Blood-brain barrier modeling with co-cultured neural progenitor cell-derived astrocytes and neurons.

In vitro blood-brain barrier (BBB) models often consist of brain microvascular endothelial cells (BMECs) that are co-cultured with other cells of the neurovascular unit, such as astrocytes and neurons, to enhance BBB properties. Obtaining primary astrocytes and neurons for co-culture models can be laborious, while yield and heterogeneity of primary isolations can also be limiting. Neural progenitor cells (NPCs), because of their self-renewal capacity and ability to reproducibly differentiate into tunable mixtures of neurons and astrocytes, represent a facile, readily scalable alternative. To this end, differentiated rat NPCs were co-cultured with rat BMECs and shown to induce BBB properties such as elevated trans-endothelial electrical resistance, improved tight junction continuity, polarized p-glycoprotein efflux, and low passive permeability at levels indistinguishable from those induced by primary rat astrocyte co-culture. An NPC differentiation time of 12 days, with the presence of 10% fetal bovine serum, was found to be crucial for generating NPC-derived progeny capable of inducing the optimal response. This approach could also be extended to human NPC-derived astrocytes and neurons which similarly regulated BBB induction. The distribution of rat or human NPC-derived progeny under these conditions was found to be a roughly 3 : 1 mixture of astrocytes to neurons with varying degrees of cellular maturity. BMEC gene expression analysis was conducted using a BBB gene panel, and it was determined that 23 of 26 genes were similarly regulated by either differentiated rat NPC or rat astrocyte co-culture while three genes were differentially altered by the rat NPC-derived progeny. Taken together, these results demonstrate that NPCs are an attractive alternative to primary neural cells for use in BBB co-culture models.

Regulatory T cells exhibit enhanced migratory characteristics, a feature impaired in patients with multiple sclerosis.

Migration of immune cells characterizes inflammation and plays a key role in autoimmune diseases such as MS. CD4(+)Foxp3(+) regulatory T cells (Treg) have the potential to dampen immune responses but show functional impairment in patients with MS. We here show that murine Treg exhibit higher constitutive cell motility in horizontal migration on laminin, surpass non-Treg in transwell assays through microporous membranes as well as across primary brain endothelium and are present in the naïve CNS to a significantly higher extent compared to spleen, lymph nodes and blood. Likewise, human Treg from healthy donors significantly exceed non-Treg in migratory rates across primary human brain endothelium. Finally, we investigated whether the propensity to migrate is impaired as a feature of autoimmunity and therefore tested patients with MS. Treg from patients with stable relapsing-remitting MS show significantly impaired migratory capacity under non-inflammatory conditions compared to healthy donors. We hypothesize that the enhanced propensity to migrate is a feature of Treg that allows for an equilibrium in parenchymal immune surveillance, e.g. of the CNS. Impaired Treg migration across the intact blood-brain barrier, as observed for Treg from patients with MS, indicates a broader functional deficiency hypothetically contributing to early CNS lesion development or phases of MS remissions.

Long-Term Assessment of Lurasidone in Schizophrenia: Post Hoc Analysis of a 12-Month, Double Blind, Active-Controlled Trial and 6-Month Open-Label Extension Study.

INTRODUCTION: A post hoc analysis of a double-blind (DB) active control trial and an open-label extension (OLE) study was conducted to evaluate the long-term effects of lurasidone in patients with schizophrenia. METHODS: In the DB trial, patients were randomised to receive lurasidone or risperidone for 12 months. In OLE, all patients received lurasidone for an additional 6 months. Treatment-emergent adverse events (TEAEs) were evaluated. Efficacy assessments included relapse rate (DB trial only), and Positive and Negative Syndrome Scale, Clinical Global Impression-Severity scale, and Montgomery-Åsberg Depression Rating Scale. RESULTS: In the DB trial, patients with schizophrenia were randomised to lurasidone (n = 399) and risperidone (n = 190), of whom 129 and 84 continued into OLE, respectively. During the DB trial, incidence of TEAEs was similar for lurasidone (84.1%) and risperidone (84.2%). Lurasidone was associated with minimal changes in metabolic variables and prolactin levels, whereas risperidone was associated with clinically significant increases in prolactin and fasting glucose levels. The proportion of patients with metabolic syndrome was significantly lower in patients treated with lurasidone versus risperidone at the end of the DB trial (25.5% vs 40.4%; p = 0.0177). During OLE, patients switching from risperidone to lurasidone experienced a reduction in weight and prolactin levels; those continuing treatment with lurasidone experienced minimal changes in metabolic variables and prolactin. At the end of OLE, the proportion of patients with metabolic syndrome was no longer significantly different between groups (23.5% vs 31.5%; p = not significant). Efficacy outcomes were generally similar between groups during the DB trial, and were maintained during OLE. CONCLUSION: Lurasidone was generally well tolerated and effective in clinically stable schizophrenia patients over the long term. Lurasidone was also generally well tolerated and maintained effectiveness over 6 months in patients switching from risperidone. Patients switching from risperidone experienced improvements in metabolic parameters and prolactin levels. These findings confirm lurasidone's long-term effectiveness and favourable metabolic profile in patients with schizophrenia. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT00641745.

Intracerebral dendritic cells critically modulate encephalitogenic versus regulatory immune responses in the CNS.

Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation associated with autoimmune disorders, such as multiple sclerosis, but the contribution of these cells to the outcome of disease is not yet clear. Here, we show that stimulatory or tolerogenic functional states of intracerebral DCs regulate the systemic activation of neuroantigen-specific T cells, the recruitment of these cells into the CNS and the onset and progression of experimental autoimmune encephalomyelitis (EAE). Intracerebral microinjection of stimulatory DCs exacerbated the onset and clinical course of EAE, accompanied with an early T-cell infiltration and a decreased proportion of regulatory FoxP3-expressing cells in the brain. In contrast, the intracerebral microinjection of DCs modified by tumor necrosis factor alpha induced their tolerogenic functional state and delayed or prevented EAE onset. This triggered the generation of interleukin 10 (IL-10)-producing neuroantigen-specific lymphocytes in the periphery and restricted IL-17 production in the CNS. Our findings suggest that DCs are a rate-limiting factor for neuroinflammation.

Specific central nervous system recruitment of HLA-G(+) regulatory T cells in multiple sclerosis.

OBJECTIVE: We have recently described a novel population of natural regulatory T cells (T(reg)) that are characterized by the expression of HLA-G and may be found at sites of tissue inflammation (HLA-G(pos) T(reg)). Here we studied the role of these cells in multiple sclerosis (MS), a prototypic autoimmune inflammatory disorder of the central nervous system (CNS). METHODS: Sixty-four patients with different types of MS, 9 patients with other neurological diseases, and 20 healthy donors were included in this study. Inflamed brain lesions from 5 additional untreated MS patients were examined. HLA-G(pos) T(reg) were analyzed in the cerebrospinal fluid (CSF) by flow cytometry and in inflammatory demyelinating lesions of MS brain specimens by immunohistochemistry. Functional capacity was accessed and transmigration was determined using an in vitro model of the human blood-brain barrier (BBB). RESULTS: HLA-G(pos) T(reg) were found enriched in the inflamed CSF of MS patients and in inflammatory demyelinating lesions of MS brain specimens. HLA-G(pos) T(reg) showed a strong propensity to transmigrate across BBB, which was vigorously driven by inflammatory chemokines, and associated with a gain of suppressive capacity upon transmigration. CSF-derived HLA-G(pos) T(reg) of MS patients represented a population of activated central memory activated T cells with an upregulated expression of inflammatory chemokine receptors and exhibiting full suppressive capacity. Unlike natural FoxP3-expressing T(reg), HLA-G(pos) T(reg) derived from peripheral blood were functionally unimpaired in MS. INTERPRETATION: In MS, HLA-G(pos) T(reg) may serve to control potentially destructive immune responses directly at the sites of CNS inflammation and to counterbalance inflammation once specifically recruited to the CNS.

Pericyte-endothelial cell interaction increases MMP-9 secretion at the blood-brain barrier in vitro.

The extracellular matrix (ECM) connecting brain capillary endothelial cells (BCEC) with the surrounding brain resident cells is an essential part of the blood-brain barrier (BBB). Represented by the basement membrane which joins BCEC with brain neuroglia (astrocytes and pericytes) it forms a neurovascular unit. Neuroglia-secreted matrix metalloproteinases (MMPs) control the ECM composition and are involved in the integrity and function of the BBB during cell diapedesis and BBB breakdown after ischemia and other CNS diseases. We examined the involvement of pericytes and astrocytes in endothelial MMP secretion and their effect on endothelial barrier properties in a primary cell culture model using porcine BCEC. We applied puromycin to eliminate pericyte growth and demonstrated a significant (to about 30%) reduction of endothelial MMP-9 production in pericyte-free cultures. In contrast, BCEC co-culture with pericytes resulted in an increased amount of endothelial MMP-9 and active MMPs measured by both zymography and fluorimetric assay. Astrocyte co-culture in a filter setup with BCEC allowing a cell-cell signaling via soluble factors revealed significantly reduced endothelial MMP activity. These data were directly correlated with improved BBB integrity under pericyte elimination and astrocyte co-culture conditions as indicated by transendothelial electrical resistance (TEER) values. Our data define pericyte interactions as a main inducer of endothelial MMP secretion and propose a new role for pericyte-endothelial cell crosstalk at the BBB in vitro.

Murine brain capillary endothelial cells exhibit improved barrier properties under the influence of hydrocortisone.

Hydrocortisone is known to induce barrier properties in porcine primary cultures of microvascular endothelial cells. Here we present similar effects of hydrocortisone on a serum-free in vitro model based on primary cultured mouse brain endothelial cells. These cells in culture express typical blood-brain barrier properties and the transendothelial electrical resistance is enhanced after the addition of hydrocortisone to the medium in physiological concentrations. The improvement of the barrier is accompanied by changes at the cell borders indicated by immunofluorescence staining of tight junction proteins. Transmission electron microscopy imaging indicates morphological changes at the cell-cell contact zones which correlates to the observed changes in the transendothelial electrical resistance after HC supplementation. Phalloidin staining of F-actin shows a rearrangement to "fiber-like" structures in the longitudinal direction of the cell. These findings together with additional electrical impedance analysis of the monolayer suggest that several changes including cell-cell contact alteration, cell-substrate attachment and cytoskeletal rearrangements cause enhanced barrier properties in this murine endothelial culture. The present data are consistent with earlier findings in a porcine serum-free in vitro model. Thus, evidence is given that the barrier enforcement induced by glucocorticoids is not a species-specific effect and that the barrier improvement is correlated with a change of the cell morphology rather than changes in tight junction protein expression.

T cell suppression by naturally occurring HLA-G-expressing regulatory CD4+ T cells is IL-10-dependent and reversible.

CD4(+) T cells constitutively expressing the immune-tolerogenic HLA-G have been described recently as a new type of nT(reg) (HLA-G(pos) T(reg)) in humans. HLA-G(pos) T(reg) accumulate at sites of inflammation and are potent suppressors of T cell proliferation in vitro, suggesting their role in immune regulation. We here characterize the mechanism of how CD4(+) HLA-G(pos) T(reg) influence autologous HLA-G(neg) T(resp) function. Using a suppression system free of APC, we demonstrate a T-T cell interaction, resulting in suppression of HLA-G(neg) T(resp), which is facilitated by TCR engagement on HLA-G(pos) T(reg). Suppression is independent of cell-cell contact and is reversible, as the removal of HLA-G(pos) T(reg) from the established coculture restored the proliferative capability of responder cells. Further, HLA-G(pos) T(reg)-mediated suppression critically depends on the secretion of IL-10 but not TGF-beta.

A beta-lactam antibiotic dampens excitotoxic inflammatory CNS damage in a mouse model of multiple sclerosis.

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial "Excitatory Amino Acid Transporters" (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a beta-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in "Myelin Oligodendrocyte Glycoprotein" (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFgamma and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a beta-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.

Differentiating embryonic neural progenitor cells induce blood-brain barrier properties.

The blood-brain barrier (BBB) is a multifunctional endothelial interface separating the bloodstream from the brain interior. Although the mature BBB is well characterized, the embryonic development of this complex system remains poorly understood. Embryonic neural progenitor cells (NPC) are a potential inductive cell type populating the developing brain, and their ability to influence BBB properties was therefore examined. When puromycin-purified brain microvascular endothelial cells (BMEC) were co-cultured with embryonic NPC in a two-compartment Transwell system, the BMEC exhibited enhanced barrier properties in the form of increased transendothelial electrical resistance (TEER) and decreased permeability to the small molecule tracer, sodium fluorescein. These changes required the presence of NPC in the early stages of differentiation and were accompanied by alterations in the fidelity of BMEC tight junctions as indicated by occludin, claudin 5, and zonula occluden-1 redistribution at cell-cell borders. In contrast to the findings with NPC, post-natal astrocytes elicited a delayed, but longer duration response in BMEC TEER. BMEC co-culture also suppressed neuronal differentiation of NPC indicating a reciprocal signaling between the two cell populations. This study demonstrates that NPC-BMEC interactions are prevalent and for the first time demonstrates that NPC are capable of inducing BBB properties.

Puromycin-purified rat brain microvascular endothelial cell cultures exhibit improved barrier properties in response to glucocorticoid induction.

In vitro blood-brain barrier (BBB) models using primary rat brain microvessel endothelial cells (BMEC) are often hampered by a lack of culture purity and poor barrier properties. To address these problems, the translation inhibitor puromycin was used to purify rat BMEC cultures. BMEC purities of 99.8% were routinely attained using puromycin treatment, and this technique proved to be far superior to other purification methods of similar difficulty. In contrast to cultures without puromycin treatment, purity of puromycin-treated cultures was unaffected by initial seeding density. Next, rat BMEC monolayer transendothelial electrical resistance (TEER) was increased by glucocorticoid treatment with either corticosterone (CORT) or hydrocortisone (HC), and a corresponding decrease in monolayer permeability to small molecules was observed. Importantly, cultures treated with both puromycin and glucocorticoid attained significantly higher TEER values (CORT 168 +/- 13 Omega x cm2; HC 218 +/- 66 Omega x cm2) than those treated by the glucocorticoid alone (CORT 57 +/- 5 Omega x cm2; HC 70 +/- 2 Omega x cm2). Glucocorticoid induction resulted in BMEC morphological changes that accompanied the increases in TEER, and BMEC tight junctions exhibited improved integrity as visualized by the localization of tight junction proteins zonula occluden-1, occludin and claudin-5. The combined use of puromycin and glucocorticoid therefore provides an in vitro system that is well suited for molecular level BBB investigations.

Influence of hydrocortisone on the mechanical properties of the cerebral endothelium in vitro.

Cerebral endothelial cells accomplish the barrier functions between blood and brain interstitium. Structural features are the tight junctions between adjacent endothelial cells and the formation of marginal folds at the cell-cell contacts. The glucocorticoid hydrocortisone (HC) has been reported to enforce the blood-brain-barrier in vitro measurable by an increase of the transendothelial electrical resistance. This study shows the impact of HC on the mechanical and morphological properties of confluent cell layers of brain microvascular endothelial cells. HC induces an increase in height of these marginal folds and a reduction of the intercellular contact surface. These morphological changes are accompanied by changes in cell elasticity. Staining of fibrous actin indicates that HC induces a reorganization of the actin cortex. The quantitative determination of the local elastic properties of cells reveals for the first time an HC-induced increase of the representative Young's modulus according to cytoskeletal rearrangements. For this study, cells of two different species, porcine brain capillary endothelial cells and murine brain capillary endothelial cells, were used yielding similar results, which clearly demonstrates that the HC effect on the cell elasticity is species independent.

Individual subunits of the glutamate transporter EAAC1 homotrimer function independently of each other.

Glutamate transporters are thought to be assembled as trimers of identical subunits that line a central hole, possibly the permeation pathway for anions. Here, we have tested the effect of multimerization on the transporter function. To do so, we coexpressed EAAC1(WT) with the mutant transporter EAAC1(R446Q), which transports glutamine but not glutamate. Application of 50 microM glutamate or 50 microM glutamine to cells coexpressing similar numbers of both transporters resulted in anion currents of 165 and 130 pA, respectively. Application of both substrates at the same time generated an anion current of 297 pA, demonstrating that the currents catalyzed by the wild-type and mutant transporter subunits are purely additive. This result is unexpected for anion permeation through a central pore but could be explained by anion permeation through independently functioning subunits. To further test the subunit independence, we coexpressed EAAC1(WT) and EAAC1(H295K), a transporter with a 90-fold reduced glutamate affinity as compared to EAAC1(WT), and determined the glutamate concentration dependence of currents of the mixed transporter population. The data were consistent with two independent populations of transporters with apparent glutamate affinities similar to those of EAAC1(H295K) and EAAC1(WT), respectively. Finally, we coexpressed EAAC1(WT) with the pH-independent mutant transporter EAAC1(E373Q), showing two independent populations of transporters, one being pH-dependent and the other being pH-independent. In conclusion, we propose that EAAC1 assembles as trimers of identical subunits but that the individual subunits in the trimer function independently of each other.