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2.
Antimicrob Agents Chemother ; 59(2): 890-904, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25421467

RESUMEN

Treatment of late-stage sleeping sickness requires drugs that can cross the blood-brain barrier (BBB) to reach the parasites located in the brain. We report here the synthesis and evaluation of four new N-hydroxy and 12 new N-alkoxy derivatives of bisimidazoline leads as potential agents for the treatment of late-stage sleeping sickness. These compounds, which have reduced basicity compared to the parent leads (i.e., are less ionized at physiological pH), were evaluated in vitro against Trypanosoma brucei rhodesiense and in vivo in murine models of first- and second-stage sleeping sickness. Resistance profile, physicochemical parameters, in vitro BBB permeability, and microsomal stability also were determined. The N-hydroxy imidazoline analogues were the most effective in vivo, with 4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide (14d) showing 100% cures in the first-stage disease, while 15d, 16d, and 17d appeared to slightly improve survival. In addition, 14d showed weak activity in the chronic model of central nervous system infection in mice. No evidence of reduction of this compound with hepatic microsomes and mitochondria was found in vitro, suggesting that N-hydroxy imidazolines are metabolically stable and have intrinsic activity against T. brucei. In contrast to its unsubstituted parent compound, the uptake of 14d in T. brucei was independent of known drug transporters (i.e., T. brucei AT1/P2 and HAPT), indicating a lower predisposition to cross-resistance with other diamidines and arsenical drugs. Hence, the N-hydroxy bisimidazolines (14d in particular) represent a new class of promising antitrypanosomal agents.


Asunto(s)
Tripanocidas/uso terapéutico , Trypanosoma brucei brucei/patogenicidad , Trypanosoma brucei rhodesiense/patogenicidad , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Femenino , Imidazolinas/uso terapéutico , Ratones , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei rhodesiense/efectos de los fármacos
3.
Cell Microbiol ; 15(4): 512-9, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23189983

RESUMEN

The brain and meningeal spaces are protected from bacterial invasion by the blood-brain barrier, formed by specialized endothelial cells and tight intercellular junctional complexes. However, once in the bloodstream, Neisseria meningitidis crosses this barrier in about 60% of the cases. This highlights the particular efficacy with which N. meningitidis targets the brain vascular cell wall. The first step of central nervous system invasion is the direct interaction between bacteria and endothelial cells. This step is mediated by the type IV pili, which induce a remodelling of the endothelial monolayer, leading to the opening of the intercellular space. In this review, strategies used by the bacteria to survive in the bloodstream, to colonize the brain vasculature and to cross the blood-brain barrier will be discussed.


Asunto(s)
Barrera Hematoencefálica/microbiología , Encéfalo/microbiología , Líquido Cefalorraquídeo/microbiología , Endotelio/microbiología , Neisseria meningitidis/fisiología , Barrera Hematoencefálica/inmunología , Fimbrias Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Neisseria meningitidis/crecimiento & desarrollo
4.
J Infect Dis ; 208(10): 1590-7, 2013 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-23840047

RESUMEN

Neisseria meningitidis is a strict human pathogen that closely interacts with human endothelial cells via type IV pili in vitro. To decipher whether this interaction plays a role in vivo, we set up an experimental model of fulminant meningococcemia in human skin grafted SCID mice using the wild-type strain 2C4.3. Human skin and mouse tissues were sampled 24 hours after bacterial challenge for histopathology, immunohistochemistry and ultrastructural analysis. In all infected mice, N. meningitidis targeted the human vasculature, leading to bacterial and blood thrombi, infectious vasculitis and vascular leakage. Mouse vessels, including brain vessels, remained unaffected by the infectious and thrombotic process, and a nonpiliated Δ pilE derivative of 2C4.3 failed to target human graft vessels and to induce vascular damages. These data demonstrate that N. meningitidis targets human endothelial cells in vivo and that this interaction triggers the vascular damages that characterize purpura fulminans.


Asunto(s)
Microvasos/microbiología , Neisseria meningitidis/fisiología , Púrpura Fulminante/etiología , Púrpura Fulminante/patología , Animales , Adhesión Bacteriana , Células Endoteliales/microbiología , Células Endoteliales/patología , Endotelio Vascular/microbiología , Endotelio Vascular/patología , Femenino , Fimbrias Bacterianas/fisiología , Xenoinjertos , Humanos , Infecciones Meningocócicas/complicaciones , Infecciones Meningocócicas/microbiología , Ratones , Microvasos/patología , Piel/irrigación sanguínea , Piel/patología , Trasplante de Piel
5.
Mol Pharm ; 10(1): 289-96, 2013 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-23137377

RESUMEN

Human cerebral microvascular endothelial cell line hCMEC/D3 is an established model of the human blood-brain barrier (BBB). The purpose of the present study was to determine, by means of quantitative targeted absolute proteomics, the protein expression levels in hCMEC/D3 cells of multiple transporters, receptors and junction proteins for comparison with our previously reported findings in isolated human brain microvessels. Among 91 target molecules, 12 transporters, 2 receptors, 1 junction protein and 1 membrane marker were present at quantifiable levels in plasma membrane fraction of hCMEC/D3 cells. ABCA2, MDR1, MRP4, BCRP, GLUT1, 4F2hc, MCT1, ENT1, transferrin and insulin receptors and claudin-5 were detected in both hCMEC/D3 cells and human brain microvessels. After normalization based on Na(+)/K(+) ATPase expression, the differences in protein expression levels between hCMEC/D3 cells and human brain microvessels were within 4-fold for these proteins, with the exceptions of ENT1, transferrin receptor and claudin-5. ABCA8, LAT1, LRP1 and γ-GTP were below the limit of quantification in the cells, but were found in human brain microvessels. ABCA3, ABCA6, MRP1 and ATA1 were found only in hCMEC/D3 cells. Furthermore, compared with human umbilical vein endothelial cells (HUVECs) as reference nonbrain endothelial cells, MDR1 was found only in hCMEC/D3 cells, and GLUT1 expression was 15-fold higher in hCMEC/D3 cells than in HUVECs. In conclusion, this is the first study to examine the suitability and limitations of the hCMEC/D3 cell line as a BBB functional model in terms of quantitative expression levels of transporters, receptors and tight junction proteins.


Asunto(s)
Barrera Hematoencefálica/metabolismo , Cerebro/irrigación sanguínea , Cerebro/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Proteínas de la Membrana/metabolismo , Microvasos/metabolismo , Proteoma/metabolismo , Transporte Biológico , Línea Celular , Membrana Celular/metabolismo , Circulación Cerebrovascular , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Proteómica/métodos
6.
Cell Tissue Res ; 343(3): 521-36, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21191615

RESUMEN

The enzyme tissue non-specific alkaline phosphatase (TNAP) belongs to the ectophosphatase family. It is present in large amounts in bone in which it plays a role in mineralization but little is known about its function in other tissues. Arguments are accumulating for its involvement in the brain, in particular in view of the neurological symptoms accompanying human TNAP deficiencies. We have previously shown, by histochemistry, alkaline phosphatase (AP) activity in monkey brain vessels and parenchyma in which AP exhibits specific patterns. Here, we clearly attribute this activity to TNAP expression rather than to other APs in primates (human and marmoset) and in rodents (rat and mouse). We have not found any brain-specific transcripts but our data demonstrate that neuronal and endothelial cells exclusively express the bone TNAP transcript in all species tested, except in mouse neurons in which liver TNAP transcripts have also been detected. Moreover, we highlight the developmental regulation of TNAP expression; this also acts during neuronal differentiation. Our study should help to characterize the regulation of the expression of this ectophosphatase in various cell types of the central nervous system.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Huesos/enzimología , Encéfalo/enzimología , Isoenzimas/metabolismo , Hígado/enzimología , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Vasos Sanguíneos/enzimología , Línea Celular , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/enzimología , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Primates , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Distribución Tisular
7.
Biochim Biophys Acta ; 1788(4): 842-57, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19061857

RESUMEN

Brain endothelial cells are unique among endothelial cells in that they express apical junctional complexes, including tight junctions, which quite resemble epithelial tight junctions both structurally and functionally. They form the blood-brain-barrier (BBB) which strictly controls the exchanges between the blood and the brain compartments by limiting passive diffusion of blood-borne solutes while actively transporting nutrients to the brain. Accumulating experimental and clinical evidence indicate that BBB dysfunctions are associated with a number of serious CNS diseases with important social impacts, such as multiple sclerosis, stroke, brain tumors, epilepsy or Alzheimer's disease. This review will focus on the implication of brain endothelial tight junctions in BBB architecture and physiology, will discuss the consequences of BBB dysfunction in these CNS diseases and will present some therapeutic strategies for drug delivery to the brain across the BBB.


Asunto(s)
Barrera Hematoencefálica/fisiología , Encéfalo/fisiología , Homeostasis , Enfermedades del Sistema Nervioso/fisiopatología , Uniones Estrechas/fisiología , Movimiento Celular/fisiología , Células Endoteliales/fisiología , Células Endoteliales/ultraestructura , Humanos , Leucocitos/fisiología , Meningitis Bacterianas/fisiopatología , Meningitis Viral/fisiopatología , Esclerosis Múltiple/fisiopatología , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades Neurodegenerativas/fisiopatología , Permeabilidad , Accidente Cerebrovascular/fisiopatología
8.
Neurobiol Dis ; 37(3): 656-60, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20005954

RESUMEN

Sandhoff disease is an autosomal recessive lysosomal disorder due to mutations in the beta-hexosaminidase beta-chain gene, resulting in beta-hexosaminidases A (alphabeta) and B (betabeta) deficiency and GM2 ganglioside accumulation in the brain. In this study, our aim was to demonstrate that transduction of cerebral endothelial cells cultured in two-chamber culture inserts with a lentiviral vector encoding the hexosaminidases alpha and beta chains could induce a vectorial secretion of hexosaminidases. Therefore, the human cerebral endothelial cell line hCMEC/D3 was infected with the bicistronic vector from the apical compartment, and beta-hexosaminidase activity was measured in transduced cells and in deficient fibroblasts co-cultured in the basal (i.e. brain) compartment. Induced beta-hexosaminidase secretion by transduced hCMEC/D3 cells was sufficient to allow for a 70-90% restoration of beta-hexosaminidase activity in deficient fibroblasts. On the basis of these in vitro data, we propose that brain endothelium be considered as a novel therapeutic target in Sandhoff disease.


Asunto(s)
Células Endoteliales/enzimología , Terapia Genética/métodos , Enfermedad de Sandhoff/enzimología , Enfermedad de Sandhoff/terapia , Transducción Genética/métodos , beta-N-Acetilhexosaminidasas/metabolismo , Línea Celular Transformada , Arterias Cerebrales/citología , Arterias Cerebrales/enzimología , Cerebro/irrigación sanguínea , Cerebro/enzimología , Cerebro/fisiopatología , Técnicas de Cocultivo , Cámaras de Difusión de Cultivos , Células Endoteliales/metabolismo , Fibroblastos/enzimología , Fibroblastos/metabolismo , Gangliósido G(M2)/metabolismo , Vectores Genéticos/farmacología , Vectores Genéticos/uso terapéutico , Humanos , Lentivirus/genética , Enfermedad de Sandhoff/genética , beta-N-Acetilhexosaminidasas/genética
9.
Stem Cells ; 26(7): 1673-82, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18450824

RESUMEN

Systemically injected neural precursor cells (NPCs) were unexpectedly shown to reach the cerebral parenchyma and induce recovery in various diffuse brain pathologies, including animal models of multiple sclerosis. However, the molecular mechanisms supporting NPC migration across brain endothelium remain elusive. Brain endothelium constitutes the blood-brain barrier, which uniquely controls the access of drugs and trafficking of cells, including leukocytes, from the blood to the brain. Taking advantage of the availability of in vitro models of human and rat blood-brain barrier developed in our laboratory and validated by us and others, we show here that soluble hyaluronic acid, the major ligand of the adhesion molecule CD44, as well as anti-CD44 blocking antibodies, largely prevents NPC adhesion to and migration across brain endothelium in inflammatory conditions. We present further evidence that NPCs, surprisingly, induce the formation of apical cups at the surface of brain endothelial cells, enriched in CD44 and other adhesion molecules, thus hijacking the endothelial signaling recently shown to be involved in leukocyte extravasation. These results demonstrate the pivotal role of CD44 in the trans-endothelial migration of NPCs across brain endothelial cells: we propose that they may help design new strategies for the delivery of therapeutic NPCs to the brain by systemic administration.


Asunto(s)
Encéfalo/metabolismo , Células Endoteliales/metabolismo , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/fisiología , Neuronas/citología , Células Madre/citología , Animales , Encéfalo/embriología , Adhesión Celular , Movimiento Celular , Células Endoteliales/citología , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Leucocitos/citología , Ratones , Ratas , Transducción de Señal
10.
Neurosci Lett ; 392(3): 216-20, 2006 Jan 16.
Artículo en Inglés | MEDLINE | ID: mdl-16214291

RESUMEN

Increased lymphocyte trafficking across blood-brain barrier (BBB) is a prominent and early event in inflammatory and immune-mediated CNS diseases. The adhesion molecules that control the entry of leukocytes into the brain have not been fully elucidated. Although the role of ICAM-1 and VCAM-1 has been well documented, the expression and role of selectins is still a matter of controversy. In a mouse syngenic in vitro BBB model, highly relevant for examining immunological events, mouse brain capillary endothelial cells (MBCECs) do not express selectins. Treatment of MBCECs with LPS, induced E- and P-selectin expression, whereas TNF-alpha or IFN-gamma treatments did not. Finally, P-selectin but not E-selectin expression was induced in IL-1beta treated MBCECs. Thus, our study suggests that diverse inflammatory stimuli could differentially regulate selectin expression at the BBB.


Asunto(s)
Corteza Cerebral/citología , Citocinas/farmacología , Células Endoteliales/efectos de los fármacos , Lipopolisacáridos/farmacología , Neuroglía/metabolismo , Selectinas/metabolismo , Animales , Animales Recién Nacidos , Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Inflamación/inducido químicamente , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Neuroglía/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo
11.
mBio ; 5(1): e01024-13, 2014 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-24520062

RESUMEN

UNLABELLED: Type IV pili (Tfp) are expressed by many Gram-negative bacteria to promote aggregation, adhesion, internalization, twitching motility, or natural transformation. Tfp of Neisseria meningitidis, the causative agent of cerebrospinal meningitis, are involved in the colonization of human nasopharynx. After invasion of the bloodstream, Tfp allow adhesion of N. meningitidis to human endothelial cells, which leads to the opening of the blood-brain barrier and meningitis. To achieve firm adhesion, N. meningitidis induces a host cell response that results in elongation of microvilli surrounding the meningococcal colony. Here we study the role of the major pilin subunit PilE during host cell response using human dermal microvascular endothelial cells and the pharynx carcinoma-derived FaDu epithelial cell line. We first show that some PilE variants are unable to induce a host cell response. By engineering PilE mutants, we observed that the PilE C-terminus domain, which contains a disulfide bonded region (D-region), is critical for the host cell response and that hypervariable regions confer different host cell specificities. Moreover, the study of point mutants of the pilin D-region combined with structural modeling of PilE revealed that the D-region contains two independent regions involved in signaling to human dermal microvascular endothelial cells (HDMECs) or FaDu cells. Our results indicate that the diversity of the PilE D-region sequence allows the induction of the host cell response via several receptors. This suggests that Neisseria meningitidis has evolved a powerful tool to adapt easily to many niches by modifying its ability to interact with host cells. IMPORTANCE: Type IV pili (Tfp) are long appendages expressed by many Gram-negative bacteria, including Neisseria meningitidis, the causative agent of cerebrospinal meningitis. These pili are involved in many aspects of pathogenesis: natural competence, aggregation, adhesion, and twitching motility. More specifically, Neisseria meningitidis, which is devoid of a secretion system to manipulate its host, has evolved its Tfp to signal to brain endothelial cells and open the blood-brain barrier. In this report, we investigate, at the molecular level, the involvement of the major pilin subunit PilE in host cell response. Our results indicate that the PilE C-terminal domain, which contains a disulfide bonded region (D-region), is critical for the host cell response and contains two independent regions involved in host cell signaling.


Asunto(s)
Variación Antigénica , Adhesión Celular , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Neisseria meningitidis/fisiología , Células Cultivadas , Células Endoteliales/microbiología , Células Epiteliales/microbiología , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Neisseria meningitidis/genética
12.
J Pharm Sci ; 103(8): 2509-19, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24916334

RESUMEN

To document the efficacy of clonazepam (CLZ) either free as a solution or loaded in solid lipid nanoparticles (CLZ-SLN) or mixed micelles (CLZ-MM), the in vitro blood-brain barrier permeability of the formulations was determined. Behavior and/or electroencephalograms (EEGs) of rodents receiving treatments were also studied. The in vitro permeability of CLZ increased when associated with SLN, but decreased in the case of MM. The occurrence of the pentylenetetrazole (PTZ)-induced seizures in mice was significantly prevented by CLZ, even when exposed a lower dose of CLZ-SLN after administration by the oral route. The behavioral severity and EEGs showing the PTZ-induced paroxystic activity in rats diminished significantly in the presence of CLZ alone (0.3 mg/kg), and were almost totally prevented in the rats treated with CLZ-SLN (equivalent to 0.3 mg/kg). The frequency, duration, and spreading of the spikes-wave of rats treated with CLZ-SLN decreased significantly as compared with CLZ alone, CLZ-MM, or the vehicle. These results show an in vitro-in vivo correlation in the enhanced blood-brain barrier permeability of SLN formulation, and a contribution of MM to the carrier effect of drugs toward the bloodstream and brain, where this pharmaceutical formulation of CLZ-SLN improves the anticonvulsant effect of this benzodiazepine, thus offering additional advantages after oral administration.


Asunto(s)
Anticonvulsivantes/administración & dosificación , Clonazepam/administración & dosificación , Portadores de Fármacos/química , Lípidos/química , Nanopartículas/química , Convulsiones/tratamiento farmacológico , Animales , Anticonvulsivantes/uso terapéutico , Línea Celular , Clonazepam/uso terapéutico , Electroencefalografía , Humanos , Masculino , Ratones , Micelas , Pentilenotetrazol , Ratas , Ratas Wistar , Convulsiones/inducido químicamente
13.
Virulence ; 3(2): 222-9, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22460635

RESUMEN

Homeostasis in the central nervous system (CNS) is maintained by active interfaces between the bloodstream and the brain parenchyma. The blood-brain barrier (BBB) constitutes a selective filter for exchange of water, solutes, nutrients, and controls toxic compounds or pathogens entry. Some parasites, bacteria, and viruses have however developed various CNS invasion strategies, and can bypass the brain barriers. Concerning viruses, these strategies include transport along neural pathways, transcytosis, infection of the brain endothelial cells, breaching of the BBB, and passage of infected-leukocytes. Moreover, neurotropic viruses can alter BBB functions, thus compromising CNS homeostasis. Retroviruses have been associated to human neurological diseases: HIV (human immunodeficiency virus 1) can induce HIV-associated dementia, and HTLV-1 (human T lymphotropic virus 1) is the etiological factor of tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). The present review focuses on how the different retroviruses interact with this structure, bypass it and alter its functions.


Asunto(s)
Barrera Hematoencefálica/fisiología , VIH/patogenicidad , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Infecciones por Retroviridae/inmunología , Infecciones por Retroviridae/patología , Humanos , Modelos Biológicos
14.
Invest Ophthalmol Vis Sci ; 53(4): 2292-9, 2012 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-22427552

RESUMEN

PURPOSE: The aim of the present study was the in vitro and in vivo evaluation of a novel aqueous formulation based on polymeric micelles for the topical delivery of cyclosporine A for dry eye treatment. METHODS: In vitro experiments were carried out on primary rabbit corneal cells, which were characterized by immunocytochemistry using fluorescein-labeled lectin I/isolectin B4 for the endothelial cells and mouse monoclonal antibody to cytokeratin 3+12 for the epithelial ones. Living cells were incubated for 1 hour or 24 hours with a fluorescently labeled micelle formulation and analyzed by fluorescence microscopy. In vivo evaluations were done by Schirmer test, osmolarity measurement, CyA kinetics in tears, and CyA ocular distribution after topical instillation. A 0.05% CyA micelle formulation was compared to a marketed emulsion (Restasis). RESULTS: The in vitro experiments showed the internalization of micelles in the living cells. The Schirmer test and osmolarity measurements demonstrated that micelles did not alter the ocular surface properties. The evaluation of the tear fluid gave similar CyA kinetics values: AUC = 2339 ± 1032 min*µg/mL and 2321 ± 881.63; Cmax = 478 ± 111 µg/mL and 451 ± 74; half-life = 36 ± 9 min and 28 ± 9 for the micelle formulation and Restasis, respectively. The ocular distribution investigation revealed that the novel formulation delivered 1540 ± 400 ng CyA/g tissue to the cornea. CONCLUSIONS: The micelle formulation delivered active CyA into the cornea without evident negative influence on the ocular surface properties. This formulation could be applied for immune-related ocular surface diseases.


Asunto(s)
Córnea/metabolismo , Ciclosporina/farmacocinética , Inmunosupresores/farmacocinética , Lágrimas/metabolismo , Administración Tópica , Animales , Femenino , Micelas , Microscopía Fluorescente , Soluciones Oftálmicas , Conejos , Ratas , Ratas Endogámicas Lew
15.
J Cereb Blood Flow Metab ; 31(2): 514-26, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20683453

RESUMEN

The pathogenesis of cerebral malaria (CM) remains largely unknown. There is growing evidence that combination of both parasite and host factors could be involved in blood-brain barrier (BBB) breakdown. However, lack of adequate in vitro model of human BBB so far hampered molecular studies. In this article, we propose the use of hCMEC/D3 cells, a well-established human cerebral microvascular endothelial cell (EC) line, to study BBB breakdown induced by Plasmodium falciparum-parasitized red blood cells and environmental conditions. We show that coculture of parasitized erythrocytes with hCMEC/D3 cells induces cell adhesion and paracellular permeability increase, which correlates with disorganization of zonula occludens protein 1 expression pattern. Permeability increase and modification of tight junction proteins distribution are cytoadhesion independent. Finally, we show that permeability of hCMEC/D3 cell monolayers is mediated through parasite induced metabolic acidosis, which in turns correlates with apoptosis of parasitized erythrocytes. This new coculture model represents a very useful tool, which will improve the knowledge of BBB breakdown and the development of adjuvant therapies, together with antiparasitic drugs.


Asunto(s)
Acidosis/metabolismo , Acidosis/parasitología , Barrera Hematoencefálica/parasitología , Eritrocitos/parasitología , Malaria Cerebral/sangre , Malaria Cerebral/parasitología , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Plasmodium falciparum , Anexina A5/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular , Células Endoteliales/fisiología , Citometría de Flujo , Humanos , Concentración de Iones de Hidrógeno , L-Lactato Deshidrogenasa/metabolismo , Lentivirus/genética , Merozoítos/parasitología , Merozoítos/fisiología , Microscopía Confocal , Permeabilidad , Interferencia de ARN , Uniones Estrechas/metabolismo , Transducción Genética , Tripsina/farmacología
16.
J Med Chem ; 54(2): 485-94, 2011 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-21175162

RESUMEN

To improve the blood-brain barrier permeability of the trypanocidal lead compound 4,4'-bis(imidazolinylamino)diphenylamine (1), five N-alkoxy analogues were synthesized from bis(4-isothiocyanatophenyl)amine and N-alkoxy-N-(2-aminoethyl)-2-nitrobenzenesulfonamides following successive chemical reactions in just one reactor ("one-pot procedure"). This involved: (a) formation of a thiourea intermediate, (b) removal of the amine protecting groups, and (c) intramolecular cyclization. The blood-brain barrier permeability of the compounds determined in vitro by transport assays through the hCMEC/D3 human cell line, a well-known and characterized human cellular blood-brain barrier model, showed that the N-hydroxy analogue 16 had enhanced blood-brain barrier permeability compared with the unsubstituted lead compound. Moreover, this compound displayed low micromolar IC(50) against Trypanosoma brucei rhodesiense and Plasmodium falciparum and moderate activity by intraperitoneal administration in the STIB900 murine model of acute sleeping sickness.


Asunto(s)
Barrera Hematoencefálica/metabolismo , Difenilamina/análogos & derivados , Difenilamina/síntesis química , Imidazolinas/síntesis química , Tripanocidas/síntesis química , Animales , Antimaláricos/síntesis química , Antimaláricos/farmacología , Línea Celular , Difenilamina/farmacología , Humanos , Imidazolinas/farmacología , Leishmania donovani/efectos de los fármacos , Ratones , Permeabilidad , Plasmodium falciparum/efectos de los fármacos , Relación Estructura-Actividad , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei gambiense , Trypanosoma cruzi/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico
17.
J Neuroimmunol ; 223(1-2): 131-4, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20400187

RESUMEN

It has been recently shown that systemically injected neural precursor cells (NPCs) could cross brain endothelium and favor functional recovery in animal models of multiple sclerosis (MS). Here we show that human NPCs express receptors of the chemokines IL8 and CXCL13 (CXCR1 and CXCR5, respectively) and migrate across brain endothelial cells in vitro, in response to these chemokines. Considering that these chemokines have been found overexpressed in MS in active, but not inactive areas of demyelination, our data suggest that systemically injected human NPCs may be considered for targeting active areas of demyelination in therapeutic approaches of MS.


Asunto(s)
Encéfalo/inmunología , Quimiocina CXCL13/fisiología , Quimiotaxis de Leucocito/inmunología , Células Madre Embrionarias/inmunología , Células Endoteliales/inmunología , Interleucina-8/fisiología , Neuronas/inmunología , Encéfalo/metabolismo , Células Cultivadas , Quimiocina CXCL13/biosíntesis , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Células Endoteliales/metabolismo , Células Endoteliales/trasplante , Humanos , Interleucina-8/biosíntesis , Neuronas/metabolismo , Neuronas/trasplante
18.
Biochem Pharmacol ; 77(5): 897-909, 2009 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-19041851

RESUMEN

We investigated the expression of genes encoding ABC transporters, cytochromes P450 (CYPs) and some transcription factors in the hCMEC/D3 immortalized human cerebral microvascular endothelial cell line, a promising in vitro model of the human BBB, and we compared these expressions to a non-brain endothelial cell line (HUVEC) and freshly human brain microvessels. qRT-PCR showed that the MDR1, BCRP, MRP1, MRP3, MRP4 and MRP5 genes were expressed and that the main CYP gene was CYP2U1 in hCMEC/D3. The pattern of ABC and CYPs gene expression in hCMEC/D3 differed from HUVEC which did not express MDR1. Moreover, expression of P-gp and BCRP was lower in hCMEC/D3 than in human brain microvessels but remain functional as shown by rhodamine 123 efflux assay. The gene encoding the aryl hydrocarbon receptor (AhR), a transcription factor that regulates the expression of some ABC and CYPs was highly expressed in hCMEC/D3 and HUVEC, while the pregnane-X-receptor (PXR) and the constitutive androstane receptor (CAR) were barely detected. We investigated the function of the AhR-mediated regulatory pathway in hCMEC/D3 by treating them with the AhR agonist TCDD. The expressions of two AhR-target genes, CYP1A1 and CYP1B1, were increased 26-fold and 28-fold. But the expressions of ABC transporter genes were not significantly altered. We have thus determined the pattern of expression of the genes encoding ABC transporters, CYPs and three transcription factors in hCMEC/D3 and shown that the AhR pathway might afford an original functional transport and metabolic pattern in cerebral endothelial cells that is different from other peripheral endothelial cells.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Encéfalo/irrigación sanguínea , Sistema Enzimático del Citocromo P-450/genética , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Secuencia de Bases , Western Blotting , Línea Celular Transformada , Cartilla de ADN , Dronabinol/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Humanos , Dibenzodioxinas Policloradas/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rifampin/farmacología
19.
Science ; 325(5936): 83-7, 2009 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-19520910

RESUMEN

Type IV pili mediate the initial interaction of many bacterial pathogens with their host cells. In Neisseria meningitidis, the causative agent of cerebrospinal meningitis, type IV pili-mediated adhesion to brain endothelial cells is required for bacteria to cross the blood-brain barrier. Here, type IV pili-mediated adhesion of N. meningitidis to human brain endothelial cells was found to recruit the Par3/Par6/PKCzeta polarity complex that plays a pivotal role in the establishment of eukaryotic cell polarity and the formation of intercellular junctions. This recruitment leads to the formation of ectopic intercellular junctional domains at the site of bacteria-host cell interaction and a subsequent depletion of junctional proteins at the cell-cell interface with opening of the intercellular junctions of the brain-endothelial interface.


Asunto(s)
Adhesión Bacteriana , Barrera Hematoencefálica/microbiología , Encéfalo/irrigación sanguínea , Endotelio Vascular/microbiología , Fimbrias Bacterianas/fisiología , Uniones Intercelulares/metabolismo , Neisseria meningitidis/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Encéfalo/microbiología , Cadherinas/metabolismo , Cateninas , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Polaridad Celular , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Humanos , Uniones Intercelulares/microbiología , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/metabolismo , Neisseria meningitidis/patogenicidad , Fosfoproteínas/metabolismo , Proteína Quinasa C/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Catenina delta
20.
J Leukoc Biol ; 86(5): 1049-63, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19696154

RESUMEN

Mechanisms of T lymphocyte trafficking in the brain remain unclear in MS. We hypothesized that MS is associated with increased CD4+ and CD8+ T lymphocyte trafficking across the BBB. To test this hypothesis, we calculated the frequency of PSGL-1+/CD4+ and PSGL-1+CD8+ or LFA-1+/CD4+/CD8+ T cells in the PBMC of 27 patients with a RR-MS (21 untreated and six IFN-beta-treated) and 18 HI. Next, we measured their ex vivo TR across resting and TNF-alpha-activated human BBB-derived hCMEC/D3 endothelial layers under static conditions. The frequency of PSGL-1+CD4+ T lymphocytes was significantly higher in treated or untreated MS patients than HI. Furthermore, resting hCMEC/D3 TR of CD4+ lymphocytes (purified or in PBMC) from treated or untreated MS patients were significantly higher than those of HI and associated with significant enrichments of CD4+PSGL+ or CD4+PSGL-1+CD45RO+ T cells in their transmigrating fractions. The TR of CD4+ and CD8+ from MS patients across TNF-alpha-activated hCMEC/D3 were also significantly higher than that observed in HI. Resting hCMEC/D3 transmigration was blocked significantly by anti-PSGL-1/anti-LFA-1 in all groups, and anti-VLA-4 inhibited transmigration of MS T cells specifically. Purified PSGL-1-negative CD4+ lymphocytes transmigrated resting hCMEC/D3 with <10% of transmigrating cells re-expressing PSGL-1, suggesting PSGL-1-independent transmigration mechanisms. The frequency of PSGL-1 was unchanged in CD8+ cells from MS patients, whereas CD8+LFA-1(high) were reduced significantly in IFN-beta-treated patients specifically. Collectively, MS is associated with an expanding pool of PSGL-1+CD4+ T lymphocytes able to transmigrate the BBB endothelium in vitro and possibly contributing to brain pathology.


Asunto(s)
Barrera Hematoencefálica/inmunología , Linfocitos T CD4-Positivos/inmunología , Glicoproteínas de Membrana/genética , Esclerosis Múltiple/inmunología , Linfocitos T/inmunología , Adulto , Encéfalo/inmunología , División Celular , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Femenino , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Interferón beta/uso terapéutico , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/tratamiento farmacológico , Valores de Referencia , Adulto Joven
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