Advancing human induced pluripotent stem cell-derived blood-brain barrier models for studying immune cell interactions.

Human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier (BBB) models established to date lack expression of key adhesion molecules involved in immune cell migration across the BBB in vivo. Here, we introduce the extended endothelial cell culture method (EECM), which differentiates hiPSC-derived endothelial progenitor cells to brain microvascular endothelial cell (BMEC)-like cells with good barrier properties and mature tight junctions. Importantly, EECM-BMEC-like cells exhibited constitutive cell surface expression of ICAM-1, ICAM-2, and E-selectin. Pro-inflammatory cytokine stimulation increased the cell surface expression of ICAM-1 and induced cell surface expression of P-selectin and VCAM-1. Co-culture of EECM-BMEC-like cells with hiPSC-derived smooth muscle-like cells or their conditioned medium further increased the induction of VCAM-1. Functional expression of endothelial ICAM-1 and VCAM-1 was confirmed by T-cell interaction with EECM-BMEC-like cells. Taken together, we introduce the first hiPSC-derived BBB model that displays an adhesion molecule phenotype that is suitable for the study of immune cell interactions.

GRP78 antibodies damage the blood-brain barrier and relate to cerebellar degeneration in Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the neuromuscular junction caused by autoantibodies binding to P/Q-type voltage-gated calcium channels. Breakdown of the blood-brain barrier and diffusion of cerebellar granule/Purkinje cell-reactive autoantibodies into the CNS are critical for the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome. We recently found evidence that glucose-regulated protein 78 (GRP78) autoantibodies in the plasma of patients with neuromyelitis optica promote the CNS access of AQP4 autoantibodies. In the present study, we investigated whether the GRP78 autoantibodies in PCD-LEMS IgG boost the brain uptake of cerebellar cell-reactive antibodies across the blood-brain barrier and facilitate cerebellar dysfunction. We first evaluated the effects of purified IgG from PCD-LEMS or PCD patients on the blood-brain barrier function in human brain microvascular endothelial cells using a high content imaging system with nuclear factor κB p65 and intracellular adhesion molecule 1 (ICAM1) immunostaining. Next, we identified GRP78 autoantibodies causing blood-brain barrier permeability in PCD-LEMS IgG by co-immunoprecipitation and the living cell-based antibody binding assays. Exposure of brain microvascular endothelial cells to IgG from PCD-LEMS patients induced nuclear factor κB p65 nuclear translocation, ICAM1 upregulation, reduced claudin-5 expression, increased permeability and increased autocrine IL-1ß and IL-8 secretion; the IgG from patients with Lambert-Eaton myasthenic syndrome did not have these effects. We detected GRP78 autoantibodies in the IgG of LEMS-PCD (83.3%, n = 18), but observed fewer in patients with LEMS (6.6%, n = 15) and none were observed in the control subjects (n = 8). The depletion of GRP78 autoantibodies reduced the biological effect of LEMS-PCD IgG on brain microvascular endothelial cells. These findings suggest that GRP78 autoantibodies play a role beyond neuromyelitis optica and that they have direct implications in the phenotypic differences between PCD-LEMS and LEMS.

Serotonin/5-HT1A Signaling in the Neurovascular Unit Regulates Endothelial CLDN5 Expression.

We previously reported that site-selective claudin-5 (CLDN5) breakdown and protein kinase A (PKA) activation are observed in brain microvessels of schizophrenia, but the underlying molecular basis remains unknown. The 5-HT1 receptors decline the intracellular cAMP levels and inactivate the major downstream PKA, and the 5-HT1A receptor is a promising target for schizophrenia. Therefore, we elucidated the involvement of serotonin/5-HT1A signaling in the endothelial CLDN5 expression. We demonstrate, by immunohistochemistry using post-mortem human brain tissue, that the 5-HT1A receptor is expressed in brain microvascular endothelial cells (BMVECs) and mural cells of the normal prefrontal cortex (PFC) gray matter. We also show that PKA is aberrantly activated not only in BMVECs but also in mural cells of the schizophrenic PFC. We subsequently revealed that the endothelial cell-pericyte tube-like structure was formed in a novel two-dimensional co-culture of human primary BMVECs and a human brain-derived pericyte cell line, in both of which the 5-HT1A receptor was expressed. Furthermore, we disclose that the serotonin/5-HT1A signaling enhances endothelial CLDN5 expression in BMVECs under two-dimensional co-culture conditions. Our findings provide novel insights into the physiological and pathological significance of serotonin/5-HT1A signaling in the region-specific regulation of the blood-brain barrier.

Increased IP-10 production by blood-nerve barrier in multifocal acquired demyelinating sensory and motor neuropathy and multifocal motor neuropathy.

OBJECTIVE: Dysfunction of the blood-nerve barrier (BNB) plays important roles in chronic inflammatory demyelinating polyneuropathy (CIDP) and multifocal motor neuropathy (MMN). The aim of the present study was to identify the candidate cytokines/chemokines that cause the breakdown of the BNB using sera from patients with CIDP and MMN. METHODS: We determined the levels of 27 cytokines and chemokines in human peripheral nerve microvascular endothelial cells (PnMECs) after exposure to sera obtained from patients with CIDP variants (typical CIDP and multifocal acquired demyelinating sensory and motor neuropathy [MADSAM]), MMN and amyotrophic lateral sclerosis (ALS), and healthy controls (HC), using a multiplexed fluorescent bead-based immunoassay system. RESULTS: The induced protein (IP)10 level in the cells in both the MADSAM and MMN groups was markedly increased in comparison with the typical CIDP, ALS and HC groups. The other cytokines, including granulocyte colony-stimulating factor,vascular endothelial growth factor (VEGF) and interleukin-7, were also significantly upregulated in the MADSAM group. The increase of IP-10 produced by PnMECs was correlated with the presence of conduction block in both the MADSAM and MMN groups. CONCLUSION: The autocrine secretion of IP-10 induced by patient sera in PnMECs was markedly upregulated in both the MADSAM and MMN groups. The overproduction of IP-10 by PnMECs leads to the focal breakdown of the BNB and may help to mediate the transfer of pathogenic T cells across the BNB, thereby resulting in the appearance of conduction block in electrophysiological studies of patients with MADSAM and MMN.

Establishment of a new conditionally immortalized human skeletal muscle microvascular endothelial cell line.

In skeletal muscle, the capillaries have tight junctions (TJs) that are structurally similar to those in the blood-brain barrier (BBB) and blood-nerve barrier (BNB). Although many findings have been clarified in the territory of BBB and BNB, few have so far examined the TJs of capillaries in the skeletal muscle. In addition, no in vitro human skeletal muscle microvasculature models have been reported thus far. We newly established a new human skeletal muscle microvascular endothelial cell (HSMMEC) line. HSMMECs were isolated from human skeletal muscle and were infected with retroviruses harboring temperature-sensitive SV40 T antigen and telomerase genes. This cell line, termed TSM15, showed a spindle fiber-shaped morphology, an immunoreactivity to anti-factor VIII and anti-VE-cadherin antibodies, and a temperature-sensitive growth. TSM15 cells grew stably for more than 40 passages when they were cultured at 33°C, thereby retaining their spindle fiber-shaped morphology and contact inhibition at confluence. The cells expressed tight junctional molecules such as claudin-5, occludin, and zonula occludens-1, as well as transporters such as a glucose transporter 1. The transendothelial electrical resistance of TSM15 was as high as those of the human brain microvascular endothelial cell line. This novel cell line might facilitate the analyses of the pathophysiology of inflammatory myopathy, such as dermatomyositis, and can improve our understanding of the physiological and biochemical properties of the microvasculature in human skeletal muscle.

Astrocytes contribute to Aß-induced blood-brain barrier damage through activation of endothelial MMP9.

The blood-brain barrier (BBB) plays an important role in the maintenance of the brain homeostasis, and its proper functions are warranted by the interplay between different cellular components (endothelial cells, astrocytes and pericytes). BBB dysfunctions in pathological conditions, and particularly in Alzheimer's disease, have been documented. Here, using an in vitroBBB model, the interaction between endothelial cells and astrocytes exposed to Aß1-42 was investigated. Human endothelial cells, cultured in monolayer or co-cultured with astrocytes, were exposed to Aß1-42 (2 µM for 18 h). Aß induced dysfunction of endothelial barrier, as assessed by enhanced permeability to FITC-conjugated dextran and reduced expression of claudin-5; these modifications were observed in the co-culture model, but not in endothelial cells cultured in monolayer. Similarly, Aß-induced damage at the barrier was observed when endothelial cells were challenged in the presence of conditioned medium generated by astrocytes previously exposed to Aß (ACM Aß). Endothelial barrier damages were associated with enhanced matrix metalloprotease 9 (MMP9) activity, known to mediate claudin-5 disruption. These events were not related to the direct effects played by Aß on endothelial cells, but they were rather the consequence of Aß-induced vascular endothelial growth factor (VEGF) expression in astrocytes. Indeed, when vascular endothelial growth factor expression was down-regulated in astrocytes, neither barrier properties or MMP9 expression in endothelial cells were affected after Aß exposure both in the co-culture model or in the presence of ACM Aß. These data point out the importance of astrocytes' mediation in inducing endothelial sensitivity to Aß1-42.

Identification of galectin-3 as a possible antibody target for secondary progressive multiple sclerosis.

BACKGROUND: Recent studies have revealed that the disruption of the blood-brain barrier (BBB) might contribute to the induction of neurodegeneration in the progressive stage of multiple sclerosis (MS). OBJECTIVE: We investigated a potential target for the serum auto-antibodies responsible for the BBB impairment in patients with secondary progressive MS (SPMS). METHODS: We identified undetermined target antigens in human brain microvascular endothelial cells (BMECs) that reacted with auto-antibodies in sera from SPMS patients using a proteomic approach. In addition, we examined how the identified auto-antibodies compromise the BBB integrity. RESULTS: We found that 10 of 11 SPMS sera had auto-antibodies against galectin-3, although the patients with other neurological diseases did not have these antibodies. Downregulation of galectin-3 led to elevated intercellular adhesion molecule-1 (ICAM-1) and phospho-nuclear factor-kappa (NFκ) B p65 expression in the BMECs. Exposure to SPMS patients' sera also increased the protein levels of ICAM-1 and phospho-NFκB p65 in BMECs, but these effects induced by anti-galectin-3 immunoreactivity were canceled by the downregulation of galectin-3. CONCLUSION: Galectin-3 is a possible immunological target molecule of the pathogenic auto-antibodies and contributes to the persistent BBB breakdown in patients with SPMS. These antibodies may also serve as a novel biomarker for SPMS.

Pertussis Toxin Exploits Specific Host Cell Signaling Pathways for Promoting Invasion and Translocation of Escherichia coli K1 RS218 in Human Brain-derived Microvascular Endothelial Cells.

Pertussis toxin (PTx), an AB5 toxin and major virulence factor of the whooping cough-causing pathogen Bordetella pertussis, has been shown to affect the blood-brain barrier. Dysfunction of the blood-brain barrier may facilitate penetration of bacterial pathogens into the brain, such as Escherichia coli K1 (RS218). In this study, we investigated the influence of PTx on blood-brain barrier permissiveness to E. coli infection using human brain-derived endothelial HBMEC and TY10 cells as in vitro models. Our results indicate that PTx acts at several key points of host cell intracellular signaling pathways, which are also affected by E. coli K1 RS218 infection. Application of PTx increased the expression of the pathogen binding receptor gp96. Further, we found an activation of STAT3 and of the small GTPase Rac1, which have been described as being essential for bacterial invasion involving host cell actin cytoskeleton rearrangements at the bacterial entry site. In addition, we showed that PTx induces a remarkable relocation of VE-cadherin and ß-catenin from intercellular junctions. The observed changes in host cell signaling molecules were accompanied by differences in intracellular calcium levels, which might act as a second messenger system for PTx. In summary, PTx not only facilitates invasion of E. coli K1 RS218 by activating essential signaling cascades; it also affects intercellular barriers to increase paracellular translocation.

The contribution of suilysin to the pathogenesis of Streptococcus suis meningitis.

BACKGROUND: Streptococcus suis is an emerging zoonotic pathogen, and causes sepsis and meningitis in humans. Although sequence type (ST) 1 and ST104 strains are capable of causing sepsis, ST1 strains commonly cause meningitis. In this study, we investigated the role of suilysin, a member of cholesterol-dependent cytolysins, in differential pathogenicity between ST1 and ST104 strains. METHODS: The levels of transcription and translation of the sly gene and messenger RNA of both ST strains were compared by means of quantitative polymerase chain reaction and Western blotting. Survival rates and bacterial densities in brain were compared between mice infected with wild-type and sly-knockout ST1 strain. ST104 infections with or without complementation of suilysin were also assessed. RESULTS: The amounts of suilysin produced by ST1 strains were much higher than those produced by ST104 strains. Lower production of suilysin by ST104 strains were attributed to the attenuated sly gene expression, which seemed to be associated with 2 nucleotide insertions in sly promoter region. Furthermore, suilysin contributed to the higher bacterial density and enhanced inflammation in brain and increased mortality. CONCLUSIONS: Our data may explain why ST1 strains, but not ST104 strains, commonly cause meningitis and also suggest the contribution of suilysin to the pathogenesis of meningitis in humans.

Autocrine MMP-2/9 secretion increases the BBB permeability in neuromyelitis optica.

OBJECTIVE: Pathological breakdown of the blood-brain barrier (BBB) is thought to constitute the beginning of the disease process in neuromyelitis optica (NMO). In the current study, we investigated possible molecular mechanisms responsible for the breakdown of BBB using NMO sera. METHODS: We analysed the effects of sera obtained from anti-aquaporin 4 (AQP4) antibody-positive NMO spectrum disorder (NMOSD) patients, multiple sclerosis (MS) patients and control subjects on the production of claudin-5, matrix-metalloproteinases (MMPs)-2/9, and vascular cell adhesion protein-1 (VCAM-1) in human brain microvascular endothelial cells (BMECs). We also examined whether immunoglobulin G (IgG) purified from NMOSD sera influences the claudin-5 or VCAM-1 protein expression. RESULTS: The disturbance of BBB properties in BMECs following exposure to NMOSD sera was restored after adding the MMP inhibitor, GM6001. The secretion of MMP-2/9 by BMECs significantly increased after applying the NMOSD sera. The sera from NMOSD patients also increased both the MMP-2/9 secretion and the VCAM-1 protein level by BMECs. The IgG purified from NMOSD sera did not influence the BBB properties or the amount of MMP-2/9 proteins, although it did increase the amount of VCAM-1 proteins in BMECs. Reduction in anti-AQP4 antibody titre was not correlated with a reduction in VCAM-1 expression. CONCLUSIONS: The autocrine secretion of MMP-2/9 by BMECs induced by humoral factors, other than IgG, in sera obtained from NMOSD patients potentially increases BBB permeability. IgG obtained from NMOSD sera, apart from anti-AQP4 antibodies, affect the BBB by upregulating VCAM, thereby facilitating the entry of inflammatory cells into the central nervous system.

Sera from patients with multifocal motor neuropathy disrupt the blood-nerve barrier.

OBJECTIVE: In multifocal motor neuropathy (MMN), the destruction of the blood-nerve barrier (BNB) has been considered to be the key step in the disease process. The purpose of the present study was to ascertain whether sera from patients with MMN can open the BNB, and which component of patient sera is the most important for this disruption. METHODS: We evaluated the effects of sera from patients with MMN, patients with amyotrophic lateral sclerosis, and control subjects on the expression of tight junction proteins and vascular cell adhesion molecule-1 (VCAM-1), and on the transendothelial electrical resistance (TEER) in human peripheral nerve microvascular endothelial cells (PnMECs). RESULTS: The sera from patients with MMN decreased the claudin-5 protein expression and the TEER in PnMECs. However, this effect was reversed after application of an anti-vascular endothelial growth factor (anti-VEGF) neutralising antibody. The VEGF secreted by PnMECs was significantly increased after exposure to the sera from patients with MMN. The sera from patients with MMN also increased the VCAM-1 protein expression by upregulating the nuclear factor kappa-B (NF-κB) signalling. The immunoglobulin G purified from MMN sera decreased the expression of claudin-5 and increased the VCAM-1 expression in PnMECs. CONCLUSIONS: The sera from MMN patients may disrupt the BNB function via the autocrine secretion of VEGF in PnMECs, or the exposure to autoantibodies against PnMECs that are contained in the MMN sera. Autoantibodies against PnMECs in MMN sera may activate the BNB by upregulating the VCAM-1 expression, thereby allowing for the entry of a large number of circulating inflammatory cells into the peripheral nervous system.

Blood-brain barrier destruction determines Fisher/Bickerstaff clinical phenotypes: an in vitro study.

OBJECTIVE: To ascertain the hypothesis that the phenotypic differences between Bickerstaff's brainstem encephalitis (BBE) and Miller Fisher syndrome (MFS) are derived from the differences in the effects of sera on blood-brain barrier (BBB) and blood-nerve barrier. BACKGROUND: Antibodies against GQ1b are frequently detected in BBE and MFS, and these two disorders may share the same pathogenesis, but the clinical phenotypes of BBE and MFS are substantially different. METHODS: The effects of sera obtained from BBE patients, MFS patients and control subjects were evaluated with regard to the expression of tight junction proteins and transendothelial electrical resistance in human brain microvascular endothelial cells (BMECs) and human peripheral nerve microvascular endothelial cells. RESULTS: The sera obtained from BBE patients decreased the transendothelial electrical resistance values and claudin-5 protein expression in BMECs, although the sera obtained from MFS patients had no effect on BMECs or peripheral nerve microvascular endothelial cells. This effect was reversed after the application of matrix metalloproteinase (MMP) inhibitor, GM6001. The presence or absence of anti-GQ1b antibodies did not significantly influence the results. MMP-9 secreted by BMECs was significantly increased after exposure to the sera obtained from BBE patients, whereas it was not changed after exposure to the sera obtained from MFS patients. CONCLUSIONS: Only the sera obtained from BBE patients destroyed BBB and it might explain the phenotypical differences between BBE and MFS. BBE sera disrupted BBB, possibly via the autocrine secretion of MMP-9 from BBB-composing endothelial cells.

Sequential changes in the non-coding control region sequences of JC polyomaviruses from the cerebrospinal fluid of patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is caused by JC polyomavirus (JCV) infection in the brain. JCV isolates from PML patients have variable mutations in the non-coding control region (NCCR) of the genome. This study was conducted to examine sequential changes in NCCR patterns of JCV isolates obtained from the cerebrospinal fluid (CSF) of PML patients. CSF specimens were collected from PML patients at different time points, the NCCR sequences were determined, and their compositions were assessed by computer-based analysis. In patients showing a marked increase in JCV load, the most frequent NCCR sequences in the follow-up specimens were different from those in the initial samples. In contrast, the dominant NCCRs in the CSF remained unaltered during the follow-up of individuals in whom the viral load decreased after therapeutic intervention. These data demonstrate that the majority of JCV variants emerge with the progression of PML and that these changes are suppressed when the viral load is decreased.

Autocrine TNF-α Increases Penetration of Myelin-Associated Glycoprotein Antibodies Across the Blood-Nerve Barrier in Anti-MAG Neuropathy.

BACKGROUND AND OBJECTIVES: Deposition of myelin-associated glycoprotein (MAG) immunoglobulin M (IgM) antibodies in the sural nerve is a key feature in anti-MAG neuropathy. Whether the blood-nerve barrier (BNB) is disrupted in anti-MAG neuropathy remains elusive.We aimed to evaluate the effect of sera from anti-MAG neuropathy at the molecular level using our in vitro human BNB model and observe the change of BNB endothelial cells in the sural nerve of anti-MAG neuropathy. METHODS: Diluted sera from patients with anti-MAG neuropathy (n = 16), monoclonal gammopathies of undetermined significance (MGUS) neuropathy (n = 7), amyotrophic lateral sclerosis (ALS, n = 10), and healthy controls (HCs, n = 10) incubated with human BNB endothelial cells to identify the key molecule of BNB activation using RNA-seq and a high-content imaging system, and exposed with a BNB coculture model to evaluate small molecule/IgG/IgM/anti-MAG antibody permeability. RESULTS: RNA-seq and the high-content imaging system showed the significant upregulation of tumor necrosis factor (TNF-α) and nuclear factor-kappa B (NF-κB) in BNB endothelial cells after exposure to sera from patients with anti-MAG neuropathy, whereas the serum TNF-α concentration was not changed among the MAG/MGUS/ALS/HC groups. Sera from patients with anti-MAG neuropathy did not increase 10-kDa dextran or IgG permeability but enhanced IgM and anti-MAG antibody permeability. Sural nerve biopsy specimens from patients with anti-MAG neuropathy showed higher TNF-α expression levels in BNB endothelial cells and preservation of the structural integrity of the tight junctions and the presence of more vesicles in BNB endothelial cells. Neutralization of TNF-α reduces IgM/anti-MAG antibody permeability. DISCUSSION: Sera from individuals with anti-MAG neuropathy increased transcellular IgM/anti-MAG antibody permeability via autocrine TNF-α secretion and NF-κB signaling in the BNB.

Establishment and characterization of human peripheral nerve microvascular endothelial cell lines: a new in vitro blood-nerve barrier (BNB) model.

The blood-nerve barrier (BNB) is a highly specialized unit that maintains the microenvironments of the peripheral nervous system. Since the breakdown of the BNB has been considered a key step in autoimmune neuropathies such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyraduculoneuropathy, it is important to understand the cellular properties of the peripheral nerve microvascular endothelial cells (PnMECs) which constitute the BNB. For this purpose, we established an immortalized cell line derived from human PnMECs. The human PnMECs were transduced with retroviral vectors encoding the temperature-sensitive SV40 large T antigen and human telomerase. This cell line, termed FH-BNB, showed a spindle fiber-shaped morphology, expression of von Willebrand factor and uptake of acetylated low density lipoprotein. These cells expressed tight junction proteins including occludin, claudin-5, ZO-1 and ZO-2 at the cell-cell boundaries. P-glycoprotein and GLUT-1 were also detected by a Western blot analysis and the cells exhibited the functional expression of p-glycoprotein. In addition, transendothelial electrical resistance experiments and paracellular permeabilities of sodium fluorescein and fluorescein isothiocyanate-labeled dextran of molecular weight 4 kDa across these cells demonstrated that FH-BNBs had functional tight junctions. These results indicated that FH-BNBs had highly specialized barrier properties and they might therefore be a useful tool to analyze the pathophysiology of various neuropathies.

Sera from neuromyelitis optica patients disrupt the blood-brain barrier.

OBJECTIVE: In neuromyelitis optica (NMO), the destruction of the blood-brain barrier (BBB) has been considered to be the first step of the disease process. It is unclear whether sera from patients with NMO can open the BBB, and which component of patient sera is most important for this disruption. METHODS: The effects of sera from antiaquaporin4 (AQP4) antibody positive NMO patients, multiple sclerosis patients and control subjects were evaluated for expression of tight junction proteins and for transendothelial electrical resistance (TEER) of human brain microvascular endothelial cells (BMECs). Whether antibodies against human BMECs as well as anti-AQP4 antibodies exist in NMO sera was also examined using western blot analysis. RESULTS: Expression of tight junction proteins and TEER in BMECs was significantly decreased after exposure to NMO sera. However, this effect was reversed after application of an antivascular endothelial growth factor (VEGF) neutralising antibody. Antibodies against BMECs other than anti-AQP4 antibodies were found in the sera of NMO patients whereas no specific bands were detected in the sera of healthy and neurological controls. These antibodies apparently disrupt the BBB by increasing the autocrine secretion of VEGF by BMECs themselves. Absorption of the anti-AQP4 antibody by AQP4 transfected astrocytes reduced AQP4 antibody titres but was not associated with a reduction in BBB disruption. CONCLUSIONS: Sera from NMO patients reduce expression of tight junction proteins and disrupt the BBB. Autoantibodies against BMECs other than anti-AQP4 antibodies may disrupt the BBB through upregulation of VEGF in BMECs.

Pericyte-derived glial cell line-derived neurotrophic factor increase the expression of claudin-5 in the blood-brain barrier and the blood-nerve barrier.

The destruction of blood-brain barrier (BBB) and blood-nerve barrier (BNB) has been considered to be a key step in the disease process of a number of neurological disorders including cerebral ischemia, Alzheimer's disease, multiple sclerosis, and diabetic neuropathy. Although glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) facilitate neuronal or axonal regeneration in the brain or peripheral nerves, their action in the BBB and BNB remains unclear. The purpose of the present study was to elucidate whether these neurotrophic factors secreted from the brain or peripheral nerve pericytes increase the barrier function of the BBB or BNB, using our newly established human brain microvascular endothelial cell (BMEC) line or peripheral nerve microvascular endothelial cell (PnMEC) line. GDNF increased the expression of claudin-5 and the transendothelial electrical resistance (TEER) of BMECs and PnMECs, whereas BDNF did not have this effect. Furthermore, we herein demonstrate that the GDNF secreted from the brain and peripheral nerve pericytes was one of the key molecules responsible for the up-regulation of claudin-5 expression and the TEER value in the BBB and BNB. These results indicate that the regulation of GDNF secreted from pericytes may therefore be a novel therapeutic strategy to modify the BBB or BNB functions and promote brain or peripheral nerve regeneration.

[An 11 year old woman with myelin-oligodendrocyte glycoprotein antibody showing various phenotypes of central nervous system disorders in one year].

An 11-year-old woman with myelin-oligodendrocyte glycoprotein (MOG) antibody developed cortical encephalitis twice, followed by acute disseminated encephalomyelitis (ADEM) and optic neuritis in one year. Although optic neuritis was refractory after corticosteroid therapy, plasma exchange was effective and complete remission was achieved. We considered that episodes of cortical encephalitis, ADEM and optic neuritis occurred in the present patient can be included in MOG IgG-associated disorders. Also, we recommend plasma exchange for refractory MOG IgG-associated optic neuritis, even in pediatric patient.

Decreased Astrocytic CCL2 Accounts for BAF-312 Effect on PBMCs Transendothelial Migration Through a Blood Brain Barrier in Vitro Model.

Disruption of the blood brain barrier (BBB) is a common event in several neurological diseases and in particular, in multiple sclerosis (MS), it contributes to the infiltration of the central nervous system by peripheral inflammatory cells. Sphingosine-1-phosphate (S1P) is a bioactive molecule with pleiotropic effects. Agonists of S1P receptors such as fingolimod and siponimod (BAF-312) are in clinical practice for MS and have been shown to preserve BBB function in inflammatory conditions. Using an in vitro BBB model of endothelial-astrocytes co-culture exposed to an inflammatory insult (tumor necrosis factor-α and interferon-γ; T&I), we show that BAF-312 reduced the migration of peripheral blood mononuclear cells (PBMCs) through the endothelial layer, only in the presence of astrocytes. This effect was accompanied by decreased expression of the adhesion molecule ICAM-1. BAF-312 also reduced the activation of astrocytes, by controlling NF-kB and NLRP3 induction and preventing the increase of proinflammatory cytokine and chemokines. Reduction of CCL2 by BAF-312 may be responsible for the observed effects and, accordingly, addition of exogenous CCL2 was able to counteract BAF-312 effects and rescued T&I responses on PBMC migration, ICAM-1 expression and astrocyte activation. The present results further point out BAF-312 effects on BBB properties, suggesting also the key role of astrocytes in mediating drug effects on endothelial function.