Donor Pigs Research Articles

Preconditioning of donors with interleukin-10 reduces hepatic ischemia-reperfusion injury after liver transplantation in pigs.

Graft ischemia-reperfusion injury (IRI) resulting from postreperfusion inflammatory reaction remains a major cause of complications after liver transplantation. In this article, the authors investigated the effect of anti-inflammatory cytokine interleukin (IL)-10 on IRI, in a preclinical model of liver transplantation in pigs. Donor pigs received IL-10 or saline at the start of liver graft harvesting. After 5 hr of cold ischemia, liver grafts were transplanted into untreated recipient pigs. IRI severity was measured in recipients by transaminase release and by cellular infiltration and necrosis on liver biopsy specimens. Donor IL-10 administration attenuated IRI, as indicated by significant reduction of mean peak of transaminase in recipients of grafts from IL-10-treated donors. In contrast, no significant differences in cell infiltration or amount of necrosis were observed on liver biopsy specimens between groups. Donor preconditioning with IL-10 may constitute an interesting pharmacologic approach to reduce IRI severity after liver transplantation.

Lysine synthesized by the gastrointestinal microflora of pigs is absorbed, mostly in the small intestine

This study used a digesta transfer protocol to determine the site of absorption of lysine synthesized by the gastrointestinal microflora of pigs. Eight pigs were used, four with reentrant cannulas in the terminal ileum, two with simple T cannulas in the terminal ileum, and two intact. All pigs were given, for 5 days, the same low-protein diet that included fermentable carbohydrates. The diet of two pigs with reentrant cannulas (donor) and of the two intact (control) pigs was supplemented with (15)NH(4)Cl. The two other pigs with reentrant cannulas (acceptor pigs) and those with simple cannulas (used to supply unlabeled digesta) were given the same diet but unlabeled NH(4)Cl. Ileal digesta were collected continuously from all of the reentrant cannulas and kept on ice. All digesta from each donor pig were reheated and returned to the distal cannula of its companion acceptor, whose ileal digesta were discarded. Unlabeled ileal digesta from the pigs with simple cannulas were instilled into the distal cannulas of the donor pigs. At the end of the experiment, the average (15)N enrichment in the plasma free lysine of control pigs was 0.0407 atom % excess (APE); that of donor pigs was 0.0322 APE (79% of controls), whereas that of acceptor pigs was only 0.0096 APE (24% of controls). Due to nitrogen recycling, acceptor pigs had labeled lysine in the digesta of the stomach and small intestine, and donor pigs had labeled lysine in the digesta of the large intestine. If account is taken of the higher (15)N enrichment of microbial lysine in the large compared with the small intestine, it can be estimated that >90% of the absorption of microbial lysine took place in the small intestine.

Successful liver transplantation from agonal non-heart-beating donors in pigs

Abstract An effective way to overcome shortage of donors in livertransplantation (LTx) is to consider such from non-heart-beating donors (NHBDs). We investigated how a liver graft should be treated before and/or after procurement for successful LTx from an NHBD. Porcine LTx was performed with FR167653 (FR), a dual inhibitor of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and/or prostaglandin E1 (PG). Animals were allocated to an FR group (n=4, donors and recipients were treated with FR), a PG group (n=4, donors and recipients were treated with PG), or an FRPG group (n=4, donors and recipients were treated with both FR and PG). No recipients in the FR group and only two of four recipients in the PG group survived, whereas all recipients in the FRPG group survived. Suppression of TNF-α and IL-1β and maintenance of microcirculation are the key to successful transplantation from NHBDs.

Transmission of porcine reproductive and respiratory syndrome virus by houseflies

Three hundred houseflies were allowed to feed on donor pigs viraemic with porcine reproductive and respiratory syndrome virus (PRRSV) on the fifth, sixth and seventh days after the pigs had...

Pre-transplant analysis of accommodation in donor pigs.

Accommodation could lead to xenograft acceptance without the need for severe immune suppression. Generally graft accommodation is appreciated in the sensitized recipient, after transplantation. By inducing accommodation in chimeric donors, however, the risk and cost of inducing accommodation in the recipient would be reduced. An indirect assay of accommodation in the donor pig is needed for screening donors prior to procurement of the xenograft. The resistance of peripheral blood lymphocytes to cytolysis by antibody and complement was assessed in chimeric pigs and compared with control pigs. Peripheral blood lymphocytes (PBL) from chimeric pigs demonstrated a wide range of cytolysis (0 to 85%, median 13%) whereas PBL from control pigs were consistently lysed with these conditions (86 to 99%, median 96.5%, P < 0.0001). Accommodation or reduction in cytolysis did not correlate with the amount of chimerism. A longitudinal study demonstrated persistent accommodation of the PBL for as long as 15 weeks, when the donors averaged 68 kg in weight. Accommodation has been induced by low levels of antibodies interacting with the target tissue. An ELISA for sheep IgG was developed and the serum from newborn pigs assessed. Sheep IgG (up to 4.6 microg/ml) was detected in four of seven piglets with chimerism detectable by flow cytometry and in one of four piglets with minimal chimerism, detectable only by PCR. Lymphocyte accommodation was observed in all pigs with detectable sheep IgG. Of four pigs without accommodation, none had sheep IgG. Three pigs without detectable sheep IgG also had accommodation, suggesting that factors other than sheep IgG may induce accommodation. Acute vascular rejection was not observed in the heterotopic heart transplants from six donors with PBL accommodation. Only one incident of moderate diffuse cellular rejection (grade 3) was observed.

Successful liver transplantation from agonal non-heart-beating donors in pigs.

An effective way to overcome shortage of donors in liver transplantation (LTx) is to consider such from non-heart-beating donors (NHBDs). We investigated how a liver graft should be treated before and/or after procurement for successful LTx from an NHBD. Porcine LTx was performed with FR167653 (FR), a dual inhibitor of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and/or prostaglandin E(1) (PG). Animals were allocated to an FR group (n=4, donors and recipients were treated with FR), a PG group (n=4, donors and recipients were treated with PG), or an FRPG group (n=4, donors and recipients were treated with both FR and PG). No recipients in the FR group and only two of four recipients in the PG group survived, whereas all recipients in the FRPG group survived. Suppression of TNF-alpha and IL-1beta and maintenance of microcirculation are the key to successful transplantation from NHBDs.

The preload recruitable stroke work relationship as a measure of left ventricular contractile dysfunction in porcine cardiac allografts.

Paradoxically, it has been reported that after 1.5-4 h of hypothermic ischaemic preservation there is complete recovery of contractile function in canine cardiac allografts, as assessed by the preload recruitable stroke work (PRSW) relationship. This raises questions about the suitability of the canine heart as a model for preservation research and the PRSW relationship as an end-point. The aim of the present study was to evaluate the PRSW relationship as an index of left ventricular contractility in porcine cardiac allografts. Eighteen orthotopic heart transplants were performed in inbred Westran pigs. Brain death was induced in the donor pigs 1 h prior to explantation. The donor hearts were arrested with extracellular cardioplegia, which was stored in ice prior to administration. On explantation, the donor hearts were immersed in cardioplegia and stored in ice. The donor hearts were subjected to either 4 (IT4, n = 6), 6 (IT6, n = 9) or 14 (IT14, n = 3) h of ischaemia. Post-transplant, all hearts were supported with dobutamine (10 mcg/kg per min). The PRSW relationship was derived from pressure-volume loops obtained by epicardial sonomicrometry and transmyocardial micromanometry. Multiple linear regression was used to describe and compare the PRSW relationship before brain death in the donor and after weaning from bypass in the recipient. Eleven hearts were weaned successfully from cardiopulmonary bypass: IT4 100% (6/6), IT6 56% (5/9) and IT14 0% (0/3) (IT4 versus IT14: P = 0.012). Analysis of the PRSW relationship revealed a reduction in contractility in both the IT4 and IT6 groups (both P < 0.0001), but a greater reduction in the IT6 group (P < 0.0001). Notably, the volume-axis intercept of the PRSW relationship was found to be a better discriminator of post-preservation contractile dysfunction than the slope of the PRSW relationship. The porcine heart's susceptibility to ischaemic injury makes it ideal for evaluating the effect of different preservation strategies on contractile recovery. The PRSW relationship can be used to evaluate the differences in contractile recovery, though the nature of the effect of ischaemic preservation necessitates analysis by multiple linear regression.

Prediction of Graft Viability from Non-Heart-Beating Donor Pigs Using Hepatic Microdialysate Hypoxanthine Levels

Background. Non-heart-beating donors (NHBDs) are not yet acceptable in orthotopic liver transplantation (OLTX) because of the high frequency of primary graft nonfunction. In this study, we aimed to develop a new predictive method of graft viability in OLTX from NHBDs. Materials and methods. (1) Pigs were subjected to 15 min of hepatic ischemia and reperfusion (I/R). (2) Porcine OLTX was performed using grafts obtained from NHBDs subjected to in situ warm ischemia (0, 30, and 60 min). During both operations, hepatic hypoxanthine levels were measured by a microdialysis method. Results. In the I/R model, hypoxanthine accumulated during ischemia and decreased after reperfusion, whereas marked xanthine and uric acid production were observed after reperfusion. In the NHBDs model, all of the 0-min group, 6 of 13 pigs in the 30-min group, and 1 of 6 pigs in the 60-min group survived more than 7 days. Significant increases of hypoxanthine levels were seen dependent on warm ischemic time. In the 30-min group, hypoxanthine levels were significantly higher in the pigs that died than in those that survived, and correlated with aspartate aminotransferase, lactate dehydrogenase, and adenosine triphosphate levels of recipients. Histological examination revealed that graft injury was severe in the pigs with higher hypoxanthine levels. Conclusions. It is suggested that hepatic microdialysate hypoxanthine levels during warm ischemia in NHBDs were correlated with graft viability in the recipient. By using of this technique, prediction of the graft viability may be possible during donor operation.

Treatment of acute liver failure in pigs reduces hepatocyte function in a bioartificial liver support system.

Several different types of bioartificial liver (BAL) support systems have been developed to bridge patients suffering from acute liver failure (ALF) to transplantation or liver regeneration. In this study we assessed the effects of ALF plasma on hepatocyte function in the BAL system that has been developed in our center. Pigs (40-60 kg) were anaesthetised and a total hepatectomy was performed. Cells were isolated from the resected livers and were transferred to the bioreactor of the BAL system. Twenty hours after cell isolation, hepatocytes in the BAL were tested for cell viability and functional activity by using a recirculating test medium in which assessment of LDH leakage, ammonia clearance, urea synthesis, 7-ethoxycoumarin O-deethylase (ECOD) activity and pseudocholine esterase production was performed. Subsequently, two groups were studied. In one group (I, n=5), the cell-loaded bioreactor was used to treat the donor pig, rendered anhepatic, for 24 hours. In the second group (II, n=5) the bioreactor was cultured for 24 h and served as a control. After 24 hours treatment or culturing, the cell viability count and functional activity tests were repeated. The results show that hepatocytes in the BAL remained viable after 24 h treatment of anhepatic pigs, as shown by the LDH release and pseudocholine esterase production. However, metabolic functions such as ammonia clearance, ECOD and urea synthesis were reduced after 24 h exposure of hepatocytes to autologous ALF plasma, whereas these functions were unaltered after 24 h culturing of the cells in the bioreactor.

Anti-CD45RB antibody deters xenograft rejection by modulating T cell priming and homing.

Pancreatic islet xenotransplantation has been advocated as a way of overcoming the shortage of human donor tissue for the treatment of type 1 diabetes. However, the potent immune response against xenografts is a major barrier to their use. We show that a short course of the anti-CD45RB antibody, MB23G2, prolongs survival of fetal pig pancreas grafts in mice. To investigate this effect further we used an i.p. xenograft model in which both donor pig cells and host inflammatory cells can be expediently recovered and analyzed. Graft prolongation was associated with reduced T cell and macrophage infiltration, and reduced production of both T(h)1 and T(h)2 cytokines at the graft site. Graft survival was further increased and T cell infiltration further reduced by combining anti-CD45RB antibody with co-stimulation blockade. The primary effect of anti-CD45RB antibody may be on CD4 T cells, in keeping with the marked reduction in T cell cytokine production in both spleen and graft sites. This concurs with previous studies in allogeneic models that indicate that this antibody perturbs T cell responses by modifying signaling via the TCR. In addition, anti-CD45RB treatment led to reduced expression of LFA-1 and CD62 ligand (CD62L) on CD4 T cells, independent of antigenic challenge. LFA-1 may enhance co-stimulation, and both LFA-1 and CD62L are involved in T cell trafficking. Their reduced expression provides an explanation why the T cell pool is reduced in lymph nodes. We conclude that modulation of inflammation against xenografts by anti-CD45RB antibody is due to effects on both T cell priming and trafficking.

Endoventricular transplantation of allogenic skeletal myoblasts in a porcine model of myocardial infarction.

To assess the technical feasibility of percutaneous endoventricular injection of skeletal myoblasts into an infarcted porcine myocardium. A skeletal muscle biopsy was obtained from a donor pig and processed for myoblast expansion in vitro. Myocardial infarction was induced in a host pig via fibrin coil placement in the left anterior descending artery. Four weeks later, electromechanical mapping of the left ventricle identified the infarction site, into which approximately 200 million allogenic cells obtained from the muscle biopsy were directly injected (0.1 mL/injection at 25 sites) from inside the ventricular cavity via a needle injection catheter inserted through the femoral artery. Ten days after transplantation, the injected heart was evaluated histologically for the presence of myoblasts. Electrocardiography, echocardiography, left ventricular angiography, and electromechanical mapping confirmed the myocardial infarction. During the cell transfer procedure, premature ventricular contractions confirmed needle placement in the endocardium. Histological evaluation of the host heart 10 days after cell transfer revealed living myoblasts and multinucleated myotubes in the infarcted region, indicating successful transplantation. Direct myoblast transplantation into infarcted porcine myocardium using an endoventricular injection was technically feasible. The results in this model show that transplanted myoblasts were able to survive for 10 days after transplantation.

Further studies to quantify the dose of natural aerosols of foot-and-mouth disease virus for pigs.

Foot-and-mouth disease virus (FMDV) can be spread by a variety of mechanisms, including wind. Simulation models, developed to predict the risk of airborne spread, have played an important part in decision making in some outbreaks. The amount of airborne virus excreted as well as the minimal infectious dose (MID) of FMDV for different species are important determinants of airborne spread. The objective of this study was to obtain data for the O1 Lausanne, O SKR 2000 and O UKG 2001 strains of FMDV to enhance the capability of such models. Pigs were exposed to naturally generated aerosols of the three strains using an experimental design which delivered high doses of the two strains O1 Lausanne and O SKR 2000 over a short period, or of the O UKG 2001 strain over an extended period. The average excretion of the O1 Lausanne strain was 10(6.4) TCID50 per pig per hour. The excretion of the O SKR 2000 strain averaged 10(5.8) and the O UKG 2001 strain 10(6.1) TCID per pig per 24 h. The results show that the previous estimate of 'above' 800 TCID50 as the MID50 for the O1 Lausanne strain is a considerable under-estimate and that the real dose may be as high as 6000 TCID50. A dose of around 650 TCID50 of the O SKR 2000 strain failed to infect any pigs. Thus, the aerosol MID50 for pigs for this isolate is at least 1000 TCID50 and likely to be as high or higher than the O1 Lausanne strain. The exposure of pairs of recipient pigs kept physically separated from donor pigs in a series of rooms to aerosol exposure doses of the O UKG 2001 strain of around 50 TCID50 per min for 24-48 h failed to infect any of eight pigs. Thus, the present experiment confirms our previous findings that pigs, compared to cattle and sheep, are relatively resistant to infection with airborne FMDV.

Single vs Dual Vessel Porcine Extracorporeal Liver Perfusion

Background. The use of porcine extracorporeal liver perfusion (PECLP) to provide temporary hepatic support for patients in fulminant hepatic failure has been limited by the fact that individual perfusions can be sustained for only a few hours. Inadequate liver function and/or hemodynamic instability are the major contributing factors for early interruption of PECLP. Recent reports suggest that the choice of single (portal vein only) vs dual (portal vein and hepatic artery) vessel perfusion may influence the duration of perfusion. We hypothesize that PECLP with single vessel perfusion (SVP) is associated with worse liver function and greater hemodynamic instability than PECLP with dual vessel perfusion (DVP).Materials and methods. To eliminate the potentially confounding influences of liver failure and xenograft rejection, liver isografts procured from White-Landrace pig donors were perfused by either SVP or DVP via an extracorporeal circuit established with normal White-Landrace pig recipients. The function of perfused livers was evaluated by measuring production of bile and Factors V and VIII, clearance of ammonia and lactate, and extraction of O2 at baseline and at 0, 1, 3, 6, 12, and 24 h after initiation of PECLP. The impact of PECLP on recipient hemodynamic status was assessed by monitoring BP, heart rate, urine output, O2 saturation, etc. Among other parameters evaluated were serum albumin and total protein and hepatic release of IL-1β and nitric oxide to assess their possible contributions to hemodynamic instability.Results. DVP and SVP livers cleared ammonia and lactate similarly. Both approaches were associated with progressive hypoalbuminemia and hypoproteinemia. DVP livers produced more bile and Factor V and were associated with less recipient hypotension and IL-1β and NO release than SVP livers.Conclusions. Livers with DVP function better than livers with SVP. The duration of PECLP can be limited by recipient hypotension, although this complication is less severe with DVP than with SVP.

Sensitisation to Swine Leukocyte Antigens in Patients with Broadly Reactive HLA Specific Antibodies

We have previously shown that IgG HLA specific antibodies in the sera of highly sensitised patients awaiting renal transplantation can cross-react with swine leukocyte antigens (SLA). In this study we determined the frequency of patient serum IgG HLA specific antibody binding to a porcine lymphocyte panel and the likelihood of locating a cross-match negative pig donor for sensitised patients. Serum samples (n = 82) were obtained from 35 sensitised [current IgG panel reactive antibodies (PRA) > 10%] and seven nonsensitised patients awaiting renal transplantation at Addenbrooke's Hospital, Cambridge, UK. Fifty sera had IgG HLA specific PRA of 11–84%, 20 had IgG PRA of > 84% and 12 had 0% PRA (negative controls). Sera were absorbed with porcine erythrocytes to remove xenoreactive natural antibodies and tested for cross-reactive IgG HLA specific antibody binding by flow cytometry against a panel of porcine lymphocytes obtained from 23 human decay accelerating factor (hDAF) transgenic pigs. A total of 1884 cross-match combinations were tested and 369 (20%) gave a positive porcine lymphocyte cross-match. For sera from sensitised patients with IgG PRA (11–64%), only 6 of 805 (0.75%) cross-match tests were positive. In contrast, for sera from patients with high IgG PRA (> 64%), 363 of 805 (45%) cross-match tests were positive (p < 0.0001). There was no difference in the frequency of positive cross-matches between patient sera with IgG PRA 65–84% and highly sensitised patient sera with IgG PRA 85–100% [156/345 (45%) vs. 207/460 (45%)]. This study demonstrates that only patient sera with broadly reactive IgG HLA specific PRA (> 64%) cross-react with porcine lymphocytes. If future clinical trials of xenotransplantation are undertaken, it may be of value to select a cross-match-negative pig organ donor for such patients.

Transgenic pigs designed to express human CD59 and H-transferase to avoid humoral xenograft rejection.

Research in pig-to-primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell- and tissue-based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human alpha1,2-fucosyltransferase (H transferase, HT) in pigs modifies the cell-surface carbohydrate phenotype resulting in reduced Galalpha1,3-Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum-mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24 h to pro-inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.

Cytomegalovirus early promoter induced expression of hCD59 in porcine organs provides protection against hyperacute rejection.

The critical shortage of human donor organs has generated growing interest for porcine to human xenotransplantation. The major immunological barrier to xenotransplantation is the hyperacute rejection (HAR) response that is mediated by preformed xenoreactive antibodies and complement. A promising strategy to control the complement activation, is the expression of human complement regulatory proteins in transgenic animals. We have used the human early cytomegalovirus (CMV) promoter to drive expression of the human complement regulatory protein CD59 (hCD59) in transgenic pigs. A total of eight live transgenic founder animals was born from which five transgenic lines could be established. mRNA analysis and Western blotting revealed high expression of hCD59 in heart, kidney, skeletal muscle, and skin in animals of lines 1 and 5, as well as in the pancreas of four lines. This pattern of expression was confirmed by immunhistological staining. A cell-specific expression in heart and kidney tissue of transgenic lines 1 and 5 was determined. Primary fibroblasts and endothelial cell cultures derived from the aorta of transgenic pigs showed a significantly diminished sensitivity against the challenge with xenoreactive human antibodies and complement whereas non-transgenic control cells were highly susceptible to complement mediated lysis. Ex vivo perfusion of kidneys with pooled human blood revealed a significant protective effect of hCD59 against HAR. The average survival of transgenic kidneys was significantly extended (P<0.05) over nontransgenic controls (207.5+/-54.6 vs. 57.5+/-64.5 min). These data support the concept that hCD59 protects nonprimate cells against human complement mediated lysis and suggest that donor pigs transgenic for hCD59 could play a crucial role in clinical xenotransplantation. Two of five hCD59 transgenic lines showed strong hCD59 expression in several organs relevant for xenotransplantation and a protective effect against HAR. This indicates that the use of the CMV-promoter can facilitate the selection process for optimized transgene expression.

Highly disparate xenogeneic skin graft tolerance induction by fetal pig thymus in thymectomized mice: Conditioning requirements and the role of coimplantation of fetal pig liver.

Highly disparate xenogeneic pig skin graft tolerance and efficient repopulation of mouse CD4+ T cells are achieved in thymectomized (ATX) B6 mice that receive T cell and natural killer (NK) cell depletion by injection of a mixture of monoclonal antibodies (mAbs) (GK1.5, 2.43, 30-H12, and PK136) on days -6, -1, +7, and +14 and 3 Gy total body irradiation (TBI) followed by implantation of fetal pig thymus/liver (FP THY/LIV) grafts on day 0. The requirements for each treatment in this model to achieve pig skin graft tolerance have not previously been defined. Therefore, we performed a series of experiments to address the role of each treatment in achieving maximal skin graft tolerance. Peripheral mouse CD4+ T-cell repopulation and pig skin graft survival were followed in this pig-to-mouse model in which recipient B6 mice were treated with modified regimens that omitted thymectomy, 3 Gy TBI, anti-Thy1.2, and anti-NK1.1 mAbs, injection of a mixture of mAbs on day +14, or coimplantation of FP LIV, respectively. Prolongation but not permanent survival of donor MHC-matched pig skin grafts was observed in euthymic B6 mice that received T and NK cell depletion, 3 Gy TBI, and 7 Gy thymic irradiation and FP THY/LIV in the mediastinum, suggesting that full xenogeneic tolerance was not achieved in euthymic mice. However, after grafting FP THY alone to ATX B6 mice treated either with the "standard" regimen, or with a conditioning regimen that omitted all components of the conditioning regimen except treatment with anti-CD4 and anti-CD8 mAbs, efficient peripheral repopulation of mouse CD4+ T cells and long-term donor MHC-matched pig skin graft acceptance were observed. Highly disparate xenogeneic pig skin graft tolerance can be achieved by grafting FP THY alone in anti-CD4 and anti-CD8 mAb-treated ATX B6 mice, but not in euthymic B6 mice. Additional treatment of ATX recipient mice with anti-Thy1.2 and NK1.1 mAbs and 3 Gy TBI is not essential for donor pig skin graft tolerance induction.

Enhanced CD4 reconstitution by grafting neonatal porcine tissue in alternative locations is associated with donor-specific tolerance and suppression of preexisting xenoreactive T cells.

Donor-specific xenograft tolerance can be achieved by grafting fetal porcine thymus tissue to thymectomized (ATX) mice treated with natural killer (NK) and T-cell-depleting monoclonal antibodies plus 3 Gy of total body irradiation (TBI). Grafting of neonatal, instead of fetal, thymus, along with neonatal pig spleen, leads to a lower level of mouse CD4 cell reconstitution, with less reliable tolerance induction. For a number of reasons, it would be advantageous to use neonatal rather than fetal pigs as donors. We therefore investigated the possibility that grafting larger amounts of neonatal porcine thymus tissue to different sites could allow improved outcomes to be achieved. Multiple or single fragments of neonatal porcine thymus tissue were grafted with a splenic fragment to different sites (mediastinum, mesentery, and kidney capsule) of ATX B6 mice treated with T- and NK-cell-depleting antibodies and 3Gy TBI. Mice also received an intraperitoneal injection containing 1 x 10(7) donor splenocytes. Donor-specific skin graft tolerance was evaluated, and CD4 reconstitution and mouse anti-donor xenoantibodies were followed by flow cytometry. Peripheral repopulation of CD4+ cells occurred by 7 weeks after transplantation in mice grafted with four fragments of neonatal porcine tissue in either the mediastinum or the mesentery, but not in mice grafted under both kidney capsules with the same amount of tissue. The level of CD4 reconstitution correlated with skin graft tolerance and an absence of induced anti-donor xenoantibodies. Seventy-five percent of mice with >20% of CD4+ cells among peripheral blood lymphocytes (PBL) by 13 weeks posttransplantation accepted donor porcine skin, while rejecting either non-donor neonatal porcine or mouse BALB/c skin allografts. In contrast, only 29% of grafted mice with <20% CD4+ cells in the peripheral blood at 13 weeks accepted donor porcine skin. Grafted mice with poor reconstitution showed either low or high levels of anti-pig xenoantibodies of the IgM, IgG1, and IgG2a isotypes. Grafted mice with >20% CD4+ cells all had low levels of anti-pig xenoantibodies of these isotypes and displayed mixed lymphocyte reaction (MLR) tolerance to donor pig major histocompatibility complex (MHC), with responsiveness to allogeneic mouse stimulators. Grafting neonatal porcine thymus into either the mediastinum or mesentery provides earlier and more efficient reconstitution of the CD4 compartment than does grafting under the kidney capsule. Good CD4 reconstitution was associated with optimal donor-specific skin graft tolerance and avoidance of the anti-donor xenoantibody responses observed in mice with poor CD4 reconstitution. These results also suggest that there is a suppressive component to the porcine xenograft tolerance induced with this approach.

Chimeric Pig Hearts Resist Hyperacute Rejection in ex vivo Perfusion Model

With surrogate tolerogenesis, the recipient immune system is engrafted within the donor pig before organ transplant. Chimeric pig hearts may resist hyperacute rejection by inducing accommodation. This hypothesis was tested using an ex vivo isolated piglet heart perfusion model. Processed sheep marrow was infused into fetal pigs at 45 days gestation. Heart explants from chimeric or nonchimeric pigs were suspended in a Langendorff apparatus and perfused with plasma from unsensitized sheep or sensitized sheep. Nonchimeric hearts perfused with plasma from unsensitized functioned for 240 min (N = 3). Nonchimeric hearts perfused with sensitized plasma deteriorated rapidly, functioning at 19 ± 12 min (N = 6); Immunohistochemistry of heart graft revealed extensive deposition of IgG, IgM in the microvascular. In contrast, chimeric hearts perfused with sensitized plasma functioned for 183 ± 46 min (N = 3)(p &lt;.001); Deposition of IgG, IgM had substantially less. Heart grafts procured from chimeric pigs survived in the presence of antidonor IgG, IgM, and complement, demonstrating that chimeric pig hearts resist hyperacute rejection.

The in vitro activity and specificity of human endothelial cell-specific promoters in porcine cells.

The chronic shortage of human organs, tissues and cells for transplantation has inspired research on the possibility of using animal donor tissue instead. Transplantation over a species barrier is associated with rejections which are difficult to control. Therefore, it is generally agreed that successful pig to human xenotransplantation requires donor pigs to be genetically modified. Vascular endothelium is the most immediate barrier between the xenogeneic donor organ and host immune and nonimmune defense systems. Thus, these cells are the prime targets for such genetic modifications. Luciferase assays were used to evaluate the activity and specificity of human endothelial-cell specific promoters in porcine aortic-, microvascular- and nonendothelial cells. The promoters for human Flk-1 (fetal liver kinase-1), Flt-1 (fms-like tyrosine kinase), ICAM-2 (intercellular adhesion molecule-2), thrombomodulin and vWf (von Willebrand factor) supported similar levels of luciferase expression in human and porcine aortic endothelial cells, with the Flk-1 promoter being the strongest followed by the thrombomodulin promoter. Relative to the activity of the CMV promoter, the human endothelial cell-specific promoters all showed less activity in porcine kidney microvascular endothelial cells than in liver or brain microvascular endothelial cells. The thrombomodulin and Flk-1 promoters exhibited similar activity in liver and kidney microvascular endothelial cells, whereas the Flk-1 promoter was stronger in aortic and brain microvascular endothelial cells. Human endothelial cell-specific promoters also showed some degree of specificity in pig, because they supported less luciferase activity in porcine nonendothelial cell lines. Based on the in vitro data and previously published in vivo data, the human Flk-1 and thrombomodulin promoters are good candidate promoters for strong endothelial cell-specific gene expression in transgenic pigs.