Inducción de tolerancia a endotoxina en un modelo de trasplante de tejido compuesto / Tolerance induction to endotoxin in a composite tissue transplant

Introducción y Objetivo: La tolerancia a endotoxina (TE) es un fenómeno biológico que consiste en la desensibilización de las células del sistema inmune ante exposiciones bajas al lipopolisacárido (LPS), haciendo que entren en un estado de anergia ante otros estímulos. El objetivo de este trabajo es valorar el efecto inmunomodulador del precondicionamiento con lipopolisacárido en el contexto de un trasplante de tejido compuesto. Material y Método: Realizamos transferencias de patas traseras entre ratas cruzando el Complejo Mayor de Histocompatibilidad. Los animales se dividieron en 2 grupos según el precondicionamiento que recibieron, LPS y suero salino fisiológico. Resultados: El grupo control presentó una mediana de supervivencia del alotrasplante menor a 15 días tras el cese del tratamiento inmunosupresor. El grupo precondicionado con LPS presentó una mediana de supervivencia superior a 30 días (p= 0.001). Conclusiones: El mecanismo de tolerancia a endotoxina aumenta la supervivencia del alotrasplante de tejido compuesto

Background and Objetive: Endotoxin tolerance is a biological phenomenon which consists in desensitization of immune system cells to low exposures to lipopolysaccharide (LPS). It leads to antigens anergy. The aim of this study is to asses the development of tolerance after precondicioning with LPS in the context of a composite tissue transplant. Methods: Transferences of hind legs were made between rats crossing the Main Histocompatibility Complex (MHC). The animals were divided into 2 groups according to the preconditioning they received, LPS and physiological saline. Results: The control group presented a median survival of the allograft less tan 15 days after the cessation of inmunosupressive treatment. The group preconditioned with LPS presented median survival greater than 30 days (p=0.001). Conclusions: The mechanism of tolerance to endotoxin increases the survival of composite tissue allotransplantation

Estudio sobre quimerismo reverso y el efecto de la movilización de células madre de médula ósea sobre la expresión de citoquinas en un modelo experimental de trasplante de tejidos compuestos / Study on reverse chimerism and the effect of mobilization of bone marrow stem cells on the expression of cytokines in an experimental model of composite tissue transplantation

Introducción y Objetivo: Los trasplantes de tejidos compuestos sufren rechazo crónico modulado entre otros factores por citoquinas. El quimerismo reverso o quimerismo del aloinjerto se define como la repoblación del tejido trasplantado por células circulantes del receptor. Plerixafor produce la movilización de células madre de médula ósea CD34+ hacia la sangre periférica. El objetivo del estudio fue conocer los mecanismos moleculares que intervienen en el rechazo crónico y el quimerismo reverso tras la administración de plerixafor. Material y Método: Realizamos 16 trasplantes osteomusculares heterotópicos de pata posterior entre ratas Brown-Norway hembra y Wistar Lewis macho bajo inmunosupresión subterapéutica con tacrólimus. Establecimos 2 grupos de estudio según la administración postoperatoria de plerixafor. Transcurridas 9 semanas estudiamos la expresión de citoquinas y el infiltrado leucocitario en distintas localizaciones musculares, así como el grado de rechazo crónico y porcentaje de quimerismo reverso en diferentes tejidos del aloinjerto. Resultados: Encontramos diferencias estadísticas en la expresión de factor estimulante de colonias granulocíticas e interleucina 12 a nivel de los tercios medio y distal del aloinjerto, y de interleucina 6 a nivel del tercio medio del aloinjerto. La intensidad del infiltrado leucocitario fue mayor en el grupo que no recibió plerixafor. Ambos grupos desarrollaron rechazo crónico y pudimos observar la aparición de quimerismo reverso. Sin embargo no observamos diferencias significativas en el infiltrado leucocitario, el rechazo crónico ni el quimerismo reverso. Conclusiones: La movilización de células madre de médula ósea CD34+ se asoció con una menor expresión de factor estimulante de colonias granulocíticas, interleucina 6 e interleucina 12. Estos hallazgos contribuyen a elucidar los mecanismos moleculares que podrían conducir a la creación de quimeras en el aloinjerto

Background and Objective: Vascularized composite allotransplantation suffer chronic rejection modulated by cytokines. Reverse chimerism or allograft chimerism is defined as the repopulation of the transplanted tissue by circulating cells of the recipient. Plerixafor mobilizes CD34+ bone marrow stem cells to the peripheral blood. The aim of the study was to know the molecular mechanisms involved in chronic rejection and reverse chimerism after plerixafor administration. Methods: Sixteen heterotopic osteomuscular hindlimb transplants were performed between female Brown-Norway rats as donors and male Wistar Lewis rats as recipients under subtherapeutic immunosuppression with tacrolimus. Two groups were established according to the postoperative administration of plerixafor. After 9 weeks, expression of cytokines and leukocyte infiltration were studied in different muscle locations, as well as the degree of chronic rejection and percentage of reverse chimerism in different tissues of the allograft. Results: Statistical differences were found in granulocyte colony stimulating factor and interleukin 12 expression at middle and distal allograft thirds, and interleukin 6 expression at middle allograft third. The intensity of leukocyte infiltration was greater in the group that did not receive plerixafor. Both groups developed chronic rejection and the appearance of reverse chimerism could be observed. However, no significant differences were observed in leukocyte infiltration, chronic rejection or reverse chimerism. Conclusions: The mobilization of CD34+ bone marrow stem cells was associated with a lower expression of granulocytic colony stimulating factor, interleukin 6 and interleukin 12. These findings contribute to elucidate the molecular mechanisms that could lead to the creation of chimeras in the allograft

Protective effects of mitochondrion-targeted peptide SS-31 against hind limb ischemia-reperfusion injury

Hind limb ischemia-reperfusion injury is an important pathology in vascular surgery. Reactive oxygen species are thought to be involved in the pathogenesis of hind limb ischemia-reperfusion injury. SS-31, which belongs to a family of mitochondrion-targeted peptide antioxidants, was shown to reduce mitochondrial reactive oxygen species production. In this study, we investigated whether the treatment of SS-31 could protect hind limb from ischemia-reperfusion injury in a mouse model. The results showed that SS-31 treatment either before or after ischemia exhibited similar protective effects. Histopathologically, SS-31 treatment prevented the IR-induced histological deterioration compared with the corresponding vehicle control. SS-31 treatment diminished oxidative stress revealed by the reduced malondialdehyde level and increased activities and protein levels of Sod and catalase. Cellular ATP contents and mitochondrial membrane potential increased and the level of cytosolic cytC was decreased after SS-31 treatment in this IR model, demonstrating that mitochondria were protected. The IR-induced increase of levels of inflammatory factors, such as Tnf-α and Il-1β, was prevented by SS-31 treatment. In agreement with the reduced cytosolic cytC, cleaved-caspase 3 was kept at a very low level after SS-31 treatment. Overall, the effect of SS-31 treatment before ischemia is mildly more effective than that after ischemia. In conclusion, our results demonstrate that SS-31 confers a protective effect in the mouse model of hind limb ischemia-reperfusion injury preventatively and therapeutically

Aerobic training and l-arginine supplementation promotes rat heart and hindleg muscles arteriogenesis after myocardial infarction

Arteriogenesis is a main defense mechanism to prevent heart and local tissues dysfunction in occlusive artery disease. TGF-β and angiostatin have a pivotal role in arteriogenesis. We tested the hypothesis that aerobic training and l-arginine supplementation promotes cardiac and skeletal muscles arteriogenesis after myocardial infarction (MI) parallel to upregulation of TGF-β and downregulation of angiostatin. For this purpose, 4 weeks after LAD occlusion, 50 male Wistar rats were randomly distributed into five groups: (1) sham surgery without MI (sham, n = 10), (2) control-MI (Con-MI, n = 10), (3) l-arginine-MI (La-MI, n = 10), (4) exercise training-MI (Ex-MI, n = 10), and (5) exercise and l-arginine-MI (Ex + La-MI). Exercise training groups running on a treadmill for 10 weeks with moderate intensity. Rats in the l-arginine-treated groups drank water containing 4 % l-arginine. Arteriolar density with different diameters (11-25, 26-50, 51-75, and 76-150 μm), TGF-β, and angiostatin gene expression were measured in cardiac (area at risk) and skeletal (soleus and gastrocnemius) muscles. Smaller arterioles decreased in cardiac after MI. Aerobic training and l-arginine increased the number of cardiac arterioles with 11-25 and 26-50 μm diameters parallel to TGF-β overexpression. In gastrocnemius muscle, the number of arterioles/mm2 was only increased in the 11 to 25 μm in response to training with and without l-arginine parallel to angiostatin downregulation. Soleus arteriolar density with different size was not different between experimental groups. Results showed that 10 weeks aerobic exercise training and l-arginine supplementation promotes arteriogenesis of heart and gastrocnemius muscles parallel to overexpression of TGF-β and downregulation of angiostatin in MI rats (AU)

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Preservación en frío y rechazo crónico, desarrollo de un modelo de trasplante de extremidad posterior en ratas / Cold preservation and chronic rejection, development of a rat hind limb transplantation model

Los trasplantes de tejidos compuestos son una realidad clínica con más de 150 casos realizados con éxito en los últimos 15 años. La vasculopatía crónica del aloinjerto es una causa de pérdida tardía del órgano observada en 3 trasplantes de tejidos compuestos. Histológicamente se caracteriza por estenosis de la luz vascular causada por hiperplasia de la íntima y fibrosis cicatricial de la adventicia. La vasculopatía está causada por factores inmunitarios y no inmunes, entre los que se encuentra el daño por frío. Presentamos el desarrollo de un modelo experimental de vasculopatía crónica del aloinjerto en el contexto del trasplante de pata posterior entre ratas tipo Lewis Brown-Norway como donantes y ratas tipo Wistar-Lewis como receptoras, tras 7 horas de isquemia fría a 4ºC. Todos los animales recibieron dosis infraterapéuticas de ciclosporina A y fueron sacrificados trascurridos 2 meses desde el trasplante. El análisis histológico permitió identificar los hallazgos característicos de la vasculopatía crónica del aloinjerto en los tercios musculares medio y distal de cada aloinjerto. Encontramos diferencias estadísticamente significativas al comparar el número de vasos patológicos de pequeño y mediano calibre observados por campo de aumento en el tercio medio y el tercio muscular distal (P = 0,007 y 0,004 respectivamente). El presente estudio establece un modelo experimental reproducible de vasculopatía crónica del aloinjerto en trasplante de tejidos compuestos. Pone en evidencia también la asociación entre la preservación en isquemia fría y la vasculopatía en diferentes segmentos vasculares de aloinjertos de pata posterior en ratas (AU)

Composite tissue allotransplantation has become a clinical reality nowadays, with more than 150 transplants performed in the last 15 years. Chronic allograft vasculopathy has been detected in 3 cases of composite tissue allotransplantation, being responsible of late graft failure. Decreased vascular lumina caused by intimal hyperplasia and constrictive remodeling of the adventitia, are the main histological features of this entity. Immune and not immune factors are related with the development of the vasculopathy, being cold damage between the later. We present the development of an experimental model of chronic allograft vasculopathy in a model of rat hind limb allotransplantation after 7 hours of cold ischemia at 4ºC, between Lewis Brown-Norway rats as donors and Wistar-Lewis rats as receptors. All animals were under low dose immunosuppression with cyclosporine A and were sacrificed 2 months post transplantation. The characteristic changes of chronic vasculopathy were found in the middle muscular and distal thirds at the histological analysis. Statistically differences were found when comparing the number of pathological small and medium size vessels of the middle and distal muscular thirds (P = 0,007 and 0,004 respectively). The present study establishes a reproducible experimental model of chronic allograft vasculopathy in composite tissue allotransplantation. We have verified the association of cold ischemia preservation and chronic vasculopathy at different vascular segments of the rat hind limb (AU)