Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors

Uncontrolled insertion of gene transfer vectors into the human genome is raising significant safety concerns for their clinical use. The wild-type adeno-associated virus (AAV) can insert its genome at a specific site in human chromosome 19 (AAVS1) through the activity of a specific replicase/integrase protein (Rep) binding both the AAVS1 and the viral inverted terminal repeats (ITRs). AAV-derived vectors, however, do not carry the rep gene and cannot maintain site-specific integration properties. We describe a novel hybrid vector carrying an integration cassette flanked by AAV ITRs and a tightly regulated, drug-inducible Rep expression cassette in the framework of a high-capacity, helper-dependent adenoviral (Ad) vector. Rep-dependent integration of ITR-flanked cassettes of intact size and function was obtained in human primary cells and cell lines in the absence of selection. The majority of integrations were site specific and occurred within a 1000-bp region of the AAVS1. Genome-wide sequencing of integration junctions indicates that nonspecific integrations occurred predominantly in intergenic regions. Site-specific integration was obtained also in vivo, in an AAVS1 transgenic mouse model: upon a single tail vein administration of a nontoxic dose of Ad/AAV vectors, AAVS1-specific integrations were detected and sequenced in DNA obtained from the liver of all animals in which Rep expression was induced by drug treatment. Nonrandom integration of double-stranded DNA can therefore be obtained ex vivo and in vivo by the use of hybrid Ad/AAV vectors, in the absence of toxicity and with efficiency compatible with gene therapy applications

Application of advanced brain positron emission tomography-based molecular imaging for a biological framework in neurodegenerative proteinopathies

IntroductionA rapid transition from a clinical‐based classification to a pathology‐based classification of neurodegenerative conditions, largely promoted by the increasing availability of imaging biomarkers, is emerging. The Framework for Innovative Multi‐tracer molecular Brain Imaging, funded by the EU Joint Program ‐ Neurodegenerative Disease Research 2016 “Working Groups for Harmonisation and Alignment in Brain Imaging Methods for Neurodegeneration,” aimed at providing a roadmap for the applications of established and new molecular imaging techniques in dementia.MethodsWe consider current and future implications of adopting a pathology‐based framework for the use and development of positron emission tomography techniques.ResultsThis approach will enhance efforts to understand the multifactorial etiology of Alzheimer's disease and other dementias.DiscussionThe availability of pathology biomarkers will soon transform clinical and research practice. Crucially, a comprehensive understanding of strengths and caveats of these techniques will promote an informed use to take full advantage of these tools.</p

Time-dependent recovery of brain hypometabolism in neuro-COVID-19 patients

Purpose We evaluated brain metabolic dysfunctions and associations with neurological and biological parameters in acute, subacute and chronic COVID-19 phases to provide deeper insights into the pathophysiology of the disease.Methods Twenty-six patients with neurological symptoms (neuro-COVID-19) and [F-18]FDG-PET were included. Seven patients were acute (&lt; 1 month (m) after onset), 12 subacute (4 &gt;= 1-m, 4 &gt;= 2-m and 4 &gt;= 3-m) and 7 with neuro-post-COVID-19 (3 &gt;= 5-m and 4 &gt;= 7-9-m). One patient was evaluated longitudinally (acute and 5-m). Brain hypo- and hypermetabolism were analysed at single-subject and group levels. Correlations between severity/extent of brain hypo- and hypermetabolism and biological (oxygen saturation and C-reactive protein) and clinical variables (global cognition and Body Mass Index) were assessed.Results The "fronto-insular cortex" emerged as the hypometabolic hallmark of neuro-COVID-19. Acute patients showed the most severe hypometabolism affecting several cortical regions. Three-m and 5-m patients showed a progressive reduction of hypometabolism, with limited frontal clusters. After 7-9 months, no brain hypometabolism was detected. The patient evaluated longitudinally showed a diffuse brain hypometabolism in the acute phase, almost recovered after 5 months. Brain hypometabolism correlated with cognitive dysfunction, low blood saturation and high inflammatory status. Hypermetabolism in the brainstem, cerebellum, hippocampus and amygdala persisted over time and correlated with inflammation status.Conclusion Synergistic effects of systemic virus-mediated inflammation and transient hypoxia yield a dysfunction of the fronto-insular cortex, a signature of CNS involvement in neuro-COVID-19. This brain dysfunction is likely to be transient and almost reversible. The long-lasting brain hypermetabolism seems to reflect persistent inflammation processes

Occipital hypometabolism is a risk factor for conversion to Parkinson’s disease in isolated REM sleep behaviour disorder

Purpose: Isolated REM sleep behaviour disorder (iRBD) patients are at high risk of developing clinical syndromes of the α-synuclein spectrum. Progression markers are needed to determine the neurodegenerative changes and to predict their conversion. Brain imaging with 18F-FDG PET in iRBD is promising, but longitudinal studies are scarce. We investigated the regional brain changes in iRBD over time, related to phenoconversion.Methods: Twenty iRBD patients underwent two consecutive 18F-FDG PET brain scans and clinical assessments (3.7 ± 0.6 years apart). Seventeen patients also underwent 123I-MIBG and 123I-FP-CIT SPECT scans at baseline. Four subjects phenoconverted to Parkinson’s disease (PD) during follow-up. 18F-FDG PET scans were compared to controls with a voxel-wise single-subject procedure. The relationship between regional brain changes in metabolism and PD-related pattern scores (PDRP) was investigated.Results: Individual hypometabolism t-maps revealed three scenarios: (1) normal 18F-FDG PET scans at baseline and follow-up (N = 10); (2) normal scans at baseline but occipital or occipito-parietal hypometabolism at follow-up (N = 4); (3) occipital hypometabolism at baseline and follow-up (N = 6). All patients in the last group had pathological 123I-MIBG and 123I-FP-CIT SPECT. iRBD converters (N = 4) showed occipital hypometabolism at baseline (third scenario). At the group level, hypometabolism in the frontal and occipito-parietal regions and hypermetabolism in the cerebellum and limbic regions were progressive over time. PDRP z-scores increased over time (0.54 ± 0.36 per year). PDRP expression was driven by occipital hypometabolism and cerebellar hypermetabolism.Conclusions: Our results suggest that occipital hypometabolism at baseline in iRBD implies a short-term conversion to PD. This might help in stratification strategies for disease-modifying trials.</p

Unfavourable gender effect of high body mass index on brain metabolism and connectivity

The influence of Body Mass Index (BMI) on neurodegeneration in dementia has yet to be elucidated. We aimed at exploring the effects of BMI levels on cerebral resting-state metabolism and brain connectivity, as crucial measures of synaptic function and activity, in a large group of patients with Alzheimer\u2019s Dementia (AD) (n = 206), considering gender. We tested the correlation between BMI levels and brain metabolism, as assessed by18F-FDG-PET, and the modulation of the resting-state functional networks by BMI. At comparable dementia severity, females with high BMI can withstand a lower degree of brain metabolism dysfunction, as shown by a significant BMI-brain metabolism correlation in the temporal-parietal regions, which are typically vulnerable to AD pathology (R = 0.269, p = 0.009). Of note, high BMI was also associated with reduced connectivity in frontal and limbic brain networks, again only in AD females (p < 0.05 FDR-corrected, k = 100 voxels). This suggests a major vulnerability of neural systems known to be selectively involved in brain compensatory mechanisms in AD females. These findings indicate a strong gender effect of high BMI and obesity in AD, namely reducing the available reserve mechanisms in female patients. This brings to considerations for medical practice and health policy

Simultaneous PET-MRI Studies of the Concordance of Atrophy and Hypometabolism in Syndromic Variants of Alzheimer's Disease and Frontotemporal Dementia: An Extended Case Series

Background: Simultaneous PET-MRI is used to compare patterns of cerebral hypometabolism and atrophy in six different dementia syndromes. Objectives: The primary objective was to conduct an initial exploratory study regarding the concordance of atrophy and hypometabolism in syndromic variants of Alzheimer’s disease (AD) and frontotemporal dementia (FTD). The secondary objective was to determine the effect of image analysis methods on determination of atrophy and hypometabolism. Method: PET and MRI data were acquired simultaneously on 24 subjects with six variants of AD and FTD (n = 4 per group). Atrophy was rated visually and also quantified with measures of cortical thickness. Hypometabolism was rated visually and also quantified using atlas- and SPM-based approaches. Concordance was measured using weighted Cohen’s kappa. Results: Atrophy-hypometabolism concordance differed markedly between patient groups; kappa scores ranged from 0.13 (nonfluent/agrammatic variant of primary progressive aphasia, nfvPPA) to 0.49 (posterior cortical variant of AD, PCA). Heterogeneity was also observed within groups; the confidence intervals of kappa scores ranging from 0–0.25 for PCA to 0.29–0.61 for nfvPPA. More widespread MRI and PET changes were identified using quantitative methods than on visual rating. Conclusion: The marked differences in concordance identified in this initial study may reflect differences in the molecular pathologies underlying AD and FTD syndromic variants but also operational differences in the methods used to diagnose these syndromes. The superior ability of quantitative methodologies to detect changes on PET and MRI, if confirmed on larger cohorts, may favor their usage over qualitative visual inspection in future clinical diagnostic practic

Optimising EEG-fMRI for Localisation of Focal Epilepsy in Children

BACKGROUND: Early surgical intervention in children with drug resistant epilepsy has benefits but requires using tolerable and minimally invasive tests. EEG-fMRI studies have demonstrated good sensitivity for the localization of epileptic focus but a poor yield although the reasons for this have not been systematically addressed. While adults EEG-fMRI studies are performed in the "resting state"; children are commonly sedated however, this has associated risks and potential confounds. In this study, we assessed the impact of the following factors on the tolerability and results of EEG-fMRI in children: viewing a movie inside the scanner; movement; occurrence of interictal epileptiform discharges (IED); scan duration and design efficiency. This work's motivation is to optimize EEG-fMRI parameters to make this test widely available to paediatric population. METHODS: Forty-six children with focal epilepsy and 20 controls (6-18) underwent EEG-fMRI. For two 10 minutes sessions subjects were told to lie still with eyes closed, as it is classically performed in adult studies ("rest sessions"), for another two sessions, subjects watched a child friendly stimulation i.e. movie ("movie sessions"). IED were mapped with EEG-fMRI for each session and across sessions. The resulting maps were classified as concordant/discordant with the presumed epileptogenic focus for each subject. FINDINGS: Movement increased with scan duration, but the movie reduced movement by ~40% when played within the first 20 minutes. There was no effect of movie on the occurrence of IED, nor in the concordance of the test. Ability of EEG-fMRI to map the epileptogenic region was similar for the 20 and 40 minute scan durations. Design efficiency was predictive of concordance. CONCLUSIONS: A child friendly natural stimulus improves the tolerability of EEG-fMRI and reduces in-scanner movement without having an effect on IED occurrence and quality of EEG-fMRI maps. This allowed us to scan children as young as 6 and obtain localising information without sedation. Our data suggest that ~20 minutes is the optimal length of scanning for EEG-fMRI studies in children with frequent IED. The efficiency of the fMRI design derived from spontaneous IED generation is an important factor for producing concordant results

EGFR oligomerization organizes kinase-active dimers into competent signalling platforms

Epidermal growth factor receptor (EGFR) signalling is activated by ligand-induced receptor dimerization. Notably, ligand binding also induces EGFR oligomerization, but the structures and functions of the oligomers are poorly understood. Here, we use fluorophore localization imaging with photobleaching to probe the structure of EGFR oligomers. We find that at physiological epidermal growth factor (EGF) concentrations, EGFR assembles into oligomers, as indicated by pairwise distances of receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand distances correspond well with the predictions of our structural model of the oligomers constructed from molecular dynamics simulations. The model suggests that oligomerization is mediated extracellularly by unoccupied ligand-binding sites and that oligomerization organizes kinase-active dimers in ways optimal for auto-phosphorylation in trans between neighbouring dimers. We argue that ligand-induced oligomerization is essential to the regulation of EGFR signalling

The epidemiology of Varicella Zoster Virus infection in Italy

<p>Abstract</p> <p>Background</p> <p>The epidemiological importance of varicella and zoster and the availability of an efficacious and safe vaccine have led to an important international debate regarding the suitability of mass vaccination. The objective of the study was to describe the epidemiology of varicella and zoster in Italy and to determine whether there have been changes with respect to observations provided by an analogous study conducted 8 years ago, in order to define the most appropriate vaccination strategy.</p> <p>Methods</p> <p>A number of data sources were evaluated, a cross-sectional population-based seroprevalence study was conducted on samples collected in 2004, and the results were compared with data obtained in 1996.</p> <p>Results</p> <p>The data from active and passive surveillance systems confirm that varicella is a widespread infectious disease which mainly affects children. VZV seroprevalence did not substantially differ from that found in the previous study. The sero-epidemiological profile in Italy is different from that in other European countries. In particular, the percentage of susceptible adolescents is at least nearly twice as high as in other European countries and in the age group 20–39 yrs, approximately 9% of individuals are susceptible to VZV.</p> <p>Conclusion</p> <p>The results of this study can contribute to evaluating the options for varicella vaccination. It is possible that in a few years, in all Italian Regions, there will exist the conditions necessary for implementing a mass vaccination campaign and that the large-scale availability of MMRV tetravalent vaccines will facilitate mass vaccination.</p