Natural Anti-pig Antibodies Research Articles

Pretransplant Screening for Prevention of Hyperacute Graft Loss in Pig-to-primate Kidney Xenotransplantation.

Xenotransplantation using pig organs is now a clinical reality. However, the process for xenograft recipient screening lacks clarity and scientific rigor: no established thresholds exist to determine which levels of preformed antipig natural antibodies (Nabs) will be safe for clinical xenograft transplantation, and hyperacute rejection (HAR) or acute humoral xenograft rejection (AHXR), which still impacts pig-to-primate kidney xenograft survivals, may impede broader application of pig-to-human clinical xenograft transplantation. We retrospectively examined 28 cases of pig-to-baboon kidney xenotransplantation using GalTKO±human complement regulatory protein (hCRP)-transgenic (Tg) pig donors, as well as 6 cases of triple-KO multi-Tg (10GE) pig donors, and developed screening algorithms to predict risk of HAR/AHXR based on recipient antipig Nab levels. Preformed Nabs were evaluated using both complement-dependent cytotoxicity and antibody (IgM and IgG) binding flow-cytometry assays. High complement-dependent cytotoxicity was associated with HAR/AHXR as expected. However, we also found that high levels of IgG were independently associated with HAR/AHXR, and we developed 2 indices to interpret and predict the risk of IgG-mediated HAR/AHXR. Based on the data in this study, we have established a new 2-step screening, which will be used for future clinical kidney xenotransplantation trials.

The effect of SLA expression on genetically-engineered pig renal cells on the human T cell xenoresponse

Abstract Background Xenotransplantation using genetically-engineered (GE) pig organs offers an unlimited supply of organs for patients with end-stage organ failure. Disrupting the porcine GGTA1/CMAH/β4GalNT2 genes (TKO) markedly reduces binding of human natural anti-pig antibodies in human to carbohydrate antigens expressed on the pig organ. However, more evident have demonstrated that T cell responses play a major role in xenogeneic rejection, and that immunosuppression alone is likely incapable of completely suppressing these responses. The object of this study is at an organ-specific level to evaluate human T cell-mediated response against GE pig renal microvascular endothelial cells (RMEC) with or without swine leukocyte antigen (SLA) class I or II. Methods Primary RMEC were isolated from the GGTA1/CMAH knockout (DKO) pigs, and immortalized using Lenti-SV40 T. The gene editing was performed to create specific TKO RMEC cell lines expressing SLA class II. Stimulation of human T cell activation or proliferation by RMEC was performed flow cytometry-based mixed lymphocyte reaction (MLR). Results The specific GE cells lacking carbohydrates or expressing SLA class I or II were confirmed. MLR data shows that human CD4+ T cell proliferation by CD31+ RMEC was higher than that by fibroblast-like renal cells (RC), and was significantly inhibited by immunosuppressive drugs. Furthermore, both of CD69+ and CD25+ T cells were significantly enhanced by RMEC with expression of SLA class I and II compared with SLA class I alone. Conclusion Porcine RMEC express SLA class I/II and known costimulatory molecules and have the ability to simulate human T cell proliferation. RMEC will be a useful reagent for the further study of xenotransplantation.

Intra-bone Bone Marrow Transplantation in Pig-to-Nonhuman Primates for the Induction of Tolerance Across Xenogeneic Barriers.

Mixed chimerism and thymic tissue transplantation strategies have achieved xenogeneic tolerance in pig-to-mouse models, and both have been extended to pig-to-baboon models. A mixed chimerism strategy has shown promise toward inducing tolerance in allogeneic models in mice, pigs, nonhuman primates (NHP), humans, and a rat-to-mouse small animal xeno-model. However, even though α-1,3-galactosyltransferase gene knockout (GalTKO) pigs have been used as bone marrow (BM) donors, direct intravenous injection of porcine BM cells was detected for only up to 4days (peripheral macro-chimerism) in one case, and the rest lost chimerism within 2days.Recent data in allogeneic models demonstrated that direct injection of donor BM cells into recipient BM spaces (intra-bone bone marrow transplantation: IBBMTx) produces rapid reconstitution and a higher survival rate compared to i.v. injection. In order to minimize the loss of injected porcine BM peripherally before reaching the BM space, Yamada developed a xeno-specific regimen including IBBMTx coated with a collagen gel matrix in a preclinical pig-to-baboon model (Yamada IBBMTx). This strategy aims to achieve improved, persistent macro-chimerism as well as engraftment of BM across a xenogeneic barrier. The initial study published in 2015 demonstrated that this IBBMTx strategy leads to markedly prolonged peripheral macro-chimerism detectable for up to 23days. Furthermore, a more recent study using human CD47-transgenic (Tg) GalTKO pigs as xeno-donors achieved long-lasting macro-chimerism >60days with evidence of reduction of anti-pig natural antibodies (nAb). This is the longest macro-chimerism that has ever been achieved in a preclinical large animal xenotransplant model to date. In this chapter, we introduce a brief summary of our achievements in regard to successful tolerance induction by utilizing our novel strategy of IBBMTx as well as describe the step-by-step methodology of surgical and in vitro procedures that are required for this project.

An approach to induction of tolerance to pig cardiac xenografts in neonates.

There is a continuing need for donor hearts for infants with complex congenital heart defects. The transplantation of hearts from neonatal pigs would be an alternative to human organs, particularly if donor-specific immunological tolerance could be achieved. The great majority of infant humans do not make natural (preformed) antibodies against triple-knockout (TKO) pigs (that do not express any of the three known pig antigens against which humans have natural anti-pig antibodies). The transplantation of a heart from a TKO pig into an infant would therefore minimize any risk of early antibody-mediated rejection, and, with adequate immunosuppressive therapy, prolonged graft survival may well be achieved. Total host thymectomy (commonly carried out at the time of orthotopic heart transplantation in this age group)±residual T-cell depletion and donor-specific pig thymus tissue transplantation might induce T-cell tolerance and allow immunosuppressive therapy to be discontinued (if there is in vitro evidence of T-cell and B-cell nonresponsiveness to donor-specific pig cells). Even if tolerance were not achieved, with continuing immunosuppressive therapy, the graft would likely "bridge" the patient until a suitable allograft became available or be associated with prolonged xenograft function.

The pathobiology of pig-to-primate xenotransplantation: a historical review.

The immunologic barriers to successful xenotransplantation are related to the presence of natural anti-pig antibodies in humans and non-human primates that bind to antigens expressed on the transplanted pig organ (the most important of which is galactose-α1,3-galactose [Gal]), and activate the complement cascade, which results in rapid destruction of the graft, a process known as hyperacute rejection. High levels of elicited anti-pig IgG may develop if the adaptive immune response is not prevented by adequate immunosuppressive therapy, resulting in activation and injury of the vascular endothelium. The transplantation of organs and cells from pigs that do not express the important Gal antigen (α1,3-galactosyltransferase gene-knockout [GTKO] pigs) and express one or more human complement-regulatory proteins (hCRP, e.g., CD46, CD55), when combined with an effective costimulation blockade-based immunosuppressive regimen, prevents early antibody-mediated and cellular rejection. However, low levels of anti-non-Gal antibody and innate immune cells and/or platelets may initiate the development of a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. This pathogenic process is accentuated by the dysregulation of the coagulation-anticoagulation systems between pigs and primates. The expression in GTKO/hCRP pigs of a human coagulation-regulatory protein, for example, thrombomodulin, is increasingly being associated with prolonged pig graft survival in non-human primates. Initial clinical trials of islet and corneal xenotransplantation are already underway, and trials of pig kidney or heart transplantation are anticipated within the next few years.

Rituximab Treatment Prevents the Early Development of Proteinuria following Pig-to-Baboon Xeno-Kidney Transplantation

We previously reported life-supporting α1,3-galactosyltransferase knockout (GalTKO) thymokidney xenograft survival of >2 months in baboons. However, despite otherwise normal renal function, recipients developed proteinuria with morphologic changes (podocyte effacement), a condition that presents a major obstacle to long-term studies in this model. A recent clinical study showed that rituximab therapy after allogeneic transplant prevented proteinuria possibly associated with loss of sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b). Here, we demonstrate that rituximab prevents the disruption of pig podocytes in an SMPDL-3b-dependent manner in vitro and the early development of proteinuria after xenogeneic kidney transplantation in baboons. Immunofluorescence showed SMPDL-3b expression in pig glomerular epithelium; immunoprecipitation demonstrated rituximab binding to SMPDL-3b in glomeruli. Culture of isolated pig podocytes with naive baboon sera, which has preformed antipig natural antibodies, reduced SMPDL-3b expression, disrupted podocyte morphology, and decreased podocyte proliferation, whereas pretreatment with rituximab prevented these effects. Six baboons received rituximab before transplantation to deplete B cells and again in the peri-transplant period; 18 baboons treated only before transplantation served as historical controls. The onset of post-transplant proteinuria was significantly delayed in a B cell-independent manner in the animals that received peri-transplant rituximab treatment. Although further optimization of this protocol is required, these data provide intriguing clues to the mechanisms of post-transplant proteinuria in xenogeneic kidney transplantation and a potential strategy for its prevention.

Minimal effect of bortezomib in reducing anti‐pig antibodies in human leukocyte antigen‐sensitized patients: a pilot study

Bortezomib, a proteasome inhibitor used to treat multiple myeloma, has been administered (± plasma exchange ± intravenous immunoglobulin [IVIg]) in attempts to reduce antibodies against human leukocyte antigens (HLA) in sensitized patients undergoing organ transplantation. To our knowledge, bortezomib has not been investigated for its effect on natural anti-pig antibodies. If bortezomib could reduce the production of anti-pig antibodies, this would likely be beneficial to the outcome of pig organ grafts in primates. Nine patients received bortezomib either to reduce anti-HLA antibody levels before organ allotransplantation or to treat antibody-mediated rejection. Patients at the Mayo Clinic (Group 1; n=4) received bortezomib alone, whereas at the UPMC (Group 2; n=5), this was combined with plasmaphereses ± IVIg in some cases. Anti-pig IgM and IgG levels against wild-type (WT) and α1,3-galactosyltransferase gene knockout (GTKO) pig aortic endothelial cells (flow cytometry-relative mean fluorescence intensity) and anti-Gal IgM and IgG (ELISA-OD480nm ) were measured pre- and post-bortezomib therapy. Mean anti-pig IgM levels were 11.2 (WT) and 1.9 (GTKO) pre-bortezomib treatment and 9.4 (WT: P=0.02) and 1.7 (GTKO: P=0.33) post-bortezomib treatment, respectively. Mean anti-pig IgG levels were 4.3 (WT) and 1.5 (GTKO) pre-bortezomib treatment and 3.6 (WT: P=0.21) and 1.4 (GTKO: P=0.20) post-bortezomib treatment, respectively. Mean anti-Gal IgM and IgG levels were 0.7 and 1.1, respectively, pre-treatment, and 0.6 (P=0.03) and 1.1 (NS), respectively, post-treatment. When the data were analyzed in Groups 1 and 2 separately, there were no significant differences between the pre- and post-bortezomib levels of anti-pig, anti-non-Gal, or anti-Gal IgM or IgG. From this limited study, we conclude that bortezomib might reduce anti-Gal IgM levels in primates, but, in this respect alone, is unlikely to have any significant effect on the outcome of GTKO pig organ transplantation.

The potential of genetically-engineered pigs in providing an alternative source of organs and cells for transplantation

There is a critical shortage of organs, cells, and corneas from deceased human donors worldwide. There are also shortages of human blood for transfusion. A potential solution to all of these problems is the transplantation of organs, cells, and corneas from a readily available animal species, such as the pig, and the transfusion of red blood cells from pigs into humans. However, to achieve these ends, major immunologic and other barriers have to be overcome. Considerable progress has been made in this respect by the genetic modification of pigs to protect their tissues from the primate immune response and to correct several molecular incompatibilities that exist between pig and primate. These have included knockout of genes responsible for the expression of major antigenic targets for primate natural anti-pig antibodies, insertion of human complement- and coagulation-regulatory transgenes, and knockdown of swine leukocyte antigens that stimulate the primate's adaptive immune response. As a result of these manipulations, the administration of novel immunosuppressive agents, and other innovations, pig hearts have now functioned in baboons for 6-8 months, pig islets have maintained normoglycemia in diabetic monkeys for > 1 year, and pig corneas have maintained transparency for several months. Clinical trials of pig islet transplantation are already in progress. Future developments will involve further genetic manipulations of the organ-source pig, with most of the genes that are likely to be beneficial already identified.

Outwitting evolution*

The author pays tribute to those who stimulated him to follow a career in clinical cardiothoracic surgery and experimental transplantation. Highlights of his career have been in contributing to (i) the first hypothermic perfusion storage of the donor heart to be used successfully in clinical practice, (ii) hormonal therapy in the management of the brain-dead potential organ donor, (iii) the identification of Gal alpha 1,3Gal as the major antigenic target for primate natural anti-pig antibodies, (iv) the 2- to 6-month survival of alpha1,3-galactosyltransferase gene-knockout hearts transplanted into baboons, and (v) the 3- to 12-month normoglycemia following the transplantation of islets from CD46-transgenic pigs into diabetic monkeys. Many friends have been made through a mutual interest in transplantation, particularly through the activities of the International Xenotransplantation Association (IXA). The author thanks the many mentors, colleagues, and research fellows with whom it has been his great privilege and pleasure to work during the past several decades, and readily acknowledges that it is largely their contributions that have enabled him to receive the honor of Honorary Membership of the IXA.

Complement activation and coagulation in xenotransplantation

Xenotransplantation using porcine donors holds tremendous promise for alleviating the chronic shortage of human organ donors. However, transplantation from pigs into higher primates triggers a vigorous immune response involving complement activation and thrombosis. Hyperacute rejection can be prevented by using donors in which GalT, the gene responsible for the predominant target of anti-pig natural antibodies, has been deleted. Unfortunately, the adaptive response to GalT knockout (KO) organs has been difficult to immunosuppress. The focus has shifted to identifying additional genetic modifications that will extend xenograft survival beyond the several months currently achievable. Which gene(s) should be added to the GalT KO background is open to debate. Overexpression of a complement regulatory factor(s) seems a logical first choice, supported by recent in vitro data but not yet validated in preclinical models. The next obvious candidate would be an anticoagulant protein(s) to deal with the dysregulated coagulation that is almost invariably associated with xenograft rejection. Several potentially useful molecules have been identified, although this study is yet to be translated to the pig. Our understanding of the mechanisms of GalT KO xenograft rejection continues to grow, providing a rational basis for tailoring further genetic modification of the donor and immunosuppression of the recipient.

Porcine cell microchimerism but lack of productive porcine endogenous retrovirus (PERV) infection in naive and humanized SCID-beige mice treated with porcine peripheral blood mononuclear cells

Pigs are considered a suitable source of cells and organs for xenotransplantation. All known strains of pigs contain porcine endogenous retrovirus (PERV) and PERV released by porcine cells may infect human cells in vitro and severe-combined immunodeficient (SCID) mice in vivo. Humanized SCID (hu-SCID) mice develop immune response to porcine antigens. Here we investigated PERV transmission in humanized SCID-beige mice using porcine peripheral blood mononuclear cells (PBMC) as the donor tissue (and the source of PERV). Mice were infused in the peritoneal cavity with 1.5–3.0×10 7 unfractionated human PBMC. Unfractionated porcine PBMC (1.5–3.0×10 7 cell/mouse) were infused to the mice simultaneously with human PBMC or 3 weeks after human PBMC infusion. The treated mice were monitored for weight and skin changes, donor cell chimerism, anti-pig antibodies and PERV transmission. All humanized mice tested 5–12 weeks after human PBMC transplantation were macrochimeric (up to 40% of cells in blood) for human cells, where 99% of the human cells were T-lymphocytes. Although human B lymphocytes were very rare in the blood of humanized mice at that point, the mice were positive for human anti-pig natural antibodies. The control SCID-beige mice or mice treated with porcine PBMC alone were negative for anti-porcine antibodies. Approximately 70% of the humanized mice treated with porcine PBMC were also microchimeric for porcine cells. Although some tissue samples of these mice were positive for PERV DNA in the absence of porcine DNA indicating PERV infection, the infection was non-productive as PERV transcripts were not detectable in those tissues. PERV infection of human and mouse cells in vitro by co-culturing with porcine PBMC was also non-productive. Humanized SCID-beige mice suffered weight loss and occasional minor skin changes due to graft vs. host disease caused by human PBMC but none of the mice showed observable effect attributable to the apparent PERV infection alone.

Depletion of natural anti-pig antibodies by the continuous infusion of oligosaccharides in a pig-to-baboon model

Depletion of natural anti-pig antibodies by the continuous infusion of oligosaccharides in a pig-to-baboon model

Long-term discordant xenogeneic (porcine-to-primate) bone marrow engraftment in a monkey treated with porcine-specific growth factors.

Mixed allogeneic hematopoietic chimerism has previously been reliably achieved and shown to induce tolerance to fully MHC-mismatched allografts in mice and monkeys. However, the establishment of hematopoietic chimerism has been difficult to achieve in the discordant pig-to-primate xenogeneic model. To address this issue, two cynomolgus monkeys were conditioned by whole body irradiation (total dose 300 cGy) 6 and 5 days before the infusion of pig bone marrow (BM). Monkey anti-pig natural antibodies were immunoadsorbed by extracorporeal perfusion of monkey blood through a pig liver, immediately before the intravenous infusion of porcine BM (day 0). Cyclosporine was administered for 4 weeks and 15-deoxyspergualin for 2 weeks. One monkey received recombinant pig cytokines (stem cell factor and interleukin 3) for 2 weeks, whereas the other received only saline as a control. Both monkeys recovered from pancytopenia within 4 weeks of whole body irradiation. Anti-pig IgM and IgG antibodies were successfully depleted by the liver perfusion but returned to pretreatment levels within 12-14 days. Methylcellulose colony assays at days 180 and 300 revealed that about 2% of the myeloid progenitors in the BM of the cytokine-treated recipient were of pig origin, whereas no chimerism was detected in the BM of the untreated control monkey at similar times. The chimeric animal was less responsive by mixed lymphocyte reaction to pig-specific stimulators than the control monkey and significantly hyporesponsive when compared with a monkey that had rejected a porcine kidney transplant. To our knowledge, this is the first report of long-term survival of discordant xenogeneic BM in a primate recipient.

Nitric oxide production by pig endothelial cells in response to human-derived injury.

The hyperacute rejection induced by natural antibodies is the first barrier to the success of pig to human organ xenotransplantation. When this barrier is overcome, an infiltrate of mainly monocytes and natural killer cells can be observed. Nitric oxide (NO) has been described to be involved in allo- and xenorejection, and to participate in the regulation of monocyte infiltration in other models. We have studied the capacity of human monocytes and natural antibodies to induce the production of NO by pig endothelial cells, by measuring NO2, a stable end product of NO. Human monocytes can induce HuProVim (HUP), a pig endothelial line, and "in situ, ex vivo" pig endothelial cells to produce NO2. This NO2 production was inhibited by NG-nitro-L-arginine-methylester and NG-monomethyl-L-arginine, inhibitors of NO production. This induction can be observed even if cells are separated by a semipermeable membrane, which indicates that it is a result of soluble factors. Activated cells continue producing NO after triggering for 1 hr. No NO2 production was observed after activation of HUP cells with recombinant human interleukin (IL)-1alpha, IL-1beta, IL-6, IL-10, transforming growth factor-beta1, IL-2, IL-4, interferon-gamma, or recombinant human tumor necrosis factor-alpha (rhTNF-alpha) alone. Only the combination of rhTNF-alpha+rIL-1alpha or rIL-1beta, and rhTNF-alpha+rIL-1alpha+IFN-gamma induces some NO production. Human natural anti-pig antibodies, which had been described to induce cytoskeleton changes on endothelial cells, do not induce NO production. Our data show that human monocytes induce the production of NO by pig endothelial cells. The inducing signal is soluble and cannot be provided by human anti-pig natural antibodies.

DEMONSTRATION OF IgM ANTIBODIES OF HIGH AFFINITY WITHIN THE ANTI-GAL??1-3GAL ANTIBODY REPERTOIRE

Human anti-Galalpha1-3Gal IgG and IgM xenoantibodies can distinguish between very similar epitopes with a high degree of selectivity. Anti-Galalpha1-3Gal antibodies were affinity isolated using two separate Galalpha1-3Gal-based immunoadsorbents, Galalpha1-3Gal itself and Galalpha1-3Galbeta1-4Glc. IgG and IgM were separated using a protein G column. Antibody purity was achieved by serial adsorption/elutions from the columns. By this means, different antibody fractions were prepared that contained either IgG or IgM, reactive with either Galalpha1-3Gal, Galalpha1-3Galbeta1-4Glc, or both. The dissociation equilibrium constants (Kd) of these antibodies were then measured using an IAsys biosensor. Sera from two individuals were used and Kd values for one IgG (fraction 1A) and two IgM (fractions 1B and 2A) fractions were obtained. The Kd for the IgG was 4.85 x 10(-7) M (fraction 1A). For IgM, the Kd values were higher at 7.8x10(-10) M (fraction 1B) and 1.07x10(-10) M (fraction 2A). Natural anti-pig antibodies include high affinity IgM that continue to be produced without class switch. The B cell mechanism behind this is not known. It may be possible to exploit this mechanism in future xenotransplantation strategies.

B-cell reconstitution and xenoreactive anti-pig natural antibody production in severe combined immunodeficient mice reconstituted with immunocompetent B cells from varying sources.

Little is known about the B-cell subsets that produce xenoreactive natural antibodies (NAb). This study was undertaken to investigate the potential role of varying B-cell populations in anti-pig NAb production in mice. Severe combined immunodeficient (scid) mice were reconstituted with bone marrow or splenic or peritoneal B cells from immunocompetent mice. B-cell reconstitution and anti-pig NAb were evaluated by flow cytometric analysis. Adult marrow failed to reconstitute normal numbers of CD5+ B1a cells, but fully reconstituted CD5- Mac1- B2 and CD5- Mac1+ B1b cell populations in scid mice. Recipients of peritoneal B cells showed poor reconstitution of B2 cells, and an overshoot of B1 cells in the peritoneal cavity. Although B2 cells predominate in the adult spleen, splenic B cells from immunocompetent mice preferentially reconstituted B cells, including B1 cells, in the peritoneal cavity, but did not reconstitute splenic B2 cells. Therefore, neither adult marrow, splenocytes nor peritoneal cells can fully reconstitute scid mice with all B-cell subpopulations. Nevertheless, serum anti-pig NAb in marrow-reconstituted mice recovered to normal levels by 3 weeks, and were maintained for at least 30 weeks. Serum NAb in scid mice receiving peritoneal B cells reached normal levels by 4-7 weeks after transfer. However, NAb in sera of scid mice receiving splenic B cells took longer (>25 weeks) to reach normal levels. These results indicate that adult marrow-derived B cells can efficiently produce anti-pig NAb, and that peritoneal B cells have greater NAb-producing ability than splenic B cells or their immediate progeny.

α-Galactosyl Oligosaccharides Conjugated with Polyethylene Glycol as Potential Inhibitors of Hyperacute Rejection upon Xenotransplantation

Antibodies to an α-galactosyl saccharide structure are mainly responsible for hyperacute rejection after pig-to-primate xenotransplantation. The beneficial effect of α-galactosyl oligosaccharides has been shown on the inhibition of anti-pig natural antibodies. We synthesized polyethylene glycol (PEG)-conjugates of α-galactosyl disaccharide (Di) and trisaccharide (Tri) as potential inhibitors of the rejection reaction. The half lives of Di, Tri, PEG-conjugated Di (Di-PEG) and PEG-conjugated Tri (Tri-PEG) were 18.1 ± 2.3 min, 20.2 ± 0.9 min, 38.7 ± 2.8 min and 35.8 ± 1.6 min, respectively. Furthermore, Di-PEG and Tri-PEG showed biphasic clearance, and their half lives at the second phase were longer than 10 hours. PEG-conjugated oligosaccharides (Di-PEG, Tri-PEG) markedly inhibited cytotoxic action of human sera to pig kidney cell line (PK15) compared to unconjugated oligosaccharides (Di, Tri). The binding of IgM antibodies to PK15 cells, however, was more strongly blocked by unconjugated oligosaccharides than PEG-conjugated oligosaccharides. This phenomenon can be explained by the finding that PEG has anti-complement activity and masks antigenic sites of oligosaccharides. In conclusion, conjugation of PEG to oligosaccharides provided two beneficial effects; prolonged intravascular retention time and anti-complement activity, upon systemic application of the oligosaccharides. The present findings opened a new approach to treatment of hyperacute rejection after xenotransplantation.

Old World News: This Little Pig Went to Market

The creatures outside looked from pig to man, and from man to pig, and from pig to man again; but already it was impossible to say, which was which. --George Orwell, Animal Farm George Orwell's vision will come one stage closer to reality later this year. At least that is the rather optimistic hope of Imutran, a company researching the genetic manipulation of pigs in an effort to make their organs transplantable into humans. Imutran's spokesmen in England have said several times that the company expects to carry out the first human trial of its genetically modified xenografts in late 1996. The company's enthusiasm has not been widely shared, and various groups have been examining the desirability of developing animal-to-human transplants. The Nuffield Council on Bioethics reported[1] in March and the British Medical Association's ethics committee[2] in May. The government set up a committee as soon as it got wind of Imutran's intention to start human trials: the committee will report to ministers in July, but there is no indication of when its report will be published. The central problem with transplanting animal organs into humans is hyperacute rejection, which can destroy an organ's function within an hour. The interaction of natural antipig antibodies in the human with antigenic molecules in the donor organ initiates the with blood clotting and vasoconstriction. Imutran hopes to prevent the cascade by genetically engineering pigs to express specifically human regulators of complement activity. If it succeeds, however, it will only have overcome the hyperacute rejection. Other forms of rejection are still likely to happen; it is hoped that they can be suppressed by cyclosporin. One as vet unquantifiable risk is that microorganisms that are harmless to pigs but pathogenic in man may also be transplanted. The Nuffield Council addressed whether it is ever acceptable to use animals to provide donor organs and, if so, whether primates could be used. They concluded that the special protection afforded to primates ruled out their use, but approved the use of transgenic pigs, as long as they were subject to the same protections as other animals used in research. The council also took up questions concerning the timing of human trials, protection of patients' welfare, possible impacts on public willingness to donate human organs, as well as potential health risks to humans. The council recommended that several safeguards be put in place before human trials begin, including the establishment of a governmental advisory committee with the power to regulate xenografting, a code of practice, and standards and mechanisms for monitoring human recipients. …