Search

Purpose This study aims to explore the overlap between symptoms of depression, anxiety, irritability and aggressiveness in autism spectrum disorder (ASD), to measure specific and idiosyncratic emotional responses. Design/methodology/approach A total of 42 high functioning adolescents and adults, between 12 and 39 years old, meeting the diagnostic and statistical manual of mental disorders – 5 criteria for ASD were selected from the InFoR Autism cohort. Data were analyzed in an exploratory way using Hill and Smith and K-medoids cluster analysis. Findings The authors found an aggregation of anxiety, depression, aggressive behaviors and irritability. Cluster analysis was maximized for two groups with 17 and 25 participants, respectively. The first group was characterized by high levels of symptoms of irritability, aggressiveness, hyperactivity and intermediate levels of anxiety and depression. In the first group, participants had significantly higher levels of autistic symptoms considering the social responsiveness scale and repetitive behavior scale-revised scales (relatives’ reports) suggesting that a particular group of subjects with a high level of ASD specific symptoms may express anxiety and depression in a specific way based on externalizing behaviors in addition to the common mood and anxiety symptoms. Research limitations/implications Improved understanding of the aggregation of externalized symptoms with symptoms of anxiety and mood disorders in ASD should lead to a better understanding of the underlying mechanisms related to emotion dysregulation in ASD. Practical implications Improved knowledge of the symptoms could lead to enhanced detection of psychiatric comorbidities in ASD. Originality/value The study was based on a transdiagnostic approach of psychiatric symptoms in individuals with ASD. Aggregation and clustering analysis was used to explore naive patterns of these psychiatric symptoms.

Autism spectrum disorder (ASD) is a developmental disorder underdiagnosed in adults. To date, no consistent evidence of alterations in brain structure has been reported in adults with ASD and few studies were conducted at that age. We analyzed structural magnetic resonance imaging data from 167 high functioning adults with ASD and 195 controls. We ran our analyses on a discovery (n = 301) and a replication sample (n = 61). The right caudal anterior cingulate cortical thickness was significantly thinner in adults with ASD compared to controls in both the discovery and the replication sample. Our work underlines the relevance of studying the brain anatomy of an adult ASD population.

Attention Deficit and Hyperactive Disorder (ADHD) and Autism Spectrum Disorders (ASD) are frequent comorbid neurodevelopmental conditions and the overlap between both disorders remains to be delineated. A more complete understanding of the shared genetic and environmental factors is needed. Using a family-based method, we evaluated the risk of ADHD in a group of relatives with an ASD proband (ASD-) and a group of relatives with an ASD and ADHD proband (ASD+). We enrolled 1245 individuals in the study: 499 probands, their 746 first-degree relatives and 140 controls. We used a multivariate generalized estimating equation (GEE) model, in which the dependent variable was the ADHD diagnosis in the relatives and the independent variable the ASD+ or ASD- in probands. We adjusted for sociodemographic factors (age, sex, IQ) and for the nature of the familial relationship with the affected proband (parent or sibling). Among the probands, there were 287 ASD- and 212 ASD+ individuals. ADHD was more frequent in relatives (19%) than in the control group (7%) (p = 0.001). The risk of ADHD was higher in the ASD+ relatives group than in the ASD- relatives group (GEE model OR 1.58 [95% CI 1.04-2.38], p = 0.032). This result was found in parents (OR 1.96 [95% CI 1.14-3.36], but not in siblings (OR 1.28 [95% CI 0.84-1.94], p = 0.434). Our study provides a representative estimate of the family distribution of ADHD in relatives of ASD probands but supports the modest effect of shared genetic and environmental factors between both disorders.

Objective The posterior superior temporal sulcus (pSTS) plays a critical role in the ‘social brain’. Its neurodevelopment and relationship with the social impairment in autism spectrum disorders (ASD) are not well understood. We explored the relationship between social cognition and the neurodevelopment of the pSTS in ASD. Method We included 44 adults with high-functioning ASD and 36 controls. We assessed their performances on the ‘Reading the mind in the eyes’ test (for 34 of 44 subjects with ASD and 30 of 36 controls), their fixation time on the eyes with eye tracking (for 35 of 44 subjects with ASD and 30 of 36 controls) and the morphology of the caudal branches of the pSTS (length and depth), markers of the neurodevelopment, with structural MRI. Results The right anterior caudal ramus of the pSTS was significantly longer in patients with ASD compared with controls (52.6 mm vs. 38.3 mm; P = 1.4 × 10−3; Cohen's d = 0.76). Its length negatively correlated with fixation time on the eyes (P = 0.03) in the ASD group and with the ‘Reading the mind in the eyes’ test scores in both groups (P = 0.03). Conclusion Our findings suggest that the neurodevelopment of the pSTS is related to the ASD social impairments.

The cerebellum is implicated in social cognition and is likely to be involved in the pathophysiology of autism spectrum disorder (ASD). The goal of our study was to explore cerebellar morphology in adults with ASD and its relationship to eye contact, as measured by fixation time allocated on the eye region using an eye-tracking device. Two-hundred ninety-four subjects with ASD and controls were included in our study and underwent a structural magnetic resonance imaging scan. Global segmentation and cortical parcellation of the cerebellum were performed. A sub-sample of 59 subjects underwent an eye tracking protocol in order to measure the fixation time allocated to the eye region. We did not observe any difference in global cerebellar volumes between ASD patients and controls; however, regional analyses found a decrease of the volume of the right anterior cerebellum in subjects with ASD compared to controls. There were significant correlations between fixation time on eyes and the volumes of the vermis and Crus I. Our results suggest that cerebellar morphology may be related to eye avoidance and reduced social attention. Eye tracking may be a promising neuro-anatomically based stratifying biomarker of ASD.

Background: Gene electrotrotransfer describes the use of electric pulses to transfer DNA to cells. Particularly skeletal muscle has potential for systemic secretion of therapeutic proteins. Gene electrotransfer to muscle using the integrin inhibitor plasmid AMEP (Antiangiogenic MEtargidin Peptide) was investigated in a phase I dose escalation study. Primary objective was safety. Material and methods: Patients with metastatic or locally advanced solid tumors, without further standard treatments available, were treated with once-only gene electrotransfer of plasmid AMEP to the femoral muscle. Safety was monitored by adverse events registration, visual analog scale (VAS) after procedure and magnetic resonance imaging (MRI) of treated muscles. Pharmacokinetics of plasmid AMEP in plasma and urine was determined by quantitative polymerase chain reaction. Response was evaluated by positron emission tomography–computed tomography (PET–CT) scans. Results: Seven patients were enrolled and treated at dose levels from 50 to 250 μg of plasmid AMEP, the study was terminated early due to cessation of plasmid production. Minimal systemic toxicity was observed and only transient mild pain was associated with the delivery of the electric pulses. MRI of the treated muscles revealed discrete intramuscular edema 24 h after treatment. The changes in the muscle tissue resolved within 2 weeks after treatment. Peak concentrations of plasmid AMEP was detected only in plasma within the first 24 hours after injection. Protein AMEP could not be detected, which could be due to the limit of detection. No objective responses were seen. Conclusions: Gene electrotransfer of plasmid AMEP was found to be safe and tolerable. No objective responses were observed but other DNA drugs may be tested in the future using this procedure.

Importance Intravitreal gene therapy is regarded as generally safe with limited mild adverse events, but its systemic effects remain to be investigated. Objective To examine the association between immune response and intraocular inflammation after ocular gene therapy with recombinant adeno-associated virus 2 carrying the ND4 gene (rAAV2/2-ND4). Design, setting, and participants This secondary analysis of an open-label, dose-escalation phase 1/2 randomized clinical trial of rAAV2/2-ND4 included data from February 13, 2014 (first patient visit), to March 30, 2017 (last patient visit at week 96), the first 2 years after injection. Patients older than 15 years with diagnosed ND4 Leber hereditary optic neuropathy (LHON) and visual acuity of at least counting fingers were enrolled in 1 of 5 cohorts. Four dose cohorts of 3 patients each were treated sequentially. An extension cohort of 3 patients received the dose of 9 × 1010 viral genomes per eye. Interventions Patients received increasing doses of rAAV2/2-ND4 (9 × 109, 3 × 1010, 9 × 1010, and 1.8 × 1011 viral genomes per eye) as a single unilateral intravitreal injection. Patients were monitored for 96 weeks after injection; ocular examinations were performed regularly, and blood samples were collected for immunologic testing. Main outcomes and measures A composite ocular inflammation score (OIS) was calculated based on grades of anterior chamber cells and flare, vitreous cells, and haze according to the Standardization of Uveitis Nomenclature. The systemic immune response was quantified by enzyme-linked immunospot (cellular immune response), enzyme-linked immunosorbent assay (IgG titers), and luciferase assay (neutralizing antibody [NAb] titers). Results The present analysis included 15 patients (mean [SD] age, 47.9 [17.2] years; 13 men and 2 women) enrolled in the 5 cohorts of the clinical trial. Thirteen patients experienced intraocular inflammation after rAAV2/2-ND4 administration. Mild anterior chamber inflammation and vitritis were reported at all doses, and all cases were responsive to treatment. A maximum OIS of 9.5 was observed in a patient with history of idiopathic uveitis. Overall, OIS was not associated with the viral dose administered. No NAbs against AAV2 were detected in aqueous humor before treatment. Two patients tested positive for cellular immune response against AAV2 at baseline and after treatment. Humoral immune response was not apparently associated with the dose administered or with the immune status of patients at baseline. No association was found between OISs and serum NAb titers. Conclusions and relevance In this study, intravitreal administration of rAAV2/2-ND4 in patients with LHON was safe and well tolerated. Further investigations may shed light into the local immune response to rAAV2/2-ND4 as a potential explanation for the observed intraocular inflammation.

Despite the high clinical burden, little is known about pathophysiology underlying autism spectrum disorder (ASD). Recent resting-state functional magnetic resonance imaging (rs-fMRI) studies have found atypical synchronization of brain activity in ASD. However, no consensus has been reached on the nature and clinical relevance of these alterations. Here, we addressed these questions in four large ASD cohorts. Using rs-fMRI, we identified functional connectivity alterations associated with ASD. We tested for associations of these imaging phenotypes with clinical and demographic factors such as age, sex, medication status, and clinical symptom severity. Our results showed reproducible patterns of ASD-associated functional hyper- and hypoconnectivity. Hypoconnectivity was primarily restricted to sensory-motor regions, whereas hyperconnectivity hubs were predominately located in prefrontal and parietal cortices. Shifts in cortico-cortical between-network connectivity from outside to within the identified regions were shown to be a key driver of these abnormalities. This reproducible pathophysiological phenotype was partially associated with core ASD symptoms related to communication and daily living skills and was not affected by age, sex, or medication status. Although the large effect sizes in standardized cohorts are encouraging with respect to potential application as a treatment and for patient stratification, the moderate link to clinical symptoms and the large overlap with healthy controls currently limit the usability of identified alterations as diagnostic or efficacy readout.

Autism spectrum disorder (ASD) is a developmental disorder underdiagnosed in adults. To date, no consistent evidence of alterations in brain structure has been reported in adults with ASD and few studies were conducted at that age. We analyzed structural magnetic resonance imaging data from 167 high functioning adults with ASD and 195 controls. We ran our analyses on a discovery (n = 301) and a replication sample (n = 61). The right caudal anterior cingulate cortical thickness was significantly thinner in adults with ASD compared to controls in both the discovery and the replication sample. Our work underlines the relevance of studying the brain anatomy of an adult ASD population.

International audience; No abstract available

The current theory implying local, short-range overconnectivity in autism spectrum disorder, contrasting with long-range underconnectivity, is based on heterogeneous results, on limited data involving functional connectivity studies, on heterogeneous paediatric populations and non-specific methodologies. In this work, we studied short-distance structural connectivity in a homogeneous population of males with high-functioning autism spectrum disorder and used a novel methodology specifically suited for assessing U-shaped short-distance tracts, including a recently developed tractography-based atlas of the superficial white matter fibres. We acquired diffusion-weighted MRI for 58 males (27 subjects with high-functioning autism spectrum disorder and 31 control subjects) and extracted the mean generalized fractional anisotropy of 63 short-distance tracts. Neuropsychological evaluation included Wechsler Adult Intelligence Scale IV (WAIS-IV), Communication Checklist-Adult, Empathy Quotient, Social Responsiveness Scale and Behaviour Rating Inventory of Executive Function-Adult (BRIEF-A). In contradiction with the models of short-range over-connectivity in autism spectrum disorder, we found that patients with autism spectrum disorder had a significantly decreased anatomical connectivity in a component comprising 13 short tracts compared to controls. Specific short-tract atypicalities in temporal lobe and insula were significantly associated with clinical manifestations of autism spectrum disorder such as social awareness, language structure, pragmatic skills and empathy, emphasizing their importance in social dysfunction. Short-range decreased anatomical connectivity may thus be an important substrate of social deficits in autism spectrum disorder, in contrast with current models.

Despite the high clinical burden little is known about pathophysiology underlying autism spectrum disorder (ASD). Recent resting state functional magnetic resonance imaging (rs-fMRI) studies have found atypical synchronization of brain activity in ASD. However, no consensus has been reached on the nature and clinical relevance of these alterations. Here we address these questions in the most comprehensive, large-scale effort to date comprising evaluation of four large ASD cohorts. We followed a strict exploration and replication procedure to identify core rs-fMRI functional connectivity (degree centrality) alterations associated with ASD as compared to typically developing (TD) controls (ASD: N=841, TD: N=984). We then tested for associations of these imaging phenotypes with clinical and demographic factors such as age, sex, medication status and clinical symptom severity. We find reproducible patterns of ASD-associated functional hyper- and hypo-connectivity with hypo-connectivity being primarily restricted to sensory-motor regions and hyper-connectivity hubs being predominately located in prefrontal and parietal cortices. We establish shifts in between-network connectivity from outside to within the identified regions as a key driver of these abnormalities. The magnitude of these alterations is linked to core ASD symptoms related to communication and social interaction and is not affected by age, sex or medication status. The identified brain functional alterations provide a reproducible pathophysiological phenotype underlying the diagnosis of ASD reconciling previous divergent findings. The large effect sizes in standardized cohorts and the link to clinical symptoms emphasize the importance of the identified imaging alterations as potential treatment and stratification biomarkers for ASD.

Gene therapy with Plasmid AMEP (antiangiogenic metargidin peptide) has recently been studied as a potential targeted therapy for melanoma. This plasmid is designed to downregulate α5β1 and αvβ3 integrins. In our study, electroporation was used as a nonviral delivery system. We investigated the antiangiogenic and direct antitumor effectiveness of this gene therapy on low and highly metastatic B16 melanoma variants. In vitro, the antiangiogenic effectiveness as determined by tube formation assay on endothelial cells was predominantly dependent on AMEP expression levels. In vivo, antitumor effectiveness was mediated by the inhibition of proliferation, migration and invasion of melanoma cells and correlated with the expression of integrins on tumor cells after intratumor delivery. In addition, reduced metastatic potential was shown. Intramuscular gene electrotransfer of Plasmid AMEP, for AMEP systemic distribution, had no antitumor effect with this specific preventive treatment protocol, confirming that direct tumor delivery was more effective. This study confirms our previous in vitro data that the expression levels of integrins on melanoma cells could be used as a biomarker for antitumor effectiveness in integrin-targeted therapies, whereas the expression levels of AMEP peptide could be a predictive factor for antiangiogenic effectiveness of Plasmid AMEP in the treatment of melanoma.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by dysfunctions in social interactions resulting from a complex interplay between immunogenetic and environmental risk factors. Autoimmunity has been proposed as a major etiological component of ASD. Whether specific autoantibodies directed against brain targets are involved in ASD remains an open question. Here, we identified within a cohort an ASD patient with multiple circulating autoantibodies, including the well-characterized one against glutamate NMDA receptor (NMDAR-Ab). The patient exhibited alexithymia and previously suffered from two major depressive episodes without psychotic symptoms. Using a single molecule-based imaging approach, we demonstrate that neither NMDAR-Ab type G immunoglobulin purified from the ASD patient serum, nor that from a seropositive healthy subject, disorganize membrane NMDAR complexes at synapses. These findings suggest that the autistic patient NMDAR-Abs do not play a direct role in the etiology of ASD and that other autoantibodies directed against neuronal targets should be investigated.El trastorno del espectro autista (TEA) es un trastorno del neurodesarrollo caracterizado por dísfunciones en las interacciones sociales que se traducen en un complejo interjuego entre factores de riesgo ambientales e inmunogenéticos. Se ha propuesto a la autoinmunidad como un componente etiológico importante en el TEA. Sigue pendiente saber si hay autoanticuerpos específicos dirigidos contra blancos cerebrales involucrados en el TEA. En este artículo se identificó, dentro de una cohorte, un paciente con TEA con múltiples autoanticuerpos circulantes, incluyendo uno bien caracterizado contra el receptor de glutamato NMDA (NMDAR-Ab). El paciente presentaba alexitimia y previamente había tenido dos episodios depresivos mayores sin síntomas psicóticos. Mediante técnica de imágenes de molécula única se demostró que ni la γ inmunoglobulina purificada del NMDAR-Ab del suero del paciente con TEA, ni la de un sujeto sano seropositivo desorganizaban los complejos de membrana del NMDAR en las sinapsis. Estos hallazgos sugieren que los auto-anticuerpos del NMDAR de pacientes autistas no juegan un papel directo en la etiología del TEA y que se deben investigar otros autoanticuerpos dirigidos contra blancos neuronales.Les troubles du spectre autistique (TSA) sont des maladies neurodéveloppementales caracterisées par des dysfonctions des interactions sociales provoquées par un jeu complexe entre des facteurs de risque immunogénétiques et environnementaux. L'auto-immunité a été proposée comme composant étiologique majeur des TSA. Il reste à savoir si des auto-anticorps spécifiques dirigés contre des cibles cérébrales sont impliqués dans les TSA. Dans cet article, nous identifions au sein d'une cohorte, un patient TSA ayant de nombreux auto-anticorps circulants dont celui très connu contre le récepteur NMDA du glutamate (NMDAR-Ab). Ce patient présente une alexithymie et a eu antérieurement deux épisodes dépressifs caractérisés sans symptômes psychotiques. Grâce à l'utilisation d'une technique d'imagerie de molécule unique, nous démontrons que ni l'immunoglobuline γ purifiée NMDAR-Ab sérique du patient TSA ni celle d'un patient sain ayant le même anticorps ne désorganisent les complexes membranaires NMDAR au niveau synaptique. Ces résultats semblent indiquer que les auto-anticorps NMDAR-Ab de patients autistes ne jouent pas de rôle direct dans I'étiologie des TSA et que d'autres autoanticorps dirigés contre des cibles neuronales devraient faire l'objet de recherches.

The data on the biological responsiveness of melanoma and endothelial cells that are targeted by Anti- angiogenic MEtargidin Peptide (AMEP) are limited; therefore, the antiproliferative, antimetastatic and antian- giogenic effects of AMEP were investigated in murine melanoma and human endothelial cells after plasmid AMEP gene electrotransfer into the cells in vitro. Plasmid AMEP, a plasmid coding for the disintegrin domain of metargidin targeting specific integrins, had cytotoxic and antiproliferative effects on murine melanoma and human endothelial cells. Among the metastatic properties of cells, migration, invasion and adhesion were investigated. Plas- mid AMEP strongly affected the migration of murine melanoma and human endothelial cell lines and also affected the invasion of highly metastatic murine melanoma B16F10 and human endothelial cell lines. There was no effect on cell adhesion on Matrigel TM or fibronectin in all cell lines. The antiangiogenic effect was shown with tube formation assay, where human microvascular endothelial cell line (HMEC-1) proved to be more sensitive to plasmid AMEP gene electrotransfer than the human umbilical vein endothelial cell line (HUVEC). The study indicates that antiproliferative and antimetastatic biological responses to gene electrotransfer of plasmid AMEP in murine melanoma cells were dependent on the integrin quantity on melanoma cells and not on the expression level of AMEP. The strong antiangiogenic effect expressed in human endothelial cell lines was only partly dependent on the quantity of integrins and seemed to be plasmid AMEP dose dependent.

Gene electrotransfer of Plasmid AMEP is a new antiangiogenic approach in cancer treatment. While the local effects after intratumoral administration have been already studied, systemic effects still need to be elucidated. Therefore intramtumoral or intramuscular gene therapy with Plasmid AMEP was performed and effects on distant tumors or metastases were observed and compared. Only 8% of the mice developed distant metastases after Plasmid AMEP gene electrotransfer, compared to 40% in the control groups. Additionally, intramuscular gene elctrotransfer performed before the tumor implantation, has no effect on the tumor outgrowth regardless of the GET protocol used. Study showed that gene therapy with Plasmid AMEP has antimetastatic potential only after intratumoral, but not after intramuscular gene electrotransfer. Most probably circulating AMEP peptide produced in muscle cells may be rapidly biodegraded and could not be able to reach tumor site in high amounts needed for AMEP effectiveness.

International audience; Metachromatic leukodystrophy (MLD) is a lethal neurodegenerative disease caused by a deficiency in the lysosomal arylsulfatase A (ARSA) enzyme leading to the accumulation of sulfatides in glial and neuronal cells. We previously demonstrated in ARSA-deficient mice that intracerebral injection of a serotype 5 adeno-associated vector (AAV) encoding human ARSA corrects the biochemical, neuropathological and behavioral abnormalities. However, before considering a potential clinical application, scaling-up issues should be addressed in large animals. Therefore, we performed intracerebral injection of the same AAV vector (total dose of 3.8 3 1011 or 1.9 3 1012 vector genome, three sites of injection in the right hemisphere, two deposits per site of injection) into three selected areas of the centrum semiovale white matter, or in the deep gray matter nuclei (caudate nucleus, putamen, thalamus) of six non-human primates to evaluate vector distribution, as well as expression and activity of human ARSA. The procedure was perfectly tolerated, without any adverse effect or change in neurobehavioral examination. AAV vector was detected in a brain volume of 12–15 cm3 that corresponded to 37–46% of the injected hemisphere. ARSA enzyme was expressed in multiple interconnected brain areas over a distance of 22–33 mm. ARSA activity was increased by 12–38% in a brain volume that corresponded to 50–65% of injected hemisphere. These data provide substantial evidence for potential benefits of brain gene therapy in patients with MLD.

International audience; The aim of the present study was to examine modifications of anti-tumor activity and toxicity of paclitaxel (PLX) when given p.o. after recombinant interleukin-2 (rIL-2) to Lewis lung carcinoma-bearing mice. PLX was given orally to mice at the dose of 15 mg/kg on day 8 and 30 mg/kg on day 15, either alone or after 16.5 microg of rIL-2 given i.p. twice a day either 1 or 3 days before. The anti-tumor activity was higher and PLX hematological toxicity not increased if orally administered PLX was given after a 3-day rIL-2 pre-treatment rather than if given alone. Lung metastasis was significantly lower and s.c. tumors were smaller in the PLX+rIL-2 group than in the PLX or rIL-2 or non-treated groups. In addition, a decrease in lung P-glycoprotein expression (investigated by Western blot analysis) was observed 1 h after the last administration of rIL-2 on day 7.

At present, the gold standard to evaluate tumor necrosis is histology. We described here a new method to quantify the degree of tumor necrosis by ultrasonography. This technique combines ultrasound exploration of tissue and post-treatment of the numerical sequences using a dedicated software to evaluate backscattered power within the tumor.In order to establish that the backscattered power could be considered as a relevant marker of tumor necrosis, we performed (1) intra- and interoperator reproducibility in estimation of tumor dimensions obtained on sonographic scans; and (2) intra- and interoperator reproducibility in quantification of backscattered power in postprocessing using the HDILab software. The third part of the study consisted of correlating the degree of tumor necrosis estimated by histology and the ultrasound backscattered power, both obtained on xenografted melanomas at different days after tumor transplantation.Results concerning tumor size estimations and quantification of echogenicity were reproducible (coefficient of variation4.33%). The degree of necrosis measured in histology and echogenicity were significantly negatively correlated (P0.003).In conclusion, backscattered power could be considered as a relevant parameter to quantify tumor necrosis in vivo.

GenSight Biologics develops a rAAV2/2-ND4 vector (GS010) administered via intravitreal route (IVI) against vision loss in LHON patients bearing ND4 mutation. Gene therapy Adeno Associated Viral vectors are known to trigger host immune response which may interfere with safety and efficacy. Therefore, relationship between anti-AAV2 antibody systemic levels and eye inflammatory reactions following GS010 administration in non-human primates and LHON patients has been investigated. 36 non-human primates (NHP) and 15 LHON-ND4 patients (Phase I/IIa; ClinicalTrials. gov NCT02064569) underwent IVI of GS010 with two (4.3E10; 3.1E11 vg/eye) and four dose levels (9E9; 3E10; 9E10; 1.8E11 vg/eye) respectively. Ocular examinations were performed up to 6 months post-injection in NHP and up to 48 weeks in patients. NHP eye histopathology was performed and sera collected for neutralizing antibodies (Nabs) analysis (using a seroneutralization luciferase cell-based assay). Serum was collected from all patients and aqueous humor samples prior to injection in the last 8 patients, for quantification of anti-AAV2 IgG by ELISA and NAbs using the same method principle than for NHP. At baseline, NAbs were undetectable in 55% of NHPs and ranged between 1:5 and 1:400 titers for 45% of them. All animals showed titer increase up to 1:12800 starting week 2 post-injection. Between 2 and 6 months titers remained stable. Ocular inflammation (corneal punctuate focal opacity, vitreal haze, and superficial retinal mononuclear infiltration in histopathology) was observed in up to 80% of NHPs without deleterious effects on retinas. Preliminary Phase I/IIa data showed that, at baseline, 7 out of the 15 patients had undetectable serum NAb levels and 2 patients had titers above 1:1000. Prior treatment, none of the 8 tested patients had NAbs in aqueous humor, even the one showing a titer >2000 in serum. Two weeks after injection, IgG titers increased up to 19 times their baseline level in 7 patients and NAbs up to 39 times in 10 patients. This humoral response increase was not correlated to the dose level. At week 8, NAb titers remained above 1:1000 titer for 6 out of 15 patients; then titers tended to decrease progressively overtime. 13 out of 15 patients experienced mild to moderate ocular inflammation (anterior chamber inflammation and/or vitritis), responsive to standard medication, without apparent correlation with the humoral serum response. In conclusion, a humoral serum response against AAV2 was observed from 2 weeks post-injection of GS010 in both NHP and LHON patients. Ocular inflammation was reported, but currently no consistent correlation with anti-AAV2 antibody levels (at baseline or at the time-points when inflammation was recorded) were noted. Additional follow-up of humoral and cellular immunogenicity in upcoming nonclinical study and Phase III trials will help to confirm these observations and delineate the potential predictable impact for contralateral eye injection.

Introduction: Expression of a light-sensitive opsin in retinal ganglion cells (RGCs) is an attractive strategy to restore vision. We evaluated the ability of ChrimsonR-tdTomato (ChrR-tdT), derived from the algal light-gated cation channel ChrimsonR (Ed Boyden, MIT), to convert light insensitive RGCs into photoactivatable cells in normal macaques. A photostimulation device (GS030-MD) is developed in parallel to complement the biologics. This GS030 combination treatment is intended to treat blindness caused by retinal degenerative diseases such as retinitis pigmentosa. Methods: Cynomolgus macaques were injected intravitreally with the AAV2.7m8 vector encoding ChrR-tdT under the control of the CAG promoter (GS030-DP; 5×1011 vg/eye). Electrophysiological measurements by microelectrode array (MEA) and patch clamp as well as expression of the ChrR-tdT protein by immunofluorescence were assessed on explanted retinas 2 months after injection. Local tolerance was evaluated by ophthalmic examination and histology at 2 and 6 months post administration. Results: ChR-tdT was essentially expressed in RGCs and its expression restricted to the perifoveal area. MEA recordings showed light responses in all treated retinas, with 3 out of 4 retinas displaying high amplitudes of electrical responses to light stimulation (up to 360 Hz). One retina was less responsive (50 Hz). In patch clamp experiments, conducted by targeting tdT-expressing RGCs, large photocurrents were recorded in 3 out of 4 retinas in response to illumination, and according to the expected action spectrum for ChR-tdT. An exploratory study was conducted in parallel in monkeys, which received a single bilateral intravitreal administration of GS030-DP (5×1011 vg/eye) to assess local tolerance, systemic toxicity and immunogenicity at 2 and 6 months. No clinical signs indicative of systemic toxicity or local intolerance were observed. No adverse effects were seen by ophthalmic or histological examinations, especially no retinal structural modifications, inflammation or necrosis. Anti-AAV2 neutralizing antibodies (NAbs) measured in serum at 2, 3 and 6 months slightly increased at month 2 (≤ 1:100) and then returned to baseline levels at month 6. No NAbs were detected in aqueous humor at necropsy (at 2 or 6 months). In parallel, in preparation of a first-in-man clinical trial, a complete prototype of the photostimulation device (“goggles”) was developed with a full functional optical chain, an electronic subsystem, and firmware and software architecture. These goggles (GS030-MD) capture external scenes through an event-based camera and deliver visual stimuli onto the transduced retina at irradiances shown to activate ChrR-tdT expressing RGCs in monkey retinas. Conclusion: GS030 vector (GS030-DP) transduced efficiently and safely RGCs in vivo after intravitreal administration and induced light responses in normal monkey retinas under pharmacological block of endogenous phototransducion. The GS030 treatment combining the AAV2.7m8-ChrR-tdT vector and the photo stimulation goggles represents a valuable treatment for vision restoration in retinitis pigmentosa.

We have shown that the loss of p53 function contributed to resistance of tumor cells to TNF-induced cytotoxicity. In the present study, we evaluated the effect of wild-type p53 (wt-p53) expression on TNF sensitivity, by introducing wt-p53 into MCF7/Adr cells in which p53 was deleted, via a recombinant adenovirus encoding p53 (Ad-p53). Our results indicate that infection with Ad-p53 (50-100 viral particles per cell) resulted in pronounced cytotoxicity, whereas infection with 10 viral particles per cell, which was weakly toxic for the MCF7/Adr cells, sensitized these cells to TNF-induced cell death. Moreover, expression of wt-p53 in MCF7/Adr cells induced the production of reactive oxygen intermediates (ROIs) and caused glutathione (GSH) depletion, indicating disturbances in the cellular redox state. Additional treatment of cells with the anti-oxidant and glutathione (GSH) precursor N-acetylcysteine (NAC) resulted in inhibition of p53-induced ROIs production and in partial restoration of intracellular GSH levels, which was associated with the ability of NAC to inhibit p53-modulated TNF-induced cytotoxicity. Interestingly, Ad-p53 was able to inhibit TNF-induced MnSOD mRNA expression in MCF7/Adr cells, which might contribute to the sensitization of cells to the cytotoxic action of TNF. Taken together, our data strongly suggest that wt-p53 expression sensitizes TNF-resistant MCF7 cells with p53 deletion to TNF-induced cell death by a pathway that is dependent on ROIs production.

Antiangiogenic metargidin peptide (AMEP) is a novel anticancer agent exerting antiproliferative and antiangiogenic effects by binding to αvβ3 and α5β1 integrins. Electrotransfer designates the use of electric pulses (electroporation) to transfer plasmid DNA into tissues. This first-in-man phase I study investigated safety and tolerability of intratumoral plasmid AMEP electrotransfer into cutaneous metastatic melanoma. Secondary objectives were efficacy and pharmacokinetics. Five patients with disseminated melanoma without further treatment options were treated at two dose levels (1 and 2 mg DNA). In each patient, two cutaneous lesions were identified (one treated and one control). At day 1 and day 8, plasmid AMEP was injected intratumorally followed by electrotransfer. Patients were monitored weekly until day 29, and at day 64. Local efficacy was assessed at day 29 by direct measurement, and posttreatment biopsies for AMEP mRNA levels were evaluated by reverse transcriptase quantitative polymerase chain reaction. Plasmid copy number in blood and urine was determined by quantitative polymerase chain reaction. Minimal systemic toxicity was observed, including transient fever and transitory increase in C-reactive protein. No related serious adverse events occurred. Plasmid AMEP was detected in plasma but not in urine. AMEP mRNA was found in three of five treated lesions and none of the control lesions. At day 29, all five treated lesions were stable in diameter, whereas four of five control lesions increased more than 20%. No response occurred in distant lesions. This first-in-man study on electrotransfer of plasmid AMEP into cutaneous melanoma shows that the procedure and drug are safe and that local transfection was obtained.

Introduction Leber Hereditary Optic Neuropathy (LHON) is a rare mitochondrial genetic disorder predominantly affecting young males. Affected patients experience bilateral severe central vision loss. Currently no therapy is approved in the United States to prevent, halt or reverse vision loss due to LHON. Preliminary safety and pharmacodynamic results of a first-in-man trial of GS010, a gene therapy candidate for patients with LHON carrying the ND4 mutation will be presented. Methods GS010 is a recombinant adeno-associated viral vector, serotype 2, carrying the wild-type ND4 gene (rAAV2/2-ND4) and is an experimental gene therapy for the treatment of LHON due to the G11778A ND4 mitochondrial mutation. GS010 has received orphan drug designation in EU & USA. GS010 contains a Mitochondrial Targeting Sequence (MTS) that allows localization of the wild-type protein to the mitochondrion, enabling restoration of mitochondrial function. An open-label Phase I/IIa safety study (NCT02064569) included patients with vision loss due to ND4 LHON and has completed recruitment. Four dose escalation cohorts and an extension cohort were comprised of 3 patients each. Patients received a single intra-vitreal injection of rAAV2/2-ND4 in their worse seeing eye. Primary outcome was the occurrence of adverse events (AE). Secondary outcomes included immune response to AAV2 and evaluation of visual function. Results Systemic safety was excellent as no unexpected adverse events occurred. Ocular tolerability was good with mostly mild inflammation that were responsive to and resolve with standard therapies. Of the first 9 patients with 48 week follow up, preliminary results indicate that symptom duration could impact magnitude of treatment effect. Additionally, baseline vision status at time of treatment also indicate a relation with potential greater magnitude of effect which was noted with relatively shorter disease duration (< 2 years). These data confirm the importance of treating early from onset of vision loss. The RESCUE (NCT02652767) and REVERSE (NCT02652780) Phase III studies of GS010 have been initiated in the United States and some European Union countries. Both studies are randomized, double-masked, sham-controlled trials and will specifically include patients up to 6 months and one year after the onset of vision loss.

BACKGROUND AND PURPOSE: We hypothesized that electrotransfer of a plasmid encoding an antiangiogenic factor, the recombinant disintegrin domain of ADAM-15, (pRDD) could modify the tumor microenvironment and radiosensitize tumor. MATERIALS AND METHODS: pRDD was injected in the TLT tumor or FSaII fibrosarcomas before electroporation. pO2 in tumors and oxygen consumption in vitro were measured by electronic paramagnetic resonance (EPR) oximetry. Tumor perfusion was assessed by laser doppler imaging and patent blue assay. RESULTS: pRDD electrotransfer caused a significant delay in TLT growth and an anti-angiogenic effect. It significantly increased tumor pO2 in TLT and FSaII for at least 4days. pRDD electrotransfer and radiotherapy were more effective than either treatment alone. Modifications of tumor microenvironment were evaluated: tumor perfusion and interstitial fluid pressure were not modified. Oxygen consumption by the cells was decreased resulting both from a decrease in oxygen consumption rate and from a decrease in cell viability. CONCLUSION: The combination of localized antiangiogenic gene therapy and radiotherapy applied in the time of maximal oxygenation could be a promising alternative for cancer treatment.

Aims Interferon-alpha (IFN-α) was shown to reduce P-glycoprotein (P-gp) expression and activity in several tissues. The purpose of this study was to evaluate the impact of IFN-α pretreatment on the antitumoral and antimetastatic, Docetaxel (DTX, P-gp substrate), on Lewis Lung Cancer (3LL) bearing mice and to correlate it to DTX pharmacokinetics. Main methods Six groups of C57/Bl6 mice received subcutaneous (s.c.) 2.10 6 3LL cells, then IFN-α 4 MIU/kg for 7 days, then received or did not receive i.v. or oral DTX (30 mg/kg). Pharmacokinetic studies were done on a part of the mice: DTX concentrations were assessed in plasma and tumors, where AUC were estimated with the Bailer method, and half-lives and MRT were determined with a non-compartmental analysis. Tumor growth was assessed more than 21 days: animals were then sacrificed and lung metastases number was counted. Kaplan–Meier analysis was made to analyze survival data during the survey period. Key findings DTX i.v. associated with IFN-α significantly improved mouse survival (19.6 ± 0.6 days vs. 17.1 ± 0.8 days for control mice, p = 0.047) with greater antimetastatic effects (87.5% reduction in the number of metastases compared to control mice). The effect on tumor growth was not modified within the IFN-α/DTX i.v. treated groups when compared to mice receiving DTX i.v. alone. The pharmacokinetic analysis showed an increase of DTX concentrations in tumors at 30 min after DTX i.v. administration and an increase in the oral bioavailability of orally given DTX following an IFN-α treatment. Significance Our study established that IFN-α increases DTX uptake in tumors, improves its antitumoral efficiency and improves animals' survival.

BACKGROUND: Despite the discovery of novel inhibitors of tumor angiogenesis, protein-based antiangiogenic cancer therapy suffers some limitations that antiangiogenic gene therapy could overcome. We investigated whether intra-tumoral electrotransfer of three angiogenic plasmids could inhibit tumor growth and metastasis. METHODS: Plasmids encoding recombinant disintegrin domain of ADAM-15 (RDD), thrombospondin 1 (TSP-1), and the soluble isoform of the VEGF receptor 1 (sFlt-1) were injected into B16F10 melanoma-bearing C57BL/6 mice followed by electroporation. Tumor volume was measured daily using a digital caliper. Metastasis was monitored by in vivo bioluminescence after surgical removal of the primary luciferase-encoding B16F10 tumor 5days after intra-tumoral electrotransfer. Markers of vascularization and cell proliferation were quantified by immunohistochemistry. RESULTS: Intra-tumoral electrotransfer of the antiangiogenic plasmids induced a significant inhibition of tumor growth, doubling of mean survival time and long-term survivors (∼40% vs 0% in control). When the tumor was removed by surgery after intra-tumoral plasmid electrotransfer, a significant decrease in tumor metastasis was observed leading to long-term tumor-free survival especially after treatment with pRDD plasmid (84% vs 0% in control). Unlike pTSP-1 and psFlt-1, pRDD significantly decreased cell proliferation in B16F10 primary tumors which express αvβ3 and α5β1 integrins. No effect of antiangiogenic plasmid electrotransfer on normal skin blood flow was detected. CONCLUSION: The intra-tumoral electrotransfer of the three antiangiogenic plasmids is a promising method for the treatment of melanoma. The plasmid encoding RDD seems to be particularly effective due to its direct antitumoral activity combined with angiogenesis suppression, and its marked inhibition of metastasis.

Dysregulated angiogenesis is a hallmark of chronic inflammatory diseases, including psoriasis, a common skin disorder that affects approximately 2% of the population. Studying both human psoriasis in 2 complementary xenotransplantation models and psoriasis-like skin lesions in transgenic mice with epidermal expression of human TGF-β1, we have demonstrated that antiangiogenic non-viral somatic gene therapy reduces the cutaneous microvasculature and alleviates chronic inflammatory skin disorders. Transient muscular expression of the recombinant disintegrin domain (RDD) of metargidin (also known as ADAM-15) by in vivo electroporation reduced cutaneous angiogenesis and vascularization in all 3 models. As demonstrated using red fluorescent protein–coupled RDD, the treatment resulted in muscular expression of the gene product and its deposition within the cutaneous hyperangiogenic connective tissue. High-resolution ultrasound revealed reduced cutaneous blood flow in vivo after electroporation with RDD but not with control plasmids. In addition, angiogenesis- and inflammation-related molecular markers, keratinocyte proliferation, epidermal thickness, and clinical disease scores were downregulated in all models. Thus, non-viral antiangiogenic gene therapy can alleviate psoriasis and may do so in other angiogenesis-related inflammatory skin disorders. peerReviewed

International audience; Adenovirus (Ad)-mediated delivery of anti-angiogenic molecules into tumors constitutes an appealing approach for growth inhibition. However, lack of expression on tumors of Ad receptors leads to weak tumor transduction. Therefore, to provide Ad with a new entry pathway into tumors, an NGR peptide was inserted into either fiber (AdFNGR) or hexon (AdHNGR) capsid proteins. This strategy provided Ad with a very efficient entry pathway in both endothelial cells and tumor cells, with the highest efficacy observed for AdHNGR. Using pharmacological, biochemical and genetic approaches, AdHNGR and AdFNGR were shown to bind not only to CD13 receptor, but also to alphavbeta3 integrins. Both vectors were efficient tools to deliver angiostatin K1-5 cDNA into endothelial cells, thus leading to a dramatic inhibition of their proliferation and increased cell death. Although AdHNGR and Adwt were found to display similar gene transduction efficacy in Lewis lung carcinoma (LLC), pseudotyping AdHNGR with an Ad3-fiber unmasked the ability of NGR-peptide to target these tumors. As a result, delivery of angiostatin K1-5 cDNA into highly aggressive tumors translated into a stronger inhibition of their growth. Altogether, our results suggest that NGR-bearing Ad are valuable tools to realize the potential of this anti-angiogenic approach to anti-tumor therapy.Gene Therapy advance online publication, 30 July 2009; doi:10.1038/gt.2009.97.

Prostate cancer metastasis to bone results in mixed osteolytic and osteoblastic lesions associated with high morbidity, and there is mounting evidence that the urokinase-type plasminogen system is causatively involved in the progression of prostate cancer. Adult mesenchymal stem cells (MSCs) are promising tools for cell-mediated gene therapy with the advantage of osteogenic potential, a critical issue in the case of osteolytic metastases. In this study, we evaluated the therapeutic use of engineered murine MSCs for in vivo delivery of the urokinase-type plasminogen antagonist amino-terminal fragment (hATF) to impair osteolytic prostate cancer cell progression in bone and to repair bone lesions. Bioluminescence imaging revealed that both primary MSCs and the MSC line C3H10T1/2 (C3) expressing hATF (MSC-hATF) significantly inhibited intratibial PC-3 Luciferase (Luc) growth following coinjection in SCID mice. Furthermore, microcomputed tomography imaging of vascular network clearly demonstrated a significant decrease in tumor-associated angiogenesis and a protection from tumor-induced osteolysis in MSC-hATF-treated mice. Importantly, the osteogenic potential of MSC-hATF cells was unaffected, and an area of new bone formation was evidenced in 60% of animals. Together, these data support the concept of MSC-based therapy of tumor osteolysis disease, indicating that MSCs may combine properties of vehicle for angiostatic agent with osteogenic potential. Disclosure of potential conflicts of interest is found at the end of this article.

Outgrowth of axons during neuronal development, as well as their regeneration after injury, of the adult nervous system is controlled by specific extracellular cues which are diffusible, or bound to cell membranes or extracellular matrix. The exact molecular mechanisms through which these extracellular signals are integrated by the growing axon, are not yet well defined. However, it is widely accepted that most, if not all, signaling cascades triggered by guidance cues eventually converge onto the cytoskeleton. The action of extracellular guidance factors is thus modulated not only by specific membrane receptors, but also by cytoskeletal and cytoskeleton-associated molecules within the axon. In fact, the cytoskeleton represents a point of convergence and integration of both neuron-intrinsic and extrinsic factors. Moreover, in recent years, there has been increasing evidence for the involvement of a coordinated cross-talk between actin filaments and microtubules, the two main components of the growth cone cytoskeleton. Their reorganization is complex and involves numerous cytoskeleton-associated proteins whose function is regulated via activation or inhibition of particular signaling pathways.

KiSS1 is a putative metastasis suppressor gene in melanoma and breast cancer-encoding kisspeptins, which are also described as neuroendocrine regulators of the gonadotropic axis. Negative as well as positive regulation of KiSS1 gene expression by estradiol (E2) has been reported in the hypothalamus. Estrogen receptor α (ERα level is recognized as a marker of breast cancer, raising the question of whether expression of KiSS1 and its G-protein-coupled receptor (GPR54) is down- or upregulated by estrogens in breast cancer cells. KiSS1 was found to be expressed in MDA-MB-231, MCF7, and T47D cell lines, but not in ZR75-1, L56Br, and MDA-MB-435 cells. KiSS1 mRNA levels decreased significantly in ERα-negative MDA-MB-231 cells expressing recombinant ERα. In contrast, tamoxifen (TAM) treatment of ERα-positive MCF7 and T47D cells increased KiSS1 and GPR54 levels. The clinical relevance of this negative regulation of KiSS1 and GPR54 by E2 was then studied in postmenopausal breast cancers. KiSS1 mRNA increased with the grade of the breast tumors. ERα-positive invasive primary tumors expressed sevenfold lower KiSS1 levels than ERα-negative tumors. Among ERα-positive breast tumors from postmenopausal women treated with TAM, high KiSS1 combined with high GPR54 mRNA tumoral levels was unexpectedly associated with shorter relapse-free survival (RFS) relative to tumors expressing low tumoral mRNA levels of both genes. The contradictory observation of putative metastasis inhibitor role of kisspeptins and RFS to TAM treatment suggests that evaluation of KiSS1 and its receptor tumoral mRNA levels could be new interesting markers of the tumoral resistance to anti-estrogen treatment.

Outgrowth of axons during neuronal development, as well as their regeneration after injury, of the adult nervous system is controlled by specific extracellular cues which are diffusible, or bound to cell membranes or extracellular matrix. The exact molecular mechanisms through which these extracellular signals are integrated by the growing axon, are not yet well defined. However, it is widely accepted that most, if not all, signaling cascades triggered by guidance cues eventually converge onto the cytoskeleton. The action of extracellular guidance factors is thus modulated not only by specific membrane receptors, but also by cytoskeletal and cytoskeleton-associated molecules within the axon. In fact, the cytoskeleton represents a point of convergence and integration of both neuron-intrinsic and extrinsic factors. Moreover, in recent years, there has been increasing evidence for the involvement of a coordinated cross-talk between actin filaments and microtubules, the two main components of the growth cone cytoskeleton. Their reorganization is complex and involves numerous cytoskeleton-associated proteins whose function is regulated via activation or inhibition of particular signaling pathways.

We previously described the function of MAP1B in both turning and branching of regenerating neurites. Our results suggested implication of MAP1B in coupling of actin and microtubule movements, a hypothesis investigated here using DRG neurons and Schwann cells (SCs), which also transiently express MAP1B. Cell motility and cytoskeletal rearrangements were assessed before and after addition of lysophosphatidic acid (LPA), an extracellular signaling phospholipid triggering changes in actin distribution and cell morphology. First, we show that MAP1B is required for SC migration in vitro, extending our previous work on its function in growth cone motility. Second, LPA stimulation induces drastic retraction of processes from MAP1B-expressing cells in a two-step process: actin contraction, which is followed by microtubule backfolding. More importantly, we provide evidence that MAP1B is required for microtubule backfolding, thereby unravelling an important molecular mechanism implicated in coupling the movements of actin and microtubules during process retraction of neural cells.

International audience; Angiogenesis is essential for tumor growth and metastatic dissemination. We have previously shown that human angiotensinogen (AGT) can in vitro inhibit endothelial cell proliferation and migration, capillary-like tube formation, and neovascularization. To determine whether AGT can exert an antitumoral effect through its antiangiogenic properties, we constructed a recombinant adenovirus carrying the human angiotensinogen gene under the control of the cytomegalovirus promoter (AdAGT). In vitro studies showed that AdAGT selectively inhibited endothelial cell proliferation. In vivo, injections of AdAGT into preestablished human MDA-MB-231 mammary carcinomas in nude mice inhibited tumor growth by 70% compared to controls, with 21% total regression. This effect was associated with the suppression of intratumoral vascularization and marked necrosis. Furthermore, in vitroAdAGT infection of MDA-MB-231 and murine melanoma B16F10 cells strongly blocked their in vivo tumorigenicity. Then, in mice expressing high levels of AGT (i.e., either iv injected with AdAGT or HuAGT transgenic mice), the number of B16F10 pulmonary metastases was 85% lower than in control C57BL/6 mice. Our data demonstrate that AGT is a very potent antiangiogenic factor in vivo, independent of angiotensin II generation. Its delivery by gene transfer represents a promising new strategy to block primary tumor growth and to prevent metastasis.

Canstatin, the noncollagenous domain of collagen type IV α-chains, belongs to a series of collagen-derived angiogenic inhibitors. We have elucidated the functional receptors and intracellular signaling induced by canstatin that explain its strong antitumor efficacy in vivo. For this purpose, we generated a canstatin-human serum albumin (CanHSA) fusion protein, employing the HSA moiety as an expression tag. We show that CanHSA triggers a crucial mitochondrial apoptotic mechanism through procaspase-9 cleavage in both endothelial and tumor cells, which is mediated through cross-talk between αvβ3- and αvβ5-integrin receptors. As a point of reference, we employed the first three kringle domains of angiostatin (K1-3), fused with HSA, which, in contrast to CanHSA, act only on endothelial cells through αvβ3-integrin receptor–mediated activation of caspase-8 alone, without ensuing mitochondrial damage. Taken together, these results provide insights into how canstatin might exert its strong anticancer effect.

Expression of the cell adhesion molecule TAG-1 is down-regulated in adult brain, with the exception of certain areas exhibiting structural plasticity. Here, we present evidence that TAG-1 expression persists also in adult rat spinal cord and dorsal root ganglia (DRG), and can be up-regulated after injury. On Western blots of adult tissue, TAG-1 is detected as a 135-kDa band, with an additional specific 90-kDa band, not present in developing tissue. TAG-1 expression is found both in DRG neurons and in Schwann cells, particularly those associated with the peripherally projecting DRG processes. Quantitative in situ hybridization revealed that TAG-1 expression is significantly higher in small neurons that give rise to unmyelinated fibers, than in large DRG neurons. The regulation of TAG-1 was then examined in two different lesion paradigms. After a sciatic nerve lesion, TAG-1 expression is not up-regulated in DRG neurons, but decreases with time. At the lesion site, reactive Schwann cells up-regulate TAG-1, as demonstrated by both immunohistochemistry and in situ hybridization. In a second paradigm, we injected kainic acid into the spinal cord that kills neurons but spares glia and axons. TAG-1 is up-regulated in the spinal neuron-depleted area as well as in the corresponding dorsal and ventral roots, associated with both target-deprived afferent fibers and with the non-neuronal cells that invade the lesion site. These results demonstrate a local up-regulation of TAG-1 in the adult that is induced in response to injury, suggesting its involvement in axonal re-modelling, neuron-glia interactions, and glial cell migration.

During development, microtubule-associated protein 1B (MAP1B) is one of the earliest MAPs, preferentially localized in axons and growth cones, and plays a role in axonal outgrowth. Although generally downregulated in the adult, we have shown that MAP1B is constitutively highly expressed in adult dorsal root ganglia (DRGs) and associated with central sprouting and peripheral regeneration of these neurons. Mutant mice with a complete MAP1B null allele that survive until adulthood exhibit a reduced myelin sheath diameter and conductance velocity of peripheral axons and lack of the corpus callosum. Here, to determine the function of MAP1B in axonal regeneration, we used cultures of adult DRG explants and/or dissociated neurons derived from thismap1b-/- mouse line. Whereas the overall length of regenerating neurites lacking MAP1B was similar to wild-type controls, our analysis revealed two main defects. First,map1b-/- neurites exhibited significantly (twofold) higher terminal and collateral branching. Second, the turning capacity of growth cones (i.e., “choice” of a proper orientation) was impaired. In addition, lack of MAP1B may affect the post-translational modification of tubulin polymers: quantitative analysis showed a reduced amount of acetylated microtubules within growth cones, whereas the distribution of tyrosinated or detyrosinated microtubules was normal. Both growth cone turning and axonal branch formation are known to involve local regulation of the microtubule network. Our results demonstrate that MAP1B plays a role in these processes during plastic changes in the adult. In particular, the data suggest MAP1B implication in the locally coordinated assembly of cytoskeletal components required for branching and straight directional axon growth.

Currently available murine models to evaluate mesenchymal stem cell (MSC) differentiation are based on cell injection at ectopic sites such as muscle or skin. Due to the importance of environmental factors on the differentiation capacities of stem cells in vivo, we investigated whether the peculiar synovial/cartilaginous environment may influence the lineage specificity of bone morphogenetic protein (BMP)-2-engineered MSCs. To this aim, we used the C3H10T1/2-derived C9 MSCs that express BMP-2 under control of the doxycycline (Dox)-repressible promoter, Tet-Off, and showed in vitro, using the micropellet culture system that C9 MSCs kept their potential to differentiate toward chondrocytes. Implantation of C9 cells, either into the tibialis anterior muscles or into the joints of CB17-severe combined immunodeficient bg mice led to the formation of cartilage and bone filled with bone marrow as soon as day 10. However, no differentiation was observed after injection of naive MSCs or C9 cells that were repressed to secrete BMP-2 by Dox addition. The BMP-2-induced differentiation of adult MSCs is thus independent of soluble factors present in the local environment of the synovial/cartilaginous tissues. Importantly, we demonstrated that a short-term expression of the BMP-2 growth factor is necessary and sufficient to irreversibly induce bone formation, suggesting that a stable genetic modification of MSCs is not required for stem cell-based bone/cartilage engineering.

Human neuroblastoma (NB) is a highly heterogeneous childhood cancer secreting a high level of vascular endothelial growth factor (VEGF). Its vascularization has been clearly correlated with metastatic progression and poor outcome. Thus, molecules that target the vascular endothelium are regarded as new therapeutics of clinical interest. Angiostatin, an internal fragment of plasminogen containing the first four kringle structures, has been described as a powerful angiogenic inhibitor. We used a recombinant adenovirus encoding the human angiostatin kringle 1-3 directly fused to human serum albumin HSA (AdK3-HSA). Coupling to HSA has been previously shown to increase the in vivo half-life of this angiostatic factor, and to lead to tumor growth inhibition in the MDA-MB-231 carcinoma model. For the assessment of antiangiogenic gene therapy in the human NB IGR-N835 tumor model, 5 x 10(9) PFU of AdK3-HSA were intravenously injected in tumor-bearing athymic mice presenting either of the following experimental settings: early stage, established, and minimal residual tumors. No delay in tumor growth was observed in animals treated with AdK3-HSA as compared to those treated with the empty virus AdCO1. In early-stage tumors, kinetics of tumor occurrence and tumor growth were similar in AdK3-HSA- and AdCO1-treated animals. K3-HSA was found to be expressed at high levels (the mean value for the three experiments being 19.4+/-15.9 microg/ml) in the circulation of all animals up to 21-35 days after virus injection. In addition, IGR-N835 tumors were found to be highly vascularized and to release high amounts of angiogenic factors, in particular VEGF (665+/-370 pg/mg total protein). Thus, in spite of high circulating levels, K3-HSA may be unable to displace the NB proangiogenic switch. In this regard, a more promising target to inhibit NB angiogenesis seems to be the VEGF/VEGFR system.

Diabetic retinopathy and retinopathy of prematurity are among the leading causes of vision impairment throughout the world. Both diseases are characterized by pathological angiogenesis, which severely impairs vision. Extracellular proteinases play important roles in endothelial cell migration during angiogenesis. Amino-terminal fragment (ATF) is an angiostatic molecule that targets the uPA/uPAR system and inhibits endothelial cell migration. The angiostatic effect of ATF has been demonstrated in models of cancer, but has never been assessed in pathological retinal neovascularization. Endostatin also has angiostatic effects on tumor growth and retinal neovascularization. We used an adenoviral vector carrying the murine ATF (AdATFHSA) or endostatin gene coupled to human serum albumin (HSA) (AdEndoHSA) to increase the half-life of the therapeutic protein in the circulation. We induced retinopathy by exposing 7-day-old mice to high levels of oxygen. They were intravitreally injected with the vectors. Local injection of AdATFHSA or AdEndoHSA reduced retinal neovascularization by 78.1 and 79.2%, respectively. Thus, the adenovirus-mediated delivery of ATFHSA or EndoHSA reduces retinal neovascularization in a mouse model of hypoxia-induced neovascularization.

We previously reported that intratumoral injection of AdK3, a recombinant adenovirus encoding human angiostatin kringle (K) 1 to 3, inhibits tumor vascularization and tumor growth. To reduce the serum clearance of this factor, we constructed an adenovirus (AdK3-HSA) that carries a chimeric gene encoding a fusion protein between angiostatin K1-3 and human serum albumin (HSA). This conjugate inhibited endothelial cell proliferation as efficiently as K1-3. K3-HSA serum concentrations in immunodeficient mice systemically injected with AdK3-HSA were dramatically higher than in AdK3-injected mice. Furthermore, the growth of MDA-MB-231 tumors grafted into nude mice that had been injected intravenously with AdK3-HSA was inhibited by 79% (versus 17% with AdK3). In TRP-1/SV40 Tag transgenic mice, which spontaneously develop eye tumors with brain metastases, intravenous injections of AdK3-HSA in newborn mice blocked metastatic dissemination efficiently and significantly, and prolonged survival by 3 weeks. After 2 months, only 46% of AdK3-HSA-treated animals developed micrometastases, whereas 94% of the AdCO1-injected group displayed numerous macrometastases. Nevertheless, ocular tumor growth was not modified because of impaired diffusion of the conjugate in the eye compartment. Our results show that HSA genetic coupling is an efficient way to increase the pharmacokinetics of circulating angiogenic inhibitors and thus their antitumoral activity.

An adaptive immune response, is now considered as a favorable prognostic factor in primary colorectal cancer (CRC). It is defined by a dense lymphocytic CD8+/CD45+ infiltrate located within and at the periphery of the tumor. FoxP3+ regulatory T lymphocytes inhibit this adaptive response. However, data about immune response in CRC liver metastases (LM) are lacking, especially after preoperative systemic and targeted treatments, which can modulate the recruitment of immune cells. The aim of our study was to analyze the immune response in CRC LM after different types of pre-operative treatments. Materials and methods. 107 CRC LM were selected and divided in 4 groups: chemotherapy alone (CT, n= 29), CT + anti-VEGF (bevacizumab) (n= 28), CT + anti-EGFR (cetuximab) (n= 21), surgery alone (control group with no treatment, n= 29). All cases corresponded to LM treated in first-line and to a conventional lieberkuhnian adenocarcinoma. Histologic response was assessed according to Rubbia-Brandt classification, based on the Tumor Regression Grade (TRG). LM were classified as TRG1-2 (major response), TRG3 (partial response) and TRG4-5 (minor or no response). Immune microenvironnement was evaluated in the center (IT, intratumoral) and the periphery (PT, peritumoral) of LM, classified as minor or major and assessed by immunohistochemistry to characterize: i)T lymphocytes: CD8+ (cytotoxic) , CD 45+ (memory), Tbet+ (T helper 1), FoxP3+ (regulators) ii) macrophages: CD68+, CD163+. Results. A major immune infiltrate was more frequently associated with LM displaying major or partial responses than with LM with no or minor response, with the following markers and locations: CD8+ (PT, IT; p = 0.049, p = 0.004), CD45+ (IT; p = 0.006), Tbet+ (PT; p= 0.015), CD 68+ (IT; p= 0.049), CD163 (PT, IT; p = 0.001, p = 0.022). Conversely, a major FoxP3+ IT immune infiltrate was more frequently associated with LM displaying no or minor response, than with LM showing major or partial responses (p = 0.033). Moreover, a major immune infiltrate was more frequently associated with treated LM than with untreated LM with the following markers and locations: CD8+ (IT; p = 0.014), TBet+ (PT, IT; p = 0.017 p= 0.004), CD 68+ (IT; p = 0.041), CD163+ (IT; p = 0.009). Among treated LM, a major PT CD45+ immune infiltrate was more frequently associated with CT+anti-EGFR than with others treatments (PT; p = 0.016), and a major IT CD45+ immune infiltrate was more frequently associated with CT+anti-EGFR and chemotherapy alone than with CT+anti-VEGF (IT; p = 0.048). Conclusion. These original data suggest that pre-operative treatments have an impact on CRC LM immune microenvironnement. Indeed, treated LM displaying histologic response are characterized by a CD8+/CD45+ adaptive immune response. Moreover, they simultaneously harbor a stimulated T helper 1 pathway and a down regulation of Treg lymphocytes. Such data suggest immune therapeutic approaches for CRC LM. Citation Format: Frederic Bibeau, Hugo Gil, Florence Castan, Magalie Pedot, François Quenet, Marc Ychou, Celine Bouquet, Charrier Véronique, Vaios Karanikas, Michael Cannarile, Solange Romagnoli, Fabien Gaire, Florence Boissiere. Impact of preoperative treatments on the immune microenvironnement of colorectal liver metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 141. doi:10.1158/1538-7445.AM2014-141

Plasmin is essential for metalloproteases activation, endothelial cell migration and degradation of the extracellular matrix. The process is common to neoangiogenesis pannus formation and cartilage degradation within arthritic joints. Since 80% of synovial cells express urokinase plasminogen activator receptor (uPAR), we investigated the inhibition of plasmin activation in a collagen-induced arthritis (CIA) mice model, by expressing a uPA/uPAR antagonist molecule (ATF) fused to human serum albumin (HSA) to extend its serum half-life. Overexpression was obtained with an adenoviral vector expressing the chimeric murine ATF-HSA. We showed that the genetic coupling did not significantly reduce the ability of the ATF moiety to interact with its receptor uPAR. The chimeric protein was detectable in the sera of injected mice 7 days following Ad-mATF-HSA injection, then decreased in parallel with the anti-HSA titer increase. Systemic Ad-mATF-HSA injection performed on day 25 following CIA induction decreased the incidence of arthritis and the severity of the disease. Moreover, synovial angiogenesis in arthritic paws was decreased after Ad-mATF-HSA gene transfer, as assessed by smooth muscle actin immunostaining. The preventive effect observed on arthritis was related to the decrease in angiogenesis, rather than inhibition of extracellular matrix degradation.