Specific Expression of Glial-Derived Neurotrophic Factor in Muscles as Gene Therapy Strategy for Amyotrophic Lateral Sclerosis.

Glial cell line-derived neurotrophic factor (GDNF) is a powerful neuroprotective growth factor. However, systemic or intrathecal administration of GDNF is associated with side effects. Here, we aimed to avoid this by restricting the transgene expression to the skeletal muscle by gene therapy. To specifically target most skeletal muscles in the mouse model of amyotrophic lateral sclerosis (ALS), SOD1G93A transgenic mice were intravenously injected with adeno-associated vectors coding for GDNF under the control of the desmin promoter. Treated and control SOD1G93A mice were evaluated by rotarod and nerve conduction tests from 8 to 20 weeks of age, and then histological and molecular analyses were performed. Muscle-specific GDNF expression delayed the progression of the disease in SOD1G93A female and male mice by preserving the neuromuscular function; increasing the number of innervated neuromuscular junctions, the survival of spinal motoneurons; and reducing glial reactivity in treated SOD1G93A mice. These beneficial actions are attributed to a paracrine protective mechanism from the muscle to the motoneurons by GDNF. Importantly, no adverse secondary effects were detected. These results highlight the potential of muscle GDNF-targeted expression for ALS therapy.

Central nervous system delivery of helper-dependent canine adenovirus corrects neuropathology and behavior in mucopolysaccharidosis type VII mice.

Canine adenovirus type 2 vectors (CAV-2) are promising tools to treat global central nervous system (CNS) disorders because of their preferential transduction of neurons and efficient retrograde axonal transport. Here we tested the potential of a helper-dependent CAV-2 vector expressing ß-glucuronidase (HD-RIGIE) in a mouse model of mucopolysaccharidosis type VII (MPS VII), a lysosomal storage disease caused by deficiency in ß-glucuronidase activity. MPS VII leads to glycosaminoglycan accumulation into enlarged vesicles in peripheral tissues and the CNS, resulting in peripheral and neuronal dysfunction. After intracranial administration of HD-RIGIE, we show long-term expression of ß-glucuronidase that led to correction of neuropathology around the injection site and in distal areas. This phenotypic correction correlated with a decrease in secondary-elevated lysosomal enzyme activity and glycosaminoglycan levels, consistent with global biochemical correction. Moreover, HD-RIGIE-treated mice show significant cognitive improvement. Thus, injections of HD-CAV-2 vectors in the brain allow a global and sustained expression and may have implications for brain therapy in patients with lysosomal storage disease.

Intrathecal administration of IGF-I by AAVrh10 improves sensory and motor deficits in a mouse model of diabetic neuropathy.

Different adeno-associated virus (AAV) serotypes efficiently transduce neurons from central and peripheral nervous systems through various administration routes. Direct administration of the vectors to the cerebrospinal fluid (CSF) could be an efficient and safe strategy. Here, we show that lumbar puncture of a nonhuman AAV leads to wide and stable distribution of the vector along the spinal cord in adult mice. AAVrh10 efficiently and specifically infects neurons, both in dorsal root ganglia (60% total sensory neurons) and in the spinal cord (up to one-third of α-motor neurons). As a proof of concept, we demonstrate the efficacy of AAVrh10 in a mouse model of diabetic neuropathy, in which intrathecal delivery of the vector coding for insulin-like growth factor (IGF-I) favored the release of the therapeutic protein into the CSF through its expression by sensory and motor neurons. IGF-I-treated diabetic animals showed increased vascular endothelial growth factor expression, activation of Akt/PI3K pathway, and stimulated nerve regeneration and myelination in injured limbs. Moreover, we achieved restoration of nerve conduction velocities in both sensory and motor nerves by AAVrh10, whereas we reached only sensory nerve improvement with AAV1. Our results indicate that intrathecal injection of AAVrh10 is a promising tool to design gene therapy approaches for sensorimotor diseases.

Comparative study of peripheral neuropathy and nerve regeneration in NOD and ICR diabetic mice.

The non-obese diabetic (NOD) mouse was suggested as an adequate model for diabetic autonomic neuropathy. We evaluated sensory-motor neuropathy and nerve regeneration following sciatic nerve crush in NOD males rendered diabetic by multiple low doses of streptozotocin, in comparison with similarly treated Institute for Cancer Research (ICR) mice, a widely used model for type I diabetes. Neurophysiological values for both strains showed a decline in motor and sensory nerve conduction velocity at 7 and 8 weeks after induction of diabetes in the intact hindlimb. However, amplitudes of compound muscle and sensory action potentials (CMAPs and CNAPs) were significantly reduced in NOD but not in ICR diabetic mice. Morphometrical analysis showed myelinated fiber loss in highly hyperglycemic NOD mice, but no significant changes in fiber size. There was a reduction of intraepidermal nerve fibers, more pronounced in NOD than in ICR diabetic mice. Interestingly, aldose reductase and poly(ADP-ribose) polymerase (PARP) activities were increased already at 1 week of hyperglycemia, persisting until the end of the experiment in both strains. Muscle and nerve reinnervation was delayed in diabetic mice following sciatic nerve crush, being more marked in NOD mice. Thus, diabetes of mid-duration induces more severe peripheral neuropathy and slower nerve regeneration in NOD than in ICR mice.

Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants.

In this study, we enlarged our previous investigation focusing on the biodiversity of chlamydiae and amoebae in a drinking water treatment plant, by the inclusion of two additional plants and by searching also for the presence of legionellae and mycobacteria. Autochthonous amoebae were recovered onto non-nutritive agar, identified by 18S rRNA gene sequencing, and screened for the presence of bacterial endosymbionts. Bacteria were also searched for by Acanthamoeba co-culture. From a total of 125 samples, we recovered 38 amoebae, among which six harboured endosymbionts (three chlamydiae and three legionellae). In addition, we recovered by amoebal co-culture 11 chlamydiae, 36 legionellae (no L. pneumophila), and 24 mycobacteria (all rapid-growers). Two plants presented a similar percentage of samples positive for chlamydiae (11%), mycobacteria (20%) and amoebae (27%), whereas in the third plant the number of recovered bacteria was almost twice higher. Each plant exhibited a relatively high specific microbiota. Amoebae were mainly represented by various Naegleria species, Acanthamoeba species and Hartmannella vermiformis. Parachlamydiaceae were the most abundant chlamydiae (8 strains in total), and in this study we recovered a new genus-level strain, along with new chlamydiae previously reported. Similarly, about 66% of the recovered legionellae and 47% of the isolated mycobacteria could represent new species. Our work highlighted a high species diversity among legionellae and mycobacteria, dominated by putative new species, and it confirmed the presence of chlamydiae in these artificial water systems.

[Prevention of pressure ulcers in heels]. / Prevención de UPP en talones. Impacto clínico y económico en una unidad de medicina interna.

Heels are, for all assistance levels, one of the most frequent locations for the development of pressure ulcers (PU). In this study we deal to investigate in order to determine in patients at risk in an Internal Medicine Unit, the PU incidence on heels, after applying a specific prevention protocol. This protocol particularly designed for pressure ulcers on heels included a combined application of special hydrocellular dressings specially shaped for heels (Allevyn Heel), hyper-oxygenated fatty acids (Mepentol) and special surfaces for pressure management (Aerocare); afterwards, we attempted a comparison of our results with those from previous similar studies. We designed a prospective study which lasted from May 1-2002 until June 30-2003, with a sample of 100 patients without PU included in the study when admitted to the unit. The cumulated incidence established for PU in heels is a 4% which means an incidence rate of 2.06 PU in heels per 1000 persons/day. After observing the results we may affirm that applying the protocol is, under a clinical point of view, as effective as other measures used in previous studies. If we focus on the cost-benefit, the protocol studied represents an option with an excellent cost-efficiency relationship.

Prevención de UPP en talones. Impacto clínico y económico en una unidad de medicina interna / Prevention of pressure ulcers in heels

Los talones constituyen, en todos los niveles asistenciales, una de las localizaciones más frecuentes de úlceras por presión (upp). Nos planteamos como objetivos de investigación determinar, en pacientes de riesgo de una Unidad de Medicina Interna, la incidencia de upp en los talones, tras la aplicación de un protocolo específico de prevención que incluía la aplicación combinada de apósitos hidrocelulares especiales con forma de talón (Allevyn¸ Heel), ácidos grasos hiperoxigenados (Mepentol®) y superficies especiales para el manejo de la presión (SEMP) y comparar los resultados con estudios previos semejantes. Se diseñó un estudio prospectivo, desde el 1-05-2002 hasta 30-06-2003, con una muestra de 100 pacientes sin upp incluidos según ingresaban en la unidad. Se estableció una incidencia acumulada de aparición de upp en los talones del 4 por ciento, lo que supone una tasa de incidencia de 2,06 upp en talón por 1.000 personas-día. A la vista de los resultados podemos afirmar que la aplicación del protocolo resulta, bajo un punto de vista clínico, tan efectiva como otras medidas utilizadas en estudios previos; si nos centramos en la dimensión de coste-beneficio el protocolo en estudio representa una opción con una excelente relación (AU)