Inducción de tolerancia en modelo de autotrasplante / Induction of tolerance in allogeneic transplant model

Introduction: Vascularized composite allotransplantation (VCA) involves the transplantation of complex anatomical structures including different kinds of tissue. The aim was to study the effect of a treatment with immature dendritic cells in a model of VCA. Materials and Methods: The rat hind limb allotransplantation model was used. Due to the high antigenic mistmatch Brown Norway rats were used as donors and Lewis rats as recipients. The bone marrow derived immature dendritic cells were cultured under GM-CSF stimuli and donor tissue. The rejection grade and the survival of the graft were assessed. Experimental groups: group I (n = 3): no treatment; Group II (n = 6): tacrolimus 10 mg/kg one day before the transplantation (day -1); Group III (n = 3): tacrolimus 10 mg/kg on day -1 and 6 mg/kg from day 0 to 14, plus intravenous saline infusion on days 7 and 14; Group IV (n = 3): tacrolimus 10 mg/kg on day -1 and 6 mg/kg from day 0 to 14, plus intravenous immature dendritic cells on days 7 and 14. Results: All 15 allografts developed rejection. The mean allograft survival was 14 days in group I, 15 days in group II, 34 days in group III and 58 days in groups IV (p < 0.05). Conclusions: In the rat hind limb allotransplantation model under tacrolimus monotherapy, the treatment with immature bone marrow derived dendritic cells pulsed with alloantigens increases the survival of the graft.

Introducción: El alotrasplante compuesto vascularizado (ACV) involucra el trasplante de estructuras anatómicas complejas que pueden contener distintos tipos de tejidos. El objetivo de este estudio fue evaluar el efecto del tratamiento con células dendríticas inmaduras derivadas de médula ósea del receptor y cargadas con aloantígenos como potencial inductor de tolerancia en un modelo de ACV. Animales y Métodos: Para realizar el modelo de alotrasplante de extremidad posterior de la rata, se utilizaron como donantes ratas Brown Norway y como receptoras ratas Lewis. Las células dendríticas se diferenciaron a partir de precursores de médula ósea que se cargaron con lisado de tejido del donante. Grupos experimentales: Grupo I (n = 3): sin tratamiento; Grupo II (n = 6): tacrolimus 10 mg/kg vía oral el día previo al trasplante (día -1); Grupo III (n = 3): tacrolimus 10 mg/kg el día -1 y 6 mg/kg desde el día 0 al 14 post operatorio como mantención; Grupo IV (n = 3): mismo esquema de tacrolimus que grupo III, pero además infusión intravenosa de células dendríticas los días 7 y 14. Se evaluó la sobrevida de los implantes y el grado de rechazo. Resultados: Los 15 animales trasplantados presentaron rechazo. La sobrevida media del ACV fue de 14 días en el grupo I, 15 días en el grupo II, 34 días en el grupo III y 58 días en el grupo IV (p < 0,05). Conclusión: En un modelo de ACV bajo tratamiento con tacrolimus, la infusión de células dendríticas inmaduras derivadas de médula ósea y pulsadas con aloantígeno aumentan la sobrevida del implante.

Entrenamiento microquirúrgico básico para realizar un modelo animal de alotrasplante compuesto vascularizado / Basic microsurgical training for a vascularized composite allotransplantation animal model

Introduction: rat hind limb transplantation is a complex animal model of vascularized composite allo-transplantation (VCA). A basic microsurgical training is required prior to the implementation of this model. Aim: to propose a training program for the acquisition of basic skills to perform a microsurgical VCA model. Animals and Methods: the training program was conducted in two stages. First, at the dry lab, basic suturing skills with 9-0 to 11-0 nylon sutures were practiced, reproduced from surgical videos performed by experts. In a second stage, at the wet lab using 13 Lewis rats, 6 hind limb microsurgical dissections were performed and the important steps for transplantation were identified: 10 end to end femoral artery anastomoses with 10-0 nylon interrupted suture; 10 end to end femoral vein anastomoses with 10-0 nylon suture; 3 femoral vein interposition in the femoral artery; 6 end to end sciatic nerve neurorrhaphy; 4 femur osteosynthesis with 21g needle and wire cerclage. Anastomotic patency rate and anastomotic surgical time were recorded. Results: arterial and venous patency rate was 100 and 90 percent respectively. Surgical time decreased from 49 to 24 minutes on arterial anastomoses and from 55 to 25 minutes on venous anastomoses after completion of the training program. When a vein interposition was performed, an immediate patency rate of 100 percent was obtained. Conclusion: a successful staged training model of basic microsurgical skills was performed, in order to perform a VCA model.

Introducción: el trasplante de extremidad posterior de la rata es un modelo microquirúrgico de alotras-plante compuesto vascularizado (ACV), que requiere para su implementación de un entrenamiento microquirúrgico básico sistematizado. Objetivo: comunicar un modelo de entrenamiento microquirúrgico básico para adquirir las habilidades y destrezas que permitan realizar un modelo microquirúrgico de ACV Animales y Métodos: el entrenamiento se realizó en 2 etapas: la primera, en laboratorio en seco. A partir de vídeos de microcirugía, se practicó las destrezas para maniobrar suturas de nylon de 9-0 a 11-0. En la segunda etapa in vivo con animales, (13 ratas Lewis), se realizaron: 6 disecciones microquirúrgicas de extremidad posterior para reconocimiento de estructuras anatómicas; 10 anastomosis término-terminal (T-T) de arteria femoral con sutura interrumpida 10-0; 10 anastomosis T-T de vena femoral; 3 interposiciones de vena femoral en arteria femoral; 6 neurorrafias T-T de nervio ciático; 4 fijaciones óseas de fémur con aguja 21 g y cerclaje con alambre. Se evaluó la permeabilidad de las anastomosis y los tiempos quirúrgicos. Resultados: anastomosis arteriales: la permeabilidad inmediata fue de un 100 por ciento. Los tiempos de anastomosis disminuyeron de 49 a 24 min con el entrenamiento. Anastomosis venosas: la permeabilidad inmediata fue de un 90 por ciento. Los tiempos de anastomosis disminuyeron paulatinamente de 55 a 25 min. Interposiciones venosas: se logró una permeabilidad de 100 por ciento. Conclusión: una estrategia de entrenamiento por etapas, es una forma factible de entrenamiento en microcirugía. Este entrenamiento permite obtener una aceptable permeabilidad anastomótica, paso crítico para realizar modelos de ACV.

Células dendríticas como determinantes del equilibrio entre inmunidad y autoinmunidad / Dendritic cells like determinants of the balance between immunity and autoimmunity

Autoinmune diseases occur when the immunological system loses its capability to recognize between self and foreign, and develops a response against healthy tissues. Both molecular bases that trigger these pathologies and genetic factors of susceptibility are unknown. Identification of these factors is fundamental for understanding and developing specific therapies for autoimmune diseases. Dendritic cells (DCs) - professional antigen presenting cells - play a fundamental roll in the development and modulation of the acquired immune response because they have the unique capacity to active naïve T lymphocytes. In order to activate T lymphocytes, the DCs must supply simultaneously two molecular signals which correspond to the antigen (or peptid complex MHC) plus costimulating signals. In absence of costimulation, the DCs induce inactivation of T lymphocytes, mechanism by which they are able to maintain peripheral tolerance against self antigens. In this way, it has been proposed that the immature DCs present antigens in absence of costimulation and provoke tolerance. On the contrary, mature DCs present antigens together with costimulating signals, which lead to immunity. In self-immune diseases, the process of tolerance in the presence of autoantigens could be deficient due to alterations in the normal function of DCs. One of the causes of this defect could be that there is an unbalance in the expression of activating and inhibiting Fcy receptors on the surface of DCs. This would generate DCs with a constitutive phenotype of maturity that would interfere with its tolerogenic activity, and would favor the permanent activation of T lymphocytes, including those that are autoreactive.