Validation of a qPCR method for determination of viral genome titres of AAV2-based vector preparations.

The viral genome titre is universally used for the dosing of adeno-associated virus (AAV)-based vectors used for gene therapy. To standardise this determination, the development of a common method would be valuable to facilitate comparison of viral doses used in the clinic and in the subsequent quality control of the products. A collaborative study was initiated by the Gene Therapy Working Group of the General European Official Medicines Control Laboratories Network in order to validate a qPCR-based method targeting the ITR2 sequence common to a broad variety of AAV vectors, independently from the serotype of the capsid or from the specific transgene. Five preparations of AAV vectors from various serotypes, including the AAV2/2 (RSS2) and AAV2/8 (RSS8) Reference Standard Stocks (American Type Culture Collection, USA) were used in the study. A plasmid carrying the ITR2 sequence was used to prepare standard curves. Its digestion outside the ITR regions facilitated melting of the hairpin ITR sequence during PCR, allowing better accessibility to the DNA polymerase. The results show that this qPCR method is satisfactory in terms of accuracy and precision. The reproducibility is also acceptable when compared with other similar studies, as it was shown previously that titres obtained by qPCR generally show higher inter-laboratory variability. The use of RSS2 or RSS8 as normalisation control in each assay demonstrated a promising help to identify potential sources of variation in a given laboratory or to smooth out inter-laboratory variations, thus improving reproducibility.

Setting the scene for Mediterranean litterscape management: The first basin-scale quantification and mapping of floating marine debris.

Plastic pollution has become one of the biggest environmental concerns of the Anthropocene as it represents a major threat to both wildlife and human health. Garbage patches in the world's oceans are well documented, but quantitative assessments of floating debris are still lacking in some major areas. The Mediterranean Sea is one such area, despite being one of the most plastic polluted environments. We used data from the first international basin-scale survey of the Mediterranean Sea to provide the first abundance estimate of floating mega-debris (>30 cm) and map their distribution over the entire Mediterranean Sea. We estimated the total number of floating mega-debris at 2.9 million items, taking into account imperfect detection. Items larger than 30 cm represent only one fourth of the complete load of anthropogenic debris (>2 cm) in the Mediterranean, which scales up the estimate to 11.5 million floating debris. The highest densities were observed in the central Mediterranean, and the lowest in the eastern basin. This acute marine pollution might threaten to disrupt entire ecosystems through its impact on marine fauna (entanglement, ingestion, contamination), eventually impacting the tourism industry and the well-being of Mediterranean populations.

The effect of a multi-target protocol on cetacean detection and abundance estimation in aerial surveys.

A double-platform protocol was implemented in the Bay of Biscay and English Channel during the SCANS-III survey (2016). Two observation platforms using different protocols were operating on board a single aircraft: the reference platform (Scans), targeting cetaceans, and the 'Megafauna' platform, recording all the marine fauna visible at the sea surface (jellyfish to seabirds). We tested for a potential bias in small cetacean detection and density estimation when recording all marine fauna. At a small temporal scale (30 s, roughly 1.5 km), our results provided overall similar perception probabilities for both platforms. Small cetacean perception was higher following the detection of another cetacean within the previous 30 s in both platforms. The only prior target that decreased small cetacean perception during the subsequent 30 s was seabirds, in the Megafauna platform. However, at a larger scale (study area), this small-scale perception bias had no effect on the density estimates, which were similar for the two protocols. As a result, there was no evidence of lower performance regarding small cetacean population monitoring for the multi-target protocol in our study area. Because our study area was characterized by moderate cetacean densities and small spatial overlap of cetaceans and seabirds, any extrapolation to other areas or time requires caution. Nonetheless, by permitting the collection of cost-effective quantitative data for marine fauna, anthropogenic activities and marine litter at the sea surface, the multi-target protocol is valuable for optimizing logistical and financial resources to efficiently monitor biodiversity and study community ecology.

Stable isotopes document the trophic structure of a deep-sea cephalopod assemblage including giant octopod and giant squid.

Although deep-sea cephalopods are key marine organims, their feeding ecology remains essentially unknown. Here, we report for the first time the trophic structure of an assemblage of these animals (19 species) by measuring the isotopic signature of wings of their lower beaks, which accumulated in stomachs of stranded sperm whales. Overall, the species encompassed a narrow range in delta(13)C values (1.7 per thousand), indicating that they lived in closely related and overlapping habitats. delta(13)C values can be interpreted in terms of distribution with the more (13)C-depleted species (e.g. Stigmatoteuthis arcturi, Vampyroteuthis infernalis) having a more pelagic habitat than the more (13)C-enriched, bathyal species (e.g. Todarodes sagittatus and the giant squid Architeuthis dux). The cephalopods sampled had delta(15)N values ranging 4.6 per thousand, which is consistent with the species spanning approximately 1.5 trophic levels. Neither the giant octopod (Haliphron atlanticus) nor the giant squid reached the highest trophic position. Species delta(15)N was independent of body size, with large squids having both the highest (Taningia danae) and lowest (Lepidoteuthis grimaldii) delta(15)N values. Their trophic position indicates that some species share the top of the food web, together with other megacarnivores such as the sperm whale.

Bioaccumulation of persistent organic pollutants in female common dolphins (Delphinus delphis) and harbour porpoises (Phocoena phocoena) from western European seas: geographical trends, causal factors and effects on reproduction and mortality.

Concentrations of polychlorinated biphenyls (PCBs) in blubber of female common dolphins and harbour porpoises from the Atlantic coast of Europe were frequently above the threshold at which effects on reproduction could be expected, in 40% and 47% of cases respectively. This rose to 74% for porpoises from the southern North Sea. PCB concentrations were also high in southern North Sea fish. The average pregnancy rate recorded in porpoises (42%) in the study area was lower than in the western Atlantic but that in common dolphins (25%) was similar to that of the western Atlantic population. Porpoises that died from disease or parasitic infection had higher concentrations of persistent organic pollutants (POPs) than animals dying from other causes. Few of the common dolphins sampled had died from disease or parasitic infection. POP profiles in common dolphin blubber were related to individual feeding history while those in porpoises were more strongly related to condition.

Refined localization of the gene for Clouston syndrome (hidrotic ectodermal dysplasia) in a large French family.

Hidrotic ectodermal dysplasia (HED) or Clouston syndrome is a rare autosomal dominant disorder characterized by nail dystrophy, alopecia and palmoplantar hyperkeratosis, which maps to chromosome 13q11-q12.1. We confirmed linkage of HED to this region in a large French family. To define the critical region for HED, detailed haplotypes were constructed with new pericentromeric polymorphic markers. A recombination event in the family indicates that the HED locus maps centromeric to D13S1832. Our French family does not share a common haplotype with other pedigrees previously published (particularly French-Canadian), indicating that the mutations in these families are likely to be of different origin.

Epidermal diseases in bottlenose dolphins: impacts of natural and anthropogenic factors.

Experimental studies have highlighted the potential influence of contaminants on marine mammal immune function and anthropogenic contaminants are commonly believed to influence the development of diseases observed in the wild. However, estimates of the impact of contaminants on wild populations are constrained by uncertainty over natural variation in disease patterns under different environmental conditions. We used photographic techniques to compare levels of epidermal disease in ten coastal populations of bottlenose dolphins (Tursiops truncatus) exposed to a wide range of natural and anthropogenic conditions. Epidermal lesions were common in all populations (affecting > 60% of individuals), but both the prevalence and severity of 15 lesion categories varied between populations. No relationships were found between epidermal disease and contaminant levels across the four populations for which toxicological data were available. In contrast, there were highly significant linear relationships with oceanographic variables. In particular, populations from areas of low water temperature and low salinity exhibited higher lesion prevalence and severity. Such conditions may impact on epidermal integrity or produce more general physiological stress, potentially making animals more vulnerable to natural infections or anthropogenic factors. These results show that variations in natural environmental factors must be accounted for when investigating the importance of anthropogenic impacts on disease in wild marine mammals.

Recurrent seizures and hippocampal sclerosis following intrahippocampal kainate injection in adult mice: electroencephalography, histopathology and synaptic reorganization similar to mesial temporal lobe epilepsy.

Human mesial temporal lobe epilepsy is characterized by hippocampal seizures associated with pyramidal cell loss in the hippocampus and dispersion of dentate gyrus granule cells. A similar histological pattern was recently described in a model of extensive neuroplasticity in adult mice after injection of kainate into the dorsal hippocampus [Suzuki et al. (1995) Neuroscience 64, 665-674]. The aim of the present study was to determine whether (i) recurrent seizures develop in mice after intrahippocampal injection of kainate, and (ii) the electroencephalographic, histopathological and behavioural changes in such mice are similar to those in human mesial temporal lobe epilepsy. Adult mice receiving a unilateral injection of kainate (0.2 microg; 50 nl) or saline into the dorsal hippocampus displayed recurrent paroxysmal discharges on the electroencephalographic recordings associated with immobility, staring and, occasionally, clonic components. These seizures started immediately after kainate injection and recurrid for up to eight months. Epileptiform activities occurred most often during sleep but occasionally while awake. The pattern of seizures did not change over time nor did they secondarily generalize. Glucose metabolic changes assessed by [14C]2-deoxyglucose autoradiography were restricted to the ipsilateral hippocampus for 30 days, but had spread to the thalamus by 120 days after kainate. Ipsilateral cell loss was prominent in hippocampal pyramidal cells and hilar neurons. An unusual pattern of progressive enlargement of the dentate gyrus was observed with a marked radial dispersion of the granule cells associated with reactive astrocytes. Mossy fibre sprouting occurred both in the supragranular molecular layer and infrapyramidal stratum oriens layer of CA3. The expression of the embryonic form of the neural cell adhesion molecule coincided over time with granule cell dispersion. Our data describe the first histological, electrophysiological and behavioural evidence suggesting that discrete excitotoxic lesions of the hippocampus in mice can be used as an isomorphic model of mesial temporal lobe epilepsy.

Prevention of kainic acid-induced limbic seizures and Fos expression by the GABA-A receptor agonist muscimol.

Fos oncoprotein expression has been shown to be a sensitive marker for sequential neuronal activation in response to a specific stimulus. The present study investigated the effect of the gamma-aminobutyric acid (GABA)-A receptor agonist muscimol on kainic acid (KA)-induced limbic seizures and Fos expression in the rat forebrain. One hour after KA injection, a substantial Fos expression was observed in the hippocampal dentate gyrus, whereas only a low level of Fos induction was seen in CA1-3 fields. Six hours post-injection a prominent increase of Fos expression occurred in most forebrain structures, including the whole hippocampus. Following 0.5 mg/kg muscimol treatment a remarkable decrease of Fos expression occurred but only in the caudate putamen and core of the accumbens nucleus. Treatment with 1 mg/kg muscimol led to further significant decreases of Fos expression in CA1-3 pyramidal neurons and the disappearance of Fos induction in the cerebral cortex above the rhinal fissure, reticular thalamic nucleus, claustrum, fundus striati, ventral pallidum, septal nucleus, lateral habenular nucleus, and lateral amygdaloid nucleus. When 2 mg/kg muscimol was injected, animals exhibited "absence seizures' instead of limbic seizures, and Fos expression in the hippocampus was effectively blocked. These results suggest that a reduction of GABAergic inhibition plays a crucial role not only in limbic seizure genesis in the dentate gyrus, but also in the seizure spread mechanism in many brain structures, among which the hippocampal CA1-3 fields are most markedly involved, less marked in the cerebral cortex and some other structures, and least marked in the caudate putamen and core of the accumbens nucleus.

Adenovirus-mediated transfer of a functional GAD gene into nerve cells: potential for the treatment of neurological diseases.

The recent development of efficient virus-mediated gene transfer into nerve cells allows the prospect of new strategies for the treatment of drug-resistant neurological diseases. Some forms of epilepsy may be amenable to gene therapy. Although there is no obvious candidate gene, the consensual GABA hypothesis of epilepsy suggests that the GAD gene may be beneficial. GAD gene expression may be useful in supplying the inhibitory neurotransmitter GABA to particular critical brain territories. We show herein that a nonreplicative recombinant adenovirus carrying the GAD67 gene under the control of Rous sarcoma virus long terminal repeat promoter is able to express the transgene in primary cultures of neurons and glial cells. Expression of the GAD67 gene was assessed by immunoblotting and immunohistochemical analysis. We demonstrated the functionality of the transgene, the expression of which resulted in production of large amounts of GABA in neuronal and glial cell cultures. Substantial production of the enzyme was also detected for several weeks in infected organotypic slices cultured from new-born rat hippocampal tissues. The virally encoded GAD67 was also expressed in vivo in various brain areas involved in various neurological disorders and thus may be of value for the development of gene therapies.

Fos oncoprotein expression in the rat forebrain following muscimol-induced absence seizures.

Fos oncoprotein expression is a marker of neuronal activation following seizures. Here, using this method we examined the anatomical locations of muscimol-induced absence seizures in the rat forebrain. Six hours after a systemic injection of muscimol a massive Fos immunoreactivity appeared in the olfactory system, retrosplenial cortex and paraventricular thalamic nucleus, whereas other cortical areas contained low level of Fos expression. These results provide the first functional morphological evidence suggesting that these forebrain structures with Fos expression may play an important role in the pathophysiology of muscimol-induced absence seizures.

Adenovirus mediated gene transfer in organotypic brain slices.

A replication-defective adenovirus vector carrying the reporter gene encoding beta-galactosidase was used to transfect organotypic slices maintained in culture for up to 1 month. Three different delivery systems were used to inoculate the viral solution, either into the culture medium, or directly onto the surface of the slices or by microinjection into the tissue. Using the two first paradigms beta-galactosidase expressing cells were mostly of glial phenotype and distributed throughout the slices without any specific regional pattern. In contrast, microinjection of the adenovirus resulted in a large number of both infected neurones and glia, concentrated at the site of injection. This method thus appears to be able to circumvent some of the constraints and limitations associated with in vivo gene transfer.

Adenoviral vectors as functional retrograde neuronal tracers.

Adenoviruses have been recently recognized as a highly efficient system for gene delivery to various tissues. The ability of replication-defective recombinant adenovirus to transfer the lacZ reporter gene encoding beta-galactosidase to nerve cells in various brain structures has been demonstrated. Here, on the continuation of these studies, we present evidence that the adenovirus can be transported in a retrograde manner to nerve cell bodies from axonal terminals. This method may be of great value for infecting selected subsets of specific neurons for either anatomo-functional studies or even therapeutic purposes.

The use of adenovirus vectors for intracerebral grafting of transfected nervous cells.

Grafting genetically-modified cells into the brain is a promising approach to address fundamental and clinical issues in neurobiology. Despite recent substantial progress, most of the methods used for introducing DNA sequences into donor cells result in weak efficacy or transient gene expression after transplantation. We tested whether the use of adenovirus as the vector for foreign genes avoided these problems. A replication-defective adenovirus vector carrying a reporter gene encoding for beta-galactosidase was used to transfect primary astrocytes. After grafting into various brain structures, transfected cells exhibited robust survival and expressed the transgene for at least five months. These results demonstrate the advantage of adenovirus-mediated gene transfer for prolonged transgene expression in grafted primary cells.

Ex vivo culture of adult microglial cells from previously lesioned rat brains.

In an attempt to study more precisely the glial cells involved in reactions following specific brain injuries, we tried to culture cells derived from surgically-lesioned rat brains or adult rat hippocampus previously treated with kainic acid, a convulsant which induces status epilepticus associated with structural modifications. We find that, contrary to cultures derived from normal adult rat brain, cultures from lesioned rat brains can survive and proliferate in vitro. Characterization of the cell types using double labeling with isolectin B4 for microglia and GFAP antisera for astrocytes shows that cultures from KA-treated adult rats consist of nearly 100% macrophagic-microglial cells, whereas those obtained from surgically-lesioned brains are composed of a mixed population of microglial cells and astrocytes. These models are proposed as suitable for the further study of microglial-neuronal interactions involved in brain damage and repair.

An adenovirus vector for gene transfer into neurons and glia in the brain.

The efficient introduction of genetic material into quiescent nerve cells is important in the study of brain function and for gene therapy of neurological disorders. A replication-deficient adenoviral vector that contained a reporter gene encoding beta-galactosidase infected rat nerve cells in vitro and in vivo. beta-Galactosidase was expressed in almost all sympathetic neurons and astrocytes in culture. After stereotactic inoculations into the rat hippocampus and the substantia nigra, beta-galactosidase activity was detected for 2 months. Infected cells were identified as microglial cells, astrocytes, or neurons with anatomical, morphological, and immunohistochemical criteria. No obvious cytopathic effect was observed.