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1
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Lee S, Dohlman TH, Dana R. Immunology in corneal transplantation-From homeostasis to graft rejection. Transplant Rev (Orlando) 2025; 39:100909. [PMID: 39798206 PMCID: PMC11975484 DOI: 10.1016/j.trre.2025.100909] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2024] [Revised: 12/19/2024] [Accepted: 01/08/2025] [Indexed: 01/15/2025]
Abstract
Immunology depends on maintaining a delicate balance within the human body, and disruptions can result in conditions such as autoimmune diseases, immunodeficiencies, and hypersensitivity reactions. This balance is especially crucial in transplantation immunology, where one of the primary challenges is preventing graft rejection. Such rejection can lead to organ failure, increased patient mortality, and higher healthcare costs due to the limited availability of donor tissues relative to patient needs. Xenotransplantation, like using porcine corneas for human transplants, offers a potential solution to the donor tissue shortage but faces substantial immunological rejection issues. To prevent rejection in both allo- and xenotransplantation, a deep understanding of how the body maintains immunological balance is essential, particularly since achieving tolerance to non-self tissues is considered the "holy grail" of the field. The cornea, the most frequently transplanted solid organ, has a high acceptance rate due to its immune-privileged status and serves as an ideal model for studying graft rejection mechanisms that disrupt tolerance. However, multiple immune pathways complicate our understanding of these mechanisms. This review examines the rejection mechanisms in corneal transplantation, identifying key cells involved and potential therapeutic strategies to induce and maintain immunological tolerance in both allo- and xenografts across various transplants.
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Affiliation(s)
- Seokjoo Lee
- Laboratory of Ocular Immunology, Transplantation, and Regeneration, Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA
| | - Thomas H Dohlman
- Laboratory of Ocular Immunology, Transplantation, and Regeneration, Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA
| | - Reza Dana
- Laboratory of Ocular Immunology, Transplantation, and Regeneration, Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.
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2
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Zor F, Kapaj R, Kulahci Y, Karslioglu Y, Gorantla VS. Composite tissue xenopreservation: Preliminary results of staged VCA in rat to mouse model. Microsurgery 2023; 43:823-830. [PMID: 37354047 DOI: 10.1002/micr.31079] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2023] [Revised: 05/02/2023] [Accepted: 06/16/2023] [Indexed: 06/26/2023]
Abstract
BACKGROUND The time between procurement and transplantation of composite tissues, especially regarding the limited donor pool, is a challenge effecting the outcomes of the transplantation. Current preservation techniques mainly include either cold preservation with a solution or machine perfusion using blood or certain oxygen-carrying solutions. However, none enables preservation beyond 24 h. Increasing this time to several days will provide better usage of the donor pool, safer transplantation of VCA with significant muscle content, and gives time to stabilize a patient before long surgical procedures. Herein, we described a novel strategy of xenopreservation (preservation via xenotransplantation) to preserve composite tissues for 7 days, followed by staged transplantation. MATERIALS AND METHODS We used two concordant species, female Sprague Dawley rats (n = 10) and female CF-1 mice (n = 10) in this study. Four of pair of animals are used for anatomical study. The groin flap of the rat was used as a xenograft and xenotransplanted to the neck area of the carrier mouse. Cyclosporine (CsA) was administered used as immunosuppressant. After 7 days of preservation on the mouse neck, xenotransplanted groin flap (called xenopreserved flap) was re-harvested, skin and vessels samples were collected for histopathological evaluation, and the xenopreserved flap was transplanted to the donor rat's opposite groin area. Anastomoses were performed between the flap's pedicle and the femoral vessels. Clinical observation regarding inflammation and tissue perfusion of the xenopreserved flap was monitored daily. Fifteen days after the second surgical procedure, the rats were euthanized, and skin and vessel samples were collected. Histologic evaluation, including inflammatory cell numbers, was performed. Wilcoxon test was used to compare the changes in inflammation severity and p < .05 was set for statistical significance. RESULTS All xenopreserved groin flaps except one survived. Mean lymphocyte count before the second operation (at the end of the xenopreservation procedure) was 20,22 ± 0.44 and reduced to 13,14 ± 0.47 at the end of 15 days, and the difference was statistically significant (p < .05). CONCLUSION This proof-of-concept study with preliminary results showed that xenotransplantation might be a novel strategy for preservation of VCA for a certain period of time. However, additional translational studies are needed to modulate the tissue changes following xenopreservation.
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Affiliation(s)
- Fatih Zor
- Department of Surgery, Wake Forest University Health Sciences, Wake Forest Institute for Regenerative Medicine, Winston Salem, North Carolina, USA
- Department of Plastic and Reconstructive Surgery, Gulhane Military Medical Academy, Ankara, Turkey
| | - Rezarta Kapaj
- Department of Plastic and Reconstructive Surgery, Gulhane Military Medical Academy, Ankara, Turkey
| | - Yalcin Kulahci
- Department of Surgery, Wake Forest University Health Sciences, Wake Forest Institute for Regenerative Medicine, Winston Salem, North Carolina, USA
- Department of Hand and Upper Extremity Surgery, Gulhane Military Medical Academy, Ankara, Turkey
| | | | - Vijay S Gorantla
- Department of Surgery, Wake Forest University Health Sciences, Wake Forest Institute for Regenerative Medicine, Winston Salem, North Carolina, USA
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Baik J, Ortiz-Cordero C, Magli A, Azzag K, Crist SB, Yamashita A, Kiley J, Selvaraj S, Mondragon-Gonzalez R, Perrin E, Maufort JP, Janecek JL, Lee RM, Stone LH, Rangarajan P, Ramachandran S, Graham ML, Perlingeiro RCR. Establishment of Skeletal Myogenic Progenitors from Non-Human Primate Induced Pluripotent Stem Cells. Cells 2023; 12:1147. [PMID: 37190056 PMCID: PMC10137227 DOI: 10.3390/cells12081147] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Revised: 04/01/2023] [Accepted: 04/06/2023] [Indexed: 05/17/2023] Open
Abstract
Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied.
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Affiliation(s)
- June Baik
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | | | - Alessandro Magli
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | - Karim Azzag
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | - Sarah B. Crist
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | - Aline Yamashita
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | - James Kiley
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | - Sridhar Selvaraj
- Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
| | | | - Elizabeth Perrin
- Stem Cell Resources and the Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI 53715, USA
| | - John P. Maufort
- Stem Cell Resources and the Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI 53715, USA
| | - Jody L. Janecek
- Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA
| | - Rachael M. Lee
- Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA
| | - Laura Hocum Stone
- Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA
| | | | | | - Melanie L. Graham
- Department of Surgery, University of Minnesota, Minneapolis, MN 55455, USA
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Rodrigues Costa M, Fischer N, Gronewold A, Gulich B, Godehardt AW, Tönjes RR. Isolation of an Ecotropic Porcine Endogenous Retrovirus PERV-C from a Yucatan SLA D/D Inbred Miniature Swine. J Virol 2023; 97:e0006223. [PMID: 36883860 PMCID: PMC10062142 DOI: 10.1128/jvi.00062-23] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2023] [Accepted: 02/06/2023] [Indexed: 03/09/2023] Open
Abstract
Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, confirming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 5'-proviral flanking sequences. Full-length PCR using 5'-and 3'-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals.
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Affiliation(s)
| | - Nicole Fischer
- Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany
| | - Antonia Gronewold
- Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany
| | - Barbara Gulich
- Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany
| | - Antonia W. Godehardt
- Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany
| | - Ralf R. Tönjes
- Division of Hematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, Langen, Germany
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5
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Choe YH, Sorensen J, Garry DJ, Garry MG. Blastocyst complementation and interspecies chimeras in gene edited pigs. Front Cell Dev Biol 2022; 10:1065536. [PMID: 36568986 PMCID: PMC9773398 DOI: 10.3389/fcell.2022.1065536] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2022] [Accepted: 11/17/2022] [Indexed: 12/13/2022] Open
Abstract
The only curative therapy for many endstage diseases is allograft organ transplantation. Due to the limited supply of donor organs, relatively few patients are recipients of a transplanted organ. Therefore, new strategies are warranted to address this unmet need. Using gene editing technologies, somatic cell nuclear transfer and human induced pluripotent stem cell technologies, interspecies chimeric organs have been pursued with promising results. In this review, we highlight the overall technical strategy, the successful early results and the hurdles that need to be addressed in order for these approaches to produce a successful organ that could be transplanted in patients with endstage diseases.
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Affiliation(s)
- Yong-ho Choe
- Lillehei Heart Institute, University of Minnesota, Minneapolis, MN, United States
- Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, MN, United States
| | - Jacob Sorensen
- Lillehei Heart Institute, University of Minnesota, Minneapolis, MN, United States
- Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, MN, United States
| | - Daniel J. Garry
- Lillehei Heart Institute, University of Minnesota, Minneapolis, MN, United States
- Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, MN, United States
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States
- Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN, United States
| | - Mary G. Garry
- Lillehei Heart Institute, University of Minnesota, Minneapolis, MN, United States
- Cardiovascular Division, Department of Medicine, University of Minnesota, Minneapolis, MN, United States
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, United States
- Paul and Sheila Wellstone Muscular Dystrophy Center, University of Minnesota, Minneapolis, MN, United States
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Zhou Q, Li T, Wang K, Zhang Q, Geng Z, Deng S, Cheng C, Wang Y. Current status of xenotransplantation research and the strategies for preventing xenograft rejection. Front Immunol 2022; 13:928173. [PMID: 35967435 PMCID: PMC9367636 DOI: 10.3389/fimmu.2022.928173] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2022] [Accepted: 07/07/2022] [Indexed: 12/13/2022] Open
Abstract
Transplantation is often the last resort for end-stage organ failures, e.g., kidney, liver, heart, lung, and pancreas. The shortage of donor organs is the main limiting factor for successful transplantation in humans. Except living donations, other alternatives are needed, e.g., xenotransplantation of pig organs. However, immune rejection remains the major challenge to overcome in xenotransplantation. There are three different xenogeneic types of rejections, based on the responses and mechanisms involved. It includes hyperacute rejection (HAR), delayed xenograft rejection (DXR) and chronic rejection. DXR, sometimes involves acute humoral xenograft rejection (AHR) and cellular xenograft rejection (CXR), which cannot be strictly distinguished from each other in pathological process. In this review, we comprehensively discussed the mechanism of these immunological rejections and summarized the strategies for preventing them, such as generation of gene knock out donors by different genome editing tools and the use of immunosuppressive regimens. We also addressed organ-specific barriers and challenges needed to pave the way for clinical xenotransplantation. Taken together, this information will benefit the current immunological research in the field of xenotransplantation.
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Affiliation(s)
- Qiao Zhou
- Department of Rheumatology and Immunology, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, China
- Clinical Immunology Translational Medicine Key Laboratory of Sichuan Province, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, China
- Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu, China
| | - Ting Li
- Department of Rheumatology, Wenjiang District People’s Hospital, Chengdu, China
| | - Kaiwen Wang
- School of Medicine, Faculty of Medicine and Health, The University of Leeds, Leeds, United Kingdom
| | - Qi Zhang
- School of Medicine, University of Electronics and Technology of China, Chengdu, China
| | - Zhuowen Geng
- School of Medicine, Faculty of Medicine and Health, The University of Leeds, Leeds, United Kingdom
| | - Shaoping Deng
- Clinical Immunology Translational Medicine Key Laboratory of Sichuan Province, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, China
- Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu, China
- Institute of Organ Transplantation, Sichuan Academy of Medical Science and Sichuan Provincial People’s Hospital, Chengdu, China
| | - Chunming Cheng
- Department of Radiation Oncology, James Comprehensive Cancer Center and College of Medicine at The Ohio State University, Columbus, OH, United States
- *Correspondence: Chunming Cheng, ; Yi Wang,
| | - Yi Wang
- Department of Critical Care Medicine, Sichuan Academy of Medical Science and Sichuan Provincial People's Hospital, Chengdu, China
- *Correspondence: Chunming Cheng, ; Yi Wang,
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7
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Eisenson DL, Hisadome Y, Yamada K. Progress in Xenotransplantation: Immunologic Barriers, Advances in Gene Editing, and Successful Tolerance Induction Strategies in Pig-To-Primate Transplantation. Front Immunol 2022; 13:899657. [PMID: 35663933 PMCID: PMC9157571 DOI: 10.3389/fimmu.2022.899657] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2022] [Accepted: 04/20/2022] [Indexed: 02/05/2023] Open
Abstract
Organ transplantation is the most effective treatment for end stage organ failure, but there are not enough organs to meet burgeoning demand. One potential solution to this organ shortage is xenotransplantation using pig tissues. Decades of progress in xenotransplantation, accelerated by the development of rapid genome editing tools, particularly the advent of CRISPR-Cas9 gene editing technologies, have enabled remarkable advances in kidney and heart xenotransplantation in pig-to-nonhuman primates. These breakthroughs in large animal preclinical models laid the foundation for three recent pig-to-human transplants by three different groups: two kidney xenografts in brain dead recipients deemed ineligible for transplant, and one heart xenograft in the first clinical grade study of pig-to-human transplantation. However, despite tremendous progress, recent data including the first clinical case suggest that gene-modification alone will not overcome all xenogeneic immunologic barriers, and thus an active and innovative immunologic strategy is required for successful xenotransplantation. This review highlights xenogeneic immunologic barriers, advances in gene editing, and tolerance-inducing strategies in pig-to-human xenotransplantation.
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Affiliation(s)
- Daniel L Eisenson
- Columbia Center for Translational Immunology, Department of Medicine, Columbia University Irving Medical Center, New York, NY, United States.,Department of Surgery, The Johns Hopkins Hospital, Baltimore, MD, United States
| | - Yu Hisadome
- Columbia Center for Translational Immunology, Department of Medicine, Columbia University Irving Medical Center, New York, NY, United States
| | - Kazuhiko Yamada
- Columbia Center for Translational Immunology, Department of Medicine, Columbia University Irving Medical Center, New York, NY, United States.,Department of Surgery, Columbia University Irving Medical Center, New York, NY, United States
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8
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Birdwhistell KE, Hurley DJ, Heins B, Peroni JF. Evaluation of equine xenogeneic mixed lymphocyte reactions using 5-ethynyl-2'-deoxyuridine (EdU). Vet Immunol Immunopathol 2022; 249:110430. [PMID: 35525064 DOI: 10.1016/j.vetimm.2022.110430] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Revised: 04/29/2022] [Accepted: 05/01/2022] [Indexed: 11/19/2022]
Abstract
Allogeneic solid organ transplantation is currently the only treatment option for end stage organ disease. The shortage of available donor organs has driven efforts to utilize xenogeneic organs for transplantation. In vitro methods for evaluating immune-compatibility are a quick and low cost means of screening novel tissue products prior to more involved, expensive, and invasive live animal studies. Recently, a new analog of the DNA base thymidine, 5-ethynyl-2'-deoxyuridine (EdU), was developed. It may be used in a fast, efficient and specific means of evaluating cell proliferation via flow cytometry. This study was designed to test and optimize this platform for assessing equine xenogeneic one-way mixed lymphocyte reaction (MLR) to porcine stimulator cells. Furthermore, it was hypothesized that an enriched T-lymphocyte (T-cell) population would generate a stronger proliferative response to stimulation, and higher levels of cytokine production when compared to unfractionated peripheral blood mononuclear cells (PBMCs). PBMCs and T-cells were isolated from 3 horses and 4 pigs. Equine xenogeneic MLRs were set up using porcine allogeneic MLRs as a reference for clinically acceptable levels of cell proliferation. Equine T-cells showed significantly greater EdU incorporation in one-way xenogeneic MLRs than equine PBMCs. However, there was no significant difference in cell proliferation between porcine T-cell and PBMC as responders in allogenic one-way MLRs. Given the results of this study, we consider that enriched equine T-cells should be used in preference to unfractionated PBMCs when attempting to evaluate the equine xenogeneic response using the EdU assay as an indicator of suitability for transplant in vivo.
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Affiliation(s)
- Kate E Birdwhistell
- Department of Large Animal Medicine and Surgery, University of Georgia College of Veterinary Medicine, 2200 College Station Rd, Athens, GA 30602, USA.
| | - David J Hurley
- Department of Population Health, University of Georgia College of Veterinary Medicine, 2200 College Station Rd, Athens, GA 30602, USA
| | - Bradley Heins
- Department of Population Health, University of Georgia College of Veterinary Medicine, 2200 College Station Rd, Athens, GA 30602, USA
| | - John F Peroni
- Department of Large Animal Medicine and Surgery, University of Georgia College of Veterinary Medicine, 2200 College Station Rd, Athens, GA 30602, USA
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Tao C, Zhu W, Iqbal J, Xu C, Wang DA. Stabilized albumin coatings on engineered xenografts for attenuation of acute immune and inflammatory responses. J Mater Chem B 2021; 8:6080-6091. [PMID: 32555888 DOI: 10.1039/d0tb01111h] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Xenogeneic grafts are promising candidates for transplantation therapy due to their easily accessible sources. Nevertheless, the immune and inflammatory responses induced by xenografts need to be addressed for clinical use. A novel and facile method was introduced for the attenuation of immune and inflammatory responses by extending the immune evasion potential of albumin to the tissue engineering field and coating albumin, which could passivate biomaterial surfaces, onto xenografts. Albumin was first modified by dopamine to enhance its adhesion on graft surfaces. Porcine chondrocytes derived living hyaline cartilage graft (LhCG) and decellularized LhCG (dLhCG) were applied as xenograft models implanted in the omentum of rats. Both LhCG which contained porcine chondrocytes as well as secreted ECM and dLhCG which was mainly composed of the porcine source ECM showed alleviated immune and inflammatory responses after being coated with albumin at cell, protein and gene levels, respectively. Significantly less inflammatory cells including neutrophils, macrophages and lymphocytes were recruited according to pathological analysis and immunohistochemistry staining with lower gene expression encoding inflammation-related cytokines including MCP-1, IL-6 and IL-1β after employing LhCG and dLhCG with albumin passivation coating.
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Affiliation(s)
- Chao Tao
- School of Chemical and Biomedical Engineering, Nanyang Technological University, 50 Nanyang Ave, 639798, Singapore
| | - Wenzhen Zhu
- School of Chemical and Biomedical Engineering, Nanyang Technological University, 50 Nanyang Ave, 639798, Singapore
| | - Jabed Iqbal
- Department of Pathology, Singapore General Hospital, 20 College Road, Academia, Diagnostics Tower, Level 10, Singapore 169856, Singapore
| | - Chenjie Xu
- School of Chemical and Biomedical Engineering, Nanyang Technological University, 50 Nanyang Ave, 639798, Singapore and City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong.
| | - Dong-An Wang
- City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong.
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10
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Black C, Kanczler JM, de Andrés MC, White LJ, Savi FM, Bas O, Saifzadeh S, Henkel J, Zannettino A, Gronthos S, Woodruff MA, Hutmacher DW, Oreffo ROC. Characterisation and evaluation of the regenerative capacity of Stro-4+ enriched bone marrow mesenchymal stromal cells using bovine extracellular matrix hydrogel and a novel biocompatible melt electro-written medical-grade polycaprolactone scaffold. Biomaterials 2020; 247:119998. [PMID: 32251928 PMCID: PMC7184676 DOI: 10.1016/j.biomaterials.2020.119998] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2019] [Revised: 03/20/2020] [Accepted: 03/20/2020] [Indexed: 12/15/2022]
Abstract
Many skeletal tissue regenerative strategies centre around the multifunctional properties of bone marrow derived stromal cells (BMSC) or mesenchymal stem/stromal cells (MSC)/bone marrow derived skeletal stem cells (SSC). Specific identification of these particular stem cells has been inconclusive. However, enriching these heterogeneous bone marrow cell populations with characterised skeletal progenitor markers has been a contributing factor in successful skeletal bone regeneration and repair strategies. In the current studies we have isolated, characterised and enriched ovine bone marrow mesenchymal stromal cells (oBMSCs) using a specific antibody, Stro-4, examined their multipotential differentiation capacity and, in translational studies combined Stro-4+ oBMSCs with a bovine extracellular matrix (bECM) hydrogel and a biocompatible melt electro-written medical-grade polycaprolactone scaffold, and tested their bone regenerative capacity in a small in vivo, highly vascularised, chick chorioallantoic membrane (CAM) model and a preclinical, critical-sized ovine segmental tibial defect model. Proliferation rates and CFU-F formation were similar between unselected and Stro-4+ oBMSCs. Col1A1, Col2A1, mSOX-9, PPARG gene expression were upregulated in respective osteogenic, chondrogenic and adipogenic culture conditions compared to basal conditions with no significant difference between Stro-4+ and unselected oBMSCs. In contrast, proteoglycan expression, alkaline phosphatase activity and adipogenesis were significantly upregulated in the Stro-4+ cells. Furthermore, with extended cultures, the oBMSCs had a predisposition to maintain a strong chondrogenic phenotype. In the CAM model Stro-4+ oBMSCs/bECM hydrogel was able to induce bone formation at a femur fracture site compared to bECM hydrogel and control blank defect alone. Translational studies in a critical-sized ovine tibial defect showed autograft samples contained significantly more bone, (4250.63 mm3, SD = 1485.57) than blank (1045.29 mm3, SD = 219.68) ECM-hydrogel (1152.58 mm3, SD = 191.95) and Stro-4+/ECM-hydrogel (1127.95 mm3, SD = 166.44) groups. Stro-4+ oBMSCs demonstrated a potential to aid bone repair in vitro and in a small in vivo bone defect model using select scaffolds. However, critically, translation to a large related preclinical model demonstrated the complexities of bringing small scale reported stem-cell material therapies to a clinically relevant model and thus facilitate progression to the clinic.
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Affiliation(s)
- C Black
- Bone & Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Human Development & Health, Institute of Developmental Sciences, University of Southampton, SO16 6YD, UK
| | - J M Kanczler
- Bone & Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Human Development & Health, Institute of Developmental Sciences, University of Southampton, SO16 6YD, UK
| | - M C de Andrés
- Bone & Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Human Development & Health, Institute of Developmental Sciences, University of Southampton, SO16 6YD, UK; Cartilage Epigenetics Group, Rheumatology Division, Biomedical Research Institute of A Coruña (INIBIC), Hospital Universitario de A Coruña-CHUAC, 15006 A Coruña ,Spain
| | - L J White
- School of Pharmacy, Biodiscovery Institute, University Park, University of Nottingham, Nottingham, NG7 2RD, UK
| | - F M Savi
- ARC Industrial Transformation Training Centre in Additive Biomanufacturing, Queensland University of Technology (QUT), Brisbane, QLD, 4059, Australia; Institute of Health Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, 4059, Australia
| | - O Bas
- ARC Industrial Transformation Training Centre in Additive Biomanufacturing, Queensland University of Technology (QUT), Brisbane, QLD, 4059, Australia; Institute of Health Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, 4059, Australia
| | - S Saifzadeh
- ARC Industrial Transformation Training Centre in Additive Biomanufacturing, Queensland University of Technology (QUT), Brisbane, QLD, 4059, Australia
| | - J Henkel
- ARC Industrial Transformation Training Centre in Additive Biomanufacturing, Queensland University of Technology (QUT), Brisbane, QLD, 4059, Australia
| | - A Zannettino
- Myeloma Research Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia and Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, Australia and Central Adelaide Local Health Network, Adelaide, South Australia, Australia
| | - S Gronthos
- Mesenchymal Stem Cell Laboratory, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, Australia and Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, Australia
| | - M A Woodruff
- ARC Industrial Transformation Training Centre in Additive Biomanufacturing, Queensland University of Technology (QUT), Brisbane, QLD, 4059, Australia
| | - D W Hutmacher
- ARC Industrial Transformation Training Centre in Additive Biomanufacturing, Queensland University of Technology (QUT), Brisbane, QLD, 4059, Australia; Institute of Health Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, 4059, Australia
| | - R O C Oreffo
- Bone & Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Human Development & Health, Institute of Developmental Sciences, University of Southampton, SO16 6YD, UK; College of Biomedical Engineering, China Medical University, Taichung, 40402, Taiwan.
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11
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Kim JK, Jang MJ, Kim BH, Choi KR, Song G, Shin HC, Suh N, Kim YM, You D, Ahn TY, Kim CS. Establishment of NOAEL for intracavernous injections of human bone marrow-derived mesenchymal stem cells in rats. Investig Clin Urol 2019; 61:88-98. [PMID: 31942468 PMCID: PMC6946817 DOI: 10.4111/icu.2020.61.1.88] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2019] [Accepted: 08/21/2019] [Indexed: 01/09/2023] Open
Abstract
Purpose To assess the possible negative health effects of human bone marrow-derived mesenchymal stem cells (hBMSCs) on fertility and early embryonic development following intracavernous injections in rats. Materials and Methods A total of 88 Crl:CD(SD) male and female rats were equally divided into 4 groups in a random manner: control group (normal saline), low-dose group (2×105 hBMSCs), moderate-dose group (1×106 hBMSCs), and high-dose group (2×106 hBMSCs). hBMSCs or normal saline was injected into the penis of the rats 3 times at 2-week-intervals prior to mating. We compared each group with respect to parameters of reproduction and histopathology. Results For male rats, various degrees of flushing and swelling were observed at the penile injection site in all the groups, although the severity increased in a dose-dependent manner in the hBMSC injection groups. There were no statistically significant differences in mean body weights and food consumption among all the groups of both sexes. There were no statistically significant differences in reproductive parameters among all the groups of both sexes. The absolute and relative organ weights did not significantly differ among the groups. At the time of necropsy, no remarkable findings were observed in gross examinations in all groups. On histopathological analysis, minimal mononuclear cell infiltration was observed in the right epididymis of each rat in the moderate- and high-dose groups. Conclusions The non-toxic amount of hBMSCs for male fertility and early embryogenesis in rats under the test conditions was determined to be 2×106 cells/head.
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Affiliation(s)
- Jong Keun Kim
- Department of Urology, Hallym University Dongtan Sacred Heart Hospital, Hwaseong, Korea
| | - Myoung Jin Jang
- Asan Institute for Life Science, Asan Medical Center, Seoul, Korea
| | - Bo Hyun Kim
- Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | | | - Geehyun Song
- Department of Urology, Kangwon National University School of Medicine, Chuncheon, Korea
| | | | - Nayoung Suh
- Department of Pharmaceutical Engineering, College of Medical Sciences, Soon Chun Hyang University, Asan, Korea
| | | | - Dalsan You
- Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Tai Young Ahn
- Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
| | - Choung-Soo Kim
- Department of Urology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea
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12
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Platt JL, Cascalho M, Piedrahita JA. Xenotransplantation: Progress Along Paths Uncertain from Models to Application. ILAR J 2019; 59:286-308. [PMID: 30541147 DOI: 10.1093/ilar/ily015] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2017] [Revised: 08/23/2018] [Indexed: 12/18/2022] Open
Abstract
For more than a century, transplantation of tissues and organs from animals into man, xenotransplantation, has been viewed as a potential way to treat disease. Ironically, interest in xenotransplantation was fueled especially by successful application of allotransplantation, that is, transplantation of human tissue and organs, as a treatment for a variety of diseases, especially organ failure because scarcity of human tissues limited allotransplantation to a fraction of those who could benefit. In principle, use of animals such as pigs as a source of transplants would allow transplantation to exert a vastly greater impact than allotransplantation on medicine and public health. However, biological barriers to xenotransplantation, including immunity of the recipient, incompatibility of biological systems, and transmission of novel infectious agents, are believed to exceed the barriers to allotransplantation and presently to hinder clinical applications. One way potentially to address the barriers to xenotransplantation is by genetic engineering animal sources. The last 2 decades have brought progressive advances in approaches that can be applied to genetic modification of large animals. Application of these approaches to genetic engineering of pigs has contributed to dramatic improvement in the outcome of experimental xenografts in nonhuman primates and have encouraged the development of a new type of xenograft, a reverse xenograft, in which human stem cells are introduced into pigs under conditions that support differentiation and expansion into functional tissues and potentially organs. These advances make it appropriate to consider the potential limitation of genetic engineering and of current models for advancing the clinical applications of xenotransplantation and reverse xenotransplantation.
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Affiliation(s)
- Jeffrey L Platt
- Surgery, Microbiology & Immunology, and Transplantation Biology, University of Michigan, Ann Arbor, Michigan
| | - Marilia Cascalho
- Surgery, Microbiology & Immunology, and Transplantation Biology, University of Michigan, Ann Arbor, Michigan
| | - Jorge A Piedrahita
- Translational Medicine and The Comparative Medicine Institute, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina
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13
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Breimer ME, Holgersson J. The Structural Complexity and Animal Tissue Distribution of N-Glycolylneuraminic Acid (Neu5Gc)-Terminated Glycans. Implications for Their Immunogenicity in Clinical Xenografting. Front Mol Biosci 2019; 6:57. [PMID: 31428616 PMCID: PMC6690001 DOI: 10.3389/fmolb.2019.00057] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Accepted: 07/04/2019] [Indexed: 12/13/2022] Open
Abstract
N-Glycolylneuraminic acid (Neu5Gc)-terminated glycans are present in all animal cells/tissues that are already used in the clinic such as bioprosthetic heart valves (BHV) as well as in those that potentially will be xenografted in the future to overcome end stage cell/organ failure. Humans, as a species lack this antigen determinant and can react with an immune response after exposure to Neu5Gc present in these products/cells/tissues. Genetically engineered source animals lacking Neu5Gc has been generated and so has animals that in addition lack the major αGal xenoantigen. The use of cells/tissues/organs from such animals may improve the long-term performance of BHV and allow future xenografting. This review summarizes the present knowledge regarding structural complexity and tissue distribution of Neu5Gc on glycans of cells/tissue/organs already used in the clinic or intended for treatment of end stage organ failure by xenografting. In addition, we briefly discuss the role of anti-Neu5Gc antibodies in the xenorejection process and how knowledge about Neu5Gc structural complexity can be used to design novel diagnostics for anti-Neu5Gc antibody detection.
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Affiliation(s)
- Michael E Breimer
- Department of Surgery, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
| | - Jan Holgersson
- Laboratory Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
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14
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Chen YF, Yang X, Yang HJ. Heterologous Antibodies Adsorption in Xenotransplantation of a Landrace Piglet Kidney Into a Rhesus Monkey. Transplant Proc 2019; 51:987-992. [PMID: 30979492 DOI: 10.1016/j.transproceed.2019.01.032] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2018] [Accepted: 01/04/2019] [Indexed: 11/29/2022]
Abstract
BACKGROUND To explore the adsorption of heterologous antibodies in 6 xenotransplants of Landrace piglet kidneys into rhesus monkeys. METHODS The Landrace piglets and rhesus monkeys were used as donors and recipients, respectively. The donor kidney was the left kidney excised from each Landrace piglet and lavaged with University of Wisconsin solution through the renal artery and vein ex vivo. The renal arteriovenous end of the recipient was preserved. After anastomosis of the renal artery and vein with the arteriovenous end of the recipient for reperfusion, a cross-lymphocyte cytotoxicity test of the heterogeneous kidney was performed. RESULTS All 6 Landrace piglet kidneys absorbed heterologous antibodies that were pre-existing in the rhesus macaques' kidneys. The cross-lymphocyte toxicity test was performed after the kidney were completely blackened. The cross-lymphocyte toxicity in all each heterogeneous kidney changed from strong positive to weak positive. CONCLUSIONS Heterologous antibodies were adsorbed in xenotransplants of Landrace piglet kidneys into rhesus monkeys. Xenotransplanted kidney can adsorb heterologous antibodies and consume relevant complements, which is a good model for research of hyperacute rejection in xenotransplantation.
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Affiliation(s)
- Y-F Chen
- Organ Transplant Center and Hepatobiliary Ward 3, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, Sichuan, China
| | - X Yang
- Wenjiang District People's Hospital, Chengdu, Sichuan, China
| | - H-J Yang
- Organ Transplant Center and Hepatobiliary Ward 3, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, Sichuan, China.
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15
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Piscopo NJ, Mueller KP, Das A, Hematti P, Murphy WL, Palecek SP, Capitini CM, Saha K. Bioengineering Solutions for Manufacturing Challenges in CAR T Cells. Biotechnol J 2018; 13:10.1002/biot.201700095. [PMID: 28840981 PMCID: PMC5796845 DOI: 10.1002/biot.201700095] [Citation(s) in RCA: 52] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2017] [Revised: 07/26/2017] [Indexed: 12/13/2022]
Abstract
The next generation of therapeutic products to be approved for the clinic is anticipated to be cell therapies, termed "living drugs" for their capacity to dynamically and temporally respond to changes during their production ex vivo and after their administration in vivo. Genetically engineered chimeric antigen receptor (CAR) T cells have rapidly developed into powerful tools to harness the power of immune system manipulation against cancer. Regulatory agencies are beginning to approve CAR T cell therapies due to their striking efficacy in treating some hematological malignancies. However, the engineering and manufacturing of such cells remains a challenge for widespread adoption of this technology. Bioengineering approaches including biomaterials, synthetic biology, metabolic engineering, process control and automation, and in vitro disease modeling could offer promising methods to overcome some of these challenges. Here, we describe the manufacturing process of CAR T cells, highlighting potential roles for bioengineers to partner with biologists and clinicians to advance the manufacture of these complex cellular products under rigorous regulatory and quality control.
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Affiliation(s)
- Nicole J Piscopo
- Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA
- Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI, USA
| | - Katherine P Mueller
- Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA
- Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI, USA
| | - Amritava Das
- Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA
- Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI, USA
| | - Peiman Hematti
- Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA
- University of Wisconsin Carbone Cancer Center, Madison, WI, USA
| | - William L Murphy
- Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA
- Department of Orthopedics and Rehabilitation, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA
| | - Sean P Palecek
- Department of Chemical and Biological Engineering, University of Wisconsin, Madison, WI, USA
| | - Christian M Capitini
- University of Wisconsin Carbone Cancer Center, Madison, WI, USA
- Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA
| | - Krishanu Saha
- Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA
- Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI, USA
- University of Wisconsin Carbone Cancer Center, Madison, WI, USA
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16
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Li H, Song X, Yang F, Bao H, Lu X, Perez-Campo FM, Zhao J. Application of oligonucleotides to construct a conditional targeting vector for porcine IκBα. Mol Med Rep 2018; 17:653-659. [PMID: 29115518 DOI: 10.3892/mmr.2017.7917] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2016] [Accepted: 03/31/2017] [Indexed: 11/06/2022] Open
Abstract
Conditional gene targeting at porcine IκBα may be a solution to delayed xenograft rejection, the main barrier to xenotransplantation. An oligonucleotide‑based method was applied to construct the vector for conditional targeting of porcine IκBα. This method was free from PCR amplification during the assembling of the different vector elements, avoiding introduction of unwanted mutations. With the help of short double‑stranded DNA fragments produced by annealing oligonucleotides, nondirectional cloning has also been avoided. By making the best of directional cloning, a highly complex targeting vector was built within 3 weeks. The present study also explained why the two recombination‑based methods (recombineering and gateway recombination), although having demonstrated to be highly efficient in constructing ordinary targeting vectors, were not appropriate in this context. The description in the present study of an additional method to efficiently construct targeting vectors is suggested to introduce more flexibility in the field therefore helping to meet the different needs of the researchers.
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Affiliation(s)
- Hegang Li
- College of Animal Science, Qingdao Agricultural University, Qingdao, Shandong 266109, P.R. China
| | - Xiaona Song
- College of Animal Science, Qingdao Agricultural University, Qingdao, Shandong 266109, P.R. China
| | - Feng Yang
- College of Animal Science, Qingdao Agricultural University, Qingdao, Shandong 266109, P.R. China
| | - Hanxun Bao
- Jiaozhou Bureau of Animal Husbandry and Veterinary Medicine, Qingdao, Shandong 266300, P.R. China
| | - Xiaolong Lu
- Jiaozhou Bureau of Animal Husbandry and Veterinary Medicine, Qingdao, Shandong 266300, P.R. China
| | - Flor M Perez-Campo
- Stem Cell Biology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK
| | - Jinshan Zhao
- College of Animal Science, Qingdao Agricultural University, Qingdao, Shandong 266109, P.R. China
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17
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Brevig T, Meyer M, Kristensen T, Zimmer J. Neural Xenotransplantation: Pretreatment of Porcine Embryonic Nigral Tissue with Anti-Gal Antibodies and Complement is not Toxic for the Dopaminergic Neurons. Cell Transplant 2017. [DOI: 10.3727/000000001783986954] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Affiliation(s)
- Thomas Brevig
- Department of Anatomy and Neurobiology, University of Southern Denmark, DK-5000 Odense C, Denmark
- Department of Clinical Immunology, Odense University Hospital, DK-5000 Odense C, Denmark
| | - Morten Meyer
- Department of Anatomy and Neurobiology, University of Southern Denmark, DK-5000 Odense C, Denmark
| | - Tom Kristensen
- Department of Clinical Immunology, Odense University Hospital, DK-5000 Odense C, Denmark
| | - Jens Zimmer
- Department of Anatomy and Neurobiology, University of Southern Denmark, DK-5000 Odense C, Denmark
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18
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Costa C, Pizzolato MC, Shen Y, Wang Y, Fodor WL. CD86 Blockade in Genetically Modified Porcine Cells Delays Xenograft Rejection by Inhibiting T-Cell and NK-Cell Activation. Cell Transplant 2017; 13:75-87. [PMID: 15040608 DOI: 10.3727/000000004772664923] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Porcine xenografts transplanted into primates are rejected in spite of immunosuppression. Identification of the triggering mechanisms and the strategies to overcome them is crucial to achieve long-term graft survival. We hypothesized that porcine CD86 (pCD86) contributes to xenograft rejection by direct activation of host T cells and NK cells. Formerly, we designed the human chimeric molecule hCD152-hCD59 to block pCD86 in cis. To test the efficacy in vivo, we have utilized a pig-to-mouse xenotransplant model. First, we showed that hCD152-hCD59 expression prevents the binding of murine CD28Ig to pCD86 on porcine aortic endothelial cells (PAEC) and dramatically reduces IL-2 secretion by Con A-stimulated mouse splenocytes in coculture. Moreover, IFN-γ secretion by IL-12-stimulated mouse NK cells was averted after coculture with hCD152-hCD59 PAEC. In vivo, control PAEC implanted under the kidney capsule were rapidly rejected (2–4 weeks) in BALB/c and BALB/c SCID mice. Rejection of hCD152-hCD59 PAEC was significantly delayed in both cases. Signs of immune modulation in the hCD152-hCD59-PAEC BALB/c recipients were identified such as early hyporesponsiveness and diminished antibody response. Thus, simply modifying the donor xenogeneic cell can diminish both T cell and NK cell immune responses. We specifically demonstrate that pCD86 contributes to rejection of porcine xenografts.
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MESH Headings
- Animals
- Antigens, CD/analysis
- Antigens, CD/blood
- Antigens, CD/immunology
- Antigens, Differentiation/analysis
- Aorta
- B7-2 Antigen
- CD59 Antigens/analysis
- CTLA-4 Antigen
- Coculture Techniques
- Concanavalin A
- Endothelium, Vascular/immunology
- Endothelium, Vascular/pathology
- Endothelium, Vascular/transplantation
- Graft Rejection/immunology
- Graft Rejection/prevention & control
- Humans
- Interleukin-2/metabolism
- Kidney Transplantation/immunology
- Kidney Transplantation/pathology
- Killer Cells, Natural/immunology
- Lymphocyte Activation/immunology
- Membrane Glycoproteins/blood
- Membrane Glycoproteins/immunology
- Mice
- Mice, Inbred BALB C
- Mice, SCID
- Swine
- T-Lymphocytes/immunology
- Transplantation, Heterologous/immunology
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Affiliation(s)
- Cristina Costa
- Department of Molecular Science, Alexion Pharmaceuticals Inc., 352 Knotter Drive, Cheshire, CT 06410, USA.
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19
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Stevens S. Synthetic Biology in Cell and Organ Transplantation. Cold Spring Harb Perspect Biol 2017; 9:cshperspect.a029561. [PMID: 28003184 DOI: 10.1101/cshperspect.a029561] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The transplantation of cells and organs has an extensive history, with blood transfusion and skin grafts described as some of the earliest medical interventions. The speed and efficiency of the human immune system evolved to rapidly recognize and remove pathogens; the human immune system also serves as a barrier against the transplant of cells and organs from even highly related donors. Although this shows the remarkable effectiveness of the immune system, the engineering of cells and organs that will survive in a host patient over the long term remains a steep challenge. Progress in the understanding of host immune responses to donor cells and organs, combined with the rapid advancement in synthetic biology applications, allows the rational engineering of more effective solutions for transplantation.
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Affiliation(s)
- Sean Stevens
- Mammalian Synthetic Biology, Synthetic Genomics, Inc., La Jolla, California 92037
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20
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Rajalingam R. The Impact of HLA Class I-Specific Killer Cell Immunoglobulin-Like Receptors on Antibody-Dependent Natural Killer Cell-Mediated Cytotoxicity and Organ Allograft Rejection. Front Immunol 2016; 7:585. [PMID: 28066408 PMCID: PMC5165035 DOI: 10.3389/fimmu.2016.00585] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2016] [Accepted: 11/25/2016] [Indexed: 12/21/2022] Open
Abstract
Natural killer (NK) cells of the innate immune system are cytotoxic lymphocytes that play an important roles following transplantation of solid organs and hematopoietic stem cells. Recognition of self-human leukocyte antigen (HLA) class I molecules by inhibitory killer cell immunoglobulin-like receptors (KIRs) is involved in the calibration of NK cell effector capacities during the developmental stage, allowing the subsequent recognition and elimination of target cells with decreased expression of self-HLA class I (due to virus infection or tumor transformation) or HLA class I disparities (in the setting of allogeneic transplantation). NK cells expressing an inhibitory KIR-binding self-HLA can be activated when confronted with allografts lacking a ligand for the inhibitory receptor. Following the response of the adaptive immune system, NK cells can further destroy allograft endothelium by antibody-dependent cell-mediated cytotoxicity (ADCC), triggered through cross-linking of the CD16 Fc receptor by donor-specific antibodies bound to allograft. Upon recognizing allogeneic target cells, NK cells also secrete cytokines and chemokines that drive maturation of dendritic cells to promote cellular and humoral adaptive immune responses against the allograft. The cumulative activating and inhibitory signals generated by ligation of the receptors regulates mature NK cell killing of target cells and their production of cytokines and chemokines. This review summarizes the role of NK cells in allograft rejection and proposes mechanistic concepts that indicate a prominent role for KIR-HLA interactions in facilitating NK cells for Fc receptor-mediated ADCC effector function involved in antibody-mediated rejection of solid organ transplants.
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Affiliation(s)
- Raja Rajalingam
- Immunogenetics and Transplantation Laboratory, Department of Surgery, University of California San Francisco , San Francisco, CA , USA
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21
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Gaudet JM, Hamilton AM, Chen Y, Fox MS, Foster PJ. Application of dual19F and iron cellular MRI agents to track the infiltration of immune cells to the site of a rejected stem cell transplant. Magn Reson Med 2016; 78:713-720. [DOI: 10.1002/mrm.26400] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2016] [Revised: 08/07/2016] [Accepted: 08/09/2016] [Indexed: 01/05/2023]
Affiliation(s)
- Jeffrey M. Gaudet
- Imaging Research Laboratories, Robarts Research Institute; London Ontario Canada
- Department of Medical Biophysics; University of Western Ontario; London Ontario Canada
| | - Amanda M. Hamilton
- Imaging Research Laboratories, Robarts Research Institute; London Ontario Canada
| | - Yuanxin Chen
- Imaging Research Laboratories, Robarts Research Institute; London Ontario Canada
| | - Matthew S. Fox
- Imaging Research Laboratories, Robarts Research Institute; London Ontario Canada
- Department of Medical Biophysics; University of Western Ontario; London Ontario Canada
| | - Paula J. Foster
- Imaging Research Laboratories, Robarts Research Institute; London Ontario Canada
- Department of Medical Biophysics; University of Western Ontario; London Ontario Canada
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22
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Pawelec-Wojtalik M, Mrówczyński W, Wodziński A, Wojtalik M, Henschke J, Sharma GK. Mid-Term Experience with Valved Bovine Jugular Vein Conduits. Asian Cardiovasc Thorac Ann 2016; 13:361-5. [PMID: 16304226 DOI: 10.1177/021849230501300414] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
From June 1999 to January 2004, 43 children underwent implantation of a valved bovine jugular vein conduit and correction of complex congenital heart defects. Median age was 1.98 years (range, 11 days – 13.3 years). There were 7 early deaths (16.3%) unrelated to conduit failure or thrombosis. Median follow-up of 36 survivors was 24 months (range, 1–48 months, quartile range, 12–48 months), total follow-up was 78 patient-years. There were 3 late deaths (8.3%) due to infection, pulmonary thromboembolism, and sudden cardiac arrest after re-operation to repair a right ventricular outflow tract aneurysm. There were 2 conduit explantations due to dysfunction and suspected endocarditis. Three patients underwent balloon dilatation of distal stenoses. The mean peak gradient through the pulmonary anastomosis was 15 mm Hg (range, 3–42 mm Hg) among patients free from re-intervention. No severe valve regurgitation was observed. Freedom from re-intervention was 72% at 48 months. This conduit remains a good alternative to homografts. Causes of distal stenosis must be clarified, guidelines for prophylactic anticoagulation must be created, and the role of percutaneous balloon dilatation established.
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Affiliation(s)
- Malgorzata Pawelec-Wojtalik
- , Department of Pediatric Cardiac Surgery, Poznan University of Medical Sciences, 27/33 Szpitalna, Poznan 60-572, Poland.
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23
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Mahou R, Passemard S, Carvello M, Petrelli A, Noverraz F, Gerber-Lemaire S, Wandrey C. Contribution of polymeric materials to progress in xenotransplantation of microencapsulated cells: a review. Xenotransplantation 2016; 23:179-201. [PMID: 27250036 DOI: 10.1111/xen.12240] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2016] [Accepted: 05/09/2016] [Indexed: 12/13/2022]
Abstract
Cell microencapsulation and subsequent transplantation of the microencapsulated cells require multidisciplinary approaches. Physical, chemical, biological, engineering, and medical expertise has to be combined. Several natural and synthetic polymeric materials and different technologies have been reported for the preparation of hydrogels, which are suitable to protect cells by microencapsulation. However, owing to the frequent lack of adequate characterization of the hydrogels and their components as well as incomplete description of the technology, many results of in vitro and in vivo studies appear contradictory or cannot reliably be reproduced. This review addresses the state of the art in cell microencapsulation with special focus on microencapsulated cells intended for xenotransplantation cell therapies. The choice of materials, the design and fabrication of the microspheres, as well as the conditions to be met during the cell microencapsulation process, are summarized and discussed prior to presenting research results of in vitro and in vivo studies. Overall, this review will serve to sensitize medically educated specialists for materials and technological aspects of cell microencapsulation.
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Affiliation(s)
- Redouan Mahou
- Interfaculty Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,Institute for Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada
| | - Solène Passemard
- Interfaculty Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Michele Carvello
- Department of Surgery, San Raffaele Scientific Institute, Milan, Italy
| | | | - François Noverraz
- Interfaculty Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Sandrine Gerber-Lemaire
- Interfaculty Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Christine Wandrey
- Interfaculty Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.,Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
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24
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Abdolghafoorian H, Farnia P, Sajadi Nia RS, Bahrami A, Dorudinia A, Ghanavi J. Effect of Heart Valve Decellularization on Xenograft Rejection. EXP CLIN TRANSPLANT 2016; 15:329-336. [PMID: 27210227 DOI: 10.6002/ect.2015.0321] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
OBJECTIVES Endothelial cells harbor many antigenic determinants that may be targets for the immune system. The aim of this study was to determine the immunologic effects of decellularization, using 3 different methods, on xenograft rejection. MATERIALS AND METHODS In a sterile plate containing phosphate-buffered saline, fresh sheep aortic heart valves were decellularized using 3 different enzymatic methods: with 900 μg/mL of collagenase at 40°C (method A), with 450 μg/mL of collagenase at 4°C (method B), and with 900 μg/mL of collagenase at 4°C (method C). Intact and decellularized valves were implanted subdermally into inbred male albino rabbits and extracted after 21 days (extra valve pieces were also extracted after 60 days, as control samples, for assessing chronic rejection). Valves were histologically analyzed for inflammatory cell infiltration. Subendothelial structure integrity was determined using surface electron microscope. RESULTS No inflammatory cell infiltration was seen around the decellularized valve with method A, and no subendothelial structure change was observed by surface electron microscope. Infiltration of immune cells involved in rejection was not seen around valves decellularized with method B, although the subendothelial structure was relatively preserved and valve stiffness was increased. With method C, we observed a foreign body-type reaction around the intact valve and the decellularized valve. CONCLUSIONS Method A is considered the optimal method of decellularization in our study, as this method significantly reduced the immune response to xenograft tissue, while maintaining subendothelial tissue.
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Affiliation(s)
- Hamed Abdolghafoorian
- From the Shahid Beheshti University of Medical Sciences, Tehran, Iran and the Experimental Medicine and Tissue Engineering Center at Masih Daneshvari Hospital, Tehran, Iran
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TRAN HLB, DINH TTH, NGUYEN MTN, TO QM, PHAM ATT. Preparation and characterization of acellular porcinepericardium for cardiovascular surgery. Turk J Biol 2016. [DOI: 10.3906/biy-1510-44] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
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Characterization of N-glycosylation and amino acid sequence features of immunoglobulins from swine. Glycoconj J 2015; 33:79-91. [DOI: 10.1007/s10719-015-9637-z] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2015] [Revised: 11/02/2015] [Accepted: 11/03/2015] [Indexed: 12/29/2022]
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Tansaz S, Boccaccini AR. Biomedical applications of soy protein: A brief overview. J Biomed Mater Res A 2015; 104:553-69. [PMID: 26402327 DOI: 10.1002/jbm.a.35569] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2015] [Accepted: 09/17/2015] [Indexed: 12/25/2022]
Abstract
Soy protein (SP) based materials are gaining increasing interest for biomedical applications because of their tailorable biodegradability, abundance, being relatively inexpensive, exhibiting low immunogenicity, and for being structurally similar to components of the extracellular matrix (ECM) of tissues. Analysis of the available literature indicates that soy protein can be fabricated into different shapes, being relatively easy to be processed by solvent or melt based techniques. Furthermore soy protein can be blended with other synthetic and natural polymers and with inorganic materials to improve the mechanical properties and the bioactive behavior for several demands. This review discusses succinctly the biomedical applications of SP based materials focusing on processing methods, properties and applications highlighting future avenues for research.
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Affiliation(s)
- Samira Tansaz
- Institute of Biomaterials, University of Erlangen-Nuremberg, Cauerstr.6, 91058, Erlangen, Germany
| | - Aldo R Boccaccini
- Institute of Biomaterials, University of Erlangen-Nuremberg, Cauerstr.6, 91058, Erlangen, Germany
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Feng Y, Wang W. In vivo confocal microscopic observation of lamellar corneal transplantation in the rabbit using xenogenic acellular corneal scaffolds as a substitute. Chin Med J (Engl) 2015; 128:933-40. [PMID: 25836615 PMCID: PMC4834011 DOI: 10.4103/0366-6999.154301] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
BACKGROUND The limiting factor to corneal transplantation is the availability of donors. Research has suggested that xenogenic acellular corneal scaffolds (XACS) may be a possible alternative to transplantation. This study aimed to investigate the viability of performing lamellar corneal transplantation (LCT) in rabbits using canine XACS. METHODS Fresh dog corneas were decellularized by serial digestion, and LCT was performed on rabbit eyes using xenogeneic decellularized corneal matrix. Cellular and morphological changes were observed by slit-lamp, light, and scanning electron microscopy at 7, 30 and 90 days postoperatively. Immunocytochemical staining for specific markers such as keratin 3, vimentin and MUC5AC, was used to identify cells in the graft. RESULTS Decellularized xenogenic corneal matrix remained transparent for about 1-month after LCT. The recipient cells were able to survive and proliferate into the grafts. Three months after transplantation, grafts had merged with host tissue, and graft epithelialization and vascularization had occurred. Corneal nerve fibers were able to grow into the graft in rabbits transplanted with XACS. CONCLUSIONS Xenogenic acellular corneal scaffolds can maintain the transparency of corneal grafts about 1-month and permit growth of cells and nerve fibers, and is, therefore, a potential substitute or carrier for a replacement cornea.
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Affiliation(s)
| | - Wei Wang
- Department of Ophthalmology, Peking University Third Hospital, Peking University Eye Center, Beijing 100191, China
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The Role of Liver Sinusoidal Endothelial Cells in Induction of Carbohydrate Reactive B Cells Tolerance Through the Programmed Death 1/Programmed Death Ligand 1 Pathway. Transplantation 2015; 99:2325-36. [PMID: 26247556 DOI: 10.1097/tp.0000000000000831] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND A spontaneous tolerance of B cells responding to blood group antigens frequently develops in ABO-incompatible pediatric liver transplantation (LT). Liver sinusoidal endothelial cells (LSECs), which exclusively express blood group antigens in the liver, possess a capacity to induce alloantigen-specific tolerance. In this study, we elucidated the role of LSECs in the tolerance induction of blood group antigen-reactive B cells after ABO-incompatible LT using mice that lack galactose-α(1,3)galactose (Gal) epitopes resembling blood group carbohydrate antigens. METHODS Using adoptive transfer of LSECs from wild type (WT) C57BL/6J mice to congenic α1,3-galactosyltransferase gene knockout (GalT) mice, we established orthotropic GalT → GalT LSEC chimera mice. Anti-Gal Ab (antibody) production was evaluated after immunization of GalT → GalT LSEC chimera mice with Gal rabbit RBC. RESULTS Adoptive transfer of LSECs isolated from WT GalT mice via the portal vein resulted in persistent engraftment of Gal LSECs in congenic GalT mouse livers. Only when GalT mice were splenectomized before LSEC inoculation, the GalT → GalT LSEC chimera lost the ability to produce anti-Gal Abs. The administration of blocking monoclonal Abs (mAbs) against programmed death ligand 1 to the splenectomized GalT → GalT LSEC chimera resulted in the recovery of anti-Gal Ab production. CONCLUSIONS These findings suggest that LSECs take a part in tolerization of immature but not mature B cells specifically for Gal. Furthermore, the programmed death 1/programmed death ligand 1 pathway likely plays a crucial role in the mechanisms underlying spontaneous tolerization of B cells responding to ABO-blood group antigens in LT.
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Controlling immune rejection is a fail-safe system against potential tumorigenicity after human iPSC-derived neural stem cell transplantation. PLoS One 2015; 10:e0116413. [PMID: 25706286 PMCID: PMC4338009 DOI: 10.1371/journal.pone.0116413] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2014] [Accepted: 12/09/2014] [Indexed: 02/07/2023] Open
Abstract
Our previous work reported functional recovery after transplantation of mouse and human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs) into rodent models of spinal cord injury (SCI). Although hiPSC-NS/PCs proved useful for the treatment of SCI, the tumorigenicity of the transplanted cells must be resolved before they can be used in clinical applications. The current study sought to determine the feasibility of ablation of the tumors formed after hiPSC-NS/PC transplantation through immunoregulation. Tumorigenic hiPSC-NS/PCs were transplanted into the intact spinal cords of immunocompetent BALB/cA mice with or without immunosuppressant treatment. In vivo bioluminescence imaging was used to evaluate the chronological survival and growth of the transplanted cells. The graft survival rate was 0% in the group without immunosuppressants versus 100% in the group with immunosuppressants. Most of the mice that received immunosuppressants exhibited hind-limb paralysis owing to tumor growth at 3 months after iPSC-NS/PC transplantation. Histological analysis showed that the tumors shared certain characteristics with low-grade gliomas rather than with teratomas. After confirming the progression of the tumors in immunosuppressed mice, the immunosuppressant agents were discontinued, resulting in the complete rejection of iPSC-NS/PC-derived masses within 42 days after drug cessation. In accordance with the tumor rejection, hind-limb motor function was recovered in all of the mice. Moreover, infiltration of microglia and lymphocytes was observed during the course of tumor rejection, along with apoptosis of iPSC-NS/PC-generated cells. Thus, immune rejection can be used as a fail-safe system against potential tumorigenicity after transplantation of iPSC-NS/PCs to treat SCI.
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Abstract
Natural killer (NK) cells are effector cells of the innate immune system that can lyse target cells without prior sensitization and have an important role in host defense to pathogens and transformed cells. A balance between negative and positive signals transmitted via germ line-encoded inhibitory and activating receptors controls the function of NK cells. Although the concept of "missing-self" would suggest that NK cells could target foreign allografts, the prevailing dogma has been that NK cells are not active participants in the mechanisms that culminate in the rejection of solid organ allografts. Recent studies, however, challenge this conclusion and instead implicate NK cells in contributing to both graft rejection and tolerance to an allograft. In this review, we highlight recent studies with the goal of understanding the complex NK cell interactions that impact alloimmunity.
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Affiliation(s)
- Uzi Hadad
- Division of Abdominal Transplantation, Department of Surgery and Stanford Immunology, Stanford University School of Medicine, Stanford, CA, USA
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Kourtzelis I, Magnusson PU, Kotlabova K, Lambris JD, Chavakis T. Regulation of Instant Blood Mediated Inflammatory Reaction (IBMIR) in Pancreatic Islet Xeno-Transplantation: Points for Therapeutic Interventions. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2015; 865:171-88. [DOI: 10.1007/978-3-319-18603-0_11] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
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Teramura Y, Asif S, Ekdahl KN, Nilsson B. Cell Surface Engineering for Regulation of Immune Reactions in Cell Therapy. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2015; 865:189-209. [PMID: 26306451 DOI: 10.1007/978-3-319-18603-0_12] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Transplantation of the pancreatic islets of Langerhans (islets) is a promising cell therapy for treating insulin-dependent type 1 diabetes mellitus. Islet transplantation is a minimally-invasive technique involving relatively simple surgery. However, after intraportal transplantation, the transplanted islets are attacked by the recipient's immune system, because they activate a number of systems, including coagulation, complement response, inflammation, immune rejection, and recurrence of autoimmune disease. We have developed a surface modification and microencapsulation technique that protects cells and islets with biomaterials and bioactive substances, which may be useful in clinical settings. This approach employs amphiphilic polymers, which can interact with lipid bilayer membranes, without increasing cell volume. Molecules attached to these polymers can protect transplanted cells and islets from attack by the host immune system. We expect that this surface modification technique will improve graft survival in clinical islet transplantation.
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Affiliation(s)
- Yuji Teramura
- Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan,
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Tsujikura M, Nagasawa T, Ichiki S, Nakamura R, Somamoto T, Nakao M. A CD46-like molecule functional in teleost fish represents an ancestral form of membrane-bound regulators of complement activation. THE JOURNAL OF IMMUNOLOGY 2014; 194:262-72. [PMID: 25452563 DOI: 10.4049/jimmunol.1303179] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
In the complement system, the regulators of complement activation (RCA) play crucial roles in controlling excessive complement activation and in protecting host cell from misdirected attack of complement. Several members of RCA family have been cloned from cyclostome and bony fish species and classified into soluble and membrane-bound type as in mammalian RCA factors. Complement-regulatory functions have been described only for soluble RCA of lamprey and barred sand bass; however, little is known on the biological function of the membrane-bound RCA proteins in the lower vertebrates. In this study, a membrane-bound RCA protein, designated teleost complement-regulatory membrane protein (Tecrem), was cloned and characterized for its complement-regulatory roles. Carp Tecrem, an ortholog of a zebrafish type 2 RCA, ZCR1, consists of four short consensus repeat modules, a serine/threonine/proline-rich domain, a transmembrane region, and a cytoplasmic domain, from the N terminus, as does mammalian CD46. Tecrem showed a ubiquitous mRNA expression in carp tissues, agreeing well with the putative regulatory role in complement activation. A recombinant Chinese hamster ovary cell line bearing carp Tecrem showed a significantly higher tolerance against lytic activity of carp complement and less deposition of C3-S, the major C3 isotypes acting on the target cell, than control Chinese hamster ovary (mock transfectant). Anti-Tecrem mAb enhanced the depositions of carp C3 and two C4 isotypes on autologous erythrocytes. Thus, the present findings provide the evidence of complement regulation by a membrane-bound group 2 RCA in bony fish, implying the host-cell protection is an evolutionarily conserved mechanism in regulation of the complement system.
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Affiliation(s)
- Masakazu Tsujikura
- Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
| | - Takahiro Nagasawa
- Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
| | - Satoko Ichiki
- Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
| | - Ryota Nakamura
- Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
| | - Tomonori Somamoto
- Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
| | - Miki Nakao
- Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
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Cao JS, Qi F, Lu CY, Gu YC, Zhu LW. Effects of interfering RNA of α-1,3-galactosyltransferase and nuclear factor-kappa B on cardiac xenotransplantation. Transpl Immunol 2014; 31:173-82. [PMID: 25128705 DOI: 10.1016/j.trim.2014.07.002] [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: 04/30/2014] [Revised: 07/24/2014] [Accepted: 07/24/2014] [Indexed: 10/24/2022]
Abstract
BACKGROUND Both α1,3-galactosyltransferase (α1,3GT) and nuclear factor kappa B (NF-κB) play an important role in the immune response of xenotransplantation. The purpose of this study is to investigate the effect of RNAi of α1,3GT and NF-κB on xenotransplantation. METHODS Lentiviral vectors with shRNA focusing on α1, 3GT and RelA were constructed. The effect of RNAi on α1, 3GT and RelA was examined in vitro and in vivo. Additionally, we established a mouse-to-rat heterotopic cardiac xenotransplantatic model (donor hearts transplanted to the right side of the neck in rat) using a modified cuff technique. The survival time of donor hearts in each group was monitored. The expressions of α1, 3GT and RelA mRNA, Galα1,3Gal antigen, and RelA protein were detected by RT-PCR, immunofluorescence, and Western blot respectively. The expressions of C3, IgM, IgG, NK, macrophages, ICAM-1 on donor hearts were examined by immunohistochemistry. RESULTS High titer lentiviral vectors carrying α1, 3GT and RelA shRNA plasmids had a high and stable transfection rate on EOMA in vitro. In vivo, heart tissue showed a much stronger GFP expression and significant decrease in target gene mRNA expression and protein expression in shRNA interfering groups (p < 0.01). The survival time of α1,3GTi-3 and dual lentiviral vector groups was significantly longer than other groups. The mRNA expression levels of α1,3GT and RelA, as well as Galα(1,3)Gal and RelA proteins, in α1,3GTi-3, RelAi-3, and dual lentiviral vector groups were downregulated and compared to other groups (p < 0.01). The depositions of C3, IgM, IgG in α1,3GTi-3 group and dual lentiviral vector group were less than other groups (p < 0.01). The infiltration of NK, macrophages and ICAM-1 in α1,3GTi-3 group and dual lentiviral vector group was more than other groups (p < 0.01), but the infiltration of NK, macrophages and ICAM-1 in dual lentiviral vector group was less than α1,3GTi-3 group (p < 0.01). CONCLUSIONS Our results indicate that RNAi technology with lentiviral vectors is an effective method to transmit exogenous genes into living bodies and stably inhibit the expression of target genes. Moreover, siRNA targeting the α1,3GT gene was found to control the immune process and obviously prolong the survival time of donors, whereas knocking down NF-κB alone showed no differences. However, the RNAi of NF-κB can make the infiltration of macrophages and natural killer cells decrease, and the expression of ICAM-1 in the xenografts also decreases, contributing to the restraining of AVR.
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Affiliation(s)
- Ji Sen Cao
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China
| | - Feng Qi
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China.
| | - Cheng Yu Lu
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China
| | - Ya Chuan Gu
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China
| | - Li Wei Zhu
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China
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Itakura G, Kobayashi Y, Nishimura S, Iwai H, Takano M, Iwanami A, Toyama Y, Okano H, Nakamura M. Control of the Survival and Growth of Human Glioblastoma Grafted Into the Spinal Cord of Mice by Taking Advantage of Immunorejection. Cell Transplant 2014; 24:1299-311. [PMID: 24818989 DOI: 10.3727/096368914x681711] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Recent studies have demonstrated that transplantation of induced pluripotent stem cell-derived neurospheres can promote functional recovery after spinal cord injury in rodents, as well as in nonhuman primates. However, the potential tumorigenicity of the transplanted cells remains a matter of apprehension prior to clinical applications. As a first step to overcome this concern, this study established a glioblastoma multiforme xenograft model mouse. The feasibility of controlling immune suppression to ablate the grafted cells was then investigated. The human glioblastoma multiforme cell line U251 MG was transplanted into the intact spinal cords of immunodeficient NOD/SCID mice or into those of immunocompetent C57BL/6J H-2kb mice treated with or without immunosuppressants [FK506 plus anticluster of differentiation (CD) 4 antibody (Ab), or FK506 alone]. In vivo bioluminescent imaging was used to evaluate the chronological survival of the transplanted cells. The graft survival rate was 100% (n = 9/9) in NOD/SCID mice, 0% (n = 6/6) in C57BL/6J mice without immunosuppressant treatment, and 100% (n = 37/37) in C57BL6/J mice with immunosuppressant treatment. After confirming the growth of the grafted cells in the C57/BL6J mice treated with immunosuppressants, immune suppression was discontinued. The grafted cells were subsequently rejected within 3 days in C57BL/6J mice treated with FK506 alone, as opposed to 26 days in C57BL/6J mice treated with FK506 plus anti-CD4 Ab. Histological evaluation confirmed the ablation of the grafted cells. Although this work describes a xenograft setting, the results suggest that this immunomodulatory strategy could provide a safety lock against tumor formation stemming from transplanted cells.
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Affiliation(s)
- Go Itakura
- Department of Orthopaedic Surgery, Keio University School of Medicine, Shinjuku, Tokyo, Japan
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Recognition of Foreign Antigen and Foreign Major Histocompatibility Complex. Xenotransplantation 2014. [DOI: 10.1128/9781555818043.ch9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Meier RPH, Seebach JD, Morel P, Mahou R, Borot S, Giovannoni L, Parnaud G, Montanari E, Bosco D, Wandrey C, Berney T, Bühler LH, Muller YD. Survival of free and encapsulated human and rat islet xenografts transplanted into the mouse bone marrow. PLoS One 2014; 9:e91268. [PMID: 24625569 PMCID: PMC3953382 DOI: 10.1371/journal.pone.0091268] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2013] [Accepted: 02/10/2014] [Indexed: 01/19/2023] Open
Abstract
Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.
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Affiliation(s)
- Raphael P. H. Meier
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
| | - Jörg D. Seebach
- Division of Clinical Immunology and Allergology, Department of Internal Medicine, University Hospital and Medical Faculty, Geneva, Switzerland
| | - Philippe Morel
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
| | - Redouan Mahou
- Institut d’Ingénierie Biologique et Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Sophie Borot
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
| | - Laurianne Giovannoni
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
| | - Geraldine Parnaud
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
| | - Elisa Montanari
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
| | - Domenico Bosco
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
| | - Christine Wandrey
- Institut d’Ingénierie Biologique et Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Thierry Berney
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
| | - Leo H. Bühler
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
| | - Yannick D. Muller
- Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals and University of Geneva, Geneva, Switzerland
- Division of Clinical Immunology and Allergology, Department of Internal Medicine, University Hospital and Medical Faculty, Geneva, Switzerland
- * E-mail:
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Hogenes M, Huibers M, Kroone C, de Weger R. Humanized mouse models in transplantation research. Transplant Rev (Orlando) 2014; 28:103-10. [PMID: 24636846 DOI: 10.1016/j.trre.2014.02.002] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2014] [Accepted: 02/09/2014] [Indexed: 11/30/2022]
Abstract
The interest in the use of humanized mouse models for research topics like Graft versus Host Disease (GvHD), allograft studies and other studies to the human immune system is growing. The design of these models is still improving and enables even more complicated studies to these topics. For researchers it can be difficult to choose the best option from the current pool of available models. The decision will depend on which hypothesis needs to be tested, in which field of interest, and therefore 'the best model' will differ from one to another. In this review, we provide a guide to the most common available humanized mouse models, with regards to different mouse strains, transplantation material, transplantation techniques, pre- and post-conditioning and references to advantages and disadvantages. Also, an evaluation of experiences with humanized mouse models in studies on GvHD and allograft rejection is provided.
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Affiliation(s)
- Marieke Hogenes
- Department of Pathology, University Medical Centre Utrecht, PO box 85500, 3508 GA Utrecht, The Netherlands
| | - Manon Huibers
- Department of Pathology, University Medical Centre Utrecht, PO box 85500, 3508 GA Utrecht, The Netherlands
| | - Chantal Kroone
- Department of Pathology, University Medical Centre Utrecht, PO box 85500, 3508 GA Utrecht, The Netherlands
| | - Roel de Weger
- Department of Pathology, University Medical Centre Utrecht, PO box 85500, 3508 GA Utrecht, The Netherlands.
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Costa MR, Fischer N, Gulich B, Tönjes RR. Comparison of porcine endogenous retroviruses infectious potential in supernatants of producer cells and in cocultures. Xenotransplantation 2014; 21:162-73. [PMID: 24447212 DOI: 10.1111/xen.12081] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2013] [Accepted: 11/25/2013] [Indexed: 11/26/2022]
Abstract
BACKGROUND Porcine endogenous retroviruses (PERV) pose a zoonotic risk potential in pig-to-human xenotransplantation given that PERV capacity to infect different human cell lines in vitro has been clearly shown in the past. However, PERV infectious potential for human peripheral blood mononuclear cells (huPBMC) has been also demonstrated, albeit with controversial results. As productive PERV infection of huPBMC involves immune suppression that may attract opportunistic pathogens as shown for other retroviruses, it is crucial to ascertain unequivocally huPBMC susceptibility for PERV. To address this question, we first investigated in vitro infectivity of PERV for huPBMC using supernatants containing highly infectious PERV-A/C. Second, huPBMC were cocultivated with PERV-A/C producer cells to come a step closer to the in vivo situation of xenotransplantation. In addition, cocultivation of huPBMC with porcine PBMC (poPBMC) isolated from German landrace pigs was performed to distinguish PERV replication competence when they were constitutively produced by immortalized cells or by primary poPBMC. METHODS Supernatants containing recombinant highly infectious PERV-A/C were used to infect PHA-activated huPBMC in the presence or absence of polybrene. Next, PERV-producing cell lines such as human 293/5° and primary mitogenically activated poPBMC of three German landrace pigs were cocultivated with huPBMC as well as with susceptible human and porcine cell lines as controls. PERV infection was monitored by using three test approaches. The presence of provirus DNA in putatively infected cells was detected via sensitive nested PCR. Viral expression was determined by screening for the activity of gammaretroviral reverse transcriptase (RT) in cell-free supernatants of infected cells. Virus release was monitored by counting the number of packaged RNA particles in supernatants via PERV-specific quantitative one-step real-time reverse transcriptase PCR. RESULTS Porcine endogenous retroviruses-A/C in supernatants of human producer 293/5° cells was not able to infect huPBMC. Neither RT activity nor PERV copies were detected. Even provirus could not be detected displaying the inability of PERV-A/C to induce a productive infection in huPBMC. In cocultivation experiments only non-productive infection of huPBMC with PERV derived from 293/5° cell line and from PHA-activated poPBMC was observed by detection of provirus DNA in infected cells. CONCLUSION Recombinant PERV-A/C in supernatants of producer cells failed to infect huPBMC, whereas coculture experiments with producer cell lines lead to non-productive infection of huPBMC. PERV in supernatants seem to have not sufficient infectious potential for huPBMC. However, extensive PERV exposure to huPBMC via cocultivation enabled at least virus cell entry as provirus was detected by nested PCR. Furthermore, results presented support previous data showing German landrace pigs as low producers with negligible infectious potential due to the absence of replication-competent PERV in the genome. The low PERV expression profile and the lack of significant replication competence of German landrace pigs raise hope for considering these animals as putative donor animals in future pig-to-human xenotransplantation. Nonetheless, data imply that PERV still represent a virological risk in the course of xenotransplantation, as the presence of PERV provirus in host cells may lead to a provirus integration resulting in insertional mutagenesis and chromosomal rearrangements.
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Ruan J, Liu N, Ouyang H, Yang S, Li K. Spatiotemporal control of porcine p65RHD expression by advanced Tet-On system in PIEC cells helps regulate NFкB activity. Mol Biol Rep 2014; 41:1753-61. [PMID: 24443222 DOI: 10.1007/s11033-014-3024-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2013] [Accepted: 01/03/2014] [Indexed: 11/24/2022]
Abstract
NFкB transcription activation leads to malfunction of endothelial cells, which is the main reason for pig xenograft rejection. Overexpression of a dominant negative mutant of porcine NFκB p65 (pp65RHD) could inhibit NFкB activation in endothelial cells. This study presents an advanced tetracycline-regulated system for pp65RHD spatiotemporal expression in porcine iliac endothelial cell line. In this system, an endothelial specific promoter ICAM-2 is used to improve pTet-On and internal ribosome entry site as well as enhanced green fluorescent protein (EGFP) elements are used to facilitate the result observation in pTRE-Tight. Through transfection and drug selection, we obtained 7 single cell clones containing the advanced Tet-On system, in which pp65RHD expression is under tight regulated by doxycycline and can be visualized easily through EGFP. The distribution of induced pp65RHD was verified by immunocytochemical assays test. Then, NFкB activity was tested. Luciferase reporter assays showed that NFкB activity in two clones was influenced by the Dox-induced pp65RHD expression, but other clones weren't influenced. Therefore, we picked up 2 cell clones from the uninfluenced clones for further investigation by immunocytochemical assays and RT-PCR detection. The final results supported the overexpression of pp65RHD in one clone could successfully inhibit NFкB activity. The success of pp65RHD spatiotemporal expression system is helpful to regulate NFкB activity and conquer cell-mediated immunity and could be used for preparation of transgenic pig, contributing to xenotransplantation.
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Affiliation(s)
- Jinxue Ruan
- Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science, Jilin University, Changchun, 130012, People's Republic of China
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Abstract
Beta cell replacement therapy has been proposed as a novel therapy for the treatment of type 1 diabetes. The proof of concept has been demonstrated with successful islet allotransplantation. Islet xenotransplantation has been proposed as an alternative, more reliable, and infinite source of beta cells. The advantages of islet xenotransplantation are the ability to transplant a well differentiated cell that is responsive to glucose and the potential for genetic modification which focuses the treatment on the donor rather than the recipient. The major hurdle remains overcoming the severe cellular rejection that affects xenografts. This review will focus on the major advances that have occurred with genetic modification and the successful therapeutic strategies that have been demonstrated in nonhuman primates. Novel approaches to overcome cell-mediated rejection including biological agents that target selectively costimulation molecules, the development of local immunosuppression through genetic manipulation, and encapsulation will be discussed. Overall, there has been considerable progress in all these areas, which eventually should lead to clinical trials.
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Affiliation(s)
- Philip J O'Connell
- Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney at Westmead Hospital, Westmead, NSW, 2145, Australia,
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Ro H, Lee EW, Hong JH, Han KH, Yeom HJ, Kim HJ, Kim MG, Jung HS, Oh KH, Park KS, Ahn C, Yang J. Roles of Islet Toll-Like Receptors in Pig to Mouse Islet Xenotransplantation. Cell Transplant 2013; 22:1709-22. [DOI: 10.3727/096368912x657684] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Although innate immunity plays important roles in xenograft rejection, there have been few studies on the role of toll-like receptors (TLRs) in xenotransplantation. Furthermore, most studies focused on the recipient's TLRs. Therefore, we investigated whether TLRs in porcine islets can contribute to islet xenograft rejection. Adult porcine islets were isolated and stimulated by polyinosinic/polycytidylic acid (poly I:C) or lipopolysaccharide (LPS). Both poly I:C and LPS stimulation in porcine islets induced expression of chemokines (RANTES, MCP-1, IP-10, and IL-8), cytokines (IL-6 and type I interferons), and adhesion molecules (VCAM-1 and ICAM-1). Porcine islet supernatants stimulated by TLR agonists induced chemotaxis of human leukocytes. They also induced procoagulant activation (tissue factor and fgl-2). However, TLR stimulation did not influence insulin secretion. When porcine MyD88 was knocked down using shRNA lentivirus, TLR-mediated induction of proinflammatory mediators and procoagulants was attenuated. When LPS was injected to MyD88 or TLR4 knockout mice after porcine islet transplantation, LPS stimulation on donor islets interfered with islet xenograft tolerance induction by anti-CD154 antibodies. Inflammatory cell infiltration and expression of proinflammatory chemokines and cytokines in islet xenografts also increased. In conclusion, TLR activation in porcine islets induced both a proinflammatory and procoagulant response and thereby contributed to xenograft rejection.
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Affiliation(s)
- Han Ro
- Transplantation Center, Seoul National University Hospital, Seoul, Republic of Korea
- Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Eun Won Lee
- Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Joo Ho Hong
- Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Kyu Hyun Han
- Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Hye-Jung Yeom
- Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Hwa Jung Kim
- Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Myung-Gyu Kim
- Transplantation Center, Seoul National University Hospital, Seoul, Republic of Korea
| | - Hye Seung Jung
- Division of Endocrinology and Metabolism, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Kook-Hwan Oh
- Division of Nephrology, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Kyong Soo Park
- Division of Endocrinology and Metabolism, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Curie Ahn
- Transplantation Center, Seoul National University Hospital, Seoul, Republic of Korea
- Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
- Division of Nephrology, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Jaeseok Yang
- Transplantation Center, Seoul National University Hospital, Seoul, Republic of Korea
- Transplantation Research Institute, Seoul National University College of Medicine, Seoul, Republic of Korea
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del Rio ML, Seebach JD, Fernández-Renedo C, Rodriguez-Barbosa JI. ITIM-dependent negative signaling pathways for the control of cell-mediated xenogeneic immune responses. Xenotransplantation 2013; 20:397-406. [PMID: 23968542 DOI: 10.1111/xen.12049] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2013] [Accepted: 07/31/2013] [Indexed: 12/24/2022]
Abstract
Xenotransplantation is an innovative field of research with the potential to provide us with an alternative source of organs to face the severe shortage of human organ donors. For several reasons, pigs have been chosen as the most suitable source of organs and tissues for transplantation in humans. However, porcine xenografts undergo cellular immune responses representing a major barrier to their acceptance and normal functioning. Innate and adaptive xenogeneic immunity is mediated by both the recognition of xenogeneic tissue antigens and the lack of inhibition due to molecular cross-species incompatibilities of regulatory pathways. Therefore, the delivery of immunoreceptor tyrosine-based inhibitory motif (ITIM)-dependent and related negative signals to control innate (NK cells, macrophages) and adaptive T and B cells might overcome cell-mediated xenogeneic immunity. The proof of this concept has already been achieved in vitro by the transgenic overexpression of human ligands of several inhibitory receptors in porcine cells resulting in their resistance against xenoreactivity. Consequently, several transgenic pigs expressing tissue-specific human ligands of inhibitory coreceptors (HLA-E, CD47) or soluble competitors of costimulation (belatacept) have already been generated. The development of these robust and innovative approaches to modulate human anti-pig cellular immune responses, complementary to conventional immunosuppression, will help to achieve long-term xenograft survival. In this review, we will focus on the current strategies to enhance negative signaling pathways for the regulation of undesirable cell-mediated xenoreactive immune responses.
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Affiliation(s)
- Maria-Luisa del Rio
- Transplantation Immunobiology Section, Institute of Biomedicine, University of Leon, Leon, Spain; Leon University Hospital, Castilla and Leon Transplantation Regional Agency, Leon, Spain
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Jang KS, Kim YG, Adhya M, Park HM, Kim BG. The sweets standing at the borderline between allo- and xenotransplantation. Xenotransplantation 2013; 20:199-208. [PMID: 23551837 DOI: 10.1111/xen.12030] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2012] [Accepted: 02/28/2013] [Indexed: 01/06/2023]
Abstract
Animal cells are densely covered with glycoconjugates, such as N-glycan, O-glycan, and glycosphingolipids, which are important for various biological and immunological events at the cell surface and in the extracellular matrix. Endothelial α-Gal carbohydrate epitopes (Galα3Gal-R) expressed on porcine tissue or cell surfaces are such glycoconjugates and directly mediate hyperacute immunological rejection in pig-to-human xenotransplantation. Although researchers have been able to develop α1,3-galactosyltransferase (GalT) gene knockout (KO) pigs, there remain unclarified non-Gal antigens that prevent xenotransplantation. Based on our expertise in the structural analysis of xenoantigenic carbohydrates, we describe the immunologically significant non-human carbohydrate antigens, including α-Gal antigens, analyzed as part of efforts to assess the antigens responsible for hyperacute immunological rejection in pig-to-human xenotransplantation. The importance of studying human, pig, and GalT-KO pig glycoprofiles, and of developing adequate pig-to-human glycan databases, is also discussed.
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Affiliation(s)
- Kyoung-Soon Jang
- Institute of Molecular Biology and Genetics, Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul, Korea
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Pan J, Yung Chan S, Common JEA, Amini S, Miserez A, Birgitte Lane E, Kang L. Fabrication of a 3D hair follicle-like hydrogel by soft lithography. J Biomed Mater Res A 2013; 101:3159-69. [PMID: 23554315 DOI: 10.1002/jbm.a.34628] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2012] [Revised: 01/08/2013] [Accepted: 01/22/2013] [Indexed: 12/13/2022]
Abstract
Hair follicle transplantation is often used in the treatment of androgenetic alopecia (AGA). However, the only source of hair follicles is from human donors themselves, which limits the application of this approach. One possible solution is to reconstitute hair follicle from dissociated cells. Currently, a number of microscale technologies have been developed to create size and shape controlled microenvironments in tissue engineering. Photopolymerizable PEGDA hydrogels are often selected as promising scaffolds in engineered microtissues due to their biocompatibility and adjustable mechanical properties. Here, we fabricated an array of PEGDA microwells with center islets that mimic the architecture of human hair follicles using soft lithography. Dermal and epithelial cells were seeded in different compartments of the microstructured mould to mimic mesenchymal and epithelial compartmentalization in native hair follicles. We demonstrated that these compartmentalized microstructures support cell proliferation and cell survival over 14 days, and spreading of dermal fibroblasts was observed. This hydrogel micromould provides a potentially useful tool for engineering 3D hair follicle-mimicking complex cultures in vitro.
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Affiliation(s)
- Jing Pan
- Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore, 117543, Singapore
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47
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Hirao A, Kawarasaki T, Konno K, Enya S, Shibata M, Kangawa A, Kobayashi E. Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs. ACTA ZOOL-STOCKHOLM 2013. [DOI: 10.1111/azo.12029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Atsushi Hirao
- Department of Anatomy; Division of Anatomy & Embryology; Jichi Medical University; Tochigi 329-0498 Japan
| | - Tatsuo Kawarasaki
- Department of Animal Science; School of Agriculture; Tokai University; Minamiaso Kumamoto 869-1404 Japan
| | - Kenjiro Konno
- Laboratory of Animal Science and Medicine; Department of Animal Medical Sciences; Faculty of Science; Kyoto Sangyo University; Kyoto 603-8555 Japan
| | - Satoko Enya
- Shizuoka Swine & Poultry Research Center; Shizuoka Prefectural Research Institute of Animal Industry; Shizuoka 439-0037 Japan
| | - Masatoshi Shibata
- Shizuoka Swine & Poultry Research Center; Shizuoka Prefectural Research Institute of Animal Industry; Shizuoka 439-0037 Japan
| | - Akihisa Kangawa
- Shizuoka Swine & Poultry Research Center; Shizuoka Prefectural Research Institute of Animal Industry; Shizuoka 439-0037 Japan
| | - Eiji Kobayashi
- Division of Development of Advanced Treatment; Center for Development of Advanced Medical Technology; Jichi Medical University; Tochigi 329-0498 Japan
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Dufrane D, Gianello P. Macro- or microencapsulation of pig islets to cure type 1 diabetes. World J Gastroenterol 2012; 18:6885-93. [PMID: 23322985 PMCID: PMC3531671 DOI: 10.3748/wjg.v18.i47.6885] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/18/2012] [Revised: 06/14/2012] [Accepted: 06/28/2012] [Indexed: 02/06/2023] Open
Abstract
Although allogeneic islet transplantation can successfully cure type 1 diabetes, it has limited applicability. For example, organs are in short supply; several human pancreas donors are often needed to treat one diabetic recipient; the intrahepatic site may not be the most appropriate site for islet implantation; and immunosuppressive regimens, which are associated with side effects, are often required to prolong survival of the islet graft. An alternative source of insulin-producing cells would therefore be of major interest. Pigs represent a possible alternative source of beta cells. Grafting of pig islets may appear difficult because of the immunologic species barrier, but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression. Therefore, a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation. Although several groups have shown that encapsulated pig islets are functional in small-animal models, less is known about the use of bioartificial pancreases in large-animal models. In this review, we summarize current knowledge of encapsulated pig islets, to determine obstacles to implantation in humans and possible solutions to overcome these obstacles.
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Abstract
The complement cascade is a major contributor to the innate immune response. It has now been well accepted that complement plays a critical role in hyperacute rejection and acute antibody-mediated rejection of transplanted organ. There is also increasing evidence that complement proteins contribute to the pathogenesis of organ ischemia-reperfusion injury, and even to cell-mediated rejection. Furthermore, the chemoattractants C3a and C5a and the terminal membrane attack complex that are generated by complement activation can directly or indirectly mediate tissue injury and trigger adaptive immune responses. Here, we review recent findings concerning the role of complement in graft ischemia-reperfusion injury, antibody-mediated rejection and accommodation, and cell-mediated rejection. We also discuss the current status of complement intervention therapies in clinical transplantation and describe potential new therapeutic strategies for clinical application.
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Affiliation(s)
- Gang Chen
- Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
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50
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Butler JE, Wertz N, Sun XZ, Lunney JK, Muyldermans S. Resolution of an immunodiagnostic dilemma: heavy chain chimeric antibodies for species in which plasmocytomas are unknown. Mol Immunol 2012; 53:140-8. [PMID: 22922509 DOI: 10.1016/j.molimm.2012.07.008] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2012] [Revised: 07/10/2012] [Accepted: 07/21/2012] [Indexed: 01/30/2023]
Abstract
The immunoglobulin (Ig) genes of many vertebrates have been characterized but IgG subclasses, IgD and IgE proteins are only available for three species in which plasmacytomas occur. This creates a major problem in the production and specificity verification of diagnostic anti-Ig reagents for the vast majority of mammals. We describe a novel solution using the swine system with its eleven different variants of IgG. It involves the in vitro synthesis of chimeric porcine-camelid heavy chain antibodies (HCAbs) that do not require light chains and therefore only a single transfection vector. The expressed chimeric HCAbs are comprised of the camelid VHH domain encoding specificity for lysozyme and the hinge, CH2 and CH3 domains of the various porcine IgGs. These HCAb retain their antigenic integrity and their ability to recognize lysozyme. The engineered specificity assures that these HCAb can be immobilized in native configuration when used for testing the specificity of anti-swine IgG antibodies. Comparative data to illustrate the importance of this point are provided. These are now available for use in hybridoma selection and as reference standards for evaluating the specificity of currently available anti-swine IgG antibodies.
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Affiliation(s)
- J E Butler
- Department of Microbiology and Interdisciplinary Immunology Program, University of Iowa, Iowa City, IA 52242, United States.
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