Effective GDNF brain delivery using microspheres-A promising strategy for Parkinson's disease

Glial cell line-derived neurotrophic factor (GDNF) has shown promise in the treatment of neurodegenerative disorders of basal ganglia origin such us Parkinson\u27s disease (PD). In this study, we investigated the neurorestorative effect of controlled GDNF delivery using biodegradable microspheres in an animal model with partial dopaminergic lesion. Microspheres were loaded with N-glycosylated recombinant GDNF and prepared using the Total Recirculation One-Machine System (TROMS). GDNF-loaded microparticles were unilaterally injected into the rat striatum by stereotaxic surgery two weeks after a unilateral partial 6-OHDA nigrostriatal lesion. Animals were tested for amphetamine-induced rotational asymmetry at different times and were sacrificed two months after microsphere implantation for immunohistochemical analysis. The putative presence of serum IgG antibodies against rat glycosylated GDNF was analyzed for addressing safety issues. The results demonstrated that GDNF-loaded microspheres, improved the rotational behavior induced by amphetamine of the GDNF-treated animals together with an increase in the density of TH positive fibers at the striatal level. The developed GDNF-loaded microparticles proved to be suitable to release biologically active GDNF over up to 5 weeks in vivo. Furthermore, none of the animals developed antibodies against GDNF demonstrating the safety of glycosylated GDNF use

Inactivation of Listeria monocytogenes by pulsed light in packaged and sliced salpicão, a ready-to-eat traditional cured smoked meat sausage

The efficacy of pulsed light (PL) for the surface decontamination of a sliced ready-to-eat cured meat product, salpicão, was studied. The surface of the slices was inoculated with Listeria monocytogenes (107 cfu/g), and then PL treatment was applied. Microbial analyses (L. monocytogenes, coagulase-negative Staphylococci (CNS), Lactic acid bacteria (LAB) and Enterobacteriaceae) were performed immediately after the treatment. pH, aw, colour and volatile profile were also evaluated. Response Surface experimental Design was applied regarding factors voltage (ranging from 1828 to 3000 V) and distance (ranging from 2.6 to 5 cm) to the light source, and a maximum of 16.11 J/cm2 energy dose was achieved. A reduction of 1.58 log cfu/g in L. monocytogenes count was obtained when a fluence of 5.31 J/cm2 was applied. Enterobacteriaceae, LAB and CNS endogenous microbial populations were not influenced by PL treatment. PL application reduced a* values and influenced b* values without impacting the sample ΔE or L* values. The potential oxidative effect of this technology was evaluated by solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS), and no significant changes were verified after its application in this sliced cured meat product.info:eu-repo/semantics/publishedVersio

CNN disruption predictor at JET: Early versus late data fusion approach

This work focuses on the development of a data driven model, based on Convolutional Neural Networks (CNNs), for the real-time detection of disruptive events at JET. The predictor exploits the ability of CNNs in learning relevant spatiotemporal information straight from 1D plasma profiles, avoiding hand-engineered feature extraction procedures. In this paper, the radiation profiles from both the bolometer horizontal and vertical cameras have been considered amongst the predictor inputs, with the aim of discriminating between the core radiation due to impurity accumulations and the outboard radiation phenomena. Moreover, an innovative predictor architecture is proposed, where two separate CNNs are trained to focus on events with different timescales, that is, the destabilization of radiation, electron density and temperature profiles, and the mode-locking and current profile variations. The outputs of the two CNNs are combined with a logic OR function to provide the disruption alarm trigger. The advantages of this data fusion approach impact on the predictor performance, with a very limited number of false alarms (only 1 in the considered test set), and on the model output interpretability as the two different branches are triggered by different types of events

Expresión y purificación de GDNF para su microencapsulación y aplicación en la enfermedad de Parkinson

La enfermedad de Parkinosn (EP) es un proceso neurodegenerativo del sistema nervioso central que afecta a las neuronas de dopamina de la sustancia negra, núcleo mesoencefálico del control motor. La perdida en el cerebro de este neurotransmisor vital causa los síntomas de la enfermedad. La EP afecta actualmente a 200 de cada 100.000 personas y a 2 de cada 100 entre los mayores de 60 años. En España hay unos 110.000 enfermos. Además, hoy por hoy no se conoce nada que pueda prevenir o curar la enfermedad, ni existe ninguna prueba de laboratorio que permita diagnosticarla. Recentiemente se ha demostrado que el GDNF, factor neurotrófico derivado de las células gliales, es capaz de proteger las neuronas dopaminérgicas e incluso inducir la regeneración del tejido dopaminérgico dañado in vivo. El objetivo del trabajo fue diseñar y desarrolar un método de expresión y purificación de GDNF bioactivo para su posterior microencapsulación y aplicación en la enfermedad de Parkison. El sistema escogido para expresar el GDNF fue el sistema de células eucariotas de mamífero. El vector utilizado para la producción del GDNF en células eucariotas fue el pDEST26 (Tecnología Gateway de Invitrogen). Como sistema de expresión de GDNF se utilizaron las líneas celulares eucariota BHK, 293 y COS 7. Estas células fueron cultivadas en medio D-MEM (Invitrogen) complementado con un 10% de suero fetal bovino (FBS) y Penicilina/Streptomicina (100u/ml) (Invitrogen). La transfección se realizó con Lipofectamine Plus (Invitrogen). Se analizó la expresión de GDNF a nivel de mRNA mediante PCR y a nivel de proteína mediante Western Blot del medio condicionado. Los clones positivos se crecieron en botellas de cultivo de 850 cm2 (Corning) y se realizaron ciclos de recolección del medio. Cada ciclo fue analizado por SDS-PAGE y Western Blot. Para evaluar la actividad de la proteína se ha desarrollado un ensayo de actividad en el que se demuestra la diferenciación morfológica de células PC-12 inducida por GDNF. La presencia de los receptores GFRa1 y RET, necesarios para que el GDNF ejerza su acción, fue determinada por PCR. Las conclusiones obtenidas de este estudio son la obtención de GDNF recombinante a partir de un sistema de expresión en células eucariotas, el desarrollo de un protocolo para su posterior purificación y la obtención de GDNF recombinante biológicamente activo

Terapias neuroprotectoras y neurorestauradoras en el tratamiento de la enfermedad de Parkinson

Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Current therapies are symptomatic and, although these therapies are efficacious during the early stages of the disease, they present important side effects when they are used for a long time. The ideal therapy would be the one that would slow down or stop the progression of the disease. This can be achieved, for instance, with neuroprotective and neurorestorative therapies. Among them, cell therapy and therapy with trophic factors such as glial cell line derived neurotrophic factor (GDNF) are the most challenging and promising ones for the scientific community. Although the use of GDNF as a treatment for Parkinson s disease was proposed several years ago, it is necessary to develop alternative strategies to deliver GDNF appropriately to concrete areas of the brain. Here, the use of microspheres as the most suitable tool for the administration of this neurotrophic factor is discussed

Microencapsulación de factores neurotróficos: aplicación al tratamiento de la enfermedad de Parkinson

La enfermedad de Parkinson (EP) es una enfermedad degenerativa, lentamente progresiva caracterizada por temblor de reposo, cara inexpresiva, rigidez, lentitud al iniciar y practicar movimientos voluntarios. Neuropatológicamente se caracteriza por pérdida de células dopaminérgicas en la sustancia nigra lo cual conlleva déficit en el suministro de dopamina a nivel de ganglios basales. Los factores de crecimiento nervioso, o factores neurotróficos, que respaldan la supervivencia, crecimiento y desarrollo de las células cerebrales, son un tipo de terapia prometedora para la enfermedad de Parkinson. Se ha demostrado que el GDNF, factor neurotrófico derivado de la línea celular glial, protege las neuronas de dopamina y promueve su supervivencia en los modelos animales de la enfermedad de Parkinson. Sin embargo, la administración de proteínas en el cerebro no está exenta de dificultades, por ello, el sistema elegido para administrar el GDNF en el cerebro será uno de los puntos clave para el éxito del tratamiento. En este sentido, el uso de micropartículas formuladas a partir de polímeros biodegradables parece ser la estrategia más apropia. En nuestro grupo de investigación hemos desarrollado un protocolo de expresión y purificación de GDNF en células eucariótas de mamífero. El objetivo de este estudio es la microencapsulación de la proteína en partículas biodegradables

Long-term neuroprotection and neurorestoration by glial cell-derived neurotrophic factor microspheres for the treatment of parkinson's disease

BACKGROUND: Glial cell-derived neurotrophic factor is a survival factor for dopaminergic neurons and a promising candidate for the treatment of Parkinson's disease. However, the delivery issue of the protein to the brain still remains unsolved. Our aim was to investigate the effect of long-term delivery of encapsulated glial cell-derived neurotrophic factor within microspheres. METHODS: A single dose of microspheres containing 2.5 μg of glial cell-derived neurotrophic factor was implanted intrastriatally in animals 2 weeks after a 6-hydroxydopamine lesion. RESULTS: The amphetamine test showed a complete behavioral recovery after 16 weeks of treatment, which was maintained until the end of the study (week 30). This effect was accompanied by an increase in dopaminergic striatal terminals and neuroprotection of dopaminergic neurons. CONCLUSIONS: The main achievement was the long-term neurorestoration in parkinsonian animals induced by encapsulated glial cell-derived neurotrophic factor, suggesting that microspheres may be considered as a means to deliver glial cell-derived neurotrophic factor for Parkinson's disease treatment. © 2011 Movement Disorder Society

Production of highly pure human glycosylated GDNF in a mammalian cell line

The administration of glial cell line-derived neurotrophic factor (GDNF) has emerged as a promising strategy for the treatment of several diseases of the nervous system as Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury and nerve regeneration as well as ocular diseases and drug addictions. A procedure for the purification of human recombinant glycosylated GDNF using a mammalian expression system as the source of the protein is discussed in the present paper. The neurotrophic factor was purified using cation exchange chromatography and gel filtration. A human cell line was chosen as the source of therapeutic protein, since a recombinant protein with a structure and glycosylation pattern equivalent to the native form is desirable for its prospective therapeutic utilization. The activity of the highly pure protein obtained was confirmed with a cell-based bioassay. The purified protein is suitable for its in vivo evaluation in animals and for possible subsequent clinical application

Sustained release of bioactive glycosylated glial cell-line derived neurotrophic factor from biodegradable polymeric microspheres

Glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophic factor for dopaminergic neurons, appeared as a promising candidate for treating Parkinson's disease. GDNF microencapsulation could ensure protection against degradation due to the fragile nature of the protein. Poly(lactide-co-glycolide) (PLGA) microparticles loaded with recombinant glycosylated GDNF obtained in a mammalian cell line were prepared by TROMS, a semi-industrial technique capable of encapsulating fragile molecules maintaining their native properties. The effects of several parameters as PLGA copolymer type, PEG 400 quantity co-encapsulated with GDNF or drug loading, on the properties of the particles were investigated. Microparticles showed a mean diameter between 8 and 30 μm, compatible with their stereotaxic implantation. The drug entrapment efficiency ranged from 50.6 to 100% depending on the microsphere composition. GDNF was better encapsulated using hydrophilic polymers with high molecular weight such as RG 503H. In vitro drug release was influenced by the polymer type as well as by the amount of PEG 400 co-encapsulated with GDNF. Microparticles prepared using PLGA RG 503H released 67% of the total protein content within 40 days. Moreover, very low concentrations of poly (vinyl alcohol) were detected after microparticles washing and freeze-drying. Finally, a PC-12 bioassay demonstrated that the in vitro GDNF released was bioactive