Brain Microvascular Endothelial Cell Monolayers Research Articles

OspA-CD40 dyad: ligand-receptor interaction in the translocation of neuroinvasive Borrelia across the blood-brain barrier

Lyme borreliosis is the most widespread vector-borne disease in temperate zones of Europe and North America. Although the infection is treatable, the symptoms are often overlooked resulting in infection of the neuronal system. In this work we uncover the underlying molecular mechanism of borrelial translocation across the blood-brain barrier (BBB). We demonstrate that neuroinvasive strain of Borrelia readily crosses monolayer of brain-microvascular endothelial cells (BMECs) in vitro and BBB in vivo. Using protein-protein interaction assays we found that CD40 of BMECs and OspA of Borrelia are the primary molecules in transient tethering of Borrelia to endothelium. OspA of neuroinvasive Borrelia, but not of non-neuroinvasive strain, binds CD40. Furthermore, only the neuroinvasive Borrelia and its recombinant OspA activated CD40-dependent pathway in BMECs and induced expression of integrins essential for stationary adhesion. Demonstration of the CD40-ligand interactions may provide a new possible perspective on molecular mechanisms of borrelial BBB translocation process.

Expression of ornithine decarboxylase during the transport of saquinavir across the blood–brain barrier using composite polymeric nanocarriers under an electromagnetic field

The expression of human ornithine decarboxylase (ODC) and permeability of saquinavir (SQV) across the blood–brain barrier were studied using nanoparticles (NPs) composed of poly(lactide - co-glycolide) (PLGA), poly-(γ-glutamic acid) (γ-PGA), and polyethyleneimine (PEI). SQV was encapsulated in the particle core to traverse a monolayer of human brain-microvascular endothelial cells (HBMECs) with the regulation of human astrocytes under an electromagnetic field (EMF). An increase in the weight percentage of PEI enhanced the particle size, zeta potential, and permeability of SQV. However, the viability of HBMECs reduced when the weight percentage of PEI increased. In addition, an increment in the molecular weight of γ-PGA enhanced the particle size and viability of HBMECs, and reduced the zeta potential. The permeability of SQV and expression of ODC were in the order: an EMF with amplitude modulation (AM) > an EMF with frequency modulation > no EMF. At 0.04% PEI, the AM EMF increased 2.38 times the uptake of NPs and 2.72 times the expression of ODC. The combination of PEI/γ-PGA/PLGA NPs and EMF can be an innovative strategy for delivering SQV into the brain.

Mesenchymal stem cells transmigrate across brain microvascular endothelial cell monolayers through transiently formed inter-endothelial gaps

Mesenchymal stem cells (MSCs) hold much promise for cell therapy for neurological diseases such as cerebral ischemia and Parkinson's disease. Intravenously administered MSCs accumulate in lesions within the brain parenchyma, but little is known of the details of MSC transmigration across the blood–brain barrier (BBB). To study MSC transmigration across the BBB, we developed an in vitro culture system consisting of rat brain microvascular endothelial cells (BMECs) and bone marrow-derived MSCs using Transwell or Millicell culture inserts. Using this system, we first investigated the influence of the number of MSCs added to the upper chamber on BMEC barrier integrity. The addition of MSCs at a density of 1.5 × 10 5 cells/cm 2 led to disruption of the BMEC monolayer structure and decreased barrier function as measured by the transendothelial electrical resistance (TEER). When applied at a density of 1.5 × 10 4 cells/cm 2, neither remarkable disruption of the BMEC monolayers nor a significant decrease in TEER was observed until at least 12 h. After cultivation for 24 h under this condition, MSCs were found in the subendothelial space or beneath the insert membrane, suggesting that MSCs transmigrate across BMEC monolayers. Time-lapse imaging revealed that MSCs transmigrated across the BMEC monolayers through transiently formed intercellular gaps between the BMECs. These results show that our in vitro culture system consisting of BMECs and MSCs is useful for investigating the molecular and cellular mechanisms underlying MSC transmigration across the BBB.

Transport of saquinavir across human brain-microvascular endothelial cells by poly(lactide- co-glycolide) nanoparticles with surface poly-(γ-glutamic acid)

Poly(lactide- co-glycolide) (PLGA) nanoparticles (NPs) with surface poly-(γ-glutamic acid) (γ-PGA) were applied to enhance the transport of saquinavir (SQV) across the blood–brain barrier (BBB). PLGA NPs encapsulated SQV and grafted with γ-PGA to form drug carriers (γ-PGA/SQV-PLGA NPs) for crossing through a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated with human astrocytes. The results revealed that a lower molecular weight of γ-PGA yielded a higher grafting efficiency of γ-PGA on PLGA NPs. In addition, γ-PGA with a low molecular weight accelerated the dissolution of SQV from γ-PGA/SQV-PLGA NPs. A higher grafting efficiency (more didecyl dimethylammonium bromide) and a lower molecular weight of γ-PGA increased the permeability of SQV across the BBB, in general. When the grafting efficiency was 85.2% at 6 kDa of γ-PGA, γ-PGA/SQV-PLGA NPs reached about 6 times the permeability of free SQV (the maximal permeability). γ-PGA could also promote the endocytosis of NPs and expression of ornithine decarboxylase by HBMECs. γ-PGA/SQV-PLGA NPs are efficacious nanoparticulate carriers in delivering antiretroviral drug across the BBB.

Regulation of endocytosis into human brain-microvascular endothelial cells by inhibition of efflux proteins

The expressions of P-glycoprotein (P-gp) and multidrug resistance-associated proteins (MRPs) in the blood-brain barrier (BBB) were regulated by verapamil and probenecid. The two protein interveners inhibited the efflux-pumping capability for permeating calcein-AM in the monolayer of human brain-microvascular endothelial cells (HBMECs). The immunochemical staining revealed that the order in the integrity of tight junction was human astrocyte (HA)-regulated HBMECs>HBMECs cultured with 100% astrocyte-conditioned medium (ACM)>HBMECs cultured with 50% ACM>HBMECs. The viability of HBMECs was higher than 94% when the concentrations of verapamil and probenecid were lower than 50 μM and 1000 μM, respectively. The culture using ACM negligibly affected the activity of P-gp and MRPs on HBMECs after the suppression with verapamil and/or probenecid. However, the double culture of HA-regulated HBMECs promoted the quantity of P-gp and MRPs and reduced the endocytosis of calcein-AM. The inhibitive and endocytotic analysis can unveil the role of HAs in the protein expressions on HBMECs for establishing a reliable BBB model in vitro.

Contribution of Lethal Toxin and Edema Toxin to the Pathogenesis of Anthrax Meningitis

Bacillus anthracis is a Gram-positive spore-forming bacterium that causes anthrax disease in humans and animals. Systemic infection is characterized by septicemia, toxemia, and meningitis, the main neurological complication associated with high mortality. We have shown previously that B. anthracis Sterne is capable of blood-brain barrier (BBB) penetration, establishing the classic signs of meningitis, and that infection is dependent on the expression of both major anthrax toxins, lethal toxin (LT) and edema toxin (ET). Here we further investigate the contribution of the individual toxins to BBB disruption using isogenic toxin mutants deficient in lethal factor, ΔLF, and edema factor, ΔEF. Acute infection with B. anthracis Sterne and the ΔLF mutant resulted in disruption of human brain microvascular endothelial cell (hBMEC) monolayer integrity and tight junction protein zona occludens-1, while the result for cells infected with the ΔEF mutant was similar to that for the noninfected control. A significant decrease in bacterial invasion of BBB endothelium in vitro was observed during infection with the ΔLF strain, suggesting a prominent role for LT in promoting BBB interaction. Further, treatment of hBMECs with purified LT or chemicals that mimic LT action on host signaling pathways rescued the hypoinvasive phenotype of the ΔLF mutant and resulted in increased bacterial uptake. We also observed that toxin expression reduced bacterial intracellular survival by inducing the bulk degradative autophagy pathway in host cells. Finally, in a murine model of anthrax meningitis, mice infected with the ΔLF mutant exhibited no mortality, brain bacterial load, or evidence of meningitis compared to mice infected with the parental or ΔEF strains.

VE-cadherin and PECAM-1 enhance ALL migration across brain microvascular endothelial cell monolayers

Infiltration of the central nervous system (CNS) by leukemia is a problematic disease manifestation of acute lymphoblastic leukemia (ALL). The mechanisms by which leukocytes interact with human brain-derived microvasculature endothelial cells (HBMEC) and enter the CNS are largely derived from models of inflammation. However, our data indicate that ALL cells do not elicit an inflammatory phenotype by HBMEC. Our current investigation focuses on the contribution of the unique coexpression of vascular endothelial (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1) by ALL in mediating leukemic cell interactions with HBMEC as an in vitro model of the blood-brain barrier. Primary ALL and ALL cell lines were evaluated for VE-cadherin and PECAM-1 expression. Lentiviral-mediated transduction of VE-cadherin and PECAM-1 into REH cells and antibody neutralization of VE-cadherin and PECAM-1 in SUP-B15 cells was used to delineate the role of these two proteins in mediating ALL adhesion to, and migration through, HBMEC monolayers. Although cell line models indicate that VE-cadherin and PECAM-1 expression is found on the surface Philadelphia chromosome-positive ALL, evaluation of primary ALL demonstrates that VE-cadherin and PECAM-1 are expressed independent of Philadelphia status. Expression of VE-cadherin and PECAM-1 by ALL enhanced the adhesion of ALL to HBMEC, while expression of PECAM-1 enhanced ALL adhesion to, and migration through, HBMEC. Expression of VE-cadherin and PECAM-1 by ALL cells positions them to interact with HBMEC. By increasing our understanding of molecular mechanisms through which ALL cells gain entry into the CNS, new strategies may be designed to prevent leukemia cell entry into the CNS.

Inhibition of Glycogen Synthase Kinase 3β (GSK3β) Decreases Inflammatory Responses in Brain Endothelial Cells

Immune mediators and leukocyte engagement of brain microvascular endothelial cells (BMVECs) contribute to blood-brain barrier impairment during neuroinflammation. Glycogen synthase kinase 3beta (GSK3beta) was recently identified as a potent regulator of immune responses in in vitro systems and animal models. However, the role of GSK3beta in regulation of immune endothelial functions remains undetermined. Here we evaluated the effect of GSK3beta inhibition on the regulation of inflammatory responses in BMVECs. A focused PCR gene array of 84 genes was performed to identify the cytokine and chemokine gene expression profile in tumor necrosis factor (TNF) alpha-stimulated BMVECs after GSK3beta inactivation by specific inhibitors. Fifteen of 39 genes induced by TNFalpha stimulation were down-regulated after GSK3beta inhibition. Genes known to contribute to neuroinflammation that were most negatively affected by GSK3beta inactivation included IP-10/CXCL10, MCP-1/CCL2, IL-8/CXCL8, RANTES/CCL5, and Groalpha/CXCL1. GSK3beta suppression resulted in diminished secretion of these proinflammatory mediators by inflamed BMVECs detected by ELISA. GSK3beta inhibition in BMVECs reduced adhesion molecule expression as well as monocyte adhesion to and migration across cytokine stimulated BMVEC monolayers. Interactions of monocytes with TNFalpha-activated BMVECs led to barrier disruption, and GSK3beta suppression in the endothelium restored barrier integrity. GSK3beta inhibition in vivo substantially decreased leukocyte adhesion to brain endothelium under inflammatory conditions. In summary, inhibition of GSK3beta emerges as an important target for stabilization of the blood-brain barrier in neuroinflammation.

Methamphetamine Disrupts Blood–Brain Barrier Function by Induction of Oxidative Stress in Brain Endothelial Cells

Methamphetamine (METH), a potent stimulant with strong euphoric properties, has a high abuse liability and long-lasting neurotoxic effects. Recent studies in animal models have indicated that METH can induce impairment of the blood-brain barrier (BBB), thus suggesting that some of the neurotoxic effects resulting from METH abuse could be the outcome of barrier disruption. In this study, we provide evidence that METH alters BBB function through direct effects on endothelial cells and explore possible underlying mechanisms leading to endothelial injury. We report that METH increases BBB permeability in vivo, and exposure of primary human brain microvascular endothelial cells (BMVEC) to METH diminishes the tightness of BMVEC monolayers in a dose- and time-dependent manner by decreasing the expression of cell membrane-associated tight junction (TJ) proteins. These changes were accompanied by the enhanced production of reactive oxygen species, increased monocyte migration across METH-treated endothelial monolayers, and activation of myosin light chain kinase (MLCK) in BMVEC. Antioxidant treatment attenuated or completely reversed all tested aspects of METH-induced BBB dysfunction. Our data suggest that BBB injury is caused by METH-mediated oxidative stress, which activates MLCK and negatively affects the TJ complex. These observations provide a basis for antioxidant protection against brain endothelial injury caused by METH exposure.

Effect of curcumin on the adhesion of platelets to brain microvascular endothelial cellsin vitro1

To determine whether curcumin prevents the adhesion of platelets to brain microvascular endothelial cells (BMECs) cultured in vitro. [3H]Adenine- labeled platelets were incubated with BMECs to investigate the role of curcumin in the adhesion of platelets to BMECs. The number of platelets adhering to the BMECs monolayer was determined by liquid scintillation spectroscopy. The thrombin-induced expression of platelets P-selectin, glycoprotein IIb (GPIIb), and glycoprotein IIIa (GPIIIa) on the cell surface, was measured by flow cytometry. P-selectin mRNA levels of BMECs were determined by RT-PCR. The TNF-alpha- induced expressions of P-selectin and E-selectin on the surface of BMECs were determined by Western blotting. The adhesion between thrombin-activated platelets and normal BMECs, and that of TNF-alpha-activated BMECs and normal platelets were significantly increased, and this increase could be inhibited by curcumin (30-240 micromol/L) in a concentration-dependant manner. The platelets activated with thrombin and BMECs stimulated by TNF-alpha demonstrated an upregulated expressions of P-selectin and E-selectin, and this increase, when pretreated with curcumin for 30 min, could be restrained dose dependently. Curcumin also inhibited the increase of the GPIIb/GPIIIa expression of thrombinactivated platelets in a concentration-dependent manner. Curcumin can inhibit the platelets to BMECs. This effect may be related to the decreased expressions of P-selectin, E-selectin, and GPIIb/GPIIIa on platelets and BMECs.

LFA1 Mediated Attachment of Primary Human Monocytes to Activated Brain Microvascular Endothelial Cells Is Regulated by SDF-1α through the SRC Family Kinase Lyn; Potential Involvement of This Signaling Pathway in HIV-Associated Dementia.

Infiltration of activated monocytes into the brain of HIV-infected patients is a prerequisite for the development of HIV-associated dementia (HAD). The chemokine stromal derived factor-1α (SDF-1α) is expressed at increased levels in the central nervous system (CNS) of HAD patients and elicits chemotaxis and other cellular effects through its receptor CXCR4. In this project, we investigated the intracellular signaling pathway by which SDF-1α mediates the movement and attachment of monocytes to brain microvascular endothelia, and which may contribute to their infiltration into the CNS in HAD. We demonstrated that SDF-1α stimulates migration of primary human monocytes through its receptor CXCR4, and decreases monocyte adherence to surfaces coated with ICAM-1. SDF-1α also decreases monocyte adherence to brain microvascular endothelial cells (BMVEC) activated with the pro-inflammatory cytokines TNF-α or IL-1β, or with recombinant HIV-1 envelope glycoprotein (gp120), which increase endothelial cells expression of ICAM-1. The decreased monocyte adherence was linked to down regulation of the activation-dependent epitope of the β2 integrin LFA-1 which is a ligand for ICAM-1. We then demonstrated that the Src family kinase Lyn is a central modulator of migration and LFA-1-mediated adhesion of SDF-1α-stimulated primary monocytes. Using siRNA knockdown we achieved 80% down regulation of Lyn kinase in human monocytes. Lyn down regulation decreased SDF-1α-mediated migration and prevented its inhibition of monocyte attachment to ICAM-1 coated surfaces and activated BMVEC. These data indicate that in SDF-1α-stimulated primary human monocytes Lyn is a positive regulator of cell migration, and a negative regulator of cell adhesion to BMVEC by inhibiting the ICAM-1 binding activity of the LFA-1 integrin. Thus, CXCR4-triggered inside-out integrin signaling, through Lyn, inhibits adherence and stimulates movement of monocytes towards SDF-1α gradient on BMVEC monolayers. These results provide new insight into the intracellular signaling cascade that controls primary human monocytes movement and attachment at the blood brain barrier.

Unconjugated bilirubin efflux by bovine brain microvascular endothelial cells in vitro

The passage of unconjugated bilirubin (UCB) across the blood-brain barrier into the central nervous system is a crucial first step in the development of kernicterus. The objective of the current study was to characterize the passage of UCB across primary bovine brain microvascular endothelial cell (BBMVEC) monolayers in vitro. Experimental study. Research institute. BBMVECs. Tritiated UCB (H-UCB) transport at 60, 80, 100, 200, 300, and 400 nM concentrations was tested in both the apical to basolateral (A--> B) and basolateral to apical (B-->A) directions in BBMVEC monolayers in vitro with or without preincubation with pharmacologic active transport inhibitors cyclosporine A, indomethacin, or MK571. The rate of H-UCB transport in the B-->A direction was 6.2- to 7.3-fold higher than in the A-->B direction, suggesting active efflux of UCB. Cyclosporine A (5 microM), a model inhibitor of P-glycoprotein, enhanced A-->B while decreasing B-->A UCB transport, resulting in an overall decrease in BBMVEC UCB efflux of between 46% and 54%. Indomethacin (10 microM) and MK-571 (50 microM), respectively a substrate and potent inhibitor of multidrug resistance-associated protein-1, had no effect. We conclude that 1) UCB is transported by BBMVEC monolayers in vitro in a net B-->A direction (i.e., active efflux); and 2) cyclosporine A partially inhibits such transport. We speculate that the blood-brain barrier limits the passage and central nervous system retention of UCB by active transport and that this may be accounted in part by P-glycoprotein.

Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells

AbstractRecent evidence from this laboratory indicated that reduced expression of caveolin-1 accompanied the diminished expression of tight junction (TJ)–associated proteins occludin and zonula occludens-1 (ZO-1) following stimulation of brain microvascular endothelial cells (BMECs) with the chemokine CCL2 (formerly called MCP-1). Because attenuated caveolin-1 levels have also been correlated with heightened permeability of other endothelia, the objective of this study was to test the hypothesis that reduced caveolin-1 expression is causally linked to the action of CCL2 on BMEC junctional protein expression and barrier integrity. This was achieved using adenovirus to nondestructively deliver caveolin-1 siRNA (Ad-siCav-1) to BMEC monolayers, which model the blood-brain barrier (BBB). Treatment with siRNA reduced the caveolin-1 protein level as well as occludin and ZO-1. Additionally, occludin exhibited dissociation from the cytoskeletal framework. These changes were attended by comparable alterations in adherens junction (AJ)–associated proteins, VE-cadherin and β-catenin, increased BMEC paracellular permeability, and facilitated the ability of CCL2 to stimulate monocytic transendothelial migration. Furthermore, treating BMECs with cavtratin, a synthetic cell-permeable peptide encoding the caveolin-1 scaffolding domain, antagonized effects of both Ad-siCav-1 and CCL2. These results collectively highlight caveolin-1 loss as a critical step in CCL2-induced modulation of BMEC junctional protein expression and integrity, and possibly serve a crucial role in regulating inflammation at the BBB.

Gene Expression Pattern in Human Brain Endothelial Cells in Response to Neisseria meningitidis

To extend our knowledge of target proteins in endothelial cells infected with the meningitis-causing pathogen Neisseria meningitidis, we characterized the interaction between the bacterial and human brain microvascular endothelial cell (HBMEC) monolayers. By use of human cDNA microarrays, transcriptional analysis revealed distinct responses to 4 and 8 h of infection. We also addressed the question of whether the major virulence factor of meningococci, i.e., the capsule, influences the host cell response. Of the 1,493 (at 4 h postinfection) and 1,246 (at 8 h postinfection) genes with altered expression upon bacterial contact, about 49.4% and 45%, respectively, depended on capsule expression. In particular, we identified an increase of expression for genes encoding proteins involved in bacterial adhesion and invasion. High levels of apoptosis-related gene (bad, bak, asp, and immediate-early response gene 1) expression could also be detected in infected cells. Further analyses confirmed that HBMECs displayed several hallmarks of apoptosis in response to N. meningitidis infection, namely, phosphatidylserine translocation and activation of caspase 3 and AMP-activated protein kinase alpha. Moreover, several differentially regulated genes not previously known to respond to meningococcal infection were identified. Of these, genes encoding cell adhesion proteins (CD44, CD98, and CD99), genes involved in downstream signaling of integrins (integrin-linked kinase, mitogen-activated protein kinase kinase 1, and mitogen-activated protein kinase kinase kinase 10) as well as negative regulators of these pathways (dual-specificity phosphatases 1, 5, and 14 and G protein pathway suppressor 2), and genes involved in cytoskeleton reorganization (those encoding Arp2/3, p34-arc, actinin alpha 1, vasodilatator-stimulated protein, and Wiskott-Aldrich syndrome protein) were the most prominent. This global transcriptional analysis creates a new platform for further molecular and cellular analysis of the interaction between N. meningitidis and target cells.

Alcohol‐induced blood–brain barrier dysfunction is mediated via inositol 1,4,5‐triphosphate receptor (IP3R)‐gated intracellular calcium release

The blood-brain barrier (BBB) formed by brain microvascular endothelial cells (BMVEC), pericytes and astrocytes controls the transport of ions, peptides and leukocytes in and out of the brain. Tight junctions (TJ) composed of TJ proteins (occludin, claudins and zonula occludens) ensure the structural integrity of the BMVEC monolayer. Neuropathologic studies indicated that the BBB was impaired in alcohol abusers; however, the underlying mechanism of BBB dysfunction remains elusive. Using primary human BMVEC, we previously demonstrated that oxidative stress induced by ethanol (EtOH) metabolism in BMVEC activated myosin light chain kinase (MLCK), resulting in the enhanced phosphorylation of either cytoskeletal or TJ proteins, and in BBB impairment. We proposed that EtOH metabolites stimulated inositol 1,4,5-triphosphate receptor (IP(3)R)-operated intracellular calcium (Ca(2+)) release, thereby causing the activation of MLCK in BMVEC. Indeed, treatment of primary human BMVEC with EtOH or its metabolites resulted in the increased expression of IP(3)R protein and IP(3)R-gated intracellular Ca(2+) release. These functional changes paralleled MLCK activation, phosphorylation of cytoskeletal/TJ proteins, loss of BBB integrity, and enhanced leukocyte migration across BMVEC monolayers. Inhibition of either EtOH metabolism or IP(3)R activation prevented BBB impairment. These findings suggest that EtOH metabolites act as signaling molecules for the activation of MLCK via the stimulation of IP(3)R-gated intracellular Ca(2+) release in BMVEC. These putative events can lead to BBB dysfunction in the setting of alcoholism, and to neuro-inflammatory disorders promoting leukocyte migration across the BBB.

Adrenomedullin improves the blood-brain barrier function through the expression of claudin-5.

Brain vascular endothelial cells secret Adrenomedullin (AM) has multifunctional biological properties. AM affects cerebral blood flow and blood-brain barrier (BBB) function. We studied the role of AM on the permeability and tight junction proteins of brain microvascular endothelial cells (BMEC). BMEC were isolated from rats and a BBB in vitro model was generated. The barrier functions were studied by measuring the transendothelial electrical resistance (TEER) and the permeability of sodium fluorescein and Evans' blue albumin. The expressions of tight junction proteins were analyzed using immunocytochemistry and immunoblotting. AM increased TEER of BMEC monolayer dose-dependently. Immunocytochemistry revealed that AM enhanced the claudin-5 expression at a cell-cell contact site in a dose-dependent manner. Immunoblotting also showed an overexpression of claudin-5 in AM exposure. AM therefore inhibits the paracellular transport in a BBB in vitro model through claudin-5 overexpression.

Transport of Zidovudine-and Lamivudine-Loaded Polybutylcyanoacrylate and Methylmethacrylate-Sulfopropylmethacrylate Nanoparticles across the In Vitro Blood-Brain Barrier: Characteristics of the Drug-D

Transport study of zidovudine (AZT) and lamivudine (3TC) loaded on polybutylcyanoacrylate (PBCA) and methylmethacrylate-sulfopropylmethacrylate (MMA-SPM) nanoparticles (NPs) across the in vitro blood-brain barrier (BBB) model system was presented. Through primary culture, confluent monolayer of bovine brain-microvascular endothelial cells (BBMECs) with the feature of cellular homogeneity and tight intercellular junction was demonstrated by immunocytochemical fluorescent method. Besides, for the colloidal drug-carrier system, average diameters of the two nearly monodispersed PBCA and MMA-SPM nanospheres were, respectively, about 92 nm and 78 nm. The amount of AZT and 3TC across the current BBMEC monolayer in carrier-incorporated system was much higher than that in non-carrier system, suggesting sufficient amelioration in drug transport by employing the NP carriers for brain-targeting delivery. Comparing PBCA NPs with MMA-SPM NPs, the amount of drug (AZT and 3TC) across the in vitro BBB system for the former was in excess of 1.5 times that for the latter.

Th1 and Th2 lymphocyte migration across the human BBB is specifically regulated by interferon β and copolymer-1

Lymphocyte migration into the central nervous system is a central event in lesion formation in MS. Both interferon β (IFNβ) and copolymer-1 (Cop-1) reduce the overall lymphocyte entry into the brain through the blood–brain barrier (BBB) as judged by MRI based studies. In this study, we used a modified Boyden chamber assay in which human brain microvascular endothelial cell (HBEC) monolayers are grown on a fibronectin coated transwell membrane to evaluate in vitro migration of allo-antigen Th1 and Th2 lymphocytes across brain endothelium. We confirmed previous observations showing that migration rates of Th2 lymphocytes across HBECs were higher than migration rates of Th1 cells. When HBECs were pre-treated with IFNβ (100 U/ml) 30 min prior to migration, the migration rate of Th1 was significantly decreased (45% reduction) while the migration of Th2 remained unchanged. Addition of Cop-1 (30 μg/ml) to HBEC monolayers 30 min prior to migration significantly increased the migration rate of Th2 cells and did not affect the migration of Th1 cells. We did not observe any changes in (1) the expression of adhesion molecules on the surface of HBECs and (2) the pattern of chemokine production by HBECs after IFNβ or Cop-1 treatment. The changes in cellular migration rates were not paralleled with changes in diffusion of large molecular weight tracers across brain ECs. Our data support the notion that immuno-modulators used for the treatment of MS selectively and differentially regulate the migration of T helper lymphocyte subsets and that Cop-1 promotes trans-endothelial migration of Th2 cells across the BBB.

In-vitro-Effekte von An�sthetika auf die Blut-Hirn-Schranke

The blood-brain barrier (BBB) forms a selective barrier between blood and brain and regulates the passage of most molecules. Pathological conditions such as ischemia lead to breakdown of the BBB. Vascular endothelial growth factor (VEGF) has been shown to be responsible for hypoxia-induced hyperpermeability of the BBB in vivo as well as in vitro. To eliminate factors which alter the permeability of the BBB in vivo, an in vitro model was used to test the effects of intravenous and volatile anesthetics on the permeability and on VEGF expression during normoxia and hypoxia. The in vitro model of the BBB consisted of primary cultures of porcine brain microvascular endothelial cells (BMEC). The permeability was measured by the paracellular passage of [3H]inulin across the BMEC monolayer and the expression of VEGF was determined by northern blot analysis. All intravenous and volatile anesthetics tested (etomidate, ketamine, fentanyl, propofol, midazolam, sodium-gamma-hydroxybutyrate as well as halothane, enflurane, isoflurane, sevoflurane, desflurane) did not alter the permeability of the BBB or the expression of VEGF in vitro. Hypoxia (2 vol%) increased the permeability and the VEGF expression significantly which was not altered in the presence of the anesthetics. The in vitro model represents a suitable model of the BBB to investigate direct effects of anesthetics on functions of the BBB independent of hemodynamic factors.

AFRICAN TRYPANOSOME INTERACTIONS WITH AN IN VITRO MODEL OF THE HUMAN BLOOD–BRAIN BARRIER

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood-brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly-infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstreamform T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.