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Resumen Objetivo Optimizar el radiomarcaje con 99mTc y 67Ga de nanoparticulas de albumina recubiertas con 4 polimeros sinteticos distintos y evaluar su estabilidad in vivo e in vitro, asi como su biodistribucion in vivo tras su administracion intravenosa. Material y metodos Las nanoparticulas se prepararon empleando albumina y albumina modificada con NOTA mediante el metodo de desolvatacion y se recubrieron con 4 polimeros distintos; HPMC, GMN2, GPM2 y GTM2. Se purificaron, liofilizaron y caracterizaron. El marcaje con 99mTc se realizo con 74 MBq de pertecnetato [99mTc] sodico previamente reducido con una disolucion acida de cloruro de estano a diferentes concentraciones (0,003; 0,005; 0,007; 0,01; 0,05 y 0,1 mg/ml), a distintos tiempos (5, 10, 15, 30 y 60 min) y temperaturas (temperatura ambiente, 40 °C y 60 °C). El marcaje con 67Ga se llevo a cabo mediante incubacion de las nanoparticulas con 37 MBq de cloruro de 67Ga (obtenido a partir de citrato de 67Ga comercial) a distintos tiempos (10 y 30 min) y temperaturas (temperatura ambiente, 30 °C y 60 °C) y posterior purificacion con microconcentradores. La pureza radioquimica de ambos marcajes se evaluo mediante TLC. Se llevaron a cabo estudios de estabilidad de las nanoparticulas marcadas en suero fisiologico y plasma sanguineo. Los estudios de biodistribucion de las nanoparticulas recubiertas con el polimero GPM2 se llevaron a cabo en ratas Wistar tras la administracion intravenosa de las nanoparticulas. Se realizaron animales control con pertecnetato [99mTc] sodico y cloruro de 67Ga. Posteriormente, los animales fueron sacrificados y se midio la actividad de los organos en un contador gamma. Resultados El marcaje con 99mTc se llevo a cabo de forma optima con una concentracion de estano de 0,007 mg/ml para las nanoparticulas GPM2 y de 0,005 mg/ml para el resto de formulaciones, con un tiempo de marcaje de 10 min y a temperatura ambiente. En el caso del 67Ga el marcaje se optimizo a 30 °C de temperatura y 30 min de incubacion. En ambos casos, la pureza radioquimica obtenida fue superior al 97%. Las nanoparticulas presentaron una elevada estabilidad in vitro pasadas las 48 h del marcaje (70% las nanoparticulas marcadas con 99mTc y 90% las marcadas con 67Ga). Los estudios de biodistribucion de las nanoparticulas [99mTc]-GPM2 y [67Ga]-NOTA-GPM2 mostraron una elevada acumulacion de actividad en el higado tanto a las 2 h como a las 24 h de la administracion intravenosa. Conclusion El procedimiento de marcaje con 99mTc y 67Ga de nanoparticulas de albumina y albumina modificada con NOTA permite la obtencion de nanoparticulas con elevados rendimientos de marcaje y una adecuada estabilidad in vitro, permitiendo su utilizacion para la realizacion de estudios in vivo.

Id1 promotes carcinogenesis and metastasis, and predicts prognosis of non-small cell lung cancer (NSCLC)-adenocarcionoma patients. We hypothesized that Id1 may play a critical role in lung cancer colonization of the liver by affecting both tumor cells and the microenvironment. Depleted levels of Id1 in LLC (Lewis lung carcinoma cells, LLC shId1) significantly reduced cell proliferation and migration in vitro. Genetic loss of Id1 in the host tissue (Id1(-/-) mice) impaired liver colonization and increased survival of Id1 animals. Histologically, the presence of Idl in tumor cells of liver metastasis was responsible for liver colonization. Microarray analysis comparing liver tumor nodules from Id1(+/+) mice and Idl(-/-) mice injected with LLC control cells revealed that Id1 loss reduces the levels of EMT-related proteins, such as vimentin. In tissue microarrays containing 532 NSCLC patients' samples, we found that Idl significantly correlated with vimentin and other EMT-related proteins. Idl loss decreased the levels of vimentin, integrin beta 1, TGF beta 1 and snail, both in vitro and in vivo. Therefore, Id1 enables both LLC and the host microenvironment for an effective liver colonization, and may represent a novel therapeutic target to avoid NSCLC liver metastasis. (C) 2017 Elsevier B.V. All rights reserved.

PRGF is a platelet concentrate within a plasma suspension that forms an in situ-generated fibrin-matrix delivery system, releasing multiple growth factors and other bioactive molecules that play key roles in tissue regeneration. This study was aimed at exploring the angiogenic and myogenic effects of PRGF on in vitro endothelial cells (HUVEC) and skeletal myoblasts (hSkMb) as well as on in vivo mouse subcutaneously implanted matrigel and on limb muscles after a severe ischemia. Human PRGF was prepared and characterized. Both proliferative and anti-apoptotic responses to PRGF were assessed in vitro in HUVEC and hSkMb. In vivo murine matrigel plug assay was conducted to determine the angiogenic capacity of PRGF, whereas in vivo ischemic hind limb model was carried out to demonstrate PRGF-driven vascular and myogenic regeneration. Primary HUVEC and hSkMb incubated with PRGF showed a dose dependent proliferative and anti-apoptotic effect and the PRGF matrigel plugs triggered an early and significant sustained angiogenesis compared with the control group. Moreover, mice treated with PRGF intramuscular infiltrations displayed a substantial reperfusion enhancement at day 28 associated with a fibrotic tissue reduction. These findings suggest that PRGF-induced angiogenesis is functionally effective at expanding the perfusion capacity of the new vasculature and attenuating the endogenous tissue fibrosis after a severe-induced skeletal muscle ischemia.

Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector.

Resumen Objetivo Este trabajo evalua la idoneidad del radiotrazador 11C-(+)-α-dihidrotetrabenazina (11C-(+)DTBZ) para cuantificar mediante tomografia de emision de positrons (PET) la inervacion dopaminergica en rata y mono, especies utilizadas como modelos animales en el estudio de la enfermedad de Parkinson. Material y metodos En ratas se estudio una poblacion control sana (n = 10) y el efecto del neurotoxico 6-hidroxidopamina (6-OHDA), ademas de realizarse estudios PET con 6-[(18)F]-fluoro-L-DOPA (18F-DOPA) y de autorradiografia digital cuantitativa. El estudio en Macaca fascicularis se realizo en animales control y tratados con el toxico 1-metil-4-fenil- 1,2,3,6-tetrahidropiridina (MPTP). Resultados En ambas especies se obtuvieron imagenes de gran calidad donde se observo una alta captacion de 11C-(+) DTBZ en el estriado. La cuantificacion se realizo mediante la creacion de imagenes parametricas y el calculo del potencial de union (BP). La medida del BP en la poblacion control de ratas arrojo un valor de 1,10 ± 0,16 (media ± error estandar [EE]), mientras que los estriados danados con 6-OHDA mostraron una captacion disminuida en funcion del grado de la lesion. Las imagines obtenidas con 18F-DOPA no fueron aptas para el analisis al no discriminar los estriados, mientras que el estudio mediante autorradiografia digital cuantitativa confirmo la elevada afinidad de la 11C-(+)DTBZ por estas estructuras. En monos, el valor final de BP fue de 1,31 y 1,06 mientras que el tratamiento con MPTP disminuyo la captacion en un 40%. Conclusiones La calidad de las imagenes PET y la disminucion de la captacion en las lesiones con 6-OHDA y MPTP indican que la 11C-(+)DTBZ es un radiotrazador adecuado para el estudio de la inervacion dopaminergica en estas especies animales.

Summary Aim This study evaluates the utility of 11 C-(+)-α-dihydrotetrabenazine ( 11 C-(+)DTBZ) in the quantification of dopaminergic innervation by positron emission tomography (PET) in rat and monkey, two animal species used as animal models of Parkinson's disease. Material and methods Healthy control animals (n = 10) and the effect of 6-hydroxidopamine (6-OHDA) neurotoxic were studied in rats. 18 F-DOPA PET studies and digital quantitative autoradiography were also carried out. Studies with Macaca fascicularis were performed in control and 1-methyl 4-phenyl 1, 2, 3, 6-tetrahydropyridine (MPTP) treated animals. Results In both species high quality images were generated in which clear uptake of 11 C-(+)DTBZ was found in the striatum. 11 C-(+)DTBZ uptake quantification was estimated by creating parametric images and binding potential (BP) calculation. BP in control rats was 1.10 ± 0.16 (mean ± standard deviation [SD], whereas 6-OHDA produced a decrease in the uptake depending on the lesion degree. Images obtained with 18 F-DOPA were not adequate for the analysis as they did not discriminate the stratum whereas digital quantitative autoradiography studies confirmed the high affinity of striatum by 11 C-(+)DTBZ. In monkeys, final BP values were 1.31 and 1.06 and MPTP treatment educed uptake by 40%. Conclusions The quality of PET images and the decrease of uptake in 6-OHDA and MPTP lesions show that 11 C-(+)DTBZ is an adequate radiotracer for the study of dopaminergic innervation in these animal models.

Resumen La dihidrotetrabenazina (2-hidroxi-3-isobutil- 9,10-dimetoxi-1,3,4,6,7-hexahidro-11bH-benzo[a]-quinolizina, DTBZ) se ha convertido en el ligando ideal de los transportadores presinapticos de monoaminas (VMAT2) debido a su elevada afinidad de union y su lipofilicidad. Objetivo Desarrollar un procedimiento de sintesis automatico para el marcaje con carbono-11 de la DTBZ para utilizarla como marcador en el estudio in vivo mediante tomografia por emision de positrones de perdidas neuronales en modelos animals de enfermedad de Parkinson. Material y metodos Se ha disenado un nuevo metodo de sintesis totalmente automatizado para la obtencion de 11C-(+)DTBZ. La reaccion de metilacion del precursor -(+)desmetildihidrotetrabenazina– se lleva a cabo a temperatura ambiente, a partir de la obtencion de 11CH3I que utilizamos como precursor primario, en presencia de dimetilsulfoxido e hidroxido de potasio. Para los procesos de purificacion se han utilizado cartuchos de extraccion en fase solida alumina y los disolventes residuales del producto final se eliminaron mediante evaporacion bajo flujo de helio. Resultados De las 54 sintesis realizadas se han obtenido, con un tiempo de bombardeo de 5 minutos, y 6 minutos de sintesis tras la obtencion de 11CH3I, unas producciones medias de 1,94 ± 0,13 GBq de 11C-(+)DTBZ, esteril, apirogeno y con una pureza radioquimica > 99%. Conclusiones Este nuevo procedimiento de sintesis es rapido y simple, ya que para la purificacion final se han optimizado tecnicas que permitieran la eliminacion de los disolventes residuales basandonos en su polaridad y es aplicable a otras sintesis automaticas para la obtencion de otros compuestos marcados mediante reacciones de metilacion.

Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9,10-dimethoxy-1,3,4,6,7-hexahydro-11bH-benzo[a]-quinolizine, DTBZ) has become the ideal radioligand for the presynaptic vesicular monoamine transporter VMAT2 based on its high binding affinity and optimal lipophilicity. Objective To develop an automatic procedure for labelling DTBZ with carbon-11, which has been shown to be a highly effective marker for in vivostudies of neuronal losses in animal models with Parkinson's disease using positron emission tomography (PET). Materials and methods We have developed a new fully automated synthesis procedure to obtain 11 C-(+)DTBZ quickly and simply through labelling the precursor –(+)desmethyldihydrotetrabenazine– at room temperature in the presence of dimethyl sulfoxide (DMSO) and potassium hydroxide (KOH), using 11 CH 3 I as primary precursor. The final purification was carried out by solid phase extraction using commercially available cartridges and the residual solvents (DMSO and ethyl ether) were eliminated by evaporation. Results The whole procedure was automated, and after 54 syntheses, an average production of 1.94 GBq of sterile, pyrogen- free 11 C-(+)DTBZ with a radiochemical purity > 99% was obtained with 5 minutes irradiation and 6 minutes of synthesis after 11 CH 3 I production. 11 C-(+)DTBZ binding to presynaptic dopamine nerve terminals has been demonstrated by MicroPET studies in Wistar rats and M. Fascicularismonkeys. Conclusions This new synthesis procedure is quick and simple, due to optimised techniques, which have allowed elimination of residual solvents based on their polarity for the final purification. It is also applicable to other automatic syntheses for obtaining compounds labelled by methylation reactions.

We herein describe a simple setup for the automated simultaneous synthesis of l -[methyl-11C]methionine and N-[methyl-11C]choline by solid-supported methylation . The setup is extremely simple and easy to adapt to other automated systems and due to its versatility, the method can be utilized for the production of other radiopharmaceuticals requiring a simple [11C]methylation step. Furthermore, it can be used for multiple simultaneous synthesis.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two multifunctional peptides processed from a common precursor which have been described in numerous mammalian organs, including the pituitary gland. Previous studies have found AM immunoreactivity in neurohypophysis nerve fibers of amphibian pituitary. In the present study, immunocytochemical and Western blot analysis in the pituitary gland of the amphibian Rana perezi demonstrated in the adenohypophysis both AM and PAMP. AM-like immunoreactivity was found in a moderate number of endocrine cells of the pars distalis. In the neurohypophysis, AM was observed not only in nerve fibers of pars nervosa and axonal projections innervating the pars intermedia, but also in the outer zone of the median eminence. PAMP staining was observed in numerous endocrine cells scattered all over the pars distalis and in some cells of the pars tuberalis, but not in the neurohypophysis. In order to compare the quantity of AM and PAMP immunoreactivity between pars distalis of female and male specimens, an image analysis study was done. Significant differences for AM immunoreactivity (p

This study focuses on the occupational doses of technologists working at an Animal Research Unit using PET radiotracers and on the environmental dose rates produced by the animals (mice, rats and monkeys). In particular, whole body and extremity monitoring is reported and related with the workload. The study shows that doses not only depend on the amount of activity injected but also on the type of animals and radiotracers managed. The extremities, with a great variability of the doses received, are the limiting organs as far as regulatory dose limits for workers are concerned. Mean H∗(10) rates in contact and at 20 cm from the animals, when they are handled by the technologist, range from around 1 mSv/h to 20 μSv/h, respectively.

Aim: Around 30% of non-small cell lung cancer (NSCLC) patients present LM during the disease course causing a negative clinical impact on survival and quality of life. The expression of certain genes in cancer cells and also TME gene expression might be crucial for allowing tumor cells to spread to the liver. According to this hypothesis Id1 and Id3 genes, part of the signature that facilitates breast cancer cells to disseminate to the lungs, might be determinant for NSCLC LM development. Methods: First we validated Id1 as a significant and independent prognostic factor among patients with adenocarcinoma of the lung. Recently, we have communicated the development of a mouse model of LM from lung cancer in which the only absence of Id1 expression in TME of Id1-knockout (KO) animals was sufficient to avoid LM formation by 50%. Now we aimed to evaluate the additional impact of Id1 and Id3 genes deficiency in tumor cells on LM generation. Therefore, 2 cohorts including 23 mice were compared; Id1 wild-type (WT) female mice (n = 12) vs. KO female animals (n = 11). In both groups of mice 500,000 Lewis Lung Carcinoma cells (LLC) either Id1 homozygously deficient (Id1-/-) and Id3 WT (Id3 + /+) or Id1-/- and Id3 heterozygously deficient (Id3 + /-), generated through gene silencing, were intrasplenically injected. Thereafter, both groups of mice were weekly monitored with FDG-micro-positron emission tomography scans (mPET) for LM formation. Animals were sacrificed (and tissues microscopically analyzed) by the time LM were developed and clinical deterioration was evident, according to our Animal Ethics Committee approved protocol. Mice genotype Positive PET at week 2 p value WT 41.7% KO 0% 0.02 Microscopic LM at necropsy WT 83.3% KO 36.4% 0.03 Results: LM were significantly more likely to appear among Id1 WT mice compared to Id1 KO mice with a relative risk of 2.29 (IC95% 1.01-5.22). Results are summarized in Table 1 Conclusions: In this model, the absence of Id1 in TME of KO mice in combination with the lack of Id1/Id3 expression in intrasplenically injected lung cancer cells showed to be a more significantly efficient strategy to prevent LM formation. Disclosure: All authors have declared no conflicts of interest.