Left common carotid artery to left innominate vein arteriovenous fistula after transvenous laser lead extraction.

Transvenous lead extraction (TLE) is used for lead infection, lead debulking, venous recanalization and device upgrades. Lead extraction is performed using specialized tools including locking stylets, mechanical or rotating sheaths, femoral snares or laser sheaths. The most feared complications associated with lead extraction are bleeding, vascular tear, cardiac avulsion and tamponade. Despite technological progress, the incidence of major procedural complications including death remains slightly above 1%. This case depicts an asymptomatic left common carotid artery (LCCA) to left innominate vein arteriovenous fistula (AVF) after laser-assisted TLE successfully treated with an endovascular covered stent.

VGluT2 Expression in Dopamine Neurons Contributes to Postlesional Striatal Reinnervation.

A subset of adult ventral tegmental area dopamine (DA) neurons expresses vesicular glutamate transporter 2 (VGluT2) and releases glutamate as a second neurotransmitter in the striatum, while only few adult substantia nigra DA neurons have this capacity. Recent work showed that cellular stress created by neurotoxins such as MPTP and 6-hydroxydopamine can upregulate VGluT2 in surviving DA neurons, suggesting the possibility of a role in cell survival, although a high level of overexpression could be toxic to DA neurons. Here we examined the level of VGluT2 upregulation in response to neurotoxins and its impact on postlesional plasticity. We first took advantage of an in vitro neurotoxin model of Parkinson's disease and found that this caused an average 2.5-fold enhancement of Vglut2 mRNA in DA neurons. This could represent a reactivation of a developmental phenotype because using an intersectional genetic lineage-mapping approach, we find that >98% of DA neurons have a VGluT2+ lineage. Expression of VGluT2 was detectable in most DA neurons at embryonic day 11.5 and was localized in developing axons. Finally, compatible with the possibility that enhanced VGluT2 expression in DA neurons promotes axonal outgrowth and reinnervation in the postlesional brain, we observed that DA neurons in female and male mice in which VGluT2 was conditionally removed established fewer striatal connections 7 weeks after a neurotoxin lesion. Thus, we propose here that the developmental expression of VGluT2 in DA neurons can be reactivated at postnatal stages, contributing to postlesional plasticity of dopaminergic axons.SIGNIFICANCE STATEMENT A small subset of dopamine neurons in the adult, healthy brain expresses vesicular glutamate transporter 2 (VGluT2) and thus releases glutamate as a second neurotransmitter in the striatum. This neurochemical phenotype appears to be plastic as exposure to neurotoxins, such as 6-OHDA or MPTP, that model certain aspects of Parkinson's disease pathophysiology, boosts VGluT2 expression in surviving dopamine neurons. Here we show that this enhanced VGluT2 expression in dopamine neurons drives axonal outgrowth and contributes to dopamine neuron axonal plasticity in the postlesional brain. A better understanding of the neurochemical changes that occur during the progression of Parkinson's disease pathology will aid the development of novel therapeutic strategies for this disease.

A plant-derived quadrivalent virus like particle influenza vaccine induces cross-reactive antibody and T cell response in healthy adults.

Recent issues regarding efficacy of influenza vaccines have re-emphasized the need of new approaches to face this major public health issue. In a phase 1-2 clinical trial, healthy adults received one intramuscular dose of a seasonal influenza plant-based quadrivalent virus-like particle (QVLP) vaccine or placebo. The hemagglutination inhibition (HI) titers met all the European licensure criteria for the type A influenza strains at the 3µg/strain dose and for all four strains at the higher dosages 21days after immunization. High HI titers were maintained for most of the strains 6months after vaccination. QVLP vaccine induced a substantial and sustained increase of hemagglutinin-specific polyfunctional CD4 T cells, mainly transitional memory and TEMRA effector IFN-γ(+) CD4 T cells. A T cells cross-reactive response was also observed against A/Hong-Kong/1/1968 H3N2 and B/Massachusetts/2/2012. Plant-based QVLP offers an attractive alternative manufacturing method for producing effective and HA-strain matching seasonal influenza vaccines.

Excessive Wnt/beta-catenin signaling promotes midbrain floor plate neurogenesis, but results in vacillating dopamine progenitors.

The floor plate (FP), a ventral midline structure of the developing neural tube, has differential neurogenic capabilities along the anterior-posterior axis. The midbrain FP, unlike the hindbrain and spinal cord floor plate, is highly neurogenic and produces midbrain dopaminergic (mDA) neurons. Canonical Wnt/beta-catenin signaling, at least in part, is thought to account for the difference in neurogenic capability. Removal of beta-catenin results in mDA progenitor specification defects as well as a profound reduction of neurogenesis. To examine the effects of excessive Wnt/beta-catenin signaling on mDA specification and neurogenesis, we have analyzed a model wherein beta-catenin is conditionally stabilized in the Shh+domain. Here, we show that the Foxa2+/Lmx1a+ domain is extended rostrally in mutant embryos, suggesting that canonical Wnt/beta-catenin signaling can drive FP expansion along the rostrocaudal axis. Although excess canonical Wnt/beta-catenin signaling generally promotes neurogenesis at midbrain levels, less tyrosine hydroxylase (Th)+, mDA neurons are generated, particularly impacting the Substantia Nigra pars compacta. This is likely because of improper progenitor specification. Excess canonical Wnt/beta-catenin signaling causes downregulation of net Lmx1b, Shh and Foxa2 levels in mDA progenitors. Moreover, these progenitors assume a mixed identity to that of Lmx1a+/Lmx1b+/Nkx6-1+/Neurog1+ progenitors. We also show by lineage tracing analysis that normally, Neurog1+ progenitors predominantly give rise to Pou4f1+ neurons, but not Th+ neurons. Accordingly, in the mutant embryos, Neurog1+ progenitors at the midline generate ectopic Pou4f1+ neurons at the expense of Th+ mDA neurons. Our study suggests that an optimal dose of Wnt/beta-catenin signaling is critical for proper establishment of the mDA progenitor character. Our findings will impact embryonic stem cell protocols that utilize Wnt pathway reagents to derive mDA neuron models and therapeutics for Parkinson's disease.

Influenza virus-like particle vaccines made in Nicotiana benthamiana elicit durable, poly-functional and cross-reactive T cell responses to influenza HA antigens.

Cell-mediated immunity plays a major role in long-lived, cross-reactive protection against influenza virus. We measured long-term poly-functional and cross-reactive T cell responses to influenza hemagglutinin (HA) elicited by a new plant-made Virus-Like Particle (VLP) vaccine targeting either H1N1 A/California/7/09 (H1) or H5N1 A/Indonesia/5/05 (H5). In two independent clinical trials, we characterized the CD4(+) and CD8(+) T cell homotypic and heterotypic responses 6 months after different vaccination regimens. Responses of VLP-vaccinated subjects were compared with placebo and/or a commercial trivalent inactivated vaccine (TIV:Fluzone™) recipients. Both H1 and H5 VLP vaccines elicited significantly greater poly-functional CD4(+) T cell responses than placebo and TIV. Poly-functional CD8(+) T cell responses were also observed after H1 VLP vaccination. Our results show that plant-made HA VLP vaccines elicit both strong antibody responses and poly-functional, cross-reactive memory T cells that persist for at least 6 months after vaccination.

Enkephalin downregulation in the nucleus accumbens underlies chronic stress-induced anhedonia.

Restraint and immobilization have been extensively used to study habituation of the neuroendocrine response to a repeated stressor, but behavioral consequences of this stress regimen remain largely uncharacterized. In this study, we used sucrose preference and the elevated-plus maze to probe behavioral alterations resulting from 14 days of restraint in rats. We observed a decrease in sucrose preference in stressed animals, particularly in a subgroup of individuals, but no alteration in anxiety behaviors (as measured in the elevated-plus maze) four days following the last restraint. In these low-sucrose preference animals, we observed a downregulation of the expression of preproenkephalin mRNA in the nucleus accumbens. Furthermore, we observed a strong correlation between enkephalin expression and sucrose preference in the shell part of the nucleus accumbens, with a lower level of enkephalin expression being associated with lower sucrose preference. Interestingly, quantification of the corticosterone response revealed a delayed habituation to restraint in the low-sucrose preference population, which suggests that vulnerability to stress-induced deficits might be associated with prolonged exposure to glucocorticoids. The induction of ΔFosB is also reduced in the nucleus accumbens shell of the low-sucrose preference population and this transcription factor is expressed in enkephalin neurons. Taken together, these results suggest that a ΔFosB-mediated downregulation of enkephalin in the nucleus accumbens might underlie the susceptibility to chronic stress. Further experiments will be needed to determine causality between these two phenomena.

Molecular and spatial transcriptomic classification of midbrain dopamine neurons and their alterations in a LRRK2G2019S model of Parkinson's disease.

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using single cell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson's disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2G2019S knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.

Enkephalin knockdown in the central amygdala nucleus reduces unconditioned fear and anxiety.

The endogenous opioid enkephalins (ENK) are highly expressed in the central nucleus of the amygdaloid complex (CeA) where several lines of evidence point to a potential role in the modulation of fear and anxiety. In this study, we aimed to assess the role of CeA ENK using local injections of a lentiviral vector expressing a short hairpin RNA (shRNA) targeting ENK in Sprague-Dawley rats. We injected this vector in the CeA and a 56% downregulation of ENK mRNA was observed in animals when compared with scrambled shRNA animals. Anxiety-like behaviors were also assessed using the elevated plus maze and social interaction test. There was an increase in exploration of open arms of the elevated plus maze in ENK knockdown animals compared with controls, but no change in social interaction. In addition, we used the contextual fear conditioning procedure to assess fear expression and learning in these animals. There was a reduction in freezing induced by acute shocks during the training procedure. Interestingly, associative learning was not affected, and ENK knockdown animals displayed an equivalent freezing when re-exposed to the conditioning chamber 48 h later. These results contrast with knockout mice studies, which ascribed anxiolytic properties to ENK, and they demonstrate the need for a thorough understanding and characterization of neuroanatomically distinct ENK pathways.

Sox6 expression distinguishes dorsally and ventrally biased dopamine neurons in the substantia nigra with distinctive properties and embryonic origins.

Dopamine (DA) neurons in the ventral tier of the substantia nigra pars compacta (SNc) degenerate prominently in Parkinson's disease, while those in the dorsal tier are relatively spared. Defining the molecular, functional, and developmental characteristics of each SNc tier is crucial to understand their distinct susceptibility. We demonstrate that Sox6 expression distinguishes ventrally and dorsally biased DA neuron populations in the SNc. The Sox6+ population in the ventral SNc includes an Aldh1a1+ subset and is enriched in gene pathways that underpin vulnerability. Sox6+ neurons project to the dorsal striatum and show activity correlated with acceleration. Sox6- neurons project to the medial, ventral, and caudal striatum and respond to rewards. Moreover, we show that this adult division is encoded early in development. Overall, our work demonstrates a dual origin of the SNc that results in DA neuron cohorts with distinct molecular profiles, projections, and functions.

Cysteamine, the molecule used to treat cystinosis, potentiates the antimalarial efficacy of artemisinin.

Malaria continues to be a major threat to global health. Artemisinin combination therapy (ACT) is the recommended treatment for clinical malaria; however, recent reports of parasite resistance to artemisinin in certain areas where malaria is endemic have stressed the need for developing more efficacious ACT. We report that cysteamine (Cys), the aminothiol used to treat nephropathic cystinosis in humans, strongly potentiates the efficacy of artemisinin against the Plasmodium parasite in vivo. Using a mouse model of infection with Plasmodium chabaudi AS, we observe that Cys dosing used to treat cystinosis in humans can strongly potentiate (by 3- to 4-fold) the antimalarial properties of the artemisinin derivatives artesunate and dihydroartemisinin. Addition of Cys to suboptimal doses of artemisinin delays the appearance of blood parasitemia, strongly reduces the extent of parasite replication, and significantly improves survival in a model of lethal P. chabaudi infection. Cys, the natural product of the enzyme pantetheinase, has a history of safe use for the clinical management of cystinosis. Our findings suggest that Cys could be included in novel ACTs to improve efficacy against Plasmodium parasite replication, including artemisinin-resistant isolates. Future work will include clinical evaluation of novel Cys-containing ACTs and elucidation of the mechanism underlying the potentiation effect of Cys.

Classification of Midbrain Dopamine Neurons Using Single-Cell Gene Expression Profiling Approaches.

Dysfunctional dopamine (DA) signaling has been associated with a broad spectrum of neuropsychiatric disorders, prompting investigations into how midbrain DA neuron heterogeneity may underpin this variety of behavioral symptoms. Emerging literature indeed points to functional heterogeneity even within anatomically defined DA clusters. Recognizing the need for a systematic classification scheme, several groups have used single-cell profiling to catalog DA neurons based on their gene expression profiles. We aim here not only to synthesize points of congruence but also to highlight key differences between the molecular classification schemes derived from these studies. In doing so, we hope to provide a common framework that will facilitate investigations into the functions of DA neuron subtypes in the healthy and diseased brain.

PRISM: A Progenitor-Restricted Intersectional Fate Mapping Approach Redefines Forebrain Lineages.

Lineage tracing aims to identify the progeny of a defined population of dividing progenitor cells, a daunting task in the developing central nervous system where thousands of cell types are generated. In mice, lineage analysis has been accomplished using Cre recombinase to indelibly label a defined progenitor population and its progeny. However, the interpretation of historical recombination events is hampered by the fact that driver genes are often expressed in both progenitors and postmitotic cells. Genetically inducible approaches provide temporal specificity but are afflicted by mosaicism and toxicity. Here, we present PRISM, a progenitor-restricted intersectional fate mapping approach in which Flp recombinase expression is both dependent on Cre and restricted to neural progenitors, thus circumventing the aforementioned confounds. This tool can be used in conjunction with existing Cre lines making it broadly applicable. We applied PRISM to resolve two developmentally important, but contentious, lineages-Shh and Cux2.

Immunogenicity and safety of a quadrivalent plant-derived virus like particle influenza vaccine candidate-Two randomized Phase II clinical trials in 18 to 49 and ≥50 years old adults.

BACKGROUND: New influenza vaccines eliciting more effective protection are needed, particularly for the elderly who paid a large and disproportional toll of hospitalization and dead during seasonal influenza epidemics. Low (≤15 µg/strain) doses of a new plant-derived virus-like-particle (VLP) vaccine candidate have been shown to induce humoral and cellular responses against both homologous and heterologous strains in healthy adults 18-64 years of age. The two clinical trials reported here addressed the safety and immunogenicity of higher doses (≥15 µg/strain) of quadrivalent VLP candidate vaccine on 18-49 years old and ≥50 years old subjects. We also investigated the impact of alum on the immunogenicity induced by lower doses of the vaccine candidate. METHOD: In the first Phase II trial reported here (NCT02233816), 18-49 year old subjects received 15, 30 or 60 µg/strain of a hemagglutinin-bearing quadrivalent virus-like particle (QVLP) vaccine or placebo. In the second trial (NCT02236052), ≥50 years old subjects received QVLP as above or placebo with additional groups receiving 7.5 or 15 µg/strain with alum. Along with safety recording, the humoral and cell-mediated immune responses were analyzed. RESULTS: Local and systemic side-effects were similar to those reported previously. The QVLP vaccine induced significant homologous and heterologous antibody responses at the two higher doses, the addition of alum having little-to-no effect. Serologic outcomes tended to be lower in ≥50 years old subjects previously vaccinated. The candidate vaccine also consistently elicited both homologous and heterologous antigen-specific CD4+ T cells characterized by their production of interferon-gamma (IFN-γ), interleukine-2 (IL-2) and/or tumor-necrosis factor alpha (TNF-α) upon ex vivo antigenic restimulation. CONCLUSION: Overall, the 30 µg dose produced the most consistent humoral and cellular responses in both 18-49 and ≥50 years old subjects, strongly supporting the clinical development of this candidate vaccine. That particular dose was chosen to test in the ongoing Phase III clinical trial.

Enkephalin co-expression with classic neurotransmitters in the amygdaloid complex of the rat.

This study aimed at characterizing the neurotransmitter phenotype of enkephalin neurons in the rat amygdaloid complex. We first established the detailed distribution of vesicular glutamate transporters 1 and 2 (VGLUT1 and -2) and glutamate decarboxylase 65 (GAD65) in the amygdala by using in situ hybridization. In the amygdaloid complex, GAD65 is strongly expressed in striatal-like divisions, namely, the anterior amygdaloid area, the central nucleus (CEA), the intercalated nuclei, and the dorsal part of the medial nucleus (MEA). VGLUT1 and -2 expression is mostly segregated to specific divisions of the amygdale, with VGLUT2 being expressed only in the MEA, the anterior cortical nucleus (COAa), and the anterior basomedial nucleus (BMAa), whereas VGLUT1 is expressed in all other divisions of the amygdala. Second, we assessed the co-expression of preproenkephalin (ppENK) with GAD65, VGLUT1, or VGLUT2 by using double fluorescent in situ hybridization. We found that ppENK mRNA co-localized exclusively with GAD65 in all striatal-like structures of the amygdaloid complex with the exception of the MEA, where ENK also co-localized with VGLUT2 mRNA. This co-localization is most apparent in the posteroventral part of the MEA, where 70% of ENKergic cells expressed VGLUT2. In addition, ppENK also co-localized with VGLUT1 because more than 95% of ENK cells in the basolateral amygdala expressed VGLUT1. In contrast, less than 25% of ENKergic cells expressed VGLUT1 in the lateral nucleus of the amygdale, with the majority of ENK cells expressing GAD65 mRNA in this nucleus. These results have broad implications for understanding the functional roles of enkephalinergic neurotransmission in the amygdaloid complex.

Impact of feed solution flow rate on Peptide fractionation by electrodialysis with ultrafiltration membrane.

Recently, processes combining an electrical field as a driving force to porous membranes have been developed for the separation of protein or peptide mixtures to obtain more purified products with higher functionality or nutritional value. The objective of this work was to evaluate the influence of the flow rate on the productivity and selectivity as well as on the electrodialytic parameters of electrodialysis with an ultrafiltration membrane (EDUF) during the fractionation of peptides from a beta-lactoglobulin tryptic hydrolysate. It appeared that the feed solution flow rate had no impact on the yield of the process but induced changes in the selectivity. In fact, increases in the flow rate decreased the migration of the peptides with limited electrophoretic mobility.

Humoral and cell-mediated immune responses to H5N1 plant-made virus-like particle vaccine are differentially impacted by alum and GLA-SE adjuvants in a Phase 2 clinical trial.

The hemagglutinination inhibition (HI) response remains the gold standard used for the licensure of influenza vaccines. However, cell-mediated immunity (CMI) deserves more attention, especially when evaluating H5N1 influenza vaccines that tend to induce poor HI response. In this study, we measured the humoral response (HI) and CMI (flow cytometry) during a Phase II dose-ranging clinical trial (NCT01991561). Subjects received two intramuscular doses, 21 days apart, of plant-derived virus-like particles (VLP) presenting the A/Indonesia/05/2005 H5N1 influenza hemagglutinin protein (H5) at the surface of the VLP (H5VLP). The vaccine was co-administrated with Alhydrogel® or with a glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE). We demonstrated that low doses (3.75 or 7.5 µg H5VLP) of GLA-SE-adjuvanted vaccines induced HI responses that met criteria for licensure at both antigen doses tested. Alhydrogel adjuvanted vaccines induced readily detectable HI response that however failed to meet licensure criteria at any of three doses (10, 15 and 20 µg) tested. The H5VLP also induced a sustained (up to 6 months) polyfunctional and cross-reactive HA-specific CD4+ T cell response in all vaccinated groups. Interestingly, the frequency of central memory Th1-primed precursor cells before the boost significantly correlated with HI titers 21 days after the boost. The ability of the low dose GLA-SE-adjuvanted H5VLP to elicit both humoral response and a sustained cross-reactive CMI in healthy adults is very attractive and could result in significant dose-sparing in a pandemic situation.

Dopamine neuron glutamate cotransmission evokes a delayed excitation in lateral dorsal striatal cholinergic interneurons.

Dopamine neurons have different synaptic actions in the ventral and dorsal striatum (dStr), but whether this heterogeneity extends to dStr subregions has not been addressed. We have found that optogenetic activation of dStr dopamine neuron terminals in mouse brain slices pauses the firing of cholinergic interneurons in both the medial and lateral subregions, while in the lateral subregion the pause is shorter due to a subsequent excitation. This excitation is mediated mainly by metabotropic glutamate receptor 1 (mGluR1) and partially by dopamine D1-like receptors coupled to transient receptor potential channel 3 and 7. DA neurons do not signal to spiny projection neurons in the medial dStr, while they elicit ionotropic glutamate responses in the lateral dStr. The DA neurons mediating these excitatory signals are in the substantia nigra (SN). Thus, SN dopamine neurons engage different receptors in different postsynaptic neurons in different dStr subregions to convey strikingly different signals. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Mapping projections of molecularly defined dopamine neuron subtypes using intersectional genetic approaches.

Midbrain dopamine (DA) neurons have diverse functions that can in part be explained by their heterogeneity. Although molecularly distinct subtypes of DA neurons have been identified by single-cell gene expression profiling, fundamental features such as their projection patterns have not been elucidated. Progress in this regard has been hindered by the lack of genetic tools for studying DA neuron subtypes. Here we develop intersectional genetic labeling strategies, based on combinatorial gene expression, to map the projections of molecularly defined DA neuron subtypes. We reveal distinct genetically defined dopaminergic pathways arising from the substantia nigra pars compacta and from the ventral tegmental area that innervate specific regions of the caudate putamen, nucleus accumbens and amygdala. Together, the genetic toolbox and DA neuron subtype projections presented here constitute a resource that will accelerate the investigation of this clinically significant neurotransmitter system.

Enkephalinergic afferents of the centromedial amygdala in the rat.

The connectivity of the amygdaloid complex has been extensively explored with both anterograde and retrograde tracers. Even though the afferents of the centromedial amygdala [comprising the central (CEA) and medial (MEA) amygdaloid nuclei] are well established, relatively little is known about the neuropeptide phenotype of these connections. In this study, we first examined the distribution of mu-opioid receptor (MOR) and delta-opioid receptor (DOR) in the amygdala via in situ hybridization and immunohistochemistry. We then investigated the distribution of Met-enkephalin (ENK) and Leu-ENK fibers with immunohistochemistry and examined the distribution of preproenkephalin mRNA in the amygdala by using in situ hybridization. Finally, we examined the ENK projections to the CEA and MEA by using stereotaxic injections of the retrograde tracer cholera toxin subunit B or fluorogold revealed by immunohistochemistry combined with in situ hybridization to identify ENKergic neurons. Our results indicate that the centromedial amygdala receives ENK afferents, as indicated by the presence of MOR, DOR, and ENK fibers in the CEA and MEA, originating primarily from the bed nucleus of the stria terminalis (BST) and from other amygdaloid nuclei. The posterior BST, the basomedial nucleus (BMA), and the cortical nucleus of the amygdala (COA) were found to be the major ENK afferents of the MEA, whereas the anterolateral BST, the COA, the MEA, and the BMA provided the main ENKergic innervation of the CEA. In addition, we found that the ventromedial nucleus of the hypothalamus and the pontine parabrachial nucleus provide a moderate ENK input to the CEA and MEA. The functional implications of these connections in stress, anxiety, and nociception are discussed.