RESUMO
BACKGROUND: Cellular therapies for immunomodulation in vascularized composite allotransplantation (VCA) have gained importance due to their potential for minimization of immunosuppression. Adipose-derived (AD) mesenchymal stem cells (MSCs) especially have shown encouraging potential. We investigated the influence of timing and frequency of AD-MSC treatment on immunologic and graft survival as well as graft vasculopathy outcomes after VCA. METHODS: Lewis rats received full-mismatched Brown Norway rat hindlimb transplants. Recipient animals were assigned to groups receiving donor-derived AD-MSCs (10 cells/animal) either on postoperative day (POD) 1, POD 4, or repeatedly on POD 4, 8, and 15, and compared to untreated controls. RESULTS: Although AD-MSC administration on POD 1 or POD 4, 8, and 15 resulted in 50% long-term graft acceptance, recipients treated on POD 4, and controls rejected before POD 50. All treated animals revealed peripheral blood chimerism (4 weeks), most pronounced after repetitive cell administration (12.92% vs 5.03% [POD 1] vs 6.31% [POD 4]; P < 0.05; all P < 0.01 vs control 1.45%). Chimerism was associated with the generation of regulatory T cells (CD4CD25FoxP3). In vitro mixed lymphocyte reactions revealed modulation of the recipient immune response after AD-MSC treatment. Graft arteries at end point revealed significant differences of arterial intimal thickness between rejecting and AD-MSC-treated animals (P < 0.01). CONCLUSIONS: Taken together, our results point to the potential for repetitive AD-MSC administration in improving outcomes after VCA. Future studies are warranted into optimization of the dosing and frequency of AD-MSC therapy, either alone or used in, combination with other cell therapies (such as hematopoietic stem cells or bone marrow-derived MSC or dendritic cells) for optimization of appropriate conditioning or maintenance regimens.
Assuntos
Tecido Adiposo/citologia , Aloenxertos Compostos/irrigação sanguínea , Aloenxertos Compostos/transplante , Sobrevivência de Enxerto , Membro Posterior/irrigação sanguínea , Membro Posterior/transplante , Imunoterapia/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/imunologia , Alotransplante de Tecidos Compostos Vascularizados/métodos , Animais , Proliferação de Células , Células Cultivadas , Aloenxertos Compostos/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Membro Posterior/imunologia , Imunoterapia/efeitos adversos , Ativação Linfocitária , Masculino , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Modelos Animais , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Linfócitos T Reguladores/imunologia , Fatores de Tempo , Quimeras de Transplante , Tolerância ao Transplante , Doenças Vasculares/imunologia , Doenças Vasculares/prevenção & controle , Alotransplante de Tecidos Compostos Vascularizados/efeitos adversosRESUMO
BACKGROUND: Induction of tolerance and minimizing the toxicity of immunosuppression are two fundamental goals in vascularized composite allotransplantation. Accumulating data indicate that triptolide is an agent that may have the capacity to suppress inflammation and immunologic rejection. METHODS: A heterotopic hindlimb allotransplantation model from Brown Norway to Lewis rats was established and treated with different doses of tacrolimus combined with or without triptolide. Mean survival time of the transplants was monitored, and histopathologic examination of the skin was performed. The level of inflammatory cytokine interleukin-1ß, interleukin-6, and tumor necrosis factor-á in peripheral blood was assayed. The percentage of T lymphocytes and its subsets was measured using flow cytometry. The level of recipient peripheral chimerism and the apoptosis of donor bone marrow cells were evaluated. The apoptotic related genes bcl-2 and Bax were detected by real-time polymerase chain reaction. RESULTS: The authors' results showed that triptolide not only reduces the dose of tacrolimus required for immunosuppression, but also decreased drug side effects in terms of weight gain and diarrhea. Triptolide had an obvious effect on proinflammatory cytokine expression and T-lymphocyte proliferation in the peripheral blood. Interestingly, triptolide could increase the mixed chimerism level of recipients, possibly by inhibiting the apoptosis of transplanted bone marrow cells by means of regulation of the apoptotic genes bcl-2 and Bax. CONCLUSIONS: Triptolide reduces the dose of tacrolimus required for immunosuppression by attenuating inflammation and by T-cell suppression. Furthermore, triptolide increases the chimerism level, which might contribute to acceptance of the allografts.
Assuntos
Quimerismo/efeitos dos fármacos , Diterpenos/farmacologia , Membro Posterior/transplante , Tolerância Imunológica/efeitos dos fármacos , Imunossupressores/farmacologia , Fenantrenos/farmacologia , Tacrolimo/administração & dosagem , Alotransplante de Tecidos Compostos Vascularizados , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Citocinas/metabolismo , Diterpenos/uso terapêutico , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Compostos de Epóxi/farmacologia , Compostos de Epóxi/uso terapêutico , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Imunossupressores/uso terapêutico , Masculino , Fenantrenos/uso terapêutico , Distribuição Aleatória , Ratos , Ratos Endogâmicos Lew , Linfócitos T/metabolismo , Tacrolimo/uso terapêuticoRESUMO
BACKGROUND: Strategies aiming at minimization or elimination of systemic immunosuppression are key immediate goals for clinical expansion of vascularized composite allotransplantation (VCA). We compared the in vitro and in vivo immunomodulatory efficacy of adipose-derived mesenchymal stem cells (AD-MSCs) and bone marrow (BM)-derived MSCs in a rat VCA model. METHODS: Both cell types were tested in vitro for suppressor function using mixed lymphocyte reactivity assays. AD-MSCs or BM-MSCs were administered intravenously (1 × 10 or 5 × 10 cells/animal) to Lewis rat recipients of mismatched Brown Norway hindlimb transplants. Short course tacrolimus (FK-506) monotherapy was withdrawn at postoperative day 21. In vivo regulatory T-cell induction, peripheral blood chimerism, and microchimerism in lymphatic organs were analyzed. RESULTS: AD-MSCs and BM-MSCs exhibited strong dose-dependent suppressor function in vitro, which was significantly more pronounced for AD cells. In vivo, all animals revealed peripheral multi-lineage chimerism at four weeks (P < 0.01) independent of cell type and dosage. Regulatory T-cell levels were increased with both cell types, the most in AD-MSC groups. These immunomodulatory effects were only transient. MSC treatment resulted in long-term (>120 day) allograft survival in 47% of the animals, which correlated with durable microchimerism in BM and spleen. CONCLUSIONS: AD-MSCs and BM-MSCs exert immunomodulatory effects that prolong survival of immunogenic skin-bearing VCA grafts with short course (21 day) tacrolimus induction therapy. The in vivo findings in terms of allograft survival did not reflect superior immunomodulatory characteristics of AD-MSCs found in vitro.
Assuntos
Tecido Adiposo/citologia , Transplante de Medula Óssea , Aloenxertos Compostos/irrigação sanguínea , Aloenxertos Compostos/transplante , Sobrevivência de Enxerto , Membro Posterior/irrigação sanguínea , Membro Posterior/transplante , Terapia de Imunossupressão/métodos , Transplante de Células-Tronco Mesenquimais , Transplante de Pele , Alotransplante de Tecidos Compostos Vascularizados , Animais , Células Cultivadas , Aloenxertos Compostos/efeitos dos fármacos , Esquema de Medicação , Sobrevivência de Enxerto/efeitos dos fármacos , Membro Posterior/efeitos dos fármacos , Imunossupressores/administração & dosagem , Masculino , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Linfócitos T Reguladores/imunologia , Tacrolimo/administração & dosagem , Fatores de Tempo , Quimeras de Transplante , Tolerância ao TransplanteRESUMO
Vascularized composite allotransplantation (VCA) has emerged as a treatment option for treating nonlife-threatening conditions. Therefore, in order to make VCA a safe reconstruction option, there is a need to minimize immunosuppression, develop tolerance-inducing strategies and elucidate the mechanisms of VCA rejection and tolerance. In this study we explored the effects of hIL-2/Fc (a long-lasting human IL-2 fusion protein), in combination with antilymphocyte serum (ALS) and short-term cyclosporine A (CsA), on graft survival, regulatory T cell (Treg) proliferation and tolerance induction in a rat hind-limb transplant model. We demonstrate that hIL-2/Fc therapy tips the immune balance, increasing Treg proliferation and suppressing effector T cells, and permits VCA tolerance as demonstrated by long-term allograft survival and donor-antigen acceptance. Moreover, we observe two distinct types of acute rejection (AR), progressive and reversible, within hIL-2/Fc plus ALS and CsA treated recipients. Our study shows differential gene expression profiles of FoxP3 versus GzmB, Prf1 or interferon-γ in these two types of AR, with reversible rejection demonstrating higher Treg to Teff gene expression. This correlation of gene expression profile at the first clinical sign of AR with VCA outcomes can provide the basis for further inquiry into the mechanistic aspects of VCA rejection and future drug targets.
Assuntos
Membro Posterior/transplante , Interleucina-2/química , Proteínas Recombinantes de Fusão/química , Tolerância ao Transplante/efeitos dos fármacos , Alotransplante de Tecidos Compostos Vascularizados/métodos , Animais , Proliferação de Células , Ciclosporina/química , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Rejeição de Enxerto , Sobrevivência de Enxerto , Granzimas/metabolismo , Humanos , Sistema Imunitário , Tolerância Imunológica , Masculino , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Transplante HomólogoRESUMO
Foi realizado o estudo comparativo do efeito imunossupressor de três tratamentos, utilizando-se a combinação de ciclosporina A e metilprednisolona, um ciclo curto e um ciclo longo de FK506 por 12 semanas no transplante de membro inferior entre ratos Sprague-Dawley e Wistar. O tempo médio de instalação da rejeição foi de 6,6 dias nos animais que não receberam imunossupressão, 34 dias nos que receberam ciclosporina A e metilprednisolona, 58 dias para os que receberam FK506 em ciclo curto e 77.75 para os que receberam FK506 em ciclo longo. Os grupos que receberam FK506 apresentaram tempo prolongado de sobrevivência do transplante. /The immunosuppressive effect of combined therapy using cyclosporine A and metilprednisolone, a short course and a long course of FK506 for 12 weeks in a Sprague-Dawley/Wistar rat limb allotransplantation model was tested. 36 right hindlimb transplantations were performed. Median time for onset of rejection was 6,6 days in animals without immunosuppression, 34 days receiving cyclosporine A and metilprednisolone, 58 days receiving FK506 short course and 77.75 days in the long course group. The FK506 groups presented survival time of the allograft longer than the cyclosporine A and metilprednisolone, and the FK506 long course was the most efficient among the three treatments preventing rejection. The mortality of the animal was increased in the cyclosporine A and metilprednisolone compared to the FK506 groups...
Assuntos
Animais , Masculino , Adulto , Membro Posterior/transplante , Transplante Homólogo/estatística & dados numéricos , Ciclosporina/efeitos adversos , Ciclosporina/uso terapêutico , Modelos Animais de Doenças , Hemissuccinato de Metilprednisolona/uso terapêutico , Ratos Sprague-Dawley , Ratos Wistar , Rejeição de Enxerto/tratamento farmacológico , Transplante Homólogo/efeitos adversos , Transplante Homólogo/mortalidadeRESUMO
OBJETIVO: Analisar as complicaçöes pós-operatórias dos transplantes microcirúrgicos alógenos de membro, bem como determinar fatores associados à toxicidade das drogas. MÉTODO. O estudo foi realizado entre combinaçöes de maior histocompatibilidade, utilizando 39 ratos Brown-Norway (doadores) e 78 ratos Fischer 344 (receptores), submetidos ao transplante microcirúrgico alógenode membro e ao tratamento imunossupressor com ciclosporina e RS-61443. RESULTADOS. Um (1/78: 1,28 por cento) foi sacrificado devido à trombose; dois (2/78:2,56 por cento) por enterite; um (1/78:1,28 por cento) devido à autofagia e oito (8/78:10,42 por cento) morreram por causa indeterminada. CONCLUSÄO. Dentre os 78 (78/78:100 por cento) ratos submetidos ao transplante alógeno microcirúrgico de membro, 12 (15,5 por cento) ratos morreram ou foram sacrificados devido às complicaçöes pós-operatórias agudas näo relacionadas à rejeiçäo, toxicidade dos agentes imunossupressores ou infecçäo oportunista
Assuntos
Animais , Masculino , Ratos , Ciclosporina/administração & dosagem , Membro Posterior/transplante , Imunossupressores/administração & dosagem , Ácido Micofenólico/análogos & derivados , Histocompatibilidade , Microcirurgia , Ácido Micofenólico/administração & dosagem , Complicações Pós-Operatórias , Ratos Endogâmicos BN , Transplante Autólogo , Transplante Homólogo/efeitos adversos , Transplante Homólogo/mortalidadeRESUMO
PURPOSE: To analyze the composite tissue vascularized allotransplantation post operative complications and to identify factors associated with agents toxicity. METHOD: The study was done across a strong histocompatibility barrier using 39 Brown-Norway rats as donors and 78 Fischer 344 as hindlimb allotransplantation recipients treated with cyclosporine and RS-61443. RESULTS: 1 (1/78: 1.28%) was eliminated owing to thrombosis; 2 (2/78: 2.56%) owing to enteritis; 1 (1/78: 1.28%) owing to autophagia and 8 (8/78: 10.42%) died owing to undiscovered cause. CONCLUSION: Among 78 (78/78: 100%) rat limb vascularized allotransplantations, 12 (15.5%) rats died or were eliminated because of acute postoperative complications that were not related to rejection, to immunosuppressive toxicity or to opportunistic infection.
Assuntos
Ciclosporina/administração & dosagem , Membro Posterior/transplante , Imunossupressores/administração & dosagem , Ácido Micofenólico/análogos & derivados , Complicações Pós-Operatórias/etiologia , Animais , Masculino , Microcirurgia , Ácido Micofenólico/administração & dosagem , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Transplante Homólogo/efeitos adversos , Transplante Homólogo/mortalidadeAssuntos
Transplante de Medula Óssea/imunologia , Medula Óssea/irrigação sanguínea , Quimera , Doença Enxerto-Hospedeiro/prevenção & controle , Reação Enxerto-Hospedeiro/imunologia , Membro Posterior/transplante , Transfusão de Linfócitos , Linfócitos T/imunologia , Transplante Homólogo/imunologia , Animais , Citometria de Fluxo , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/imunologia , Isoantígenos/análise , Linfonodos/imunologia , Masculino , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T alfa-beta/análiseRESUMO
Using the triploid cell marker, the cellular contribution from graft and stump to the supernumerary limbs which result from controlateral grafts of limb buds and regeneration blastemas in the axolotl has been analyzed. Grafts were made so as to appose anterior and posterior limb positions. Overall, the contribution from graft and stump tissue was found to be approximately equal although the position of the boundary between the two was variable from limb to limb. This result is consistent with models which suggest that intercalary regeneration is the driving force for patterning of the vertebrate limb. In addition, the pattern of cellular contribution to supernumerary limbs was consistently found to be asymmetrical in the dorsal-ventral axis. Hence, posterior limb tissue predominantly contributed cells to the posterior and dorsal part of the supernumerary limb whereas anterior limb tissue predominantly contributed cells to the anterior and ventral part of the supernumerary limb. The reason for this asymmetrical pattern remains unknown, but we suggest that it might result from a directional bias in intercalary regeneration, similar to that observed during intercalation in the proximal-distal axis of the urodele limb. Using the triploid cell marker in conjunction with a black/white pigmentation marker, the relationship between the cellular contribution boundary and the pigmentation boundary in supernumerary limbs has also been analyzed. It has been found that the positions of the two boundaries do not coincide, a result which suggests that the eventual location of pigment cells is not a good indicator of the location of nonpigment cells derived from graft and stump.