Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

β1-integrin/matrix interactions support blood-brain barrier integrity

Brain microvascular endothelium forms an active permeability barrier, the blood-brain barrier (BBB). In neurologic disorders, barrier properties of the BBB are often lost indicating their dependance on molecular cues of the brain microenvironment. In this issue, Osada et al demonstrate that the endothelial extracellular matrix (ECM) provides one of these cues. Their study shows that β1-integrin-mediated adhesion of brain endothelial cells to the surrounding ECM is critical for stabilizing claudin-5 in BBB tight junctions (TJs) and BBB integrity. These observations point to a novel intracellular signaling pathway from β1-integrin/ECM endothelial adhesions to BBB TJs contributing to BBB integrity.

TET inducible expression of the α4β7-integrin ligand MAdCAM-1 on the blood-brain barrier does not influence the immunopathogenesis of experimental autoimmune encephalomyelitis

Inhibiting the α4 subunit of the integrin heterodimers α4β1 and α4β7 with the mab natalizumab is an effective treatment of multiple sclerosis (MS). Which of the two α4 heterodimers is involved in disease pathogenesis has, however, remained controversial. Whereas the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, is ameliorated in β7-integrin-deficient C57BL/6 mice, neutralizing antibodies against the β7-integrin subunit or the α4β7-integrin heterodimer fail to interfere with EAE pathogenesis in the SJL mouse. To facilitate α4β7-integrin-mediated immune-cell trafficking across the blood-brain barrier (BBB), we established transgenic C57BL/6 mice with endothelial cell-specific, inducible expression of the α4β7-integrin ligand mucosal addressin cell adhesion molecule (MAdCAM)-1 using the tetracycline (TET)-OFF system. Although TET-regulated MAdCAM-1 induced α4β7-integrin mediated interaction of α4β7(+) /α4β1(-) T cells with the BBB in vitro and in vivo, it failed to influence EAE pathogenesis in C57BL/6 mice. TET-regulated MAdCAM-1 on the BBB neither changed the localization of central nervous system (CNS) perivascular inflammatory cuffs nor did it enhance the percentage of α4β7-integrin(+) inflammatory cells within the CNS during EAE. In conclusion, our study demonstrates that ectopic expression of MAdCAM-1 at the BBB does not increase α4β7-integrin-mediated immune cell trafficking into the CNS during MOG(aa35-55)-induced EAE.

Review: leucocyte-endothelial cell crosstalk at the blood-brain barrier: a prerequisite for successful immune cell entry to the brain

Leucocyte migration into the central nervous system is a key stage in the development of multiple sclerosis. While much has been learnt regarding the sequential steps of leucocyte capture, adhesion and migration across the vasculature, the molecular basis of leucocyte extravasation is only just being unravelled. It is now recognized that bidirectional crosstalk between the immune cell and endothelium is an essential element in mediating diapedesis during both normal immune surveillance and under inflammatory conditions. The induction of various signalling networks, through engagement of cell surface molecules such as integrins on the leucocyte and immunoglobulin superfamily cell adhesion molecules on the endothelial cell, play a major role in determining the pattern and route of leucocyte emigration. In this review we discuss the extent of our knowledge regarding leucocyte migration across the blood-brain barrier and in particular the endothelial cell signalling pathways contributing to this process.

S-0 and S-1 State Structure, Methyl Torsional Barrier Heights, and Fast Intersystem Crossing Dynamics of 5-Methyl-2-hydroxypyrimidine

We report the analysis of the SI So rotational band contours of jet-cooled 5-methyl-2-hydroxypyrimidine (5M2HP), the enol form of deoxythymine. Unlike thymine, which exhibits a structureless spectrum, the vibronic spectrum of 5M2HP is well structured, allowing us to determine the rotational constants and the methyl group torsional barriers in the S-0 and S-1 states. The 0(0)(0), 6a(0)(1), 6b(0)(1), and 14(0)(1) band contours were measured at 900 MHz (0.03 cm(-1)) resolution using mass-specific two-color resonant two-photon ionization (2C-R2PI) spectroscopy. All four bands are polarized perpendicular to the pyrimidine plane (>90% c type), identifying the S-1 <- S-0 excitation of 5M2HP as a 1n pi* transition. All contours exhibit two methyl rotor subbands that arise from the lowest 5-methyl torsional states 0A '' and 1E ''. The S-0 and S-1 state torsional barriers were extracted from fits to the torsional subbands. The 3-fold barriers are V-3 '' = 13 cm(-1) and V3' = SI cm(-1); the 6-fold barrier contributions V-6 '' and V-6' are in the range of 2-3 cm(-1) and are positive in both states. The changes of A, B, and C rotational constants upon S-1 <- S-0 excitation were extracted from the contours and reflect an "anti-quinoidal" distortion. The 0(0)(0) contour can only be simulated if a 3 GHz Lorentzian line shape is included, which implies that the S-1(1n pi*) lifetime is similar to 55 ps. For the 6a(0)(1) and 6b(0)(1) bands, the Lorentzian component increases to 5.5 GHz, reflecting a lifetime decrease to similar to 30 ps. The short lifetimes are consistent with the absence of fluorescence from the 1n pi* state. Combining these measurements with the previous observation of efficient intersystem crossing (ISC) from the Si state to a long-lived T-1((3)n pi*) state that lies similar to 2200 cm(-1) below [S. Lobsiger, S. et al. Phys. Chem. Chem. Phys. 2010, 12, 5032] implies that the broadening arises from fast intersystem crossing with k(ISC) approximate to 2 x 10(10) s(-1). In comparison to 5-methylpyrimidine, the ISC rate is enhanced by at least 10 000 by the additional hydroxy group in position 2.

Osteoblast proliferation and differentiation on a barrier membrane in combination with BMP2 and TGFβ1

Bioresorbable collagen membranes are routinely utilized in guided bone regeneration to selectively direct the growth and repopulation of bone cells in areas of insufficient volume. However, the exact nature by which alveolar osteoblasts react to barrier membranes as well as the effects following the addition of growth factors to the membranes are still poorly understood. The objective of the present study was therefore to investigate the effect of a bioresorbable collagen membrane soak-loaded in growth factors bone morphogenetic protein 2 (BMP2) or transforming growth factor β1 (TGFβ1) on osteoblast adhesion, proliferation, and differentiation.

Development and remodeling of the vertebrate blood-gas barrier

During vertebrate development, the lung inaugurates as an endodermal bud from the primitive foregut. Dichotomous subdivision of the bud results in arborizing airways that form the prospective gas exchanging chambers, where a thin blood-gas barrier (BGB) is established. In the mammalian lung, this proceeds through conversion of type II cells to type I cells, thinning, and elongation of the cells as well as extrusion of the lamellar bodies. Subsequent diminution of interstitial tissue and apposition of capillaries to the alveolar epithelium establish a thin BGB. In the noncompliant avian lung, attenuation proceeds through cell-cutting processes that result in remarkable thinning of the epithelial layer. A host of morphoregulatory molecules, including transcription factors such as Nkx2.1, GATA, HNF-3, and WNT5a; signaling molecules including FGF, BMP-4, Shh, and TFG- β and extracellular proteins and their receptors have been implicated. During normal physiological function, the BGB may be remodeled in response to alterations in transmural pressures in both blood capillaries and airspaces. Such changes are mitigated through rapid expression of the relevant genes for extracellular matrix proteins and growth factors. While an appreciable amount of information regarding molecular control has been documented in the mammalian lung, very little is available on the avian lung.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

Going against the tide--how encephalitogenic T cells breach the blood-brain barrier

During multiple sclerosis or its animal model, experimental autoimmune encephalomyelitis, circulating immune cells enter the central nervous system (CNS) causing neuroinflammation. Extravasation from the blood circulation across the vessel wall occurs through a multistep process regulated by adhesion and signal transducing molecules on the immune cells and on the endothelium. Since the CNS is shielded by the highly specialized blood-brain barrier (BBB), immune cell extravasation into the CNS requires breaching this particularly tight endothelial border. Consequently, travelling into the CNS demands unique adaptations which account for the extreme tightness of the BBB. Modern imaging tools have shown that after arresting on BBB endothelium, in vivo or in vitro encephalitogenic effector/memory T cells crawl for long distances, possibly exceeding 150 µm along the surface of the BBB endothelium before rapidly crossing the BBB. Interestingly, in addition to the distance of crawling, the preferred direction of crawling against the flow is unique for T cell crawling on the luminal surface of CNS microvessels. In this review, we will summarize the cellular and molecular mechanisms involved in the unique T cell behavior that is obviously required for finding a site permissive for diapedesis across the unique vascular bed of the BBB.