposed to long-term hyperglycemia with those implanted under euglycemic conditions. METHODS: mice Neonatal porcine islets were transplanted under the left renal capsule of diabetic SCID mice (group H), or in diabetic SCID mice who were also implanted with 500 BALB/c islets under the right renal capsule (group N). On day 42, the right kidneys were removed in both groups. RESULTS: No animals in group H achieved euglycemia within 3 weeks after transplantation. Thus, these mice were exposed to long-term hyperglycemia. Mice in group N became euglycemic immediately after transplantation, however after removal of BALB/c grafts on day 42 they exhibited significantly higher blood glucose levels than in group H and showed glucose intolerance after glucose administration. Cellular insulin content of NPI grafts harvested on day 58 or 72 was significantly lower in group N mice compared to group H. CONCLUSIONS: These results suggest that tight control of glycemia reduces the functional maturation of NPI grafts.     

HMGB1 release in co-cultures of porcine endothelial and human T cells

High mobility group box-1 (HMGB1) protein, primarily from the nucleus, is released into the extracellular milieu either passively by necrotic or damaged cells, or actively by secretion from monocytes/macrophages. Extracellular HMGB1 acts as a potent inflammatory stimulator by promoting cytokine (for example, tumor necrosis factor-alpha) production, and also has pro-coagulant activity. The signaling pathway initiated by receptor for advanced glycation end-product (RAGE), which is the HMGB1 receptor, also induces complement activation. Recent studies have implicated HMGB1 in acute cardiac allograft rejection, and have identified infiltrating T cells and other damaged cells as its main sources. HMGB1 blockade using the anti-HMGB1 antibody HMGB1 box-A (amino-terminal region) and soluble RAGE rescues mice from acute rejection. We therefore studied the release of HMGB1 in co-cultures of porcine aortic endothelial cells (PAEC) and human leukocytes. Human T cells, but not B cells, monocytes or neutrophils, stimulated significant HMGB1 release in culture with PAEC; this activity required cell-cell contact and was dose-dependent, as determined by Western blotting. The released HMGB1 originated from both cell types, as immunofluorescent microscopy showed that it was present in the cytosol of PAEC in contact with T cells, and had disappeared from the T-cell nuclei. These results demonstrate that direct interactions between PAEC and T cells might be a key factor in triggering HMGB1 release, which suggests that HMGB1 is associated with graft rejection in the early phase.     

Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation

BACKGROUND: The long-term viability and function of transplanted encapsulated neonatal porcine islets was examined in a diabetic patient. METHODS AND RESULTS: A 41-yr-old Caucasian male with type 1 diabetes for 18 yr was given an intraperitoneal transplant of alginate-encapsulated porcine islets at the dose of 15,000 islet equivalents (IEQs)/kg bodyweight (total dose 1,305,000 IEQs) via laparoscopy. By 12 weeks following the transplant, his insulin dose was significantly reduced by 30% (P = 0.0001 by multiple regression tests) from 53 units daily prior to transplant. The insulin dose returned to the pre-transplant level at week 49. Improvement in glycaemic control continued as reflected by total glycated haemoglobin of 7.8% at 14 months from a pre-transplant level of 9.3%. Urinary porcine C-peptide peaked at 4 months (9.5 ng/ml) and remained detectable for 11 months (0.6 ng/ml). The patient was followed as part of a long-term microbiologic monitoring programme which subsequently showed no evidence of porcine viral or retroviral infection. At laparoscopy 9.5 yr after transplantation, abundant nodules were seen throughout the peritoneum. Biopsies of the nodules showed opacified capsules containing cell clusters that stained as live cells under fluorescence microscopy. Immunohistology noted sparse insulin and moderate glucagon staining cells. The retrieved capsules produced a small amount of insulin when placed in high glucose concentrations in vitro. An oral glucose tolerance test induced a small rise in serum of immuno-reactive insulin, identified as porcine by reversed phase high pressure liquid chromatography. CONCLUSION: This form of xenotransplantation treatment has the potential for sustained benefit in human type 1 diabetics.     

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Executive summary

The International Xenotransplantation Association has updated its original “Consensus Statement on Conditions for Undertaking Clinical Trials of Porcine Islet Products in Type 1 Diabetes,” which was published in Xenotransplantation in 2009. This update is timely and important in light of scientific progress and changes in the regulatory framework pertinent to islet xenotransplantation. Except for the chapter on “informed consent,” which has remained relevant in its 2009 version, all other chapters included in the initial consensus statement have been revised for inclusion in this update. These chapters will not provide complete revisions of the original chapters; rather, they restate the key points made in 2009, emphasize new and under‐appreciated topics not fully addressed in 2009, suggest relevant revisions, and communicate opinions that complement the consensus opinion. Chapter 1 provides an update on national regulatory frameworks addressing xenotransplantation. Chapter 2 a, previously Chapter 2, suggests several important revisions regarding the generation of suitable source pigs from the perspective of the prevention of xenozoonoses. The newly added Chapter 2b discusses conditions for the use of genetically modified source pigs in clinical islet xenotransplantation. Chapter 3 reviews porcine islet product manufacturing and release testing. Chapter 4 revisits the critically important topic of preclinical efficacy and safety data required to justify a clinical trial. The main achievements in the field of transmission of all porcine microorganisms, the rationale for more proportionate recipient monitoring, and response plans are reviewed in Chapter 5. Patient selection criteria and circumstances where trials of islet xenotransplantation would be both medically and ethically justified are examined in Chapter 6 in the context of recent advances in available and emerging alternative therapies for serious and potentially life‐threatening complications of diabetes. It is hoped that this first update of the International Xenotransplantation Association porcine islet transplant consensus statement will assist the islet xenotransplant scientific community, sponsors, regulators, and other stakeholders actively involved in the clinical translation of islet xenotransplantation.     

Long‐term culture and in vitro maturation of macroencapsulated adult and neonatal porcine islets

Encapsulated porcine islets could be used to treat type I diabetes without necessitating severe immunosuppression. Islet survival and secretory function in the encapsulation device need to be preserved to ensure efficient insulin output in response to surrounding stimuli. In the present study, we evaluated stimulated insulin secretion from adult and neonatal pig islets seeded on an acellular collagen matrix and encapsulated in alginate during long‐term culture. Pig islets survived longer and secreted more insulin when cultured on acellular porcine dermis compared to human fascia. Islets from neonatal pigs could survive up to 33 weeks in vitro, and their insulin secretion increased during the first 5 weeks of culture in a beta‐cell maturation medium. In fact, by the 4th week of culture, insulin secretion from neonatal islets attained the same level as adult islets and even surpassed it by the 18th week. Our results show that in vitro maturation of encapsulated neonatal porcine islets is possible and can actually compensate the initial low insulin secretion from these islets while allowing enough time to perform complete functional and biosafety characterization of islets before transplantation.     

ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro

Paris LL, Chihara RK, Reyes LM, Sidner RA, Estrada JL, Downey SM, Milgrom DA, Joseph Tector A, Burlak C. ASGR1 expressed by porcine enriched liver sinusoidal endothelial cells mediates human platelet phagocytosis in vitro. Xenotransplantation 2011; 18: 245–251. © 2011 John Wiley & Sons A/S. Abstract: Background: Porcine liver xenografts represent a potential solution to the organ shortage, but thrombocytopenia occurs within minutes to hours after xenotransplantation, preventing clinical application. Recently, it was discovered that porcine liver sinusoidal endothelial cells (LSEC) bind and phagocytose human platelets. We examined the role of ASGR1 in binding and removing human platelets by the pig liver endothelium. Methods: Primary porcine enriched LSEC (eLSEC) were characterized by flow cytometry, immunoblot, quantitative PCR, and immunohistochemistry using confocal microscopy. Phagocytosis inhibition assays using anti‐ASGR1 and an ASGR1 substrate were performed. ASGR1 was targeted for siRNA knockdown, and ASGR1‐reduced cells were tested for human platelet binding and phagocytosis. Results: ASGR1 is expressed by eLSEC. Human platelet binding and phagocytosis by porcine eLSEC was inhibited by asialofetuin, but not fetuin, suggesting an interaction with galactose β1‐4 N‐acetyl glucosamine. Anti‐ASGR1 antibodies inhibited human platelet binding in a dose‐dependent manner. Knockdown experiments using siRNA reduced ASGR1 expression in asynchronous primary eLSEC by 40%–80%. There was a 20% reduction in translated protein significantly correlated with a 21% decrease in human platelet binding. Conclusions: ASGR1 on porcine eLSEC mediates phagocytosis of xenogeneic platelets.     

Engraftment of Adult Porcine Islet Xenografts in Diabetic Nonhuman Primates Through Targeting of Costimulation Pathways

Recent advances in human allogeneic islet transplantation have established beta-cell replacement therapy as a potentially viable treatment option for individuals afflicted with Type 1 diabetes. Two recent successes, one involving neonatal porcine islet xenografts transplanted into diabetic rhesus macaques treated with a costimulation blockade-based regimen and the other involving diabetic cynomolgus monkeys transplanted with adult porcine islet xenografts treated with an alternative multidrug immunosuppressive regimen have demonstrated the feasibility of porcine islet xenotransplantation in nonhuman primate models. In the current study, we assessed whether transplantation of adult porcine islet xenografts into pancreatectomized macaques, under the cover of a costimulation blockade-based immunosuppressive regimen (CD28 and CD154 blockade), could correct hyperglycemia. Our findings suggest that the adult porcine islets transplanted into rhesus macaques receiving a costimulation blockade-based regimen are not uniformly subject to hyperacute rejection, can engraft (2/5 recipients), and have the potential to provide sustained normoglycemia. These results provide further evidence to suggest that porcine islet xenotransplantation may be an attainable strategy to alleviate the islet supply crisis that is one of the principal obstacles to large-scale application of islet replacement therapy in the treatment of Type 1 diabetes.     

Effects of collagenase concentration on the purity and viability of isolated porcine pancreatic islets for use in xenotransplantation studies

Abstract: The use of lower concentrations of collagenase (0.8 mg/ml as compared to 1.25 and 1.5 mg/ml) resulted in isolation of porcine pancreatic islets retaining their original compact, wholesome appearance. In in vitro experiments, 100% of islet batches isolated with the low collagenase concentration responded to glucose stimulation, while only 63.1% of islet batches isolated with the high concentration of the enzyme did. The response to high glucose challenge of islets isolated with the low enzyme concentration was considerably higher compared to high collagenase concentration islets.
In in vivo experiments, intraperitoneal transplants of microencapsulated islets isolated using 0.8 mg/ml of collagenase reversed diabetes in diabetic BALB-c mice in a significantly larger proportion of recipients (87.5%) as compared to 33.3% of recipients grafted with 1.5 mg/ml collagenase islets.
The results of this study demonstrate that the use of a low collagenase concentration is conducive to isolation of islets of a superior morphology, purity, viability, and functioning ability.     

Downregulation of Gabarapl1 significantly attenuates antibody binding to porcine aortic endothelial cells

After hyperacute rejection in pig‐to‐primate xenotransplantation had been overcome by the introduction of α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs, acute and chronic antibody‐mediated rejection became one of the major barriers to long‐term graft survival. This was associated with exposure of non‐Gal antigens to the recipient's immune system and indicated that further genetic engineering of the pigs would be necessary. We here report that Gabarapl1, a regulator of tumorigenesis, plays a role in the regulation of immunogenicity of porcine aortic endothelial cells (PAECs). Knockdown of Gabarapl1 in PAECs results in a remarkable reduction in binding of serum antibody from PAEC‐immunized monkeys, associated with decreased serum cytotoxicity of pig cells. Expression of swine leukocyte antigens (SLA) II DR was downregulated by Gabarapl1 knockdown. However, suppression of expression of SLA II is associated with less reduction of antibody binding than achieved by Gabarapl1 knockdown, suggesting that other Gabarapl1‐regulated xenoantigens may be more important. These findings indicate a hitherto unknown relationship between Gabarapl1 and xenoimmunogenicity, suggesting a potential new strategy to reduce rejection initiated by the presence of non‐Gal antigens.     

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes – Chapter 3: Porcine islet product manufacturing and release testing criteria

In the 2009 IXA consensus, the requirements for the quality and control of manufacturing of porcine islet products were based on the U.S. regulatory framework where the porcine islet products fall within the definition of somatic cell therapy under the statutory authority of the U.S. Food and Drug Administration (FDA). In addition, porcine islet products require pre‐market approval as a biologic product under the Public Health Services Act and they meet the definition of a drug under the Federal Food, Drug, and Cosmetic Act (FD&C Act). Thus, they are subject to applicable provisions of the law and as such, control of manufacturing as well as reproducibility and consistency of porcine islet products, safety of porcine islet products, and characterization of porcine islet products must be met before proceeding to clinical trials. In terms of control of manufacturing as well as reproducibility and consistency of porcine islet products, the manufacturing facility must be in compliance with current Good Manufacturing Practices (cGMP) guidelines appropriate for the initiation of Phase 1/2 clinical trials. Sponsors intending to conduct a Phase 1/2 trial of islet xenotransplantation products must be able to demonstrate the safety of the product through the establishment of particular quality assurance and quality control procedures. All materials (including animal source and pancreas) used in the manufacturing process of the porcine islet products must be free of adventitious agents. The final porcine islet product must undergo tests for the presence of these adventitious agents including sterility, mycoplasma (if they are cultured), and endotoxin. Assessments of the final product must include the safety specifications mentioned above even if the results are not available until after release as these data would be useful for patient diagnosis and treatment if necessary. In addition, a plan of action must be in place for patient notification and treatment in case the sterility culture results are positive. In terms of the characterization of porcine islet products and product release criteria, the information on the porcine islet products should be acquired from a sample of the final product to be used for transplantation and must include the morphology of the islets, specific identity, purity, viability, and potency of the product. In addition, information on the quantity of the islet products should also be provided in a standardized fashion and this should be in terms of islet equivalents and/or cell numbers. The current consensus was created to provide guidelines that manufacturing facilities may find helpful in the manufacture of and the release criteria for porcine islet products including encapsulated islets and combined islet products. Our intent with the above recommendations is to provide a framework for individual porcine islet manufacturing facilities to ensure a high level of safety for the initiation of Phase 1/2 clinical trials on porcine islet xenotransplantation.