Donor Pigs Research Articles

Chapter 2: Source pigs

An islet xenotransplantation product includes live cells from a non-human source, in this study, a pig source. A live product cannot be subjected to conventional disinfection, and therefore the source pig must be depleted of infectious agents that can transmit to the recipient and cause disease. Among other requirements, regulatory guidances specify that donor animals fulfill the designated pathogen-free (DPF) status. Donor pigs fulfilling DPF status are generally bred and maintained in biosecure facilities, where they are shielded from the outside by filtered air, disinfected water, and irradiated food that is certified free of any mammalian protein. All materials are autoclaved upon entry, and personnel enter by shower-in/shower-out, and are wearing special clothes. The operation of such facilities is in compliance with Current Good Manufacturing Practices. To ensure DPF status, pigs are brought into such facilities via Cesarean section, and the source pigs are kept as a closed herd. The DPF status cannot be realized for endogenous viruses, such as porcine endogenous retrovirus. Therefore, regulatory authorities require patient monitoring after xenotransplantation. Considering the infectious pathogen status and necessary regulatory compliance, it is recommended that organ procurement be conducted at the animal facility and that cell manufacturing facilities be located nearby. To enable assessment of as-yet unknown pathogens long after xenotransplantation, regulatory guidances mandate archiving donor materials for at least 50 yr. As this is essentially a public health issue, governmental institutions are urged to be responsible for the archive.

People’s attitude toward xenotransplantation: affective reactions and the influence of the evaluation context

One of the major issues in transplantation is to find a strategy to overcome the scarcity of human organs. One of the interventions under investigation is represented by xenotransplantation. The present study aimed to understand the role of psychological factors on people's perception of xenotransplantation. In particular, we tested a condition in which different alternatives (e.g., human vs. pig donors) are presented together allowing people to compare among them (joint evaluation) and two conditions in which people are presented with only one of the two alternatives and cannot compare them (separate evaluation). The study was conducted with three different groups of participants: patients waiting for liver transplantation (N = 31 in joint evaluation and N = 30 in each of the two separate evaluation conditions); students (N = 30 in join evaluation and N = 30 in each of the two separate evaluation conditions); and healthy adults (N = 30 in joint evaluation and N = 30 in each of the two separate evaluation conditions). Participants were presented with hypothetical scenarios and asked how good (or bad) were their feelings toward one or two types of donor (e.g., human and pig). Patients showed a skeptical attitude toward xenotransplantation both when it was evaluated together with the human donor (P < 0.01) or when it was evaluated separately (P < 0.01). Differently, when asked to evaluate each donor separately healthy adults and students showed similar affective reactions toward the two alternatives (human organ and xenograft). The present study demonstrates that the evaluation context may increase the impact of affective reactions and reduce healthy people's ability to use information on the potential benefit of a novel biomedical technology. Regardless of the evaluation context, patients always rely on affective reactions and show an overall preference for the human organ.

Evidence forIn VivoGrowth Potential and Vascular Remodeling of Tissue-Engineered Artery

Nondegradable synthetic polymer vascular grafts currently used in cardiovascular surgery have no growth potential. Tissue-engineered vascular grafts (TEVGs) may solve this problem. In this study, we developed a TEVG using autologous bone marrow-derived cells (BMCs) and decellularized tissue matrices, and tested whether the TEVGs exhibit growth potential and vascular remodeling in vivo. Vascular smooth muscle-like cells and endothelial-like cells were differentiated from bone marrow mononuclear cells in vitro. TEVGs were fabricated by seeding these cells onto decellularized porcine abdominal aortas and implanted into the abdominal aortas of 4-month-old, bone marrow donor pigs (n = 4). Eighteen weeks after implantation, the dimensions of TEVGs were measured and compared with those of native abdominal aortas. Expression of molecules associated with vascular remodeling was examined with reverse transcription-polymerase chain reaction assay and immunohistochemistry. Eighteen weeks after implantation, all TEVGs were patent with no sign of thrombus formation, dilatation, or stenosis. Histological and immunohistochemical analyses of the retrieved TEVGs revealed regeneration of endothelium and smooth muscle and the presence of collagen and elastin. The outer diameter of three of the four TEVGs increased in proportion to increases in body weight and outer native aorta diameter. Considerable extents of expression of molecules associated with extracellular matrix (ECM) degradation (i.e., matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase) and ECM precursors (i.e., procollagen I, procollagen III, and tropoelastin) occurred in the TEVGs, indicating vascular remodeling associated with degradation of exogenous ECMs (implanted decellularized matrices) and synthesis of autologous ECMs. This study demonstrates that the TEVGs with autologous BMCs and decellularized tissue matrices exhibit growth potential and vascular remodeling in vivo of tissue-engineered artery.

Dose-Dependent Effects of Intravenous Allogeneic Mesenchymal Stem Cells in the Infarcted Porcine Heart

Intravenous delivery of mesenchymal stem cells (MSCs) preserves myocardial function after infarction. This dose-escalating study was performed to examine pathologic remodeling and scar formation in a pig model of permanent coronary occlusion without restoration of reperfusion. MSCs labeled with fluorescent dye 48 h or saline (negative control, n = 8) were given intravenously 48 h post proximal left anterior descending artery occlusion. Animals received either autologous or allogeneic MSCs in doses from 1 x 10(3) up to 1 x 10(6) per kg bodyweight from an unrelated donor pig. Infarct size and myocardial function were assessed after 1 month. Morphologic analysis revealed that labeled autologous MSCs migrated in the peri-infarct region resulting in smaller infarct size (19 +/- 7% vs. 32 +/- 7%, p < 0.008) and higher fractional area shortening (33 +/- 7% vs. 21 +/- 3%, p < 0.001). Similarly, allogeneic MSCs had dose-dependent beneficial effects on cardiac function, statistically significant at 1 x 10(5) and 1 x 10(6) cells per kg bodyweight. Autologous as well as allogeneic MSCs specifically "home" to the heart after systemic delivery, leading to limited myocardial infarct size and improved functional outcome, even without coronary reperfusion. Therefore, intravenously administration of MSCs is an attractive minimal-invasive approach for cardiac tissue repair.

Circulatory Load During Hypoxia Impairs Post-transplant Myocardial Functional Recovery in Donation After Cardiac Death

Circulatory load during hypoxia is unavoidable in donation after cardiac death (DCD) hearts, but it causes severe myocardial damage. The impact of circulatory load on donor heart function has not been investigated. The purpose of this study was to evaluate its effect on post-transplant functional recovery of DCD hearts. Twelve donor pigs (20 kg) were used. Cardiac arrest was induced by asphyxiation (turning off the ventilator) in the load group (n = 6) and by exsanguination (dividing the vena cava) in the unload group (n = 6). Left ventricle end-diastolic volume (LDEDV) and end-systolic pressure (LVESP) were monitored until cardiac arrest. Orthotopic transplantation was performed after 30-minute warm ischemia following cardiac arrest. After weaning from cardiopulmonary bypass, left ventricular end-diastolic pressure-volume ratio (LV Emax) and creatine kinase (CK-MB) were measured while on 0.1 microg/kg/min epinephrine. During the agonal period, the maximum LVEDV and LVESP in the load group were 132 +/- 1% of baseline at 10 minutes and 148 +/- 16% of baseline at 4 minutes, respectively. Recovery rates of post-transplant cardiac function in the load group were worse than in the unload group (LV Emax: 64 +/- 8 vs 84 +/- 5%, p < 0.05). Levels of post-transplant CK-MB in the load group were higher than in the unload group (639 +/- 119 vs 308 +/- 70 IU/liter, p < 0.05). Cardiac arrest with circulatory load by asphyxiation caused more myocardial damage than unloaded arrest. This difference between the modes of death should be considered when evaluating the DCD hearts for clinical application.

Failure of IL-8 to assess early reperfusion injury following lung transplantation of cardiac death donor pigs

Although interleukins (IL) 8 and 10 predict lung viability in lung transplantation from heart beating donors (HBD) and IL-1beta is a marker of ex vivo performance from after cardiac death donors (ACDD), IL expression in the recipient remains unknown. This study assessed IL-1beta, IL-8 and IL-10 as indicators of functional performance in single-lung transplantation from ACDD pigs. Animals were divided into: (i) HBD: immediate lung excision; (ii) ACDD: fibrillation, 30 min warm ischemia and 3 h topical cooling. Left lungs of both groups were then flushed with Perfadex and stored at 3-4 degrees C for 3 h. IL in bronchoalveolar lavage fluid (BAL) and hemodynamic and graft function were measured in the donor and during the 2 h reperfusion period in the recipient. Myeloperoxidase, nuclear factor kappa beta, wet/dry weight ratio and a histologic injury score were assessed from biopsies in basal conditions in the donor and at the end of reperfusion. Despite similar pulmonary function and histologic markers of injury in both groups and higher IL-1beta in the donor of ACDD, IL-8 during reperfusion was significantly lower in ACDD (119 +/- 33% of basal) than in HBD (306 +/- 238%, P < 0.05) recipients. The paradoxical behavior of IL-8 makes it an unreliable predictor of ACDD early outcome in this transplantation model.

Comparison of the age-related porcine endogenous retrovirus (PERV) expression using duplex RT-PCR

Porcine endogenous retroviruses (PERVs) are members of family Retroviridae, genus Gamma retrovirus, and transmitted by both horizontally and vertically like other endogenous retroviruses (ERVs). PERV was initially described in the 1970s having inserted its gene in the host genome of different pig breeds, and three classes, PERV-A, PERV-B, and PERV-C are known. The therapeutic use of living cells, tissues, and organs from animals called xenotransplantation might relieve the limited supply of allografts in the treatment of organ dysfunction. Because of ethical considerations, compatible organ sizes, and physiology, the pig has been regarded as an alternative source for xenotransplantation. Sensitive duplex reverse transcription-polymerase chain reaction protocols for simultaneously detecting PERV gag mRNA and porcine glyceraldehydes 3-phosphate dehydrogenase mRNA in one tube was established. To compare the age-related PERV expression patterns of the lung, liver, spleen, kidney, heart, and pancreas in commercial pigs, 20 pigs from four age groups (5 heads each in 10 days-, 40 days-, 70 days-, and 110 days-old, respectively) were used in this study. The expression patterns of PERV were statistically different among age groups in lung, liver, and kidney (ANOVA, p < 0.05). These data may support in the selection of appropriate donor pigs expressing low levels of PERV mRNA.

Production of Cloned Miniature Pigs by Enucleation using the Spindle View System

Porcine somatic cell nuclear transfer (NT) has been successfully performed, but its efficiency remains quite low. In this study, we improvised on the enucleation method to enhance the development of NT embryos. Initially, an experiment was performed to determine the location relationship between the metaphase plate and the first polar body, where the results showed that the metaphase plate may frequently be displaced during the varying period of maturation process. When the metaphase plates were removed using the 'blind' enucleation method, the enucleation rate was affected by the maturation time; however, when the spindle view system was used, an enucleation rate of 100% was achieved. In the next experiment, these two methods were used to construct embryos: the fusion efficiency was significantly higher (p < 0.01) in the spindle view system group and the development rates of the reconstructed embryos were significantly higher in the spindle view system group compared with the 'blind' enucleation group (p < 0.01). An average of 174 (141-210) cloned embryos from the spindle view system group were transferred into five surrogate pigs and one piglet was delivered at 114 days after embryo transfer by caesarean section. DNA analysis confirmed that the piglet was genetically identical to the male donor pig. We showed that enucleation by the spindle view system is the another new technique compare the handmade cloning method [Theriogenology 2007: 68, 1104] to promote the development of the reconstructed embryos, and that a full-term cloned pig could be produced using this method.

Development of a recombinant antigen‐based ELISA for the sero‐detection of porcine lymphotropic herpesviruses

Xenotransplantation using pigs as donor species carries a risk for the activation of latent porcine herpesviruses and potential transmission to the human recipient. The porcine lymphotropic herpesviruses (PLHV-1, -2, -3) are widespread in domestic pigs and closely related to the human gammaherpesviruses, Epstein-Barr virus and Kaposi sarcoma herpesvirus, causing lymphoproliferative disorders. PLHV-1 has been associated with a porcine post-transplantation lymphoproliferative disorder (PTLD), affecting miniature swine after experimental transplantation. In human xenotransplantation, PLHV might be transferred to the transplant recipient and cause PTLD or related diseases. The elimination of PLHV from donor pigs is therefore necessary, and requires the availability of nucleic-acid- and antibody-based detection methods. The N- and C-terminal parts (gB1 and gB2) of the glycoprotein B gene of PLHV-1, -2 and -3 were cloned and expressed in Escherichia coli. Antisera were raised in mice. PLHV PCR was performed as published earlier. An ELISA was developed, using recombinant glycoprotein B of PLHV-1 as the antigen, and used for the analysis of groups of pigs, differing by age and origin. Seropositivity ranged from 38% (piglets) to 90% (gilts) and 100% (breeding sows, miniature pigs and pigs for slaughter). In comparison, PCR products of PLHV were found in the blood of 0 to 80% of the pig groups. Additionally, a group of 12 piglets was tested repeatedly after birth until the age of 156 days. A decline of antibodies was found during the first 3 weeks after birth, followed by a rise in most pigs during the weeks thereafter. PLHV PCR products in the blood were only observed later than 3 weeks after birth. Newborn pigs may be passively protected by maternal antibodies against PLHV infection during the first 3 weeks post partum. The rise of antibody titers thereafter and the appearance of PLHV sequences in the blood possibly indicates de novo infection by contact to the infected mother sow. The PLHV-ELISA may aid in breeding PLHV-free pigs.

TOWARD IMPROVED SURGICAL TECHNIQUES FOR PIG PANCREAS PROCUREMENT

Background: Warm ischemia time (WIT) and elevated temperature during pancreas procurement can significantly affect the viability of transplanted tissue. Quantitative information on these parameters is currently lacking and needed. Aim: To evaluate and optimize the efficiency, effectiveness, and practicality of existing and emergent pancreas procurement techniques in non-heart beating donor pigs by measuring the WIT and the organ temperature in situ. Methods: Pancreata were removed by en bloc viscerectomy from 81 female Landrace pigs. The first procurement method (27%) utilized only crushed ice for cooling. The second method (25%) utilized crushed ice and a vascular flush of cold preservation solution by cannulation of the suprarenal aorta. A third method (48%) was developed to selectively flush through the celiac trunk (CT) and the superior mesenteric artery (SMA). Ductal infusion with preservation solution was also investigated in conjunction with all three methods. Temperature measurements were taken by needle probe in several organs. Results: Vascular flush (methods 2 and 3) resulted in improved cooling and macroscopic pancreas morphology compared to surface cooling (method 1) which can take >30 min to cool the core to 4 °C. Method 3 eliminates solution waste without increasing WIT. Ductal infusion improved organ cooling but was not as effective as the vascular flush. Conclusions: Selective CT and SMA flush with cold storage solution in conjunction with ductal infusion promotes rapid cooling and improves pancreas preservation.

Abstract 2329: Donor Pretreatment with Hypertonic Saline Attenuates Primary Cardiac Allograft Dysfunction

Objective: Hypertonic saline (HTS) has been previously demonstrated to have immune modulatory and vascular protective effects. We assessed the effect of donor pretreatment with HTS on allograft myocardial function in a porcine model of orthotopic heart transplantation. We hypothesized that HTS infusion prior to donor heart arrest and storage would limit ischemia-reperfusion injury and improve myocardial performance following transplantation. Methods: Orthotopic transplants were performed after 6 hours of ischemic allograft storage. Donor pigs were randomly assigned to pretreatment with (n = 7) or without (n = 6) HTS (4.5 ml/kg of 7.5% NaCl) administered 1 hour prior to donor heart arrest. Left ventricular performance was determined after caval occlusion using a Millar micromanometer and conductance catheter. Hemodynamic measurements were obtained using a Swan-Ganz catheter. Results: Administration of hypertonic saline increased serum sodium level from 138 ± 2 mmol/L to 154 ± 4 mmol/L, which normalized to 144 ± 3 mmol/L 1 hour post-infusion. Weaning from CPB and LV performance after transplantation was improved after donor HTS treatment. Successful weaning from CPB was significantly greater in HTS treated hearts (6/7 versus 1/6, p &lt; 0.05). Preload recruitable stroke work post-transplantation was improved compared to control (88 ± 21% versus 35 ± 8% of baseline, p &lt; 0.01). Similarly, maximal elastance was improved compared to control (85 ± 17% versus 42 ± 12% of baseline, p = 0.03). No significant differences in post-transplant central venous, pulmonary artery or pulmonary capillary wedge pressures were observed between groups. However, post-transplant systolic blood pressure was significantly higher in the donor HTS group (60 ± 9 mmHg versus 35 ± 6 mmHg, p = 0.04). Conculsions: Donor HTS pretreatment attenuates post-transplant cardiac allograft myocardial function and improves post-transplant systemic hemodynamic function. HTS may be a novel organ donor intervention to prevent primary graft dysfunction. Further clinical evaluation of this simple and potentially cost-effective intervention is warranted.

Pig to Canine Auxiliary Hepatic Xenotransplantation Model: Prevention of Hyperacute Rejection Via Kupffer Cell Blockade and Complement Regulation

Objectives Large animal experiment models are a critical prerequisite to preclinical trials. However, the pig-to-primate model is expensive and proper experimental conditions are difficult to establish. Several pig-to-canine lung xenotransplantation experiments have shown hyperacute rejection. Therefore, we designed a pig-to-canine liver xenotransplantation model to study the diverse immunologic and hemodynamic consequences after xenotransplantation and hyperacute rejection. Methods Animals were divided into two groups of 3 each: a cobra venom factor plus gadolinium trichloride (GdCl 3) treatment group (CVF+Gd group) and a control group. Whole livers from 15-kg donor pigs were harvested and perfused with histidine-tryptophan-ketoglutarate solution. Seventy percent of the left lobe of the livers of 17-kg recipient dogs was resected. The harvested pig whole liver was transplanted using the canine left hepatic vein, left portal vein, and common hepatic artery. After graft reperfusion, blood samples and aliquots of liver, lung, and kidney tissues were obtained at 1 hour after reperfusion. Results We successfully completed 6 pig-to-canine auxiliary hepatic xenotransplantations. In the control group, the grafts showed a patchy hypoperfused liver surface that was rubbery solid compared with the CVF+Gd group. Serum total protein, albumin, fibrinogen, and platelet counts decreased abruptly; however, there were no significant differences between the two groups. There were no identifiable changes in blood urea nitrogen and creatinine concentrations. Serum prothrombin time, partial thromboplastin time, and further degradation product were increased in both groups; however, in the CVF+Gd group, the slope was more obtuse than in the control group. At microscopy, the graft at 20, 40, and 60 minutes after reperfusion, no intravascular pathologic changes were noted. Only scant intravascular fibrin deposition was observed. Hepatocellular vacuolization and sinusoidal dilatation were seen. There was patch necrosis without a zonal distribution, and intrasinusoidal neutrophil sequestration and interstitial hemorrhage. These findings were milder in the CVF+Gd group. Conclusion A pig-to-canine partial auxiliary liver xenotransplantation model is feasible. In the CVF+Gd treatment group, pathologic findings of patch hepatocyte necrosis were less severe. Inasmuch there was no corresponding vascular pathologic finding, these abnormalities are not a direct effect of CVF+Gd treatment. Other factors such as ischemia-reperfusion injury should be considered.

Analysis of Heterogeneous Mitochondria Distribution in Somatic Cell Nuclear Transfer Porcine Embryos

We previously reported that translocation of mitochondria from the oocyte cortex to the perinuclear area indicates positive developmental potential that was reduced in porcine somatic cell nuclear transfer (SCNT) embryos compared to in vitro220.). The present study is focused on distribution of donor cell mitochondria in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using either pig fibroblasts with mitochondria-stained MitoTracker CMXRos or YFP-mitochondria 3T3 cells (pPhi-Yellow-mito) as donor cells. Transmission electron microscopy was employed for ultrastructural analysis of pig oocyte and donor cell mitochondria. Our results revealed donor cell mitochondrial clusters around the donor nucleus that gradually dispersed into the ooplasm at 3 h after SCNT. Donor-derived mitochondria distributed into daughter blastomeres equally (82.8%) or unequally (17.2%) at first cleavage. Mitochondrial morphology was clearly different between donor cells and oocytes in which various complex shapes and configurations were seen. These data indicate that (1) unequal donor cell mitochondria distribution is observed in 17.2% of embryos, which may negatively influence development; and (2) complex mitochondrial morphologies are observed in IVF and SCNT embryos, which may influence mitochondrial translocation and affect development.

Emerging infectious diseases and xenotransplantation

A total of 335 infectious diseases was reported in the global human population between 1940 and 2004, the majority of which were caused by zoonotic pathogens [1]. Although viral pathogens constitute only 25%, some have spread worldwide with most starting from Central Africa. These include human immunodeficiency virus (HIV) causing acquired immunodeficiency syndromes (AIDS), chikungunya virus and West Nile virus, which also cause severe diseases in humans. HIV-1 and HIV-2, for example, are the result of trans-species transmission from non-human primates [2] to humans sometime in the last century. The spread of two henipaviruses causing fatal diseases in horses, pigs and humans has been observed in Asia and Australia, and although these viruses represent transspecies transmissions from bats, secondary transmissions from pigs to humans have also occurred. These and many other examples of emerging infectious diseases call for strong safety considerations in the field of xenotransplantation. Whereas known viruses can easily be eliminated from donor pigs, strategies should be developed to detect new zoonotic pathogens. In addition, all pigs carry porcine endogenous retroviruses (PERVs) in their genome. Two of these, PERV-A and PERV-B, as wells as recombinant PERV-A/C are able to infect human cells. The greatest threat appears to come from the recombinant PERV-A/C viruses as they appear to have an increased infectivity [3,4]. An increase in PERV expression was not observed in multitransgenic pigs expressing DAF, TRAIL and HLAE, generated to prevent immune rejection [5]. Our laboratory has developed a variety of strategies to prevent PERV transmission following xenotransplantation: (i) selection of animals that do not harbour PERV-C genomes in order to prevent recombination, (ii) selection of PERV-A and PERV-B low-producers [6], (iii) development of an antiviral vaccine to protect xenotransplant recipients [7] and (iv) generation of transgenic pigs in which PERV expression is inhibited via RNA interference. Inhibition of PERV expression using either synthetic small interfering (si) RNA or short hairpin (sh) RNA was demonstrated in PERV infected human cells [8], in primary pig cells [9] and in all transgenic piglets born [10]. A second generation of pigs expressing PERV-specific siRNA is now under study and experiments have been started to introduce multiple shRNA. Supported by Deutsche Forschungsgemeinschaft, DFG, DE729/4.

Porcine cells express more than one functional ligand for the human lymphocyte activating receptor NKG2D

Xenotransplantation could ameliorate the severe shortage of donor organs. The initial results of transplantation from genetically-modified pig donors to primate recipients suggest that hyperacute rejection can be overcome, but thrombotic microangiopathy and the human anti-pig cellular immune response remain as significant impediments to successful clinical xenotransplantation. NKG2D is an activating immunoreceptor found on human natural killer (HuNK) cells, CD8(+) and gammadelta T cells. Signaling through NKG2D mediates cytotoxicity and cytokine secretion by NK cells and co-stimulation of T cells. Chinese hamster ovary P (CHOP) cells were transfected with human NKG2D and used in cell-cell binding studies with porcine epithelial, and endothelial cell lines. Soluble recombinant NKG2D-Fc was used to stain various porcine cells and tissues to indicate ligand expression. Porcine cells were used as targets in cytotoxicity assays with the HuNK cell lines NKL and YT, with and without enzymatic removal of pULBP1 and antibody blockade of NKG2D signaling. In this study, we demonstrate the expression of ligands for human NKG2D on porcine cell lines of endothelial and epithelial origin, islet cell clusters and rejecting kidney. HuNK cells were activated to kill pig cells expressing NKG2D ligands, and cytotoxicity was inhibited by antibody blockade of NKG2D. A previous study identified pULBP1 as the principal ligand for human NKG2D on pig aortic endothelial cells. In the current study, renal epithelial and intestinal endothelial cells each expressed high surface levels of pULBP1, but binding of soluble recombinant NKG2D and NKG2D-dependent cytotoxicity against these cells persisted after the enzymatic removal of pULBP1, strongly suggesting the presence of at least one additional functional ligand for human NKG2D in these cell types.

Porcine Endogenous Retrovirus Transmission Characteristics From a Designated Pathogen-Free Herd

Previously, a strategy for monitoring pigs intended for cell transplantation was developed and successfully applied to several representative herds in New Zealand. A better understanding of porcine viruses’ epidemiology in New Zealand has been achieved, and, as a result, a designated pathogen-free (DPF) herd has been chosen as a good candidate for xenotransplantation. This herd is free of all infectious agents relevant to xenotransplantation. The presented study of pig endogenous retrovirus (PERV) transmission with cocultures in vitro has shown no evidence of PERV transmission from DPF pig tissue. Additionally, in PERV-C–positive DPF donor pigs tested, a specific locus for PERV-C present in miniature swine possibly associated with the transmission of PERV was absent. The data on PERV transmission allowed classifying the DPF potential donors as “null” or noninfectious pigs.

Indirect Transmission of Escherichia coli O157:H7 Occurs Readily among Swine but Not among Sheep

Transmission of Escherichia coli O157:H7 among reservoir animals is generally thought to occur either by direct contact between a naïve animal and an infected animal or by consumption of food or water containing the organism. Although ruminants are considered the major reservoir, there are two reports of human infections caused by E. coli O157:H7 linked to the consumption of pork products or to the contamination of fresh produce by swine manure. The objective of this study was to determine whether E. coli O157:H7 could be transmitted to naïve animals, both sheep and swine, that did not have any direct contact with an infected donor animal. We recovered E. coli O157:H7 from 10/10 pigs with nose-to-nose contact with the infected donor or animals adjacent to the donor and from 5/6 naïve pigs that were penned in the same room as the donor pig but 10 to 20 ft away. In contrast, when the experiment was repeated with sheep, E. coli O157:H7 was recovered from 4/6 animals that had nose-to-nose contact with the infected donor or adjacent animals and from 0/6 naïve animals penned 10 to 20 ft away from the donor. These results suggest that E. coli O157:H7 is readily transmitted among swine and that transmission can occur by the creation of contaminated aerosols.

Aggression in cataract-bearing α-1,3-galactosyltransferase knockout mice

The Galalpha1-3Galbeta1-4GlcNAc epitope is the key antigen in the hyperacute rejection of pig-to-man xenotransplantation. In the alpha-1,3-galactosyltransferase knockout (alpha-1,3GT-KO) mouse - a model for xenograft donor pigs - a targeted mutation of the alpha-1,3 galactosyltransferase gene (Ggta1) has been constructed. These mice are depleted of the carbohydrate antigen and besides the mice are also known to develop cortical cataracts. The present study aimed at evaluating the morphology and the degree of the cataract in a population of alpha-GT KO mice, its age of onset, its progression and the impact the cataract may have on aggression, anxiety and perception of light. The alpha-gal epitope could be shown in the lenses with lectin GS1 B4 in all wild-type and none of the alpha-GT KO mice. Histology showed apparent cataract in all alpha-GT KO mice from six weeks of age. Apart from a single wild-type mouse with a small degree of microscopically visible cataract without epithelial involvement at the age of 30 weeks none of the wild-type mice showed signs of cataract. Behavioural testing demonstrated significantly more mounting behaviour and a longer duration of attacking in the alpha-GT KO mice. Apart from this, the agonistic behaviour was not influenced by genotype. Neither did the genotype affect anxiety or perception of light.

Effect of Preservation Solution on Graft Viability in Single-Lung Transplantation From Heart-Beating Donors in Pigs

Background Low-potassium-dextran preservation solution Perfadex (PER) may provide better outcome of transplanted lungs than high-potassium Euro-Collins (EC) solution. However, there are no comparative studies of the recipient inflammatory response to the graft. Purpose The purpose of this study was to compare EC versus PER as preservation solutions with respect to the functional performance and inflammatory response in single-lung transplantation from heart-beating donors in pigs. Materials and Methods The donor left lung flushed with the corresponding cold preservation solution was stored at 3°C for 3 hours. We assessed hemodynamic values and pulmonary function in the recipient over a 2-hour reperfusion period calculated as percent of basal values, and expressed as mean of the reperfusion period. Interleukin-8 (IL-8) concentration in the donor was estimated in bronchoalveolar lavage fluid 2 hours after recipient reperfusion. Biopsies of the donor right lung and the transplanted lung were obtained to measure myeloperoxidase (MPO) activity. IL-8 and MPO values were expressed as percent of the donor value. We evaluated the wet/dry pulmonary weight ratio (W/D), polymorphonuclear neutrophil count (PMN), and a score of histological damage in the transplanted graft. Results Pulmonary function evaluated by % static: 66.6 ± 6.8 (EC), 82.3 ± 10.2 (PER), and dynamic: 74.0 ± 7.3 (EC), 89.3 ± 7.7 (PER) compliances, as well as % IL-8: 562.5 ± 168.6 (EC), 232.3 ± 148.7 (PER), % MPO: 485.9 ± 194.9 (EC), 140.8 ± 21.1 (PER), W/D: 9.9 ± 3.1 (EC), 6.8 ± 1.4 (PER), PMN 13.5 ± 6.8 (EC), 5.5 ± 3.3 (PER) and the histological damage score: 3.0 ± 1.5 (EC), 0.7 ± 0.4 (PER) showed significant differences between the EC and the PER ( P < .01). Conclusions PER affords good lung preservation with early graft function and modest evidences of inflammation, lung injury, and edema compared with the EC perfused lung.

Whole pancreatoduodenal allotransplantation model with jejunum drainage and portal vein drainage for treatment of type I diabetes in pigs

Objective To establish the model of pancreatoduodenal transplantation in pigs with enteric drainage （ED） and portal venous drainage （PVD）. Methods Forty hybrid landraces were randomized into the donor group and recipient group. Pancreatoduodenal allotransplantation was then performed. The donor pigs were perfused via abdominal aorta without clamping the portal venous outflow with UW solution at 80- 100 crnH20 after heparinization. Whole pancreatoduodenal graft was harvested with segments of abdominal aorta and portal vein and shaped under cold UW solution. Then the end-to-end anastomosis was performed with the donor iliac artery bifurcation Y graft to the recipient superior mesenteric arteries and celiac artery. Furthermore, type Ⅰ diabetes model was established by removal of the recipient pancreas. The venous anastomosis was reconstructed between the donor portal vein and the recipient superior mesenteric vein. Meanwhile, the end-to-side anastomosis was performed with the donor common iliac artery bifurcation Y graft to the recipient abdominal aorta and the side-to-side intestinal anastomosis was done between the donor duodenum and the recipient jejunum. External jugular vein was intubated for transfusion. The levels of sugar, insulin and glucagons in blood were determined during the operation and 1, 3, 5 and 7 d after the operation. Results Twenty pigs in the recipient group received pancreatoduodenal allotransplantation. One died from complication of anesthesia. The successful rate was 95.6%. Operative complications occurred in 3 pigs, in which phlebothrombosis accounted for 4. 6%, duodenojejunal anastomotic leakage for 4. 6%. The blood level of sugar decreased and those of insulin and glucagons increased within 30 min and recovered on the 2nd d after removal of the pancreas. Rejection occurred on the 1st d and reached the worst level on the 7th d after transplantation without the changes in blood levels of insulin and glucagons and symptoms of rejection. Conclusions Pancreatoduodenal transplantation in pigs can be used to treat type Ⅰ diabetes. ED and PVD can keep the endocrine function. The technique of duodenal transplantation with ED and PVD may pave the way for further development of pancreatic transplantation in clinical practice. Key words: Transplantation homologous; Pancreas; Operation; Animal model