A new nonpolar N-hydroxy imidazoline lead compound with improved activity in a murine model of late-stage Trypanosoma brucei brucei infection is not cross-resistant with diamidines.

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.

Nanoparticle formulation improves the anticonvulsant effect of clonazepam on the pentylenetetrazole-induced seizures: behavior and electroencephalogram.

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.

The hypervariable region of meningococcal major pilin PilE controls the host cell response via antigenic variation.

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.

Meningococcal interaction to microvasculature triggers the tissular lesions of purpura fulminans.

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.

Neisseria meningitidis colonization of the brain endothelium and cerebrospinal fluid invasion.

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.

Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model.

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.

A novel cyclosporin a aqueous formulation for dry eye treatment: in vitro and in vivo evaluation.

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.

Blood-brain barrier and retroviral infections.

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.

Synthesis and antiprotozoal activity of N-alkoxy analogues of the trypanocidal lead compound 4,4'-bis(imidazolinylamino)diphenylamine with improved human blood-brain barrier permeability.

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.

Differential expression of the bone and the liver tissue non-specific alkaline phosphatase isoforms in brain tissues.

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.

Metabolic acidosis induced by Plasmodium falciparum intraerythrocytic stages alters blood-brain barrier integrity.

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.

IL8 and CXCL13 are potent chemokines for the recruitment of human neural precursor cells across brain endothelial cells.

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.

Induced secretion of beta-hexosaminidase by human brain endothelial cells: a novel approach in Sandhoff disease?

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.

Peripheral blood CD4+ T lymphocytes from multiple sclerosis patients are characterized by higher PSGL-1 expression and transmigration capacity across a human blood-brain barrier-derived endothelial cell line.

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.

Meningococcal type IV pili recruit the polarity complex to cross the brain endothelium.

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.

The blood-brain barrier in brain homeostasis and neurological diseases.

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.

Expression and transcriptional regulation of ABC transporters and cytochromes P450 in hCMEC/D3 human cerebral microvascular endothelial cells.

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.

Physiological pathway for low-density lipoproteins across the blood-brain barrier: transcytosis through brain capillary endothelial cells in vitro.

Although an immense knowledge has accumulated concerning regulation of cholesterol homeostasis in the body, this does not include the brain, where details are just emerging. Using an in vitro blood-brain barrier model, the authors have demonstrated that low-density lipoprotein (LDL) underwent transcytosis through the endothelial cells (ECs) by a receptor-mediated process, bypassing the lysosomal compartment. Moreover, caveolae might be involved in these blood-borne molecule transports from the blood to the brain. Although several ligands are known to be internalized through cell surface caveolae, the subsequent intracellular pathways have remained elusive. By cell fractionation experiment and Western blot, the authors have demonstrated that the LDL receptor is located in the caveolae membrane fraction. Then, LDLs internalized were detected by electron microscopy in multivesicular bodies. The authors identified in brain capillary ECs a novel endosomal compartment, mildly acidic, positive for marker Lamp-1 but devoid of any degradative capability. From the point of view of pH, cellular location, and caveolae-derived formation, the multivesicular organelles described here can be related to the caveosome structure. These results could provide clues to physiological functions of caveolae-caveosome transcellular pathway in brain capillary ECs and may help in the rational design of more effective therapeutic drugs to the brain.

Molecular mechanism of systemic delivery of neural precursor cells to the brain: assembly of brain endothelial apical cups and control of transmigration by CD44.

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.