Title Participants Abstract "Procarboxypeptidase U (proCPU, TAFI, proCPB2) in cerebrospinal fluid during ischemic stroke is associated with stroke progression, outcome and blood-brain barrier dysfunction" "Joachim C Mertens, Dorien Leenaerts, Raf Brouns, Sebastiaan Engelborghs, Margareta Ieven, Peter De Deyn, Anne-Marie Lambeir, Dirk Hendriks" "Essentials Little is known of procarboxypeptidase U (proCPU) in cerebrospinal fluid (CSF) of stroke patients. ProCPU levels were studied in CSF of controls and non-thrombolyzed acute ischemic stroke patients. ProCPU is elevated in CSF of stroke patients compared with controls. ProCPU in CSF correlates with stroke progression, outcome, and blood-brain barrier dysfunction. Summary: Background Procarboxypeptidase U (proCPU, TAFI, proCPB2), the zymogen of CPU, which is a potent antifibrinolytic enzyme and a modulator of inflammation, has previously been investigated in plasma of stroke patients, but so far, no information on the proCPU levels in cerebrospinal fluid (CSF) during acute ischemic stroke (AIS) is available. Objectives This case–control observational study investigates proCPU in CSF of AIS patients compared with controls with an intact blood–brain barrier (BBB) and evaluates the relationship of CSF/plasma proCPU ratios with stroke parameters. Methods A sensitive HPLC-based enzymatic assay was used to determine proCPU levels in CSF of non-thrombolyzed patients in the hyperacute phase (< 24 h after onset) of AIS (n = 72). Individuals (n = 32) without stroke, an intact BBB and no apparent abnormalities in biochemical and microbiological tests, served as controls. Relations between the CSF/plasma proCPU ratio and (i) stroke severity, (ii) stroke progression/recurrence, (iii) stroke outcome and (iv) BBB dysfunction (CSF/serum albumin ratio) were assessed. Results Mean (SEM) proCPU levels were elevated in the CSF of stroke patients compared with controls (4.36 (0.23) U L −1 vs. 3.50 (0.23) U L −1). Higher median [IQR] CSF/plasma proCPU ratios were found in patients with stroke progression ((6.0 [4.2–6.9]) × 10 −3) and poor outcome ((6.4 [3.9–7.0]) × 10 −3) after 3 months (modified Rankin Scale; mRS > 3) compared with patients without progression ((3.9 [2.7–5.4]) × 10 −3) or better outcome ((4.0 [2.8–5.0]) × 10 −3). In stroke patients with a disrupted BBB, proCPU ratios were higher compared with stroke patients with an intact BBB ((6.4 [5.8–9.0]) × 10 −3 vs. (3.7 [2.8–5.0]) × 10 −3). Conclusions ProCPU is increased in CSF during hyperacute ischemic stroke and is associated with stroke progression and outcome after 3 months, most likely due to BBB dysfunction in the hyperacute phase of ischemic stroke." "Glial regulation of the blood-brain barrier in health and disease" "Bieke BROUX, Elizabeth Gowing, Alexandre Prat" "The brain is the organ with the highest metabolic demand in the body. Therefore, it needs specialized vasculature to provide it with the necessary oxygen and nutrients, while protecting it against pathogens and toxins. The blood-brain barrier (BBB) is very tightly regulated by specialized endothelial cells, two basement membranes, and astrocytic endfeet. The proximity of astrocytes to the vessel makes them perfect candidates to influence the function of the BBB. Moreover, other glial cells are also known to contribute to either BBB quiescence or breakdown. In this review, we summarize the knowledge on glial regulation of the BBB during development, in homeostatic conditions in the adult, and during neuroinflammatory responses." "To what extent do cell-penetrating peptides selectively cross the blood-brain barrier?" "Sofie Stalmans" "The blood-brain barrier protects the brain from toxic compounds. Its selective permeability is essential for the optimal function of the central nervous system. Some peptides can cross the blood-brain barrier. On the other hand, cell-penetrating peptides are able to overcome the cell membrane. During this research project, it was investigated whether these cell-penetrating peptides also can cross the blood-brain barrier. The chemical diversity of the already reported cell-penetrating peptides was investigated and a unified response for the extent of cellular uptake of peptides was introduced. Based on this study, a set of cell-penetrating peptides was rationally selected for further research. In order to more objectively compare the quantitative data on the blood-brain barrier influx of peptides, a classification system for blood-brain barrier influx was established. The purity of the selected synthetized cell-penetrating peptides was also investigated, which is essential for obtaining reliable research conclusions. Different chromatographic systems were compared for the analysis of the selected peptides. The investigated cell-penetrating peptides crossed the blood-brain barrier to a different extent. The influx varied from very low to very high and some peptides showed efflux out of the brain. There was no correlation observed between the blood-brain barrier transport kinetics and the extent of cellular uptake. During the aging process, the blood-brain barrier shows an increased permeability and, together with other age-related functional changes, should be taken into account during the development of medicines used by the elderly. Therefore, the current regulatory status of the development of geriatric medicines was investigated." "Using microdialysis to analyse the passage of monovalent nanobodies through the blood-brain barrier" "Guy Caljon, Vicky Caveliers, Tony Lahoutte, Benoit Stijlemans, Gholamreza Hassanzadeh Ghassabeh, Jan Van Den Abbeele, Ilse Smolders, Patrick De Baetselier, Yvette Michotte, Serge Muyldermans, Stefan Magez, Ralph Clinckers" "BACKGROUND AND PURPOSE: Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes because of their favourable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the CNS remains to be demonstrated.EXPERIMENTAL APPROACH: We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei variant-specific surface glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, single photon emission computed tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabelled and (99m) Tc-labelled nanobodies using, respectively, a sensitive VSG-based nanobody-detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis.KEY RESULTS: The combined read-out methodologies showed that Nb_An33 was detected in the brain of healthy rats following i.v. injection, inflammation-induced damage to the blood-brain barrier, as in the late encephalitic stage of trypanosomiasis, significantly increased the efficiency of passage of the nanobody through this barrier. Complementing SPECT analyses with intracerebral microdialysis improved analysis of brain disposition. There is clear value in assessing penetration of the blood-brain barrier by monovalent nanobodies in models of CNS inflammation. Our data also suggest that rapid clearance from blood might hamper efficient targeting of specific nanobodies to the CNS.CONCLUSIONS AND IMPLICATIONS: Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood-brain barrier integrity is compromised." "Blood-Brain Barrier transport kinetics of the neuromedin peptides NMU, NMN, NMB and NT." "Bert Gevaert, Evelien Wynendaele, Sofie Stalmans, Nathalie Bracke, Matthias D'hondt, Ilse Smolders, Ann Van Eeckhaut, Bart De Spiegeleer" "The neuromedin peptides are peripherally and centrally produced, but until now, it is generally believed that they only function as locally acting compounds without any quantitative knowledge about their blood-brain barrier (BBB) passage. Here, we characterize the transport kinetics of four neuromedins (NMU, NMN, NMB and NT) across the BBB, as well as their metabolization profile, and evaluate if they can act as endocrine hormones. Using the in vivo mouse model, multiple time regression (MTR), capillary depletion (CD) and brain efflux studies were performed. Data was fitted using linear (NMU, NT and NMB) or biphasic modeling (NMU and NMN). Three of the four investigated peptides, i.e. NMU, NT and NMN, showed a significant influx into the brain with unidirectional influx rate constants of 1.31 and 0.75 μL/(g × min) for NMU and NT respectively and initial influx constants (K1) of 72.14 and 7.55 μL/(g × min) and net influx constants (K) of 1.28 and 1.36 x 10(-16) μL/(g x min) for NMU and NMN respectively. The influx of NMB was negligible. Only NMN and NT showed a significant efflux out of the brain with an efflux constant (kout) of 0.042 min(-1) and -0.053 min(-1) respectively. Our results indicate that locally produced neuromedin peptides and/or fragments can be transported through the whole body, including passing the BBB, and taken up by different organs/tissues, supporting the idea that the neuromedins could have a much bigger role in the regulation of biological processes than currently assumed." "Blood-brain barrier transport kinetics of NOTA-modified proteins : the somatropin case" "Nathalie Bracke, Yorick Janssens, Evelien Wynendaele, Liesa Tack, Alex Maes, Christophe Van De Wiele, Mike Sathekge, Bart De Spiegeleer" "BACKGROUND: Chemical modifications such as PEG, polyamine and radiolabeling on proteins can alter their pharmacokinetic behaviour and their blood-brain barrier (BBB) transport characteristics. NOTA, i.e. 1,4,7-triazacyclononane-1,4,7-triacetic acid, is a bifunctional chelating agent that has attracted the interest of the scientific community for its high complexation constant with metals like gallium. Until now, the comparative BBB transport characteristics of NOTA-modified proteins versus unmodified proteins are not yet described. METHODS: Somatropin (i.e. recombinant human growth hormone), NOTA-conjugated somatropin and gallium-labelled NOTA-conjugated somatropin were investigated for their brain penetration characteristics (multiple time regression and capillary depletion) in an in vivo mice model to determine the blood-brain transfer properties. RESULTS: The three compounds showed comparable initial brain influx, with Kin = 0.38 ± 0.14 µL/(g×min), 0.36 ± 0.16 µL/(g×min) and 0.28 ± 0.18 µL/(g×min), respectively. Capillary depletion indicated that more than 80% of the influxed compounds reached the brain parenchyma. All three compounds were in vivo stable in serum and brain during the time frame of the experiments. CONCLUSIONS: Our results show that modification of NOTA as well as gallium chelation onto proteins, in casu somatropin, does not lead to a significantly changed pharmacokinetic profile at the blood-brain barrier." "Skin, mucosal and blood-brain barrier kinetics of model cyclic depsipeptides : the mycotoxins beauvericin and enniatins" "Lien Taevernier" "This research takes off by giving an introduction to the wonderful world of the chemically very diverse cyclic depsipeptides (CDPs). This family of compounds is biosynthetically produced by an extreme variety of natural organisms, such as bacteria and fungi, which can be found all over the world. CDPs exhibit multiple biological activities, serving as interesting drug leads for the development of medicines. For the first time a classification system for CDPs was introduced. Some CDPs synthesised by fungi, however, can also be toxic and are potentially harmful. In scientific literature, there is a lack of a clear definition for these so-called mycotoxins, although very important for risk analyses. Therefore, we presented a new, quantitative expressed U+2018mycotoxinU+2019 definition, by means of the philosophical concept of explication. Recently, beauvericin and enniatins are recognised as U+2018emergingU+2019 mycotoxins. Exposure to these CDPs should not be considered as trivial, given their potential biological effects and presence as food and feed contaminants. Subsequent to the successful development of a bioanalytical method for the determination of beauvericin and enniatins in diverse matrices, their kinetic transport across the skin, oral mucosal and blood-brain barrier was quantitatively evaluated. Finally, a pharmaceutical preparation, containing enniatins as active ingredient and used in the treatment of upper respiratory tract infections, was investigated. Because of the negative risk-benefit ratio, we questioned its use in these innocent respiratory infections. For the same reason, the European Medicines Agency (EMA) has very recently endorsed the recall of these medicines from the European market." "Liver X Receptor Alpha Is Important in Maintaining Blood-Brain Barrier Function" "Elien WOUTERS, Nienke M. de Wit, Jasmine VANMOL, Susanne M. A. van der Pol, Bert van Het Hof, Daniela SOMMER, Melanie LOIX, Dirk Geerts, Jan Ake Gustafsson, Knut R. Steffensen, Tim VANMIERLO, Jeroen BOGIE, Jerome HENDRIKS, Helga E. de Vries" "Dysfunction of the blood-brain barrier (BBB) contributes significantly to the pathogenesis of several neuroinflammatory diseases, including multiple sclerosis (MS). Potential players that regulate BBB function are the liver X receptors (LXRs), which are ligand activated transcription factors comprising two isoforms, LXR alpha, and LXR beta. However, the role of LXR alpha, and LXR beta in regulating BBB (dys)function during neuroinflammation remains unclear, as well as their individual involvement. Therefore, the goal of the present study is to unravel whether LXR isoforms have different roles in regulating BBB function under neuroinflammatory conditions. We demonstrate that LXR alpha, and not LXR beta, is essential to maintain barrier integrity in vitro. Specific knockout of LXR alpha in brain endothelial cells resulted in a more permeable barrier with reduced expression of tight junctions. Additionally, the observed dysfunction was accompanied by increased endothelial inflammation, as detected by enhanced expression of vascular cell adhesion molecule (VCAM-1) and increased transendothelial migration of monocytes toward inflammatory stimuli. To unravel the importance of LXR alpha in BBB function in vivo, we made use of the experimental autoimmune encephalomyelitis (EAE) MS mouse model. Induction of EAE in a constitutive LXR alpha knockout mouse and in an endothelial specific LXR alpha knockout mouse resulted in a more severe disease score in these animals. This was accompanied by higher numbers of infiltrating leukocytes, increased endothelial VCAM-1 expression, and decreased expression of the tight junction molecule claudin-5. Together, this study reveals that LXR alpha is indispensable for maintaining BBB integrity and its immune quiescence. Targeting the LXR alpha isoform may help in the development of novel therapeutic strategies to prevent BBB dysfunction, and thereby neuroinflammatory disorders." "Imaging blood-brain barrier dysfunction : a state-of-the-art review from a clinical perspective" "Paulien Moyaert, Beatriz E Padrela, Catherine A Morgan, Jan Petr, Jan Versijpt, Frederik Barkhof, Michael T Jurkiewicz, Xingfeng Shao, Olujide Oyeniran, Tabitha Manson, Danny J J Wang, Matthias Günther, Eric Achten, Henk J M M Mutsaerts, Udunna C Anazodo" "The blood-brain barrier (BBB) consists of specialized cells that tightly regulate the in- and outflow of molecules from the blood to brain parenchyma, protecting the brain's microenvironment. If one of the BBB components starts to fail, its dysfunction can lead to a cascade of neuroinflammatory events leading to neuronal dysfunction and degeneration. Preliminary imaging findings suggest that BBB dysfunction could serve as an early diagnostic and prognostic biomarker for a number of neurological diseases. This review aims to provide clinicians with an overview of the emerging field of BBB imaging in humans by answering three key questions: (1. Disease) In which diseases could BBB imaging be useful? (2. Device) What are currently available imaging methods for evaluating BBB integrity? And (3. Distribution) what is the potential of BBB imaging in different environments, particularly in resource limited settings? We conclude that further advances are needed, such as the validation, standardization and implementation of readily available, low-cost and non-contrast BBB imaging techniques, for BBB imaging to be a useful clinical biomarker in both resource-limited and well-resourced settings." "Oncostatin M triggers brain inflammation by compromising blood-brain barrier integrity" "Doryssa HERMANS, Evelien HOUBEN, Paulien BAETEN, Helena Slaets, Kris JANSSENS, Cindy HOEKS, Baharak HOSSEINKHANI, Gayel DURAN, Seppe BORMANS, Elizabeth Gowing, Chloe Hoornaert, Lien BECKERS, Wing Ka Fung, Horst Schroten, Hiroshi Ishikawa, Judith FRAUSSEN, Ronald THOELEN, Helga E. de Vries, Gijs Kooij, Stephanie Zandee, Alexandre Prat, Niels HELLINGS, Bieke BROUX" "Oncostatin M (OSM) is an IL-6 family member which exerts neuroprotective and remyelination-promoting effects after damage to the central nervous system (CNS). However, the role of OSM in neuro-inflammation is poorly understood. Here, we investigated OSM's role in pathological events important for the neuro-inflammatory disorder multiple sclerosis (MS). We show that OSM receptor (OSMR beta) expression is increased on circulating lymphocytes of MS patients, indicating their elevated responsiveness to OSM signalling. In addition, OSM production by activated myeloid cells and astrocytes is increased in MS brain lesions. In experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, OSMR beta-deficient mice exhibit milder clinical symptoms, accompanied by diminished T helper 17 (Th17) cell infiltration into the CNS and reduced BBB leakage. In vitro, OSM reduces BBB integrity by downregulating the junctional molecules claudin-5 and VE-cadherin, while promoting secretion of the Th17-attracting chemokine CCL20 by inflamed BBB-endothelial cells and reactive astrocytes. Using flow cytometric fluorescence resonance energy transfer (FRET) quantification, we found that OSM-induced endothelial CCL20 promotes activation of lymphocyte function-associated antigen 1 (LFA-1) on Th17 cells. Moreover, CCL20 enhances Th17 cell adhesion to OSM-treated inflamed endothelial cells, which is at least in part ICAM-1 mediated. Together, these data identify an OSM-CCL20 axis, in which OSM contributes significantly to BBB impairment during neuro-inflammation by inducing permeability while recruiting Th17 cells via enhanced endothelial CCL20 secretion and integrin activation. Therefore, care should be taken when considering OSM as a therapeutic agent for treatment of neuro-inflammatory diseases such as MS."