Cellular xenotransplantation of animal cells into people: benefits and risk.

The main benefit of xenotransplantation is its potential to overcome the worldwide organ shortage experienced in allotransplantation. Allogeneic transplantation is the only successful therapy for several life-threatening diseases, with cell, tissue or organ donation only partially meeting the demand and many patients dying while waiting for treatment. With supply falling short of demand, it is foreseen that the use of porcine material may at some stage overcome the existing gap between organ availability and clinical need. Recently, pig islet cells have been utilised in clinical trials, with safety being demonstrated. Indeed, pig-derived cells present several advantages: i) porcine cells have a stable function and differentiation pattern and are not tumorigenic; ii) pig cells have been shown to meet the physiological needs in large animal models; iii) the source of pig cells can be scaled up to meet demands in a highly standardised manner, and with respect to animal welfare regulations; iv) 'designated-pathogen-free' (DPF) pig lines can be produced, which could result in a higher safety profile than allotransplantation itself; v) the risk of zoonosis, which was raised years ago as the major hurdle, has been recently circumvented and is actually viewed as a controlled risk; and vi) immune risks are being circumvented via the use of genetically modified donor animals and encapsulation of porcine cells, particularly for the treatment of diabetes. Overall, the benefit appears to outweigh potential risks with respect to cellular xenotransplantation and this is discussed further in this review.

La xénotransplantation (ou hétérogreffe) a pour principal avantage de contourner le problème de la pénurie d'organes disponibles dans le monde pour réaliser des allogreffes. En effet, la transplantation allogénique est la seule thérapie qui permet de traiter avec succès certaines maladies potentiellement mortelles, mais les dons de cellules, de tissus et d'organes ne satisfont qu'une partie de la demande, de sorte que nombre de patients meurent dans l'attente d'un traitement. L'offre étant inférieure à la demande, on peut prévoir que le recours à des organes porcins puisse s'imposer dans un avenir plus ou moins proche afin de réduire l'écart entre les organes disponibles et les besoins cliniques. Récemment, des cellules d'îlots pancréatiques porcins ont été utilisées dans le cadre d'essais cliniques et leur innocuité a été démontrée. En effet, les cellules d'origine porcine présentent plusieurs avantages : i) les cellules porcines ont un fonctionnement et une différenciation cellulaires stables et ne sont pas tumorigènes ; ii) il a été démontré que les cellules porcines sont physiologiquement compatibles avec celles de modèles de grands animaux ; iii) le recours aux cellules porcines peut être échelonné en suivant des normes précises, en fonction de la demande et dans le respect de la réglementation applicable au bien-être animal ; iv) il est possible de produire des lignées cellulaires exemptes de microorganismes pathogènes spécifiques, ce qui offre encore plus de garanties de sécurité qu'une allogreffe ; v) le risque de zoonose, qui constituait le principal obstacle il y a quelques années a été récemment surmonté et on le considère aujourd'hui comme maîtrisé ; vi) les risques pour le système immunitaire du receveur ont été surmontés grâce à l'utilisation d'animaux génétiquement modifiés en tant que donneurs et à l'encapsulation des cellules porcines, en particulier pour les greffes destinées à des patients diabétiques. Les auteurs approfondissent l'examen des avantages de la xénotransplantation, qui l'emportent largement sur ses risques potentiels.

La principal ventaja del xenotrasplante reside en las posibilidades que ofrece para poner remedio a la penuria mundial de órganos destinados a alotrasplantes. El trasplante alogénico es la única terapia eficaz para muchas enfermedades potencialmente mortales, pero las donaciones de células, tejidos y órganos cubren solo una parte de la demanda y muchos pacientes mueren en espera de recibir tratamiento. Ante una oferta que no alcanza a cubrir la demanda, es previsible que en algún momento se recurra a material porcino como medio de subsanar el déficit de órganos disponibles para atender las necesidades clínicas existentes. En fechas recientes se han realizado ensayos clínicos con células de islote pancreático de cerdo y se ha demostrado que resultan seguras. De hecho, el uso de células de origen porcino presenta varias ventajas: i) las células porcinas tienen un patrón estable de funcionamiento y diferenciación y no son tumorígenas; ii) en modelos de animales de gran tamaño está demostrado que las células de cerdo responden a las necesidades fisiológicas; iii) es posible multiplicar las fuentes de células porcinas para responder a la demanda de modo sumamente normalizado y respetando las reglamentaciones de bienestar animal; iv) es posible generar linajes porcinos certificados como «exentos de patógenos¼, lo que podría ofrecer niveles de seguridad incluso mayores que los del propio alotrasplante; v) últimamente se ha podido conjurar el riesgo de zoonosis, que hace unos años parecía constituir el principal obstáculo y actualmente se considera un riesgo controlado; y vi) actualmente ya se evita el riesgo inmunitario gracias al uso de animales donantes genéticamente modificados y al encapsulamiento de las células porcinas, en especial para tratar la diabetes. Globalmente, por lo que respecta al xenotrasplante celular, los beneficios parecen pesar más que los eventuales riesgos, como indican los autores en su examen detallado.

Improved yield of canine islet isolation from deceased donors.

BACKGROUND: Canine diabetes is a strikingly prevalent and growing disease, and yet the standard treatment of a twice-daily insulin injection is both cumbersome to pet owners and only moderately effective. Islet transplantation has been performed with repeated success in canine research models, but has unfortunately not been made available to companion animals. Standard protocols for islet isolation, developed primarily for human islet transplantation, include beating-heart organ donation, vascular perfusion of preservation solutions, specialized equipment. Unfortunately, these processes are prohibitively complex and expensive for veterinary use. The aim of the study was to develop a simplified approach for isolating canine islets that is compatible with the financial and logistical restrictions inherent to veterinary medicine for the purpose of translating islet transplantation to a clinical treatment for canine diabetes. RESULTS: Here, we describe simplified strategies for isolating quality islets from deceased canine donors without vascular preservation and with up to 90 min of cold ischemia time. An average of more than 1500 islet equivalents per kg of donor bodyweight was obtained with a purity of 70% (N = 6 animals). Islets were 95% viable and responsive to glucose stimulation for a week. We found that processing only the body and tail of the pancreas increased isolation efficiency without sacrificing islet total yield. Islet yield per gram of tissue increased from 773 to 1868 islet equivalents when the head of the pancreas was discarded (N = 3/group). CONCLUSIONS: In summary, this study resulted in the development of an efficient and readily accessible method for obtaining viable and functional canine islets from deceased donors. These strategies provide an ethical means for obtaining donor islets.

Pancreatic islet transplantation: from dogs to humans and back again.

Pancreatic islet transplantation is a cell-based therapy that provides a potential cure for type 1 diabetes mellitus. After the introduction of an automated method for islet isolation and steroid-free immunosuppressive protocols, reversal of diabetes by islet transplantation is now performed at major human medical centers around the world. Despite extensive use of animal models in islet transplantation research, practical concerns have slowed the introduction of the technique into clinical veterinary practice and only a small number of studies have reported results of transplantation in dogs with spontaneously occurring diabetes mellitus; however, recent advances in islet isolation and encapsulation may make it possible to perform islet transplantation without immunosuppression in companion animals. This review summarizes experimental and clinical studies of pancreatic islet transplantation in dogs, including future directions for cell therapy in animals with naturally occurring disease.

Transgenic pigs with pancreas-specific expression of green fluorescent protein.

The development and regeneration of the pancreas is of considerable interest because of the role of these processes in pancreatic diseases, such as diabetes. Here, we sought to develop a large animal model in which the pancreatic cell lineage could be tracked. The pancreatic and duodenal homeobox-1 (Pdx1) gene promoter was conjugated to Venus, a green fluorescent protein, and introduced into 370 in vitro-matured porcine oocytes by intracytoplasmic sperm injection-mediated gene transfer. These oocytes were transferred into four recipient gilts, all of which became pregnant. Three gilts were sacrificed at 47-65 days of gestation, and the fourth was allowed to farrow. Seven of 16 fetuses obtained were transgenic (Tg) and exhibited pancreas-specific green fluorescence. The fourth recipient gilt produced a litter of six piglets, two of which were Tg. The founder Tg offspring matured normally and produced healthy first-generation (G1) progeny. A postweaning autopsy of four 27-day-old G1 Tg piglets confirmed the pancreas-specific Venus expression. Immunostaining of the pancreatic tissue indicated the transgene was expressed in ß-cells. Pancreatic islets from Tg pigs were transplanted under the renal capsules of NOD/SCID mice and expressed fluorescence up to one month after transplantation. Tg G1 pigs developed normally and had blood glucose levels within the normal range. Insulin levels before and after sexual maturity were within normal ranges, as were other blood biochemistry parameters, indicating that pancreatic function was normal. We conclude that Pdx1-Venus Tg pigs represent a large animal model suitable for research on pancreatic development/regeneration and diabetes.

Oral immunosuppressive medication for growing pigs in transplantation studies.

Immunosuppressive (IS) medication is needed to avoid graft rejection in porcine transplantation models. An ideal IS therapy should have no side-effects, but increased susceptibility to infections, disturbed intestinal microflora and toxic effects on organs and tissues are commonly reported. The aim of the present study was to design an IS protocol with tacrolimus and mycophenolic acid to be used for maintenance therapy in the post-transplant period. An eligible whole blood trough value for tacrolimus was 5-15 µg/L. Conventional specific pathogen-free pigs were fitted with an indwelling catheter under general anaesthesia, and after the acclimatization period three groups were formed: group A (n= 4) received 0.15 mg/kg body weight (BW) twice daily tacrolimus and 500 mg twice daily mycophenolic acid; group B (n= 4) received 0.3 mg/kg BW twice daily tacrolimus and 500 mg twice daily mycophenolic acid; group C (n= 2) did not receive any medication. Daily clinical examinations and analyses of blood concentrations of tacrolimus and glucose were performed. Total and differential white blood cell counts, enzyme activities, bilirubin and electrolyte concentrations were measured every fourth day. At the end of the experiment, the pigs were killed with an overdose of pentobarbital intravenously and a necropsy was performed immediately. All animals seemed to tolerate the IS treatment well. No alterations in their clinical state of health were observed throughout the study and daily weight gain was similar for the three groups. The necropsy did not reveal any pathological findings related to medication. The study showed that 0.25 mg/kg BW twice daily tacrolimus and 500 mg twice daily mycophenolic acid would be an appropriate maintenance dosage for conventional pigs.

Successful implementation of cooperative handling eliminates the need for restraint in a complex non-human primate disease model.

BACKGROUND: Streptozotocin-induced diabetic non-human primates are used to study efficacy and safety of innovative immunosuppression after islet transplantation. We implemented a training program for medical management of a chronic disease state. METHODS: Cooperation with hand feeding and drinking, shifting, and limb presentation were trained utilizing predominately positive but also negative reinforcement in 52 animals compared with 28 macaques subjected to conventional physical and/or chemical restraint. The success and timing of behavior acquisition was evaluated in a representative subset of 14 animals. RESULTS: Over 90% of animals were successful in behavior acquisition. Programmatically this resulted in complete elimination of chair restraint and negligible requirement for sedation. About half of the trained animals had no-to-moderate thymic involution, indicative of a substantial reduction in stress. CONCLUSION: Cooperative handling enhances animal well-being. This contributes to validity of scientific results and eliminates model-induced confounding that can obstruct interpretation of safety and efficacy data.

Pancreas anatomy and surgical procedure for pancreatectomy in rhesus monkeys.

BACKGROUND: The aim of this study was to investigate the pancreas anatomy and surgical procedure for harvesting pancreas for islet isolation while performing pancreatectomy to induce diabetes in rhesus monkeys. METHODS: The necropsy was performed in three cadaveric monkeys. Two monkeys underwent the total pancreatectomy and four underwent partial pancreatectomy (70-75%). RESULTS: The greater omentum without ligament to transverse colon, the cystic artery arising from the proper hepatic artery and the branches supplying the paries posterior gastricus from the splenic artery were observed. For pancreatectomy, resected pancreas can be used for islet isolation. Diabetes was not induced in the monkeys undergoing partial pancreatectomy (70-75%). CONCLUSIONS: Pancreas anatomy in rhesus monkeys is not the same as in human. Diabetes can be induced in rhesus monkeys by total but not partial pancreatectomy (70-75%). Resected pancreas can be used for islet isolation while performing pancreatectomy to induce diabetes.

Porcine marginal mass islet autografts resist metabolic failure over time and are enhanced by early treatment with liraglutide.

Although insulin independence is maintained in most islet recipients at 1 yr after transplant, extended follow-up has revealed that many patients will eventually require insulin therapy. Previous studies have shown that islet autografts are prone to chronic failure in large animals and humans, suggesting that nonimmunological events contribute to islet graft functional decay. Early intervention with therapies that promote graft stability should provide a measurable benefit over time. In this study, the efficacy of the long-acting glucagon-like peptide-1 analog liraglutide was explored in a porcine marginal mass islet autograft transplant model. Incubation with liraglutide enhanced porcine islet survival and function after prolonged culture. Most vehicle-treated (83%) and liraglutide-treated (80%) animals became insulin independent after islet autotransplantation. Although liraglutide therapy did not improve insulin independence rates or blood glucose levels after transplant, a significant increase in insulin secretion and acute-phase insulin response was observed in treated animals. Surprisingly, no evidence for deterioration of graft function was observed in any of the transplanted animals over more than 18 months of follow-up despite significant weight gain; in fact, an enhanced response to glucose developed over time even in control animals. Histological analysis showed that intraportally transplanted islets remained highly insulin positive, retained alpha-cells, and did not form amyloid deposits. This study demonstrates that marginal mass porcine islet autografts have stable long-term function, even in the presence of an increasing metabolic demand. These results are discrepant with previous large animal studies and suggest that porcine islets may be resistant to metabolic failure.

Acute gastric dilatation after porcine islet transplantation in a cynomolgus monkey - case history and review of the literature.

We report a streptozotocin-induced diabetic cynomolgus monkey that developed acute gastric dilatation within the first week after intraportal porcine islet transplantation. The monkey presented with increasing anorexia and somnolence, associated with persistent metabolic acidosis. On physical examination, a diffuse mass was palpable in the abdomen. Because of its deteriorating clinical condition, the monkey was euthanized. Necropsy revealed acute gastric dilatation. Potential etiological factors, possible methods of prevention, and guidelines for management are reviewed, with special emphasis on the incidence and management of acute gastric dilatation in non-human primates.

Prevalence of porcine endogenous retrovirus in domestic pigs in Japan and its potential infection in dogs xenotransplanted with porcine pancreatic islet cells.

The prevalence of porcine endogenous retrovirus (PERV) proviral DNA among various pig breeds raised in Japan was investigated by polymerase chain reaction (PCR). Moreover, potential infection of PERV was investigated by PCR and reverse transcriptase-polymerase chain reaction (RT-PCR) in experimentally induced diabetic dogs (n=5) implanted with the diffusion chamber type bio-artificial endocrine pancreas (Bio-AEP) containing porcine pancreatic endocrine (PE) cells. No immunosuppressant was used after the transplantation. PERV gag, pol, env-A and env-B genes were detected in any pigs examined. In two of three Landrace breeds, env-C gene was absent. PERV proviral DNAs and viral RNAs were also detected from the cultured porcine PE-cells. In the peripheral blood mononuclear cells and the spleen obtained at 6, 30, 32, 36, 79 weeks of xenotransplantation in dogs, however, no evidence of microchimerism, infection and viremia were confirmed. These results suggested that the risk of PERV infection through xenotransplantation of Bio-AEP containing porcine islet cells without immunosuppressants may be quite low.

Xenotransplantation of porcine pancreatic endocrine cells to total pancreatectomized dogs.

Xenotransplantation of porcine pancreatic endocrine (PE) cells in a diffusion chamber, a bioartificial endocrine pancreas (Bio-AEP), was conducted to total pancreatectomized dogs. Six pancreatectomized dogs were divided into two groups of 3 dogs each. In three dogs of the control group, exogenous insulin was administered twice a day for 30 weeks to maintain fasting blood glucose (FBG) levels within the normal range. The remaining three dogs were implanted with Bio-AEPs (implantation group), in addition to daily insulin administration. In the implantation group, Bio-AEPs containing 1.3 to 1.8 x 10(7) cells per kg of body weight of the recipient were implanted without fixation into the abdominal cavity. In the control group, exogenous insulin requirements did not decrease during the experimental period, whereas it significantly decreased for a certain period (3, 11, 17 weeks) after implantation in all implanted dogs. In the implantation group, laparotomy was performed after FBG and the exogenous insulin requirement increased again and Bio-AEPs were removed. Two Bio-AEPs were completely destroyed, and the remaining one was encapsulated by thin fibrous tissue. In this dog, effusion was present within the capsule, but the Bio-AEP was not destroyed. Histopathologically, the necrosis, presumably caused by hypoxia, of the PE-cells was observed on transmission electron microscopy. In conclusion, Bio-AEP could function for a certain period after implantation in this study. However, more preclinical researches should be needed to apply this technique for the treatment of diabetic dogs.

Glycosylated hemoglobin concentrations in the blood of healthy dogs and dogs with naturally developing diabetes mellitus, pancreatic beta-cell neoplasia, hyperadrenocorticism, and anemia.

OBJECTIVE: To characterize glycosylated hemoglobin (GHb) concentrations in the blood of dogs with disorders that may affect serum glucose or blood GHb concentrations, and to determine whether changes in GHb concentration correlate with changes in control of diabetes in dogs. DESIGN: Prospective study. ANIMALS: 63 healthy dogs, 9 dogs with anemia, 24 dogs with untreated hyperadrenocorticism, 12 dogs with pancreatic beta-cell neoplasia, 23 dogs with newly diagnosed diabetes mellitus, and 77 diabetic dogs treated with insulin. PROCEDURE: Control of diabetes in dogs treated with insulin was classified as good or poor on the basis of history, physical examination findings, changes in body weight, and measurement of serum glucose concentrations Sequential evaluations of control were performed and GHb concentration in blood was measured, by means of affinity chromatography, for 5 untreated diabetic dogs before and after initiating insulin treatment, for 10 poorly controlled diabetic dogs before and after increasing insulin dosage, and for 5 diabetic dogs before and after pancreatic islet cell transplantation. RESULTS: Mean (+/-SD) GHb concentration was 3.3 +/- 0.8% in the blood of healthy dogs. Compared with results from healthy dogs, mean GHb concentration was significantly lower in the blood of dogs with anemia and pancreatic beta-cell neoplasia and significantly higher in the blood of untreated diabetic dogs. Mean GHb concentration was significantly higher in the blood of 46 poorly controlled diabetic dogs, compared with 31 well-controlled diabetic dogs (7.3 +/- 1.8 vs 5.7 +/- 1.7%, respectively). Mean GHb concentration in blood decreased significantly in 5 untreated diabetic dogs after treatment (8.7 +/- 1.9 vs 5.3 +/- 1.9%). Mean GHb concentration in blood also decreased significantly in 10 poorly controlled diabetic dogs after control was improved and in 5 diabetic dogs after they had received a pancreatic islet cell transplant. CLINICAL IMPLICATIONS: Measurement of GHb concentration in blood may assist in monitoring control of diabetes in dogs.

Effect of pancreatectomy on plasma activities of amylase, isoamylase, lipase and trypsin-like immunoreactivity in dogs.

The contribution of the pancreas to the plasma activities of amylase, isoamylase, lipase and the concentration of trypsin-like immunoreactivity (TLI) in the dog was examined by measuring the activities of these enzymes before and after total pancreatectomy. Pancreatectomy was followed by a decrease in the concentration of TLI (from 6.2 +/- 0.3 micrograms litre-1 to 1.2 +/- 0.3 micrograms litre-1; P less than 0.001) and activity of isoamylase peak 4 (from 1257 +/- 105 iu litre-1 to 894 +/- 171 iu litre-1; P less than 0.05). Though significantly reduced, the activities of peak 4 isoamylase were still within the normal range for control dogs. Pancreatectomy did not significantly alter the activities of amylase, lipase or isoamylase peaks 1, 2 and 3. These findings provide strong evidence that the pancreas is not the sole source of circulating amylase, isoamylase and lipase activities. In contrast the marked reductions in TLI to values close to the limits of assay sensitivity suggest that TLI is derived from the pancreas alone. The results indicate that assay of circulating TLI provides a more sensitive and specific indicator of pancreatic exocrine mass than plasma amylase, lipase or isoamylase activities.