Claudin-12 is not required for blood-brain barrier tight junction function.

BACKGROUND The blood-brain barrier (BBB) ensures central nervous system (CNS) homeostasis by strictly controlling the passage of molecules and solutes from the bloodstream into the CNS. Complex and continuous tight junctions (TJs) between brain endothelial cells block uncontrolled paracellular diffusion of molecules across the BBB, with claudin-5 being its dominant TJs protein. However, claudin-5 deficient mice still display ultrastructurally normal TJs, suggesting the contribution of other claudins or tight-junction associated proteins in establishing BBB junctional complexes. Expression of claudin-12 at the BBB has been reported, however the exact function and subcellular localization of this atypical claudin remains unknown. METHODS We created claudin-12-lacZ-knock-in C57BL/6J mice to explore expression of claudin-12 and its role in establishing BBB TJs function during health and neuroinflammation. We furthermore performed a broad standardized phenotypic check-up of the mouse mutant. RESULTS Making use of the lacZ reporter allele, we found claudin-12 to be broadly expressed in numerous organs. In the CNS, expression of claudin-12 was detected in many cell types with very low expression in brain endothelium. Claudin-12lacZ/lacZ C57BL/6J mice lacking claudin-12 expression displayed an intact BBB and did not show any signs of BBB dysfunction or aggravated neuroinflammation in an animal model for multiple sclerosis. Determining the precise localization of claudin-12 at the BBB was prohibited by the fact that available anti-claudin-12 antibodies showed comparable detection and staining patterns in tissues from wild-type and claudin-12lacZ/lacZ C57BL/6J mice. CONCLUSIONS Our present study thus shows that claudin-12 is not essential in establishing or maintaining BBB TJs integrity. Claudin-12 is rather expressed in cells that typically lack TJs suggesting that claudin-12 plays a role other than forming classical TJs. At the same time, in depth phenotypic screening of clinically relevant organ functions of claudin-12lacZ/lacZ C57BL/6J mice suggested the involvement of claudin-12 in some neurological but, more prominently, in cardiovascular functions

Multicentric Atrial Strain COmparison between Two Different Modalities: MASCOT HIT Study

Two methods are currently available for left atrial (LA) strain measurement by speckle tracking echocardiography, with two different reference timings for starting the analysis: QRS (QRS-LASr) and P wave (P-LASr). The aim of MASCOT HIT study was to define which of the two was more reproducible, more feasible, and less time consuming. In 26 expert centers, LA strain was analyzed by two different echocardiographers (young vs senior) in a blinded fashion. The study population included: healthy subjects, patients with arterial hypertension or aortic stenosis (LA pressure overload, group 2) and patients with mitral regurgitation or heart failure (LA volume–pressure overload, group 3). Difference between the inter-correlation coefficient (ICC) by the two echocardiographers using the two techniques, feasibility and analysis time of both methods were analyzed. A total of 938 subjects were included: 309 controls, 333 patients in group 2, and 296 patients in group 3. The ICC was comparable between QRS-LASr (0.93) and P-LASr (0.90). The young echocardiographers calculated QRS-LASr in 90% of cases, the expert ones in 95%. The feasibility of P-LASr was 85% by young echocardiographers and 88% by senior ones. QRS-LASr young median time was 110 s (interquartile range, IR, 78-149) vs senior 110 s (IR 78-155); for P-LASr, 120 s (IR 80-165) and 120 s (IR 90-161), respectively. LA strain was feasible in the majority of patients with similar reproducibility for both methods. QRS complex guaranteed a slightly higher feasibility and a lower time wasting compared to the use of P wave as the reference

Étude in vitro du rôle de la barrière hémato-encéphalique dans les pathologies cérébrales à caractère inflammatoire (développement d'un modèle de barrière hémato-encéphalique chez la souris)

Les cellules endothéliales (CE) des capillaires cérébraux, support anatomique de la barrière hémato-encéphalique (BHE), présentent des caractéristiques structurales et métaboliques restreignant les échanges sang/cerveau. Les pathologies cérébrales s'accompagnent d'une rupture de la BHE, ayant pour conséquence une augmentation de la perméabilité de l'endothélium aux molécules plasmatiques et aux cellules sanguines. Dans l'étude de ces phénomènes, nous avons développé un modèle syngénique de BHE in vitro chez la souris, qui consiste en une coculture de CE de capillaires cérébraux et de cellules gliales (CG). Les CE montrent des caractéristiques de BHE in vivo: expression et localisation périphérique des protéines des jonctions serrées, expression et activité biologique de la P-glycoprotéine. La perméabilité de la monocouche de CE à divers composés a été évaluée et reproduit les données in vivo. Le recrutement des cellules immunitaires vers le SNC implique l'expression de molécules d'adhérence à la surface des CE. Nous observons ICAM-1, ICAM-2 et VCAM-1 à la surface des CE et, comme in vivo, une augmentation de l'expression d'ICAM-1 et de VCAM-1 par des agents inflammatoires est rencontrée. Une controverse subsiste quant à l'implication des sélectines. A l'état basal, les CE de notre modèle n'expriment ni E- ni P-sélectine. Par contre, selon le stimulus inflammatoire, une modulation différentielle de l'expression des sélectines est observée. Ce modèle syngénique de BHE in vitro chez la souris reproduit au plus proche la situation in vivo et s'avèrera un outil précieux dans l'étude du rôle de l'endothélium cérébral dans les phénomènes inflammatoires affectant le SNC.Blood-brain exchanges are regulated by the blood-brain barrier (BBB). Highly specialized endothelial cells form the BBB and restrict paracellular diffusion of molecules, while selective transports occur using receptor or transporter-mediated transcytosis. Central nervous system (CNS) disorders are associated with BBB breakdown which leads to the increase in BBB permeability to blood-borne molecules and immune cells. We developed a mouse syngenic in vitro BBB model, consisting in a primary coculture of mouse brain capillary endothelial cells (MBCECs) and glial cells. MBCECs exhibit in vivo BBB properties (cell border localisation of tight junction proteins, P-glycoprotein expression and efficiency). compound permeability through MBCEC monolayer was performed and compared to in vivo data. As immune cell recruitment across the BBB involves cell surface molecules. We investigated the expression of ICAM-1, ICAM-2 and VCAM-1 on MBCECs and demonstrated cell surface upregulation of ICAM-1 and VCAM-1 during inflammatory events. In vivo, a controversy remains concerning the role of selectins. MBCECs were shown to be negative for E and P-selectins under basal condition, while a differential expression of selectins is observed under inflammatory stimuli. This well-characterised mouse in vitro BBB model which mimics in vivo situation will be a valuable tool to study BBB permeability changes during CNS inflammatory events.LENS-BU Sciences (624982102) / SudocSudocFranceF

Tight junctions in brain barriers during central nervous system inflammation

Homeostasis within the central nervous system (CNS) is a prerequisite to elicit proper neuronal function. The CNS is tightly sealed from the changeable milieu of the blood stream by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB). Whereas the BBB is established by specialized endothelial cells of CNS microvessels, the BCSFB is formed by the epithelial cells of the choroid plexus. Both constitute physical barriers by a complex network of tight junctions (TJs) between adjacent cells. During many CNS inflammatory disorders, such as multiple sclerosis, human immunodeficiency virus infection, or Alzheimer's disease, production of pro-inflammatory cytokines, matrix metalloproteases, and reactive oxygen species are responsible for alterations of CNS barriers. Barrier dysfunction can contribute to neurological disorders in a passive way by vascular leakage of blood-borne molecules into the CNS and in an active way by guiding the migration of inflammatory cells into the CNS. Both ways may directly be linked to alterations in molecular composition, function, and dynamics of the TJ proteins. This review summarizes current knowledge on the cellular and molecular aspects of the functional and dysfunctional TJ complexes at the BBB and the BCSFB, with a particular emphasis on CNS inflammation and the role of reactive oxygen species

Preclinical testing of strategies for therapeutic targeting of human T-cell trafficking in vivo

Naive T cells are migratory cells that continuously recirculate between blood and lymphoid tissues. Antigen-specific stimulation of T cells within the lymph nodes reprograms the trafficking properties of T cells by inducing a specific set of adhesion molecules and chemokine receptors on their surface which allow these activated and effector T cells to effectively and specifically home to extralymphoid organs. The observations of organ-specific homing of T cells initiated the development of therapeutic strategies targeting adhesion receptors for organ-specific inhibition of chronic inflammation. As most adhesion receptors have additional immune functions besides mediating leukocyte trafficking, these drugs may have additional immunomodulatory effects. Therapeutic targeting of T-cell trafficking to the central nervous system is the underlying concept of a novel treatment of relapsing remitting multiple sclerosis with the humanized anti-alpha-4-integrin antibody natalizumab. In this chapter, we describe a possible preclinical in vivo approach to directly visualize the therapeutic efficacy of a given drug in inhibiting T-cell homing to a certain organ at the example of the potential of natalizumab to inhibit the trafficking of human T cells to the inflamed central nervous system in an animal model of multiple sclerosis

Live cell imaging techniques to study T cell trafficking across the blood-brain barrier <it>in vitro</it> and <it>in vivo</it>

<p>Abstract</p> <p>Background</p> <p>The central nervous system (CNS) is an immunologically privileged site to which access for circulating immune cells is tightly controlled by the endothelial blood–brain barrier (BBB) located in CNS microvessels. Under physiological conditions immune cell migration across the BBB is low. However, in neuroinflammatory diseases such as multiple sclerosis, many immune cells can cross the BBB and cause neurological symptoms. Extravasation of circulating immune cells is a multi-step process that is regulated by the sequential interaction of different adhesion and signaling molecules on the immune cells and on the endothelium. The specialized barrier characteristics of the BBB, therefore, imply the existence of unique mechanisms for immune cell migration across the BBB.</p> <p>Methods and design</p> <p>An <it>in vitro</it> mouse BBB model maintaining physiological barrier characteristics in a flow chamber and combined with high magnification live cell imaging, has been established. This model enables the molecular mechanisms involved in the multi-step extravasation of T cells across the <it>in vitro</it> BBB, to be defined with high-throughput analyses. Subsequently these mechanisms have been verified <it>in vivo</it> using a limited number of experimental animals and a spinal cord window surgical technique. The window enables live observation of the dynamic interaction between T cells and spinal cord microvessels under physiological and pathological conditions using real time epifluorescence intravital imaging. These <it>in vitro</it> and <it>in vivo</it> live cell imaging methods have shown that the BBB endothelium possesses unique and specialized mechanisms involved in the multi-step T cell migration across this endothelial barrier under physiological flow. The initial T cell interaction with the endothelium is either mediated by T cell capture or by T cell rolling. Arrest follows, and then T cells polarize and especially CD4⁺ T cells crawl over long distances against the direction of flow to find the rare sites permissive for diapedesis through the endothelium.</p> <p>Discussion</p> <p>The sequential use of <it>in vitro</it> and <it>in vivo</it> live cell imaging of T cells interacting with the BBB allows us to delineate the kinetics and molecular determinants involved in multistep extravasation of encephalitogenic T cells across the BBB.</p

Therapeutic targeting of leukocyte trafficking across the blood-brain barrier

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS not to disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses are mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly regulating immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier control immune cell entry into the CNS, which is rare under physiological conditions. During a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis (MS), immunocompetent cells readily traverse the BBB and subsequently enter the CNS parenchyma. Most of our current knowledge on the molecular mechanisms involved in immune cell entry into the CNS has been derived from studies performed in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Thus, a large part of our current knowledge on immune cell entry across the BBBs is based on the results obtained in this animal model. Similarly, knowledge on the benefits and potential risks associated with therapeutic targeting of immune cell recruitment across the BBB in human diseases are mostly derived from such treatment regimen in MS. Other mechanisms of immune cell entry into the CNS might therefore apply under different pathological conditions such as bacterial meningitis or stroke and need to be considered

Cutting edge: Natalizumab blocks adhesion but not initial contact of human T cells to the blood-brain barrier in vivo in an animal model of multiple sclerosis

The humanized anti-alpha(4) integrin Ab Natalizumab is an effective treatment for relapsing-remitting multiple sclerosis. Natalizumab is thought to exert its therapeutic efficacy by blocking the alpha(4) integrin-mediated binding of circulating immune cells to the blood-brain barrier (BBB). As alpha(4) integrins control other immunological processes, natalizumab may, however, execute its beneficial effects elsewhere. By means of intravital microscopy we demonstrate that natalizumab specifically inhibits the firm adhesion but not the rolling or capture of human T cells on the inflamed BBB in mice with acute experimental autoimmune encephalomyelitis (EAE). The efficiency of natalizumab to block T cell adhesion to the inflamed BBB was found to be more effective in EAE than in acute systemic TNF-alpha-induced inflammation. Our data demonstrate that alpha(4) integrin-mediated adhesion of human T cells to the inflamed BBB during EAE is efficiently blocked by natalizumab and thus provide the first direct in vivo proof of concept of this therapy in multiple sclerosis

Comparison of immortalized bEnd5 and primary mouse brain microvascular endothelial cells as in vitro blood–brain barrier models for the study of T cell extravasation

Important insights into the molecular mechanism of T cell extravasation across the blood–brain barrier (BBB) have already been obtained using immortalized mouse brain endothelioma cell lines (bEnd). However, compared with bEnd, primary brain endothelial cells have been shown to establish better barrier characteristics, including complex tight junctions and low permeability. In this study, we asked whether bEnd5 and primary mouse brain microvascular endothelial cells (pMBMECs) were equally suited as in vitro models with which to study the cellular and molecular mechanisms of T cell extravasation across the BBB. We found that both in vitro BBB models equally supported both T cell adhesion under static and physiologic flow conditions, and T cell crawling on the endothelial surface against the direction of flow. In contrast, distances of T cell crawling on pMBMECs were strikingly longer than on bEnd5, whereas diapedesis of T cells across pMBMECs was dramatically reduced compared with bEnd5. Thus, both in vitro BBB models are suited to study T cell adhesion. However, because pMBMECs better reflect endothelial BBB specialization in vivo, we propose that more reliable information about the cellular and molecular mechanisms of T cell diapedesis across the BBB can be attained using pMBMECs