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Yoon CH, Choi HJ, Kim MK. Corneal xenotransplantation: Where are we standing? Prog Retin Eye Res 2021; 80:100876. [PMID: 32755676 PMCID: PMC7396149 DOI: 10.1016/j.preteyeres.2020.100876] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2020] [Revised: 05/23/2020] [Accepted: 06/04/2020] [Indexed: 02/08/2023]
Abstract
The search for alternatives to allotransplants is driven by the shortage of corneal donors and is demanding because of the limitations of the alternatives. Indeed, current progress in genetically engineered (GE) pigs, the introduction of gene-editing technology by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, and advanced immunosuppressants have made xenotransplantation a possible option for a human trial. Porcine corneal xenotransplantation is considered applicable because the eye is regarded as an immune-privileged site. Furthermore, recent non-human primate studies have shown long-term survival of porcine xenotransplants in keratoplasty. Herein, corneal immune privilege is briefly introduced, and xenogeneic reactions are compared with allogeneic reactions in corneal transplantation. This review describes the current knowledge on special issues of xenotransplantation, xenogeneic rejection mechanisms, current immunosuppressive regimens of corneal xenotransplantation, preclinical efficacy and safety data of corneal xenotransplantation, and updates of the regulatory framework to conduct a clinical trial on corneal xenotransplantation. We also discuss barriers that might prevent xenotransplantation from becoming common practice, such as ethical dilemmas, public concerns on xenotransplantation, and the possible risk of xenozoonosis. Given that the legal definition of decellularized porcine cornea (DPC) lies somewhere between a medical device and a xenotransplant, the preclinical efficacy and clinical trial data using DPC are included. The review finally provides perspectives on the current standpoint of corneal xenotransplantation in the fields of regenerative medicine.
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Affiliation(s)
- Chang Ho Yoon
- Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Republic of Korea; Laboratory of Ocular Regenerative Medicine and Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Biomedical Research Institute, Seoul, Republic of Korea
| | - Hyuk Jin Choi
- Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Republic of Korea; Laboratory of Ocular Regenerative Medicine and Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Biomedical Research Institute, Seoul, Republic of Korea; Department of Ophthalmology, Seoul National University Hospital Healthcare System Gangnam Center, Seoul, Republic of Korea
| | - Mee Kum Kim
- Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Republic of Korea; Laboratory of Ocular Regenerative Medicine and Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Biomedical Research Institute, Seoul, Republic of Korea.
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Fishman JA, Sachs DH, Yamada K, Wilkinson RA. Absence of interaction between porcine endogenous retrovirus and porcine cytomegalovirus in pig-to-baboon renal xenotransplantation in vivo. Xenotransplantation 2018; 25:e12395. [PMID: 29624743 PMCID: PMC6158079 DOI: 10.1111/xen.12395] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2017] [Revised: 02/23/2018] [Accepted: 03/09/2018] [Indexed: 12/30/2022]
Abstract
BACKGROUND Studies of xenotransplantation from swine have identified porcine viruses as potential barriers to clinical trials. The biology of these viruses has not been extensively investigated in the in vivo xeno-environment. Enhancement of viral gene expression by viral and cellular factors acting in trans has been demonstrated for certain viruses, including bidirectional interactions between human herpesviruses and endogenous (HERV) and exogenous (HIV) retroviruses. Both porcine cytomegalovirus (PCMV) and porcine endogenous retrovirus (PERV) infections have been identified in xenografts from swine. PERV receptors exist on human cells with productive infection in vitro in permissive human target cell lines. PCMV is largely species-specific with infection restricted to the xenograft in pig-to-baboon transplants. It is unknown whether coinfection by PCMV affects the replication of PERV within xenograft tissues which might have implications for the risk of retroviral infection in the human host. METHODS A series of 11 functioning, life-supporting pig-to-baboon kidney xenografts from PERV-positive miniature swine were studied with and without PCMV co-infection. Frozen biopsy samples were analyzed using quantitative, real-time PCR with internal controls. RESULTS PERV replication was not altered in the presence of PCMV coinfection (P = .70). The absence of variation with coinfection was confirmed when PERV quantitation was expressed relative to simultaneous cellular GAPDH levels with or without PCMV coinfection (P = .59). CONCLUSIONS PCMV coinfection does not alter the replication of PERV in life-supporting renal xenotransplantation in vivo in baboons.
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Affiliation(s)
- Jay A Fishman
- Infectious Disease Division and MGH Transplant Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
| | - David H Sachs
- Columbia Center for Translational Immunology, Departments of Medicine and Surgery, Columbia University, New York, NY, USA
| | - Kazuhiko Yamada
- Columbia Center for Translational Immunology, Departments of Medicine and Surgery, Columbia University, New York, NY, USA
| | - Robert A Wilkinson
- Infectious Disease Division and MGH Transplant Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
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Pakhomov O, Martignat L, Honiger J, Clémenceau B, Saï P, Darquy S. AN69 Hollow Fiber Membrane will Reduce but Not Abolish the Risk of Transmission of Porcine Endogenous Retroviruses. Cell Transplant 2017; 14:749-56. [PMID: 16454349 DOI: 10.3727/000000005783982468] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
As the risk of porcine endogenous retrovirus (PERV) infection is a major obstacle to the xenotransplantation of porcine tissue, we investigated whether an AN69 hollow fibre membrane, used for islets of Langerhans transplantation, could prevent the transfer of PERVs and thus reduce the risk of PERV infection. PK15 cells were used as a PERV source. A specific and highly sensitive RCR was used for detection of a PERV provirus DNA (gag region) and a porcine mtDNA. Human U293 cells were incubated in vitro with encapsulated PK15 cells, concentrated encapsulated PK15 supernatant, or concentrated PK15 supernatant as a control. CD1 mice were implanted in vivo with encapsulated PK15 cells or injected with PK15 supernatant. We found no infection in human cells incubated with either encapsulated PK15 supernatant or in 10 out of 11 samples after coincubation with encapsulated PK15 cells. Infection of human cells was, however, detected in 1 out of 11 samples after coincubation with encapsulated PK15 cells. The presence of PERV provirus DNA and porcine mtDNA was detected in all the investigated tissues of the mice injected with PK15 supernatant and in various tissues of the mice implanted with encapsulated PK15 cells. Four weeks after the last injection of PK15 supernatant or a fiber explantation, no mouse showed any presence of PERV provirus DNA or porcine mtDNA. Our results demonstrate that AN69 hollow fiber membrane will reduce but not abolish the risk of PERV infection. Because the real risk of PERV infection still remains unknown, it is necessary to investigate further the real protection that could be provided by hollow fibers to ensure the safety of clinical xenotransplantation.
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Affiliation(s)
- Oleg Pakhomov
- Biology of Nutrition, Paris 5 University Faculty of Pharmacy, France
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Choi HJ, Kim J, Kim JY, Lee HJ, Wee WR, Kim MK, Hwang ES. Long-term safety from transmission of porcine endogenous retrovirus after pig-to-non-human primate corneal transplantation. Xenotransplantation 2017; 24. [PMID: 28503733 DOI: 10.1111/xen.12314] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2016] [Revised: 04/07/2017] [Accepted: 04/10/2017] [Indexed: 12/31/2022]
Abstract
BACKGROUND The risk of xenozoonosis mainly by porcine endogenous retrovirus (PERV) has been considered as one of the main hurdles in xenotransplantation and therefore should be elucidated prior to the clinical use of porcine corneal grafts. Accordingly, an investigation was performed to analyze the infectivity of PERVs from porcine keratocytes to human cells, and the long-term risk of transmission of PERVs was determined using pig-to-non-human primate (NHP) corneal transplantation models. METHODS The infectivity of PERVs from the SNU miniature pig keratocytes was investigated by coculture with a human embryonic kidney cell line. Twenty-two rhesus macaques underwent xenocorneal transplantation as follows: (i) group 1 (n=4): anterior lamellar keratoplasty (LKP) with freshly preserved porcine corneas, (ii) group 2 (n=5): anterior LKP with decellularized porcine corneas followed by penetrating keratoplasty (PKP) with allografts, (iii) group 3 (n=3): PKP under steroid-based immunosuppression, (iv) group 4 (n=4): PKP under anti-CD154 antibody-based immunosuppression, (v) group 5 (n=4): deep anterior LKP with freshly preserved porcine corneas under anti-CD40 antibody-based immunosuppression, and (vi) group 6 (n=2): PKP under anti-CD40 antibody-based immunosuppression. Postoperative blood samples were serially collected, and tissue samples were obtained from thirteen different organs at the end of each experiment. The existence of PERV DNA and RNA was investigated using PCR and RT-PCR. RESULTS Using two independent in vitro infectivity tests, neither PERV pol nor pig mitochondrial cytochrome oxidase II was detected after 41 and 92 days of coculture, respectively. After xenocorneal transplantation, a total of 257 serial peripheral blood mononuclear cell samples, 34 serial plasma samples, and 282 tissue samples were obtained from the NHP recipients up to 1176 days post-transplantation. No PERV transmission was evident in any samples. CONCLUSIONS Within the limits of this study, there is no evidence to support any risk of PERV transmission from porcine corneal tissues to NHP recipients, despite the existence of PERV-expressing cells in porcine corneas.
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Affiliation(s)
- Hyuk Jin Choi
- Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea.,Laboratory of Ocular Regenerative Medicine and Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Biomedical Research Institute, Seoul, Korea.,Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Korea
| | - Jiyeon Kim
- Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Korea.,Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Korea
| | - Jae Young Kim
- Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea.,Laboratory of Ocular Regenerative Medicine and Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Biomedical Research Institute, Seoul, Korea.,Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Korea
| | - Hyun Ju Lee
- Laboratory of Ocular Regenerative Medicine and Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Biomedical Research Institute, Seoul, Korea.,Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Korea
| | - Won Ryang Wee
- Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea.,Laboratory of Ocular Regenerative Medicine and Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Biomedical Research Institute, Seoul, Korea.,Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Korea
| | - Mee Kum Kim
- Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea.,Laboratory of Ocular Regenerative Medicine and Immunology, Seoul Artificial Eye Center, Seoul National University Hospital Biomedical Research Institute, Seoul, Korea.,Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Korea
| | - Eung Soo Hwang
- Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Korea.,Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Korea
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Timsit MO, Branchereau J, Thuret R, Kleinclauss F. [Renal transplantation in 2046: Future and perspectives]. Prog Urol 2016; 26:1132-1142. [PMID: 27665406 DOI: 10.1016/j.purol.2016.08.023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2016] [Revised: 08/24/2016] [Accepted: 08/25/2016] [Indexed: 11/25/2022]
Abstract
OBJECTIVES To report major findings that may build the future of kidney transplantation. MATERIAL AND METHODS Relevant publications were identified through Medline (http://www.ncbi.nlm.nih.gov) and Embase (http://www.embase.com) database from 1960 to 2016 using the following keywords, in association, "bio-engineering; heterotransplantation; immunomodulation; kidney; regenerative medicine; xenotransplantation". Articles were selected according to methods, language of publication and relevance. A total of 5621 articles were identified including 2264 for xenotransplantation, 1058 for regenerative medicine and 2299 for immunomodulation; after careful selection, 86 publications were eligible for our review. RESULTS Despite genetic constructs, xenotransplantation faces the inevitable obstacle of species barrier. Uncertainty regarding xenograft acceptance by recipients as well as ethical considerations due to the debatable utilization of animal lives, are major limits for its future. Regenerative medicine and tridimensional bioprinting allow successful implantation of organs. Bioengineering, using decellularized tissue matrices or synthetic scaffold, seeded with pluripotent cells and assembled using bioreactors, provide exciting results but remain far for reconstituting renal complexity and vascular patency. Immune tolerance may be achieved through a tough initial T-cell depletion or a combined haplo-identical bone marrow transplant leading to lymphohematopoietic chimerism. CONCLUSION Current researches aim to increase the pool of organs available for transplantation (xenotransplants and bio-artificial kidneys) and to increase allograft survival through the induction of immune tolerance. Reported results suggest the onset of a thrilling new era for renal transplantation providing end-stage renal disease-patients with an improved survival and quality of life.
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Affiliation(s)
- M-O Timsit
- Service d'urologie, hôpital européen Georges-Pompidou, AP-HP, 20, rue Leblanc, 75015 Paris, France; Université Paris-Descartes, 75006 Paris, France.
| | - J Branchereau
- Service d'urologie et transplantation, CHU de Nantes, 44000 Nantes, France
| | - R Thuret
- Service d'urologie et transplantation rénale, CHU de Montpellier, 34090 Montpellier, France; Université de Montpellier, 34090 Montpellier, France
| | - F Kleinclauss
- Service d'urologie et transplantation rénale, CHRU de Besançon, 25000 Besançon, France; Université de Franche-Comté, 25000 Besançon, France; Inserm UMR 1098, 25000 Besançon, France
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Luca G, Mancuso F, Calvitti M, Arato I, Falabella G, Bufalari A, De Monte V, Tresoldi E, Nastruzzi C, Basta G, Fallarino F, Lilli C, Bellucci C, Baroni T, Aglietti MC, Giovagnoli S, Cameron DF, Bodo M, Calafiore R. Long-term stability, functional competence, and safety of microencapsulated specific pathogen-free neonatal porcine Sertoli cells: a potential product for cell transplant therapy. Xenotransplantation 2015; 22:273-83. [PMID: 26134468 DOI: 10.1111/xen.12175] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2015] [Accepted: 06/04/2015] [Indexed: 01/13/2023]
Abstract
BACKGROUND Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti-inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen-free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts. METHODS Specific pathogen free neonatal pigs underwent testis excision under sterility. pSCs were isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function [namely, production of anti-müllerian hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGβ-1)]. After microencapsulation in barium alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentrations were evaluated at 1, 24 throughout 40 h to establish pre-transplant storage conditions. RESULTS The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior to and after microencapsulation, maintained high function and full viability up to 24 h of storage. At 40 h post-encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and was stable for 4 months in mice with no evident side effects. CONCLUSIONS Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantation.
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Affiliation(s)
- Giovanni Luca
- Department of Experimental Medicine, University of Perugia, Perugia, Italy.,Division of Medical Andrology and Endocrinology of Reproduction, Saint Mary Hospital, Terni, Italy
| | - Francesca Mancuso
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Mario Calvitti
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Iva Arato
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Giulia Falabella
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Antonello Bufalari
- Department of Pathology, Diagnostic and Clinical Veterinary Medicine, University of Perugia, Perugia, Italy
| | - Valentina De Monte
- Department of Pathology, Diagnostic and Clinical Veterinary Medicine, University of Perugia, Perugia, Italy
| | - Enrico Tresoldi
- Experimental Zooprophylactic Institute of Lombardia and Emilia Romagna, Brescia, Italy
| | - Claudio Nastruzzi
- Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy
| | - Giuseppe Basta
- Department of Medicine, University of Perugia, Perugia, Italy
| | | | - Cinzia Lilli
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Catia Bellucci
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Tiziano Baroni
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | | | - Stefano Giovagnoli
- Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy
| | - Don F Cameron
- Department of Internal Medicine, University of South Florida, Tampa, FL, USA
| | - Maria Bodo
- Department of Experimental Medicine, University of Perugia, Perugia, Italy
| | - Riccardo Calafiore
- Division of Medical Andrology and Endocrinology of Reproduction, Saint Mary Hospital, Terni, Italy.,Department of Medicine, University of Perugia, Perugia, Italy
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DIAO YUMEI, HONG JING. Feasibility and safety of porcine Descemet’s membrane as a carrier for generating tissue-engineered corneal endothelium. Mol Med Rep 2015; 12:1929-34. [DOI: 10.3892/mmr.2015.3665] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2014] [Accepted: 12/02/2014] [Indexed: 11/06/2022] Open
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Boksa M, Zeyland J, Słomski R, Lipiński D. Immune modulation in xenotransplantation. Arch Immunol Ther Exp (Warsz) 2014; 63:181-92. [PMID: 25354539 PMCID: PMC4429136 DOI: 10.1007/s00005-014-0317-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2014] [Accepted: 07/22/2014] [Indexed: 01/17/2023]
Abstract
The use of animals as donors of tissues and organs for xenotransplantations may help in meeting the increasing demand for organs for human transplantations. Clinical studies indicate that the domestic pig best satisfies the criteria of organ suitability for xenotransplantation. However, the considerable phylogenetic distance between humans and the pig causes tremendous immunological problems after transplantation, thus genetic modifications need to be introduced to the porcine genome, with the aim of reducing xenotransplant immunogenicity. Advances in genetic engineering have facilitated the incorporation of human genes regulating the complement into the porcine genome, knockout of the gene encoding the formation of the Gal antigen (α1,3-galactosyltransferase) or modification of surface proteins in donor cells. The next step is two-fold. Firstly, to inhibit processes of cell-mediated xenograft rejection, involving natural killer cells and macrophages. Secondly, to inhibit rejection caused by the incompatibility of proteins participating in the regulation of the coagulation system, which leads to a disruption of the equilibrium in pro- and anti-coagulant activity. Only a simultaneous incorporation of several gene constructs will make it possible to produce multitransgenic animals whose organs, when transplanted to human recipients, would be resistant to hyperacute and delayed xenograft rejection.
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Affiliation(s)
- Magdalena Boksa
- Department of Biochemistry and Biotechnology, Poznań University of Life Sciences, Dojazd 11, 60-632, Poznań, Poland,
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Lin X, Qi L, Li Z, Chi H, Lin W, Wang Y, Jiang Z, Pan M, Gao Y. Susceptibility of human liver cells to porcine endogenous retrovirus. EXP CLIN TRANSPLANT 2013; 11:541-5. [PMID: 23901808 DOI: 10.6002/ect.2012.0213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
OBJECTIVES The risk of porcine endogenous retrovirus infection is a major barrier for pig-to-human xenotransplant. Porcine endogenous retrovirus, present in porcine cells, can infect many human and nonhuman primate cells in vitro, but there is no evidence available about in vitro infection of human liver cells. We investigated the susceptibility of different human liver cells to porcine endogenous retrovirus. MATERIALS AND METHODS The supernatant from a porcine kidney cell line was added to human liver cells, including a normal hepatocyte cell line (HL-7702 cells), primary hepatocytes (Phh cells), and a liver stellate cell line (Lx-2 cells), and to human embryonic kidney cells as a reference control. Expression of the porcine endogenous retrovirus antigen p15E in the human cells was evaluated with polymerase chain reaction, reverse transcription-polymerase chain reaction, and Western blot. RESULTS The porcine endogenous retrovirus antigen p15E was not expressed in any human liver cells (HL-7702, Phh, or Lx-2 cells) that had been exposed to supernatants from porcine kidney cell lines. Porcine endogenous retrovirus-specific fragments were amplified in human kidney cells. CONCLUSIONS Human liver cells tested were not susceptible to infection by porcine endogenous retrovirus. Therefore, not all human cells are susceptible to porcine endogenous retrovirus.
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Affiliation(s)
- Xinzi Lin
- Department of Hepatobiliary Surgery, Zhujiang Hospital, China
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Denner J, Tönjes RR. Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses. Clin Microbiol Rev 2012; 25:318-43. [PMID: 22491774 PMCID: PMC3346299 DOI: 10.1128/cmr.05011-11] [Citation(s) in RCA: 148] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Xenotransplantation may be a solution to overcome the shortage of organs for the treatment of patients with organ failure, but it may be associated with the transmission of porcine microorganisms and the development of xenozoonoses. Whereas most microorganisms may be eliminated by pathogen-free breeding of the donor animals, porcine endogenous retroviruses (PERVs) cannot be eliminated, since these are integrated into the genomes of all pigs. Human-tropic PERV-A and -B are present in all pigs and are able to infect human cells. Infection of ecotropic PERV-C is limited to pig cells. PERVs may adapt to host cells by varying the number of LTR-binding transcription factor binding sites. Like all retroviruses, they may induce tumors and/or immunodeficiencies. To date, all experimental, preclinical, and clinical xenotransplantations using pig cells, tissues, and organs have not shown transmission of PERV. Highly sensitive and specific methods have been developed to analyze the PERV status of donor pigs and to monitor recipients for PERV infection. Strategies have been developed to prevent PERV transmission, including selection of PERV-C-negative, low-producer pigs, generation of an effective vaccine, selection of effective antiretrovirals, and generation of animals transgenic for a PERV-specific short hairpin RNA inhibiting PERV expression by RNA interference.
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Sakuma T, Tonne JM, Malcolm JA, Thatava T, Ohmine S, Peng KW, Ikeda Y. Long-term infection and vertical transmission of a gammaretrovirus in a foreign host species. PLoS One 2012; 7:e29682. [PMID: 22235324 PMCID: PMC3250474 DOI: 10.1371/journal.pone.0029682] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2011] [Accepted: 12/01/2011] [Indexed: 11/19/2022] Open
Abstract
Increasing evidence has indicated natural transspecies transmission of gammaretroviruses; however, viral-host interactions after initial xeno-exposure remain poorly understood. Potential association of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome has attracted broad interests in this topic. Although recent studies have indicated that XMRV is unlikely a human pathogen, further understanding of XMRV xenoinfection would allow in vivo modeling of the initial steps of gammaretroviral interspecies transmission, evolution and dissemination in a new host population. In this study, we monitored the long-term consequences of XMRV infection and its possible vertical transmission in a permissive foreign host, wild-derived Mus pahari mice. One year post-infection, XMRV-infected mice showed no notable pathological changes, while proviral DNA was detected in three out of eight mice. XMRV-infected mice remained seropositive throughout the study although the levels of gp70 Env- and p30 capsid-specific antibodies gradually decreased. When vertical XMRV transmission was assessed, no viremia, humoral immune responses nor endogenization were observed in nine offspring from infected mothers, yet one offspring was found PCR-positive for XMRV-specific sequences. Amplified viral sequences from the offspring showed several mutations, including one amino acid deletion in the receptor binding domain of Env SU. Our results therefore demonstrate long-term asymptomatic infection, low incidence of vertical transmission and limited evolution of XMRV upon transspecies infection of a permissive new host, Mus pahari.
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Affiliation(s)
- Toshie Sakuma
- Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America
| | - Jason M. Tonne
- Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America
| | - Jessica A. Malcolm
- Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America
| | - Tayaramma Thatava
- Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America
| | - Seiga Ohmine
- Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America
| | - Kah-Whye Peng
- Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America
| | - Yasuhiro Ikeda
- Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota, United States of America
- * E-mail:
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Di Nicuolo G, D'Alessandro A, Andria B, Scuderi V, Scognamiglio M, Tammaro A, Mancini A, Cozzolino S, Di Florio E, Bracco A, Calise F, Chamuleau RAFM. Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver. Xenotransplantation 2011; 17:431-9. [PMID: 21158944 DOI: 10.1111/j.1399-3089.2010.00617.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Clinical use of porcine cell-based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow-up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. METHODS Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center-BAL (AMC-BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real-time polymerase chain reaction assay has been used. RESULTS Eight patients who received a liver transplant after AMC-BAL treatment are still alive under long-term pharmacological immunosuppression. The current clinical follow-up ranges from 5.6 to 8.7 yr after BAL treatment. A new q-real-time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥ 5 copies per 1 × 10(5) PBMCs. The linear dynamic range was from 5 × 10(0) to 5 × 10(6) copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. CONCLUSION Up to 8.7 yr after exposure to treatment with porcine liver cell-based BAL, no PERV infection has been found in long-term immunosuppressed patients and in HCWs by a new highly sensitive and specific q-real-time PCR assay.
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Denner J, Schuurman HJ, Patience C. Chapter 5: Strategies to prevent transmission of porcine endogenous retroviruses. Xenotransplantation 2009; 16:239-48. [DOI: 10.1111/j.1399-3089.2009.00544.x] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Specke V, Plesker R, Wood J, Coulibaly C, Suling K, Patience C, Kurth R, Schuurman HJ, Denner J. No in vivo infection of triple immunosuppressed non-human primates after inoculation with high titers of porcine endogenous retroviruses. Xenotransplantation 2009; 16:34-44. [PMID: 19243559 DOI: 10.1111/j.1399-3089.2009.00508.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
UNLABELLED Porcine endogenous retroviruses (PERVs) released from pig tissue can infect selected human cells in vitro and therefore represent a safety risk for xenotransplantation using pig cells, tissues, or organs. Although PERVs infect cells of numerous species in vitro, attempts to establish reliable animal models failed until now. Absence of PERV transmission has been shown in first experimental and clinical xenotransplantations; however, these trials suffered from the absence of long-term exposure (transplant survival) and profound immunosuppression. METHODS We conducted infectivity studies in rhesus monkeys, pig-tailed monkeys, and baboons under chronic immunosuppression with cyclosporine A, methylprednisolone, and the rapamycin derivative. These species were selected because they are close to the human species and PERVs can be transmitted in vitro to cells of these species. In addition, the animals received twice, a C1 esterase inhibitor to block complement activation before inoculation of PERV. In order to overcome the complications of microchimerism, animals were inoculated with high titers of cell-free PERV. In addition, to enable transmission via cell-cell contact, some animals also received virus-producing cells. For inoculation the primate cell-adapted strain PERV/5 degrees was used which is characterized by a high infectious titer. Produced on human cells, this virus does not express alpha 1,3 Gal epitopes, does not contain porcine antigens on the viral surface and is therefore less immunogenic in non-human primates compared with pig cell-derived virus. Finally, we present evidence that PERV/5 degrees productively infects cells from baboons and rhesus monkeys. RESULTS In a follow-up period of 11 months, no antibody production against PERV and no integration of proviral DNA in blood cells was observed. Furthermore, no PERV sequences were detected in the DNA of different organs taken after necropsy. CONCLUSION These results indicate that in a primate model, in the presence of chronic immunosuppression, neither the inoculation of cell-free nor cell-associated PERV using a virus already adapted to primate cells results in an infection; this is despite the fact that peripheral blood mononuclear cells of the same animals are infectible in vitro.
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The Baboon in Xenotransplant Research. THE BABOON IN BIOMEDICAL RESEARCH 2009. [PMCID: PMC7120791 DOI: 10.1007/978-0-387-75991-3_19] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
If cross-species transplantation is ever to become a reasonable therapeutic modality for human beings, it will be because the potential for success has been demonstrated in a nonhuman primate model. The imperative has always been to select a primate research subject from a species that is plentiful, is not endangered, readily procreates in a managed environment, and mimics the human response (immunologic homology) to both organ transplantation and potential transfer of infectious disease. Several Papio subspecies of baboons, including Papio hamadryas anubis (olive baboon), meet these important criteria. These animals remain ubiquitous throughout sub-Saharan Africa and have adapted well to the managed environments of major primate centers worldwide. A list of United States-based primate centers housing breeding colonies of baboons can be found in Table 19.1. The Surgical Research Laboratory at Loma Linda University, for instance, has maintained a salutary relationship with the Southwest National Primate Research Center in San Antonio, Texas, for the procurement of juvenile baboon research subjects.
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Louz D, Bergmans HE, Loos BP, Hoeben RC. Reappraisal of biosafety risks posed by PERVs in xenotransplantation. Rev Med Virol 2008; 18:53-65. [PMID: 17987669 DOI: 10.1002/rmv.559] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
Donor materials of porcine origin could potentially provide an alternative source of cells, tissues or whole organs for transplantation to humans, but is hampered by the health risk posed by infection with porcine viruses. Although pigs can be bred in such a way that all known exogenous microorganisms are eliminated, this is not feasible for all endogenous pathogens, such as the porcine endogenous retroviruses (PERVs) which are present in the germline of pigs as proviruses. Upon transplantation, PERV proviruses would be transferred to the human recipient along with the xenograft. If xenotransplantation stimulates or facilitates replication of PERVs in the new hosts, a risk exists for adaptation of the virus to humans and subsequent spread of these viruses. In a worst-case scenario, this might result in the emergence of a new viral disease. Although the concerns for disease potential of PERVs are easing, only limited pre-clinical and clinical data are available. Small-scale, well-designed and carefully controlled clinical trials would provide more evidence on the safety of this approach and allow a better appreciation of the risks involved. It is therefore important to have a framework of protective measures and monitoring protocols in place to facilitate such initially small scale clinical trials. This framework will raise ethical and social considerations regarding acceptability.
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Affiliation(s)
- Derrick Louz
- GMO office, Substances Expertise Centre of the National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
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17
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Denner J. Transspecies transmissions of retroviruses: new cases. Virology 2007; 369:229-33. [PMID: 17870141 DOI: 10.1016/j.virol.2007.07.026] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2007] [Revised: 06/13/2007] [Accepted: 07/25/2007] [Indexed: 11/23/2022]
Abstract
Transspecies transmission is common among retroviruses, although the consequences of the transmission are very different. Some transspecies transmissions have resulted in fatal diseases in the new host while others have remained asymptomatic. Some retroviruses are apathogenic in the original species, but pathogenic in a new host and others can be pathogenic or apathogenic in both species. In some cases, endogenization of the retrovirus in the new host has been observed but in others not, while some transmitted retroviruses exist in both forms. Although in most cases transspecies transmission has been observed in one direction only, bidirectional transmissions of caprine and ovine lentiviruses have recently been described. Studies on newly reported natural and experimental transspecies transmissions of the koala retrovirus (KoRV) may help to understand such events.
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18
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Levy MF, Argaw T, Wilson CA, Brooks J, Sandstrom P, Merks H, Logan J, Klintmalm G. No evidence of PERV infection in healthcare workers exposed to transgenic porcine liver extracorporeal support. Xenotransplantation 2007; 14:309-15. [PMID: 17669172 DOI: 10.1111/j.1399-3089.2007.00408.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
BACKGROUND Clinical xenotransplantation holds great promise by providing one solution to the shortage of human organs for transplantation, while also posing a potential public health threat by facilitating transmission of infectious disease from source animals to humans. One potential vector for infectious disease transmission is healthcare workers (HCW) who are involved in administering xenotransplantation procedures. METHODS In this study, we studied 49 healthcare workers involved in the care of two subjects who participated in a study of porcine liver perfusion as treatment of fulminant hepatic failure. We looked for serologic and virologic evidence of transmission of porcine endogenous retrovirus, and found that HCW had no evidence of infection. CONCLUSIONS Results of our survey demonstrate that application of standard precautions may be sufficient to prevent transmission of porcine endogenous retrovirus, an agent of concern in ex vivo xenotransplantation products.
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Affiliation(s)
- Marlon F Levy
- Baylor All Saints Medical Center, Fort Worth, TX 76104, USA.
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19
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Martina Y, Marcucci KT, Cherqui S, Szabo A, Drysdale T, Srinivisan U, Wilson CA, Patience C, Salomon DR. Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. J Virol 2006; 80:3135-46. [PMID: 16537582 PMCID: PMC1440412 DOI: 10.1128/jvi.80.7.3135-3146.2006] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Porcine endogenous retrovirus (PERV) is considered one of the major risks in xenotransplantation. No valid animal model has been established to evaluate the risks associated with PERV transmission to human patients by pig tissue xenotransplantation or to study the potential pathogenesis associated with PERV infection. In previous work we isolated two genes encoding functional human PERV receptors and proved that introduction of these into mouse fibroblasts allowed the normally nonpermissive mouse cells to become productively infected (T. A. Ericsson, Y. Takeuchi, C. Templin, G. Quinn, S. F. Farhadian, J. C. Wood, B. A. Oldmixon, K. M. Suling, J. K. Ishii, Y. Kitagawa, T. Miyazawa, D. R. Salomon, R. A. Weiss, and C. Patience, Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). In the present study we created mice transgenic for human PERV-A receptor 2 (HuPAR-2). After inoculation of transgenic animals with infectious PERV supernatants, viral DNA and RNA were detected at multiple time points, indicating productive replication. This establishes the role of HuPAR-2 in PERV infection in vivo; in addition, these transgenic mice represent a new model for determining the risk of PERV transmission and potential pathogenesis. These mice also create a unique opportunity to study the immune response to PERV infection and test potential therapeutic or preventative modalities.
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MESH Headings
- Animals
- Animals, Newborn
- Blotting, Western
- Cell Line
- DNA, Viral/analysis
- DNA, Viral/genetics
- DNA, Viral/isolation & purification
- Endogenous Retroviruses/genetics
- Endogenous Retroviruses/isolation & purification
- Endogenous Retroviruses/physiology
- Humans
- Mice
- Mice, Transgenic
- Microscopy, Confocal
- NIH 3T3 Cells
- RNA, Viral/analysis
- RNA, Viral/genetics
- RNA, Viral/isolation & purification
- Receptors, Virus/metabolism
- Recombinant Proteins/biosynthesis
- Recombinant Proteins/isolation & purification
- Recombinant Proteins/metabolism
- Retroviridae Infections/transmission
- Retroviridae Infections/virology
- Reverse Transcriptase Polymerase Chain Reaction
- Swine/virology
- Time Factors
- Transgenes
- Virus Replication
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Affiliation(s)
- Y Martina
- Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
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Wang HH, Wang YJ, Liu HL, Liu J, Huang YP, Guo HT, Wang YM. Detection of PERV by polymerase chain reaction and its safety in bioartificial liver support system. World J Gastroenterol 2006; 12:1287-91. [PMID: 16534887 PMCID: PMC4124445 DOI: 10.3748/wjg.v12.i8.1287] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To establish a method detecting porcine endogenous retrovirus (PERV) in China experimental minipigs and to evaluate the safety of PERV in three individuals treated with bioartificial liver support systems based on porcine hepatocytes.
METHODS: Porcine hepatocytes were isolated with two-stage perfusion method, then cultured in the bioreactor, which is separated by a semipermeable membrane (0.2 μm) from the lumen through which the patients’ blood plasma was circulated. After post-hemoperfusion, patients’ blood was obtained for screening. Additionally, samples of medium collected from both intraluminal and extraluminal compartments of the laboratory bioreactor and culture supernate in vitro was analyzed. The presence of viral sequences was estimated by polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR). Finally, the infection of virus in the supernate of common culture was ascertained by exposure to the fetal liver cells.
RESULTS: PERV-specific gag sequences were found in the porcine hepatocytes using RT-PCR. and were detected in all samples from the intraluminal, extraluminal samples and culture supernate. However, culture supernatant from primary porcine hepatocytes (cleared of cellular debris) failed to infect human fetal liver cells. Finally, RT-PCR detected no PERV infection was found in the blood samples obtained from three patients at various times post-hemoperfusion.
CONCLUSION: The assays used are specific and sensitive, identified by second PCR. PERVs could be released from hepatocytes cultured in bioreactor without the stimulation of mitogen and could not be prevented by the hollow fiber semipermeable membrane, indicating the existence of PERV safety in extracorporeal bioartificial liver support system (EBLSS).
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Affiliation(s)
- Hai-Hui Wang
- Department of Endocrine Diseases, Southwestern Hospital, Third Military Medical University, Chongqing 400038, China
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21
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Winkler ME, Winkler M, Burian R, Hecker J, Loss M, Przemeck M, Lorenz R, Patience C, Karlas A, Sommer S, Denner J, Martin U. Analysis of pig-to-human porcine endogenous retrovirus transmission in a triple-species kidney xenotransplantation model. Transpl Int 2005; 17:848-58. [PMID: 15864489 DOI: 10.1007/s00147-005-0808-x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2003] [Revised: 12/01/2003] [Accepted: 05/04/2004] [Indexed: 10/25/2022]
Abstract
Clinical pig-to-human xenotransplantation might be associated with the risk of transmission of xenozoonoses, especially porcine endogenous retroviruses (PERVs). We have established a pig-to-humanised-cynomolgus monkey xenotransplantation model allowing the analysis of potential PERV-transmission from normal or transgenic porcine organs to human vascular tissue. Pig-to-human kidney xenotransplantation was performed in cynomolgus monkeys. An interposition graft constructed from a human saphena vein replaced the porcine kidney vein. After graft rejection and/or death of the recipient (survival 2, 4, 6, 13, 16, 19 days), the human interposition grafts were removed. Human endothelial cells (huECs) were isolated from the interposition grafts and cultivated in vitro. Explanted human vascular tissue, isolated huECs, plasma and serum samples of the graft recipients were characterised by flow cytometry and immunohistochemistry and screened for indications of PERV transmission by quantitative polymerase chain reaction (PCR), reverse transcriptase-polymerase chain reaction (RT-PCR) and RT assay. PERV-specific immune response of recipients was analysed by Western blot. No evidence of PERV infection or PERV-specific immune response was detected.
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Affiliation(s)
- Monica E Winkler
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover, Germany.
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Sykes M, d'Apice A, Sandrin M. Position Paper of the Ethics Committee of the International Xenotransplantation Association. Transplantation 2004; 78:1101-7. [PMID: 15502702 DOI: 10.1097/01.tp.0000142886.27906.3e] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Xenotransplantation (XTx) provides a potential solution to the shortage of human organs and tissues, and has several advantages over other possible solutions to this problem. However, a number of scientific and ethical barriers exist, and need to be addressed in order to advance the field of XTx in a manner that optimizes its potential to benefit society and minimizes its risk. Some of the most pressing ethical issues are discussed, and the position of the Ethics Committee of the International Xenotransplantation Association is presented.
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Affiliation(s)
- Megan Sykes
- Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, MGH-East, 13th Street, Boston, MA 02129, USA.
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23
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Cunningham DA, Dos Santos Cruz GJ, Fernández-Suárez XM, Whittam AJ, Herring C, Copeman L, Richards A, Langford GA. ACTIVATION OF PRIMARY PORCINE ENDOTHELIAL CELLS INDUCES RELEASE OF PORCINE ENDOGENOUS RETROVIRUSES. Transplantation 2004; 77:1071-9. [PMID: 15087774 DOI: 10.1097/01.tp.0000114966.20491.50] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Endothelial cells form the interface between the porcine graft and the recipient and frequently become activated after xenotransplantation. To evaluate the safety of xenotransplantation further, we assessed the effect of cellular activation on the expression and release of porcine endogenous retroviruses from primary endothelial cells isolated from transgenic and nontransgenic pigs. METHODS Primary porcine endothelial cells, cultured from pigs transgenic for human decay accelerating factor, were treated with human tumor necrosis factor-alpha, porcine interferon-gamma, or lipopolysaccharide. The release of porcine endogenous retroviruses into the supernatant was monitored at 24-hr intervals (up to 72 hr) by polymerase chain reaction-based reverse transcriptase (PBRT) assay. Activated and unactivated endothelial cells were co-cultured with human cells to investigate the capacity of any virus released from the porcine cells to infect human cells. RESULTS Virus was not detected in supernatants from quiescent cells by PBRT analysis. The number of viral particles released from endothelial cells was 10 to 5 x 10 viral particles/mL after cellular activation with tumor necrosis factor-alpha, interferon-gamma, or lipopolysaccharide, as shown by PBRT analysis. In contrast, in vitro infection of human cells was observed with unactivated endothelial cells only and was not observed in co-cultures with the activated porcine cells. CONCLUSIONS Cytokine treatment of primary porcine endothelial cells results in an increase in the release of virus into the supernatant, but the observed increase in viral titer was not mirrored by an increase in infectivity toward human cells.
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Affiliation(s)
- Deirdre A Cunningham
- Department of Parasitology, National Institute for Medical Research, Mill Hill, London, United Kingdom.
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Emerich DF, Hemendinger R, Halberstadt CR. The testicular-derived Sertoli cell: cellular immunoscience to enable transplantation. Cell Transplant 2004; 12:335-49. [PMID: 12911122 DOI: 10.3727/000000003108746894] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
There is a renewed enthusiasm for the potential of cellular transplantation as a therapy for numerous clinical disorders. The revived interest is largely due to the unprecedented success of the "Edmonton protocol," which produced a 100% cure rate for type I diabetics following the transplantation of human islet allografts together with a modified immunosuppressive regimen. While these data provide a clear and unequivocal demonstration that transplantation is a viable treatment strategy, the shortage of suitable donor tissue together with the debilitating consequences of lifelong immunosuppression necessitate a concerted effort to develop novel means to enable transplantation on a widespread basis. This review outlines the use of Sertoli cells to provide local immunoprotection to cografted discordant cells, including those from xenogeneic sources. Sertoli cells are normally found in the testes where one of their functions is to provide local immunologic protection to developing germ cells. Isolated Sertoli cells 1) engraft and self-protect when transplanted into allogeneic and xenogeneic environments, 2) protect cografted allogeneic and xenogeneic cells from immune destruction, 3) protect islet grafts to reverse diabetes in animal models, 4) enable survival and function of cografted foreign dopaminergic neurons in rodent models of Parkinson's disease (PD), and 5) promote regeneration of damaged striatal dopaminergic circuitry in those same PD models. These benefits are discussed in the context of several potential underlying biological mechanisms. While the majority of work to date has focused on Sertoli cells to facilitate transplantation for diabetes and PD, the generalized ability of these unique cells to potently suppress the local immune environment opens additional clinical possibilities.
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Abstract
The shortage of human organs and tissues for transplantation and the advances in immunology of rejection and in genetic engineering have renewed interest in xenotransplantation--the transplantation of animal organs, tissues or cells to humans. Clinical trials have involved the use of non-human primate, porcine, and bovine cells/tissues/organs. In recent years, research has focused mainly on pigs as donors (especially, pigs genetically engineered to carry some human genes). One of the major concerns in xenotransplantation is the risk of transmission of animal pathogens, particularly viruses, to recipients and the possible adaptation of such pathogens for human-to-human transmission. Porcine endogenous retroviruses (PERVs) have been of special concern because of their ability to infect human cells and because, at present, they cannot be removed from the source animal's genome. To date, retrospective studies of humans exposed to live porcine cells/tissues have not found evidence of infection with PERV but more extensive research is needed. This article reviews infectious disease risks associated with xenotransplantation, some measures for minimizing that risk, and microbiological diagnostic methods that may be used in the follow-up of xenotransplant recipients.
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Affiliation(s)
- Roumiana S Boneva
- HIV and Retrovirology Branch, Division of HIV, STD and TB Laboratory Research, National Center for HIV, STD and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
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Abstract
Xenotransplantation, in particular transplantation of pig cells, tissues and organs into human patients, may alleviate the current shortage of suitable allografts available for human transplantation. This overview addresses the physiological, immunological and virological factors considered with regard to xenotransplantation. Among the issues reviewed are the merits of using pigs as xenograft source species, the compatibility of pig and human organ physiology and the immunological hindrances with regard to the various types of rejection and attempts at abrogating rejection. Advances in the prevention of pig organ rejection by creating genetically modified pigs that are more suited to the human microenvironment are also discussed. Finally, with regard to virology, possible zoonotic infections emanating from pigs are reviewed, with special emphasis on the pig endogenous retrovirus (PERV). An in depth account of PERV studies, comprising their discovery as well as recent knowledge of the virus, is given. To date, all retrospective studies on patients with pig xenografts have shown no evidence of PERV transmission, however, many factors make us interpret these results with caution. Although the lack of PERV infection in xenograft recipients up to now is encouraging, more basic research and controlled animal studies that mimic the pig to human xenotransplantation setting more closely are required for safety assessment.
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Affiliation(s)
- Saema Magre
- Wohl Virion Centre, The Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK
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27
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Affiliation(s)
- Olle Korsgren
- Department of Clinical Immunology and Transfusion Medicine, Uppsala University Hospital, Uppsala, Sweden.
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28
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Sykes M, d'Apice A, Sandrin M. Position paper of the Ethics Committee of the International Xenotransplantation Association. Xenotransplantation 2003; 10:194-203. [PMID: 12694539 DOI: 10.1034/j.1399-3089.2003.00067.x] [Citation(s) in RCA: 79] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Xenotransplantation (XTx) provides a potential solution to the shortage of human organs and tissues, and has several advantages over other possible solutions to this problem. However, a number of scientific and ethical barriers exist, and need to be addressed in order to advance the field of XTx in a manner that optimizes its potential to benefit society and minimizes its risk. Some of the most pressing ethical issues are discussed, and the position of the Ethics Committee of the International Xenotransplantation Association is presented.
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Affiliation(s)
- Megan Sykes
- Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA 02129, USA.
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29
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Ritzhaupt A, Van Der Laan LJW, Salomon DR, Wilson CA. Porcine endogenous retrovirus infects but does not replicate in nonhuman primate primary cells and cell lines. J Virol 2002; 76:11312-20. [PMID: 12388691 PMCID: PMC136785 DOI: 10.1128/jvi.76.22.11312-11320.2002] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2002] [Accepted: 08/05/2002] [Indexed: 01/06/2023] Open
Abstract
Porcine endogenous retroviruses (PERV) can infect human cell lines in vitro; hence, there is a presumed risk of viral exposure to a recipient when pig cells are transplanted into humans (xenotransplantation). Nonhuman primates (NHP) are considered a potential permissive animal model to study the risk of in vivo infection of PERV after xenotransplantation. We set out to determine whether PERV can infect and replicate in NHP primary cells or established cell lines from African green monkey, rhesus macaque, and baboon. We confirm that the NHP cell lines under investigation were infected with PERV as measured by detection of viral DNA and RNA by PCR and reverse transcription (RT)-PCR, respectively, indicating that a functional receptor must be present on the cell surface. However, the load of detectable viral DNA in infected NHP cells declined over time, and the cells never had detectable reverse transcriptase activity. Utilizing quantitative real-time TaqMan PCR we found detectable levels of unintegrated DNA intermediates, but the levels were approximately 100-fold lower compared to HEK 293 cells infected with PERV. Virions released from infected NHP cells could productively infect naïve human cell lines, HEK 293 and HeLa, as shown by RT-PCR and RT assay. However, naïve NHP cells remained negative in RT-PCR and RT assay after exposure to virions from infected NHP cells. Together our data demonstrate that NHP cells are not permissive to productive replication by PERV, presumably due to inefficient cell entry and replication. In light of these observations, the appropriateness of NHP as suitable animal models to study PERV infection in vivo needs to be reevaluated.
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Affiliation(s)
- Armin Ritzhaupt
- Laboratory of Immunology and Virology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA
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Sato H, Kobayasi T, Murakami M, Kimura T, Yamaguchi A, Nakagawara G, Iwata H. Improving function and survival of porcine islet xenografts using microencapsulation and culture preconditioning. Pancreas 2002; 25:e42-9. [PMID: 12370549 DOI: 10.1097/00006676-200210000-00020] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/10/2022]
Abstract
INTRODUCTION AND AIMS Porcine pancreatic islets have been difficult to preserve because isolated porcine islets tend to disaggregate to single cells and lose function under culture conditions. In the current study, the influence of agarose microencapsulation on the maintenance of the number and function of islets in culture preservations and the effect of culture preconditioning of microencapsulated porcine islets on xenogenic transplantation were investigated. METHODOLOGY Porcine islets were isolated and then microencapsulated in 5% agarose membrane. The percentage of naked and microencapsulated islets remaining in the culture preservations was assessed. The effect of microencapsulation and culture on secretory function was investigated in vitro. The survival of overnight-cultured and 7-days-cultured microencapsulated islets in xenogenic transplantations was examined. RESULTS A good percentage of microencapsulated islets remained in the culture preservations. They could maintain good secretory functions in vitro after 7 days of culture. In addition, we observed a significant prolongation of mean islet survival by culture preconditioning. CONCLUSIONS The present findings suggest that microencapsulation is one of the useful preserving methods for maintenance of the number and function of cultured isolated porcine islets. Moreover, culture preconditioning is effective for improving islet survival and might be a good option leading to clinical success.
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Affiliation(s)
- Hirohide Sato
- Department of Surgery, Fukui Medical University, Fukui, Japan.
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Bartosch B, Weiss RA, Takeuchi Y. PCR-based cloning and immunocytological titration of infectious porcine endogenous retrovirus subgroup A and B. J Gen Virol 2002; 83:2231-2240. [PMID: 12185278 DOI: 10.1099/0022-1317-83-9-2231] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Two pig endogenous retroviruses (PERV), PERV-A and -B, productively infect human cells and are therefore considered to constitute a potential risk in pig-to-human xenotransplantation. A PCR-based cloning technique to isolate infectious PERV proviruses was established. Overlapping 3' half and 5' halves of PERV proviral genomes were amplified using DNA extracted from human 293 cells infected with PERV-A or -B. These clones were fused at a unique restriction site in the overlapping region and tested for their infectivity. Representative constructs possessed the same infectious properties as their parent isolates. We also developed a polyclonal anti-PERV serum by using recombinant PERV capsid protein derived from one of the infectious constructs as immunogen and established an immunocytological method for detection and titration of PERV infection. This detection method proved to be more sensitive than the current method of choice (transfer of MLV-lacZ vectors) for infectivity assessment of PERV. These findings should be considered for future characterization of PERV isolates.
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Affiliation(s)
- Birke Bartosch
- Wohl Virion Centre, The Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK1
| | - Robin A Weiss
- Wohl Virion Centre, The Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK1
| | - Yasuhiro Takeuchi
- Wohl Virion Centre, The Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, UK1
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Lee JH, Webb GC, Allen RDM, Moran C. Characterizing and mapping porcine endogenous retroviruses in Westran pigs. J Virol 2002; 76:5548-56. [PMID: 11991983 PMCID: PMC137029 DOI: 10.1128/jvi.76.11.5548-5556.2002] [Citation(s) in RCA: 42] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Since porcine endogenous retroviruses (PERVs) can infect cultured human cells, they are a potential hazard to xenotransplantation. For this reason, endogenous retroviruses from the Westran (Westmead Hospital transplantation) inbred line of pigs were analyzed by using consensus primers for the type A and type B viruses to amplify 1.8-kb envelope gene fragments. After preliminary analysis with restriction enzymes KpnI and MboI, 31 clones were sequenced. Between types A and B, five recombinant clones were identified. Fifty-five percent of clones (17 of 31) had premature stop codons within the envelope protein-encoding region. Endogenous retroviruses in Westran pigs were physically mapped by fluorescence in situ hybridization (FISH) using PERV-A and PERV-B envelope clones as probes to identify at least 32 integration sites (19 PERV-A sites and 13 PERV-B sites). The chromosomal sites of integration in the Westran strain are quite different from those in the European Large White pig. The recombinant clones suggest that defective PERVs could become infective through recombination and further that PERVs might recombine with human endogenous retroviruses in xenotransplants.
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Affiliation(s)
- Jun-Heon Lee
- Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, The University of Sydney, Sydney, New South Wales 2006, Australia
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Specke V, Schuurman HJ, Plesker R, Coulibaly C, Ozel M, Langford G, Kurth R, Denner J. Virus safety in xenotransplantation: first exploratory in vivo studies in small laboratory animals and non-human primates. Transpl Immunol 2002; 9:281-8. [PMID: 12180842 DOI: 10.1016/s0966-3274(02)00039-4] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
For xenotransplantation, the transplantation of animal cells, tissues and organs into human recipients, to date, pigs are favored as potential donors. Beside ethical, immunological, physiological and technical problems, the microbiological safety of the xenograft has to be guaranteed. It will be possible to eliminate all of the known porcine microorgansims in the nearby future by vaccinating or specified pathogen-free breeding. Thus, the main risk will come from the porcine endogenous retroviruses (PERVs) which are present in the pig genome as proviruses of different subtypes. PERVs will therefore be transmitted, with the xenograft, to the human recipient. PERVs can infect numerous different types of human primary cells and cell lines in vitro and were shown to adapt to these cells by serial passaging on uninfected cells. Furthermore, PERVs have high homology to other retroviruses, such as feline leukemia virus (FeLV) or murine leukemia virus (MuLV), which are known to induce tumors or immunodeficiencies in the infected host. To evaluate the potential risk of a trans-species transmission of PERV in vivo, naive and immunosuppressed rats, guinea pigs and minks were inoculated with PERV and screened over a period of 3 months for an antibody reaction against PERV proteins or for the integration of proviral DNA into the genomic DNA of the host's cells. Furthermore, we inoculated three different species of non-human primates, rhesus monkey (Macaca mulatta), pig-tailed monkey (Macaca nemestrina) and baboon (Papio hamadryas) with high titers of a human-adapted PERV. To simulate a situation in xenotransplantation, the animals received a daily triple immunosuppression using cyclosporine A, methylprednisolone and RAD, a rapamycin derivative, presently under development by Novartis. None of the small laboratory animals or the non-human primates showed production of antibodies against PERV or evidence of integration of proviral DNA in blood cells or cells of several organs, 3 months after virus inoculation, despite the observation that cells of the animals used in the experiment were infectible in vitro. This apparent difference in the outcome of the in vitro and the in vivo data might be explained by an efficient elimination of the virus by the innate or adaptive immunity of the animals.
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Affiliation(s)
- V Specke
- Robert Koch-Institute, Berlin, Germany
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34
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Tacke SJ, Bodusch K, Berg A, Denner J. Sensitive and specific immunological detection methods for porcine endogenous retroviruses applicable to experimental
and clinical xenotransplantation. Xenotransplantation 2002. [DOI: 10.1034/j.1399-3089.2001.00080.x-i1] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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Kuddus R, Patzer JF, Lopez R, Mazariegos GV, Meighen B, Kramer DJ, Rao AS. Clinical and laboratory evaluation of the safety of a bioartificial liver assist device for potential transmission of porcine endogenous retrovirus. Transplantation 2002; 73:420-9. [PMID: 11884940 DOI: 10.1097/00007890-200202150-00017] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND The potential risk of transmission of porcine endogenous retroviruses (PERV) from xenogeneic donors into humans has been widely debated. Because we were involved in a phase I/II clinical trial using a bioartificial liver support system (BLSS), we proceeded to evaluate the biosafety of this device. MATERIALS AND METHODS The system being evaluated contains primary porcine hepatocytes freshly isolated from pathogen-free, purpose-raised herd. Isolated hepatocytes were installed in the shell, which is separated by a semipermeable membrane (100-kD nominal cutoff) from the lumen through which the patients' whole blood is circulated. Both before and at defined intervals posthemoperfusion, patients' blood was obtained for screening. Additionally, effluent collected from a clinical bioreactor was analyzed. The presence of viral particles was estimated by reverse transcriptase-polymerase chain reaction (RT-PCR) and RT assays. For the detection of pig genomic and mitochondrial DNA, sequence-specific PCR (SS-PCR) was used. Finally, the presence of infectious viral particles in the samples was ascertained by exposure to the PERV-susceptible human cell line HEK-293. RESULTS PERV transcripts, RT activity, and infectious PERV particles were not detected in the luminal effluent of a bioreactor. Culture supernatant from untreated control or mitogen-treated porcine hepatocytes (cleared of cellular debris) also failed to infect HEK-293 cell lines. Finally, RT-PCR, SS-PCR, and PERV-specific RT assay detected no PERV infection in the blood samples obtained from five study patients both before and at various times post-hemoperfusion. CONCLUSION Although longer patient follow-up is required and mandated to unequivocally establish the biosafety of this device and related bioartificial organ systems, these analyses support the conclusion that when used under standard operational conditions, the BLSS is safe.
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Affiliation(s)
- R Kuddus
- Thomas E. Starzl Transplantation Institute, and the Department of Surgery, University of Pittsburgh Medical Center-Health System, Pittsburgh, Pennsylvania, USA
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Abstract
At present, the most successful treatment of acute liver failure is orthotopic liver transplantation, with survival rates ranging from 70% to 85%. However, mortality rates for liver failure remain high because of the shortage of available donor organs. Therefore, there has been renewed interest in temporary treatment methods for patients with acute liver failure to either allow liver regeneration or await liver transplantation. It is thought that the function of the liver can only be replaced with the biological substrate, e.g. liver cells or a whole liver specimen, which requires the availability of liver tissue from xenogeneic or human sources. In this review, existing temporary liver support techniques are summarized and the potential hazards are described. These include the immunological implications of these techniques, e.g. the host versus graft reaction, which may influence the effectivity of the support system, and in the long run may sensitize the patient to subsequent allogeneic transplantation. The graft versus host reaction is also considered. At present, one of the major concerns is the threat of pig-to-human transmission of activated endogenous retrovirus present in the pig genome. An overview is given of literature concerning the transmission of retrovirus particles in vitro and in vivo. Finally, new solutions for the development of ex vivo systems for temporary treatment of patients with acute liver failure are discussed. These include the use of new immortalized human cell lines and human fetal hepatocytes, and the possibility of isolating, expanding and genetically manipulating stem cells in order to have stable differentiated and committed cells.
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Affiliation(s)
- Hein B A C Stockmann
- Department of Surgery, Erasmus University Medical Centre, Rotterdam, The Netherlands.
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Herring C, Quinn G, Bower R, Parsons N, Logan NA, Brawley A, Elsome K, Whittam A, Fernandez-Suarez XM, Cunningham D, Onions D, Langford G, Scobie L. Mapping full-length porcine endogenous retroviruses in a large white pig. J Virol 2001; 75:12252-65. [PMID: 11711616 PMCID: PMC116122 DOI: 10.1128/jvi.75.24.12252-12265.2001] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2001] [Accepted: 09/26/2001] [Indexed: 11/20/2022] Open
Abstract
Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus. In order to determine the feasibility of this, we have determined the number of potentially replication-competent full-length PERV proviruses and obtained data on their integration sites within the porcine genome. We have screened genomic DNA libraries from a Large White pig for potentially intact proviruses. We identified six unique PERV B proviruses that were apparently intact in all three genes, while the majority of isolated proviruses were defective in one or more genes. No intact PERV A proviruses were found in this pig, despite the identification of multiple defective A proviruses. Genotyping of 30 unrelated pigs for these unique proviruses showed a heterogeneous distribution. Two proviruses were uncommon, present in 7 of 30 and 3 of 30 pigs, while three were each present in 24 of 30 pigs, and one was present in 30 of 30 animals examined. Our data indicate that few PERV proviruses in Large White pigs are capable of productive infection and suggest that many could be removed by selective breeding. Further studies are required to determine if all potentially functional proviruses could be removed by breeding or whether gene knockout techniques will be required to remove the residuum.
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Affiliation(s)
- C Herring
- Imutran Ltd. (a Novartis Pharma AG Company), Cambridge CB2 2YP, United Kingdom
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38
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Gill RG. Use of small animal models for screening immunoisolation approaches to cellular transplantation. Ann N Y Acad Sci 2001; 944:35-46. [PMID: 11797684 DOI: 10.1111/j.1749-6632.2001.tb03821.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
It has been recognized for many years that immunoisolation strategies form an attractive approach to preventing the rejection of cellular allografts and xenografts. Although immunoisolation has proven dramatically successful in some cases, the results have tended to be somewhat variable. Although many advances have been made in the development of biocompatible materials for separating host immune cells from the transplanted tissues, much of the experimentation in this area has been outcome driven. That is, the nature of host reactivity and/or biomaterial design resulting in the failure of some immunoisolation strategies has mostly been undefined. A first premise of this discussion is that immunoisolation is primarily cell isolation and not antigen isolation, per se. That is, although varied membrane barriers are designed to prevent cell-cell contact between host and donor cells, soluble antigens derived from the transplant are likely to gain access to the host immune system. A key question centers on the degree and consequence of this type of antigen presentation in the host to the immunoisolated transplant. To address this and related concerns, this overview presents a simple paradigm for using defined rodent (mouse) models for systematically screening the efficacy of immunoisolated cellular transplants. The proposition is made that understanding the basis of graft failure will aid in the design of future immunoisolation technologies.
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Affiliation(s)
- R G Gill
- Barbara Davis Center for Childhood Diabetes, University of Colorado Health Sciences Center, Denver 80262, USA.
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39
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Quinn G, Langford G. The porcine endogenous retrovirus long terminal repeat contains a single nucleotide polymorphism that confers distinct differences in estrogen receptor binding affinity between PERV A and PERV B/C subtypes. Virology 2001; 286:83-90. [PMID: 11448161 DOI: 10.1006/viro.2001.0996] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Porcine endogenous retroviruses (PERV) have been shown to have zoonotic potential, both in vitro and in vivo. Once integrated into the host cell genome activation of the proviral genes is ultimately dependent upon transactivation of the long terminal repeat (LTR). Currently there is no direct evidence of host cell transcription factors interacting with PERV LTRs. Using comparative genomics we discovered a potentially functional single nucleotide polymorphism (SNP) within the U5 region downstream of the TATA box in the PERV LTR that distinguishes PERV A from PERV B and PERV C subtypes. We demonstrated that the SNP occurs within a potential hormone-responsive region where it has a profound effect, not only upon estrogen receptor binding but also upon the binding of other transcription factors at this site. These results suggest that differences in transcriptional regulation between PERV subtypes are subtle and, as for other retroviruses, transcription can be mediated by steroid hormone receptors.
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Affiliation(s)
- G Quinn
- Imutran Limited (A Novartis Pharma AG Company), Cambridge, CB2 2YP, United Kingdom.
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40
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Specke V, Rubant S, Denner J. Productive infection of human primary cells and cell lines with porcine endogenous retroviruses. Virology 2001; 285:177-80. [PMID: 11437652 DOI: 10.1006/viro.2001.0934] [Citation(s) in RCA: 99] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Porcine endogenous retroviruses (PERVs) infect human cells in vitro and therefore represent a risk for xenotransplantation. However, first clinical transplantations of pig cells into humans or ex vivo perfusions did not result in transmission of PERVs. On the other hand, recent experiments with SCID mice demonstrated infections with PERV in vivo. In order to define and characterize human target cells, we studied numerous primary human cells and cell lines. Infection with PERVs was shown for human peripheral blood mononuclear cells, primary endothelial cells, and primary aortic smooth muscle cells as well as lymphocytic, monocytic, and epithelial cell lines.
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Affiliation(s)
- V Specke
- Robert Koch-Institute, Nordufer 20, Berlin, D-13353, Germany
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41
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Cunningham DA, Herring C, Fernández-Suárez XM, Whittam AJ, Paradis K, Langford GA. Analysis of patients treated with living pig tissue for evidence of infection by porcine endogenous retroviruses. Trends Cardiovasc Med 2001; 11:190-6. [PMID: 11597830 DOI: 10.1016/s1050-1738(01)00104-9] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The use of pigs as a source of cells and organs for transplantation has the potential to reduce the current chronic shortage of organs for the treatment of many end-stage diseases. The risk of transmission of infectious agents across the species barrier (zoonoses) has to be assessed. Many such agents can be eliminated from the pig herd. However, porcine endogenous retroviruses, which are carried within the pig genome, are not easily eliminated. They can infect primary and immortalized human cells in vitro, but to date no evidence for in vivo infection has been found in retrospective studies of humans exposed to viable porcine cells. Small-scale clinical trials using porcine cells for the treatment of Parkinson's and Huntington's disease are currently in progress. The prospective monitoring of these patients in conjunction with further research into the biology of this virus will help address safety issues.
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42
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Switzer WM, Michler RE, Shanmugam V, Matthews A, Hussain AI, Wright A, Sandstrom P, Chapman LE, Weber C, Safley S, Denny RR, Navarro A, Evans V, Norin AJ, Kwiatkowski P, Heneine W. Lack of cross-species transmission of porcine endogenous retrovirus infection to nonhuman primate recipients of porcine cells, tissues, or organs. Transplantation 2001; 71:959-65. [PMID: 11349732 DOI: 10.1097/00007890-200104150-00022] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Nonhuman primates (NHPs) have been widely used in different porcine xenograft procedures inevitably resulting in exposure to porcine endogenous retrovirus (PERV). Surveillance for PERV infection in these NHPs may provide information on the risks of cross-species transmission of PERV, particularly for recipients of vascularized organ xenografts for whom data from human clinical trials is unavailable. METHODS We tested 21 Old World and 2 New World primates exposed to a variety of porcine xenografts for evidence of PERV infection. These NHPs included six baboon recipients of pig hearts, six bonnet macaque recipients of transgenic pig skin grafts, and nine rhesus macaque and two capuchin recipients of encapsulated pig islet cells. Serologic screening for PERV antibody was done by a validated Western blot assay, and molecular detection of PERV sequences in peripheral blood mononuclear cells (PBMCs) and plasma was performed using sensitive polymerase chain reaction and reverse transcriptase-polymerase chain reaction assays, respectively. Spleen and lymph node tissues available from six bonnet macaques and three rhesus macaques were also tested for PERV sequences. RESULTS All plasma samples were negative for PERV RNA suggesting the absence of viremia in these xenografted animals. Similarly, PERV sequences were not detectable in any PBMC and tissue samples, arguing for the lack of latent infection of these compartments. In addition, all plasma samples were negative for PERV antibodies. CONCLUSION These data suggest the absence of PERV infection in all 23 NHPs despite exposure to vascularized porcine organs or tissue xenografts and the use of immunosuppressive therapies in some animals. These findings suggest that PERV is not easily transmitted to these NHP species through these types of xenografts.
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Affiliation(s)
- W M Switzer
- Division of AIDS, STDs, and TB Research Laboratory, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
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Dejardin LM, Arnoczky SP, Ewers BJ, Haut RC, Clarke RB. Tissue-engineered rotator cuff tendon using porcine small intestine submucosa. Histologic and mechanical evaluation in dogs. Am J Sports Med 2001; 29:175-84. [PMID: 11292042 DOI: 10.1177/03635465010290021001] [Citation(s) in RCA: 188] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
To determine its efficacy in stimulating the regeneration of a rotator cuff tendon, an implant of 10-ply porcine small intestinal submucosa was used to replace a completely resected infraspinatus tendon in 21 adult mongrel dogs. The contralateral infraspinatus tendon was elevated and then reattached to the greater tubercle with sutures to mimic conventional repair (sham operation). Mechanical evaluations were performed at 0, 3, and 6 months (five specimens at each time period). Histologic comparisons were made at 3 and 6 months (three specimens). At both times, the gross appearance, histologic continuity, and failure mode of the constructs mimicked those of sham-operated and native infraspinatus tendons, thus suggesting host tissue ingrowth and implant remodeling with solid integration of the regenerated tissue to muscular and bony interfaces. Tissue ingrowth occurred without histologic evidence of foreign body or immune-mediated reactions or adhesions to peripheral tissues. Sham operations simulated tendon mobilization and reimplantation procedures routinely performed to treat chronic rotator cuff tendon injuries. Although the ultimate strength of small intestinal submucosa-regenerated tendons was significantly less than that of native infraspinatus tendons (P < 0.001), it was similar to that of reimplanted tendons at 3 (P > 0.05) and 6 months (P > 0.05).
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Affiliation(s)
- L M Dejardin
- Laboratory for Comparative Orthopaedic Research, College of Veterinary Medicine, Michigan State University, East Lansing 48824-1314, USA
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44
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Cross-species transmission of porcine endogenous retroviruses in xenotransplantation: a PERVerted reality? Curr Opin Organ Transplant 2001. [DOI: 10.1097/00075200-200103000-00010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Abouna GM, Ganguly P, Jabur S, Tweed W, Hamdy H, Costa G, Farid E, Sater A. Successful ex vivo liver perfusion system for hepatic failure pending liver regeneration or liver transplantation. Transplant Proc 2001; 33:1962-4. [PMID: 11267589 DOI: 10.1016/s0041-1345(00)02755-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Affiliation(s)
- G M Abouna
- MCP Hahneman University, Philadelphia, Pennsylvania, USA
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46
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Herring C, Cunningham DA, Whittam AJ, Fernández-Suárez XM, Langford GA. Monitoring xenotransplant recipients for infection by PERV. Clin Biochem 2001; 34:23-7. [PMID: 11239511 DOI: 10.1016/s0009-9120(00)00187-9] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
OBJECTIVES Concerns have been raised over the possibility of transmission of porcine endogenous retrovirus (PERV) to porcine xenograft recipients. METHODS To help assess this risk, diagnostic assays capable of detection of an active, latent or cleared PERV infection, and the presence of pig cell microchimerism have been developed by a number of groups. Retrospective studies of patients exposed to living pig tissues have been performed using these assays to look for evidence of cross species transmission. RESULTS To date no evidence of PERV infection has been found in studies of humans exposed to pig tissues, despite evidence of long lived microchimerism. CONCLUSIONS These data suggest that PERV infection has not occurred in a clinical setting. However, as infection has been seen in a small animal model further investigation of the risk from PERV is warranted.
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Affiliation(s)
- C Herring
- Porcine Endogenous Retrovirus Research Group, Imutran Ltd (A Novartis Pharma AG Co), PO Box 399, CB2 2YP, Cambridge, UK.
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Abstract
Xenotransplantation, the transplantation of living organs, tissues, or cells from one species to another, is viewed as a potential solution to the existing shortage of human organs for transplantation. While whole-organ xenotransplantation is still in the preclinical stage, cellular xenotransplantation and extracorporeal perfusion applications are showing promise in early clinical trials. Advances in immunosuppressive therapy, gene engineering, and cloning of animals bring a broader array of xenotransplantation protocols closer to clinical trials. Despite several potential advantages over allotransplantation, xenotransplantation encompasses a number of problems. Immunologic rejection remains the primary hindrance. The potential to introduce infections across species barriers, another major concern, is the main focus of this review. Nonhuman primates are unlikely to be a main source for xenotransplantation products despite their phylogenetic proximity to humans. Genetically engineered pigs, bred under special conditions, are currently envisaged as the major source. Thus far, there has been no evidence for human infections caused by pig xenotransplantation products. However, the existence of xenotropic endogenous retroviruses and the clinical evidence of long-lasting porcine cell microchimerism indicate the potential for xenogeneic infections. Thus, further trials should continue under regulatory oversight, with close clinical and laboratory monitoring for potential xenogeneic infections.
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Affiliation(s)
- R S Boneva
- HIV/AIDS and Retrovirology Branch, Division of AIDS, STD and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
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48
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Powell SK, Gates ME, Langford G, Gu ML, Lockey C, Long Z, Otto E. Antiretroviral agents inhibit infection of human cells by porcine endogenous retroviruses. Antimicrob Agents Chemother 2000; 44:3432-3. [PMID: 11083652 PMCID: PMC90217 DOI: 10.1128/aac.44.12.3432-3433.2000] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The efficacy of antiretroviral drugs against porcine endogenous retroviruses (PERV) that may be harbored in pig organs intended for transplantation was examined in human cells in vitro. The nucleoside analogs zidovudine and dideoxyinosine were found to effectively inhibit PERV replication.
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Affiliation(s)
- S K Powell
- Genetic Therapy, Inc., a Novartis Company, Gaithersburg, Maryland 20878, USA
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49
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Deng YM, Tuch BE, Rawlinson WD. Transmission of porcine endogenous retroviruses in severe combined immunodeficient mice xenotransplanted with fetal porcine pancreatic cells. Transplantation 2000; 70:1010-6. [PMID: 11045635 DOI: 10.1097/00007890-200010150-00004] [Citation(s) in RCA: 96] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
BACKGROUND Xenotransplantation using pig organs or tissues may alleviate the human donor organ shortage. However, one concern is the potential transmission of pig pathogens to humans, especially pig endogenous retroviruses (PERV), which infect human cell lines in vitro. In this report, the cross-species in vivo transmission of PERV by xenotransplantation was studied using a severe combined immunodeficient (SCID) mouse model. METHODS Twenty-one SCID mice were transplanted with fetal pig pancreatic cells and left for periods from three to 41 weeks before being killed. DNA and RNA were extracted from liver, spleen, and brain of these mice, and examined for PERV using nested polymerase chain reaction (PCR) and reverse transcriptase-PCR. The pig mitochondrial cytochrome oxidase II subunit gene (COII) was also amplified to monitor the presence of pig cell microchimerism in xenotransplanted tissues, and a housekeeping gene was included to monitor the DNA quality and quantity. RESULTS Examination of 39 DNA samples from tissues of the 21 xenografted mice identified two mouse tissues (M4-liver and M19-spleen) that were positive for PERV but negative for COII. A total of 23 (59%) of the mouse tissues were positive for both PERV and COII, 6 (16%) were negative for both, and 8 (20%) were positive for COII only. PCR and direct sequencing of the PCR products identified three PERV variants, which were different from the PERV sequence detected by PCR direct sequencing from the pig donor cells. CONCLUSIONS The PERV+/COII- results from M4-liver and M19-spleen indicated the presence of PERV transmission from pig to mouse tissue. The PERV variants detected in the mouse tissues indicated that different PERVs were transmissible from the pig to mouse tissue during xenotransplantation. The negative reverse transcriptase-PCR results for PERV from three mouse samples including M4-liver and M19-spleen suggest there was no active PERV transcription in the mouse tissues, although this would need to be studied further.
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Affiliation(s)
- Y M Deng
- Department of Endocrinology, SEALS, Prince of Wales Hospital, Sydney, NSW, Australia
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50
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Abstract
The shortage of cadaveric human organs for transplantation may, be alleviated by the use of xenografts as a therapeutic option for end-stage organ failure. Successful attempts have been made to prevent rejection of xenograft tissues in humans. The potential spread of animal-derived pathogens to the xenograft recipient is a complication of xenotransplantation, which must be addressed. This can be complicated further by, the presence of new pathogens, new clinical syndromes, and altered behaviour of these organisms in the immunocompromised recipient. There is concern over the possible activation of latent viruses, including retroviruses, from xenograft tissues. This paper discusses the possible dangers of transmission of animal viruses to humans via xenotransplantation.
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Affiliation(s)
- D K Langat
- Institute of Primate Research, National Museums of Kenya, Karen, Nairobi
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