Intraarticular and intravenous administration of 99MTc-HMPAO-labeled human mesenchymal stem cells (99MTC-AH-MSCS): In vivo imaging and biodistribution.

INTRODUCTION: Therapeutic application of intravenous administered (IV) human bone marrow-derived mesenchymal stem cells (ahMSCs) appears to have as main drawback the massive retention of cells in the lung parenchyma, questioning the suitability of this via of administration. Intraarticular administration (IAR) could be considered as an alternative route for therapy in degenerative and traumatic joint lesions. Our work is outlined as a comparative study of biodistribution of 99mTc-ahMSCs after IV and IAR administration, via scintigraphic study in an animal model. METHODS: Isolated primary culture of adult human mesenchymal stem cells was labeled with 99mTc-HMPAO for scintigraphic study of in vivo distribution after intravenous and intra-articular (knee) administration in rabbits. RESULTS: IV administration of radiolabeled ahMSCs showed the bulk of radioactivity in the lung parenchyma while IAR images showed activity mainly in the injected cavity and complete absence of uptake in pulmonary bed. CONCLUSIONS: Our study shows that IAR administration overcomes the limitations of IV injection, in particular, those related to cells destruction in the lung parenchyma. After IAR administration, cells remain within the joint cavity, as expected given its size and adhesion properties. ADVANCES IN KNOWLEDGE: Intra-articular administration of adult human mesenchymal stem cells could be a suitable route for therapeutic effect in joint lesions. IMPLICATIONS FOR PATIENT CARE: Local administration of adult human mesenchymal stem cells could improve their therapeutic effects, minimizing side effects in patients.

Estudio hemodinámico, metabólico e histopatológico de un modelo porcino de trasplante auxiliar heterotópico hepático con arterialización portal / A hemodynamic, metabolic and histopathological study of a heterotopic auxiliary swine liver graft with portal vein arterialization

INTRODUCCIÓN: El trasplante auxiliar heterotópico hepático con arterialización de la vena porta (TAHH-AVP) es un modelo poco estudiado a pesar de su potencial terapéutico. El objetivo del estudio es valorar la respuesta hemodinámica y bioquímica durante el implante y analizar la repercusión de la arterialización portal en la funcionalidad y morfología hepática. MÉTODOS: Se realizó un estudio hemodinámico y bioquímico durante el implante auxiliar en un modelo porcino (n = 15 TAHH-AVP). Además, se analizaron las consecuencias de la arterialización portal sobre la arquitectura hepática mediante un estudio ultraestructural. RESULTADOS: La reperfusión del injerto arterializado aumentó la frecuencia cardiaca (FC) respecto a los valores basales (p = 0,004) y a la fase del pinzamiento de la vena cava (p = 0,004) y disminuyó las resistencias vasculares sistémicas respecto a la fase del pinzamiento de la vena cava (p = 0,021). Al final del implante, el gasto cardiaco permaneció elevado (p = 0,001), al igual que la FC respecto a la fase basal (p = 0,002). La presión arterial media disminuyó con el pinzamiento venoso, pero no se vio afectada ni por la reperfusión del injerto ni por el cierre de la piel. Todas las muestras histológicas obtenidas a los 3, 10 y 21 días conservaron su morfología y arquitectura hepáticas. Si bien se observaron algunos focos de necrosis perilobular el día 3 (p = 0,049) y proliferación conectiva perilobular el día 10 (p = 0,007), respecto al hígado nativo. CONCLUSIONES: El trasplante del injerto hepático arterializado descrito minimiza el número de anastomosis vasculares respecto a los modelos previamente publicados, presenta una buena tolerancia hemodinámica y metabólica, y la arterialización portal del injerto no produce cambios significativos en la histología hepática

BACKGROUND: Auxiliary heterotopic liver transplantation with portal vein arterialization (AHLT-PVA) is a model that has been hardly studied, despite its therapeutic potential. METHODS: Hemodynamic and biochemical characterization was carried out during graft implantation, in a pig-to-pig model (n = 15 AHLT-PVA). Furthermore a histopathological study was performed to establish microscopic alterations due to PVA. RESULTS: Reperfusion of the arterialized graft produced an increase in heart rate (HR) vs. baseline (P=.004) and vs. inferior vena cava clamping phase (P=.004); and a decrease in systemic vascular resistance vs. cava clamping phase (P=.021). At the end of implantation, cardiac output remained elevated (P=.001), likewise HR remained increased vs. baseline phase (P=.002). Mean arterial pressure decreased with cava clamping, but was not affected by the reperfusion of the graft, nor the skin closure. The histopathological study at 3, 10, and 21 days post-PVA revealed that functional liver structure was maintained although it is common to find foci of perilobular necrosis on day 3 (P=.049), and perilobular connective tissue proliferation at day 10 (P=.007), vs. native liver. CONCLUSIONS: The described arterialized liver graft model minimizes the number of vascular anastomoses vs. previously described models. It is hemodynamically and metabolically well tolerated and the double arterial vascularization of the graft does not cause significant changes in liver histology

A hemodynamic, metabolic and histopathological study of a heterotopic auxiliary swine liver graft with portal vein arterialization.

BACKGROUND: Auxiliary heterotopic liver transplantation with portal vein arterialization (AHLT-PVA) is a model that has been hardly studied, despite its therapeutic potential. METHODS: Hemodynamic and biochemical characterization was carried out during graft implantation, in a pig-to-pig model (n=15 AHLT-PVA). Furthermore a histopathological study was performed to establish microscopic alterations due to PVA. RESULTS: Reperfusion of the arterialized graft produced an increase in heart rate (HR) vs. baseline (P=.004) and vs. inferior vena cava clamping phase (P=.004); and a decrease in systemic vascular resistance vs. cava clamping phase (P=.021). At the end of implantation, cardiac output remained elevated (P=.001), likewise HR remained increased vs. baseline phase (P=.002). Mean arterial pressure decreased with cava clamping, but was not affected by the reperfusion of the graft, nor the skin closure. The histopathological study at 3, 10, and 21 days post-PVA revealed that functional liver structure was maintained although it is common to find foci of perilobular necrosis on day 3 (P=.049), and perilobular connective tissue proliferation at day 10 (P=.007), vs. native liver. CONCLUSIONS: The described arterialized liver graft model minimizes the number of vascular anastomoses vs. previously described models. It is hemodynamically and metabolically well tolerated and the double arterial vascularization of the graft does not cause significant changes in liver histology.

Quantification of the chromosomal radiation damage induced by labelling of leukocytes with [18F]FDG.

INTRODUCTION: The aim of our work is to quantify the radiation damage in lymphocytes after labelling with [18F]FDG. Comparison with gold standard [99mTc]HMPAO labelling is established. An approach to cellular dosimetry is proposed. METHODS: Mixed leukocytes were separated from fresh venous blood and labelled with [18F]FDG and [99mTc]HMPAO following published guidelines. Cytokinesis-block micronucleus (CBMN) assay was performed for both sets of experiments. Tests for quality control of labelling described in guidelines were followed. Cellular dosimetry was calculated according to MIRD. RESULTS: MN scored after labelling with 37 MBq of [18F]FDG were 956 ± 172 and 347 ± 26 for [99mTc]HMPAO (p < 0.05). Absorbed dose in cell nucleus was of 0.23 Gy for [18F]FDG and 0.08 Gy for [99mTc]HMPAO labelling. The CBMN assay after labelling with ~290 MBq of [18F]FDG showed radiation induced inhibition of proliferation capacity of the lymphocytes, confirmed by proliferation study. CONCLUSIONS: [18F]FDG labelling of mixed leukocytes causes severe radiation damage to the cell, higher than with [99mTc]HMPAO in accordance with the absorbed dose. Labelling of mixed leukocytes for clinical purpose induces high cytotoxicity reflected in the loss of proliferation capacity in lymphocytes this statement allows us to consider a low oncogenic risk however the association between MN formation in the PBL and subsequent risk of cancer is not well established. ADVANCES IN KNOWLEDGE: This is the first work about radiation damage with [18F]FDG labelled cells. We focused on [18F]FDG labelling of leukocytes due to the growing number of research and review articles about this technique. IMPLICATIONS FOR PATIENT CARE: The possibility of an increased risk of lymphoid malignancies associated with the administration of radiolabelled lymphocytes is a very controversial subject. Studies on radiation damage on new labelling procedures as the one exposed in this work must be considered.

PDE2 activity differs in right and left rat ventricular myocardium and differentially regulates ß2 adrenoceptor-mediated effects.

The important regulator of cardiac function, cAMP, is hydrolyzed by different cyclic nucleotide phosphodiesterases (PDEs), whose expression and activity are not uniform throughout the heart. Of these enzymes, PDE2 shapes ß1 adrenoceptor-dependent cardiac cAMP signaling, both in the right and left ventricular myocardium, but its role in regulating ß2 adrenoceptor-mediated responses is less well known. Our aim was to investigate possible differences in PDE2 transcription and activity between right (RV) and left (LV) rat ventricular myocardium, as well as its role in regulating ß2 adrenoceptor effects. The free walls of the RV and the LV were obtained from Sprague-Dawley rat hearts. Relative mRNA for PDE2 (quantified by qPCR) and PDE2 activity (evaluated by a colorimetric procedure and using the PDE2 inhibitor EHNA) were determined in RV and LV. Also, ß2 adrenoceptor-mediated effects (ß2-adrenoceptor agonist salbutamol + ß1 adrenoceptor antagonist CGP-20712A) on contractility and cAMP concentrations, in the absence or presence of EHNA, were studied in the RV and LV. PDE2 transcript levels were less abundant in RV than in LV and the contribution of PDE2 to the total PDE activity was around 25% lower in the microsomal fraction of the RV compared with the LV. ß2 adrenoceptor activation increased inotropy and cAMP levels in the LV when measured in the presence of EHNA, but no such effects were observed in the RV, either in the presence or absence of EHNA. These results indicate interventricular differences in PDE2 transcript and activity levels, which may distinctly regulate ß2 adrenoceptor-mediated contractility and cAMP concentrations in the RV and in the LV of the rat heart.

Influence of radiolytic-produced hydrogen peroxide on the stability of commercial kit (99m) technetium-exametazime preparation.

99mTc-d,l-hexamethylpropylene amine oxime ((99m) Tc-d,l-HMPAO) is a widely used radiopharmaceutical that suffers from an inherent instability with a shelf life of 30 min that constrains its availability for clinical use. A protocol for improving the stability of the kit with minimal modification of manufacturer's instructions and no chemicals addition to the commercial formulation is proposed. The protocol is based on the displacement of the oxygen present in the preparation, preventing free radicals build up and free pertechnetate formation. Although the degradation of (99m) Tc-d,l-HMPAO cannot be explained solely by the radiolytic production of free radicals, it appears to be an important factor in the shelf stability of the complex.

The increase in rat ventricular automaticity induced by salbutamol is mediated through ß(1)- but not ß(2)-adrenoceptors: role of phosphodiesterases.

AIMS: While ß(2)-adrenoceptor (AR) agonists are useful bronchodilators, they also produce cardiac arrhythmias. These agents are not fully selective and also activate ß(1)-AR, but the involvement of ß(1)-AR and ß(2)-AR in the observed pro-arrhythmic effect has not been established. We studied the effect of ß(1)-AR and ß(2)-AR activation on ventricular automaticity and the role of phosphodiesterases (PDE) in regulating this effect. MAIN METHODS: Experiments were performed in the spontaneously beating isolated right ventricle of the rat heart. We also measured cAMP production in this tissue. KEY FINDINGS: The ß(2)-AR agonist salbutamol (1-100 µM) produced a concentration-dependent increase in ventricular automaticity that was not affected by 50nM of the ß(2)-AR antagonist ICI 118551. This effect was enhanced by the non-selective PDE inhibitor theophylline (100 µM) and by the selective PDE4 inhibitors rolipram (1 µM) and Ro 201724 (2 µM), but not modified by the selective PDE3 inhibitors cilostamide (0.3 µM) or milrinone (0.2 µM). The effects of salbutamol alone and in the presence of either theophylline or rolipram were virtually abolished by 0.1 µM ß(1)-AR antagonist CGP 20712A. Salbutamol (10 µM) increased the cAMP concentration, and this effect was abolished by CGP 20712A (0.1 µM) but enhanced by theophylline (100 µM) or rolipram (1 µM). Cilostamide (0.3 µM) failed to modify the effect of salbutamol on cAMP concentration. SIGNIFICANCE: These results indicate that the increase of ventricular automaticity elicited by salbutamol was exclusively mediated through ß(1)-AR and enhanced by non-selective PDE inhibition with theophylline or selective PDE4 inhibition. However, PDE3 did not appear to regulate this effect.

Phosphodiesterases inhibition unmask a positive inotropic effect mediated by beta2-adrenoceptors in rat ventricular myocardium.

The effects of salbutamol on contractility and cAMP levels were investigated in rat right ventricular myocardium. Salbutamol (1-300 microM), produced a concentration-dependent positive inotropic effect which was not affected by ICI 118551 (50 nM), a beta2-adrenoceptor antagonist but was abolished by CGP 20712A (1 microM) a beta1-adrenoceptor antagonist. However, in rats pretreated with pertussis toxin (30 microg/kg intraperitoneal injection) salbutamol increases contractility (Emax = 9.8 +/- 1.8%, - log EC50 = 6.25 +/- 0.07, n = 5). The combination of salbutamol + CGP 20712A, also produces a concentration-dependent enhancement of contractility (Emax = 43.0 +/- 7.5%, - log EC50 = 6.3 +/- 0.04, n = 6), in the presence of 30 microM of the non selective phosphodiesterase (PDE) inhibitor 3-isobutylmethylxantine (IBMX) which was prevented by ICI 118551 (50 nM). Also, salbutamol + CGP 20712A fail to increase cAMP tissue levels but enhance them in the presence of IBMX. This effect was also prevented by ICI 118551. These results indicate that PDEs blunt contractility and cAMP production mediated by beta2-adrenoceptors in rat ventricular myocardium. Gi protein, although less efficiently than PDEs, also limits inotropic effects of salbutamol mediated by beta2-adrenoceptors in this tissue.

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes.

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.