Neuron-to-neuron wild-type Tau protein transfer through a trans-synaptic mechanism: relevance to sporadic tauopathies.

BACKGROUND: In sporadic Tauopathies, neurofibrillary degeneration (NFD) is characterised by the intraneuronal aggregation of wild-type Tau proteins. In the human brain, the hierarchical pathways of this neurodegeneration have been well established in Alzheimer's disease (AD) and other sporadic tauopathies such as argyrophilic grain disorder and progressive supranuclear palsy but the molecular and cellular mechanisms supporting this progression are yet not known. These pathways appear to be associated with the intercellular transmission of pathology, as recently suggested in Tau transgenic mice. However, these conclusions remain ill-defined due to a lack of toxicity data and difficulties associated with the use of mutant Tau. RESULTS: Using a lentiviral-mediated rat model of hippocampal NFD, we demonstrated that wild-type human Tau protein is axonally transferred from ventral hippocampus neurons to connected secondary neurons even at distant brain areas such as olfactory and limbic systems indicating a trans-synaptic protein transfer. Using different immunological tools to follow phospho-Tau species, it was clear that Tau pathology generated using mutated Tau remains near the IS whereas it spreads much further using the wild-type one. CONCLUSION: Taken together, these results support a novel mechanism for Tau protein transfer compared to previous reports based on transgenic models with mutant cDNA. It also demonstrates that mutant Tau proteins are not suitable for the development of experimental models helpful to validate therapeutic intervention interfering with Tau spreading.

Viral-mediated overexpression of mutant huntingtin to model HD in various species.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion of CAG repeats in the huntingtin (Htt) gene. Despite intensive efforts devoted to investigating the mechanisms of its pathogenesis, effective treatments for this devastating disease remain unavailable. The lack of suitable models recapitulating the entire spectrum of the degenerative process has severely hindered the identification and validation of therapeutic strategies. The discovery that the degeneration in HD is caused by a mutation in a single gene has offered new opportunities to develop experimental models of HD, ranging from in vitro models to transgenic primates. However, recent advances in viral-vector technology provide promising alternatives based on the direct transfer of genes to selected sub-regions of the brain. Rodent studies have shown that overexpression of mutant human Htt in the striatum using adeno-associated virus or lentivirus vectors induces progressive neurodegeneration, which resembles that seen in HD. This article highlights progress made in modeling HD using viral vector gene transfer. We describe data obtained with of this highly flexible approach for the targeted overexpression of a disease-causing gene. The ability to deliver mutant Htt to specific tissues has opened pathological processes to experimental analysis and allowed targeted therapeutic development in rodent and primate pre-clinical models.

Mutant prominin 1 found in patients with macular degeneration disrupts photoreceptor disk morphogenesis in mice.

Familial macular degeneration is a clinically and genetically heterogeneous group of disorders characterized by progressive central vision loss. Here we show that an R373C missense mutation in the prominin 1 gene (PROM1) causes 3 forms of autosomal-dominant macular degeneration. In transgenic mice expressing R373C mutant human PROM1, both mutant and endogenous PROM1 were found throughout the layers of the photoreceptors, rather than at the base of the photoreceptor outer segments, where PROM1 is normally localized. Moreover, the outer segment disk membranes were greatly overgrown and misoriented, indicating defective disk morphogenesis. Immunoprecipitation studies showed that PROM1 interacted with protocadherin 21 (PCDH21), a photoreceptor-specific cadherin, and with actin filaments, both of which play critical roles in disk membrane morphogenesis. Collectively, our results identify what we believe to be a novel complex involved in photoreceptor disk morphogenesis and indicate a possible role for PROM1 and PCDH21 in macular degeneration.

Mutant HbpR transcription activator isolation for 2-chlorobiphenyl via green fluorescent protein-based flow cytometry and cell sorting.

Mutants were produced in the A-domain of HbpR, a protein belonging to the XylR family of σ(54)-dependent transcription activators, with the purpose of changing its effector recognition specificity from 2-hydroxybiphenyl (2-HBP, the cognate effector) to 2-chlorobiphenyl (2-CBP). Mutations were introduced in the hbpR gene part for the A-domain via error-prone polymerase chain reaction, and assembled on a gene circuitry plasmid in Escherichia coli, permitting HbpR-dependent induction of the enhanced green fluorescent protein (egfp). Cells with mutant HbpR proteins responsive to 2-CBP were enriched and separated in a flow cytometry-assisted cell-sorting procedure. Some 70 mutants were isolated and the A-domain mutations mapped. One of these had acquired true 2-CBP recognition but reacted hypersensitively to 2-HBP (20-fold more than the wild type), whereas others had reduced sensitivity to 2-HBP but a gain of 2-CBP recognition. Sequencing showed that most mutants carried double or triple mutations in the A-domain gene part, and were not located in previously recognized conserved residues within the XylR family members. Further selection from a new mutant pool prepared of the hypersensitive mutant did not result in increased 2-CBP or reduced 2-HBP recognition. Our data thus demonstrate that a one-step in vitro 'evolutionary' adaptation of the HbpR protein can result in both enhancement and reduction of the native effector recognition.

A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance.

We report the study of a large American family displaying autosomal dominant retinitis pigmentosa with reduced penetrance, a form of hereditary retinal degeneration. Although the inheritance pattern and previous linkage mapping pointed to the involvement of the PRPF31 gene, extensive screening of all its exons and their boundaries failed in the past to reveal any mutation. In this work, we sequenced the entire PRPF31 genomic region by both the classical Sanger method and ultrahigh throughput (UHT) sequencing. Among the many variants identified, a single-base substitution (c.1374+654C>G) located deep within intron 13 and inside a repetitive DNA element was common to all patients and obligate asymptomatic carriers. This change created a new splice donor site leading to the synthesis of two mutant PRPF31 isoforms, degraded by nonsense-mediated mRNA decay. As a consequence, amounts of PRPF31 mRNA derived from the mutant allele were very reduced, with no evidence of mutant proteins being synthesized. Our results indicate that c.1374+654C>G causes retinitis pigmentosa via haploinsufficiency, similar to the vast majority of PRPF31 mutations described so far. We discuss the potential of UHT sequencing technologies in mutation screening and the continued identification of pathogenic splicing mutations buried deep within intronic regions.

SH3TC2, a protein mutant in Charcot-Marie-Tooth neuropathy, links peripheral nerve myelination to endosomal recycling

Patients with Charcot-Marie-Tooth neuropathy and gene targeting in mice revealed an essential role for the SH3TC2 gene in peripheral nerve myelination. SH3TC2 expression is restricted to Schwann cells in the peripheral nervous system, and the gene product, SH3TC2, localizes to the perinuclear recycling compartment. Here, we show that SH3TC2 interacts with the small guanosine triphosphatase Rab11, which is known to regulate the recycling of internalized membranes and receptors back to the cell surface. Results of protein binding studies and transferrin receptor trafficking are in line with a role of SH3TC2 as a Rab11 effector molecule. Consistent with a function of Rab11 in Schwann cell myelination, SH3TC2 mutations that cause neuropathy disrupt the SH3TC2/Rab11 interaction, and forced expression of dominant negative Rab11 strongly impairs myelin formation in vitro. Our data indicate that the SH3TC2/Rab11 interaction is relevant for peripheral nerve pathophysiology and place endosomal recycling on the list of cellular mechanisms involved in Schwann cell myelination.

The PROM1 mutation p.R373C causes an autosomal dominant bull's eye maculopathy associated with rod, rod-cone, and macular dystrophy.

PURPOSE: To characterize in detail the phenotype of five unrelated families with autosomal dominant bull's eye maculopathy (BEM) due to the R373C mutation in the PROM1 gene. METHODS: Forty-one individuals of five families of Caribbean (family A), British (families B, D, E), and Italian (family C) origin, segregating the R373C mutation in PROM1, were ascertained. Electrophysiological assessment, fundus autofluorescence (FAF) imaging, fundus fluorescein angiography (FFA), and optical coherence tomography (OCT) were performed in available subjects. Mutation screening of PROM1 was performed. RESULTS: The R373C mutant was present heterozygously in all affected patients. The age at onset was variable and ranged between 9 and 58 years, with most of the individuals presenting with reading difficulties. Subjects commonly had a mild to moderate reduction in visual acuity except for members of family C who experienced markedly reduced central vision. The retinal phenotype was characterized by macular dystrophy, with retinal pigment epithelial mottling in younger subjects, progressing to typical BEM over time, with the development of macular atrophy in older patients. In addition, all members of family C had typical features of RP. The electrophysiological findings were variable both within and between families. CONCLUSIONS: Mutations in PROM1 have been described to cause a severe form of autosomal recessive RP in two families of Indian and Pakistani descent. The results of this study have demonstrated that a distinct redundant PROM1 mutation (R373C) can also produce an autosomal dominant, fully penetrant retinopathy, characterized by BEM with little inter- and intrafamilial variability, and retinal dystrophy with variable rod or rod-cone dysfunction and marked intra- and interfamilial variability, ranging from isolated maculopathy without generalized photoreceptor dysfunction to maculopathy associated with very severe rod-cone dysfunction.

Pigs Transgenic For Human Dominant Mutations Of The GUCY2D Gene

Purpose: Studies on large animal models are an important step to test new therapeutical strategies before human application. Considering the importance of cone function for human vision and the paucity of large animal models for cone dystrophies having an enriched cone region, we propose to develop a pig model for cone degeneration. With a lentiviral-directed transgenesis, we obtained pigs transgenic for a cone-dominant mutant gene described in a human cone dystrophy.Methods: Lentiviral vectors encoding the human double mutant GUCY2DE837D/R838S cDNA under the control of a region of the pig arrestin-3 promoter (Arr3) was produced and used for lentiviral-derived transgenesis in pigs. PCR-genotyping and southern blotting determined the genotype of pigs born after injection of the vector at the zygote stage. Retina function analysis was performed by ERG and behavioral tests at 11, 24 and 54 weeks of age. OCT and histological analyses were performed to describe the retina morphology.Results: The ratio of transgenic pigs born after lentiviral-directed transgenesis was close to 50%. Transgenic pigs with 3 to 5 transgene copies per cell clearly present a reduced photopic response from 3 months of age on. Except for one pig, which has 6 integrated transgene copies, no dramatic decrease in general mobility was observed even at 6 months of age. OCT examinations reveal no major changes in the ONL structure of the 6-months old pigs. The retina morphology was well conserved in the 2 pigs sacrificed (3 and 6 months old) except a noticeable displacement of some cone nuclei in the outer segment layer.Conclusions: Lentiviral-directed transgenesis is a rapid and straightforward method to engineer transgenic pigs. Some Arr3-GUCY2DE837D/R838S pigs show signs of retinal dysfunction but further work is needed to describe the progression of the disease in this model.

Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.

Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.

Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.

BACKGROUND: NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD). The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2)). NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients.

Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin (HTT) protein and for which there is no cure. Although suppression of both wild type and mutant HTT expression by RNA interference is a promising therapeutic strategy, a selective silencing of mutant HTT represents the safest approach preserving WT HTT expression and functions. We developed small hairpin RNAs (shRNAs) targeting single nucleotide polymorphisms (SNP) present in the HTT gene to selectively target the disease HTT isoform. Most of these shRNAs silenced, efficiently and selectively, mutant HTT in vitro. Lentiviral-mediated infection with the shRNAs led to selective degradation of mutant HTT mRNA and prevented the apparition of neuropathology in HD rat's striatum expressing mutant HTT containing the various SNPs. In transgenic BACHD mice, the mutant HTT allele was also silenced by this approach, further demonstrating the potential for allele-specific silencing. Finally, the allele-specific silencing of mutant HTT in human embryonic stem cells was accompanied by functional recovery of the vesicular transport of BDNF along microtubules. These findings provide evidence of the therapeutic potential of allele-specific RNA interference for HD.

Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate.

Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport.

Dominant Role of CD80-CD86 Over CD40 and ICOSL in the Massive Polyclonal B Cell Activation Mediated by LAT(Y136F) CD4(+) T Cells.

Coordinated interactions between T and B cells are crucial for inducing physiological B cell responses. Mutant mice in which tyrosine 136 of linker for activation of T cell (LAT) is replaced by a phenylalanine (Lat(Y136F)) exhibit a strong CD4(+) T cell proliferation in the absence of intended immunization. The resulting effector T cells produce high amounts of T(H)2 cytokines and are extremely efficient at inducing polyclonal B cell activation. As a consequence, these Lat(Y136F) mutant mice showed massive germinal center formations and hypergammaglobulinemia. Here, we analyzed the involvement of different costimulators and their ligands in such T-B interactions both in vitro and in vivo, using blocking antibodies, knockout mice, and adoptive transfer experiments. Surprisingly, we showed in vitro that although B cell activation required contact with T cells, CD40, and inducible T cell costimulator molecule-ligand (ICOSL) signaling were not necessary for this process. These observations were further confirmed in vivo, where none of these molecules were required for the unfolding of the LAT CD4(+) T cell expansion and the subsequent polyclonal B cell activation, although, the absence of CD40 led to a reduction of the follicular B cell response. These results indicate that the crucial functions played by CD40 and ICOSL in germinal center formation and isotype switching in physiological humoral responses are partly overcome in Lat(Y136F) mice. By comparison, the absence of CD80-CD86 was found to almost completely block the in vitro B cell activation mediated by Lat(Y136F) CD4(+) T cells. The role of CD80-CD86 in T-B cooperation in vivo remained elusive due to the upstream implication of these costimulatory molecules in the expansion of Lat(Y136F) CD4(+) T cells. Together, our data suggest that CD80 and CD86 costimulators play a key role in the polyclonal B cell activation mediated by Lat(Y136F) CD4(+) T cells even though additional costimulatory molecules or cytokines are likely to be required in this process.

Induction of p38, tumour necrosis factor-α and RANTES by mechanical stretching of keratinocytes expressing mutant keratin 10R156H.

Background : Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma), characterized by ichthyotic, rippled hyperkeratosis, erythroderma and skin blistering, is a rare autosomal dominant disease caused by mutations in keratin 1 or keratin 10 (K10) genes. A severe phenotype is caused by a missense mutation in a highly conserved arginine residue at position 156 (R156) in K10. Objectives: To analyse molecular pathomechanisms of hyperproliferation and hyperkeratosis, we investigated the defects in mechanosensation and mechanotransduction in keratinocytes carrying the K10R156H mutation. Methods: Differentiated primary human keratinocytes infected with lentiviral vectors carrying wild-type K10 (K10wt) or mutated K10R156H were subjected to 20% isoaxial stretch. Cellular fragility and mechanosensation were studied by analysis of mitogen-activated protein kinase activation and cytokine release. Results: Cultured keratinocytes expressing K10R156H showed keratin aggregate formation at the cell periphery, whereas the filament network in K10wt cells was normal. Under stretching conditions K10R156H keratinocytes exhibited about a twofold higher level of filament collapse compared with steady state. In stretched K10R156H cells, higher p38 activation, higher release of tumour necrosis factor-alpha and RANTES but reduced interleukin-1 beta secretion compared with K10wt cells was observed. Conclusions: These results demonstrate that the R156H mutation in K10 destabilizes the keratin intermediate filament network and affects stress signalling and inflammatory responses to mechanical stretch in differentiated cultured keratinocytes.

Analysis of the esophagogastric junction using the 3D high resolution manometry

Contexte & Objectifs : La manométrie perfusée conventionnelle et la manométrie haute résolution (HRM) ont permis le développement d’une variété de paramètres pour mieux comprendre la motilité de l'œsophage et quantifier les caractéristiques de la jonction œsophago-gastrique (JOG). Cependant, l'anatomie de la JOG est complexe et les enregistrements de manométrie détectent à la fois la pression des structures intrinsèques et des structures extrinsèques à l'œsophage. Ces différents composants ont des rôles distincts au niveau de la JOG. Les pressions dominantes ainsi détectées au niveau de la JOG sont attribuables au sphincter œsophagien inférieur (SOI) et aux piliers du diaphragme (CD), mais aucune des technologies manométriques actuelles n’est capable de distinguer ces différents composants de la JOG. Lorsqu’on analyse les caractéristiques de la JOG au repos, celle ci se comporte avant tout comme une barrière antireflux. Les paramètres manométriques les plus couramment utilisés dans ce but sont la longueur de la JOG et le point d’inversion respiratoire (RIP), défini comme le lieu où le pic de la courbe de pression inspiratoire change de positif (dans l’abdomen) à négatif (dans le thorax), lors de la classique manœuvre de « pull-through ». Cependant, l'importance de ces mesures reste marginale comme en témoigne une récente prise de position de l’American Gastroenterology Association Institute (AGAI) (1) qui concluait que « le rôle actuel de la manométrie dans le reflux gastro-œsophagien (RGO) est d'exclure les troubles moteurs comme cause des symptômes présentés par la patient ». Lors de la déglutition, la mesure objective de la relaxation de la JOG est la pression de relaxation intégrée (IRP), qui permet de faire la distinction entre une relaxation normale et une relaxation anormale de la JOG. Toutefois, puisque la HRM utilise des pressions moyennes à chaque niveau de capteurs, certaines études de manométrie laissent suggérer qu’il existe une zone de haute pression persistante au niveau de la JOG même si un transit est mis en évidence en vidéofluoroscopie. Récemment, la manométrie haute résolution « 3D » (3D-HRM) a été développée (Given Imaging, Duluth, GA) avec le potentiel de simplifier l'évaluation de la morphologie et de la physiologie de la JOG. Le segment « 3D » de ce cathéter de HRM permet l'enregistrement de la pression à la fois de façon axiale et radiale tout en maintenant une position fixe de la sonde, et évitant ainsi la manœuvre de « pull-through ». Par conséquent, la 3D-HRM devrait permettre la mesure de paramètres importants de la JOG tels que sa longueur et le RIP. Les données extraites de l'enregistrement fait par 3D-HRM permettraient également de différencier les signaux de pression attribuables au SOI des éléments qui l’entourent. De plus, l’enregistrement des pressions de façon radiaire permettrait d’enregistrer la pression minimale de chaque niveau de capteurs et devrait corriger cette zone de haute pression parfois persistante lors la déglutition. Ainsi, les objectifs de ce travail étaient: 1) de décrire la morphologie de la JOG au repos en tant que barrière antireflux, en comparant les mesures effectuées avec la 3D-HRM en temps réel, par rapport à celle simulées lors d’une manœuvre de « pull-through » et de déterminer quelles sont les signatures des pressions attribuables au SOI et au diaphragme; 2) d’évaluer la relaxation de la JOG pendant la déglutition en testant l'hypothèse selon laquelle la 3D-HRM permet le développement d’un nouveau paradigme (appelé « 3D eSleeve ») pour le calcul de l’IRP, fondé sur l’utilisation de la pression radiale minimale à chaque niveau de capteur de pression le long de la JOG. Ce nouveau paradigme sera comparé à une étude de transit en vidéofluoroscopie pour évaluer le gradient de pression à travers la JOG. Méthodes : Nous avons utilisé un cathéter 3D-HRM, qui incorpore un segment dit « 3D » de 9 cm au sein d’un cathéter HRM par ailleurs standard. Le segment 3D est composé de 12 niveaux (espacés de 7.5mm) de 8 capteurs de pression disposés radialement, soit un total de 96 capteurs. Neuf volontaires ont été étudiés au repos, où des enregistrements ont été effectués en temps réel et pendant une manœuvre de « pull-through » du segment 3D (mobilisation successive du cathéter de 5 mm, pour que le segment 3D se déplace le long de la JOG). Les mesures de la longueur du SOI et la détermination du RIP ont été réalisées. La longueur de la JOG a été mesurée lors du « pull-through » en utilisant 4 capteurs du segment 3D dispersés radialement et les marges de la JOG ont été définies par une augmentation de la pression de 2 mmHg par rapport à la pression gastrique ou de l’œsophage. Pour le calcul en temps réel, les limites distale et proximale de la JOG ont été définies par une augmentation de pression circonférentielle de 2 mmHg par rapport à la pression de l'estomac. Le RIP a été déterminée, A) dans le mode de tracé conventionnel avec la méthode du « pull-through » [le RIP est la valeur moyenne de 4 mesures] et B) en position fixe, dans le mode de représentation topographique de la pression de l’œsophage, en utilisant l’outil logiciel pour déterminer le point d'inversion de la pression (PIP). Pour l'étude de la relaxation de la JOG lors de la déglutition, 25 volontaires ont été étudiés et ont subi 3 études de manométrie (10 déglutitions de 5ml d’eau) en position couchée avec un cathéter HRM standard et un cathéter 3D-HRM. Avec la 3D-HRM, l’analyse a été effectuée une fois avec le segment 3D et une fois avec une partie non 3D du cathéter (capteurs standard de HRM). Ainsi, pour chaque individu, l'IRP a été calculée de quatre façons: 1) avec la méthode conventionnelle en utilisant le cathéter HRM standard, 2) avec la méthode conventionnelle en utilisant le segment standard du cathéter 3D-HRM, 3) avec la méthode conventionnelle en utilisant le segment « 3D » du cathéter 3D-HRM, et 4) avec le nouveau paradigme (3D eSleeve) qui recueille la pression minimale de chaque niveau de capteurs (segment 3D). Quatorze autres sujets ont subi une vidéofluoroscopie simultanée à l’étude de manométrie avec le cathéter 3D-HRM. Les données de pression ont été exportés vers MATLAB ™ et quatre pressions ont été mesurées simultanément : 1) la pression du corps de l’œsophage, 2cm au-dessus de la JOG, 2) la pression intragastrique, 3) la pression radiale moyenne de la JOG (pression du eSleeve) et 4) la pression de la JOG en utilisant la pression minimale de chaque niveau de capteurs (pression du 3D eSleeve). Ces données ont permis de déterminer le temps permissif d'écoulement du bolus (FPT), caractérisé par la période au cours de laquelle un gradient de pression existe à travers la JOG (pression œsophagienne > pression de relaxation de la JOG > pression gastrique). La présence ou l'absence du bolus en vidéofluoroscopie et le FPT ont été codés avec des valeurs dichotomiques pour chaque période de 0,1 s. Nous avons alors calculé la sensibilité et la spécificité correspondant à la valeur du FPT pour la pression du eSleeve et pour la pression du 3D eSleeve, avec la vidéofluoroscopie pour référence. Résultats : Les enregistrements avec la 3D-HRM laissent suggérer que la longueur du sphincter évaluée avec la méthode du « pull-through » était grandement exagéré en incorporant dans la mesure du SOI les signaux de pression extrinsèques à l’œsophage, asymétriques et attribuables aux piliers du diaphragme et aux structures vasculaires. L’enregistrement en temps réel a permis de constater que les principaux constituants de la pression de la JOG au repos étaient attribuables au diaphragme. L’IRP calculé avec le nouveau paradigme 3D eSleeve était significativement inférieur à tous les autres calculs d'IRP avec une limite supérieure de la normale de 12 mmHg contre 17 mmHg pour l’IRP calculé avec la HRM standard. La sensibilité (0,78) et la spécificité (0,88) du 3D eSleeve étaient meilleurs que le eSleeve standard (0,55 et 0,85 respectivement) pour prédire le FPT par rapport à la vidéofluoroscopie. Discussion et conclusion : Nos observations suggèrent que la 3D-HRM permet l'enregistrement en temps réel des attributs de la JOG, facilitant l'analyse des constituants responsables de sa fonction au repos en tant que barrière antireflux. La résolution spatiale axiale et radiale du segment « 3D » pourrait permettre de poursuivre cette étude pour quantifier les signaux de pression de la JOG attribuable au SOI et aux structures extrinsèques (diaphragme et artéfacts vasculaires). Ces attributs du cathéter 3D-HRM suggèrent qu'il s'agit d'un nouvel outil prometteur pour l'étude de la physiopathologie du RGO. Au cours de la déglutition, nous avons évalué la faisabilité d’améliorer la mesure de l’IRP en utilisant ce nouveau cathéter de manométrie 3D avec un nouveau paradigme (3D eSleeve) basé sur l’utilisation de la pression radiale minimale à chaque niveau de capteurs de pression. Nos résultats suggèrent que cette approche est plus précise que celle de la manométrie haute résolution standard. La 3D-HRM devrait certainement améliorer la précision des mesures de relaxation de la JOG et cela devrait avoir un impact sur la recherche pour modéliser la JOG au cours de la déglutition et dans le RGO.