Neonatal Fc receptor FcRn is involved in intracellular transport of the Fc fusion protein aflibercept and its transition through retinal endothelial cells

AbstractRetinal endothelial cells (REC) likely contribute to the clearance of intravitreally injected IgG. Because this is of high relevance to the pharmacokinetic assessment of the widely used therapeutic Fc fusion protein aflibercept, we studied its transport through immortalized bovine REC (iBREC) in detail. For shuttling of IgG or Fc fusion proteins like aflibercept, endothelial cells use the highly conserved neonatal Fc receptor (FcRn) also expressed in iBREC where it is down regulated by serum depletion. Therefore, we focused on studying intracellular localization and transport of aflibercept under conditions affecting its interaction with the FcRn. Intracellular localization of aflibercept was assessed by Western-blot analyses of subcellular protein fractions or by immunofluorescence staining. After uptake in a temperature-dependent process, aflibercept co-localized with early endosomes, which harbor FcRn. Similar amounts of aflibercept were co-extracted with proteins from membranes/organelles irrespectively of the amount of FBS in the culture medium. Lowering the concentration of FBS resulted in a strong, but reversible association with cytoskeletal proteins suggesting a block in intracellular transport. In accordance with this finding, aflibercept's transport through an iBREC monolayer grown on porous membrane inserts was markedly delayed in the absence of FBS in the culture medium indicating that aflibercept is taken up but not exocytosed under these conditions. Transcytosis of aflibercept was also strongly delayed by inhibition of phosphatidylinositol 3-kinase with LY294002, which affects FcRn-mediated IgG transport. A similar inhibition of aflibercept's transport was observed with IgG-binding proteins (i.e. protein A or protein G) that block interaction between FcRn and aflibercept. Interfering with aflibercept's binding to the FcRn with protein A (or protein G) or the inhibitory FcRn-specific monoclonal antibody 1G3 resulted in a reduced amount of intracellular aflibercept. Taken together, our results strongly suggest that FcRn is involved in transport of aflibercept through REC in vitro

Left atrial tachycardia after circumferential pulmonary vein ablation for atrial fibrillation Electroanatomic characterization and treatment

ObjectivesThe purpose of this study was to evaluate the electroanatomic characteristics of left atrial tachycardia (AT) in a series of patients who underwent circumferential pulmonary vein ablation (CPVA) and to describe the ablation strategy and clinical outcome.BackgroundCircumferential pulmonary vein ablation is an effective treatment for atrial fibrillation. A potential midterm complication is the development of left AT. There are only isolated reports describing mapping and ablation of such arrhythmias.MethodsThirteen patients (age 57.4 ± 8.9 years, five female) underwent mapping and ablation of 14 left ATs via an electroanatomic mapping system a mean of 2.6 ± 1.6 months after CPVA.ResultsThree patients were characterized as having focal AT (cycle length: 266 ± 35.9 ms). Of 11 macro–re-entrant tachycardias studied in the remaining 10 patients (cycle length: 275 ± 75 ms), 5 showed single-loop and 6 dual-loop circuits. Re-entrant circuits used the mitral isthmus, the posterior wall, or gaps on previous encircling lines. Such gaps and all three foci occurred anterior to the left superior pulmonary vein or at the septal aspect of the right pulmonary veins. Thirteen of 14 tachycardias (93%) were successfully ablated.ConclusionsLeft AT after CPVA can be due to a macro–re-entrant or focal mechanism. Re-entry occurs most commonly across the mitral isthmus, the posterior wall, or gaps on previous ablation lines. Such gaps and foci occur most commonly at the anterior aspect of the left superior pulmonary vein and at the septal aspect of the right pulmonary veins. These arrhythmias can be successfully mapped and ablated with an electroanatomic mapping system

Off-shell superconformal nonlinear sigma-models in three dimensions

We develop superspace techniques to construct general off-shell N=1,2,3,4 superconformal sigma-models in three space-time dimensions. The most general N=3 and N=4 superconformal sigma-models are constructed in terms of N=2 chiral superfields. Several superspace proofs of the folklore statement that N=3 supersymmetry implies N=4 are presented both in the on-shell and off-shell settings. We also elaborate on (super)twistor realisations for (super)manifolds on which the three-dimensional N-extended superconformal groups act transitively and which include Minkowski space as a subspace.Comment: 67 pages; V2: typos corrected, one reference added, version to appear on JHE

A regional Bayesian POT model for flood frequency analysis

Flood frequency analysis is usually based on the fitting of an extreme value distribution to the local streamflow series. However, when the local data series is short, frequency analysis results become unreliable. Regional frequency analysis is a convenient way to reduce the estimation uncertainty. In this work, we propose a regional Bayesian model for short record length sites. This model is less restrictive than the index flood model while preserving the formalism of "homogeneous regions". The performance of the proposed model is assessed on a set of gauging stations in France. The accuracy of quantile estimates as a function of the degree of homogeneity of the pooling group is also analysed. The results indicate that the regional Bayesian model outperforms the index flood model and local estimators. Furthermore, it seems that working with relatively large and homogeneous regions may lead to more accurate results than working with smaller and highly homogeneous regions

Functional and structural findings of neurodegeneration in early stages of diabetic retinopathy:cross-sectional analyses of baseline data of the EUROCONDOR project

Cross-sectional study evaluating the relationship between: a) functional and structural measurements of neurodegeneration in initial stages of diabetic retinopathy (DR); and b) presence of neurodegeneration and early microvascular impairment. We analyzed baseline data of patients with type 2 diabetes (n=449) enrolled in the EUROCONDOR study (NCT01726075). Functional studies by multifocal ERG (mfERG) evaluated neurodysfunction and structural measurements using spectral domain optical-coherence tomography (SD-OCT) evaluated neurodegeneration. The mfERG P1 amplitude was more sensitive than the P1 implicit time (IT), and was lower in patients with ETDRS 20-35 than in patients with ETDRS <20 (p=0.005). In 58% of cases, mfERG abnormalities were present in the absence of visible retinopathy. Correspondence between SD-OCT thinning and mfERG abnormalities was shown in 67% of the eyes with ETDRS <20 and in 83% of the eyes with ETDRS 20-35. Notably, 32% of patients with ETDRS 20-35 presented no abnormalities in mfERG or SD-OCT. We conclude that there is a link between mfERG and SD-OCT measurements which increases with the presence of microvascular impairment. However, in our particular study population (ETDRS ≤ 35) a significant proportion of patients had normal GC-IPL thickness and normal mfERG findings. We raise the hypothesis that neurodegeneration may play a role in the pathogenesis of DR in many, but not in all type 2 diabetic patients

Implementation of a population-based epidemiological rare disease registry: study protocol of the amyotrophic lateral sclerosis (ALS) - registry Swabia

BACKGROUND: The social and medical impact of rare diseases is increasingly recognized. Amyotrophic lateral sclerosis (ALS) is the most prevalent of the motor neuron diseases. It is characterized by rapidly progressive damage to the motor neurons with a survival of 2–5 years for the majority of patients. The objective of this work is to describe the study protocol and the implementation steps of the amyotrophic lateral sclerosis (ALS) registry Swabia, located in the South of Germany. METHODS/DESIGN: The ALS registry Swabia started in October 2010 with both, the retrospective (01.10.2008-30.09.2010) and prospective (from 01.10.2010) collection of ALS cases, in a target population of 8.6 million persons in Southern Germany. In addition, a population based case–control study was implemented based on the registry that also included the collection of various biological materials. Retrospectively, 420 patients (222 men and 198 women) were identified. Prospectively data of ALS patients were collected, of which about 70% agreed to participate in the population-based case–control study. All participants in the case–control study provided also a blood sample. The prospective part of the study is ongoing. DISCUSSION: The ALS registry Swabia has been implemented successfully. In rare diseases such as ALS, the collaboration of registries, the comparison with external samples and biorepositories will facilitate to identify risk factors and to further explore the potential underlying pathophysiological mechanisms

First narrow-band search for continuous gravitational waves from known pulsars in advanced detector data

Spinning neutron stars asymmetric with respect to their rotation axis are potential sources of continuous gravitational waves for ground-based interferometric detectors. In the case of known pulsars a fully coherent search, based on matched filtering, which uses the position and rotational parameters obtained from electromagnetic observations, can be carried out. Matched filtering maximizes the signalto- noise (SNR) ratio, but a large sensitivity loss is expected in case of even a very small mismatch between the assumed and the true signal parameters. For this reason, narrow-band analysis methods have been developed, allowing a fully coherent search for gravitational waves from known pulsars over a fraction of a hertz and several spin-down values. In this paper we describe a narrow-band search of 11 pulsars using data from Advanced LIGO’s first observing run. Although we have found several initial outliers, further studies show no significant evidence for the presence of a gravitational wave signal. Finally, we have placed upper limits on the signal strain amplitude lower than the spin-down limit for 5 of the 11 targets over the bands searched; in the case of J1813-1749 the spin-down limit has been beaten for the first time. For an additional 3 targets, the median upper limit across the search bands is below the spin-down limit. This is the most sensitive narrow-band search for continuous gravitational waves carried out so far

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function.

The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins.Supported by Canadian Institutes of Health Research (PEF, PStGH), Alzheimer Society of Ontario (PEF, PStGH), Wellcome Trust (PStGH, MEV, CFK, GSK, DR, CEH), Medical Research Council (PStGH, MEV, CFK, GSK), National Institutes of Health Research, Alzheimer Research UK (CFK, GSK), Gates Cambridge Scholarship (JQL), Engineering and Physical Sciences Research Council (CFK, GSK), European Research Council Starting Grant RIBOMYLOME_309545 (GGT), European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement no. 322817 (CEH), and National Institute of Neurological Disorders and Stroke R01 NS07377 (NAS). The authors thank Tom Cech and Roy Parker for helpful discussions.This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.neuron.2015.10.03

Effects of Topically Administered Neuroprotective Drugs in Early Stages of Diabetic Retinopathy:Results of the EUROCONDOR Clinical Trial

The primary objective of this study was to assess whether the topical administration of two neuroprotective drugs (brimonidine and somatostatin) could prevent or arrest retinal neurodysfunction in patients with type 2 diabetes. For this purpose, adults aged between 45 and 75 years with a diabetes duration ≥5 years and an Early Treatment of Diabetic Retinopathy Study (ETDRS) level of ≤35 were randomly assigned to one of three arms: placebo, somatostatin, or brimonidine. The primary outcome was the change in implicit time (IT) assessed by multifocal electroretinography between baseline and at the end of follow-up (96 weeks). There were 449 eligible patients allocated to brimonidine (n = 152), somatostatin (n = 145), or placebo (n = 152). When the primary end point was evaluated in the whole population, we did not find any neuroprotective effect of brimonidine or somatostatin. However, in the subset of patients (34.7%) with preexisting retinal neurodysfunction, IT worsened in the placebo group (P < 0.001) but remained unchanged in the brimonidine and somatostatin groups. In conclusion, the topical administration of the selected neuroprotective agents appears useful in preventing the worsening of preexisting retinal neurodysfunction. This finding points to screening retinal neurodysfunction as a critical issue to identify a subset of patients in whom neuroprotective treatment might be of benefit