Directed integration of new insulated lentiviral vectors to heterochromatin towards safer gene transfer to stem cells

The need for better gene transfer systems towards improved risk=benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT). We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing genetic integration to heterochromatin. We have designed SIN-insulated retrovectors with two candidate GIEs and could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) (see Duros et al, abstract ibid). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested with chimeric HIV-1 derived integrases which comprise C-ter chromodomains targeting heterochromatin through either histone H3 (ML6chimera) or methylatedCpGislands (ML10). With DCaro4 only and both chimeras, a homogeneous expression is evidenced in over 20% of the cells which is sustained over time. With control lentivectors, less than 2% of cells express GFP as compared to background using a control double-mutant in both catalytic and ledgf binding-sites; in addition, a two-times increase of expression can be induced with histone deacetylase inhibitors. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity, according to X-SCIDs patients data.Work performed with the support of EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933

Endogenous opioid peptides in parasympathetic, sympathetic and sensory nerves in the guinea-pig heart.

Research has suggested that exogenous opioid substances can have direct effects on cardiac muscle or influence neurotransmitter release via presynaptic modulation of neuronal inputs to the heart. In the present study, multiple-labelling immunohistochemistry was employed to determine the distribution of endogenous opioid peptides within the guinea-pig heart. Approximately 40% of cardiac ganglion cells contained immunoreactivity for dynorphin A (1-8), dynorphin A (1-17) and dynorphin B whilst 20% displayed leu-enkephalin immunoreactivity. Different populations of opioid-containing ganglion cells were identified according to the co-existence of opioid immunoreactivity with immunoreactivity for somatostatin and neuropeptide Y. Immunoreactivity for prodynorphin-derived peptides was observed in many sympathetic axons in the heart and was also observed, though to a lesser extent, in sensory axons. Leu-enkephalin immunoreactivity was observed in occasional sympathetic and sensory axons. No immunoreactivity was observed for met-enkephalin-arg-gly-leu or for beta-endorphin. These results demonstrate that prodynorphin-derived peptides are present in parasympathetic, sympathetic and sensory nerves within the heart, but suggest that only the prodynorphin gene is expressed in guinea-pig cardiac nerves. This study has shown that endogenous opioid peptides are well placed to regulate cardiac function via both autonomic and sensory pathways.

The behavioral relevance and top-down susceptibility of early, low level multisensory interactions in humans

Abstract (English)General backgroundMultisensory stimuli are easier to recognize, can improve learning and a processed faster compared to unisensory ones. As such, the ability an organism has to extract and synthesize relevant sensory inputs across multiple sensory modalities shapes his perception of and interaction with the environment. A major question in the scientific field is how the brain extracts and fuses relevant information to create a unified perceptual representation (but also how it segregates unrelated information). This fusion between the senses has been termed "multisensory integration", a notion that derives from seminal animal single-cell studies performed in the superior colliculus, a subcortical structure shown to create a multisensory output differing from the sum of its unisensory inputs. At the cortical level, integration of multisensory information is traditionally deferred to higher classical associative cortical regions within the frontal, temporal and parietal lobes, after extensive processing within the sensory-specific and segregated pathways. However, many anatomical, electrophysiological and neuroimaging findings now speak for multisensory convergence and interactions as a distributed process beginning much earlier than previously appreciated and within the initial stages of sensory processing.The work presented in this thesis is aimed at studying the neural basis and mechanisms of how the human brain combines sensory information between the senses of hearing and touch. Early latency non-linear auditory-somatosensory neural response interactions have been repeatedly observed in humans and non-human primates. Whether these early, low-level interactions are directly influencing behavioral outcomes remains an open question as they have been observed under diverse experimental circumstances such as anesthesia, passive stimulation, as well as speeded reaction time tasks. Under laboratory settings, it has been demonstrated that simple reaction times to auditory-somatosensory stimuli are facilitated over their unisensory counterparts both when delivered to the same spatial location or not, suggesting that audi- tory-somatosensory integration must occur in cerebral regions with large-scale spatial representations. However experiments that required the spatial processing of the stimuli have observed effects limited to spatially aligned conditions or varying depending on which body part was stimulated. Whether those divergences stem from task requirements and/or the need for spatial processing has not been firmly established.Hypotheses and experimental resultsIn a first study, we hypothesized that auditory-somatosensory early non-linear multisensory neural response interactions are relevant to behavior. Performing a median split according to reaction time of a subset of behavioral and electroencephalographic data, we found that the earliest non-linear multisensory interactions measured within the EEG signal (i.e. between 40-83ms post-stimulus onset) were specific to fast reaction times indicating a direct correlation of early neural response interactions and behavior.In a second study, we hypothesized that the relevance of spatial information for task performance has an impact on behavioral measures of auditory-somatosensory integration. Across two psychophysical experiments we show that facilitated detection occurs even when attending to spatial information, with no modulation according to spatial alignment of the stimuli. On the other hand, discrimination performance with probes, quantified using sensitivity (d'), is impaired following multisensory trials in general and significantly more so following misaligned multisensory trials.In a third study, we hypothesized that behavioral improvements might vary depending which body part is stimulated. Preliminary results suggest a possible dissociation between behavioral improvements andERPs. RTs to multisensory stimuli were modulated by space only in the case when somatosensory stimuli were delivered to the neck whereas multisensory ERPs were modulated by spatial alignment for both types of somatosensory stimuli.ConclusionThis thesis provides insight into the functional role played by early, low-level multisensory interac-tions. Combining psychophysics and electrical neuroimaging techniques we demonstrate the behavioral re-levance of early and low-level interactions in the normal human system. Moreover, we show that these early interactions are hermetic to top-down influences on spatial processing suggesting their occurrence within cerebral regions having access to large-scale spatial representations. We finally highlight specific interactions between auditory space and somatosensory stimulation on different body parts. Gaining an in-depth understanding of how multisensory integration normally operates is of central importance as it will ultimately permit us to consider how the impaired brain could benefit from rehabilitation with multisensory stimula-Abstract (French)Background théoriqueDes stimuli multisensoriels sont plus faciles à reconnaître, peuvent améliorer l'apprentissage et sont traités plus rapidement comparé à des stimuli unisensoriels. Ainsi, la capacité qu'un organisme possède à extraire et à synthétiser avec ses différentes modalités sensorielles des inputs sensoriels pertinents, façonne sa perception et son interaction avec l'environnement. Une question majeure dans le domaine scientifique est comment le cerveau parvient à extraire et à fusionner des stimuli pour créer une représentation percep- tuelle cohérente (mais aussi comment il isole les stimuli sans rapport). Cette fusion entre les sens est appelée "intégration multisensorielle", une notion qui provient de travaux effectués dans le colliculus supérieur chez l'animal, une structure sous-corticale possédant des neurones produisant une sortie multisensorielle différant de la somme des entrées unisensorielles. Traditionnellement, l'intégration d'informations multisen- sorielles au niveau cortical est considérée comme se produisant tardivement dans les aires associatives supérieures dans les lobes frontaux, temporaux et pariétaux, suite à un traitement extensif au sein de régions unisensorielles primaires. Cependant, plusieurs découvertes anatomiques, électrophysiologiques et de neuroimageries remettent en question ce postulat, suggérant l'existence d'une convergence et d'interactions multisensorielles précoces.Les travaux présentés dans cette thèse sont destinés à mieux comprendre les bases neuronales et les mécanismes impliqués dans la combinaison d'informations sensorielles entre les sens de l'audition et du toucher chez l'homme. Des interactions neuronales non-linéaires précoces audio-somatosensorielles ont été observées à maintes reprises chez l'homme et le singe dans des circonstances aussi variées que sous anes- thésie, avec stimulation passive, et lors de tâches nécessitant un comportement (une détection simple de stimuli, par exemple). Ainsi, le rôle fonctionnel joué par ces interactions à une étape du traitement de l'information si précoce demeure une question ouverte. Il a également été démontré que les temps de réaction en réponse à des stimuli audio-somatosensoriels sont facilités par rapport à leurs homologues unisensoriels indépendamment de leur position spatiale. Ce résultat suggère que l'intégration audio- somatosensorielle se produit dans des régions cérébrales possédant des représentations spatiales à large échelle. Cependant, des expériences qui ont exigé un traitement spatial des stimuli ont produits des effets limités à des conditions où les stimuli multisensoriels étaient, alignés dans l'espace ou encore comme pouvant varier selon la partie de corps stimulée. Il n'a pas été établi à ce jour si ces divergences pourraient être dues aux contraintes liées à la tâche et/ou à la nécessité d'un traitement de l'information spatiale.Hypothèse et résultats expérimentauxDans une première étude, nous avons émis l'hypothèse que les interactions audio- somatosensorielles précoces sont pertinentes pour le comportement. En effectuant un partage des temps de réaction par rapport à la médiane d'un sous-ensemble de données comportementales et électroencépha- lographiques, nous avons constaté que les interactions multisensorielles qui se produisent à des latences précoces (entre 40-83ms) sont spécifique aux temps de réaction rapides indiquant une corrélation directe entre ces interactions neuronales précoces et le comportement.Dans une deuxième étude, nous avons émis l'hypothèse que si l'information spatiale devient perti-nente pour la tâche, elle pourrait exercer une influence sur des mesures comportementales de l'intégration audio-somatosensorielles. Dans deux expériences psychophysiques, nous montrons que même si les participants prêtent attention à l'information spatiale, une facilitation de la détection se produit et ce toujours indépendamment de la configuration spatiale des stimuli. Cependant, la performance de discrimination, quantifiée à l'aide d'un index de sensibilité (d') est altérée suite aux essais multisensoriels en général et de manière plus significative pour les essais multisensoriels non-alignés dans l'espace.Dans une troisième étude, nous avons émis l'hypothèse que des améliorations comportementales pourraient différer selon la partie du corps qui est stimulée (la main vs. la nuque). Des résultats préliminaires suggèrent une dissociation possible entre une facilitation comportementale et les potentiels évoqués. Les temps de réactions étaient influencés par la configuration spatiale uniquement dans le cas ou les stimuli somatosensoriels étaient sur la nuque alors que les potentiels évoqués étaient modulés par l'alignement spatial pour les deux types de stimuli somatosensorielles.ConclusionCette thèse apporte des éléments nouveaux concernant le rôle fonctionnel joué par les interactions multisensorielles précoces de bas niveau. En combinant la psychophysique et la neuroimagerie électrique, nous démontrons la pertinence comportementale des ces interactions dans le système humain normal. Par ailleurs, nous montrons que ces interactions précoces sont hermétiques aux influences dites «top-down» sur le traitement spatial suggérant leur occurrence dans des régions cérébrales ayant accès à des représentations spatiales de grande échelle. Nous soulignons enfin des interactions spécifiques entre l'espace auditif et la stimulation somatosensorielle sur différentes parties du corps. Approfondir la connaissance concernant les bases neuronales et les mécanismes impliqués dans l'intégration multisensorielle dans le système normale est d'une importance centrale car elle permettra d'examiner et de mieux comprendre comment le cerveau déficient pourrait bénéficier d'une réhabilitation avec la stimulation multisensorielle.

Binding under Conflict Conditions: State-Space Analysis of Multivariate EEG Synchronization.

Real-world objects are often endowed with features that violate Gestalt principles. In our experiment, we examined the neural correlates of binding under conflict conditions in terms of the binding-by-synchronization hypothesis. We presented an ambiguous stimulus ("diamond illusion") to 12 observers. The display consisted of four oblique gratings drifting within circular apertures. Its interpretation fluctuates between bound ("diamond") and unbound (component gratings) percepts. To model a situation in which Gestalt-driven analysis contradicts the perceptually explicit bound interpretation, we modified the original diamond (OD) stimulus by speeding up one grating. Using OD and modified diamond (MD) stimuli, we managed to dissociate the neural correlates of Gestalt-related (OD vs. MD) and perception-related (bound vs. unbound) factors. Their interaction was expected to reveal the neural networks synchronized specifically in the conflict situation. The synchronization topography of EEG was analyzed with the multivariate S-estimator technique. We found that good Gestalt (OD vs. MD) was associated with a higher posterior synchronization in the beta-gamma band. The effect of perception manifested itself as reciprocal modulations over the posterior and anterior regions (theta/beta-gamma bands). Specifically, higher posterior and lower anterior synchronization supported the bound percept, and the opposite was true for the unbound percept. The interaction showed that binding under challenging perceptual conditions is sustained by enhanced parietal synchronization. We argue that this distributed pattern of synchronization relates to the processes of multistage integration ranging from early grouping operations in the visual areas to maintaining representations in the frontal networks of sensory memory.

Looming signals reveal synergistic principles of multisensory integration.

Multisensory interactions are a fundamental feature of brain organization. Principles governing multisensory processing have been established by varying stimulus location, timing and efficacy independently. Determining whether and how such principles operate when stimuli vary dynamically in their perceived distance (as when looming/receding) provides an assay for synergy among the above principles and also means for linking multisensory interactions between rudimentary stimuli with higher-order signals used for communication and motor planning. Human participants indicated movement of looming or receding versus static stimuli that were visual, auditory, or multisensory combinations while 160-channel EEG was recorded. Multivariate EEG analyses and distributed source estimations were performed. Nonlinear interactions between looming signals were observed at early poststimulus latencies (∼75 ms) in analyses of voltage waveforms, global field power, and source estimations. These looming-specific interactions positively correlated with reaction time facilitation, providing direct links between neural and performance metrics of multisensory integration. Statistical analyses of source estimations identified looming-specific interactions within the right claustrum/insula extending inferiorly into the amygdala and also within the bilateral cuneus extending into the inferior and lateral occipital cortices. Multisensory effects common to all conditions, regardless of perceived distance and congruity, followed (∼115 ms) and manifested as faster transition between temporally stable brain networks (vs summed responses to unisensory conditions). We demonstrate the early-latency, synergistic interplay between existing principles of multisensory interactions. Such findings change the manner in which to model multisensory interactions at neural and behavioral/perceptual levels. We also provide neurophysiologic backing for the notion that looming signals receive preferential treatment during perception.

Esophagogastric cancer: integration of targeted therapies into systemic chemotherapy.

Although combination chemotherapy has been shown to be more effective than single agents in advanced esophagogastric cancer, the better response rates have not fulfilled their promise as overall survival times from best combination still range between 8 to 11 months. So far, the development of targeted therapies stays somewhat behind their integration into treatment concepts compared to other gastrointestinal diseases. Thus, the review summarizes the recent advances in the development of targeted therapies in advanced esophagogastric cancer. The majority of agents tested were angiogenesis inhibitors or agents targeting the epidermal growth factor receptors EGFR1 and HER2. For trastuzumab and bevacizumab, phase III trial results have been presented recently. While addition of trastuzumab to cisplatin/5-fluoropyrimidine-based chemotherapy results in a clinically relevant and statistically significant survival benefit in HER 2+ patients, the benefit of the addition of bevacizumab to chemotherapy was not significant. Thus, all patients with metastatic disease should be tested for HER-2 status in the tumor. Trastuzumab in combination with cisplatin/5-fluoropyrimidine-based chemotherapy is the new standard of care for patients with HER2-positive advanced gastric cancer.

Control of landslide retrogression by discontinuities: Evidence by the integration of airbone- and ground-based geophysical information

The objective of this work is to present a multitechnique approach to define the geometry, the kinematics, and the failure mechanism of a retrogressive large landslide (upper part of the La Valette landslide, South French Alps) by the combination of airborne and terrestrial laser scanning data and ground-based seismic tomography data. The advantage of combining different methods is to constrain the geometrical and failure mechanism models by integrating different sources of information. Because of an important point density at the ground surface (4. 1 points m?2), a small laser footprint (0.09 m) and an accurate three-dimensional positioning (0.07 m), airborne laser scanning data are adapted as a source of information to analyze morphological structures at the surface. Seismic tomography surveys (P-wave and S-wave velocities) may highlight the presence of low-seismic-velocity zones that characterize the presence of dense fracture networks at the subsurface. The surface displacements measured from the terrestrial laser scanning data over a period of 2 years (May 2008?May 2010) allow one to quantify the landslide activity at the direct vicinity of the identified discontinuities. An important subsidence of the crown area with an average subsidence rate of 3.07 m?year?1 is determined. The displacement directions indicate that the retrogression is controlled structurally by the preexisting discontinuities. A conceptual structural model is proposed to explain the failure mechanism and the retrogressive evolution of the main scarp. Uphill, the crown area is affected by planar sliding included in a deeper wedge failure system constrained by two preexisting fractures. Downhill, the landslide body acts as a buttress for the upper part. Consequently, the progression of the landslide body downhill allows the development of dip-slope failures, and coherent blocks start sliding along planar discontinuities. The volume of the failed mass in the crown area is estimated at 500,000 m3 with the sloping local base level method.

The value of 'positive' clinical signs for weakness, sensory and gait disorders in conversion disorder: a systematic and narrative review.

Experts in the field of conversion disorder have suggested for the upcoming DSM-V edition to put less weight on the associated psychological factors and to emphasise the role of clinical findings. Indeed, a critical step in reaching a diagnosis of conversion disorder is careful bedside neurological examination, aimed at excluding organic signs and identifying 'positive' signs suggestive of a functional disorder. These positive signs are well known to all trained neurologists but their validity is still not established. The aim of this study is to provide current evidence regarding their sensitivity and specificity. We conducted a systematic search on motor, sensory and gait functional signs in Embase, Medline, PsycINfo from 1965 to June 2012. Studies in English, German or French reporting objective data on more than 10 participants in a controlled design were included in a systematic review. Other relevant signs are discussed in a narrative review. Eleven controlled studies (out of 147 eligible articles) describing 14 signs (7 motor, 5 sensory, 2 gait) reported low sensitivity of 8-100% but high specificity of 92-100%. Studies were evidence class III, only two had a blinded design and none reported on inter-rater reliability of the signs. Clinical signs for functional neurological symptoms are numerous but only 14 have been validated; overall they have low sensitivity but high specificity and their use should thus be recommended, especially with the introduction of the new DSM-V criteria.

Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues.

Primary sensory neurons display various neuronal phenotypes which may be influenced by factors present in central or peripheral targets. In the case of DRG cells expressing substance P (SP), the influence of peripheral or central targets was tested on the neuronal expression of this neuropeptide. DRG cells were cultured from chick embryo at E6 or E10 (before or after establishment of functional connections with targets). Preprotachykinin mRNA was visualized in DRG cell cultures by either Northern blot or in situ hybridization using an antisense labeled riboprobe, while the neuropeptide SP was detected by immunostaining with a monoclonal antibody. In DRG cell cultures from E10, only 60% of neurons expressed SP. In contrast, DRG cell cultures performed at E6 showed a significant hybridization signal and SP-like immunoreactivity in virtually all the neurons (98%). The addition of extracts from muscle, skin, brain or spinal cord to DRG cells cultured at E6 reduced by 20% the percentage of neurons which express preprotachykinin mRNA and SP-like immunoreactivity. Our results indicate that factors issued from targets inhibit SP-expression by a subset of primary sensory neurons and act on the transcriptional control of preprotachykinin gene.