Severe Developmental B Lymphopoietic Defects in Foxp3-Deficient Mice are Refractory to Adoptive Regulatory T Cell Therapy

The role of Foxp3-expressing regulatory T (Treg) cells in tolerance and autoimmunity is well-established. However, although of considerable clinical interest, the role of Treg cells in the regulation of hematopoietic homeostasis remains poorly understood. Thus, we analysed B and T lymphopoiesis in the scurfy (Sf) mouse model of Treg cell deficiency. In these experiments, the near-complete block of B lymphopoiesis in the BM of adolescent Sf mice was attributed to autoimmune T cells. We could exclude a constitutive lympho-hematopoietic defect or a B cell-intrinsic function of Foxp3. Efficient B cell development in the BM early in ontogeny and pronounced extramedullary B lymphopoietic activity resulted in a peripheral pool of mature B cells in adolescent Sf mice. However, marginal zone B and B-1a cells were absent throughout ontogeny. Developmental B lymphopoietic defects largely correlated with defective thymopoiesis. Importantly, neonatal adoptive Treg cell therapy suppressed exacerbated production of inflammatory cytokines and restored thymopoiesis but was ineffective in recovering defective B lymphopoiesis, probably due to a failure to compensate production of stroma cell-derived IL-7 and CXCL12. Our observations on autoimmune-mediated incapacitation of the BM environment in Foxp3-deficient mice will have direct implications for the rational design of BM transplantation protocols for patients with severe genetic deficiencies in functional Foxp3+ Treg cells

Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats

A direct pharmacological stimulation of soluble guanylate cyclase (sGC) is an emerging therapeutic approach to the management of various cardiovascular disorders associated with endothelial dysfunction. Novel sGC stimulators, including riociguat (BAY 63-2521), have a dual mode of action: They sensitize sGC to endogenously produced nitric oxide (NO) and also directly stimulate sGC independently of NO. Little is known about their effects on tissue remodeling and degeneration and survival in experimental malignant hypertension.Mortality, hemodynamics and biomarkers of tissue remodeling and degeneration were assessed in Dahl salt-sensitive rats maintained on a high salt diet and treated with riociguat (3 or 10 mg/kg/d) for 14 weeks. Riociguat markedly attenuated systemic hypertension, improved systolic heart function and increased survival from 33% to 85%. Histological examination of the heart and kidneys revealed that riociguat significantly ameliorated fibrotic tissue remodeling and degeneration. Correspondingly, mRNA expression of the pro-fibrotic biomarkers osteopontin (OPN), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in the myocardium and the renal cortex was attenuated by riociguat. In addition, riociguat reduced plasma and urinary levels of OPN, TIMP-1, and PAI-1.Stimulation of sGC by riociguat markedly improves survival and attenuates systemic hypertension and systolic dysfunction, as well as fibrotic tissue remodeling in the myocardium and the renal cortex in a rodent model of pressure and volume overload. These findings suggest a therapeutic potential of sGC stimulators in diseases associated with impaired cardiovascular and renal functions

Liraglutide restores impaired associative learning in individuals with obesity

Survival under selective pressure is driven by the ability of our brain to use sensory information to our advantage to control physiological needs. To that end, neural circuits receive and integrate external environmental cues and internal metabolic signals to form learned sensory associations, consequently motivating and adapting our behaviour. The dopaminergic midbrain plays a crucial role in learning adaptive behaviour and is particularly sensitive to peripheral metabolic signals, including intestinal peptides, such as glucagon-like peptide 1 (GLP-1). In a single-blinded, randomized, controlled, crossover basic human functional magnetic resonance imaging study relying on a computational model of the adaptive learning process underlying behavioural responses, we show that adaptive learning is reduced when metabolic sensing is impaired in obesity, as indexed by reduced insulin sensitivity (participants: N = 30 with normal insulin sensitivity; N = 24 with impaired insulin sensitivity). Treatment with the GLP-1 receptor agonist liraglutide normalizes impaired learning of sensory associations in men and women with obesity. Collectively, our findings reveal that GLP-1 receptor activation modulates associative learning in people with obesity via its central effects within the mesoaccumbens pathway. These findings provide evidence for how metabolic signals can act as neuromodulators to adapt our behaviour to our body’s internal state and how GLP-1 receptor agonists work in clinics

Induced B Cell Development in Adult Mice

We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed

RNAi revised - Target mRNA-dependent enhancement of gene silencing

The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy. We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with highmultiple turnover rates of RNAibased gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery

Cellular delivery of small interfering RNA by a non-covalently attached cell-penetrating peptide: quantitative analysis of uptake and biological effect

Cell-penetrating peptides (CPPs) have evolved as promising new tools to deliver nucleic acids into cells. So far, the majority of these delivery systems require a covalent linkage between carrier and cargo. To exploit the higher flexibility of a non-covalent strategy, we focused on the characterisation of a novel carrier peptide termed MPGα, which spontaneously forms complexes with nucleic acids. Using a luciferase-targeted small interfering RNA (siRNA) as cargo, we optimised the conditions for MPGα-mediated transfection of mammalian cells. In this system, reporter gene activity could be inhibited up to 90% with an IC(50) value in the sub-nanomolar range. As a key issue, we addressed the cellular uptake mechanism of MPGα/siRNA complexes applying various approaches. First, transfection of HeLa cells with MPGα/siRNA complexes in the presence of several inhibitors of endocytosis showed a significant reduction of the RNA interference (RNAi) effect. Second, confocal laser microscopy revealed a punctual intracellular pattern rather than a diffuse distribution of fluorescently labelled RNA-cargo. These data provide strong evidence of an endocytotic pathway contributing significantly to the uptake of MPGα/siRNA complexes. Finally, we quantified the intracellular number of siRNA molecules after MPGα-mediated transfection. The amount of siRNA required to induce half maximal RNAi was 10 000 molecules per cell. Together, the combination of methods provided allows for a detailed side by side quantitative analysis of cargo internalisation and related biological effects. Thus, the overall efficiency of a given delivery technique as well as the mechanism of uptake can be assessed

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Development of a score to determine depth of sedation of patients on ICUs

Eines der wichtigen Säulen der intensivmedizinischen Therapie stellt die Analgosedierung von Intensivpatienten dar. Die Einschätzung der optimalen Sedierungstiefe stellt den Intensivmediziner vor eine wichtige Herausforderung. Somit bedarf es einer Überwachung der Sedierungstiefe. Diese erfolgt zu einem anhand von sekundären klinischen Parametern, wie zum Beispiel die Herzfrequenz und der arterielle Blutdruck, die aber sehr störanfällig sind. Ein Möglichkeit besteht in der Verwendung von Sedationsscores, die bislang aber nur unzureichend auf Validität, Reliabilität und Responsiveness untersucht sind. Somit erscheint es sinnvoll, bisher angewandte Scores auf die wichtigen Inhalte zu prüfen und daraus einen neuen Sedationsscore zu entwickeln, der diesen Herausforderungen gewachsen ist. Die Entwicklung des Vigilanzscores fand auf zwei Intensivstationen der Universitätsklinik Würzburg an 12 weiblichen und 42 männlichen Patienten (Alter 64 ± 5,2 Jahre) statt. Dabei wurden in unterschiedlichen Wachheitsgraden der Patienten 352 Einzelmessungen durchgeführt. Im nächsten Arbeitsschritt wurde die Endversion des Vigilanzscores auf drei Intensivstationen hinsichtlich seiner Reliabilität, Validität und Akzeptanz an 86 männlichen und 34 weiblichen Patienten mithilfe eines Vergleiches mit vier veröffentlichten Sedationsscores untersucht und bewertet. Die Prediction Probability PK war für den MAAS mit 0,95 am höchsten. Dies bedeutet, dass in 95% der Fälle eine Veränderung der Sedierungstiefe, gemessen mit dem Vigilanzscore, mit der Veränderung der Sedierungstiefe, gemessen mit der MAAS, übereinstimmt. Für die RS und SAS ergaben sich eine PK von 0,93 und für die VAS eine PK von 0,89. In der Relibitlitätsuntersuchung wurde gezeigt, dass bei der klinischen Einschätzung eines Patienten durch zwei Untersucher sich in 90% der Fälle eine exakte Übereinstimmung im Vigilanzscore ergab. In 95,8% der Fälle differierte die Einschätzung um höchstens eine Stufe. Der Cohen-Kappa-Koeffizient &#954; betrug 0,89 (p < 0,001). In der Akzeptanzuntersuchung wurde die Scores mit Schulnoten von 1 bis 6 vom zweiten Untersucher bewertet. Der Vigilanzscore erhielt eine Benotung von 2,4 ± 0,95. Mit dem Vigilanzscore wurde somit ein Score mit guter Validität und Reliabilität, sowie mit zufriedenstellender Akzeptanz entwickelt. Dies liegt möglicherweise am ausführlichen Aufbaus des Scores, der aber wiederum Voraussetzung für eine gute Responsiveness, also der Fähigkeit auch kleine Veränderungen am Patienten zu erkennen, ist.The purpose of this study was to develop a sedation score for the use on an ICU for health care providers. Formerly published sedation score lack the investigation of validity, reliability and responsiveness. In the first step we developed the “Vigilanzscore” in 352 measurements on two ICU of the University of Wuerzburg. In the second step the Vigilanzscore was examined on 120 patients concerning its validity, reliability and responsiveness by comparing with four published sedation scales (MAAS, Ramsay-Scale, SAS and VS). The prediction probability PK for MAAS was highest with 0,95 meaning that the Vigilanzscore can predict the depth of sedation measured by MAAS in 95% correctly. PK for Ramsay Scale and SAS were 0,93. On the reliability we showed that there was an exact agreement between two investigators on rating a patient’s sedation state in 90% and including a difference of one step of the Vigilance in 95,8%. Cohen’s-Kappa was 0,89 (p < 0,001). Testing the acceptance of the Vigilanzscore for health care providers the Vigilanzscore was rated with 2,4 (in German school marks). As a conclusion we developed a sedation score with good validity and reliability as well as satisfying acceptance