RESUMO
Live attenuated vaccines have proved to be mostly valuable in the prevention of infectious diseases in humans, especially in developing countries. The safety and potency of vaccine, and the consistency of vaccine batch-to-batch manufacturing, must be proven before being administrated to humans. For now, the tests used to control vaccine safety largely involve animal testing. For live viral vaccines, regulations require suppliers to demonstrate the absence of neurovirulence in animals, principally in non-human primates and mice. In a search to reduce the use of animals and embracing the 3Rs principles (Replacement, Reduction, Refinement in the use of laboratory animals), we developed a new Blood-Brain Barrier Minibrain (BBB-Minibrain) in cellulo device to evaluate the neuroinvasiveness/neurovirulence of live Yellow Fever virus (YFV) vaccines. A pilot study was performed using the features of two distinct YFV strains, with the ultimate goal of proposing a companion test to characterize YFV neurovirulence. Here, we demonstrate that the BBB-Minibrain model is a promising alternative to consider for future replacement of YFV vaccine in vivo neurovirulence testing (see graphical abstract).
Assuntos
Barreira Hematoencefálica/metabolismo , Modelos Imunológicos , Vacina contra Febre Amarela , Vírus da Febre Amarela , Barreira Hematoencefálica/virologia , Células Cultivadas , Humanos , Projetos Piloto , Controle de Qualidade , Vacina contra Febre Amarela/imunologia , Vacina contra Febre Amarela/farmacocinética , Vacina contra Febre Amarela/farmacologiaRESUMO
The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood-brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single-chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv-RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv-RVG fusion. Both ScFv and ScFv-RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv-RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant-produced ScFv-RVGP fusion could translocate across the cells. This study indicates that the plant-produced ScFv-RVGP fusion protein was able to cross the in celluloBBB and neutralize RABV.
Assuntos
Barreira Hematoencefálica , Glicoproteínas/imunologia , Fragmentos de Peptídeos/imunologia , Planticorpos/farmacologia , Vírus da Raiva/imunologia , Proteínas Virais/imunologia , Anticorpos Neutralizantes/biossíntese , Linhagem Celular , Humanos , Planticorpos/isolamento & purificação , Planticorpos/metabolismo , Plantas Geneticamente Modificadas , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusão , NicotianaRESUMO
Autophagy is a process leading to the degradation of cellular material, in organelles called lysosomes, to supply energy or generate building blocks for the synthesis of new materials. Over the past decades, its role has been evidenced in several indications, notably in neurodegenerative disorders and orphan diseases called lysosomal storage disorders and its modulation is largely envisioned as a therapeutic avenue to alleviate the symptoms and reverse the clinical courses of these indications. Identifying new chemical classes and drugs is, hence, of huge importance. In this study, we developed automated assays to assess the potential efficacy of chemical compounds on different steps of autophagy, notably its induction through the localization of a largely involved transcription factor, transcription factor EB (TFEB). These assays were then used to screen a collection of 1,520 approved drugs. This study led to the identification of five candidate hits modulating autophagy and TFEB subcellular localization. Our results suggest the repurposing potential of already approved drugs in central nervous system disorders with lysosomal storage impairments.
Assuntos
Autofagia , Lisossomos , Autofagia/fisiologia , Lisossomos/metabolismoRESUMO
TFEB is a mammalian transcription factor that binds directly to the CLEAR consensus sequence (5'-GTCACGTGAC-3') present in the regulatory regions of genes inducing autophagosome formation, autophagosome-lysosome fusion, hydrolase enzyme expression, and lysosomal exocytosis. By modulating these activities, TFEB coordinates on-demand control over each cell's degradation pathway. Thus, a nuclear signaling pathway regulates cellular energy metabolism through TFEB. Our growing understanding of the role of TFEB and CLEAR in the promotion of healthy clearance together with in vitro and in vivo preclinical findings in various animal models of disease supports the conclusion that the pharmacological activation of TFEB could clear toxic proteins to treat both rare and common forms of neurodegenerative disease.
Assuntos
Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Lisossomos/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica , Humanos , Doenças por Armazenamento dos Lisossomos , Estresse Oxidativo/fisiologia , Fosforilação/fisiologia , Transdução de SinaisRESUMO
Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that, as a consequence of the inflammatory process during JEV infection, there is disruption of the blood-brain barrier (BBB) tight junctions that in turn allows the virus access to the central nervous system (CNS). However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV (JEV RP9 and SA14-14-2, respectively) to cross an in vitro human BBB model. Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post viral addition. Furthermore, we find that almost 10 times more RP9 infectious particles than SA14-14 cross the model BBB, indicating this BBB model discriminates between the virulent RP9 and the vaccine SA14-14-2 strains of JEV. Beyond contributing to the understanding of early events in JEV neuroinvasion, we demonstrate this in vitro BBB model can be used as a system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB during early times of neuroinvasion.
Assuntos
Barreira Hematoencefálica/virologia , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Modelos Biológicos , Barreira Hematoencefálica/fisiologia , Linhagem Celular , Vírus da Encefalite Japonesa (Espécie)/genética , Vírus da Encefalite Japonesa (Espécie)/patogenicidade , Encefalite Japonesa/patologia , Encefalite Japonesa/virologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Virulência , Replicação ViralRESUMO
In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFPQL, by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.
Assuntos
Bioensaio , Conexina 43/genética , Drogas em Investigação/farmacologia , Neuroglia/efeitos dos fármacos , Medicamentos sob Prescrição/farmacologia , Automação Laboratorial , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzoxazóis/química , Cálcio/metabolismo , Carbenoxolona/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Conexina 43/antagonistas & inibidores , Conexina 43/metabolismo , Corantes Fluorescentes/química , Expressão Gênica , Humanos , Iodetos/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Ácido Meclofenâmico/farmacologia , Neuroglia/citologia , Neuroglia/metabolismo , Compostos de Quinolínio/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Imagem com Lapso de TempoRESUMO
OBJECTIVES: To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases. METHODS: Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes. RESULTS: Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1). CONCLUSIONS: We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.
Assuntos
Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Reação em Cadeia da Polimerase/métodos , beta-Lactamases/biossíntese , beta-Lactamases/genética , Antibacterianos/farmacologia , Primers do DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Sensibilidade e Especificidade , Análise de Sequência de DNA , Resistência beta-Lactâmica , beta-Lactamas/farmacologiaRESUMO
Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone's diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin's access to the brain. Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection. Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization.
Assuntos
Toxinas Bacterianas/farmacocinética , Sistema Nervoso Central/metabolismo , Macrolídeos/farmacocinética , Animais , Astrócitos/fisiologia , Toxinas Bacterianas/administração & dosagem , Barreira Hematoencefálica , Linhagem Celular , Células Endoteliais/fisiologia , Humanos , Larva , Macrolídeos/administração & dosagem , Mycobacterium ulcerans , Imagem Óptica , Análise Espaço-Temporal , Peixe-ZebraRESUMO
The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Modelos Neurológicos , Fármacos Neuroprotetores/metabolismo , Animais , Astrócitos/fisiologia , Barreira Hematoencefálica/fisiologia , Células Cultivadas , Células Endoteliais/fisiologia , Humanos , Neurônios/metabolismo , Fármacos Neuroprotetores/administração & dosagemRESUMO
The present study was performed in an attempt to develop an in vitro integrated testing strategy (ITS) to evaluate drug-induced neurotoxicity. A number of endpoints were analyzed using two complementary brain cell culture models and an in vitro blood-brain barrier (BBB) model after single and repeated exposure treatments with selected drugs that covered the major biological, pharmacological and neuro-toxicological responses. Furthermore, four drugs (diazepam, cyclosporine A, chlorpromazine and amiodarone) were tested more in depth as representatives of different classes of neurotoxicants, inducing toxicity through different pathways of toxicity. The developed in vitro BBB model allowed detection of toxic effects at the level of BBB and evaluation of drug transport through the barrier for predicting free brain concentrations of the studied drugs. The measurement of neuronal electrical activity was found to be a sensitive tool to predict the neuroactivity and neurotoxicity of drugs after acute exposure. The histotypic 3D re-aggregating brain cell cultures, containing all brain cell types, were found to be well suited for OMICs analyses after both acute and long term treatment. The obtained data suggest that an in vitro ITS based on the information obtained from BBB studies and combined with metabolomics, proteomics and neuronal electrical activity measurements performed in stable in vitro neuronal cell culture systems, has high potential to improve current in vitro drug-induced neurotoxicity evaluation.
Assuntos
Metabolômica , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurotoxinas/toxicidade , Proteômica , Animais , Barreira Hematoencefálica , Células Cultivadas , Relação Dose-Resposta a Droga , Fenômenos Eletrofisiológicos , Síndromes Neurotóxicas/diagnóstico , Neurotoxinas/administração & dosagem , RatosRESUMO
Historically, the focus has been to use in vitro BBB models to optimize rate of drug delivery to the CNS, whereas total in vivo brain/plasma ratios have been used for optimizing extent. However, these two parameters do not necessarily show good correlations with receptor occupancy data or other pharmacological readouts. In line with the free drug hypothesis, the use of unbound brain concentrations (Cu,br) has been shown to provide the best correlations with pharmacological data. However, typically the determination of this parameter requires microdialysis, a technique not ideally suited for screening in early drug development. Alternative, and less resource-demanding methodologies to determine Cu,br employ either equilibrium dialysis of brain homogenates or incubations of brain slices in buffer to determine fraction unbound brain (fu,br), which is subsequently multiplied by the total brain concentration to yield Cu,br. To determine Cu,br/Cu,pl ratios this way, still requires both in vitro and in vivo experiments that are quite time consuming. The main objective of this study was to explore the possibility to directly generate Cu,br/Cu,pl ratios in a single in vitro model of the BBB, using a co-culture of brain capillary endothelial and glial cells in an attempt to mimick the in vivo situation, thereby greatly simplifying existing experimental procedures. Comparison to microdialysis brain concentration profiles demonstrates the possibility to estimate brain exposure over time in the BBB model. A stronger correlation was found between in vitro Cu,br/Cu,pl ratios and in vivo Cu,br/Cu,pl obtained using fu,br from brain slice than with fu,br from brain homogenate for a set of 30 drugs. Overall, Cu,br/Cu,pl ratios were successfully predicted in vitro for 88% of the 92 studied compounds. This result supports the possibility to use this methodology for identifying compounds with a desirable in vivo response in the CNS early on in the drug discovery process.
Assuntos
Barreira Hematoencefálica/fisiologia , Células Endoteliais/metabolismo , Modelos Biológicos , Neuroglia/metabolismo , Plasma , Animais , Barreira Hematoencefálica/citologia , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/citologia , Neuroglia/citologia , Ratos , Ratos Sprague-DawleyRESUMO
The intracellular calcium concentration ([Ca(2+)](i)) is an important factor determining the permeability of endothelial barriers including the blood-brain barrier (BBB). However, nothing is known concerning the effect of spatially propagated intercellular Ca(2+) waves (ICWs). The propagation of ICWs relies in large part on channels formed by connexins that are present in endothelia. We hypothesized that ICWs may result in a strong disturbance of endothelial function, because the [Ca(2+)](i) changes are coordinated and involve multiple cells. Thus, we aimed to investigate the effect of ICWs on endothelial permeability. ICW activity was triggered in immortalized and primary brain endothelial cells by lowering the extracellular Ca(2+) concentration. Low extracellular Ca(2+) increased the endothelial permeability and this was significantly suppressed by buffering [Ca(2+)](i) with BAPTA-AM, indicating a central role of [Ca(2+)](i) changes. The endothelial permeability increase was furthermore inhibited by the connexin channel blocking peptide Gap27, which also blocked the ICWs, and by inhibiting protein kinase C (PKC), Ca(2+)/calmodulin-dependent kinase II (CaMKII) and actomyosin contraction. We compared these observations with the [Ca(2+)](i) changes and permeability alterations provoked by the inflammatory agent bradykinin (BK), which triggers oscillatory [Ca(2+)](i) changes without wave activity. BK-associated [Ca(2+)](i) changes and the endothelial permeability increase were significantly smaller than those associated with ICWs, and the permeability increase was not influenced by inhibition of PKC, CaMKII or actomyosin contraction. We conclude that ICWs significantly increase endothelial permeability and therefore, the connexins that underlie wave propagation form an interesting target to limit BBB alterations. This article is part of a Special Issue entitled Electrical Synapses.
Assuntos
Encéfalo/fisiologia , Sinalização do Cálcio/fisiologia , Cálcio/fisiologia , Endotélio Vascular/fisiologia , Actomiosina/fisiologia , Animais , Transporte Biológico Ativo/fisiologia , Western Blotting , Bradicinina/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Células Cultivadas , Conexinas/farmacologia , Citoesqueleto/fisiologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Eletroforese em Gel de Poliacrilamida , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/fisiologia , Junções Comunicantes/efeitos dos fármacos , Neuroimagem , Oligopeptídeos , Permeabilidade , Proteína Quinase C/fisiologia , RatosRESUMO
The cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) is an important factor determining the functional state of blood-brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca(2+)](i) changes on BBB function. We applied different agonists that trigger [Ca(2+)](i) oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca(2+)](i) oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca(2+)](i) with BAPTA, indicating that [Ca(2+)](i) oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca(2+)](i) oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca(2+)](i) oscillations provoked by exposure to adenosine 5' triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations.