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1.
Hum Immunol ; 84(1): 18-26, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35817653

RESUMO

BACKGROUND: Antibody-mediated rejection is a major cause of graft injury and contributes to failure of pig xenografts in nonhuman primates (NHPs). Most 'natural' or elicited antibodies found in humans and NHPs are directed against pig glycan antigens, but antibodies binding to swine leukocyte antigens (SLA) have also been detected. Of clinical importance is (i) whether the presence of high levels of antibodies directed towards human leukocyte antigens (HLA) (i.e., high panel-reactive antibodies) would be detrimental to the outcome of a pig organ xenograft; and (ii) whether, in the event of sensitization to pig antigens, a subsequent allotransplant would be at increased risk of graft failure due to elicited anti-pig antibodies that cross-react with human HLA or other antigens. SUMMARY: A literature review of pig-to-primate studies indicates that relatively few highly-HLA-sensitized humans have antibodies that cross-react with pigs, predicting that most would not be at increased risk of rejecting an organ xenograft. Furthermore, the existing evidence indicates that sensitization to pig antigens will probably not elicit increased alloantibody titers; if so, 'bridging' with a pig organ could be carried out without increased risk of subsequent antibody-mediated allograft failure. KEY MESSAGE: These issues have important implications for the design and conduct of clinical xenotransplantation trials.


Assuntos
Antígenos Heterófilos , Isoantígenos , Animais , Humanos , Transplante Heterólogo , Primatas , Antígenos , Antígenos HLA , Isoanticorpos , Rejeição de Enxerto
2.
Br Med Bull ; 125(1): 5-14, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29228112

RESUMO

Introduction: There is a continuing worldwide shortage of organs from deceased human donors for transplantation into patients with end-stage organ failure. Genetically engineered pigs could resolve this problem, and could also provide tissues and cells for the treatment of conditions such as diabetes, Parkinson's disease and corneal blindness. Sources of data: The current literature has been reviewed. Areas of agreement: The pathobiologic barriers are now largely defined. Research progress has advanced through the increasing availability of genetically engineered pigs and novel immunosuppressive agents. Life-supporting pig kidneys and islets have functioned for months or years in nonhuman primates. Areas of controversy: The potential risk of transfer of a pig infectious microorganism to the recipient continues to be debated. Growing points: Increased attention is being paid to selection of patients for initial clinical trials. Areas timely for developing research: Most of the advances required to justify a clinical trial have now been met.


Assuntos
Xenoenxertos , Coleta de Tecidos e Órgãos/métodos , Transplante Heterólogo , Animais , Animais Geneticamente Modificados , Sobrevivência de Enxerto , Humanos , Suínos , Transplante Heterólogo/efeitos adversos , Transplante Heterólogo/métodos
3.
Am J Transplant ; 17(6): 1476-1489, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28009481

RESUMO

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg-pulsed autologous DCreg (1.4-3.6 × 106 /kg) were administered intravenously, 1 day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag-pulsed DCreg (n = 5) was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.


Assuntos
Células Dendríticas/imunologia , Sobrevivência de Enxerto/imunologia , Isoantígenos/imunologia , Falência Renal Crônica/cirurgia , Transplante de Rim , Linfócitos T/imunologia , Doadores de Tecidos , Animais , Leucócitos Mononucleares , Macaca mulatta , Masculino , Tolerância ao Transplante , Transplante Homólogo
6.
Am J Transplant ; 16(6): 1917-22, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26718119

RESUMO

We investigated the contributions of direct and indirect T cell antigen recognition pathways to the immune response to porcine antigens in naïve baboons and baboon recipients of pig xenografts. In naïve baboons, in vitro culture of peripheral blood T cells with intact pig cells (direct xenorecognition pathway) or pig cell sonicates and baboon antigen-presenting cells (indirect xenorecognition pathway) induced the activation and expansion of xenoreactive T cells producing proinflammatory cytokines, interleukin-2 and interferon-γ. Primary indirect xenoresponses were mediated by preexisting memory T cells, whose presence is not typically observed in primary alloresponses. Next, baboons were conditioned with a nonmyeloablative regimen before short-term immunosuppression and transplantation of xenogeneic peripheral blood progenitor cells and a kidney, heart, or pancreatic islets from a miniature swine. All transplants were rejected acutely within 30 days after their placement. Posttransplantation, we observed an inhibition of the direct xenoresponse but a significant expansion of indirectly activated proinflammatory T cells. These results suggest that additional treatment to suppress indirect T cell immunity in primates may be required to achieve tolerance of pig xenografts through hematopoietic chimerism.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Tolerância Imunológica/imunologia , Transplante de Órgãos , Transplante de Células-Tronco de Sangue Periférico , Linfócitos T/imunologia , Animais , Xenoenxertos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Papio , Suínos , Porco Miniatura , Condicionamento Pré-Transplante , Transplante Heterólogo
7.
Am J Transplant ; 16(7): 1999-2015, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26700196

RESUMO

The ability of regulatory T cells (Treg) to prolong allograft survival and promote transplant tolerance in lymphodepleted rodents is well established. Few studies, however, have addressed the therapeutic potential of adoptively transferred, CD4(+) CD25(+) CD127(-) Foxp3(+) (Treg) in clinically relevant large animal models. We infused ex vivo-expanded, functionally stable, nonselected Treg (up to a maximum cumulative dose of 1.87 billion cells) into antithymocyte globulin-lymphodepleted, MHC-mismatched cynomolgus monkey heart graft recipients before homeostatic recovery of effector T cells. The monkeys also received tacrolimus, anti-interleukin-6 receptor monoclonal antibodies and tapered rapamycin maintenance therapy. Treg administration in single or multiple doses during the early postsurgical period (up to 1 month posttransplantation), when host T cells were profoundly depleted, resulted in inferior graft function compared with controls. This was accompanied by increased incidences of effector memory T cells, enhanced interferon-γ production by host CD8(+) T cells, elevated levels of proinflammatory cytokines, and antidonor alloantibodies. The findings caution against infusion of Treg during the early posttransplantation period after lymphodepletion. Despite marked but transient increases in Treg relative to endogenous effector T cells and use of reputed "Treg-friendly" agents, the host environment/immune effector mechanisms instigated under these conditions can perturb rather than favor the potential therapeutic efficacy of adoptively transferred Treg.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/imunologia , Transplante de Coração , Memória Imunológica/imunologia , Isoanticorpos/imunologia , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante/imunologia , Transferência Adotiva , Aloenxertos , Animais , Sobrevivência de Enxerto , Depleção Linfocítica , Macaca fascicularis
8.
Am J Transplant ; 15(5): 1253-66, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25783759

RESUMO

Ex vivo-expanded cynomolgus monkey CD4(+)CD25(+)CD127(-) regulatory T cells (Treg) maintained Foxp3 demethylation status at the Treg-specific demethylation region, and potently suppressed T cell proliferation through three rounds of expansion. When carboxyfluorescein succinimidyl ester- or violet proliferation dye 450-labeled autologous (auto) and nonautologous (non-auto)-expanded Treg were infused into monkeys, the number of labeled auto-Treg in peripheral blood declined rapidly during the first week, but persisted at low levels in both normal and anti-thymocyte globulin plus rapamycin-treated (immunosuppressed; IS) animals for at least 3 weeks. By contrast, MHC-mismatched non-auto-Treg could not be detected in normal monkey blood or in blood of two out of the three IS monkeys by day 6 postinfusion. They were also more difficult to detect than auto-Treg in peripheral lymphoid tissue. Both auto- and non-auto-Treg maintained Ki67 expression early after infusion. Sequential monitoring revealed that adoptively transferred auto-Treg maintained similarly high levels of Foxp3 and CD25 and low CD127 compared with endogenous Treg, although Foxp3 staining diminished over time in these nontransplanted recipients. Thus, infused ex vivo-expanded auto-Treg persist longer than MHC-mismatched non-auto-Treg in blood of nonhuman primates and can be detected in secondary lymphoid tissue. Host lymphodepletion and rapamycin administration did not consistently prolong the persistence of non-auto-Treg in these sites.


Assuntos
Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Soro Antilinfocitário/química , Fatores de Transcrição Forkhead/metabolismo , Haplótipos , Imunossupressores/química , Antígeno Ki-67/metabolismo , Macaca fascicularis , Complexo Principal de Histocompatibilidade , Masculino , Metilação , Fenótipo , Sirolimo/química
9.
Transpl Immunol ; 32(2): 99-108, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25687023

RESUMO

BACKGROUND: In the pig-to-nonimmunosuppressed baboon artery patch model, a graft from an α1,3-galactosyltransferase gene-knockout pig transgenic for human CD46 (GTKO/CD46) induces a significant adaptive immune response (elicited anti-pig antibody response, increase in T cell proliferation on MLR, cellular infiltration of the graft), which is effectively prevented by anti-CD154mAb-based therapy. METHODS: As anti-CD154mAb is currently not clinically applicable, we evaluated whether it could be replaced by CD28/B7 pathway blockade or by blockade of both pathways (using belatacept + anti-CD40mAb [2C10R4]). We further investigated whether a patch from a GTKO/CD46 pig with a mutant human MHC class II transactivator (CIITA-DN) gene would allow reduction in the immunosuppressive therapy administered. RESULTS: When grafts from GTKO/CD46 pigs were transplanted with blockade of both pathways, a minimal or insignificant adaptive response was documented. When a GTKO/CD46/CIITA-DN graft was transplanted, but no immunosuppressive therapy was administered, a marked adaptive response was documented. In the presence of CD28/B7 pathway blockade (abatacept or belatacept), there was a weak adaptive response that was diminished when compared with that to a GTKO/CD46 graft. Blockade of both pathways prevented an adaptive response. CONCLUSION: Although expression of the mutant MHC CIITA-DN gene was associated with a reduced adaptive immune response when immunosuppressive therapy was inadequate, when blockade of both the CD40/CD154 and CD28/B7 pathways was present, the response even to a GTKO/CD46 graft was suppressed. This was confirmed after GTKO/CD46 heart transplantation in baboons.


Assuntos
Artérias/transplante , Sobrevivência de Enxerto , Proteínas Nucleares , Transplante de Órgãos , Transativadores , Tolerância ao Transplante/genética , Animais , Animais Geneticamente Modificados , Sobrevivência de Enxerto/genética , Sobrevivência de Enxerto/imunologia , Xenoenxertos , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Papio , Suínos , Transativadores/genética , Transativadores/imunologia
10.
Am J Transplant ; 14(10): 2275-87, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25220221

RESUMO

The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti-CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only two of five demonstrating function beyond 5 months.


Assuntos
Transplante das Ilhotas Pancreáticas , Transplante Heterólogo , Animais , Animais Geneticamente Modificados , Glicemia/análise , Antígeno CTLA-4/imunologia , Feminino , Glucose/administração & dosagem , Imunossupressores/administração & dosagem , Fígado/patologia , Macaca fascicularis , Proteína Cofatora de Membrana/imunologia , Pâncreas/patologia , Suínos
11.
Transpl Immunol ; 29(1-4): 88-98, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24120957

RESUMO

Repopulation of memory T cells (Tmem) in allograft recipients after lymphodepletion is a major barrier to transplant tolerance induction. Ineffective depletion of naïve T cells (Tn) and Tmem may predispose to repopulation of Tmem after transplantation. Cynomolgus macaque monkeys given heart allografts were lymphodepleted using Alemtuzumab (Campath-1H; anti-CD52). Peripheral blood (PB) and lymph nodes (LN) were analyzed for CD95(-) (Tn) and CD95(+) cells (Tmem), one day, one month and up to three months after Alemtuzumab infusion. CD52 expression, susceptibility to Alemtuzumab cytotoxicity and pro-apoptotic caspase-3 were evaluated in Tn and Tmem. In vivo, Alemtuzumab induction profoundly depleted lymphocytes in PB (99% reduction) but exerted a lesser effect in LN (70% reduction), with similar depletion of Tn and Tmem subsets. After transplantation, Tmem comprised the majority of lymphocytes in PB and LN. In vitro, LN T cells were more resistant to Alemtuzumab-mediated cytotoxicity than PB lymphocytes. CD4(+) Tn and Tmem were equally susceptible to Alemtuzumab-mediated cytotoxicity, whereas CD8(+) Tn were more resistant than CD8(+) Tmem. However, no significant differences in CD52 expression between lymphocyte subsets in PB and LN were observed. Caspase-3 expression was higher in PB than LN T cells. CD4(+) and CD8(+) Tn expressed lower levels of Caspase-3 than Tmem, in both PB and LN. Thus, after Alemtuzumab infusion, residual Tn in secondary lymphoid tissue may predispose to rapid recovery of Tmem in allograft recipients.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Transplante de Coração , Depleção Linfocítica , Tecido Linfoide/imunologia , Memória de Curto Prazo/efeitos dos fármacos , Alemtuzumab , Aloenxertos , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Caspase 3/imunologia , Tecido Linfoide/patologia , Macaca fascicularis , Receptor fas/imunologia
12.
Am J Transplant ; 13(8): 1989-2005, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23758811

RESUMO

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5-10 × 10(6) /kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day -2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p < 0.05) in DCreg-treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95(+) T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.


Assuntos
Células Dendríticas/transplante , Sobrevivência de Enxerto/imunologia , Tolerância Imunológica/imunologia , Memória Imunológica/imunologia , Nefropatias/prevenção & controle , Transplante de Rim/imunologia , Linfócitos T Reguladores/imunologia , Abatacepte , Animais , Terapia Combinada , Células Dendríticas/citologia , Células Dendríticas/imunologia , Imunoconjugados/imunologia , Imunossupressores/uso terapêutico , Nefropatias/imunologia , Macaca mulatta , Masculino , Sirolimo/uso terapêutico , Transplante Homólogo
13.
Am J Transplant ; 13(8): 2169-78, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23635093

RESUMO

Alemtuzumab (Campath-1H) is a humanized monoclonal antibody (Ab) directed against CD52 that depletes lymphocytes and other leukocytes, mainly by complement-dependent mechanisms. We investigated the influence of alemtuzumab (i) on ex vivo-expanded cynomolgus monkey regulatory T cells (Treg) generated for prospective use in adoptive cell therapy and (ii) on naturally occurring Treg following alemtuzumab infusion. Treg were isolated from PBMC and lymph nodes and expanded for two rounds. CD52 expression, binding of alemtuzumab and both complement-mediated killing and Ab-dependent cell-mediated cytotoxicity (ADCC) were compared between freshly isolated and expanded Treg and effector T cells. Monkeys undergoing allogeneic heart transplantation given alemtuzumab were monitored for Treg and serum alemtuzumab activity. Ex vivo-expanded Treg showed progressive downregulation of CD52 expression, absence of alemtuzumab binding, minimal change in complement inhibitory protein (CD46) expression and no complement-dependent killing or ADCC. Infusion of alemtuzumab caused potent depletion of all lymphocytes, but a transient increase in the incidence of circulating Treg. After infusion of alemtuzumab, monkey serum killed fresh PBMC, but not expanded Treg. Thus, expanded cynomolgus monkey Treg are resistant to alemtuzumab-mediated, complement-dependent cytotoxicity. Furthermore, our data suggest that these expanded monkey Treg can be infused into graft recipients given alemtuzumab without risk of complement-mediated killing.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Eritrócitos/efeitos dos fármacos , Glicoproteínas/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Alemtuzumab , Animais , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Antineoplásicos/sangue , Antineoplásicos/sangue , Antígeno CD52 , Eritrócitos/metabolismo , Leucócitos Mononucleares/metabolismo , Macaca fascicularis , Linfócitos T Reguladores/metabolismo
14.
Am J Transplant ; 10(7): 1556-68, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20642682

RESUMO

Consumptive coagulopathy (CC) remains a challenge in pig-to-primate organ xenotransplantation (Tx). This study investigated the role of tissue factor (TF) expression on circulating platelets and peripheral blood mononuclear cells (PBMCs). Baboons (n = 9) received a kidney graft from pigs that were either wild-type (n = 2), alpha1,3-galactosyltransferase gene-knockout (GT-KO; n = 1) or GT-KO and transgenic for the complement-regulatory protein, CD46 (GT-KO/CD46, n = 6). In the baboon where the graft developed hyperacute rejection (n = 1), the platelets and PBMCs expressed TF within 4 h of Tx. In the remaining baboons, TF was detected on platelets on post-Tx day 1. Subsequently, platelet-leukocyte aggregation developed with formation of thrombin. In the six baboons with CC, TF was not detected on baboon PBMCs until CC was beginning to develop. Graft histopathology showed fibrin deposition and platelet aggregation (n = 6), but with only minor or no features indicating a humoral immune response (n = 3), and no macrophage, B or T cell infiltration (n = 6). Activation of platelets to express TF was associated with the initiation of CC, whereas TF expression on PBMCs was concomitant with the onset of CC, often in the relative absence of features of acute humoral xenograft rejection. Prevention of recipient platelet activation may be crucial for successful pig-to-primate kidney Tx.


Assuntos
Coagulação Intravascular Disseminada/patologia , Transplante de Rim/efeitos adversos , Tromboplastina/genética , Transplante Heterólogo/efeitos adversos , Animais , Anticorpos Monoclonais/uso terapêutico , Soro Antilinfocitário/uso terapêutico , Ligante de CD40/imunologia , Famotidina/uso terapêutico , Galactosiltransferases/genética , Técnicas de Inativação de Genes , Rejeição de Enxerto/patologia , Antagonistas dos Receptores H2 da Histamina/uso terapêutico , Imunossupressores/uso terapêutico , Papio/imunologia , Ativação Plaquetária , Agregação Plaquetária , Suínos , Trombina/biossíntese
15.
J Thromb Haemost ; 8(9): 2001-10, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20553382

RESUMO

BACKGROUND: Intravascular thrombosis remains a barrier to successful xenotransplantation. Tissue factor (TF) expression on porcine aortic endothelial cells (PAECs), which results from their activation by xenoreactive antibodies (Abs) to Galα1,3Gal (Gal) and subsequent complement activation, plays an important role. OBJECTIVES: The present study aimed to clarify the role of Abs directed against nonGal antigens in the activation of PAECs to express functional TF and to investigate selected methods of inhibiting TF activity. METHODS: PAECs from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GT-KO) pigs, or pigs transgenic for CD46 or tissue factor pathway inhibitor (TFPI), were incubated with naïve baboon serum (BS) or sensitized BS (with high anti-nonGal Ab levels). TF activity of PAECs was assessed. RESULTS: Only fresh, but not heat-inactivated (HI), naïve BS activated WT PAECs to express functional TF. Similarly, PAECs from CD46 pigs were resistant to activation by naïve BS, but not to activation by fresh or HI sensitized BS. HI sensitized BS also activated GT-KO PAECs to induce TF activity. TF expression on PAECs induced by anti-nonGal Abs was inhibited if serum was pretreated with (i) an anti-IgG Fab Ab or (ii) atorvastatin, or (iii) when PAECs were transgenic for TFPI. CONCLUSIONS: Anti-nonGal IgG Abs activated PAECs to induce TF activity through a complement-independent pathway. This implies that GT-KO pigs expressing a complement-regulatory protein may be insufficient to prevent the activation of PAECs. Genetic modification with an 'anticoagulant' gene (e.g. TFPI) or a therapeutic approach (e.g. atorvastatin) will be required to prevent coagulation dysregulation after pig-to-primate organ transplantation.


Assuntos
Anticorpos Anti-Idiotípicos/química , Aorta/patologia , Coagulação Sanguínea/efeitos dos fármacos , Endotélio Vascular/citologia , Ácidos Heptanoicos/farmacologia , Lipoproteínas/genética , Lipoproteínas/metabolismo , Pirróis/farmacologia , Animais , Animais Geneticamente Modificados , Anticolesterolemiantes/farmacologia , Anticoagulantes , Atorvastatina , Galactosiltransferases/genética , Proteína Cofatora de Membrana/biossíntese , Papio , Suínos , Transgenes
16.
Am J Transplant ; 10(4): 773-783, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20420638

RESUMO

As the target CD52 molecule is expressed on erythrocytes of most nonhuman primate strains, using alemtuzumab in these species would cause massive hemolysis. Six cynomolgus monkeys of Indonesian origin, screened by agglutination assay for absence of CD52 on erythrocytes, were administered alemtuzumab in a cumulative dose to a maximum of 60 mg/kg. In two monkeys, mycophenolate mofetil (MMF) was added as maintenance therapy. Complete depletion of T and B lymphocytes (>99.5%) was achieved with 20 mg/kg alemtuzumab and was more profound than in monkeys treated with antithymocyte globulin (n = 5), as quantified by flow cytometry. Repopulation was suppressed by weekly injections of 10 mg/kg. Without MMF, repopulation of CD20(+)B cells and CD8(+)T cells was complete within 2 and 3 months, respectively, and repopulation of CD4(+)T cells was 67% after 1 year. MMF significantly delayed CD4(+)T-cell repopulation. Among repopulating CD4(+) and CD8(+) T cells, a phenotypic shift was observed from CD45RA(hi)CD62L(hi) naïve cells toward CD45RA(lo)CD62L(lo) effector memory cells. In lymph nodes, the depletion of naïve cells was more profound than of memory cells, which may have initiated a proliferation of memory cells. This model offers opportunities to investigate lymphocyte depletion/repopulation phenomena, as well as the efficacy of alemtuzumab in preclinical transplantation models.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Depleção Linfocítica , Linfócitos/citologia , Alemtuzumab , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Anticorpos Antineoplásicos/farmacologia , Antígenos CD/imunologia , Citometria de Fluxo , Imunofenotipagem , Linfócitos/imunologia , Macaca fascicularis
17.
Am J Transplant ; 10(2): 273-85, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20041862

RESUMO

A lack of deceased human donor livers leads to a significant mortality in patients with acute-on-chronic or acute (fulminant) liver failure or with primary nonfunction of an allograft. Genetically engineered pigs could provide livers that might bridge the patient to allotransplantation. Orthotopic liver transplantation in baboons using livers from alpha1,3-galactosyltransferase gene-knockout (GTKO) pigs (n = 2) or from GTKO pigs transgenic for CD46 (n = 8) were carried out with a clinically acceptable immunosuppressive regimen. Six of 10 baboons survived for 4-7 days. In all cases, liver function was adequate, as evidenced by tests of detoxification, protein synthesis, complement activity and coagulation parameters. The major problem that prevented more prolonged survival beyond 7 days was a profound thrombocytopenia that developed within 1 h after reperfusion, ultimately resulting in spontaneous hemorrhage at various sites. We postulate that this is associated with the expression of tissue factor on platelets after contact with pig endothelium, resulting in platelet and platelet-peripheral blood mononuclear cell(s) aggregation and deposition of aggregates in the liver graft, though we were unable to confirm this conclusively. If this problem can be resolved, we would anticipate that a pig liver could provide a period during which a patient in liver failure could be successfully bridged to allotransplantation.


Assuntos
Transplante de Fígado/imunologia , Animais , Animais Geneticamente Modificados , Coagulação Sanguínea/imunologia , Feminino , Galactosiltransferases/imunologia , Humanos , Imunossupressores/imunologia , Fígado/imunologia , Falência Hepática/imunologia , Masculino , Papio , Sus scrofa , Trombocitopenia/imunologia
18.
Am J Transplant ; 9(12): 2716-26, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19845582

RESUMO

Xenotransplantation of porcine islets into diabetic non-human primates is characterized by (i) an initial massive graft loss possibly due to the instant blood-mediated inflammatory reaction and (ii) the requirement of intensive, clinically unfriendly immunosuppressive therapy. We investigated whether the transgenic expression of a human complement-regulatory protein (hCD46) on porcine islets would improve the outcome of islet xenotransplantation in streptozotocin-induced diabetic Cynomolgus monkeys. Immunosuppression consisted of thymoglobulin, anti-CD154 mAb for costimulation blockade, and mycophenolate mofetil. Following the transplantation of islets from wild-type pigs (n = 2) or from 1,3-galactosyltransferase gene-knockout pigs (n = 2), islets survived for a maximum of only 46 days, as evidenced by return to hyperglycemia and the need for exogenous insulin therapy. The transplantation of islets from hCD46 pigs resulted in graft survival and insulin-independent normoglycemia in four of five monkeys for the 3 months follow-up of the experiment. One normalized recipient, selected at random, was followed for >12 months. Inhibition of complement activation by the expression of hCD46 on the pig islets did not substantially reduce the initial loss of islet mass, rather was effective in limiting antibody-mediated rejection. This resulted in a reduced need for immunosuppression to preserve a sufficient islet mass to maintain normoglycemia long-term.


Assuntos
Glicemia/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Transplante das Ilhotas Pancreáticas/métodos , Proteína Cofatora de Membrana/genética , Transplante Heterólogo , Animais , Animais Geneticamente Modificados , Diabetes Mellitus Experimental/cirurgia , Feminino , Macaca fascicularis , Masculino , Suínos
19.
Am J Transplant ; 9(11): 2485-96, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19775318

RESUMO

The results of transplantation of human donor islets into the portal vein (PV) in patients with diabetes are encouraging. However, there are complications, for example, hemorrhage, thrombosis and an immediate loss of islets through the 'instant blood-mediated inflammatory reaction' (IBMIR). The gastric submucosal space (GSMS) offers potential advantages. Islets were isolated from adult pigs. Recipient pigs were made diabetic by streptozotocin. Donor islets were injected into the GSMS through a laparotomy (Group 1A, n = 4) or endoscopically (Group 1B, n = 8) or into the PV through a laparotomy (Group 2, n = 3). The pigs were followed for a maximum of 28 days. Monitoring of C-peptide in Group 1 indicated that there was minimal immediate loss of islets whereas in Group 2 there was considerable loss from IBMIR. In Group 1, there were significant reductions in mean blood glucose and mean exogenous insulin requirement between pretransplantation and 20 days posttransplantation. In Group 2, there was no significant reduction in either parameter. Insulin-positive cells were seen in the GSMS in Group 1, but not in the liver in Group 2. Endoscopic gastric submucosal transplantation of islets (ENDO-STI) offers a minimally invasive and quick approach to islet transplantation, avoids IBMIR and warrants further exploration.


Assuntos
Diabetes Mellitus Experimental/cirurgia , Endoscopia/métodos , Mucosa Gástrica/cirurgia , Transplante das Ilhotas Pancreáticas/métodos , Animais , Glicemia/metabolismo , Peptídeo C/sangue , Terapia Combinada , Diabetes Mellitus Experimental/tratamento farmacológico , Feminino , Rejeição de Enxerto/tratamento farmacológico , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Hipoglicemiantes/farmacologia , Imunossupressores/farmacologia , Insulina/farmacologia , Transplante das Ilhotas Pancreáticas/imunologia , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacologia , Pancreatectomia , Sus scrofa , Tacrolimo/farmacologia , Transplante Homólogo
20.
Am J Transplant ; 9(5): 1006-16, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19422330

RESUMO

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.


Assuntos
Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Transplante de Rim/imunologia , Transplante Heterólogo/imunologia , Sistema ABO de Grupos Sanguíneos/imunologia , Doença Aguda , Animais , DNA Complementar/genética , Rejeição de Enxerto/mortalidade , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/imunologia , Transplante de Coração/mortalidade , Transplante de Rim/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Papio , Proteínas/genética , Suínos/genética , Timo/transplante , Condicionamento Pré-Transplante/métodos
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