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.

Combined immersion and oral vaccination of Vietnamese catfish (Pangasianodon hypophthalmus) confers protection against mortality caused by Edwardsiella ictaluri.

Edwardsiella ictaluri septicemia occurs worldwide and causes high mortality and considerable economic damage to the catfish industry especially in Vietnam and the USA. To control Edwardsiella septicemia farmers extensively use antibiotics and various vaccination methods. Vaccination with inactivated vaccines has come with variable efficacy. In this trial the results of an approach of controlling Edwardsiella septicemia of Tra catfish (Pangasianodon hypophthalmus) in Vietnam through vaccination via mucosal surfaces are presented. The results show that a combination of primary vaccination by immersion with inactivated E. ictaluri followed by an oral boost with a formulated antigen preparation induces a statistically significant level of protection against mortality caused by experimental infection 4 weeks post-boost. Fish immunized by immersion only show significantly lower level of protection but significantly higher than the controls. Repeated boosts result in improved duration of immunity with a relative percent survival (RPS) of 47% at 90% control mortality. The immunization procedure provides an alternative for disease control through vaccination.

Renal and cardiac endothelial heterogeneity impact acute vascular rejection in pig-to-baboon xenotransplantation.

Xenograft outcomes are dictated by xenoantigen expression, for example, Gal alpha1, 3Gal (Gal), but might also depend on differing vascular responses. We investigated whether differential vascular gene expression in kidney and cardiac xenografts correlate with development of thrombotic microangiopathy (TM) and consumptive coagulation (CC). Immunosuppressed baboons underwent miniswine or hDAF pig kidney (n = 6) or heart (n = 7), or Gal-transferase gene-knockout (GalT-KO) (thymo)kidney transplantation (n = 14). Porcine cDNA miniarrays determined donor proinflammatory, apoptosis-related and vascular coagulant/fibrinolytic gene expression at defined time points; validated by mRNA, protein levels and immunopathology. hDAF-transgenic and GalT-KO xenografts, (particularly thymokidneys) exhibited prolonged survival. CC was seen with Gal-expressing porcine kidneys (3 of 6), only 1 of 7 baboons postcardiac xenotransplantation and was infrequent following GalT-KO grafts (1 of 14). Protective-type genes (heme oxygenase-I, superoxide dismutases and CD39) together with von Willebrand factor and P-selectin were upregulated in all renal grafts. Transcriptional responses in Gal-expressing xenografts were comparable to those seen in the infrequent GalT-KO rejection. In cardiac xenografts, fibrin deposition was associated with increased plasminogen activator inhibitor-1 expression establishing that gene expression profiles in renal and cardiac xenografts differ in a quantitative manner. These findings suggest that therapeutic targets may differ for renal and cardiac xenotransplants.

Rejection of cardiac xenografts transplanted from alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pigs to baboons.

The use of alpha1,3-galactosyltransferase gene-knockout (GalT-KO) swine donors in discordant xenotransplantation has extended the survival of cardiac xenografts in baboons following transplantation. Eight baboons received heterotopic cardiac xenografts from GalT-KO swine and were treated with a chronic immunosuppressive regimen. The pathologic features of acute humoral xenograft rejection (AHXR), acute cellular xenograft rejection (ACXR) and chronic rejection were assessed in the grafts. No hyperacute rejection developed and one graft survived up to 6 months after transplantation. However, all GalT-KO heart grafts underwent graft failure with AHXR, ACXR and/or chronic rejection. AHXR was characterized by interstitial hemorrhage and multiple thrombi in vessels of various sizes. ACXR was characterized by TUNEL(+) graft cell injury with the infiltration of T cells (including CD3 and TIA-1(+) cytotoxic T cells), CD4(+) cells, CD8(+) cells, macrophages and a small number of B and NK cells. Chronic xenograft vasculopathy, a manifestation of chronic rejection, was characterized by arterial intimal thickening with TUNEL(+) dead cells, antibody and complement deposition, and/or cytotoxic T-cell infiltration. In conclusion, despite the absence of the Gal epitope, acute and chronic antibody and cell-mediated rejection developed in grafts, maintained by chronic immunosupression, presumably due to de novo responses to non-Gal antigens.

Glycolipid studies in small intestine and pancreas of alpha1,3-galactosyltransferase knockout miniature swine: alpha1,3GALT-KO animals lack alphaGAL antigens and contain novel blood group H compounds.

BACKGROUND: To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knock-out (GalT-KO) pigs have been produced. However, Galalpha1,3Gal (Gal) determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens that might interfere with the human immune response. METHODS: Glycolipids isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig were tested for immune reactivity with antibodies on thin-layer chromatograms after separation by high-performance liquid chromatography, and selected fractions were analysed by proton NMR spectroscopy. RESULTS: Immunostaining using purified human anti-Gal Abs revealed that tissues from WT animals express large amounts of Gal-antigens whereas GalT-KO tissues lacked these antigens. Proton NMR spectroscopy on small intestine fractions revealed both linear and branched nona- and decaglycosylceramides, respectively, with terminal Gal-epitopes. In corresponding GalT-KO fractions, Gal-epitopes seemed to be replaced by terminal alpha1,2fucoses. Two novel branched blood group H compounds was found in the GalT-KO intestine. CONCLUSIONS: The structural complexity of alphaGal-terminating antigens in the WT organs is very high. Knockout of alpha1,3GalT by gene-targeting results in elimination of Gal-determinants. In addition structurally novel alpha1,2fucose-terminated blood group H compounds were identified in the GalT-KO tissue. These compounds are not expected to be recognized by the human immune system.

Acute and chronic vascular rejection in nonhuman primate kidney transplantation.

A nonhuman primate (NHP) study was designed to evaluate in nonlife-supporting kidney allografts the progression from acute rejection with transplant endarteritis (TXA) to chronic rejection (CR) with sclerosing vasculopathy. Group G1 (n = 6) received high cyclosporine A (CsA) immunosuppression and showed neither TXA nor CR during 90 days post-transplantation. Group G2 (n = 6) received suboptimal CsA immunosuppression and showed severe TXA with graft loss within 46 days (median). Arterial intimal changes included infiltration of macrophages and T lymphocytes (CD3, CD4, CD8) with few myofibroblasts, abundant fibronectin/collagen IV, scant collagens I/III, high rate of cellular proliferation and no C4d accumulation along peritubular capillaries. Group G3 (n = 12) received suboptimal CsA and anti-rejection therapy (rabbit ATG + methylprednisolone + CsA) of TXA. Animals developed CR and lost grafts within 65 days (median). As compared to G2, the arterial intimal changes showed less macrophages and T lymphocytes, an increased number of myofibroblasts, abundant fibronectin/collagen IV and scar collagens I/III, C4d deposition along capillaries in 60% of animals and transplant glomerulopathy in 80% of animals. In conclusion, CR is an immune stimulated process initiated during TXA with the accumulation and proliferation of myofibroblasts, and progressive deposition of collagens in the intima. Our experimental design appears well suited to study events leading to CR.

Hyperacute rejection is attenuated in GalT knockout swine lungs perfused ex vivo with human blood.

BACKGROUND: Hyperacute rejection (HAR) is one of the principal obstacles to successful xenotransplantation. Homozygous alpha-1,3-galactosyltransferase knockout (GalT-KO) miniature swine now offer the prospect of overcoming this barrier to xenotransplantation. In this study, the short-term function of GalT-KO swine lungs was evaluated in a well-established ex vivo model of swine-to-human lung xenotransplantation. METHODS: Lungs from homozygous GalT-KO swine (n = 3) and control lungs from pigs of the background strain used to create the GalT-KO pig line (n = 2) were perfused ex vivo with freshly collected heparinized human blood. Graft function was assessed by various physiologic measurements, serial histologic and immunohistochemical evaluation, and assays of complement and platelet activation. RESULTS: Xenoperfused control swine lungs exhibited HAR with graft survival times <5 minutes. In contrast, GalT-KO swine lungs retained their function for approximately 2 hours, on average. GalT-KO swine lungs showed decreased complement and platelet activation compared with controls. Nonetheless, activation of complement and coagulation cascades was not completely eliminated in the GalT-KO swine lungs. CONCLUSIONS: The survival of xenoperfused GalT-KO swine lungs was significantly prolonged, as compared with control lungs expressing Gal. This appears to have been due largely to substantially reduced complement activation. Nonetheless, the xenoperfused GalT-KO lungs still showed some evidence of complement fixation and intravascular coagulopathy by the time of graft demise.

Potential of aspirin to inhibit thrombotic microangiopathy in alpha1,3-galactosyltransferase gene-knockout pig hearts after transplantation in baboons.

Hearts from alpha1,3-Galactosyltransferase gene-knockout (GaIT-KO) pigs were transplanted heterotopically into 8 baboons that received an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen and heparin. Three baboons died or were euthanized with beating grafts on 16, 23, and 56 days, respectively, and the remaining 5 grafts functioned for 59-179 days. Hyperacute rejection did not occur, and classical features of acute humoral xenograft or acute cellular rejection were rare. However, thrombotic microangiopathy (TM) developed in all cases; its onset was delayed in 2 baboons that received aspirin. Function of a pig organ in a baboon for a period approaching 6 months has not been reported previously and lends encouragement that the barriers to xenotransplantation will be overcome, but TM requires investigation.

The anti-CD2 monoclonal antibody BTI-322 generates unresponsiveness by activation-associated T cell depletion.

The antihuman CD2 MoAb BTI-322 (Lo-CD2a) effectively inhibits T cell responses in vitro to allogeneic cells, which is followed by unresponsiveness to the original stimulator in secondary stimulation. We studied the xenogeneic human antiporcine mixed lymphocyte reaction (MLR), and utilized anti-T cell receptor (TCR) Vbeta family antibody-induced cell proliferation to determine the specificity and mechanism. BTI-322 and its humanized version, MEDI-507, effectively inhibited the primary xenogeneic MLR. After suboptimal primary stimulation using lower numbers of xenogeneic stimulator cells, the unresponsiveness in secondary culture was apparent only for xenogeneic stimulator cells of the original SLA haplotype, and not for third-party stimulators or allogeneic cells. The inhibition of primary MLR was not observed for nylon-wool-purified T cells, but was seen after reconstitution of purified T cells with monocytes. Similarly, anti-Vbeta family-specific stimulation showed family-specific unresponsiveness in secondary culture. This required the presence of the whole BTI-322 molecule: a F(ab')2 fragment was not effective. T cells of a distinct Vbeta family were depleted after stimulation with an anti-Vbeta family-specific antibody and BTI-322. We conclude that the inhibition by BTI-322 of a primary xenogeneic MLR or the response to an anti-TCR Vbeta antibody is associated with unresponsiveness upon restimulation, due to activation-associated cell depletion. In this process, the interaction between monocytes and the Fc part of the antibody is involved. This unique characteristic of BTI-322 suggests the potential of the antibody for tolerance induction in vivo, besides the potential use as a T cell depleting agent.

Initial experience with the human anti-human CD154 monoclonal antibody, ABI793, in pig-to-baboon xenotransplantation.

BACKGROUND: ABI793 (ABI) is a human monoclonal antibody (mAb) specific for human CD154. To assess the suitability of ABI for baboon transplantation studies, we carried out in vitro studies to determine ABI's reactivity with baboon cells expressing CD154, performed in vivo pharmacokinetic studies in two baboons, and tested the effect of ABI administration on elicited antibody production in two baboons undergoing either pig hematopoietic progenitor cell (PBPC) or heterotopic heart transplantation. METHODS: In vitro: Baboon peripheral blood mononuclear cells were activated in vitro to upregulate CD154, and binding of ABI to CD154 was measured by flow cytometry. In vivo: Serum levels of ABI were measured immediately before and 15 min after the intravenous administration of ABI (20 mg/kg) to two baboons over 28 days. Subsequently, ABI (25 mg/kg on days 0, 1, 4 and 7, and then 20 mg/kg every 5 days) was included in the immunosuppressive regimen in two pig-to-baboon transplants (PBPC or heart transplantation). RESULTS: In vitro: ABI was almost non-reactive to baboon T cells before stimulation, but bound to activated T cells. In vivo: In the pharmacokinetic study, trough levels of ABI (before the next dose) ranged between 190 and 580 microg/ml, and the estimated half-life was 10-15 days. There was no apparent toxicity. Following pig PBPC or heart transplantation, no elicited antibody was detected while ABI was being administered or during several weeks of follow-up. CONCLUSIONS: ABI functions in baboons, is well-tolerated, and prevents an elicited antibody response to pig antigens.

Bone marrow transplantation from alpha1,3-galactosyltransferase gene-knockout pigs in baboons.

BACKGROUND: Successful hematopoietic cell allotransplantation results in donor-specific tolerance, but this approach has been unsuccessful in the wild-type pig-to-baboon xenotransplantation model, as pig cells were lost from the circulation within 5 days. However, after cessation of immunosuppressive therapy on day 28, all baboons demonstrated non-specific unresponsiveness on mixed leukocyte reaction (MLR) for at least 30 days. We have now investigated the transplantation of bone marrow (BM) cells from miniature swine homozygous for alpha1,3-galactosyltransferase gene-knockout (GalT-KO). METHODS: Baboons (n = 3) were pre-treated with whole body and thymic irradiation, anti-thymocyte globulin, and splenectomy, and received immunosuppressive and supportive therapy for 28 days. BM was harvested from GalT-KO swine (n = 3). The baboons were monitored for the presence of pig cells by flow cytometry and colony-forming units (CFUs), and for cellular reactivity by MLR. RESULTS: A mean of 11 x 10(8) BM cells/kg was infused into each baboon. The mean absolute numbers and percentages of pig cells detected in the blood at 2 h and on days 1, 2 and 4, respectively, were 641/microl (9.5%), 132/microl (3.4%), 242/microl (3.9%), and 156/microl (2.9%). One baboon died (from accidental hemorrhage) on day 6, at which time chimerism was present in the blood (2.0%) and BM (6.4%); pig cell engraftment in the BM was confirmed by polymerase chain reaction (PCR) of CFUs. In the two other baboons, blood chimerism was lost after day 5 but returned at low levels (<1%) between days 9 to 16 and 7 to 17, respectively, indicating transient BM engraftment. Both surviving baboons showed non-specific unresponsiveness on MLR until they were euthanized on days 85 and 110, respectively. CONCLUSIONS: By using BM cells from GalT-KO pigs, chimerism was detected at levels comparable with previous studies when 30-fold more growth factor-mobilized peripheral blood progenitor cells had been transplanted. In addition, cellular hyporesponsiveness was prolonged. However, long-term engraftment and chimerism were not achieved.

Reduction of anti-Galalpha1,3Gal antibodies by infusion of types 2 and 6 gal trisaccharides conjugated to poly-L-lysine.

To investigate the specificity of anti-Galalpha1,3Gal (Gal) antibodies (Abs) with respect to Gal oligosaccharides of types 2 and 6, eight baboons received an intravenous infusion of either a poly-l-lysine conjugate of Gal type 2 (n = 5) or type 6 (n = 3), followed 48 h later by the alternative Gal type 6 or 2 conjugate, respectively. IgM Abs reactive to Gal type 2 were depleted by 80 to 89% by either Gal conjugate. IgM reactive to Gal type 6 was less efficiently depleted by the Gal type 2 conjugate (57% depletion) than the Gal type 6 (82% depletion). Gal-reactive IgG was depleted more slowly and less efficiently by either glycoconjugate (initially by only 28 to 54%). Our results indicate that the Gal type 6 conjugate depletes most anti-Gal IgM, but the Gal type 2 conjugate is less efficient in depleting anti-Gal IgM reactive with type 6. There remain small fractions of antibody that are unadsorbed, particularly of IgG, probably due to their low affinity and distribution in both the intra- and extra-vascular compartments.

Gal alpha 1,3Gal expression on porcine pancreatic islets, testis, spleen, and thymus.

Gal alpha 1,3Gal (Gal) is the first target in antibody-mediated rejection of pig-to-non-human primate xenograft. Its expression may vary between organs and constituents of organs. Gal expression was studied in pancreas, testis, spleen and thymus of 22 pigs, with ages ranging from 1 to 22 months. The immunoperoxidase technique using the biotinylated lectin, Griffonia simplicifolia (IB4), was used. In the pancreas, neither endocrine (islet cells) nor exocrine cells expressed Gal. The Sertoli cells in the testis were negative. The spleen capsule and trabeculae did not stain for Gal, although both splenic T and B lymphocytes expressed Gal (B > T). Thymocytes were weakly positive, whereas thymic epithelial cells were negative for Gal. No age-related differences were seen in any tissues. Porcine islets of Langerhans, Sertoli cells, and the splenic and thymic structural frameworks did not express Gal, and therefore, should be relatively resistant to anti-Gal antibody-mediated rejection. The availability of pigs deficient in Gal as a source of islets may therefore not be beneficial in extending islet graft survival in non-human primate models.

Structural and functional characteristics of a dominant-negative isoform of porcine MHC class II transactivator.

The MHC class II transactivator, CIITA, is critical for MHC class II gene expression in all species studied to date. We isolated an interferon (IFN)-gamma-inducible isoform of porcine CIITA (pCIITA') encoding a protein of 566 amino acids (aa) with significant homology to human CIITA (hCIITA). Analysis indicated that pCIITA' lacks the entire GTP-binding domain that is important for nuclear translocation and activation of target genes by hCIITA. In pCIITA' this region is replaced by a 14-aa motif with homology to several signalling peptide sequences. Expression of pCIITA' in porcine (ST-IOWA) and human (HeLa) cell lines resulted in suppression of IFN-gamma-stimulated MHC class II gene expression, at the protein and mRNA levels. We also identified two IFN-gamma-inducible variants of hCIITA, hCIITAlo and hCIITA' from Hela cells, both exhibiting dominant-negative suppression of MHC class II gene expression. Interestingly, hCIITA' encodes a predicted protein of 546 aa with a strikingly similar organization to pCIITA' including the 14-aa GTP-binding domain-replacement motif in which 10 out of 14 amino acids are identical to the pig sequence. Expression of hCIITA' and hCIITAlo sequences in Hela cells suppressed IFN-gamma-induced MHC class II gene expression. hCIITAlo, a predicted 303-aa protein with deleted GTP-binding and carboxy-terminal domain, displayed a more subtle suppression of IFN-gamma-induced MHC class II expression. These in vitro data indicate that there may be a role in vivo for isoforms of CIITA that can suppress full-length CIITA-mediated MHC class II gene expression. Both humans and now, potentially, pigs are candidate donors for organ and tissue allografts and xenografts, respectively. Regulation of MHC class II gene expression by manipulation of CIITA isoform expression in humans and pigs may provide a useful strategy for attenuation of T-cell-mediated cellular rejection.

Virus safety in xenotransplantation: first exploratory in vivo studies in small laboratory animals and non-human primates.

For xenotransplantation, the transplantation of animal cells, tissues and organs into human recipients, to date, pigs are favored as potential donors. Beside ethical, immunological, physiological and technical problems, the microbiological safety of the xenograft has to be guaranteed. It will be possible to eliminate all of the known porcine microorgansims in the nearby future by vaccinating or specified pathogen-free breeding. Thus, the main risk will come from the porcine endogenous retroviruses (PERVs) which are present in the pig genome as proviruses of different subtypes. PERVs will therefore be transmitted, with the xenograft, to the human recipient. PERVs can infect numerous different types of human primary cells and cell lines in vitro and were shown to adapt to these cells by serial passaging on uninfected cells. Furthermore, PERVs have high homology to other retroviruses, such as feline leukemia virus (FeLV) or murine leukemia virus (MuLV), which are known to induce tumors or immunodeficiencies in the infected host. To evaluate the potential risk of a trans-species transmission of PERV in vivo, naive and immunosuppressed rats, guinea pigs and minks were inoculated with PERV and screened over a period of 3 months for an antibody reaction against PERV proteins or for the integration of proviral DNA into the genomic DNA of the host's cells. Furthermore, we inoculated three different species of non-human primates, rhesus monkey (Macaca mulatta), pig-tailed monkey (Macaca nemestrina) and baboon (Papio hamadryas) with high titers of a human-adapted PERV. To simulate a situation in xenotransplantation, the animals received a daily triple immunosuppression using cyclosporine A, methylprednisolone and RAD, a rapamycin derivative, presently under development by Novartis. None of the small laboratory animals or the non-human primates showed production of antibodies against PERV or evidence of integration of proviral DNA in blood cells or cells of several organs, 3 months after virus inoculation, despite the observation that cells of the animals used in the experiment were infectible in vitro. This apparent difference in the outcome of the in vitro and the in vivo data might be explained by an efficient elimination of the virus by the innate or adaptive immunity of the animals.

Comparative efficacy of mycophenolate sodium (MPS) and mycophenolate mofetil (MMF) with and without cyclosporine in rat transplantation models.

BACKGROUND: ERL is the enteric-coated sodium salt of mycophenolic acid, presently in clinical development. The drug substance mycophenolate sodium (MPS) was evaluated in rat transplantation models and compared with mycophenolate mofetil (MMF) for therapeutic window and synergy with cyclosporine (CsA). METHODS: Allotransplantation was performed in the Dark Agouti-to-Lewis (DA-to-Lewis; kidney, heart, and aorta) and Brown Norway-to-Lewis (BN-to-Lewis; kidney) strain combinations, and hamster heart xenotransplantation was performed in athymic and euthymic Lewis rats. The compounds were administered daily orally, starting the day of transplantation. RESULTS: In kidney and heart transplantation the minimal efficacious dose of CsA was 5.0 mg/kg/d. For MPS this dose was 10 mg/kg/d in BN-to-Lewis kidney transplantation, 20 mg/kg/d in DA-to-Lewis heart transplantation, and 10 mg/kg/d in hamster-to-athymic rat heart transplantation. At these doses the first signs of adverse effects were evident, indicating a narrow therapeutic window. No window was established for MMF in these models or for MPS in DA-to-Lewis kidney transplantation. There was no potential synergy between CsA and MPS or MMF regarding efficacy, but fewer side effects were noted in efficacious combinations, in particular for MPS. In aorta transplantation, MPS and MMF dose-dependently inhibited intima thickening. The combination of 20 mg/kg/d MPS and 10 mg/kg/d CsA gave long-term survival of hamster-to-rat xenografts. CONCLUSIONS: Despite the overall comparable efficacy and narrow therapeutic window of MPS and MMF when given alone, MPS apparently is better tolerated than MMF in some of the transplant models. The combination of these agents with CsA allows fine-tuning between optimal immunosuppression and adverse side effects.

Pharmacokinetics of cyclosporine in monkeys after oral and intramuscular administration: relation to efficacy in kidney allografting.

In cynomolgus and rhesus monkeys, the dose-normalized exposure of cyclosporine administered orally as microemulsion preconcentrate (Neoral) was lower than that upon intramuscular administration. For oral administration, mean values ( +/- SD) of Cmax, 24-h area-under-the curve (AUC) and 24-h trough level, all normalized for a 1 mg/kg dose, were 20 +/- 9 ng x kg/mg x ml, 210 +/- 70 ng x h x kg/mg x ml and 2.6 +/- 0.9 ng x kg/mg x ml, respectively. For intramuscular administration, levels were about 5.5-fold, 9-fold and 22-fold higher. Based on pharmacokinetic data, the efficacy of oral cyclosporine treatment (without any other immunosuppressant) was evaluated in life-supporting cynomolgus monkey kidney allotransplantation. Rejection-free kidney allograft survival could be achieved using oral cyclosporine monotherapy with average 24-h trough concentrations above 100 ng/ml during maintenance treatment. Typically, daily oral doses of 100 mg/kg-150 mg/kg during the first two weeks post-transplantation, followed by daily 30 mg/kg-100 mg/kg dose levels during subsequent maintenance can result in long-term allograft survival, with 24-h average trough levels in individual animals during maintenance between 110 ng/ml and 700 ng/ml.

Ultrasound detection of non-Hodgkin's lymphoma in three cynomolgus monkeys after renal transplantation and cyclosporine immunosuppression.

PURPOSE: To describe the early detection of non-Hodgkin's lymphoma (NHL) with ultrasound in three clinically normal cynomolgus monkeys post-renal transplantation and immunosuppression with cyclosporine. MATERIALS AND METHODS: The monkeys in this report were treated with cyclosporine (Neoral) after receiving renal transplants. In addition to clinical and laboratory (hematology, serum chemistry) monitoring, renal allografts were monitored every 2 weeks with ultrasound and ultrasound-guided allograft biopsies were performed. RESULTS: Enlarged renal hilar and mesenteric lymph nodes were detected with ultrasound in three monkeys on days 36, 49 and 134 post-transplantation. Sonographically the lymph nodes were inhomogeneous, of low echogenity and rounded. In two animals, the spleen was sonographically enlarged and inhomogeneous. All three monkeys were symptom-free at the time of ultrasound detection and NHL was diagnosed histologically. CONCLUSION: Ultrasound provides a rapid, non-invasive means of early detection of NHL in animal transplantation models prior to the onset of clinical symptoms of disease.

Renal allograft vasculopathy: ultrasound findings in a non-human primate model of chronic rejection.

The purpose was to determine whether decreased cortical flow detected with power Doppler (PD) ultrasound in renal allografts in cynomolgus monkeys marks the presence or onset of chronic renal allograft vasculopathy. The 2D grey scale and PD ultrasound findings of 24 consecutively implanted non-life-supporting renal allografts in cynomolgus monkeys that underwent either 24 h (n=15) or 48 h (n=9) cold ischaemia times were recorded and compared with the results of histology performed every 2 weeks post-operatively. 13 allografts developed vasculopathies, 10 of which had PD scores equal to 1 (severe reduction of cortical flow). A PD score of 1 occurred in only one instance in the group of allografts without vasculopathies and this was due to necrosis. Allografts without vasculopathies otherwise had either PD scores of 3 (normal flow; n=2) or 2 (reduced flow; n=4). Allografts subjected to 48 h cold ischaemia times were smaller than those with 24 h cold ischaemia times (significant at weeks 5-11, p<0.05), but a reduction in graft size associated with vasculopathies occurred infrequently. In conclusion, the finding of reduced renal cortical flow detected by PD ultrasound during serial examination of non-life-supporting renal allografts is highly supportive of a diagnosis of graft vasculopathy due to arteriolar intimal proliferation, and illustrates an excellent method of monitoring changes in cortical perfusion in allografts in animal models. The combination of findings of reduced or absent cortical flow together with severe graft enlargement is highly suggestive of the presence of not only vasculopathies but also tissue damage and degeneration.