Receptor-mediated transcytosis is one of the major routes for drug delivery of large molecules into the brain. The aim of this study was to develop a novel model of the human blood-brain barrier ...(BBB) in a high-throughput microfluidic device. This model can be used to assess passage of large biopharmaceuticals, such as therapeutic antibodies, across the BBB.
The model comprises human cell lines of brain endothelial cells, astrocytes, and pericytes in a two-lane or three-lane microfluidic platform that harbors 96 or 40 chips, respectively, in a 384-well plate format. In each chip, a perfused vessel of brain endothelial cells was grown against an extracellular matrix gel, which was patterned by means of surface tension techniques. Astrocytes and pericytes were added on the other side of the gel to complete the BBB on-a-chip model. Barrier function of the model was studied using fluorescent barrier integrity assays. To test antibody transcytosis, the lumen of the model's endothelial vessel was perfused with an anti-transferrin receptor antibody or with a control antibody. The levels of antibody that penetrated to the basal compartment were quantified using a mesoscale discovery assay.
The perfused BBB on-a-chip model shows presence of adherens and tight junctions and severely limits the passage of a 20 kDa FITC-dextran dye. Penetration of the antibody targeting the human transferrin receptor (MEM-189) was markedly higher than penetration of the control antibody (apparent permeability of 2.9 × 10
versus 1.6 × 10
cm/min, respectively).
We demonstrate successful integration of a human BBB microfluidic model in a high-throughput plate-based format that can be used for drug screening purposes. This in vitro model shows sufficient barrier function to study the passage of large molecules and is sensitive to differences in antibody penetration, which could support discovery and engineering of BBB-shuttle technologies.
The blood-brain barrier (BBB) is the brain-specific endothelial cell barrier that is important for maintaining brain homeostasis and preventing the entry of toxic substances. Pathological BBB ...dysfunction is a critical step of the disease process in several immuno-mediated neurological diseases, including multiple sclerosis (MS), neuromyelitis optica (NMO), neuropsychiatric systemic lupus erythematosus (NPSLE) and neuro-Behçet diseases. The pathological findings from patients with secondary progressive (SP) MS, NMO and NPSLE showed leaky BBB in the active lesions. NMO is a disease with strong evidence of disease-specific and pathogenic autoantibodies (aquaporin 4 AQP4 autoantibodies). In the development of NMO, circulating AQP4 autoantibodies need to pass through the BBB in order to reach AQP4 on the astrocyte endfeet. Strong evidence suggests that NPSLE is associated with the disruption of the BBB and NPSLE patients frequently have antibodies bound to endothelial cells in their sera. We recently identified two BBB-reactive autoantibodies in immuno-mediated neurological diseases: galectin-3 autoantibodies in SPMS and GRP78 autoantibodies in NMO. In the present review article, we describe the basic structure and cellular biology of the BBB, discuss recent insights regarding the pathophysiology of the BBB breakdown in the setting of immuno-mediated neurological diseases, and describe our recent findings of autoantibody-mediated BBB breakdown.
Blood–brain barrier (BBB) dysfunction is a key feature in neuroimmunological and neurodegenerative diseases. In this study, we developed a microfluidic human BBB-on-a-chip to model barrier ...dysfunction and immune cell migration using immortalized TY10 brain endothelial cells, pericytes, and astrocytes. It was found that immortalized TY10 brain endothelial cells developed a microvascular structure under flow. Pericytes were localized on the basal side surrounding the TY10 microvascular structure, showing an in vivo-like structure. Barrier integrity increased under co-culture with pericytes. In addition, both ethylenediaminetetraacetic acid (EDTA) and anti-Claudin-5 (CLDN5) neutralizing antibody caused a decrease in the transendothelial electrical resistance (TEER). EDTA caused the leakage of 20 kDa dextran, suggesting different effects on the BBB based on the mechanism of action, whereas anti-CLDN5 antibody did not cause leakage. In the tri-culture model, human T cells migrated through endothelial vessels towards basal C-X-C motif chemokine ligand 12 (CXCL12). The live-imaging analysis confirmed the extravasation of fluorescence-labelled T cells in a CXCL12-concentration- and time-dependent manner. Our BBB model had an in vivo-like structure and successfully represented barrier dysfunction and transendothelial T cell migration. In addition, our study suggests that the inhibition of CLDN5 attenuates the BBB in humans. This platform has various potential uses in relation to the BBB in both drug discovery research and in elucidating the mechanisms of central nervous system diseases.
Background
A previous comparative study in Japan has demonstrated that the two consecutive UroVysion tests are useful tools to detect the presence of bladder cancer during follow-up after ...transurethral resection, but they also presented their high rates of false-positive results. Here, we aimed to evaluate the relationship between the UroVysion tests and subsequent intravesical recurrence.
Methods
In the previous study, patients without bladder cancer during the first analysis showed the same examination set repeated 3 months later as the second analysis. In this follow-up study, 326 patients showed negative findings confirmed on cystoscopy during the second UroVysion test. Recurrence-free survival was assessed using a median follow-up of 27 months.
Results
In the two consecutive UroVysion tests, 214 patients (65.6%) showed negative UroVysion results in both tests, whereas 91 presented a positive result on either tests and 21 patients presented positive results in both tests. During the follow-up, 40 patients (12.3%) had an intravesical recurrence with non-muscle-invasive bladder cancer. The recurrence rates in patients with negative results in both tests, those with one positive result in either tests, and those with positive results in both tests were 8.4%, 16.5%, and 33.3%, respectively. The multivariate analysis indicated that the history of bladder cancer and the consecutive UroVysion test pattern were independent risk factors for recurrence.
Conclusions
Our data confirmed the effectiveness of two consecutive UroVysion tests in predicting intravesical recurrence after TURBT. Further prospective studies would help determine an appropriate interval for cystoscopy follow-up.
Objective
To investigate the frequency of serum autoantibodies targeting contactin‐associated protein 1 (Caspr1) and its complexes with other paranode antigens, contactin‐1 (CNTN1) and neurofascin ...155 (NF155), in Japanese patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).
Methods
Sera from 26 CIDP patients, 35 patients with Guillain–Barré syndrome, and 31 healthy individuals participated. Paranodal immunoglobulin G antibodies were quantified using enzyme‐linked immunosorbent assays with commercially available recombinant proteins as antigens.
Results
Anti‐Caspr1 antibodies were present in one participant (case 1, 3.8%) of CIDP, and negative in all Guillain–Barré syndrome patients and healthy participant. Case 1 was a man who developed subacute distal limb‐predominant muscle weakness and sensory ataxia with postural hand tremor at 69 years‐of‐age, and therapeutic benefit of intravenous immunoglobulin and oral steroids was inadequate. The detected anti‐Caspr1 antibodies predominantly belonged to the immunoglobulin G4 subclass, and the addition of CNTN1 to Caspr1 as an antigen increased antibody reactivity, although the increase was just 20–30% at most. The presence of autoantibodies against paranode protein complexes, including Caspr1/CNTN1, Caspr1/NF155 and Caspr1/CNTN1/NF155, was confirmed in several CIDP patients, although they also had anti‐Caspr1 or anti‐NF155 antibodies; thus, in our cohort, there were no patients with autoantibodies that specifically recognized paranode protein complexes.
Conclusions
This is the first confirmed Japanese case of anti‐Caspr1 antibody‐positive CIDP with a clinical signature similar to that of patients of Western origin. Our preliminary study did not identify the presence of specific antibodies against the paranode protein complexes, and the primary target antigen is likely Caspr1.
The first Japanese case of anti‐contactin‐associated protein 1 antibody‐positive chronic inflammatory demyelinating polyradiculoneuropathy showed similar clinical pictures to cases reported from Western countries, including enlargement of the nerve roots with gadolinium contrast effect.
Neuroinflammation and brain lipid imbalances are observed in Alzheimer's disease (AD). Tumor necrosis factor-α (TNFα) and the liver X receptor (
) signaling pathways are involved in both processes. ...However, limited information is currently available regarding their relationships in human brain pericytes (HBP) of the neurovascular unit. In cultivated HBP, TNFα activates the
pathway and increases the expression of one of its target genes, the transporter ATP-binding cassette family A member 1 (
), while
is not expressed. Apolipoprotein E (
) synthesis and release are diminished. The cholesterol efflux is promoted, but is not inhibited, when
or
are blocked. Moreover, as for TNFα, direct
activation by the agonist (T0901317) increases
expression and the associated cholesterol efflux. However, this process is abolished when
/
are both inhibited. Neither the other ABC transporters nor the SR-BI are involved in this TNFα-mediated lipid efflux regulation. We also report that inflammation increases
expression and function. In conclusion, our data suggest that inflammation increases HBP protection against xenobiotics and triggers an
/
independent cholesterol release. Understanding the molecular mechanisms regulating this efflux at the level of the neurovascular unit remains fundamental to the characterization of links between neuroinflammation, cholesterol and HBP function in neurodegenerative disorders.
We describe here the design and implementation of an
in vitro
microvascular open model system using human brain microvascular endothelial cells. The design has several advantages over other ...traditional closed microfluidic platforms: (1) it enables controlled unidirectional flow of media at physiological rates to support vascular function, (2) it allows for very small volumes which makes the device ideal for studies involving biotherapeutics, (3) it is amenable for multiple high resolution imaging modalities such as transmission electron microscopy (TEM), 3D live fluorescence imaging using traditional spinning disk confocal microscopy, and advanced lattice light sheet microscopy (LLSM). Importantly, we miniaturized the design, so it can fit within the physical constraints of LLSM, with the objective to study physiology in live cells at subcellular level. We validated barrier function of our brain microvessel-on-a-chip by measuring permeability of fluorescent dextran and a human monoclonal antibody. One potential application is to investigate mechanisms of transcytosis across the brain microvessel-like barrier of fluorescently-tagged biologics, viruses or nanoparticles.
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.
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.
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.
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.
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