Results of Two Cases of Pig-to-Human Kidney Xenotransplantation.

BACKGROUND: Xenografts from genetically modified pigs have become one of the most promising solutions to the dearth of human organs available for transplantation. The challenge in this model has been hyperacute rejection. To avoid this, pigs have been bred with a knockout of the alpha-1,3-galactosyltransferase gene and with subcapsular autologous thymic tissue. METHODS: We transplanted kidneys from these genetically modified pigs into two brain-dead human recipients whose circulatory and respiratory activity was maintained on ventilators for the duration of the study. We performed serial biopsies and monitored the urine output and kinetic estimated glomerular filtration rate (eGFR) to assess renal function and xenograft rejection. RESULTS: The xenograft in both recipients began to make urine within moments after reperfusion. Over the 54-hour study, the kinetic eGFR increased from 23 ml per minute per 1.73 m2 of body-surface area before transplantation to 62 ml per minute per 1.73 m2 after transplantation in Recipient 1 and from 55 to 109 ml per minute per 1.73 m2 in Recipient 2. In both recipients, the creatinine level, which had been at a steady state, decreased after implantation of the xenograft, from 1.97 to 0.82 mg per deciliter in Recipient 1 and from 1.10 to 0.57 mg per deciliter in Recipient 2. The transplanted kidneys remained pink and well-perfused, continuing to make urine throughout the study. Biopsies that were performed at 6, 24, 48, and 54 hours revealed no signs of hyperacute or antibody-mediated rejection. Hourly urine output with the xenograft was more than double the output with the native kidneys. CONCLUSIONS: Genetically modified kidney xenografts from pigs remained viable and functioning in brain-dead human recipients for 54 hours, without signs of hyperacute rejection. (Funded by Lung Biotechnology.).

Successful surgical correction of anal atresia in a transgenic cloned piglet.

Inbred strains of pig become indispensable for a wide range of biological studies. In biomedical science, it is generally accepted that somatic cell nuclear transfer (SCNT) technology with inbreed strain of pig is essential for xenotransplantation. In this study, we observed the anal atresia in a cloned pig which was derived from fetal fibroblast of inbreed miniature pig. A presumptive anal site of the cloned pig was excised and the rectum was sutured to apposed skin for treatment. This cloned piglet seemed to be normal with healthy status after surgery. This report can be useful for the treatment of anal atresia of cloned piglets.

Transgenic technology: an overview of approaches useful in surgical research.

Advances in transgenic science have created powerful tools for the investigation of both genetic and protein regulatory systems. Recently, transgenic animals have been utilized in several vascular and transplantation research laboratories. The ability to specifically mutate genes important in oncologic and cardiovascular research is leading to a greater understanding of the role of gene and protein regulatory systems in cancer and cardiovascular disease. The expanding use of transgenic animals will undoubtedly increase our insight into complex problems in surgical research. This review briefly describes the various techniques utilized to create transgenic animals including: transgene design, gene-transfer utilizing transfection, microinjection and retroviral infection, as well as the use of embryonic stem cells, and methods for screening transgenic offspring.

Production of chicken chimeras by fusing blastodermal cells with electroporation.

AIM: To establish techniques for producing somatic and germline chimeric chicken by transferring blastodermal cells fused with electroporation. METHODS: Stage-X blastodermal cells isolated from freshly laid fertile unincubated white Leghorn and Rhode Island red chicken eggs were fused with electroporation. The treated cell suspension was transferred to the recovery medium (DMEM containing 10% FBS) and was injected into the subgerminal cavity of recipient unincubated embryos (stage X). RESULTS: Of 177 recipient embryos injected with the fusing blastodermal cells, 6 (3.4%) survived to hatching. Somatic chimerism was examined in the melanocyte of the feather. The presence of feathers originating from the donor cell was observed in 1 bird (16.7%) out of the 6 hatched birds. After 21 days of incubation two birds out of five embryos were subjected to polymerase chain reaction (PCR) analysis for W-chromosome-specific DNA for each tissue. One bird possessed W-chromosome-specific DNA in the stomach, and the other exhibited the same DNA in the left and right gonads and other tissues, but not the stomach. CONCLUSION: Recipient embryo having electrofused blastodermal cells yields somatic and germline chimeric chickens more successfully.

A transgenic rat with the human ATTR V30M: a novel tool for analyses of ATTR metabolisms.

Amyloidogenic transthyretin (ATTR) is the pathogenic protein of familial amyloidotic polyneuropathy (FAP). To establish a tool for analyses of ATTR metabolisms including after liver transplantations, we developed a transgenic rat model expressing human ATTR V30M and confirmed expressions of human ATTR V30M in various tissues. Mass spectrometry for purified TTR revealed that rat intrinsic TTR and human ATTR V30M formed tetramers. Congo red staining and immunohistochemistry revealed that nonfibrillar deposits of human ATTR V30M, but not amyloid deposits, were detected in the gastrointestinal tracts of the transgenic rats. At 24h after liver transplantation, serum human ATTR V30M levels in transgenic rats that received livers from normal rats became lower than detectable levels. These results thus suggest that this transgenic rat may be a useful animal model which analyzes the metabolism of human ATTR V30M including liver transplantation studies.

Production of transgenic sheep.

The production of transgenic sheep has proven difficult compared to the mouse and lower animals. The work load is far greater and the rates of success far less by most criteria. However, the benefits to human and animal health and agricultural productivity are potentially enormous (Ward and Nancarrow, Chapter 5) and support for the continuation of the work is assured. Unfortunately, the low rate of transgenesis for sheep, at about 1% of injected, transferred embryos, means that investigation of the regulation of expression of the transgenes, their phenotypic effects, and optimization of the fusion gene constructs, all of utmost importance to the agricultural industry, can seldom be addressed. We know now that the mouse may not be a good model for the sheep, an example being the ovine metallothioneinovine growth hormone fusion gene, GH9, for which expression and phenotypic effects were quite different for sheep and mice. In sheep, pronuclear microinjection of several hundred copies of the foreign gene into embryos is the only published method used to regularly produce transgenics and it will be the standard by which future methods for incorporation of the transgene are judged.

Successful surgical correction of anal atresia in a transgenic cloned piglet

Inbred strains of pig become indispensable for a wide range of biological studies. In biomedical science, it is generally accepted that somatic cell nuclear transfer(SCNT)technology with inbreed strain of pig is essential for xenotransplantation. In this study, we observed the anal atresia in a cloned pig which was derived from fetal fibroblast of inbreed miniature pig. A presumptive anal site of the cloned pig was excised and the rectum was sutured to apposed skin for treatment. This cloned piglet seemed to be normal with healthy status after surgery. This report can be useful for the treatment of anal atresia of cloned piglets.

Estudio clínico e inmunólogico del xenorrechazo en el xenotrasplante ortotópico de hígado de cerdo a babuino / Clinical and immunological study of xenorejection in orthotopic liver xenotransplantation from pig to baboon

Introducción. La experiencia de xenotrasplante hepático (Xtoh) de cerdo a primate no humano es muy limitada. Nuestros objetivos han sido: a) comprobar si el hígado de un cerdo transgénico h-DAF evita el rechazo hiperagudo; b) estudiar las funciones metabólicas del hígado porcino tras el Xtoh; y c) analizar el perfil clínico, bioquímico e inmunológico del rechazo vascular agudo retardado. Animales y métodos. Se realizaron 6 Xtoh de cerdo a babuino, 4 de cerdos no modificados y dos de cerdos transgénicos para h-DAF. Se llevaron a cabo determinaciones hematológicas, de coagulación, de xenoanticuerpos y del complemento. En el babuino que sobrevivió 8 días, se estudiaron durante los mismos las poblaciones linfocitarias y la actividad lítica de los linfocitos. Resultados. Los valores de xIgG e IgM descendieron drásticamente a los 3 min de la reperfusión, sobre todo del CH50, C3 y C4. En los hígados no modificados genéticamente apareció una coagulación intravascular diseminada por rechazo hiperagudo, con una supervivencia inferior a 12 h. Con los hígados h-DAF, la coagulación se normalizó, con una supervivencia de 8 y 4 días, falleciendo ambos por insuficiencia respiratoria, sin rechazo hiperagudo. El babuino que sobrevivió 8 días presentó a las 36 h un rechazo vascular agudo retardado, detectándose una estimulación de las HLA clase I sobre los linfocitos CD3+ y CD19+, que respondió al tratamiento. Conclusiones. El hígado transgénico h-DAAF previene el rechazo hiperagudo y mantiene la coagulación en rangos normales en el babuino. El rechazo vascular agudo provoca el cese en la producción de bilis y un patrón mixto de citólisis y colostasis. Los valores de expresión de HLA clase I en los linfocitos podrían ser útiles para diagnosticarlo (AU)