The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation

Shiga Toxin and Lipopolysaccharide Induce Platelet-Leukocyte Aggregates and Tissue Factor Release, a Thrombotic Mechanism in Hemolytic Uremic Syndrome

BACKGROUND: Aggregates formed between leukocytes and platelets in the circulation lead to release of tissue factor (TF)-bearing microparticles contributing to a prothrombotic state. As enterohemorrhagic Escherichia coli (EHEC) may cause hemolytic uremic syndrome (HUS), in which microthrombi cause tissue damage, this study investigated whether the interaction between blood cells and EHEC virulence factors Shiga toxin (Stx) and lipopolysaccharide (LPS) led to release of TF. METHODOLOGY/PRINCIPAL FINDINGS: The interaction between Stx or LPS and blood cells induced platelet-leukocyte aggregate formation and tissue factor (TF) release, as detected by flow cytometry in whole blood. O157LPS was more potent than other LPS serotypes. Aggregates formed mainly between monocytes and platelets and less so between neutrophils and platelets. Stimulated blood cells in complex expressed activation markers, and microparticles were released. Microparticles originated mainly from platelets and monocytes and expressed TF. TF-expressing microparticles, and functional TF in plasma, increased when blood cells were simultaneously exposed to the EHEC virulence factors and high shear stress. Stx and LPS in combination had a more pronounced effect on platelet-monocyte aggregate formation, and TF expression on these aggregates, than each virulence factor alone. Whole blood and plasma from HUS patients (n = 4) were analyzed. All patients had an increase in leukocyte-platelet aggregates, mainly between monocytes and platelets, on which TF was expressed during the acute phase of disease. Patients also exhibited an increase in microparticles, mainly originating from platelets and monocytes, bearing surface-bound TF, and functional TF was detected in their plasma. Blood cell aggregates, microparticles, and TF decreased upon recovery. CONCLUSIONS/SIGNIFICANCE: By triggering TF release in the circulation, Stx and LPS can induce a prothrombotic state contributing to the pathogenesis of HUS

Nat Genet

Aortic calcification is an important independent predictor of future cardiovascular events. We performed a genome-wide association meta-analysis to determine SNPs associated with the extent of abdominal aortic calcification (n = 9,417) or descending thoracic aortic calcification (n = 8,422). Two genetic loci, HDAC9 and RAP1GAP, were associated with abdominal aortic calcification at a genome-wide level (P < 5.0 x 10(-8)). No SNPs were associated with thoracic aortic calcification at the genome-wide threshold. Increased expression of HDAC9 in human aortic smooth muscle cells promoted calcification and reduced contractility, while inhibition of HDAC9 in human aortic smooth muscle cells inhibited calcification and enhanced cell contractility. In matrix Gla protein-deficient mice, a model of human vascular calcification, mice lacking HDAC9 had a 40% reduction in aortic calcification and improved survival. This translational genomic study identifies the first genetic risk locus associated with calcification of the abdominal aorta and describes a previously unknown role for HDAC9 in the development of vascular calcification

Entwicklung eines Testsystems fuer die Pruefung des biologischen Abbaus in Oberflaechengewaessern

The study presented here describes the development of a laboratory test system for the determination of aerobic biodegradability of substances at low concentrations in surface water. It was aimed to prepare a draft guideline for a biodegradation simulation test according to OECD format. The experimental approach was based on a literature study conducted within the frame of this project. Further useful information on the possible test design was derived from the German BBA guideline 5-1. Natural water and sediments were collected. Radiolabelled Lindane or 4-Nitrophenol was added. The test vessels (reactors) were aerated and incubated under controlled conditions for up to 92 days. The results showed biological stability of the sediment/water systems even without addition of nutrients and adherence to non-reducing conditions. Mineralisation of 4-Nitrophenol was influenced by the sediment type, the method of aeration and temperature. Factors affecting the mineralisation of Lindane were the method of application and again, the sediment type and temperature. Considerable amounts of the radioactivity were bound to the sediment and were to a large extent unextractable. The potential of a reactor to mineralise a test substance could not be correlated with the biological parameters measured. (orig.)Die vorliegende Studie beschreibt die Entwicklung eines Labortestverfahrens zur Pruefung des aeroben Abbaus niedrig konzentrierter Stoffe in Oberflaechengewaessern. Dabei war es ein Ziel, das Verfahren so weit abzusichern, dass ein Entwurf fuer eine Pruefrichtlinie als Simulationstest im Format der OECD-Richtlinien abgefasst werden konnte. Grundlage fuer die Konzeption war eine zuvoerderst durchgefuehrte Literaturstudie. Hinweise auf ein moegliches Testdesign ergaben sich auch aus der BBA-Richtlinie 5-1. Wasser und Sediment wurden der Natur entnommen und nach Zugabe der radioaktiven Pruefsubstanz Lindan oder 4-Nitrophenol in einem beluefteten Gefaess unter kontrollierten Bedingungen bis zu 92 Tage lang inkubiert. Die Ergebnisse zeigten, dass die Wasser/Sediment-Systeme auch ueber diesen langen Zeitraum ohne Zufuetterung biologisch stabil waren und nichtreduzierende Bedingungen auch im Sediment erhalten blieben. Die Mineralisierung von 4-Nitrophenol wurde vom Sedimenttyp, der Belueftungsart und der Temperatur beeinflusst, die von Lindan von der Applikationsart und ebenfalls vom Sedimenttyp und der Temperatur. Wesentliche Anteile der Radioaktivitaet wurden ans Sediment gebunden und waren zum grossen Teil nicht extrahierbar. Es wird schliesslich diskutiert, dass das Potential eines Sediments, Stoffe zu minimalisieren, nicht mit den gewaehlten biologischen Kenngroessen klassifiziert werden kann. Daraus koennen Probleme bei der Uebertragbarkeit der Ergebnisse auf neue Standorte erwachsen. (orig.)Available from TIB Hannover: RN 8422(1997,13) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Umwelt, Naturschutz und Reaktorsicherheit, Bonn (Germany)DEGerman