scholarly journals Trojan Horse Transit Contributes to Blood-Brain Barrier Crossing of a Eukaryotic Pathogen

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Felipe H. Santiago-Tirado ◽  
Michael D. Onken ◽  
John A. Cooper ◽  
Robyn S. Klein ◽  
Tamara L. Doering
Keyword(s):  
Experimental Evidence ◽  
Cryptococcus Neoformans ◽  
Blood Brain Barrier ◽  
Fungal Pathogen ◽  
Trojan Horse ◽  
Brain Barrier ◽  
Barrier Crossing ◽  
Blood Brain ◽  
The Brain

ABSTRACT The blood-brain barrier (BBB) protects the central nervous system (CNS) by restricting the passage of molecules and microorganisms. Despite this barrier, however, the fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that is estimated to kill over 600,000 people annually. Cryptococcal infection begins in the lung, and experimental evidence suggests that host phagocytes play a role in subsequent dissemination, although this role remains ill defined. Additionally, the disparate experimental approaches that have been used to probe various potential routes of BBB transit make it impossible to assess their relative contributions, confounding any integrated understanding of cryptococcal brain entry. Here we used an in vitro model BBB to show that a “Trojan horse” mechanism contributes significantly to fungal barrier crossing and that host factors regulate this process independently of free fungal transit. We also, for the first time, directly imaged C. neoformans-containing phagocytes crossing the BBB, showing that they do so via transendothelial pores. Finally, we found that Trojan horse crossing enables CNS entry of fungal mutants that cannot otherwise traverse the BBB, and we demonstrate additional intercellular interactions that may contribute to brain entry. Our work elucidates the mechanism of cryptococcal brain invasion and offers approaches to study other neuropathogens. IMPORTANCE The fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that kills hundreds of thousands of people each year. One route that has been proposed for this brain entry is a Trojan horse mechanism, whereby the fungus crosses the blood-brain barrier (BBB) as a passenger inside host phagocytes. Although indirect experimental evidence supports this intriguing mechanism, it has never been directly visualized. Here we directly image Trojan horse transit and show that it is regulated independently of free fungal entry, contributes to cryptococcal BBB crossing, and allows mutant fungi that cannot enter alone to invade the brain. IMPORTANCE The fungal pathogen Cryptococcus neoformans invades the brain, causing a meningoencephalitis that kills hundreds of thousands of people each year. One route that has been proposed for this brain entry is a Trojan horse mechanism, whereby the fungus crosses the blood-brain barrier (BBB) as a passenger inside host phagocytes. Although indirect experimental evidence supports this intriguing mechanism, it has never been directly visualized. Here we directly image Trojan horse transit and show that it is regulated independently of free fungal entry, contributes to cryptococcal BBB crossing, and allows mutant fungi that cannot enter alone to invade the brain.

scholarly journals Miniaturization and Automation of a Human In Vitro Blood–Brain Barrier Model for the High-Throughput Screening of Compounds in the Early Stage of Drug Discovery

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 892
Author(s):  
Elisa L. J. Moya ◽  
Elodie Vandenhaute ◽  
Eleonora Rizzi ◽  
Marie-Christine Boucau ◽  
Johan Hachani ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Blood Brain Barrier ◽  
Early Stage ◽  
Molecular Transport ◽  
Brain Barrier ◽  
Blood Brain ◽  
Cns Drugs ◽  
The Impact ◽  
The Brain

Central nervous system (CNS) diseases are one of the top causes of death worldwide. As there is a difficulty of drug penetration into the brain due to the blood–brain barrier (BBB), many CNS drugs treatments fail in clinical trials. Hence, there is a need to develop effective CNS drugs following strategies for delivery to the brain by better selecting them as early as possible during the drug discovery process. The use of in vitro BBB models has proved useful to evaluate the impact of drugs/compounds toxicity, BBB permeation rates and molecular transport mechanisms within the brain cells in academic research and early-stage drug discovery. However, these studies that require biological material (animal brain or human cells) are time-consuming and involve costly amounts of materials and plastic wastes due to the format of the models. Hence, to adapt to the high yields needed in early-stage drug discoveries for compound screenings, a patented well-established human in vitro BBB model was miniaturized and automated into a 96-well format. This replicate met all the BBB model reliability criteria to get predictive results, allowing a significant reduction in biological materials, waste and a higher screening capacity for being extensively used during early-stage drug discovery studies.

Understanding the Physiology of the Blood-Brain Barrier: In Vitro Models

Physiology ◽  
1998 ◽  
Vol 13 (6) ◽  
pp. 287-293 ◽  
Author(s):  
Gerald A. Grant ◽  
N. Joan Abbott ◽  
Damir Janigro
Keyword(s):  
Central Nervous System ◽  
Tissue Culture ◽  
Nervous System ◽  
Blood Brain Barrier ◽  
In Vitro Models ◽  
Brain Barrier ◽  
Blood Brain ◽  
Brain Tissue Culture ◽  
The Brain

Endothelial cells exposed to inductive central nervous system factors differentiate into a blood-brain barrier phenotype. The blood-brain barrier frequently obstructs the passage of chemotherapeutics into the brain. Tissue culture systems have been developed to reproduce key properties of the intact blood-brain barrier and to allow for testing of mechanisms of transendothelial drug permeation.

scholarly journals In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

2016 ◽  
Vol 36 (5) ◽  
pp. 862-890 ◽  
Author(s):  
Hans C Helms ◽  
N Joan Abbott ◽  
Malgorzata Burek ◽  
Romeo Cecchelli ◽  
Pierre-Olivier Couraud ◽  
...  
Keyword(s):  
Cell Culture ◽  
Endothelial Cell ◽  
Blood Brain Barrier ◽  
Brain Parenchyma ◽  
Brain Barrier ◽  
Blood Brain ◽  
Culture Models ◽  
Cell Culture Models ◽  
The Brain

The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This “blood-brain barrier” function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood–brain barrier models with a focus on their validation regarding a set of well-established blood–brain barrier characteristics. As an ideal cell culture model of the blood–brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described.

Anthropogenic Iron Oxide Nanoparticles Induce Damage to Brain Microvascular Endothelial Cells Forming the Blood-Brain Barrier

2021 ◽  
Author(s):  
Lorena Gárate-Vélez ◽  
Claudia Escudero-Lourdes ◽  
Daniela Salado-Leza ◽  
Armando González-Sánchez ◽  
Ildemar Alvarado-Morales ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Blood Brain ◽  
Fe3o4 Nps ◽  
Microvascular Endothelial ◽  
The Brain

Background: Iron nanoparticles, mainly in magnetite phase (Fe3O4 NPs), are released to the environment in areas with high traffic density and braking frequency. Fe3O4 NPs were found in postmortem human brains and are assumed to get directly into the brain through the olfactory nerve. However, these pollution-derived NPs may also translocate from the lungs to the bloodstream and then, through the blood-brain barrier (BBB), into the brain inducing oxidative and inflammatory responses that contribute to neurodegeneration. Objective: To describe the interaction and toxicity of pollution-derived Fe3O4 NPs on primary rat brain microvascular endothelial cells (rBMECs), main constituents of in vitro BBB models. Methods: Synthetic bare Fe3O4 NPs that mimic the environmental ones (miFe3O4) were synthesized by co-precipitation and characterized using complementary techniques. The rBMECs were cultured in Transwell® plates. The NPs-cell interaction was evaluated through transmission electron microscopy and standard colorimetric in vitro assays. Results: The miFe3O4 NPs, with a mean diameter of 8.45 ± 0.14 nm, presented both magnetite and maghemite phases, and showed super-paramagnetic properties. Results suggest that miFe3O4 NPs are internalized by rBMECs through endocytosis and that they are able to cross the cells monolayer. The lowest miFe3O4 NPs concentration tested induced mid cytotoxicity in terms of 1) membrane integrity (LDH release) and 2) metabolic activity (MTS transformation). Conclusion: Pollution-derived Fe3O4 NPs may interact and cross the microvascular endothelial cells forming the BBB and cause biological damage.

scholarly journals Characterization of the Blood–Brain Barrier Integrity and the Brain Transport of SN-38 in an Orthotopic Xenograft Rat Model of Diffuse Intrinsic Pontine Glioma

Pharmaceutics ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 399 ◽  
Author(s):  
Catarina Chaves ◽  
Xavier Declèves ◽  
Meryam Taghi ◽  
Marie-Claude Menet ◽  
Joelle Lacombe ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Brain Barrier ◽  
Brain Delivery ◽  
Pontine Glioma ◽  
Anticancer Drug Delivery ◽  
Blood Brain ◽  
The Brain

The blood–brain barrier (BBB) hinders the brain delivery of many anticancer drugs. In pediatric patients, diffuse intrinsic pontine glioma (DIPG) represents the main cause of brain cancer mortality lacking effective drug therapy. Using sham and DIPG-bearing rats, we analyzed (1) the brain distribution of 3-kDa-Texas red-dextran (TRD) or [14C]-sucrose as measures of BBB integrity, and (2) the role of major ATP-binding cassette (ABC) transporters at the BBB on the efflux of the irinotecan metabolite [3H]-SN-38. The unaffected [14C]-sucrose or TRD distribution in the cerebrum, cerebellum, and brainstem regions in DIPG-bearing animals suggests an intact BBB. Targeted proteomics retrieved no change in P-glycoprotein (P-gp), BCRP, MRP1, and MRP4 levels in the analyzed regions of DIPG rats. In vitro, DIPG cells express BCRP but not P-gp, MRP1, or MRP4. Dual inhibition of P-gp/Bcrp, or Mrp showed a significant increase on SN-38 BBB transport: Cerebrum (8.3-fold and 3-fold, respectively), cerebellum (4.2-fold and 2.8-fold), and brainstem (2.6-fold and 2.2-fold). Elacridar increased [3H]-SN-38 brain delivery beyond a P-gp/Bcrp inhibitor effect alone, emphasizing the role of another unidentified transporter in BBB efflux of SN-38. These results confirm a well-preserved BBB in DIPG-bearing rats, along with functional ABC-transporter expression. The development of chemotherapeutic strategies to circumvent ABC-mediated BBB efflux are needed to improve anticancer drug delivery against DIPG.

scholarly journals CoQ10 Deficient Endothelial Cell Culture Model for the Investigation of CoQ10 Blood–Brain Barrier Transport

2020 ◽  
Vol 9 (10) ◽  
pp. 3236
Author(s):  
Luke Wainwright ◽  
Iain P. Hargreaves ◽  
Ana R. Georgian ◽  
Charles Turner ◽  
R. Neil Dalton ◽  
...  
Keyword(s):  
Cell Culture ◽  
Endothelial Cell ◽  
Blood Brain Barrier ◽  
Advanced Glycation Endproducts ◽  
Brain Barrier ◽  
High Dose ◽  
Blood Brain ◽  
Coq10 Deficiency ◽  
The Brain

Primary coenzyme Q10 (CoQ10) deficiency is unique among mitochondrial respiratory chain disorders in that it is potentially treatable if high-dose CoQ10 supplements are given in the early stages of the disease. While supplements improve peripheral abnormalities, neurological symptoms are only partially or temporarily ameliorated. The reasons for this refractory response to CoQ10 supplementation are unclear, however, a contributory factor may be the poor transfer of CoQ10 across the blood–brain barrier (BBB). The aim of this study was to investigate mechanisms of CoQ10 transport across the BBB, using normal and pathophysiological (CoQ10 deficient) cell culture models. The study identifies lipoprotein-associated CoQ10 transcytosis in both directions across the in vitro BBB. Uptake via SR-B1 (Scavenger Receptor) and RAGE (Receptor for Advanced Glycation Endproducts), is matched by efflux via LDLR (Low Density Lipoprotein Receptor) transporters, resulting in no “net” transport across the BBB. In the CoQ10 deficient model, BBB tight junctions were disrupted and CoQ10 “net” transport to the brain side increased. The addition of anti-oxidants did not improve CoQ10 uptake to the brain side. This study is the first to generate in vitro BBB endothelial cell models of CoQ10 deficiency, and the first to identify lipoprotein-associated uptake and efflux mechanisms regulating CoQ10 distribution across the BBB. The results imply that the uptake of exogenous CoQ10 into the brain might be improved by the administration of LDLR inhibitors, or by interventions to stimulate luminal activity of SR-B1 transporters.

scholarly journals Photostimulation of Extravasation of Beta-Amyloid through the Model of Blood-Brain Barrier

Electronics ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1056
Author(s):  
Ekaterina Zinchenko ◽  
Maria Klimova ◽  
Aysel Mamedova ◽  
Ilana Agranovich ◽  
Inna Blokhina ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Beta Amyloid ◽  
Brain Barrier ◽  
Therapeutic Effects ◽  
Cervical Lymph Nodes ◽  
Blood Brain ◽  
The Central Nervous System ◽  
Vitro Model ◽  
The Brain

Alzheimer’s disease (AD) is an incurable pathology associated with progressive decline in memory and cognition. Phototherapy might be a new promising and alternative strategy for the effective treatment of AD, and has been actively discussed over two decades. However, the mechanisms of therapeutic photostimulation (PS) effects on subjects with AD remain poorly understood. The goal of this study was to determine the mechanisms of therapeutic PS effects in beta-amyloid (Aβ)-injected mice. The neurological severity score and the new object recognition tests demonstrate that PS 9 J/cm2 attenuates the memory and neurological deficit in mice with AD. The immunohistochemical assay revealed a decrease in the level of Aβ in the brain and an increase of Aβ in the deep cervical lymph nodes obtained from mice with AD after PS. Using the in vitro model of the blood-brain barrier (BBB), we show a PS-mediated decrease in transendothelial resistance and in the expression of tight junction proteins as well an increase in the BBB permeability to Aβ. These findings suggest that a PS-mediated BBB opening and the activation of the lymphatic clearance of Aβ from the brain might be a crucial mechanism underlying therapeutic effects of PS in mice with AD. These pioneering data open new strategies in the development of non-pharmacological methods for therapy of AD and contribute to a better understanding of the PS effects on the central nervous system.

scholarly journals Lysozyme transport to the brain by liposomes

2018 ◽  
Vol 1 (2) ◽  
pp. 146-161 ◽  
Author(s):  
Mirjam M Nordling-David ◽  
Elior Rachamin ◽  
Etty Grad ◽  
Gershon Golomb
Keyword(s):  
Blood Brain Barrier ◽  
White Blood Cells ◽  
Brain Barrier ◽  
Immunofluorescent Staining ◽  
Phagocytic Cells ◽  
Blood Brain ◽  
The Brain ◽  
Positively Charged

Delivery of drugs into the brain is limited due to poor penetrability of many drugs via the blood-brain barrier. Previous studies have shown that the brain is kept under close surveillance by the immune system, implying that circulating phagocytic cells, such as neutrophils and monocytes, are crossing the blood-brain barrier. We hypothesized that charged liposomes could be transported to the brain following their phagocytosis by circulating monocytes. In this work, we investigated the capacity of circulating monocytes to be exploited as a drug delivery system following IV administration of nano-sized, positively fluorescently labeled liposomes containing the protein lysozyme. Negatively charged fluorescently labeled liposomes were used for comparison. By using a modified thin-film hydration technique, the desired properties of the liposomal formulations were achieved including size, polydispersity index, high drug concentration, and stability. In vitro results showed a significant time-dependent uptake of positively charged liposomes by RAW264.7 cells. In vivo results revealed that circulating white blood cells (mainly monocytes) contained high levels of fluorescently labeled liposomes. Screening of brain sections using confocal microscopy uncovered that a substantial amount of fluorescently labeled liposomes, in contrast to the fluorescent markers in solution, was transported into the brain. In addition, anti-CD68 immunofluorescent staining of brain sections demonstrated co-localization of positively charged liposomes and macrophages in different brain sections. Furthermore, significantly higher levels of lysozyme were detected in brain lysates from rats treated with positively charged liposomes compared to rats treated with lysozyme solution. Taken together this confirms our hypothesis that the designed liposomes were transported to the brain following their phagocytosis by circulating monocytes.

scholarly journals Ketone Bodies Promote Amyloid-β1–40 Clearance in a Human in Vitro Blood–Brain Barrier Model

2020 ◽  
Vol 21 (3) ◽  
pp. 934 ◽  
Author(s):  
Romain Versele ◽  
Mariangela Corsi ◽  
Andrea Fuso ◽  
Emmanuel Sevin ◽  
Rita Businaro ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Ketone Bodies ◽  
Amyloid Β ◽  
Brain Barrier ◽  
Therapeutic Approaches ◽  
Protein Levels ◽  
Blood Brain ◽  
Aβ Clearance ◽  
The Brain

Alzheimer’s disease (AD) is characterized by the abnormal accumulation of amyloid-β (Aβ) peptides in the brain. The pathological process has not yet been clarified, although dysfunctional transport of Aβ across the blood–brain barrier (BBB) appears to be integral to disease development. At present, no effective therapeutic treatment against AD exists, and the adoption of a ketogenic diet (KD) or ketone body (KB) supplements have been investigated as potential new therapeutic approaches. Despite experimental evidence supporting the hypothesis that KBs reduce the Aβ load in the AD brain, little information is available about the effect of KBs on BBB and their effect on Aβ transport. Therefore, we used a human in vitro BBB model, brain-like endothelial cells (BLECs), to investigate the effect of KBs on the BBB and on Aβ transport. Our results show that KBs do not modify BBB integrity and do not cause toxicity to BLECs. Furthermore, the presence of KBs in the culture media was combined with higher MCT1 and GLUT1 protein levels in BLECs. In addition, KBs significantly enhanced the protein levels of LRP1, P-gp, and PICALM, described to be involved in Aβ clearance. Finally, the combined use of KBs promotes Aβ efflux across the BBB. Inhibition experiments demonstrated the involvement of LRP1 and P-gp in the efflux. This work provides evidence that KBs promote Aβ clearance from the brain to blood in addition to exciting perspectives for studying the use of KBs in therapeutic approaches.

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