Identification of circulation-derived exosomal microRNA candidates for liquid biopsy in pediatric acute lymphoblastic leukemia

MicroRNA aus Exosomen -extrazellulären Vesikeln, die auch physiologisch ubiquitär der Zell-Zell-Kommunikation dienen- wurde in vielen Neoplasien als wichtiger Bestandteil der Kommunikation von soliden Tumoren mit ihrer Ni-sche identifiziert. Auch in akuten Leukämien gibt es einige wenige Studien zu dieser Interaktion, aber besonders die Korrelation von exosomaler Beeinflus-sung und klinischen Verläufen bleibt bislang unbeleuchtet. Da Exosomen in die Blutbahn freigesetzt werden, können Informationen über diese Kommuni-kation mit einer peripheren Blutabnahme gewonnen werden, einer „liquid bi-opsy“. In dieser Arbeit wurde die exosomale microRNA-Expression aus peripheren Blutproben von Patienten mit pädiatrischer akuter lymphoblastischer Leukämie analysiert und mit ihren klinischen Verläufen korreliert, um potenzielle „liquid biopsy“-Marker zu identifizieren, schwere Verläufe vorherzusagen und zu ver-hindern. Dazu wurden 179 verschiedene microRNAs mit dem „Qiagen miRCURY Focus panel“ in 11 Plasmaproben von Patienten sowie von 3 gesunden Kon-trollpersonen analysiert. Beim Vergleich der Proben von therapierefraktären zu therapieansprechenden Patienten, die zum Zeitpunkt der Diagnosestellung und vor Therapiebeginn abgenommen wurden, zeigten die exosomale mir-23b-3p-, mir-409-3p- und mir-7-5p-Expressionen die signifikantesten Unterschiede: Eine Hochregulati-on von exosomaler mir-23b-3p (p=0.006) und mir-409-3p (p=0.005) und eine Herunterregulation von exosomaler mir-7-5p (p=0.003) in den therapierefraktä-ren Proben. Diese exosomalen microRNA-Expressionsunterschiede sind vielversprechen-de Kandidaten für eine „liquid biopsy“, um schon bei der Diagnosestellung Patienten zu identifizieren, die von einer vorzeitigen Therapieeskalation profi-tieren könnten. Im Vergleich von Patientenproben gegen gesunde Kontrollproben waren die exosomale mir-484 (p=0.0002), mir-92a-3p (p=0.0004) und mir-25-3p (p=0.0009) am signifikantesten hochreguliert. Um zu beweisen, dass diese Exosomen direkt von den Leukämiezellen stam-men, und nicht von anderen „bystander“-Zellen, wurde im Rahmen dieser Ar-beit eine neuartige Methode etabliert, in der Exosomen aus dem Serum von „patient derived xenograft“- Mäusen mit akuter Leukämie mit einem anti-CD63-Immunpräzipitationsverfahren humanspezifisch extrahiert wurden. Diese Me-thode zur Bestimmung der Speziesspezifität und somit der „cell of origin“ der Exosomen, zeigte hohe Sensitivität und konnte humane Exosomen bis zur Verdünnung 1:100 von humanem Serum zu Mausserum detektieren. Mit der Methode konnte gezeigt werden, dass die signifikanten Expressionsun-terschiede von exosomaler mir-484, mir-92a-3p und mir-25-3p im Blut von Pati-enten gegenüber Gesunden direkt durch das Aussenden aus Leukämiezellen bedingt ist. Diese Identifikation der Leukämiezellen als Ursprung ermöglicht weiterführende Studien zur funktionellen Analyse der exosomalen microRNA-Expressionen und weiterhin Studien zur Identifikation von therapeutischen Zielen gegen die von der Leukämie ausgehenden exosomal-vermittelten Ni-schenreprogrammierung.MicroRNA derived from exosomes -extracellular vesicles involved in physiolog-ical cell-to-cell communication- has been identified as a key component in tu-mor-to-niche-communication in various solid neoplasias. There is limited data about this communication for acute leukemias, especially the correlation be-tween exosomal reprogramming and the patients’ clinical course remains to be investigated. Because exosomes are released in the bloodstream, information about this communication is available via a peripheral venipuncture, a “liquid biopsy”. We analyzed the exosomal microRNA expression from patients suffering from pediatric acute lymphoblastic leukemia and correlated them to the clinical course to identify potential liquid biopsy markers to indicate and prevent ad-verse courses. Therefore, 179 microRNAs were analyzed with the Qiagen miRCURY qPCR Focus panel in 11 patients’ plasma samples and 3 samples from healthy indi-viduals. When comparing therapy refractory to therapy responding patient samples taken at the timepoint of initial diagnosis prior to therapy, exosomal mir-23b-3p, mir-409-3p and mir-7-5p expression showed the most significant difference with an upregulation of exosomal mir-23b-3p (p=0.006) and mir-409-3p (p=0.005) and a downregulation of mir-7-5p (p=0.003) in the refractory sam-ples. These exosomal microRNA expression differences represent promising candi-dates for a liquid biopsy to prevent therapy refraction by escalation of therapy a priori. Exosomal mir-484 (p=0.0002), mir-92a-3p (p=0.0004) and mir-25-3p (p=0.0009) were most significantly upregulated in patients’ samples compared to healthy controls. To prove that these exosomes originate directly from the leukemia cells and not from other “bystander” cells, we established a novel assay using a patient de-rived xenotransplant (PDX) mouse model. Therefore, we extracted exosomes from PDX mice with acute leukemia human specifically via an anti-CD63 im-munocapture. This assay to identify species specific exosomes and therefore the cells of origin of these exosomes revealed high sensitivity, detecting hu-man:mouse serum titrations down to a ratio of 1:100. With our assay, we confirmed that the significant expression differences of ex-osomal mir-484, mir-92a-3p and mir-25-3p in patients’ blood compared to healthy individuals are directly caused by the leukemia cells. By identifying the leukemia cells as cells of origin, we here lay the groundwork to allow a func-tional analysis of these exosomal microRNAs and the identification of possible therapeutic targets against leukemia-derived exosome-mediated niche repro-gramming

Command and control in the modern supply chain : Implications of emerging technologies on the supply chain of land forces

This master’s thesis studies the implications of introducing modern technology in the army’s supply chain on the command and control of this supply chain. It has been a qualitative study using both literature and a survey among a limited group of subject matter experts. The theoretical framework for this thesis is based on organisational theory, especially on the Mintzberg model of organisation configurations. Furthermore, elements of Strategic Technology Management add to this framework. In order to define emerging technologies promising for the army’s supply chain, a survey of several government and commercial documents has been done. Based on expected influence on the command and control of the supply chain two of these were chosen for further analysis. Research findings point out that command and control of the army supply chain will shift from the Army to a more centralised organisation outside of the army. It is expected that coordination and de-confliction of activity on land will remain the Army’s responsibility. Depending the technological developments, this can eventually be automated as well. Furthermore, mutual trust and integrating suppliers in comprehensive planning and training are indicated as important criteria for success in supplying the Army

L’accompagnement : un concept au coeur de l’État social actif. Le cas des pratiques d’accompagnement des personnes handicapées

peer reviewedL’accompagnement devient un concept clé pour comprendre les nouvelles politiques sociales : désormais, on accompagne les personnes handicapées, les jeunes en difficultés, les chômeurs, les patients, etc. L’intervention sociale se caractérise aujourd’hui par une approche individualisée des suivis et par une recherche constante de l’activation des potentialités et des ressources de l’individu. Cet article s’intéresse aux pratiques d’accompagnement des personnes handicapées en région wallonne, car celles- ci sont, selon nous, une illustration parfaite des nouvelles pratiques sociales relevant de l’État social actif. Nous envisagerons quelques questions essentielles que suscitent ces pratiques innovantes, concernant notamment la contractualisation de l’aide et la responsabilisation des individus mais aussi la conditionnalité de l’aide et la sélection des usagers. Autant de transformations du travail social qui annoncent, selon nous, un nouveau régime de protection à vocation particulariste et incitative

Deconjugation Kinetics of Glucuronidated Phase II Flavonoid Metabolites by B-glucuronidase from Neutrophils

Flavonoids are inactivated by phase II metabolism and occur in the body as glucuronides. Mammalian ß-glucuronidase released from neutrophils at inflammatory sites may be able to deconjugate and thus activate flavonoid glucuronides. We have studied deconjugation kinetics and pH optimum for four sources of ß-glucuronidase (human neutrophil, human recombinant, myeloid PLB-985 cells, Helix pomatia) with five flavonoid glucuronides (quercetin-3-glucuronide, quercetin-3'-glucuronide, quercetin-4'-glucuronide, quercetin-7-glucuronide, 3'-methylquercetin-3-glucuronide), 4-methylumbelliferyl-ß-D-glucuronide, and para-nitrophenol-glucuronide. All substrate-enzyme combinations tested exhibited first order kinetics. The optimum pH for hydrolysis was between 3.5-5, with appreciable hydrolysis activities up to pH 5.5. At pH 4, the Km ranged 44-fold from 22 µM for quercetin-4'-glucuronide with Helix pomatia ß-glucuronidase, to 981 µM for para-nitrophenol-glucuronide with recombinant ß-glucuronidase. Vmax (range: 0.735-24.012 µmol·min-1·unit-1 [1 unit is defined as the release of 1 µM 4-methylumbelliferyl-ß-D-glucuronide per min]) and the reaction rate constants at low substrate concentrations (k) (range: 0.002-0.062 min-1·(unit/L)-1 were similar for all substrates-enzyme combinations tested. In conclusion, we show that ß-glucuronidase from four different sources, including human neutrophils, is able to deconjugate flavonoid glucuronides and non-flavonoid substrates at fairly similar kinetic rates. At inflammatory sites in vivo the pH, neutrophil and flavonoid glucuronide concentrations seem favorable for deconjugation. However, it remains to be confirmed whether this is actually the case

Manufacturing conflict? How journalists intervene in the conflict frame building process

A considerable amount of research is devoted to the presence and effects of conflict frames in the news. However, it is unknown if journalists actively manufacture and inflate conflict in their coverage of politics, or if they merely respond to contentious politics as it happens. This study focuses on the extent to which journalists take an interventionist stance in the conflict frame building process. We conducted expert interviews (N = 16) among Dutch political journalists. Results show that journalists indeed take an active stance in conflict frame building. They contribute to the emergence of conflict frames by using exaggerating language, by orchestrating, and by amplifying possible consequences of political conflict. However, intervention in conflict framing is not merely a result of individual agency of journalists. Rather, some role conceptions seem to counter an interventionist stance. Media routines that are embedded in organizational practices were found to facilitate this active role in conflict framing. Finally, journalists are mainly found to be active when politicians or parties with political power are involved