key: cord-313072-8ndt7a2g
authors: Gazda, Lawrence S.; Collins, James; Lovatt, Archie; Holdcraft, Robert W.; Morin, Merribeth J.; Galbraith, Daniel; Graham, Melanie; Laramore, Melissa A.; Maclean, Christine; Black, John; Milne, Euan W.; Marthaler, Douglas G.; Vinerean, Horatiu V.; Michalak, Michelle M.; Hoffer, Deborah; Richter, Steven; Hall, Richard D.; Smith, Barry H.
title: A comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials
date: 2016-11-11
journal: Xenotransplantation
DOI: 10.1111/xen.12277
sha: 
doc_id: 313072
cord_uid: 8ndt7a2g

BACKGROUND: The use of porcine islets to replace insulin‐producing islet β‐cells, destroyed during the diabetogenic disease process, presents distinct challenges if this option is to become a therapeutic reality for the treatment of type 1 diabetes. These challenges include a thorough evaluation of the microbiological safety of the islets. In this study, we describe a robust porcine islet‐screening program that provides a high level of confidence in the microbiological safety of porcine islets suitable for clinical trials. METHODS: A four‐checkpoint program systematically screens the donor herd (Large White – Yorkshire × Landrace F1 hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. Molecular assays screen for more than 30 known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. RESULTS: Of 1207 monthly samples taken from random animals over a 2‐year period, only a single positive result for Transmissible gastroenteritis virus was observed, demonstrating the high level of biosecurity maintained in the source herd. Given the lack of clinical signs, positive antibody titers for Porcine reproductive and respiratory syndrome virus, Porcine parvovirus, and Influenza A confirm the efficacy of the herd vaccination program. Porcine respiratory coronavirus was found to be present in the herd, as expected for domestic swine. Tissue homogenate samples from six sentinel and 11 donor animals, over the same 2‐year period, were negative for the presence of viruses when co‐cultured with six different cell lines from four species. The absence of adventitious viruses in separate islet macrobead preparations produced from 12 individual pancreas donor animals was confirmed using validated molecular (n = 32 viruses), in vitro culture (cells from four species), and transmission electron microscopy assays (200 cell profiles per donor animal) over the same 2‐year period. There has been no evidence of viral transmission following the implantation of these same encapsulated and functional porcine islets into non‐immunosuppressed diabetic cynomolgus macaques for up to 4 years. Isolated peripheral blood mononuclear cells from all time points were negative for PCV (Type 2), PLHV, PRRSV, PCMV, and PERV‐A, PERV‐B, and PERV‐C by PCR analysis in all six recipient animals. CONCLUSION: The four‐checkpoint program is a robust and reliable method for characterization of the microbiological safety of encapsulated porcine islets intended for clinical trials.

The ultimate goal for the treatment of type 1 diabetes is the restoration of physiological blood glucose regulation without the requirement for lifelong therapy such as immunosuppression or immunoregulation. There are numerous challenges for the attainment of this goal, and diverse approaches ranging from insulin pumps to gene therapy to stem cell strategies are all being pursued. [1] [2] [3] [4] [5] In another approach, the replacement of lost islet cells through human islet allotransplantation has established the clinical utility of islet grafting. 6, 7 Although only 13% of human islet recipients remain insulin-independent at 5 years, the majority of patients (67%) retain some graft function as evidenced by persistent C-peptide secretion. 8 Importantly, islet transplantation largely eliminates hypoglycemic unawareness post-grafting. Further improvement in long-term insulin independence is likely; data from a few select Methods: A four-checkpoint program systematically screens the donor herd (Large White -Yorkshire × Landrace F1 hybrid animals), individual sentinel and pancreas donor animals and, critically, the islet macrobeads themselves. Molecular assays screen for more than 30 known viruses, while electron microscopy and in vitro studies are employed to screen for potential new or divergent (emergent) viruses. Organization institutions indicate that an increasing number of islet recipients (approaching 50%) are able to maintain external insulin independence for 5 years. 9 Nonetheless, the availability of suitable human pancreases for either whole organ or isolated islet transplantation is entirely inadequate as evidenced by the recovery of only 1293 donor pancreases in the United States in 2015. 10 The use of xenogeneic islets could provide a solution to the limited supply of human islets for transplantation. Porcine islets are particularly suitable as a source of insulin-producing cells because porcine insulin differs from human insulin by only a single amino acid and porcine insulin has been shown to provide reliable and safe glycemic control in human diabetic patients. Furthermore, only the transplantation of whole pancreas or intact islets of Langerhans can be expected to replicate normal and precise physiological glucose control, which is dependent on a variety of cell types and proteins from pancreatic islets. This enormously complex regulatory machinery is absent in all other approaches to the re-establishment of normoglycemia.

The use of animal-derived islet tissue, however, will necessitate either suppression of the patient's immune system or the physical isolation of the islets to prevent their rejection. We have previously described the ability of agarose-agarose encapsulated porcine islets to function in diabetic animal models. [11] [12] [13] [14] [15] These encapsulated islet macrobeads are at least partially immune isolated as an outer layer of dense agarose prohibits direct cell access to the xenogeneic islets.

Although small immune mediators, such as cytokines, can still enter the macrobead, this encapsulation approach has been sufficiently robust to allow spontaneously diabetic BB rats to survive for more than 6 months without immunosuppression and without exogenous insulin administration. 13 Ongoing studies in non-immunosuppressed diabetic cynomolgus macaques also demonstrate porcine islet macrobead function for more than 6 months in 5 of 6 animals (unpublished data, see Checkpoint 4 below).

The clinical use of islets from animals also requires a well-thoughtout strategy to assure the microbiological safety of the animal tissue.

A strategy to provide an aseptic product and to reduce the probability of inadvertent transmission of adventitious viruses is critical to the successful implementation of this exciting therapy. Our approach takes advantage of the unique aspects of the islet macrobead, namely its long-term viability in culture and lack of patient immunosuppression, to limit the microbiological safety risk of porcine islets for transplantation. Essential to our approach is the establishment of four checkpoints that span the macrobead production and transplantation processes. The checkpoint approach is built upon an increasing level of microbiological screening from the donor herd up to the islet product, using multiple assay formats with the ability to screen for known and unknown pathogens. Using the checkpoint approach, we provide evidence to support the use of donor animals housed in high hygienic but not HEPA-filtered environments, not only for islet xenotransplantation but also other xeno-cell therapies. In this study, we present and document the effectiveness of our microbiological safety strategy to minimize the microbiological risks of agarose encapsulated porcine islets for the treatment of type 1 diabetes.

Donor pigs were Large White -Yorkshire × Landrace F1 hybrid animals obtained from well-characterized, non-genetically modified Choice Genetics (formerly Newsham) source animals. The Source

Animal Facility is an independently owned herd located in rural Southwest Ohio. The breeding herd numbers approximately 2000 sows and utilizes a two-tiered quarantine testing system through a modern multisite production strategy. Three segregated facilities (nursery, gilt isolation, and farrowing and gestation) maintain data records and follow standard operating procedures (SOPs) for animal husbandry, vaccination, housing, cleaning and waste management, and biosecurity measures. Controlled transportation of animals, supplies and semen, feeding and watering, pest and rodent control, gravel barrier around facility perimeter, and caretaker qualifications including routine health assessments are all components of the comprehensive biosecurity program. Routine Quality Assurance site audits are used to verify compliance. Corn-based pig feed was produced exclusively with corn grown and ground onsite at the source animal facility. Gilts, gestation sows, and lactating sows were given the same base feed, consisting of corn with various supplements (eg, amino acids, vitamins, minerals, protein), with slight variations based on the breeding status of the animal. All ingredients used in the production of complete feed were certified to be free of any restricted use protein products as de- The facility holds Pork Quality Assurance Plus (PQA Plus ® ) certification, which is a producer-driven program to ensure U.S. pork products are of the highest quality and safe. 16 The PQA Plus program mandates Good Production Practices with an emphasis on biosecurity and practices to reduce the risk of introducing and spreading of infectious agents. All retired breeding sows not used as pancreas donors are sent to a local abattoir for pork products.

Source animals are routinely vaccinated for various agents ( Quarterly sentinel 

Following electrical stun and exsanguination at a USDA-regulated abattoir in compliance with all federal regulations concerning animal handling and welfare, numerous tissues including brain, heart, lung, liver, tonsil, lymph node, spleen, ileum, kidney, pancreas, bone marrow, PBMCs, feces, and serum were collected and archived as fixed and frozen aliquots at the time of pancreas procurement. Aliquots of isolated islets were also frozen and archived. Donor animal tissue samples were sent to the University of Minnesota, Veterinary Diagnostic Laboratory

(MVDL) to screen for the bacterial and viral pathogens listed in Table 3 .

The MVDL is one of the three major swine diagnostic laboratories 

Donor pancreases were procured from sows that were over 2 years of age with a history of multiple parities. Following electrical stunning and exsanguination of the donor, abdominal viscera were retrieved by a sterile-gowned technician and transferred to a sterile container for transport. The viscera were then placed in a custom built, HEPAfiltered, laminar flow work station for tissue retrieval. Surface monitoring was performed on the work surface prior to retrieval, and settle plates were placed inside the workstation during dissection to monitor for microbiological contamination. Intact pancreas was dissected from the viscera by a sterile-gowned technician and immediately to provide absolute viral sequestration although the encapsulated islets are protected from direct immune cell contact. Agarase, the enzyme necessary to break down agarose, is abundant in ocean-dwelling bacteria but has not been found in mammals. 19 The macrobeads, in the absence of trauma, remain intact indefinitely following implantation in the abdominal cavity.

Macrobeads were then cultured at 37°C in a humidified atmosphere of 5% CO 2 until collection for microbiological screening or implanta- , a colon, ileum, liver, spleen, lung, and mesenteric lymph node, b coronavirus (n = 1), caudovirales (n = 1), c small round viral particle, d dilution factor (df): 10, e kidney, lung, mesenteric lymph node, tonsil, and spleen, f Testing performed at National Veterinary Service Laboratory, g bacteriophage, h df: 512 (n = 1), 1024 (n = 2), i df:1024 (n = 2), 2048 (n = 2), 8192 (n = 2), ≥8192 (n = 1), j df: 1024 (n = 1), 2048 (n = 1), 4056 (n = 1), k df: 512 (n = 2), 2048 (n = 2), l df: 20 (n = 1), 40 (n = 1), m df: 40 (n = 1), 320 (n = 1), 640 (n = 1), n heart, intestine, liver, pancreas, kidney, lung, mesenteric lymph node, tonsil, and spleen. NY, USA; pre-screened for endotoxin <0.03 EU/mL and for sterility) containing 11 mmol/L glucose, 2.5% heat-inactivated porcine serum (Biologos, Montgomery, IL, USA; pre-screened for sterility, and the absence of mycoplasma and adventitious viruses per 9CFRs and endotoxin <50 EU/mL) and 1% antibiotic/anti-mycotic (Life Technologies) was changed weekly and 24-hour post-change media samples were taken for porcine insulin ELISA assays (Mercodia, Uppsala, Sweden).

Islet isolation and encapsulation procedures were performed in Class II biosafety cabinets within an ISO Class 5 (at rest) and ISO Class 7

(active processing) laboratory by sterile-gowned technicians. Routine environmental monitoring included particle counts, viable particle counts, settling plates, and surface monitoring and was performed for every islet isolation procedure. Macrobeads from a given donor were assigned a unique islet isolation number and cultured separately from islet macrobeads produced from other donor animals. 

Prior to islet macrobead implantation, representative samples of macrobeads were sent for sterility testing per USP <71> 22 

Six non-immunosuppressed streptozotocin-induced diabetic male cynomolgus macaques of Mauritian or Asian origin with a median age of 

Multiple checkpoints during the process of porcine islet isolation, encapsulation, culture, and transplantation were implemented to assess the microbiological safety of islet macrobeads (Figure 2 ).

The Step: each test sample is spiked with an internal extraction control (IEC; plant RNA or DNA) at the detection limit, mimicking the target nucleic acid. This process allows the level of nucleic acid recovery in the presence of the test sample to be assessed, which ensures that low levels of RNA or DNA can be recovered. 

Given evidence of satisfactory insulin production by cultured islet macrobeads (≥75 mU/macrobead/24-hour), the macrobeads were sent for viral screening to SGS Vitrology prior to use in pre-clinical animal studies. All islet macrobeads were negative for viruses screened by PCR (n = 32 viruses) and by co-culture assays using four different susceptible cell lines (n = 12 pancreas donor animals; Table 4 ).

Transmission scanning electron microscopy was incorporated as an (Table 4 and Figure 3 ).

were negative for bacterial growth and for the presence of endotoxin and mycoplasma (Table 4 ).

Although not a source material or product release screen, recipient monitoring post-macrobead implantation provides valuable data that could be used to not only treat individual patients but also as a check (Table 5) . Moreover, there has been no evidence of viral infection as observed and documented in the health status of the animals.

The four-checkpoint microbiological safety program outlined utilizes four separate assessments for the microbiological safety of agarose [40] [41] [42] [43] [44] [45] [46] [47] The focus of all these guidelines, including the IXA statements, is on the safety of xenotransplantation. 30, 33 An invited review of US xenogeneic regulations was published by FDA staff in 2010 and provides additional details on FDA guidelines. 48 Schuurman has more recently published a thorough overview of worldwide xenogeneic regulations with an emphasis on the European requirements. 49 F I G U R E 3 Transmission electron microscopy of islet cell profiles, typical of those present in the sample of cells examined. The islet cells were generally adherent with intercellular connections, cell junctions, and desmosomes observed. The cell surface appearances varied, smooth surfaces, blebs, and cell processes were observed. Various cell types were present, including α and β cells. No mitotic cells were observed by light or electron microscopy. No significant numbers of dead or dying cells were present in the population of cells examined. Nuclear profiles varied in size. In some cells, more than one nuclear profile was visible. They were often indented and invaginated or contained lacunae of cytoplasmic material. Nucleoli were prominent and sometimes more than one was observed. Heterochromatin was abundant, clumped, and found on the inner nuclear membrane and sporadically throughout the nucleus. The cells had a normal range of organelles. The mitochondria were numerous and varied in size and shape. The presence of cristae was noted although no matrix granules were seen. The Golgi body, when observed, was prominent and exhibited stacked cisternae. The rough-surfaced endoplasmic reticulum was extensive and occurred in varied lengths. Lipid bodies, peroxisomes, and multivesicular bodies were present. Free ribosomes, polysomes, and fibrils were found throughout the cytoplasm. Vacuoles, with electron dense and flocculent material, were observed. Coated, un-coated, and secretory vesicles, of varying types, were seen. Centrioles and microtubules were present. The cell structure is consistent with the morphology expected of secretory cells. No viruses, virus-like particles or extraneous agents, including mycoplasmas, yeasts, fungi, or bacteria, were found. Abbreviations: f, fibrils; mitochondria (m); desmosome (arrow); centriole (c); nucleus (Nu); Golgi bodies (g), lipid bodies (l), vacuoles (v), multivesicular body (mvb), and rough-surfaced endoplasmic reticulum (rER)

Our microbiological safety program is in-line with the issues for consideration in the FDA xenotransplantation guideline, as well as the IXA statements. In regard to donor animals, it is important to note that the guidelines do not state that source animals must be raised in HEPA-filtered environments. This is significant because, in addition to the difficulty of obtaining and maintaining pathogen-free swine, [50] [51] [52] there is no guarantee that the pancreas from individual animals will be suitable for islet isolation. In fact, it is becoming increasing clear that only about 25% of porcine pancreases yield a sufficient number of islets for clinical use. 18, [53] [54] [55] Given these limitations, our approach is to raise source animals in high hygienic environments, biopsy individual pancreases for islet isolation suitability and perform extensive microbiological screening of all selected donor animal tissues and the islet macrobeads themselves.

Our methodology, however, employs an alternative approach to the FDA-recommended 21-day quarantine period for individual donor animals that deserves some discussion. A minimum 7-day quarantine period for animals used in the processing of biological products is also stated in 21 CFR part 600. 56 The rationale for the quarantine period is to provide time for an acute infection to become clinically apparent. 57 but not as reliably in peripheral blood cells. 58 Thus, it is the islet macrobeads themselves, that is, the tissue to be implanted, that undergoes quarantine and it is the ability to thoroughly screen the islets using a wide variety of specific and non-specific assays that obviates the need to raise or quarantine donor animals in HEPA-filtered environments.

As discussed by 

Time points evaluated were: 1 month (±7 days) and 6 months (±15 days) post-transplant. Additionally, samples from 1 year (±60 days), 2 years (±60 days), 3 years (±60 days) and 4 years (±60 days) posttransplant were evaluated in a subset of recipients who reached these time points during extended follow-up.

T A B L E 5 PCR screening of implanted non-human primates treatment options in the case of suspected pathogen transmission but also manufacturing decisions as well as feedback to Checkpoints 1-3 that may result in the modification of one or more checkpoints.

As with any medical procedure, there will always be risk with the transplantation of porcine islets. The challenge we face is to understand those risks such that they can be minimized to an acceptable level in light of the expected clinical benefits. The potential risk of viral transfer from the transplanted cells to the recipient has been the major safety concern. To address this issue, a testing rationale was developed to screen sentinel and donor animals for viruses that are present in the herd, are of special interest such as known zoonotic agents, or are known to infect swine in the USA. An initial characterization of the PERV infectivity status of the source herd was not performed given the unlikely ability, at least at that time, to eliminate these endogenous retroviruses and the growing conclusion that the risk of PERV transmission to human xenotransplantation trial participants is low. 60 Additionally, co-culture and electron microscopy assays are employed to look for unknown viruses, which would then prompt efforts to identify the virus as either a known or unknown agent (eg, PRRSV inducing cpe in MA104 cells as one component of the islet macrobead screen).

Also, as part of their release criteria, the islet macrobeads are confirmed to be sterile and free from mycoplasma and any significant level of en- culture. This sequence was not found in purified islets isolated in the absence of porcine serum. Because we also detected the sequence in domestic and international porcine serum sources, PHoV screening of islet macrobeads cultured in these sera was postponed until a suitable PHoV-free source could be obtained.

With regard to PERV, the identification of this virus and its ability to cross the species barrier has been the principal concern with porcine xenotransplantation. It is worth mentioning that the NHP is not an ideal model to assess the risk of PERV transmission. Cynomolgus monkeys lack the PERV-A receptor 1 (PAR-1) that is the major receptor for PERV-A and PERV-A/C entry. 66 The actual risk of PERV infection in the clinic remains unclear, but so far there has been no transmission of PERV or other porcine microorganisms in humans that have been exposed to pig islet cells. 63, [67] [68] [69] We consider these findings, as well as the negative viral findings from diabetic dogs exposed to porcine islet macrobeads for 2.4 years, to be evidence that viral transmission following macrobead implantation is very low risk. 15 Studies with D17 canine cells have shown these cells to have the PERV-A receptor, 70 but apparently not to the same degree as human 293 cells. 71 Other, non-microbiological risks to the patient also exist and must be considered as part of the comprehensive safety evaluation. These include procedural risks that accompany pre-and post-implantation screening, as well as the general anesthesia and laparoscopic surgery required to implant the macrobeads. The procedural risks are well known and, although real, are modest. We have been implanting cancer macrobeads (encapsulated mouse renal adenocarcinoma cells) to treat patients with various malignancies for 10 years without adverse events attributable to the macrobeads either as an intraperitoneal irritant or as a microbiological hazard. 78 See also ClinicalTrials.gov Identifiers NCT00283075, NCT01053013, and NCT02046174.

A final important consideration is the immune status of the recipients. Recent progress in clinical islet allotransplantation has confirmed the potential of islet grafting to reduce exogenous insulin administration and the occurrence of hypoglycemic unawareness. 6, 7 While these results are encouraging, numerous adverse events including intrahepatic bleeding, neutropenia, mouth ulcers, anemia, diarrhea, edema, hypercholesterolemia, and pharyngitis have been reported. 79 Most of the reported adverse events are primarily associated with immunosuppressive therapy. Encapsulating porcine islets using the agaroseagarose method eliminates the need for immunosuppressive therapy, and a fully competent immune system is expected to significantly reduce the risk of xenozoonotic infection. 80 During the last 20 years, an understanding of the risks associated with porcine xenotransplantation, and methodologies to manage those risks, including theoretical risks and the potential presence of unknown pathogens, have significantly progressed. The recent IXA consensus update notes that "theoretical risk" still exists in relation to the transmission of infectious agents when employing appropriate safeguards, but that such events will likely be rare should they occur. 44 Although the actual risks of islet xenotransplantation cannot be not known in the absence of clinical trials, as stated by Fishman, "In clinical xenotransplantation, a level of safety has been developed beyond that available for human organs…". 84 

Insulin pump risks and benefits: a clinical appraisal of pump safety standards, adverse event reporting and research needs. A joint statement of the European Association for the Study of Diabetes and the American Diabetes Association Diabetes Technology Working Group

Performance and acceptability of a combined device for insulin infusion and glucose sensing in the home setting

Insulin gene therapy for type 1 diabetes mellitus

Gene therapy with neurogenin3, betacellulin and SOCS1 reverses diabetes in NOD mice

Toward beta cell replacement for diabetes

Achievement of insulin independence in three consecutive type-1 diabetic patients via pancreatic islet transplantation using islets isolated at a remote islet isolation center

Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen

The clinical impact of islet transplantation

Update on islet transplantation. Cold Spring Harb Perspect Med

Glucose control and long-term survival in biobreeding/Worcester rats after intraperitoneal implantation of hydrophilic macrobeads containing porcine islets without immunosuppression

Retrievable, replaceable, macroencapsulated pancreatic islet xenografts. Long-term engraftment without immunosuppression

Encapsulation of porcine islets permits extended culture time and insulin independence in spontaneously diabetic BB rats

Pravastatin improves glucose regulation and biocompatibility of agarose encapsulated porcine islets following transplantation into pancreatectomized dogs

No evidence of viral transmission following long-term implantation of agarose encapsulated porcine islets in diabetic dogs

Pork Quality Assurance Plus Certification

Evidence of pig-to-pig transmission of a modified-live PRRS virus vaccine

The use of pancreas biopsy scoring provides reliable porcine islet yields while encapsulation permits the determination of microbiological safety

Agarase: review of major sources, categories, purification method, enzyme characteristics and applications

Nucleic Acid Amplification Techniques. Section 2.6.21

Nucleic Acid-Based Techniques-Amplification

Diseases of Swine

Application of current statutory authorities to human somatic cell therapy products and gene therapy products. FDA: Federal Register

Source animal, product, preclinical, and clinical issues concerning the use of xenotransplantation products in humans

of the European Parliament and of the Council on advanced therapy medicinal products and amending Directive 2001/83/EC and Regulation (EC) No 726/2004. Official J of the Eur Union

Guideline on Human Cell-Based Medicinal Products. European Medicines Agency: Committee for Medicinal Products for Human Use

European Medicines Agency: Committee for Medicinal Products for Human Use

First WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials. World Health Organization

Second WHO Global Consultation on Regulatory Requirements for Xenotransplantation Clinical Trials. World Health Organization

Test procedures and acceptance criteria for biotechnological/biological products (Q6B)

Quality of biotechnological biological products: derivation and characterization of cell substrates used for production of biotechnological/biological products (Q5D). ICH Harmonised Tripartite Guideline

Safety preclinical evaluation of biotechnology: derived pharmaceuticals for biotechnological product (S6)

Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (Q5A)

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

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes -chapter 1: update on national regulatory frameworks pertinent to clinical islet xenotransplantation

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes -chapter 2a: source pigs-preventing xenozoonoses

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

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

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes. Chapter 4: pre-clinical efficacy and complication data required to justify a clinical trial

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes. Chapter 5: recipient monitoring and response plan for preventing disease transmission

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes. Chapter 6: patient selection for pilot clinical trials of islet xenotransplantation

Regulation of xenogeneic porcine pancreatic islets

Regulatory aspects of clinical xenotransplantation

Serological evidence for Hepatitis E virus infection in laboratory monkeys and pigs in animal facilities in Japan

Microbiological safety of porcine islets: comparison with source pig

Extended microbiological characterization of Gottingen minipigs in the context of xenotransplantation: detection and vertical transmission of Hepatitis E virus

Optimizing porcine islet isolation to markedly reduce enzyme consumption without sacrificing islet yield or function

The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs

Assessment of some porcine strains as donors of islets of Langerhans

Food and Drug Administration

PHS Guideline on infectious disease issues in xenotransplantation. U.S. Food and Drug Administration

A novel porcine gammaherpesvirus

The International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes-chapter 2: source pigs

Xenotransplantation: infectious risk revisited

Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses

Xenotransplantation-associated infectious risk: a WHO consultation

Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand

Identification of novel porcine and bovine parvoviruses closely related to human parvovirus 4

Phylogenetic characterization of the first Ungulate tetraparvovirus 2 detected in pigs in Brazil

Suboptimal porcine endogenous retrovirus infection in non-human primate cells: implication for preclinical xenotransplantation

Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. The XEN 111 Study Group

No evidence of infection with porcine endogenous retrovirus in recipients of encapsulated porcine islet xenografts

No evidence of porcine endogenous retrovirus in patients with type 1 diabetes after long-term porcine islet xenotransplantation

Host range and interference studies of three classes of pig endogenous retrovirus

Extended analysis of the in vitro tropism of porcine endogenous retrovirus

Antigenic relationships among homologous structural polypeptides of porcine, feline, and canine coronaviruses

Recombinant canine coronaviruses related to transmissible gastroenteritis virus of Swine are circulating in dogs

Pseudorabies virus (Aujeszky's disease). Virus Infections of Porcines

Replication and plaque assay of influenza virus in an established line of canine kidney cells

Susceptibility of different cell lines to Avian and Swine Influenza viruses

Emergence of human-like H3N2 influenza viruses in pet dogs in Guangxi

First-in-human phase 1 trial of agarose beads containing murine RENCA cells in advanced solid tumors

Benefits and risks of solitary islet transplantation for type 1 diabetes using steroid-sparing immunosuppression: the National Institutes of Health experience

Infection and xenotransplantation. Developing strategies to minimize risk

Implications of the advent of homozygous alpha l, 3-galactosyltransferase gene-deficient pigs on transmission of infectious agents

Reduced sensitivity to human serum inactivation of enveloped viruses produced by pig cells transgenic for human CD55 or deficient for the galactosyl-alpha(1-3) galactosyl epitope

Porcine endogenous retrovirus transmission characteristics of galactose alpha1-3 galactose-deficient pig cells

Assessment of infectious risk in clinical xenotransplantation: the lessons for clinical allotransplantation

A comprehensive microbiological safety approach for agarose encapsulated porcine islets intended for clinical trials