Rolling stones and tree rings: A state of research on dendrogeomorphic reconstructions of rockfall

This progress report focuses on the contribution of tree-ring series to rockfall research and on recent development and challenges in the field. Dendrogeomorphic techniques have been used extensively since the early 2000s and several approaches have been developed to extract rockfall signals from tree-ring records of conifer trees. The reconstruction of rockfall chronologies has been hampered in the past by sample sizes that decrease as one goes back in time, as well as by a paucity of studies that include broadleaved tree species, which are in fact quite common in rockfall-prone environments. In this report, we propose a new approach considering impact probability and quantification of uncertainty in the reconstruction of rockfall time series as well as a quantitative estimate of presumably missed events. In addition, we outline new approaches and future perspectives for the inclusion of woody vegetation in hazard assessment procedures, and end with future thematic perspectives.

Scheduling the production of rolling ingots: Industrial context, model, and solution method

We study a real-world scheduling problem arising in the context of a rolling ingots production. First we review the production process and discuss peculiarities that have to be observed when scheduling a given set of production orders on the production facilities. We then show how to model this scheduling problem using prescribed time lags between operations, different kinds of resources, and sequence-dependent changeovers. A branch-and-bound solution procedure is presented in the second part. The basic principle is to relax the resource constraints by assuming infinite resource availability. Resulting resource conflicts are then stepwise resolved by introducing precedence relationships among operations competing for the same resources. The algorithm has been implemented as a beam search heuristic enumerating alternative sets of precedence relationships.

PSGL-1 and E/P-selectins are essential for T-cell rolling in inflamed CNS microvessels but dispensable for initiation of EAE

T-cell migration across the blood-brain barrier is a crucial step in the pathogenesis of EAE, an animal model for MS. Live cell imaging studies demonstrated that P-selectin glycoprotein ligand-1 (PSGL-1) and its endothelial ligands E- and P-selectin mediate the initial rolling of T cells in brain vessels during EAE. As functional absence of PSGL-1 or E/P-selectins does not result in ameliorated EAE, we speculated that T-cell entry into the spinal cord is independent of PSGL-1 and E/P-selectin. Performing intravital microscopy, we observed the interaction of WT or PSGL-1(-/-) proteolipid protein-specific T cells in inflamed spinal cord microvessels of WT or E/P-selectin(-/-) SJL/J mice during EAE. T-cell rolling but not T-cell capture was completely abrogated in the absence of either PSGL-1 or E- and P-selectin, resulting in a significantly reduced number of T cells able to firmly adhere in the inflamed spinal cord microvessels, but did not lead to reduced T-cell invasion into the CNS parenchyma. Thus, PSGL-1 interaction with E/P-selectin is essential for T-cell rolling in inflamed spinal cord microvessels during EAE. Taken together with previous observations, our findings show that T-cell rolling is not required for successful T-cell entry into the CNS and initiation of EAE.

Generation of long, fully modified, and serum-resistant oligonucleotides by rolling circle amplification

Rolling Circle Amplification (RCA) is an isothermal enzymatic method generating single-stranded DNA products consisting of concatemers containing multiple copies of the reverse complement of the circular template precursor. Little is known on the compatibility of modified nucleoside triphosphates (dN*TPs) with RCA, which would enable the synthesis of long, fully modified ssDNA sequences. Here, dNTPs modified at any position of the scaffold were shown to be compatible with rolling circle amplification, yielding long (>1 kb), and fully modified single-stranded DNA products. This methodology was applied for the generation of long, cytosine-rich synthetic mimics of telomeric DNA. The resulting modified oligo-nucleotides displayed an improved resistance to fetal bovine serum.

Alpha4beta1 integrin mediates the recruitment of immature dendritic cells across the blood-brain barrier during experimental autoimmune encephalomyelitis

Dendritic cells (DCs) within the CNS are recognized to play an important role in the effector phase and propagation of the immune response in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. However, the mechanisms regulating DC trafficking into the CNS still need to be characterized. In this study, we show by performing intravital fluorescence videomicroscopy of the inflamed spinal cord white-matter microvasculature in SJL mice with EAE that immature, and to a lesser extent, LPS-matured, bone marrow-derived DCs efficiently interact with the CNS endothelium by rolling, capturing, and firm adhesion. Immature but not LPS-matured DCs efficiently migrated across the wall of inflamed parenchymal microvessels into the CNS. Blocking alpha4 integrins interfered with the adhesion but not the rolling or capturing of immature and LPS-matured DCs to the CNS microvascular endothelium, inhibiting their migration across the vascular wall. Functional absence of beta1 integrins but not of beta7 integrins or alpha4beta7 integrin similarly reduced the adhesion of immature DCs to the CNS microvascular endothelium, demonstrating that alpha4beta1 but not alpha4beta7 integrin mediates this step of immature DCs interaction with the inflamed blood-brain barrier during EAE. Our study shows that during EAE, especially immature DCs migrate into the CNS, where they may be crucial for the perpetuation of the CNS-targeted autoimmune response. Thus therapeutic targeting of alpha4 integrins affects DC trafficking into the CNS and may therefore lead to the resolution of the CNS autoimmune inflammation by reducing the number of CNS professional APCs.

Molecular mechanisms involved in T cell migration across the blood-brain barrier

In the healthy individuum lymphocyte traffic into the central nervous system (CNS) is very low and tightly controlled by the highly specialized blood-brain barrier (BBB). In contrast, under inflammatory conditions of the CNS such as in multiple sclerosis or in its animal model experimental autoimmune encephalomyelitis (EAE) circulating lymphocytes and monocytes/macrophages readily cross the BBB and gain access to the CNS leading to edema, inflammation and demyelination. Interaction of circulating leukocytes with the endothelium of the blood-spinal cord and blood-brain barrier therefore is a critical step in the pathogenesis of inflammatory diseases of the CNS. Leukocyte/endothelial interactions are mediated by adhesion molecules and chemokines and their respective chemokine receptors. We have developed a novel spinal cord window preparation, which enables us to directly visualize CNS white matter microcirculation by intravital fluorescence videomicroscopy. Applying this technique of intravital fluorescence videomicroscopy we could provide direct in vivo evidence that encephalitogenic T cell blasts interact with the spinal cord white matter microvasculature without rolling and that alpha4-integrin mediates the G-protein independent capture and subsequently the G-protein dependent adhesion strengthening of T cell blasts to microvascular VCAM-1. LFA-1 was found to neither mediate the G-protein independent capture nor the G- protein dependent initial adhesion strengthening of encephalitogenic T cell blasts within spinal cord microvessel, but was rather involved in T cell extravasation across the vascular wall into the spinal cord parenchyme. Our observation that G-protein mediated signalling is required to promote adhesion strengthening of encephalitogenic T cells on BBB endothelium in vivo suggested the involvement of chemokines in this process. We found functional expression of the lymphoid chemokines CCL19/ELC and CCL21/SLC in CNS venules surrounded by inflammatory cells in brain and spinal cord sections of mice afflicted with EAE suggesting that the lymphoid chemokines CCL19 and CCL21 besides regulating lymphocyte homing to secondary lymphoid tissue might be involved in T lymphocyte migration into the immuneprivileged CNS during immunosurveillance and chronic inflammation. Here, I summarize our current knowledge on the sequence of traffic signals involved in T lymphocyte recruitment across the healthy and inflamed blood-brain and blood-spinal cord barrier based on our in vitro and in vivo investigations.

Immune cell entry into the central nervous system: involvement of adhesion molecules and chemokines

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the highly specialized endothelial blood-brain barrier (BBB) and gain access to the central nervous system (CNS). It is well established that leukocyte recruitment across this vascular bed is unique due to the predominant involvement of alpha4-integrins in mediating the initial contact to as well as firm adhesion with the endothelium. In contrast, the involvement of the selectins, L-selectin, E- and P-selectin and their respective carbohydrate ligands such as P-selectin glycoprotein (PSGL)-1 in this process has been controversially discussed. Intravital microscopic analysis of immune cell interaction with superficial brain vessels demonstrates a role for E- and P-selectin and their common ligand PSGL-1 in lymphocyte rolling. However, E- and P-selectin-deficient SJL- or C57Bl/6 mice or PSGL-1-deficient C57Bl/6 mice develop EAE indistinguishable from wild-type mice. Considering these apparently discrepant observations, it needs to be discussed whether the molecular mechanisms involved in leukocyte trafficking across superficial brain vessels are irrelevant for EAE pathogenesis or whether the therapeutic efficacy of targeting alpha4-integrins in EAE is truly dependent on the inhibition of leukocyte trafficking across the BBB.

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.

Dreidimensionale Bewegungserfassung mit Consumer-Highspeedkameras: Eine Systementwicklung unter besonderer Berücksichtigung der Messunsicherheit

Schwerpunkt dieser Arbeit ist die Entwicklung und der Test eines auf Consumer-Highspeedkameras basierenden Bewegungserfassungssystems. Consumer-Kameras sind flexibler einsetzbar als kommerzielle Bewegungserfassungssysteme, zugleich kostengünstiger und werden daher oft in der sportwissenschaftlichen Forschung verwendet. Durch ihren Einsatz entstehen jedoch prinzipbedingt höhere Messunsicherheiten, deren Bestimmung in der vorliegenden Arbeit ein besonderer Stellenwert eingeräumt wird. Nach einem Überblick über aktuelle Bewegungserfassungssysteme und deren Genauigkeiten folgt eine Betrachtung der Messunsicherheit aus metrologischer Perspektive. Anschließend werden die Prozesse der Bilderfassung bei digitalen Consumer-Kameras sowie die zur Modellierung des Kameraabbildungsverhaltens notwendigen Parameteridentifikationsmethoden dargestellt. Diese reichen vom häufig genutzten DLT-Verfahren über Methoden mit Verzeichnungskorrektur bis zu Bündelausgleichsverfahren. Im Anschluss werden die für die verwendeten Kameras geeigneten Methoden auf Basis funktionaler Hardwaretests ausgewählt und weitere für das Bewegungserfassungssystem notwendige Softwarekomponenten diskutiert. Dazu gehören neben der automatisierten Video- und Bildverarbeitung, spezielle Verfahren zur Korrektur von Consumer-Kamera-spezifischen Abweichungen, z.B. die Korrektur von Rolling-Shutter-Verzerrungen. Im zweiten Teil der Arbeit richtet sich der Fokus auf die Simulation der Effekte von Parameterungenauigkeiten auf die Systemgenauigkeit sowie auf die Validierung und den Test des implementierten Systems. Dabei konnte die Rekonstruktionsgenauigkeit von 11.86mm bei einer Referenzrahmenkalibration durch den Einsatz der Kalibrationsmethode mit Bündelausgleichsverfahren von Svoboda u. a. (2005) auf maximal 4.126mm (M=0.073 mm; SD=1.486 mm) reduziert werden. Diese Methode erlaubt zudem eine einfachere Kalibration größerer Messvolumen ohne aufwändige Referenzrahmen und ist daher ideal für den sportwissenschaftlichen Einsatz geeignet. Ein weiteres Ergebnis der Arbeit ist die theoretische Ableitung der Fehlerfortpflanzung für die Prozessschritte der Bewegungserfassung. In Kombination mit der entwickelten Simulationsumgebung wird damit die Grundlage für eine Prädiktion der erreichbaren Messunsicherheit bereits vor der eigentlichen Messung gelegt.

Kindlin-3 regulates integrin activation and adhesion reinforcement of effector T cells

Activated T cells use very late antigen-4/α4β1 integrin for capture, rolling on, and firm adhesion to endothelial cells, and use leukocyte function-associated antigen-1/αLβ2 integrin for subsequent crawling and extravasation. Inhibition of α4β1 is sufficient to prevent extravasation of activated T cells and is successfully used to combat autoimmune diseases, such as multiple sclerosis. Here we show that effector T cells lacking the integrin activator Kindlin-3 extravasate and induce experimental autoimmune encephalomyelitis in mice immunized with autoantigen. In sharp contrast, adoptively transferred autoreactive T cells from Kindlin-3-deficient mice fail to extravasate into the naïve CNS. Mechanistically, autoreactive Kindlin-3-null T cells extravasate when the CNS is inflamed and the brain microvasculature expresses high levels of integrin ligands. Flow chamber assays under physiological shear conditions confirmed that Kindlin-3-null effector T cells adhere to high concentrations of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, albeit less efficiently than WT T cells. Although these arrested T cells polarize and start crawling, only few remain firmly adherent over time. Our data demonstrate that the requirement of Kindlin-3 for effector T cells to induce α4β1 and αLβ2 integrin ligand binding and stabilization of integrin-ligand bonds is critical when integrin ligand levels are low, but of less importance when integrin ligand levels are high.

Bone marrow-derived mesenchymal stromal cells improve vascular regeneration and reduce leukocyte-endothelium activation in critical ischemic murine skin in a dose-dependent manner

BACKGROUND AIMS Stem cells participate in vascular regeneration following critical ischemia. However, their angiogenic and remodeling properties, as well as their role in ischemia-related endothelial leukocyte activation, need to be further elucidated. Herein, we investigated the effect of bone marrow-derived mesenchymal stromal cells (BM-MSCs) in a critically ischemic murine skin flap model. METHODS Groups received either 1 × 10(5), 5 × 10(5), or 1 × 10(6) BM-MSCs or cell-free conditioned medium (CM). Controls received sodium chloride. Intravital fluorescence microscopy was performed for morphological and quantitative assessment of micro-hemodynamic parameters over 12 days. RESULTS Tortuosity and diameter of conduit-arterioles were pronounced in the MSC groups (P < 0.01), whereas vasodilation was shifted to the end arteriolar level in the CM group (P < 0.01). These effects were accompanied by angiopoietin-2 expression. Functional capillary density and red blood cell velocity were enhanced in all treatment groups (P < 0.01). Although a significant reduction of rolling and sticking leukocytes was observed in the MSC groups with a reduction of diameter in postcapillary venules (P < 0.01), animals receiving CM exhibited a leukocyte-endothelium interaction similar to controls. This correlated with leukocyte common antigen expression in tissue sections (P < 0.01) and p38 mitogen-activated protein kinase expression from tissue samples. Cytokine analysis from BM-MSC culture medium revealed a 50% reduction of pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-12, tumor necrosis factor-α, interferon-γ) and chemokines (keratinocyte chemoattractant, granulocyte colony-stimulating factor) under hypoxic conditions. DISCUSSION We demonstrated positive effects of BM-MSCs on vascular regeneration and modulation of endothelial leukocyte adhesion in critical ischemic skin. The improvements after MSC application were dose-dependent and superior to the use of CM alone.