RESUMEN
We have shown previously that cytokines IL-4 and IL-13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein-1 (SREBP-1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL-4-treated EC had a profound reduction in complement-mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP-1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL-4 stimulation, without increase in cholesterol content or cell proliferation. IL-4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL-4 induced activation of Akt/SREBP-1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.
Asunto(s)
Proteínas del Sistema Complemento/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Interleucina-4/farmacología , Lípidos/biosíntesis , Meliteno/efectos adversos , Transducción de Señal/efectos de los fármacos , Animales , Western Blotting , Permeabilidad de la Membrana Celular , Separación Celular , Complejo de Ataque a Membrana del Sistema Complemento/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Citometría de Flujo , Interleucina-4/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Electrónica de Transmisión , Mitocondrias/patología , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , PorcinosRESUMEN
BACKGROUND: Human type 1 diabetes is associated with defects in the hematopoietic stem cells. Simultaneous donor islet and bone marrow transplantation may be an ideal therapeutic approach for inducing tolerance to islet allogeneic antigens and restoring self-tolerance to islet autoimmune antigens. METHODS: Using a nonobese diabetic (NOD) mouse model of human type 1 diabetes, we investigated whether tolerance to MHC-matched allogeneic islet grafts from male nonobese diabetes-resistant (NOR) donors can be induced in female NOD recipients by simultaneous islet and bone marrow transplantation under fludarabine phosphate-based nonmyeloablative and irradiation-free conditioning therapy. Donor-specific chimerism in the peripheral blood of tolerant mice (n=7) was measured by semiquantitative polymerase chain reaction for a male-specific marker (SRY). RESULTS: Donor-specific tolerance to NOR islet grafts was induced in all diabetic NOD mice after simultaneous islet and bone marrow transplantation and treated with fludarabine phosphate, cyclophosphamide, anti-mouse lymphocyte serum, and rapamycin. At 100 days and 200 days after transplantation, the average percentage of male NOR marker in DNA derived from the peripheral blood of NOD recipients that had long-term islet graft survival was over 10%. CONCLUSION: Our data suggest that this approach may induce donor-specific tolerance in clinical islet transplantation and living-related donor solid organ transplantation.
Asunto(s)
Trasplante de Médula Ósea , Diabetes Mellitus Tipo 1/cirugía , Tolerancia Inmunológica/inmunología , Trasplante de Islotes Pancreáticos , Acondicionamiento Pretrasplante , Animales , Ciclofosfamida/farmacología , Diabetes Mellitus Tipo 1/terapia , Femenino , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Inmunosupresores/farmacología , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Quimera por Trasplante , Trasplante Homólogo , Fosfato de Vidarabina/análogos & derivados , Fosfato de Vidarabina/farmacologíaRESUMEN
BACKGROUND: In a pig model of intestinal transplantation, we previously showed that hepatic conditioning through portal donor-specific blood transfusion (pDSBT), high-dose tacrolimus (TAC), and steroids prevented rejection and increased survival Our current study tests a protocol of pDSBT, short-term mycophenolate mofetil (MMF), and low-dose TAC to eliminate the use of steroids, reduce TAC dosage, and increase the level of chimerism in the peripheral blood. MATERIALS AND METHODS: Four groups of outbred, mixed lymphocyte culture (MLC)-reactive pigs underwent bowel transplants and pDSBT. Immunosuppression (group 1, high-dose TAC and steroids; group 2, low-dose TAC and MMF; group 3, low-dose TAC, MMF, and aminoguanidine; group 4, low-dose TAC, MMF, and arginine) was discontinued after 28 days. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements. Chimerism levels were determined using a Q-PCR analysis. RESULTS: Pig survival and death rates due to rejection did not significantly differ between the four groups. Chimerism levels determined by Q-PCR analysis were not different until day 28. After discontinuation of immunosuppression, we noted a trend (P = 0.15) toward higher mean chimerism levels on day 60 for groups 2, 3, and 4 (9%) vs. group 1 (0.5%). Tissue cytokine and serum nitrate levels did not significantly differ between the four groups. Attempts to modify nitric oxide synthase activity offered no added benefit. CONCLUSIONS: The combination of pDSBT, MMF, and low-dose TAC (vs. high-dose TAC and steroids) allowed sustained levels of mixed chimerism to develop after discontinuation of immunosuppression.
Asunto(s)
Transfusión Sanguínea , Inmunosupresores/administración & dosificación , Intestinos/trasplante , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/administración & dosificación , Sistema Porta , Tacrolimus/administración & dosificación , Donantes de Tejidos , Quimera por Trasplante , Animales , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Quimioterapia Combinada , Rechazo de Injerto/mortalidad , Enfermedad Injerto contra Huésped/mortalidad , Infecciones/mortalidad , Masculino , Óxido Nítrico/metabolismo , Esteroides/administración & dosificación , Análisis de Supervivencia , PorcinosRESUMEN
Recently we showed that donor-specific tolerance to MHC-matched islet allografts in diabetic NOD mice could be induced by simultaneous islet and bone marrow transplantation. Mononuclear cell infiltration surrounding the islets was also found in tolerated grafts. In this study, we compared gene expression in the tolerated and rejected islet grafts by using Affymetrix Murine U74A oligonucleotide arrays. To confirm the results of microarray analysis, we performed real-time PCR and RNase protection assay on selected genes. Of over 12,000 genes studied, 57 genes were expressed at consistently higher levels in tolerated islet grafts, and 524 genes in rejected islet grafts. Genes from a variety of functional clusters were found to be different between rejected and tolerated grafts. In the rejected islet grafts, a number of T-cell surface markers and of cytotoxicity-related genes were highly expressed. Also in the rejected grafts, a number of cytokines and chemokines and their receptors were highly expressed. The differential expression of selected genes found by microarray analysis was also confirmed by real-time PCR and RNase protection assay. Our results indicated that gene microarray analysis can help us to detect gene expression differences representative of the biologic mechanisms of tolerance and rejection.
Asunto(s)
Perfilación de la Expresión Génica , Rechazo de Injerto/genética , Supervivencia de Injerto/genética , Trasplante de Islotes Pancreáticos , Animales , Antígenos de Diferenciación/genética , Moléculas de Adhesión Celular/genética , Quimiocinas/genética , Citocinas/genética , Femenino , Expresión Génica/inmunología , Inmunidad Innata/genética , Masculino , Ratones , Ratones Endogámicos NOD , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptores de Quimiocina/genética , Receptores de Citocinas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Mactinin, a 31 kDa fragment from the amino-terminal end of alpha-actinin, is chemotactic for monocytes and can promote monocyte/macrophage maturation. Macrophages are essential for wound healing, in which they play key roles in debridement, angiogenesis, fibroblast proliferation, and collagen metabolism. We have previously determined that urokinase is necessary to form mactinin from extracellular alpha-actinin, which may be present at sites of inflammation as a result of cell movement. Thus, urokinase knockout mice are unable to form mactinin and therefore are an ideal model to study mactinin's effects on wound healing. Saline- and mactinin-treated wounds were analyzed in a subcutaneous sponge wound model in both wild-type and urokinase knockout mice. The wounded urokinase knockout mice had markedly decreased leukocyte infiltration compared with wounded wild-type mice. In addition, production of the proinflammatory cytokine, interleukin-12, and of collagen was also decreased in knockouts. Treatment of knockout mice with mactinin resulted in leukocyte infiltration numbers, interleukin-12 levels, and hydroxyproline measurements similar to those in wild-type mice. The results suggest that impaired wound healing in urokinase-deficient mice can be restored by administration of mactinin.
Asunto(s)
Actinina/farmacología , Inflamación/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Animales , Western Blotting , Colágeno/efectos de los fármacos , Hidroxiprolina/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ratones , Ratones Noqueados , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Vascular endothelial cells (ECs) can be injured in a variety of pathologic processes that involve activated complement. We reported previously that porcine ECs incubated with exogenous IL-4 or IL-13 are protected from cytotoxicity by human complement and also from apoptosis by TNF-alpha. The resistance to complement consists of an intrinsic mechanism that is lost a few days after cytokine removal. In our current study, we investigated whether transfer of the IL-4 gene into porcine ECs in vitro and into porcine vascular tissues in vivo would induce efficient and durable protection from human complement. We found that ECs transduced with adenoIL-4 or adenoIL-13 exhibited continuous production of the cytokine and prolonged protection from complement-mediated killing. IL-4 also protected ECs from activation: ECs incubated with IL-4 did not develop cell retraction and intercellular gaps upon stimulation with sublytic complement. The endothelium and subendothelium of pig iliac arteries that were transduced with the IL-4 gene were effectively protected from complement-dependent immediate injury after perfusion with human blood. However, after similar perfusion, the endothelium was immediately lost from arteries that were transduced with a control adenovirus. The protection was not due to up-regulation of the complement regulators decay accelerating factor, membrane cofactor protein, and CD59, or to reduced complement activation, but required the participation of Akt. Although our studies model protection in pig-to-primate xenotransplantation, our findings of IL-4 induction of Akt-mediated protection may be more broadly applicable to EC injury as manifested in ischemia-reperfusion, allotransplantation, and various vascular diseases.
Asunto(s)
Proteínas del Sistema Complemento/toxicidad , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Arteria Ilíaca/inmunología , Arteria Ilíaca/metabolismo , Interleucina-4/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción Genética , Adenoviridae/genética , Animales , Sangre/inmunología , Células Cultivadas , Proteínas del Sistema Complemento/metabolismo , Citotoxicidad Inmunológica/genética , Endotelio Vascular/citología , Líquido Extracelular/inmunología , Líquido Extracelular/metabolismo , Técnicas de Transferencia de Gen , Humanos , Arteria Ilíaca/citología , Inmunidad Innata/genética , Interleucina-13/biosíntesis , Interleucina-13/genética , Interleucina-4/biosíntesis , Interleucina-4/fisiología , Perfusión , Proteínas Proto-Oncogénicas c-akt/fisiología , PorcinosRESUMEN
A quantitative analysis of zone-specific proliferation was done to determine the recovery of adrenal cortical zonation during regeneration after enucleation. Adult male rats underwent adrenal enucleation [unilateral enucleation (ULE)] or sham surgery, both accompanied by contralateral adrenalectomy. At 2, 5, 10, and 28 days, blood and adrenals were collected to assess functional recovery. Adrenal sections were immunostained for Ki67 (proliferation), cytochrome P-450 aldosterone synthase (P-450aldo, glomerulosa), and cytochrome P-450 11beta-hydroxylase (P-45011beta, fasciculata). Unbiased stereology was used to count proliferating glomerulosa and fasciculata cells. Recovery of fasciculata secretory function occurred by 28 days as reflected by plasma ACTH and corticosterone, whereas glomerulosa function reflected by plasma aldosterone remained low at 28 days. At 5 days, ULE adrenals showed increased Ki67+ cells in the glomerulosa and inner fasciculata, whereas at 10 and 28 days increased proliferation was restricted to the outer fasciculata. These data show that enucleation results in transient elevations in glomerulosa and inner fasciculata cell proliferation followed by a delayed increase in the outer fasciculata. To assess adrenal growth in enucleated adrenals previously suppressed by the presence of an intact adrenal, rats underwent ULE and sham surgery; after 4 wk, the intact adrenal was removed and enucleated adrenals were collected at 2, 5, and 10 days. Overall, proliferation was delayed in this model, but at 5 days, Ki67+ cells increased in the outer fasciculata, whereas by 10 days, increased proliferation occurred in the outer and inner fasciculata. The key novel finding of increased proliferation in the inner fasciculata suggests that the delayed growth of the enucleated adrenal results in part from a regenerative response.
Asunto(s)
Corteza Suprarrenal/fisiología , Regeneración/fisiología , Zona Fascicular/fisiología , Zona Glomerular/fisiología , Corteza Suprarrenal/citología , Corteza Suprarrenal/enzimología , Corteza Suprarrenal/cirugía , Hormona Adrenocorticotrópica/sangre , Aldosterona/sangre , Animales , Procesos de Crecimiento Celular/fisiología , Corticosterona/sangre , Citocromo P-450 CYP11B2/metabolismo , Técnica del Anticuerpo Fluorescente , Histocitoquímica , Antígeno Ki-67/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Esteroide 11-beta-Hidroxilasa , Zona Fascicular/enzimología , Zona Glomerular/enzimologíaRESUMEN
Opioids are sometimes used to treat pain in ulcerative wounds, and it is speculated that pain interferes with the healing process. Because the direct effect of opioids on this process remains unknown, we examined the effect of topically applied opioids on the healing of open ischemic wounds in rats. Topically applied opioids hastened wound closure, particularly in the first 4 days when no healing was initiated in phosphate buffered saline solution-treated wounds. After 1 week of application, fentanyl, hydromorphone, and morphine resulted in 66%, 55%, and 42% wound closure, respectively, as compared to only 15% in control wounds. Opioid-induced healing was accompanied by a 1.5- to 2.5-fold increase in nuclear density in the granulation tissue and 45-87% increase in angiogenesis as compared to phosphate buffered saline solution-treated wounds. Fentanyl showed significantly improved healing compared to morphine and hydromorphone (p < 0.05, fentanyl vs. others). Fentanyl-induced healing was inhibited by the opioid receptor antagonist naloxone, suggesting that peripheral opioid receptor(s) mediate the healing process. Opioids accelerate healing by up-regulating both endothelial and inducible nitric oxide synthase and the vascular endothelial-derived growth factor receptor Flk1 in the wounds. We envision that opioids can be used topically to accelerate wound healing in diverse clinical conditions ranging from surgical incisions to nonhealing ischemic ulcers in pathophysiological conditions and in hospice patients.
Asunto(s)
Analgésicos Opioides/administración & dosificación , Fentanilo/administración & dosificación , Isquemia/complicaciones , Cicatrización de Heridas/efectos de los fármacos , Heridas y Lesiones/tratamiento farmacológico , Administración Tópica , Analgésicos Opioides/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Fentanilo/farmacología , Tejido de Granulación/efectos de los fármacos , Hidromorfona/administración & dosificación , Hidromorfona/farmacología , Morfina/administración & dosificación , Morfina/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Ratas , Piel/efectos de los fármacos , Piel/fisiopatología , Heridas y Lesiones/complicaciones , Heridas y Lesiones/fisiopatologíaRESUMEN
Vascular endothelial cells (EC) perform critical functions that require a balance of cell survival and cell death. EC death by apoptosis and EC activation and injury by the membrane attack complex of complement are important mechanisms in atherosclerosis and organ graft rejection. Although the effects of various cytokines on EC apoptosis have been studied, little is known about their effects on complement-mediated EC injury. Therefore, we studied the abilities of various cytokines to induce protection of porcine aortic EC against apoptosis and killing by human complement, a model of pig-to-human xenotransplantation. We found that porcine EC incubated with IL-4 or IL-13, but not with IL-10 or IL-11, became protected from killing by complement and apoptosis induced by TNF-alpha plus cycloheximide. Maximal protection required 10 ng/ml IL-4 or IL-13, developed progressively from 12 to 72 h of incubation, and lasted 48-72 h after cytokine removal. Protection from complement was not associated with reduced complement activation, C9 binding, or changes in CD59 expression. Inhibition of PI3K prevented development of protection; however, inhibition of p38 MAPK or p42/44 MAPK had no effect. IL-4 and IL-13 induced rapid phosphorylation of Akt. Although protection was inhibited by an Akt inhibitor and a dominant negative Akt mutant transduced into EC, it was induced by transduction of EC with the constitutively active Akt variant, myristylated Akt. We conclude that IL-4 and IL-13 can induce protection of porcine EC against killing by apoptosis and human complement through activation of the PI3K/Akt signaling pathway.
Asunto(s)
Apoptosis/inmunología , Activación de Complemento/inmunología , Citotoxicidad Inmunológica/inmunología , Endotelio Vascular/inmunología , Interleucina-13/fisiología , Interleucina-4/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Animales , Antígenos CD59/biosíntesis , Células Cultivadas , Complemento C9/metabolismo , Cicloheximida/antagonistas & inhibidores , Cicloheximida/toxicidad , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Sistema de Señalización de MAP Quinasas/inmunología , Necrosis , Fosforilación , Unión Proteica/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Porcinos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/toxicidadRESUMEN
Cytoprotection of endothelial cells (EC) is important in EC biology and pathophysiology, including graft rejection. Using porcine aortic EC and human complement as an in vitro model of xenotransplantation, we have reported that ligation of EC Gal alpha (1-3)Gal epitopes (alpha Gal) with antibodies or lectins BS-I and IB4 induces EC resistance to injury by complement. However, before the protective response is observed, alpha Gal ligation induces an early, proinflammatory response. Using a similar model, we now investigated whether the early inflammatory response, as well as NF-kappa B activation, is required for induction of cytoprotection. Despite up-regulation of EC mRNA for many inflammatory cytokines rapidly after BS-I stimulation, recombinant cytokines or conditioned media from EC incubated with BS-I failed to induce protection when used to stimulate EC. While the lectin-induced inflammatory response was markedly reduced by inhibition of NF-kappa B, the protection from complement and apoptosis was unaffected. The lectins caused up-regulation of mRNA for protective genes A20, porcine inhibitor of apoptosis protein and hemoxygenase-1, which was not modified by NF-kappa B inhibition. These findings suggest that induction of cytoprotection in porcine EC by alpha Gal ligation results from activation of pathways that are largely independent of those that elicit NF-kappaB activation and the inflammatory response.
Asunto(s)
Apoptosis/fisiología , Proteínas del Sistema Complemento/metabolismo , Disacáridos/metabolismo , Células Endoteliales/metabolismo , FN-kappa B/metabolismo , Animales , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Inflamación/metabolismo , Proteínas Inhibidoras de la Apoptosis , Proteínas/metabolismo , Porcinos , Factores de TiempoRESUMEN
BACKGROUND: Immunomodulatory strategies such as donor-specific bone marrow or blood transfusions have been used to promote engraftment after intestinal transplants. We previously showed that delivery of donor antigen via the portal vein can effectively reduce the rate of intestinal graft rejection. The purpose of our current study was to investigate the impact of donor-specific cell augmentation (blood versus bone marrow) via the portal vein on cytokine expression in intestinal grafts versus native livers. MATERIAL AND METHODS: We performed heterotopic small intestinal transplants between male Brown-Norway (donor) and female Lewis (recipient) rats. We studied 10 groups according to the type of donor-specific cell augmentation and the use and dose of immunosuppressive therapy. For cell augmentation, donor-specific blood or bone marrow was transfused via the donor portal vein immediately before graft implantation. For immunosuppression, tacrolimus was used post-transplant at a high or low dose. Control rats received neither immunosuppression nor cell augmentation. Tissue samples for histological assessment were obtained at designated time points. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements (IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, and TNF-beta). Chimerism levels were determined using Q-PCR analysis. RESULTS: Without concurrent immunosuppression, neither portal donor-specific blood nor bone marrow transfusion reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood (versus bone marrow) transfusion. Irrespective of the type of donor-specific cell augmentation, severe rejection caused strong cytokine expression in the grafts of IL-1 alpha, IL-1 beta, IFN-gamma, and TNF-alpha; in the native livers, mainly of TNF-alpha (with IFN-gamma showing hardly any increase). In general, rejection caused stronger cytokine expression in the grafts than in the native livers. Mild rejection correlated well with strong intragraft expression of IL-6, TNF-alpha, and TNF-beta (early rejection markers); severe rejection with IL-1 alpha, IL-1 beta, IFN-gamma, and TNF-alpha (late rejection markers). In addition to cell augmentation per se, the type of cell augmentation also had an impact on cytokine expression in both grafts and native livers. Cell-augmented (versus tacrolimus-treated) rats showed hardly any differences in intragraft cytokine expression, but the expression of almost all cytokines was significantly stronger in the native livers. With immunosuppression, bone marrow infusion increased intragraft cytokine expression of IL-1 alpha, IL-1 beta, IFN-gamma, and TNF alpha, as well as liver cytokine expression of IL-1 beta, compared to blood transfusion. This finding reflected the more advanced rejection stages in the bone marrow infused group; different types of donor-specific cell augmentation had similar effects on liver cytokine expression. In the absence of myoablative therapy, chimerism levels were low, in both cell-augmented and non-cell-augmented groups. CONCLUSIONS: Rejection and donor-specific cell augmentation independently causes differences in intragraft versus native liver cytokine expression after intestinal transplants. Portal donor-specific blood transfusion, as compared with donor-specific bone marrow infusion, lowered the incidence of rejection and diminished intragraft cytokine up-regulation.
Asunto(s)
Citocinas/biosíntesis , Rechazo de Injerto/prevención & control , Intestinos/inmunología , Intestinos/trasplante , Hígado/inmunología , Inmunología del Trasplante/inmunología , Animales , Transfusión Sanguínea , Trasplante de Médula Ósea/inmunología , Quimera/inmunología , Femenino , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/etiología , Inmunosupresores/uso terapéutico , Masculino , Modelos Animales , Vena Porta , Ratas , Ratas Endogámicas Lew , Tacrolimus/uso terapéuticoRESUMEN
Recently we showed that donor-specific tolerance to MHC-matched islet allografts in diabetic NOD mice could be induced by simultaneous islet and bone marrow transplantation. Mononuclear cell infiltration surrounding the islets was also found in tolerated grafts. In this study, we compared gene expression in the tolerated and rejected islet grafts by using Affymetrix Murine U74A oligonucleotide arrays. To confirm the results of microarray analysis, we performed real-time PCR and RNase protection assay on selected genes. Of over 12,000 genes studied, 57 genes were expressed at consistently higher levels in tolerated islet grafts, and 524 genes in rejected islet grafts. Genes from a variety of functional clusters were found to be different between rejected and tolerated grafts. In the rejected islet grafts, a number of T-cell surface markers and of cytotoxicity-related genes were highly expressed. Also in the rejected grafts, a number of cytokines and chemokines and their receptors were highly expressed. The differential expression of selected genes found by microarray analysis was also confirmed by real-time PCR and RNase protection assay. Our results indicated that gene microarray analysis can help us to detect gene expression differences representative of the biologic mechanisms of tolerance and rejection.