Xenotransplantation Research Research Articles

Korean attitudes to Xenotransplantation: a survey conducted in 20091

Xenotransplantation research has been being actively conducted in Korea. However, there are numbers of socio-ethical issues involved in this research, and it is necessary to know public attitudes toward the research, inducing "public consensus" for the sound development of the technology. A telephone survey consisting of 10 questionnaire items was conducted to investigate the attitudes of Koreans on social and ethical issues related to xenotransplantation. Participants were randomly selected in proportion to the 2009 Korean population census. The response rate was 22.3%. Finally, 500 sets of data were collected and analyzed with SPSS® 12. A statistical analysis was performed using the chi-square test. In the first phase of the study, 69.8% of the respondents were positive about xenotransplantation while 29.0% were negative. However, after being informed of the specific benefit and risk, 58.4% of the respondents stated that xenotransplantation research should be continued, while 38.6% of them indicated that these studies should be prohibited; 63.0% of the respondents agreed with lifelong surveillance after xenotransplantation, while 34.0% disagreed. To avoid triggering immune response in the transplantees, 63.0% of the respondents also approved of the necessity of genetic modification of pigs. If xenotransplantation proves to be safe, 56.8% of the respondents said that they would accept it if necessary; 69.8% of the respondents would recommend it for their family members and friends as a therapeutic option if they are in need. Men were more positive than women about the necessity of animal experimentation for xenotransplantation research, the necessity of lifelong surveillance, and the necessity of genetic modification of pigs. There was no significant difference between the religious and non-religious group except about the issue of lifelong surveillance. Korean people have relatively positive attitudes toward xenotransplantation research and animal experiments regarding xenotransplantation.

ChemInform Abstract: Large Scale Synthesis of a Derivative of an α-Galactosyl Trisaccharide Epitope Involved in the Hyperacute Rejection of Xenotransplantation.

ABSTRACT A derivative of an α-galactosyl trisaccharide xenoactive antigen, (2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl)-(1→3)-(2,4,6-tri-O-acetyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl azide (5), was synthesized on a large scale (50 gram). The synthesis involved a high yielding and highly stereoselective (α/β>20:1) glycosylation reaction utilizing a thiogalactoside as the donor and a selectively protected lactose azide as the acceptor. This derivative serves as a versatile intermediate that can be transformed into a variety of α-Gal containing glycoconjugates highly desired in xenotransplantation research and pharmaceutical development.

Establishment of a resource population of SLA haplotype-defined Korean native pigs.

The highly polymorphic porcine major histocompatibility complex (MHC), or the swine leukocyte antigens (SLA), has been repeatedly associated with variations in swine immune response to pathogens and vaccines as well as with production traits. The SLA antigens are also important targets for immunological recognition of foreign tissue grafts. We recently established a resource population of Korean native pigs as models for human transplantation and xenotransplantation research. In this study, 115 animals derived from three generations of the Korean native pigs were genotyped for three SLA class I (SLA-2, SLA-3 and SLA-1) and three SLA class II loci (DRB1, DQB1, DQA) using PCR with sequence-specific primers (PCR-SSP) at the allele group resolution. A total of seven SLA haplotypes (Lr-5.34, Lr-7.23, Lr-31.13, Lr-56.23, Lr-56.30, Lr-59.1, Lr-65.34), comprising six unique class I and five unique class II haplotypes, were characterized in the founding animals. Class I haplotype Lr-65.0 and class II haplotype Lr-0.34 were novel; and together with Lr-56.0 these haplotypes appeared to be breed-specific. In the progeny population, Lr-7.23 and Lr-56.30 appeared to be the most prevalent haplotypes with frequencies of 34.7% and 31.6%, respectively; the overall homozygosity was 27.4%. This resource population of SLA-defined Korean native pigs will be useful as large animal models for various transplantation and xenotransplantation experiments, as well as for dissecting the roles of SLA proteins in swine disease resistance and production traits.

Aims and status of the German DFG ‐ Transregio Research Group Xenotransplantation

Allotransplantation is the therapy of choice for endstage diseases. Due to the organ shortage, three times respectively twice as many patients are currently waiting for kidneys or hearts as are actually transplanted annually in Germany. Consequently, the mortality rate on the heart transplant list is around 16%; if patients have to wait more than 24 months for a kidney, the graft survival after 10 years will be less than half when compared to those who are able to receive an organ within 6 months. Therefore, porcine xenotransplantation of kidneys and hearts will be a very much needed alternative clinical technique – including (porcine) xenogenic islet cell transplantation for diabetic patients with hypoglycaemic attacks – a large number of persons, if one considers that the number of people suffering from diabetes worldwide increased by nearly 50% over the last decade.Of course, much more work is needed before clinical xenotransplantation will become a safe and long lasting alternative. The α(1,3)‐galactosyltransferase KO, PERV C free pigs – held in specific pathogen free units – must express several human transgenes, like a complement regulator molecule (in order to suppress non Gal‐AB's) and HLA‐E (protection from NK‐cell cytotoxicity). Some kind of accommodation will be necessary (e.g. via Hemoxygenase‐1 or PDL‐1 – a negative costimulatory receptor which also increases the CD4+ CD25high Fox p3+ subset). An overexpression of thrombomodulin (TM) would finally be necessary in order to overcome the disparity between porcine (TM) and human (Thrombin) proteins and possibly avoid microvascular thrombotic disease.And to top all these desires of a clinical transplant specialist, all the genetic engineering should be achieved in close connection within one chromosome, preferably an artificial one, in order to make all these changes hereditary.The just mentioned strategies are the main topics of the DFG‐Transregio Research Group. The following abstracts will highlight the respective progress and will also inevitably evaluate arising problems. International leaders in our field will give their results and opinion – an interchange which is necessary within the small but very active xenotransplantation research field.It is my sincere belief that a clinical scenario of xenotransplantation will be possible in the near future.

Latrunculin A Dramatically Improves the Developmental Capacity of Nuclear Transfer Embryos Derived from Gene-Modified Clawn Miniature Pig Cells.

Cells, tissues, and organs of gene-modified miniature pigs produced by somatic cell nuclear transfer (SCNT) are useful for xenotransplantation and biomedical research. However, the efficiency of SCNT still remains low, when the gene-modified cells are used as donor nuclei. The objective of this study is to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerisation inhibitor, on in vitro and in vivo developmental ability of cloned embryos derived from gene-modified miniature pig cells. Fetal fibroblasts were obtained from a female cloned fetus (Day 21 of pregnancy) of Clawn miniature pigs derived from porcine alpha 1-3 galactosyltransferase gene knockout cells. Pig oocytes with compact cumulus mass collected from ovaries of a local slaughterhouse were matured for 40-42 h and in vitro-matured oocytes were enucleated by aspiration with a thin glass pipette. Then a single fibroblast cell was introduced into the perivitelline space of an enucleated oocyte and electrically fused using needle-type electrodes in fusion medium. Following fusion treatment, reconstructed embryos were electrically activated. After activation, reconstructed embryos were cultured for 2 h in modified PZM3 supplemented with or without a cytoskeletal inhibitor [0.5 µM LatA, 1 µM LatA, or 10.4 µM cytochalasin B (CB)]. Following postactivation treatment, cloned embryos were cultured in modified PZM3 for further development and assessed for cleavage and blastocyst formation at 2 and 7 days of culture, respectively. To determine the in vivo developmental ability of cloned embryos, some embryos exposed to 0.5 µM LatA were transferred to the oviducts of a recipient miniature pig (24 h of culture) and lapalotomy was carried out on Day 21 of pregnancy. The cleavage rate was significantly higher (P<0.05) in embryos exposed to 0.5 µM LatA (86.7%) than those in embryos exposed to CB (74.1%) and without a cytoskeletal inhibitor (70.9%). Moreover the blastocyst formation rate was significantly higher (P<0.05) in embryos exposed to 0.5 µM (33.7%) or 1 µM LatA (32.9%) than those in embryos exposed to CB (18.5%) and without a cytoskeletal inhibitor (11.4%). In addition, five fetuses were obtained from recipient uteri after embryo transfer. The results of this study show for the first time that postactivation treatment with LatA is effective to improve the in vitro developmental capacity of gene-modified cloned miniature pig embryos and the embryos treated with LatA have the ability to develop into fetuses. (poster)

Chapter 7: Informed consent and xenotransplantation clinical trials

This essay explores the meaning and implications of informed consent in xenotransplantation clinical trials from both ethically justifiable and international perspectives. In international and national codes and guidelines involving human subject research and in the laws of many nations, the informed consent of research subjects is obligatory. Its moral foundations include and also extend beyond respect for individual persons as autonomous agents in Western nations. Axioms regarding the value of human life and duties to protect innocent and vulnerable persons from harm, duress, and deceit underlie Western individualism and are broadly shared in many non-Western cultures. Accents on family and/or community consent in China and other nations are compatible with individual consent as long as family and community consent supplement, rather than replace, individual consent. Reflecting its moral foundations, informed consent in medical research is rightly characterized as "voluntary" or "freely given" informed consent because it encompasses researchers' disclosure and subjects' comprehension of all the relevant information about the protocol that reasonable persons would want to know in order to freely and affirmatively enroll in the research. The interplay between these conceptual foundations of informed consent and the realities of xenotransplantation research defines what the nature and functions of consent should be in xenotransplantation clinical trials. Because these trials involve a complex body of medical information, numerous procedures, numerous risks (associated with failure rates, immunosuppression, xenogeneic infections, and so on) and the subject's obligation to abide by extensive national and international precautionary guidelines, informed consent should be enacted as an organized, sequential, thoughtfully paced, jargon-free process of communication. The features and functions of consent forms or consent documents should accord with this process. Rather than being virtually equated with informed consent, consent documents should be utilized as templates of relevant, essential, and understandable information that contribute to comprehension and voluntary enrollment. In xenotransplantation clinical research, the consenting process must cover a large number of topics, including treatment choices, participation information, study procedures, information about risks associated with immunosuppression, xenogeneic infections, discomforts, and other matters. In addition, due to infectious risks, subjects are obliged to 10 post-protocol responsibilities. Two of the three unique moral issues regarding informed consent in xenotransplantation trials involve what to do to minimize post-protocol infectious risks and what to do about international and national guidelines that affirm the subject's right to withdraw from participation in medical research at any time. The third moral issue centers on issues involving the enrollment of children and mentally incapacitated adults. The other chapters in this consensus statement demonstrate that, morally and logically, favorable harm-benefit determinations precede considerations of informed consent. When these harm-benefit assessments are favorable enough to warrant the onset of clinical trials, informed consent emerges as a pivotal moral precondition for these trials.

Xeno's paradox

There has always been a serious shortage of transplant organs for use in patients with critical organ failure as the result of accident or disease. As such, the idea of transplanting animal organs into humans is almost a century old, having first been advanced in 1906 by the French surgeon Mathieu Jaboulay (1860–1913), who implanted a pig's kidney into one woman and a goat's liver into another, neither of whom survived. However, although xenotransplantation has promised to improve the provision of organs and tissues for sufferers from various otherwise terminal conditions, it has yet to find its way into clinical use. In fact, the 1990s seemed to sound a death knell for xenotransplantation, as growing fears that xenografts could be a source of potentially lethal zoonoses resulted in the mass withdrawal of the pharmaceutical industry from the research; a blow that came on top of the already seemingly intractable problem of immunological rejection of organ grafts. Against all odds, however, xenotransplantation research lived on in several countries, ultimately allowing the development of new techniques to address many of the problems, as well as fostering the conviction that the zoonosis threat might have been overstated. In any case, the chronic shortage of human organ donors continues, which means that, on average, about 60% of patients waiting for a transplant die before an organ becomes available. More recently, it has also become clear that it is going to take some time before artificial organs can be developed for use in the clinic. “Animal organs are, in my view, much further along than human‐made equivalents in being able to replace end‐stage failed organs,” commented Megan Sykes, Associate Director of the Transplantation Biology Research Centre at Massachusetts General Hospital (Boston, MA, USA). “The total artificial heart is farthest along, but is still far from …

Fibroblasts from the new-born male testicle of Guangxi Bama mini-pig (Sus scrofa) can support nuclear transferred embryo development in vitro

Miniature pigs are valuable for research in xenotransplantation and as models for investigating human diseases. Although many mammalian species have been cloned, the success rates have been very low, especially in the pig. In the present study, an attempt was made to optimize somatic cell nuclear transfer (SCNT) protocols for use in the production of the Guangxi Bama mini-pig. Firstly, mini-pig fibroblast cells from a new-born Guangxi Bama piglet were isolated and cultured. Cell type was identified by fluorescence immunocytochemistry (ICC); the cells expressed cimentin, but not cytoceratin and follicular stimulation hormone receptor (FSHR). Secondly, the optimal cell cycle synchronization protocol for treating fibroblast cells from the newborn piglet's testicle was investigated by contact inhibition and serum starvation. When fibroblast cells were treated by contact inhibition, a higher fusion (66.0% vs. 58.3%, p > 0.05) and blastocyst production (20.8% vs. 15.1, p > 0.05) rates were obtained than with serum starvation. Thirdly, to examine the ability of old cells to be morphologically remodelled after activation, testicular fibroblasts (passage 10-14) were introduced into enucleated oocytes; enlarged nuclei were formed in most of the reconstructed embryos at 6 h and enlarged nuclei or distinct pseudopronuclei were formed in nearly all the reconstructed embryos at 12 h. The old donor cell could be morphologically remodelled correctly and was competent to support embryo development to the blastocyst in vitro. Fourthly, the in vitro development potential of the cloned embryos was investigated using two types of donor cell: ear fibroblasts and low or high passage testicular fibroblasts. The rate of fusion was highest using low passage testicle fibroblasts (84.5% vs. 69.8% and 80.0%, p < 0.05), as was development to the blastocyst stage (14.6% vs. 7.7% and 6.3%, p < 0.05). Finally, the effect of phytohaemagglutinin (PHA) on parthenogenetic and cloned embryo development was examined. The PHA had no significant effect on the parthenogenetic embryos, but cloned embryo development to the blastocyst stage was significantly increased by PHA (10 microg/ml), (13.4% vs. 5.6% and 5.6%, p < 0.05).

Isolation and Sequencing of Pig Blm and Ubl-1/SUMO-1 Genes That Relate to the Recombination Machinery

Background The gene knockout technique is important in xenotransplantation research. Herein we have described the molecular cloning of two genes that are candidates to overcome the poor rate of homologous recombination. Methods Candidate cDNA fragments were amplified by polymerase chain reaction (PCR) with the corresponding primer sets deduced from a multiple alignment analysis of other mammalian genes from a cDNA library prepared from pig spleen tissue. To obtain the full-length cDNA, a 5′- and 3′-RACE PCR experiments was performed. Results We successfully isolated the cDNA sequences of two pig genes— BLM, a Bloom's syndrome-related gene, and UBL-1/SUMO-1—which are closely related to homologous recombination events. As a result, we verified the sequences of pig BLM and pig UBL-1/SUMO-1. The nucleic acid and amino acid coding sequence homologies of pig BLM gene with the corresponding human gene were 87.3% and 82.9%, respectively. The nucleic acid and amino acid coding sequence homologies of the pig UBL-1/SUMO-1 gene with the human gene were 96.4% and 100%, respectively. Conclusion Current research into homologous recombination provides the possibility for improvement of gene knockout efficiency by regulating the gene expression profiles of recombination-related genes. Transient interference with the expression of pig UBL-1/SUMO-1 and BLM is expected to improve gene targeting. The results of the present study provided important information to design siRNA knockdown vectors. They were also useful for ex ante evaluation of expression profiles of these genes in primary cultures of somatic cells, which may enhance the production of gene knockout pigs.

Future issues in transplantation ethics: ethical and legal controversies in xenotransplantation, stem cell, and cloning research

With little prospect of developing a sufficient supply of human transplantable organs to meet the large and growing demand, attention has turned to xenotransplantation, as well as stem cell and cloning research, as possible approaches for alleviating this allograft shortage. This article explores ethical and legal issues that surround developments in these fields.

Religious Aspects of Organ Transplantation

No religion formally forbids donation or receipt of organs or is against transplantation from living or deceased donors. Only some orthodox jews may have religious objections to “opting in.” However, transplantation from deceased donors may be discouraged by Native Americans, Roma Gypsies, Confucians, Shintoists, and some Orthodox rabbis. Some South Asia Muslim ulemas (scholars) and muftis (jurists) oppose donation from human living and deceased donors because the human body is an “amanat” (trusteeship) from God and must not be desecrated following death, but they encourage xenotransplantation research. No religion formally obliges one to donate or refuse organs. No religion formally obliges one to consider cadaveric organs “a societal resource” or considers organ donation “a religious duty” (except some rabbis and isolated Muslim and Christian scholars) No religion has a formal position on “bonus points,” which is priority on the waiting list. Living organ donation is strongly encouraged only between jesus christians (15 of 28 jesus christians worldwide have donated a kidney). No religion forbids this practice. Directed organ donation to people of the same religion has been proposed only by some Orthodox Jews and some Islamic Ulemas/Muftis. Only some Muslim Ulemas/Muftis and some Asian religions may prefer living donation over cadaveric donation. No religion prefers cadaveric over living donation. No religion formally forbids non–heart-beating donors (nhbd) cadaveric donation or cross-over donation. Due to the sacrad of human life, the Catholic Church is against donation from anencephalic donors or after active euthanasia. No religion formally forbids xenotransplantation. Addressing the participants of the First International Congress of the Society for Organ Sharing in 1991, Pope John Paul II said “There are many questions of an ethical, legal and social nature which need to be more deeply investigated. There are even shameful abuses which call for determined action on the part of medical association and donor societies, and especially of competent legislative bodies” and later on “In effect, the human body is always a personal body, the body of a person. The body cannot be treated as a merely physical or biological entity, nor can its organs and tissues ever be used as item for sale or exchange”. Addressing the participants at the XVIII International Congress of the Transplantation Society in 2000, Pope John Paul II said “Accordingly, any procedure which tends to commercialize human organs or to consider them as items of exchange or trade must be considered morally unacceptable, because to use the body as an object is to violate the dignity of the human person” and later on added “The criteria for assigning donated organs should in no way be discriminatory (i.e. based on age, sex, race, religion, social standing, etc.) or utilitarian (i.e. based on work capacity, social usefulness, etc.).” To conclude, according to the Catechism of the Catholic Church Compendium signed by Pope Benedict XVI on june 28, 2005, 476. Are allowed transplantation and organ donation, before and after death? Organ transplantation is morally acceptable with the consent of the donor and without excessive risks for him/her. For the noble act of organ donation after death, the real death of the donor must be fully ascertained.

Intragraft gene expression profile associated with the induction of tolerance

BackgroundXenotransplantation holds the promise of providing an unlimited supply of donor organs for terminal patients with organ failure. Pre-existing natural antibodies to the Galα1,3Galβ1,4GlcNac-R (αGal) carbohydrate xenoantigen, however, bind rapidly to the graft endothelium and initiate hyperacute rejection of wild type pig grafts in humans. Experimental procedures designed to prevent xenoantibody-mediated rejection have been tested in gal knockout mice. These mice produce anti-gal xenoantibodies and are widely used as small animal models for xenotransplantation research. In this model, chimerism for cells expressing the gal carbohydrate can be achieved by transplantation of mixed cells or by transduction of bone marrow cells with viral vectors expressing a functional α1,3 galactosyltransferase gene. Chimerism induces tolerance to heart grafts expressing αGal. The mechanisms by which tolerance is achieved include systemic changes such as clonal deletion and/or anergy. Intragraft changes that occur during the early stages of tolerance induction have not been characterized.ResultsCytoprotective genes heme oxygenase-1 (HO-1), Bcl2, and A20 that have been reported to contribute to long-term graft survival in various models of accommodation were not expressed at high levels in tolerant heart grafts. Intragraft gene expression at both early (Day 10) and late (>2 month) time points after heart transplant were examined by real-time PCR and microarray analysis was used to identify changes associated with the induction of tolerance. Intragraft gene expression profiling using microarray analysis demonstrated that genes identified in the functional categories of stress and immunity and signal transduction were significantly up-regulated in early tolerant grafts compared with syngeneic control grafts. Biological process classification showed lower binomial p-values in the categories of "response to biotic stimulus, defense response, and immune response" suggesting that up-regulated genes identified in these grafts promote survival in the presence of an immune response. The expression of the incompatible carbohydrate antigen (αGal) was reduced by 2 months post-transplant when compared with the expression of this gene at Day 10 post-transplant. These results suggest that the gal carbohydrate antigen is downmodulated over time in grafts that demonstrate tolerance.ConclusionOur study suggests that tolerance is associated with intragraft gene expression changes that render the heart resistant to immune-mediated rejection. Genes associated with stress and immunity are up-regulated, however cytoprotective genes HO-1, Bcl2 and A20 were not up-regulated. The expression of the gal carbohydrate, the key target initiating an immune response in this model, is down-regulated in the post-transplant period.

Current Status and Future Perspectives of Xenotransplantation and Stem Cell Research in Transplantation Field

Current Status and Future Perspectives of Xenotransplantation and Stem Cell Research in Transplantation Field

Ethical issues in xenotransplantation research with children

Ethical issues in xenotransplantation research with children

Current status of xenotransplantation research in the USA

Current status of xenotransplantation research in the USA

Xenome: A new start for xenotransplantation research in the EU

<scp>xenome</scp>: A new start for xenotransplantation research in the EU

A new start for xenotransplantation research in the European Union

A new start for xenotransplantation research in the European Union

The xenotransplantation narratives of nine type 1 diabetic patients with renal failure

Within the research project ''Treatment of Diabetes by Transplantation of Porcine Islets'' that was run by the Swedish Xenoislet Transplantation Network between 1998 and 2003, a partner project called ''Cultural Perspectives on Xenotransplantation'' investigated attitudes toward xenotransplantation development among the patients and their relatives as well as the medical personnel at a transplantation clinic in Sweden. In-depth interviews were done with 37 individuals: nurses, doctors, researchers, patients, and relatives. All interviews were taped and transcribed verbatim. In this presentation the interviews with the nine type 1 diabetic patients with renal failure that took part in the project are focused. The personal and embodied narratives of these nine patients illustrate the ambivalent situation of individuals who are both patients and (potential) experimental subjects simultaneously. Both as actual practice and as biomedical information xenotransplantation research triggers a certain kind of cultural identity of the chronically ill patients--an identity related to how the patients ''translate'' scientific information and, at the same time, counter risk and uncertainty as well as promises of remedy. A central dimension in this cultural identity is a positive but still reluctant embodied attitude toward what spearhead science can bring about.

Glycobiology relating to xenotransplantation

Purpose of reviewCarbohydrates, beginning with Galα1,3Gal (Gal) antigens, have played an important part in xenotransplantation research. Now that Gal-related problems have been resolved by the breeding of α1,3-galactosyltransferase gene-knockout pigs, additional issues are becoming clear and demandi

The Sacred and the Profane: Biotechnology, Rationality, and Public Debate

This paper explores the forms of argumentation employed by participants in a recent public engagement process in the United Kingdom around new technologies for organ transplantation, with specific reference to xenotransplantation and stem-cell research. Two forms of reasoning recur throughout participants' deliberations which challenge specialist framing of this issue. First, an often scatological humour and sense of the profane are evident in the ways in which participants discuss the bodily transformations that such technologies demand. Second, a sense of the sacred, in which new biotechnologies are viewed as against nature or in which commercial companies are ‘playing god’, is a repetitive and well-recognised concern. Such forms of reasoning are frequently dismissed by policymakers as ‘uninformed gut reactions’. Yet they also form a significant part of the repertoire of scientists themselves as they proclaim the hope of new medical breakthroughs, or seek to reconstruct ideas of the body to facilitate new biotechnological transformations. Through questioning of assumptions in Habermas's notion of discourse ethics, and exploring the importance of hybridity and corporeality as concepts in ethical thinking, the author suggests that, far from being ill-formed opinions, such reasonings perform an important function for thinking through the ontological significance of the corporealisation of these proposed new forms of human and animal bodies.