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.

Adhesion of human B cells to follicular dendritic cells involves both the lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and very late antigen 4/vascular cell adhesion molecule 1 pathways.

Presentation of antigen in the form of immune complexes to B lymphocytes by follicular dendritic cells (FDC) is considered to be a central step in the generation of memory B cells. During this process, which takes place in the microenvironment of the germinal center, B cells and FDC are in close physical contact. In the present study, we have explored the molecular basis of FDC-B cell interaction by using FDC and B cells derived from human tonsils. We found that FDC express high levels of the adhesion receptors intercellular adhesion molecule 1 (ICAM-1 [CD54]) and vascular cell adhesion molecule 1 (VCAM-1), while the B lymphocytes express lymphocyte function-associated antigen 1 (LFA-1 [CD11a/18]), very late antigen 4 (VLA-4 [CD49d], and CD44. Furthermore, we established that both the LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways are involved in FDC-B lymphocyte binding, and therefore, these pathways might be essential in affinity selection of B cells and in the formation of B memory cells.

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.

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.

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.

Epitopes of apolipoprotein B-100 and B-48 in both liver and intestine. Expression and evidence for local synthesis in recessive abetalipoproteinemia.

The presence of apolipoprotein (apo) B in liver and intestine from a patient with abetalipoproteinemia was evaluated by immunohistochemistry with a polyclonal and six monoclonal antibodies to different apo B-48 and B-100 epitopes. In normal liver, apo B was present inside and outside hepatocytes. The patients liver exhibited staining in the cytoplasm with the polyclonal and three monoclonal antibodies. By immunoelectron-microscopy, apo B was found to be present in the smooth endoplasmatic reticulum and the Golgi complex. Normal intestinal epithelium was labeled with polyclonal and all monoclonal antibodies, including those specific for apo B-100. The patients epithelium stained with polyclonal and six monoclonals, and apo B was present in the Golgi complex. Thus, normal intestinal mucosa expressed apo B-48 and B-100 epitopes, which indicates apo B-100 synthesis in the gut. The synthesis of the apo B molecule in the patient seems to be retained in both liver and gut, which suggests a posttranslational defect.

Juvenile chronic arthritis: T cell reactivity to human HSP60 in patients with a favorable course of arthritis.

Synovial fluid and peripheral blood mononuclear cell proliferative responses to the 60-kD human heat shock protein (HSP60) were studied in 23 patients with juvenile chronic arthritis (JCA) and 7 non-JCA control patients. All patients showed active arthritis at the time of study. The patients were divided into two groups according to the presence (group A) or absence (group B) of T lymphocyte reactivity to human HSP60. We show that reactivity to human HSP60 is primarily, though not exclusively, occurring in patients with a remitting course of disease, i.e., the subgroup of HLA-B27 negative JCA patients with an oligoarticular onset. Immunohistochemical analysis of HSP expression in synovial membranes showed a significantly higher intensity of staining in JCA patients than in non-JCA controls. The results suggest that, in accordance with the earlier observation made in experimental models, T lymphocyte reactivity to human HSP60 in this subgroup of JCA patients may be part of T cell regulatory mechanisms that control the development of arthritis.

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.

Implantation of cultured thymic fragments in patients with acquired immunodeficiency syndrome.

Cultured thymic fragments were implanted in one patient with acquired immunodeficiency syndrome (AIDS)-related complex (ARC) and in eight AIDS patients with opportunistic infections (OIs, four patients), Kaposi's sarcoma (KS, two patients), or both (two patients). Thereafter, objective clinical improvement was noted in one patient with OI, and a stable symptom-free condition was observed in the ARC patient and in two other patients with OIs. However, the ARC patient and two of the three patients with OIs developed infections three to six months after implantation. A fourth case of OI and the patients with KS showed progression of the disease. Peripheral blood investigations for counts of total leukocytes, lymphocytes, and T-lymphocyte subsets as well as for lymphocyte stimulation with mitogens showed no changes interpretable as an improvement of the cellular immune deficiency status. We conclude that cultured thymic fragments have no distinct in vivo effect on the course of AIDS, except for a temporary clinical improvement or a period of stable condition in some patients with OIs.

Modulation of cell surface molecules during HIV-1 infection of H9 cells. An immunoelectron microscopic study.

OBJECTIVE: To study cell surface molecules and HIV-1 proteins on H9 cells 2 days after infection by immunogold electron microscopy, either in single or in double labelling using combinations of host cell-derived molecules and HIV-1 proteins. DESIGN AND METHODS: The presence of host cell antigens CD3, CD4 and human leukocyte antigen-DR (HLA-DR) and HIV-1 antigens gag p15, p17, p24 and env gp41 was evaluated using immunocytochemistry at the light microscopic level. H9 cells 2 days after infection were processed for conventional transmission electron microscopy and cryo-ultramicrotomy. Leukocyte antigens investigated were CD2, CD3, CD4 (two antibodies), CD5, CD8, CD25, CD30, CD63 antigens and HLA-DR; HIV-1-encoded antigens were gag p24, pol reverse transcriptase, and env gp41 and gp120. Double immunogold labelling was performed using reagents with different sized gold particles. For leukocyte markers, the labelling density of the cell membrane was assessed quantitatively on uninfected and infected H9 cells. RESULTS: Infected cells revealed the presence of gag p24, pol, and env gp41 and gp120 antigens on HIV-1 virions. Uninfected H9 cells showed a random distribution of cell surface molecules, including CD4 antigen, along the plasma membrane. The CD63 antigen, a lysosomal membrane glycoprotein, was located mainly in the cytoplasm of uninfected cells. Cells 2 days after infection showed CD4 labelling on sites where virions were budding from or attached to the cell surface and on free virions. Virions also showed labelling by CD3, CD5, CD25, CD30 and CD63 antibodies and anti-HLA-DR. Compared with uninfected cells, a significantly lower density was found on infected cells in labelling for CD4, CD5 and anti-HLA-DR. A significantly higher density on cells 2 days after infection was seen in CD63 labelling. CONCLUSION: During the first phase of infection host cell molecules concentrate on budding structures and newly generated HIV-1 virions. This phenomenon might contribute to the disappearance of these molecules (like the CD4 molecule) from the cell membrane after infection.

T-cell receptor V beta-family usage in primary cutaneous and primary nodal T-cell non-Hodgkin's lymphomas.

To evaluate whether the expression of T-cell receptor (TCR) V beta families in eight cases of malignant T-cell lymphomas took place in a preferential manner, we analyzed four cases of mycosis fungoides (MF), the most common form of primary cutaneous T-cell non-Hodgkin's lymphomas (NHL), and four cases of primary nodal T-cell NHL. The usage of V beta families in T-cell populations was investigated on mRNA that was transcribed to cDNA using a C beta primer and reverse transcriptase. Subsequently, the specific usage of the families was analyzed by polymerase chain reaction (PCR) using combinations of the selected C beta-oligonucleotide primer and one of the family-specific V beta primers. Peripheral blood lymphocytes from four healthy volunteers and 1 "reactive" lymph node served as a control and expressed all 20 V beta families tested for. In T-cell lines, with restricted V beta expression, and in three patients with advanced MF, only one or two V beta families were expressed at the mRNA level. In an early MF lesion this monoclonal expression was absent: several V beta families were expressed with a weak intensity. This may indicate either a polyclonal origin of MF, or that too few monoclonal neoplastic cells were present in the tissue specimen. In the four nodal T-cell NHL, only one family could be clearly distinguished, whereas some of the other V beta families showed only a weak expression. These latter families represent the reactive T-cell component in the nodal T-cell NHL. Both in nodal T-cell NHL and in MF there was no preferential expression of a particular V beta family. There was a good correlation between PCR data and the expression of V beta-family protein products observed by immunohistochemistry on tissue sections of the T-cell lymphomas. All T-cell lines, three cases of MF, and three cases of nodal T-cell NHL showed a rearrangement of the TCR beta chain on DNA level.

Human antibodies to immunoglobulin A (IgA). A radioimmunological method for differentiation between anti-IgA antibodies and IgA in the serum of IgA deficient individuals.

In the sera of 12 out of 27 individuals with IgA deficiency (serum level below 0.02 mg IgA/ml) class-specific anti-IgA antibodies were demonstrated by haemagglutination. These sera showed false-positive results in a solid-phase inhibition radioimmunoassay (RIST) (apparent IgA concentration between 0.6 and 13.7 microgram IgA/ml) indicating that the RIST is not an appropriate test for the analysis of serum of IgA deficient individuals. A modification of the RIST is proposed (titration RIA) that permits differentiation between low levels of IgA and class-specific anti-IgA antibodies. With this test IgA deficient individuals could be classified as those with low but detectable levels of IgA and those with class-specific anti-IgA antibodies. A computer procedure was developed to calculate both the amount and the avidity (K) of the anti-IgA antibodies and to simulate the assay system. The K value calculated from experimental points proved to be an overestimation of the K value which fitted most adequately in the simulation. The comparison of the results with clinical findings indicated a possible correlation between the amount and the avidity of the anti-IgA antibodies and the appearance of anaphylactic reactions after transfusion of IgA.

Oral efficacy of the macrolide immunosuppressant SDZ RAD and of cyclosporine microemulsion in cynomolgus monkey kidney allotransplantation.

BACKGROUND: 40-O-(2-Hydroxyethyl)-rapamycin (SDZ RAD) is a novel, potent, macrolide immunosuppressant. Its efficacy in rodent transplantation models provided the rationale for us to evaluate the compound in a more relevant, large animal transplantation model. METHODS: Life-supporting kidney allotransplantation was performed in cynomolgus monkeys: rejection was inferred from a rise in serum creatinine or urea and was subsequently confirmed by histopathology. This model was validated with the microemulsion formulation of cyclosporine (i.e., Neoral). Two studies with a microemulsion formulation of SDZ RAD were performed. First, in a dose-finding study, the SDZ RAD dose was reduced in a stepwise fashion until rejection occurred, either with SDZ RAD as monotherapy, or in combination with a fixed, suboptimal dose of cyclosporine. Second, an efficacy study was performed in which two fixed SDZ RAD doses (0.75 and 1.50 mg/kg/ day) were evaluated in monotherapy and compared with the same doses of rapamycin (sirolimus). All immunosuppressants were administered once daily by gastric gavage. RESULTS: Untreated control animals rejected their grafts between 4 and 8 days after transplantation. Cyclosporine (initially at 150 mg/kg/day, reduced to 100 mg/kg/day 2 weeks after transplantation) yielded long-term (>100 days) rejection-free allograft survival in four of five animals. A 10 mg/kg/day dose of cyclosporine led to rejection between 10 and 27 days after transplantation and was considered suboptimal. In the dose-finding study with SDZ RAD monotherapy, rejection occurred in most of the cases (four of six animals) when a dose level of 0.63 mg/kg/day had been reached. Combined with suboptimal cyclosporine, this threshold SDZ RAD dose was about 2-fold lower. In the efficacy study, median graft survival with histologically proven rejection was 32 days (range 8-91 days, n=6) for 0.75 mg(kg/day SDZ RAD and 59 days (range 28-85 days, n=6) for 1.50 mg/kg/day SDZ RAD. For sirolimus, median graft survival was 43 days (range 5-103 days, n=7) for the 0.75 mg/kg/day dose and 56 days (range 8-103 days, n=8) for the 1.50 mg/kg/day dose. There was no statistically significant difference in efficacy between SDZ RAD and sirolimus. CONCLUSION: SDZ RAD, in the absence of any other immunosuppressant and at doses that do not show any overt toxicity, considerably prolongs rejection-free survival of cynomolgus monkeys after life-supporting kidney allotransplantation.

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.

Innervation of the endomyocardium in the first period after heart transplantation.

Serial endomyocardial biopsies from 5 patients during the first 3 months after heart transplantation were studied by immunohistochemistry for the neural markers neurofilament 200 kD, neuron-specific protein 9.5 (PGP9.5), S100 (Schwann cell marker), and tyrosine hydroxylase (TH). In normal endomyocardium, nerves immunoreactive for neurofilament 200 kD and PGP9.5 occurred in the interstitium around blood vessels, in close contact with myocyte fibrils. Immunoreactive fibers identified for S100 and TH were also present. In biopsies taken after transplantation, the basic nerve structure in neurofilament labeling was intact. There was a disappearance of immunolabeling for PGP9.5, S100, and TH during the first month after transplantation. Immunoreactivity reappeared during the second month, at first in the interstitium around blood vessels. This was observed for PGP9.5 and TH between 4 and 6 weeks after transplantation, and for S100 (in two of five patients) starting after 6 weeks. There was no apparent relation between reappearance and occurrence of rejection.

Effect of in vivo rapamycin treatment on de novo T-cell development in relation to induction of autoimmune-like immunopathology in the rat.

Cyclosporine (CsA) and FK506 are structurally unrelated immunosuppressants, but function in similar ways. FK506 and rapamycin (RAPA), on the other hand, have structural similarities, but act by different mechanisms to yield immunosuppression. Besides their immunosuppressive action, CsA and FK506 are known to interfere with T-cell development. CsA treatment after lethal X-irradiation and syngeneic bone marrow transplantation results in autoimmune disease, which is referred to as CsA-induced autoimmunity. In this study, we examined the effect of RAPA on T-cell development by flow cytometry and immunohistochemistry in female Lewis and Brown Norway rats. Irradiation and syngeneic bone marrow transplantation were performed before a 4-week course of RAPA administration to determine de novo T-cell development in relation to possible autoimmune phenomena. RAPA interfered with the maturation of thymocytes to the CD4+CD8+ DP stage, which resulted in a relative increase in TCRalphabeta(-) immature thymocytes, localized in a rim along the outer cortex. The thymus of RAPA-treated animals had a thinner cortex, leading to stronger thymic atrophy. In the periphery, only a few T cells were observed at the end of RAPA treatment. In the Lewis rat, a normal CD4/CD8 T-cell ratio and an increased Th1/Th2 ratio was observed within the T-cell population. Six weeks after cessation of RAPA therapy, the T-cell compartment was restored to normal, with respect to number and phenotype. In Brown Norway rats, however, T-cell areas were barely detectable at the end of RAPA treatment. The CD4/CD8 T-cell ratio was decreased as a result of a lower number of CD4 T cells; the Th1/Th2 ratio was increased but Th2 remained higher. Similar to Lewis rats, the situation was almost normalized 6 weeks after cessation of RAPA administration. However, Brown Norway rats, in contrast to Lewis rats, showed T-cell infiltration and concomitant induction of MHC class II in the submandibular salivary gland, as well as insulitis, in the pancreas. Possible relationships to Sjogren's disease and diabetes remain to be established.

Endomyocardial biopsies after heart transplantation. The presence of markers indicative of activation.

A series of 104 endomyocardial biopsies (EMB) from patients after heart transplantation was evaluated for the presence of immunological markers on graft component and infiltrating cells. This included markers for cells expressing alpha beta-T-cell receptors and gamma delta-T-cell receptors, and cytotoxic T cells with granules bearing the serine esterase Granzyme B; the presence of activation markers identified by CD25 (interleukin 2 receptor), CD30, CD69 (activation inducer molecule), CDw70; macrophages using antibody CD14 (WT14), and cells with Fc gamma-receptors type III (CD16). Almost all cells in T-cell infiltrates expressed the alpha beta-T cell receptor. Cells bearing the gamma delta-T cell receptor were scarcely found. The analysis with respect to the histopathologic diagnosis for rejection showed an absence of significance for T cell subsets, Granzyme B-positive cells, and activation markers except CD25. The numbers of macrophages labeled by CD14 and cells expressing Fc gamma RIII showed a significant relation to histopathology of rejection. Apart from leukocytes, also endothelium in EMB with rejection was labeled by the two anti-Fc gamma RIII antibodies used. In addition, in a small series of biopsies investigated, Fc gamma RI- and Fc gamma RII-positive cells were increased in EMB with rejection, and endothelium was labeled by Fc gamma RII antibodies. A cluster analysis on the basis of scores for CD25, CD14, and anti-Fc gamma RIII revealed three main clusters, one cluster comprising biopsies without abnormalities, one cluster containing EMB with the histopathology of rejection and high scores in immunophenotyping for lymphocytes and macrophages, and one cluster in between. The present data emphasize the importance of macrophage assessment in evaluating pathologic processes during rejection of heart allografts and diagnosing rejection.

SDZ RAD, a new rapamycin derivative: synergism with cyclosporine.

BACKGROUND: SDZ RAD is a new rapamycin analog with potent immunosuppressive activity. Compounds of the rapamycin class differ in their mode of action from cyclosporine, thus providing a rationale for potential synergism of these two potent immunosuppressants. METHODS: The two-way mouse mixed lymphocyte reaction (BALB/c-CBA strain combination) was applied. Orthotopic kidney and heterotopic heart allografting was performed in the stringent DA-to-Lewis rat strain combination, with administration of compounds orally as microemulsion preconcentrate (i.e., Neoral in the case of cyclosporine). RESULTS: Isobologram analysis of checkerboard titrations of SDZ RAD and cyclosporine in two-way mouse mixed lymphocyte reactions indicates a synergistic interaction in vitro. In vivo, the minimal effective dose of microemulsion cyclosporine giving long-term graft survival was 5.0 mg/kg/day; for SDZ RAD, the minimal effective dose was 5.0 mg/kg/day in kidney transplantation and >5.0 mg/kg/day in heart transplantation. Long-term allograft survival was noted for combinations of microemulsion cyclosporine administered at 1.0 or 2.0 mg/kg/day and SDZ RAD given at between 0.5 and 2.0 mg/kg/day. The index of synergy in different combinations ranged between 0.3 and 0.7. CONCLUSIONS: SDZ RAD and cyclosporine show synergism in immunosuppression, both in vitro and in vitro. They form a promising synergistic drug combination in allotransplantation.