Profiling Human <i>CD55</i> Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation
Xenotransplantation of pig organs receives substantial attention for being comparable to human’s. However, compatibility constraints involving hyper-acute rejection (HAR) still block clinical applications. Transgenesis of human complement regulatory proteins has been proposed to overcome xenorejecti...
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MDPI AG
2021-08-01
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| Online Access: | https://www.mdpi.com/2079-7737/10/8/747 |
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| author | Laura Martínez-Alarcón Sergio Liarte Juan J. Quereda Aida Sáez-Acosta Carlos de Torre-Minguela Livia Mendonça Juana M. Abellaneda María J. Majado Antonio Ríos Pablo Ramírez Antonio Muñoz Guillermo Ramis |
| author_facet | Laura Martínez-Alarcón Sergio Liarte Juan J. Quereda Aida Sáez-Acosta Carlos de Torre-Minguela Livia Mendonça Juana M. Abellaneda María J. Majado Antonio Ríos Pablo Ramírez Antonio Muñoz Guillermo Ramis |
| author_sort | Laura Martínez-Alarcón |
| collection | DOAJ |
| description | Xenotransplantation of pig organs receives substantial attention for being comparable to human’s. However, compatibility constraints involving hyper-acute rejection (HAR) still block clinical applications. Transgenesis of human complement regulatory proteins has been proposed to overcome xenorejection. Pigs expressing human-<i>CD55</i> have been widely tested in experimental surgery. Still, no standardized method has been developed to determine tissue expression of human decay-accelerating factor (DAF), <i>hCD55’s</i> product, or to predict the ability to overpass HAR. Here we describe objective procedures addressing this need. Organs and tissues from five <i>hCD55</i> transgenic pigs were collected and classified according to their xenotransplantation value. The ability to overcome HAR was assessed by classical complement pathway hemolysis assays. Quantitative PCR mRNA expression and Western blot protein level studies were performed. Real-time cytotoxicity assays (RTCA) on fibroblast cultures exposed to baboon and human sera informed on longer-term rejection dynamics. While greater <i>hCD55</i>/DAF expression correlated with better performance, the results obtained varied among specimens. Interestingly, the individual with highest mRNA and protein levels showed positive feedback for <i>hCD55</i> transcript after challenge with human and baboon sera. Moreover, <i>hCD55</i> expression correlated to DAF levels in the liver, lung and intestine, but not in the heart. Moreover, we found significant correlations among valuable and non-valuable tissues. In sum, the methodology proposed allows us to characterize the <i>hCD55</i> transgene functioning and performance. Moreover, the correlations found could allow us to predict <i>hCD55</i>/DAF expression in surrogate tissues, thus eliminating the need for direct biopsies, resulting in preservation of organ integrity before xenotransplantation. |
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| institution | Directory Open Access Journal |
| issn | 2079-7737 |
| language | English |
| last_indexed | 2024-03-10T09:00:11Z |
| publishDate | 2021-08-01 |
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| spelling | doaj.art-39c10ed32443406b8378cf5d12b0d5d92023-11-22T06:49:56ZengMDPI AGBiology2079-77372021-08-0110874710.3390/biology10080747Profiling Human <i>CD55</i> Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ XenotransplantationLaura Martínez-Alarcón0Sergio Liarte1Juan J. Quereda2Aida Sáez-Acosta3Carlos de Torre-Minguela4Livia Mendonça5Juana M. Abellaneda6María J. Majado7Antonio Ríos8Pablo Ramírez9Antonio Muñoz10Guillermo Ramis11Servicio de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, El Palmar, 30120 Murcia, SpainLaboratorio de Medicina Regenerativa, Oncología Molecular y TGFβ, Instituto Murciano de Investigación Biosanitaria, 30120 Murcia, SpainDepartamento Producción y Sanidad Animal, Salud Pública Veterinaria y Ciencia y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Cardenal Herrera-CEU, CEU Universities, 46115 Valencia, SpainGrupo de Investigación Cría y Salud Animal, Universidad de Murcia, 30100 Murcia, SpainUnidad de Inflamación y Cirugía Experimental, Instituto Murciano de Investigación Biosanitaria, 30120 Murcia, SpainEscola de Vetérinaria, Universidade Federal de Goiás, Goiánia 74001-970, BrazilGrupo de Investigación Cría y Salud Animal, Universidad de Murcia, 30100 Murcia, SpainServicio de Hematología, Hospital Clínico Universitario Virgen de la Arrixaca, 30120 Murcia, SpainServicio de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, El Palmar, 30120 Murcia, SpainServicio de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, El Palmar, 30120 Murcia, SpainInstituto Murciano de Investigación Biosanitaria, 30120 Murcia, SpainInstituto Murciano de Investigación Biosanitaria, 30120 Murcia, SpainXenotransplantation of pig organs receives substantial attention for being comparable to human’s. However, compatibility constraints involving hyper-acute rejection (HAR) still block clinical applications. Transgenesis of human complement regulatory proteins has been proposed to overcome xenorejection. Pigs expressing human-<i>CD55</i> have been widely tested in experimental surgery. Still, no standardized method has been developed to determine tissue expression of human decay-accelerating factor (DAF), <i>hCD55’s</i> product, or to predict the ability to overpass HAR. Here we describe objective procedures addressing this need. Organs and tissues from five <i>hCD55</i> transgenic pigs were collected and classified according to their xenotransplantation value. The ability to overcome HAR was assessed by classical complement pathway hemolysis assays. Quantitative PCR mRNA expression and Western blot protein level studies were performed. Real-time cytotoxicity assays (RTCA) on fibroblast cultures exposed to baboon and human sera informed on longer-term rejection dynamics. While greater <i>hCD55</i>/DAF expression correlated with better performance, the results obtained varied among specimens. Interestingly, the individual with highest mRNA and protein levels showed positive feedback for <i>hCD55</i> transcript after challenge with human and baboon sera. Moreover, <i>hCD55</i> expression correlated to DAF levels in the liver, lung and intestine, but not in the heart. Moreover, we found significant correlations among valuable and non-valuable tissues. In sum, the methodology proposed allows us to characterize the <i>hCD55</i> transgene functioning and performance. Moreover, the correlations found could allow us to predict <i>hCD55</i>/DAF expression in surrogate tissues, thus eliminating the need for direct biopsies, resulting in preservation of organ integrity before xenotransplantation.https://www.mdpi.com/2079-7737/10/8/747xenotransplantationtransgenic pigs<i>hCD55</i>RTCAgene expression |
| spellingShingle | Laura Martínez-Alarcón Sergio Liarte Juan J. Quereda Aida Sáez-Acosta Carlos de Torre-Minguela Livia Mendonça Juana M. Abellaneda María J. Majado Antonio Ríos Pablo Ramírez Antonio Muñoz Guillermo Ramis Profiling Human <i>CD55</i> Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation Biology xenotransplantation transgenic pigs <i>hCD55</i> RTCA gene expression |
| title | Profiling Human <i>CD55</i> Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation |
| title_full | Profiling Human <i>CD55</i> Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation |
| title_fullStr | Profiling Human <i>CD55</i> Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation |
| title_full_unstemmed | Profiling Human <i>CD55</i> Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation |
| title_short | Profiling Human <i>CD55</i> Transgene Performance Assist in Selecting Best Suited Specimens and Tissues for Swine Organ Xenotransplantation |
| title_sort | profiling human i cd55 i transgene performance assist in selecting best suited specimens and tissues for swine organ xenotransplantation |
| topic | xenotransplantation transgenic pigs <i>hCD55</i> RTCA gene expression |
| url | https://www.mdpi.com/2079-7737/10/8/747 |
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