The Role of Chemokines in Leukocyte Recruitment across the Blood-Brain Barrier

Migration of autoaggressive T cells across the blood-brain barrier (BBB) is critically involved in the initiation of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). The direct involvement of chemokines in this process was suggested by our recent observation that G-protein-mediated signaling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo. For chemokines to be involved in this process, they have to be either expressed by BBB endothelial cells themselves or would require a yet unknown transport mechanism from the central nervous system (CNS) parenchyma across the endothelial BBB to the luminal surface of the endothelial cells. To search for chemokines expressed by the endothelial BBB itself, in situ hybridizations and immunohistochemistry were performed and expression of the lymphoid chemokines CCL19 and CCL21 was found in venules surrounded by inflammatory cells. Their expression was paralleled by the presence of their common receptor CCR7 in inflammatory cells in brain and spinal cord sections of mice afflicted with EAE. Encephalitogenic T cells showed surface expression of CCR7 and the alternative receptor for CCL21, CXCR3. They specifically chemotaxed towards both CCL19 or CCL21 in a concentration dependent and pertussis toxin-sensitive manner comparable to naive lymphocytes in vitro. Functional involvement of CCL19 and CCL21 in adhesion strengthening of encephalitogenic T lymphocytes was demonstrated by binding assays on frozen brain sections of mice afflicted with EAE in vitro and preliminary by intravital fluorescence videomicroscopy in spinal cord of healthy mice in vivo. The moderate effect observed suggests additional, potentially unknown chemokines to be involved in lymphocyte recruitment across the endothelial BBB into the immunoprivileged CNS. Such chemokines, receptors as well as unknown molecules were identified at the level of the endothelial BBB by oligonucleotide microarrays, subtractive suppression hybridization (SSH) and proteomics. Besides the upregulation of expected genes and proteins described to be involved in leukocyte recruitment during EAE pathogenesis before, unexpected genes and proteins were identified. The latter included increased Duffy antigen / receptor for chemokines (DARC) expression suggesting its involvement in lymphocyte recruitment during EAE pathogenesis, which was proven as in DARC-deficient mice, disease onset was delayed, while clinical severity was increased. This may be explained by an ambiguous DARC function in EAE pathogenesis. Either endothelial cell expressed DARC "shuttles" chemokines to the luminal surface of the endothelial cells or erythrocyte expressed DARC removes chemokines by its "sink"-like function. This results in either increased or decreased chemokine concentrations accessible to encephalitogenic T lymphoblasts. Their encephalitogenicity was addressed by gene array analysis and SSH of encephalitogenic versus non-encephalitogenic T lymphoblasts identifying 79 differentially expressed genes. Based on the results obtained during this thesis, we would like to suggest the lymphoid chemokines CCL19 and CCL21 to be critically involved in lymphocyte recruitment across the endothelial BBB during EAE pathogenesis, while the chemokine receptor DARC may provide a "shuttle" mechanism for inflammatory chemokines from the CNS parenchyma across the endothelial BBB to the luminal surface of the endothelial cells during EAE. A large number of additional genes and proteins was identified to be differentially expressed in either endothelial cells or T lymphoblasts pointing to new mechanisms involved in leukocyte trafficking across the BBB

Ideal negative measurements in quantum walks disprove theories based on classical trajectories

We report on a stringent test of the non-classicality of the motion of a massive quantum particle, which propagates on a discrete lattice. Measuring temporal correlations of the position of single atoms performing a quantum walk, we observe a $6\sigma$ violation of the Leggett-Garg inequality. Our results rigorously excludes (i.e. falsifies) any explanation of quantum transport based on classical, well-defined trajectories. We use so-called ideal negative measurements -- an essential requisite for any genuine Leggett-Garg test -- to acquire information about the atom's position, yet avoiding any direct interaction with it. The interaction-free measurement is based on a novel atom transport system, which allows us to directly probe the absence rather than the presence of atoms at a chosen lattice site. Beyond the fundamental aspect of this test, we demonstrate the application of the Leggett-Garg correlation function as a witness of quantum superposition. We here employ the witness to discriminate different types of walks spanning from merely classical to wholly quantum dynamics.Comment: 10 pages, 4 figure

The Role of Chemokines in Leukocyte Recruitment across the Blood-Brain Barrier

Migration of autoaggressive T cells across the blood-brain barrier (BBB) is critically involved in the initiation of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). The direct involvement of chemokines in this process was suggested by our recent observation that G-protein-mediated signaling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo. For chemokines to be involved in this process, they have to be either expressed by BBB endothelial cells themselves or would require a yet unknown transport mechanism from the central nervous system (CNS) parenchyma across the endothelial BBB to the luminal surface of the endothelial cells. To search for chemokines expressed by the endothelial BBB itself, in situ hybridizations and immunohistochemistry were performed and expression of the lymphoid chemokines CCL19 and CCL21 was found in venules surrounded by inflammatory cells. Their expression was paralleled by the presence of their common receptor CCR7 in inflammatory cells in brain and spinal cord sections of mice afflicted with EAE. Encephalitogenic T cells showed surface expression of CCR7 and the alternative receptor for CCL21, CXCR3. They specifically chemotaxed towards both CCL19 or CCL21 in a concentration dependent and pertussis toxin-sensitive manner comparable to naive lymphocytes in vitro. Functional involvement of CCL19 and CCL21 in adhesion strengthening of encephalitogenic T lymphocytes was demonstrated by binding assays on frozen brain sections of mice afflicted with EAE in vitro and preliminary by intravital fluorescence videomicroscopy in spinal cord of healthy mice in vivo. The moderate effect observed suggests additional, potentially unknown chemokines to be involved in lymphocyte recruitment across the endothelial BBB into the immunoprivileged CNS. Such chemokines, receptors as well as unknown molecules were identified at the level of the endothelial BBB by oligonucleotide microarrays, subtractive suppression hybridization (SSH) and proteomics. Besides the upregulation of expected genes and proteins described to be involved in leukocyte recruitment during EAE pathogenesis before, unexpected genes and proteins were identified. The latter included increased Duffy antigen / receptor for chemokines (DARC) expression suggesting its involvement in lymphocyte recruitment during EAE pathogenesis, which was proven as in DARC-deficient mice, disease onset was delayed, while clinical severity was increased. This may be explained by an ambiguous DARC function in EAE pathogenesis. Either endothelial cell expressed DARC "shuttles" chemokines to the luminal surface of the endothelial cells or erythrocyte expressed DARC removes chemokines by its "sink"-like function. This results in either increased or decreased chemokine concentrations accessible to encephalitogenic T lymphoblasts. Their encephalitogenicity was addressed by gene array analysis and SSH of encephalitogenic versus non-encephalitogenic T lymphoblasts identifying 79 differentially expressed genes. Based on the results obtained during this thesis, we would like to suggest the lymphoid chemokines CCL19 and CCL21 to be critically involved in lymphocyte recruitment across the endothelial BBB during EAE pathogenesis, while the chemokine receptor DARC may provide a "shuttle" mechanism for inflammatory chemokines from the CNS parenchyma across the endothelial BBB to the luminal surface of the endothelial cells during EAE. A large number of additional genes and proteins was identified to be differentially expressed in either endothelial cells or T lymphoblasts pointing to new mechanisms involved in leukocyte trafficking across the BBB

DARC shuttles inflammatory chemokines across the blood-brain barrier during autoimmune central nervous system inflammation

Trafficking of T cells into the CNS is a pathophysiological hallmark of multiple sclerosis. Using an invitro model of the blood-brain barrier, Minten etal. reveal that the atypical chemokine receptor DARC shuttles inflammatory chemokines across the barrier, where they contribute to immune cell trafficking into the brai

Biocompatibility of the vital dye Acid Violet-17 on retinal pigment epithelial cells

Purpose: To examine the viability and differentiation of retinal pigment epithelial (RPE) cells after exposure to the vital dye Acid Violet-17 (AV-17). Methods: Bovine RPE cells were incubated with AV-17 (0.0625-0.5 mg/mL) for 30 seconds or 5 minutes. Viability was determined by live/dead staining, cleaved CASP3 immunostainings, and MTT test. Actin cytoskeleton was visualized by Alexa 488-phalloidin. Immunocytochemistry was performed to determine the levels of ZO-1, CTNNB1, and KRT19. Results: Exposure to AV-17 at the concentrations of 0.25-0.5 mg/mL resulted in a dose-dependent decrease in viability, the loss of ZO-1 from tight junctions, translocation of CTNNB1 into the cytoplasm and nucleus, disarrangement of the actin cytoskeleton, and a slight increase in KRT19. Conclusion: AV-17 at a concentration. 0.125 mg/mL is likely to be well tolerated by the RPE cells, whereas the concentrations from 0.25 mg/mL onward can reduce viability and induce dedifferentiation particularly after long-term exposure

Demonstration of quantum brachistochrones between distant states of an atom

Transforming an initial quantum state into a target state through the fastest possible route---a quantum brachistochrone---is a fundamental challenge for many technologies based on quantum mechanics. Here, we demonstrate fast coherent transport of an atomic wave packet over a distance of 15 times its size---a paradigmatic case of quantum processes where the target state cannot be reached through a local transformation. Our measurements of the transport fidelity reveal the existence of a minimum duration---a quantum speed limit---for the coherent splitting and recombination of matter waves. We obtain physical insight into this limit by relying on a geometric interpretation of quantum state dynamics. These results shed light upon a fundamental limit of quantum state dynamics and are expected to find relevant applications in quantum sensing and quantum computing.Comment: 6 pages, 3 figures, and supplemental materia

Does Dynamic Anterior Plate Fixation Provide Adequate Stability for Traumatic Subaxial Cervical Spine Fractures at Mid-Term Follow-Up?

Background: It remains questionable if the treatment of cervical fractures with dynamic plates in trauma surgery provides adequate stability for unstable fractures with disco-ligamentous injuries. The primary goal of this study was to assess the radiological and mid-term patient-reported outcome of traumatic subaxial cervical fractures treated with different plate systems. Patients and Methods: Patients, treated with anterior cervical discectomy and fusion (ACDF) between 2001 and 2015, using either a dynamic plate (DP: Mambo™, Ulrich, Germany) or a rigid locking plate (RP: CSLP™, Depuy Synthes, USA), were identified. For radiological evaluation, the sagittal alignment, the sagittal anterior translation and the bony consolidation were evaluated. After at least two years, the patient-reported outcome measures (PROM) were evaluated using the German Short-Form 36 (SF-36), Neck Disability Index (NDI) and the EuroQol in 5 Dimensions (EQ-5D) scores. Results: 33 patients met the inclusion criteria (DP: 13; RP:20). Twenty-six patients suffered from AO Type B or C fractures. Both the sagittal alignment and the sagittal translation could be sufficiently improved in both groups (p ≥ 0.05). No significant loss of reduction could be observed at the follow-up in both groups (p ≥ 0.05). Bony consolidation could be observed in 30 patients (DP: 12/13 (92%); RP: 18/20 (90%); (p ≥ 0.05)). In 20 patients, PROMs could be evaluated (follow-up: 71.2 ± 25.5 months). The whole cohort showed satisfactory PROM results (EQ-5D: 72.0 ± 4.9; SF-36 PCS: 41.9 ± 16.2, MCS: 45.4 ± 14.9; NDI: 11.0 ± 9.1). without significant differences between the DP and RP group (p ≥ 0.05) Conclusion: The dynamic plate concept provides enough stability without a difference in fusion rates in comparison to rigid locking plates in a population that mostly suffered fragile fractures