异种移植的病毒安全性研究进展

猪-人异种移植有望解决人源器官短缺的严重问题。然而,以前病毒（provirus）形式整合入猪基因组中的猪内源性反转录病毒（porcine endogenous retrovirus,PERV）难以去除,PERV有可能通过异种移植传播给人类,甚至产生新的病毒性疾病。本文回顾了PERV与异种移植病毒安全性及我国特有小型猪中PERV的相关研究。     

Involvement of position-147 for HLA-E expression

HLA-E functions as an inhibitory signaling molecule of natural killer (NK) cell-mediated cytolysis. However, the cell surface expression of HLA-E molecules is quite restricted because of the limited repertoire of binding peptide sequences, such as signal peptides of other HLA molecules, especially on xenogeneic cells. In this study, we successfully determined that position-147 is an important amino acid position for cell surface expression by producing point substitutions. For further studies concerning transplantation therapy, the point substitution, Ser147Cys, that resulted in a single atom change, oxygen to sulfur, designated as HLA-Ev(147), led to a much higher expression on the human and pig cell surface and a greater inhibitory function against human NK cells than wild type HLA-E in an in vitro model system of pig to human xenotransplantation. Consequently, HLA-Ev(147) might be a promising alternative gene tool for future transplantation therapy such as xenotransplantation.     

Xenotransplantation and tolerance

The application of xenotransplantation faces daunting immunological hurdles, some of which might be overcome with the induction of tolerance. Porcine organs transplanted into primates are subject to several types of rejection responses. Hyperacute rejection mediated by naturally occurring xenoreactive antibodies and complement can be overcome without tolerance. Acute vascular rejection and cellular rejection, however, may present important opportunities for immunological tolerance, and humoral rejection might be approached by various mechanisms including (i) clonal deletion, (ii) anergy, (iii) immune deviation, (iv) induction of immunoregulatory or suppressor cells, or (v) veto cells. B-cell tolerance, useful for preventing humoral rejection, might be approached through clonal anergy. It remains to be determined, however, whether tolerance induction is required for xenotransplantation and by which means the various mechanisms of tolerance can be applied in the setting of xenotransplantation. Regardless, the study of tolerance will surely expand understanding of the physiology and pathophysiology of the immune system.     

Piscine islet xenotransplantation

Tilapia, a teleost fish species with large anatomically discrete islet organs (Brockmann bodies; BBs) that can be easily harvested without expensive and fickle islet isolation procedures, make an excellent donor species for experimental islet xenotransplantation research. When transplanted into streptozotocin-diabetic nude or severe combined immunodeficient mice, BBs provide long-term normoglycemia and mammalian-like glucose tolerance profiles. However, when transplanted into euthymic recipients, the mechanism of islet xenograft rejection appears very similar to that of islets from "large animal" donor species such as the very popular fetal/neonatal porcine islet cell clusters (ICCs). Tilapia islets are more versatile than ICCs and can be transplanted (1) into the renal subcapsular space, the cryptorchid or noncryptorchid testis, or intraportally as neovascularized cell transplants; (2) as directly vascularized organ transplants; or (3) intraperitoneally after microencapsulation. Unlike the popular porcine ICCs, BBs function immediately after transplantation; thus, their rejection can be assessed on the basis of loss of function as well as other parameters. We have also shown that transplantation of tilapia BBs into nude mice can be used to study the possible implications of cross-species physiological incompatibilities in xenotransplantation. Unfortunately, tilapia BBs might be unsuitable for clinical islet xenotransplantation because tilapia insulin differs from human insulin by 17 amino acids and, thus, would be immunogenic and less biologically active in humans. Therefore, we have produced transgenic tilapia that express a "humanized" tilapia insulin gene. Future improvements on these transgenic fish may allow tilapia to play an important role in clinical islet xenotransplantation.     

哺乳动物卵母细胞异位发育技术及其研究进展

综述了哺乳动物卵巢移植在生物医学上的应用,及借助卵巢异位发育研究卵巢卵母细胞发育机制的研究进展;详细介绍了卵巢移植的不同技术方法,及移植后卵巢卵母细胞的发育、激素的调节机制,分析了卵巢移植早期卵泡生长和凋亡的分子机理及其在移植医学中的应用前景,并就新鲜和冻融卵巢如何用于动物辅助生殖进行了阐述.特别关注卵巢卵母细胞异种移植的潜在价值,卵巢异种移植不仅可以保存稀有和濒危物种,还可以为当前辅助生殖技术提供一个理想的实验手段.     

Repression of porcine endogenous retrovirus infection by human APOBEC3 proteins

It has been shown that porcine endogenous retrovirus (PERV) can infect human cells, indicating that PERV transmission poses a serious concern in pig-to-human xenotransplantation. A number of recent studies have reported on retrovirus interference by antiviral proteins. The most potent antiviral proteins are members of the APOBEC family of cytidine deaminases, which are involved in defense against retroviral attack. These proteins are present in the cytoplasm of mammalian cells and inhibit retroviral replication. To evaluate the inhibition of PERV transmission by human APOBEC3 proteins, we co-transfected 293T cells with a PERV molecular clone and human APOBEC3F or APOBEC3G expression vectors, and monitored PERV replication competency using a quantitative analysis of PERV pol genes. The replication of PERVs in cells co-expressing human APOBEC3s was reduced by 60–90% compared with PERV-only control. These results suggest that human APOBEC3G and APOBEC3F might serve a potential barrier function against PERV transmission in xenotransplantation.     

A shrewd and ethical approach to xenotransplantation

Policies surrounding xenotransplantation, and many other emerging high-technology interventions, must balance opportunity and risk. Whereas traditional stakeholders, such as the researcher community, government agencies and the commercial sector, readily contribute to the debates that influence policies, the voice of the public is seldom heard. Not only does this raise ethical concerns but also it might ultimately prove to be shortsighted. Before any country settles unilaterally on comprehensive policies governing the practice of xenotransplantation, well-informed public opinions need to be taken into account.     

Xenogeneic organ transplantation

Over the past few years, several major advances have occurred in the understanding of how the humoral and cellular immune system of humans recognizes and destroys transplant cells, tissues and organs derived from animal sources. Consequently, armed with this new knowledge, several laboratories have now developed novel immunoprotective technologies that may allow xenotransplantation to be clinically feasible.     

Infection of nonhuman primate cells by pig endogenous retrovirus

The ongoing shortage of human donor organs for transplantation has catalyzed new interest in the application of pig organs (xenotransplantation). One of the biggest concerns about the transplantation of porcine grafts into humans is the transmission of pig endogenous retroviruses (PERV) to the recipients or even to other members of the community. Although nonhuman primate models are excellently suited to mimic clinical xenotransplantation settings, their value for risk assessment of PERV transmission at xenotransplantation is questionable since all of the primate cell lines tested so far have been found to be nonpermissive for PERV infection. Here we demonstrate that human, gorilla, and Papio hamadryas primary skin fibroblasts and also baboon B-cell lines are permissive for PERV infection. This suggests that a reevaluation of the suitability of the baboon model for risk assessment in xenotransplantation is critical at this point.     

异种器官移植：前景如何？

目前异种器官移植在进入临床的过程中存在两大难题——免疫排斥和病原微生物感染.人类为防止免疫排斥而采取的措施使超急性排斥得到了很大程度的避免,但大大增加了器官移植受体感染病原微生物的机会.猪作为人类理想的器官供体,却有多种内源性病毒,其中猪内源性逆转录病毒（PERV）引起了人类较大的关注.PERV的跨种感染存在正、反两方面的证据,使得异种器官移植的前景喜忧掺半.     

Replication-competent chimeric lenti-oncovirus with expanded host cell tropism.

Baboon bone marrow was grafted into human immunodeficiency virus type 1-infected patients in the course of recent trials for AIDS treatment. Since the baboon genome harbors multiple copies of an endogenous oncovirus, chimeric lenti-oncoviruses could emerge in the xenotransplant recipient. To analyze the potential replication competence of hybrid viruses between different genera of retroviruses, we replaced most of the env gene of simian immunodeficiency virus with the env gene of an amphotropic murine leukemia virus. The hybrid virus could be propagated in human T-cell lines, in peripheral blood mononuclear cells of rhesus macaques, and in CD4- B-cell lines. Because of the expanded cell tropism, the hybrid virus might have a selective advantage in comparison to parental viruses. Therefore, emerging chimeric viruses may be considered a serious risk of xenotransplantation. A note of caution is also suggested for the use of pseudotyped lentiviral vectors for human gene therapy.     

Xenotransplantation: a tool for reproductive biology and animal conservation?

The transplantation of reproductive organs, including ovaries and ovarian tissue, was pioneered over 100 years ago. In the 1960s, ovarian grafting was used as a tool to investigate ovarian function, but with the recent development of more effective cryopreservation protocols for ovarian tissue, germline preservation and propagation have now become realistic goals. This review describes progress in ovarian banking and ovarian tissue transplantation, with emphasis on how fresh and frozen ovarian tissue can be used in assisted reproduction for both humans and animals. This paper focuses most closely on the potential value of xenotransplantation, the transplantation of gonads from one species to another, to conserve rare and endangered species. Specific attention is drawn to the use of xenotransplantation as a strategy for generating viable gametes that can be used to produce live fertile offspring. Other upcoming xenogeneic technologies that may be of potential significance in animal conservation, such as transplantation of whole ovaries or isolated growing follicles, and even male germ cells, are discussed.     

Differences in Activities of the Enzymes Of Nucleotide Metabolism and its Implications for Cardiac Xenotransplantation

Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5′nucleotidease (E5′N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.     

Xenotransplantation: current status and a perspective on the future

Xenotransplantation using pigs as the transplant source has the potential to resolve the severe shortage of human organ donors. Although the development of relatively non-toxic immunosuppressive or tolerance-inducing regimens will be required to justify clinical trials using pig organs, recent advances in our understanding of the biology of xenograft rejection and zoonotic infections, and the generation of alpha1,3-galactosyltransferase-deficient pigs have moved this approach closer to clinical application. This Review highlights the major obstacles impeding the translation of xenotransplantation into clinical therapies and the potential solutions, providing a perspective on the future of clinical xenotransplantation.     

Differences in activities of the enzymes of nucleotide metabolism and its implications for cardiac xenotransplantation

Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5' nucleotidease (E5' N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.     

异源器官移植研究进展

本文针对异源器官移植中面临的免疫排斥及病原微生物的感染等主要问题,综述了近年来国内外相关方面的最新研究进展,同时对解决这些难题的方法和技术路线进行了总结,并展望了异源器官移植领域的发展道路。     

Expression of Human Ecto 5′ Nucleotidase in Pig Endothelial Cells and Its Implication for Adenosine Production and Xenotransplantation

Human endothelial activity of ecto-5′-nucleotidase (E5′N) is several times higher than in pig endothelial cells. This may have implication for xenotransplantation due to the role this enzyme plays in conversion of pro-inflammatory and pro-aggreggatory nucleotides into anti-inflammatory and anti-aggregatory adenosine. We have shown in this study that human E5′N can be functionally expressed in pig endothelial cells leading to increased adenosine production from both extracellular AMP and ATP. We suggest that E5′N expression in transgenic pigs for xenotransplantation may help to prolong graft survival.     

Inhibition of budding/release of porcine endogenous retrovirus

PERV is integrated into the genome of all pigs. PERV‐A and PERV‐B are polytropic and can productively infect human cell lines, whereas PERV‐C is ecotropic. Recombinant PERV‐A/C can infect human cells and exhibits high titer replication. Therefore, use of pigs for human xenotransplantation raises concerns about the risks of transfer of this infectious agent from donors to xenotransplantation recipients. To establish strategies to inhibit PERV production from cells, in the present study, we investigated the mechanism of PERV budding and anti‐PERV activity of Tetherin/BST‐2. The results showed that DN mutants of WWP‐2, Tsg101, and Vps4A/B markedly reduced PERV production in human and porcine cell lines, suggesting that PERV budding uses these cellular factors and the cellular MVB sorting pathway as well as many other retroviruses. Moreover, PERV production was also reduced by human and porcine Tetherin/BST‐2. These data are useful for developing strategies to inhibit PERV production and may reduce the risk of PERV infection in xenotransplantation.