SerpinB1 protects the mature neutrophil reserve in the bone marrow

SerpinB1 is among the most efficient inhibitors of neutrophil serine proteases--NE, CG, and PR-3--and we investigated here its role in neutrophil development and homeostasis. We found that serpinB1 is expressed in all human bone marrow leukocytes, including stem and progenitor cells. Expression levels were highest in the neutrophil lineage and peaked at the promyelocyte stage, coincident with the production and packaging of the target proteases. Neutrophil numbers were decreased substantially in the bone marrow of serpinB1(-/-) mice. This cellular deficit was associated with an increase in serum G-CSF levels. On induction of acute pulmonary injury, neutrophils were recruited to the lungs, causing the bone marrow reserve pool to be completely exhausted in serpinB1(-/-) mice. Numbers of myeloid progenitors were normal in serpinB1(-/-) bone marrow, coincident with the absence of target protease expression at these developmental stages. Maturation arrest of serpinB1(-/-) neutrophils was excluded by the normal CFU-G growth in vitro and the normal expression in mature neutrophils of early and late differentiation markers. Normal absolute numbers of proliferating neutrophils and pulse-chase kinetic studies in vivo showed that the bone marrow deficit in serpinB1(-/-) mice was largely restricted to mature, postmitotic neutrophils. Finally, upon overnight culture, apoptosis and necrosis were greater in purified bone marrow neutrophils from serpinB1(-/-) compared with WT mice. Collectively, these findings demonstrate that serpinB1 sustains a healthy neutrophil reserve that is required in acute immune responses.

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.

DAPK2 positively regulates motility of neutrophils and eosinophils in response to intermediary chemoattractants

The tight regulation of granulocyte chemotaxis is crucial for initiation and resolution of inflammation. Here, we show that DAPK2, a Ca(2+)/CaM-sensitive serine/threonine kinase known to modulate cell death in various cell types, is a novel regulator of migration in granulocytes. We demonstrate that human neutrophils and eosinophils express DAPK2 but unlike other leukocytes, no DAPK1 or DAPK3 protein. When DAPK activities were blocked by inhibitors, we found that neither granulocyte lifespan nor phagocytosis was affected. However, such pharmacological inactivation of DAPK activity abolished motility of granulocytes in response to intermediary but not end-target chemoattractants ex vivo. The defect in chemotaxis in DAPK2-inactive granulocytes is likely a result of reduced polarization of the cells, mediated by a lack of MLC phosphorylation, resulting in radial F-actin and pseudopod formation. As neutrophils treated with DAPKi also showed reduced recruitment to the site of inflammation in a mouse peritonitis model, DAPK2 may be a novel target for anti-inflammatory therapies.

Rezension zu: Irini Katsirea, Public Broadcasting and European Law

Public broadcasting has always been a regulatory field somewhat zealously guarded within the nation states' sphere and kept willingly untouched by regional or international rules. Values inherent to the role of public broadcasting, such as cultural and national identity, social cohesion, pluralism and a sustained public sphere, were thought too critical and too historically connected with the particular society to allow any "outside" influence. Different regulatory models have emerged to reflect these specificities within the national boundaries of European countries. Yet, as media evolved technologically and economically, the constraints of state borders were rendered obsolete and the inner tension between culture and commerce of the television medium became more pronounced. This tension was only intensified with the formulation of a European Community (EC) layer of regulation, which had as its primary objective the creation of a single market for audiovisual services (or as the EC Directive beautifully put it, a "Television without Frontiers"), while also including some provisions catering for cultural concerns, such as the infamous quota system for European and independent productions. Against this backdrop, public broadcasting makes a particularly intriguing subject for a study of regulatory dilemmas of national versus supranational, integration versus intergovernmentalism, culture versus commerce, intervention versus liberalisation, and all this in the dynamic setting of contemporary media. The present paper reviews Irini Katsirea's book PUBLIC BROADCASTING AND EUROPEAN LAW and seeks to identify whether all elements of the complex governance puzzle of European public service broadcasting rules are analytically well fitted together.

Business as Usual? : The Implementation of the UNESCO Convention on Cultural Diversity and EU Media Law and Policies

Irrespective of the diverse stances taken on the effect of the UNESCO Convention on Cultural Diversity in the external relations context, since its wording is fairly open-ended, it is clear to all observers that the Convention’s impact will largely depend on how it is implemented domestically. The discussion on the national implementation of the Convention, both in the policy and in the academic discourses, is only just emerging, although six years the Convention’s entry into force have passed. The implementation model of the EU can set an important example for the international community and for the other State Parties that have ratified the UNESCO Convention, as both the EU and its Member States acting individually, have played a critical role in the adoption of the Convention, as well as in the longer process of promoting cultural concerns on the international scene. Against this backdrop, this article analyses the extent to which the EU internal law and policies, in particular in the key area of media, take into account the spirit and the letter of the UNESCO Convention on Cultural Diversity. Next to an assessment of the EU’s implementation of the Convention, the article also offers remarks of normative character – in the sense of what should be done to actually attain the objective of protecting and promoting cultural diversity. The article seeks to critically evaluate the present state of affairs and make some recommendations for calibration of future policies.

Inhibition of meningitis-associated neutrophil apoptosis by TNF-α depends on functional PI3-kinase in monocytes

In bacterial meningitis, neutrophils cope with bacterial infection but also lead to tissue damage. The balance of beneficial and harmful effects may depend on the lifespan of the neutrophils in the CNS. Here, we show that CSF of patients with meningococcal meningitis contains a neutrophil apoptosis-inhibiting capacity that correlates with TNF-α content. In vitro experiments show that Neisseria meningitidis as well as LPS derived from these bacteria regulated neutrophil apoptosis mainly by stimulating TNF-α production in monocytes. Whereas LPS-induced PI3K-dependent survival signals in monocytes are critical for neutrophil survival, PI3K signaling in granulocytes did not contribute to the increased lifespan of neutrophils. We conclude that LPS-driven PI3K signaling in monocytes regulates neutrophil apoptosis and thereby, may be crucial in the initiation of secondary brain damage in bacterial meningitis.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

The granulocyte orphan receptor CEACAM4 is able to trigger phagocytosis of bacteria.

Human granulocytes express several glycoproteins of the CEACAM family. One family member, CEACAM3, operates as a single-chain phagocytic receptor, initiating the detection, internalization, and destruction of a limited set of gram-negative bacteria. In contrast, the function of CEACAM4, a closely related protein, is completely unknown. This is mainly a result of a lack of a specific ligand for CEACAM4. By generating chimeric proteins containing the extracellular bacteria-binding domain of CEACAM3 and the transmembrane and cytoplasmic part of CEACAM4 (CEACAM3/4) we demonstrate that this chimeric receptor can trigger efficient phagocytosis of attached particles. Uptake of CEACAM3/4-bound bacteria requires the intact ITAM of CEACAM4, and this motif is phosphorylated by Src family PTKs upon receptor clustering. Furthermore, SH2 domains derived from Src PTKs, PI3K, and the adapter molecule Nck are recruited and associate directly with the phosphorylated CEACAM4 ITAM. Deletion of this sequence motif or inhibition of Src PTKs blocks CEACAM4-mediated uptake. Together, our results suggest that this orphan receptor of the CEACAM family has phagocytic function and prompt efforts to identify CEACAM4 ligands.

Low DICER1 expression is associated with attenuated neutrophil differentiation and autophagy of NB4 APL cells

Successful myeloid differentiation depends on the expression of a series of miRNAs. Thus, it is hardly surprising that miRNAs are globally repressed in AML, a disease mainly characterized by a block in cellular myeloid differentiation. Studies investigating the mechanisms for low miRNA expression in AML has mostly focused on altered transcriptional regulation or deletions, whereas defective miRNA processing has received less attention. In this study, we report that the expression of the key miRNA processing enzyme DICER1 is down-regulated in primary AML patient samples and healthy CD34(+) progenitor cells as compared with granulocytes. In line with these findings, Dicer1 expression was induced significantly in AML cell lines upon neutrophil differentiation. The knocking down of DICER1 in AML cells significantly attenuated neutrophil differentiation, which was paralleled by decreased expression of miRNAs involved in this process. Moreover, we found that inhibiting DICER1 attenuated the activation of autophagy, a cellular recycling process that is needed for proper neutrophil differentiation of AML cells. Our results clearly indicate that DICER1 plays a novel role in neutrophil differentiation as well as in myeloid autophagy of AML cells.

Fire ecology north and south of the Alps since the last ice age

Wildfires are very rare in central Europe, which is probably why fire effects on vegetation have been neglected by most central European ecologists and palaeoecologists. Presently, reconstructions of fire history and fire ecology are almost absent. We analysed sediment cores from lakes on the Swiss Plateau (Lobsigensee and Soppensee) for pollen and charcoal to investigate the relationship between vegetation and fire. Microscopic charcoal evidence suggests increasing regional fire frequencies during the Neolithic (7350-4150 cal. BP, 5400-2200 BC) and the subsequent prehistoric epochs at Lobsigensee, whereas at Soppensee burnings remained rather rare until modern times. Neolithic peaks of charcoal at 6200 and 5500 cal. BP (4250 and 3550 BC) coincided with declines of pollen of fire-sensitive taxa at both sites (e.g., Ulmus, Tilia, Hedera, Fagus), suggesting synchronous vegetational responses to fire at regional scales. However, correlation analysis between charcoal and pollen for the period 6600-4400 cal. BP (4650-2650 BC) revealed no significant link between fire and vegetation at Soppensee, whereas at Lobsigensee increases of Corylus and decreases of Fagus were related to fire events. Fire impact on vegetation increased during the subsequent epochs at both sites. Correlation analyses of charcoal and pollen data for the period 4250-1150 cal. BP (2300 BC -AD 800) suggest that fires were intentionally set to disrupt forests and to provide open areas for arable and pastoral farming (e.g., significant positive correlations between charcoal and Cerealia, Plantago lanceolata, Asteroideae). These results are compared with southern European records (Lago di Origlio, Lago di Muzzano), which are situated in particularly fire-prone environments. After the Mesolithic period (I1 200-7350 cal. BP, 9250-5400 BC), charcoal influx was higher by an order of magnitude in the south, suggesting more frequent fires. Neolithic fires caused similar though more pronounced responses of vegetation in the south (e.g., expansions of Corylus). Post-Neolithic land-use practices involving (controlled) burning culminated in both regions at about 2550 cal. BP (c. 600 BC). However, fire-caused disappearances of entire forest communities were confined to the southern sites. Such differences in fire effects among the sites are explained by the dissimilar importance of fire as a result of different climatic conditions and cultural activities. Our results imply that the remaining (fire-sensitive) fragments of central European vegetation north of the Alps are especially endangered by increasing fire frequencies resulting from predicted climatic change.

Retrospective evaluation of all recorded horse race starts in Switzerland during a four-year period focusing on discipline specific risk factors for clinical events

REASONS FOR PERFORMING THE STUDY: Racetrack injuries are of welfare concern and prevention of injuries is an important goal in many racing jurisdictions. Over the years this has led to more detailed recording of clinical events on racecourses. However, risk factor analyses of clinical events at race meetings have never been reported for Switzerland OBJECTIVE: To identify discipline-specific factors that influence the occurrence of clinical events during race meetings with the ultimate aim to improve the monitoring and safety on racetracks in Switzerland and optimise racehorse welfare. STUDY DESIGN: Retrospective study of horse race data collected by the Swiss horse racing association. METHODS: All race starts (n = 17,670, including 6,198 flat, 1,257 obstacle and 10,215 trot race starts) recorded over a period of four years (2009-2012) were analysed in multivariable mixed effect logistic regression models including horse and racecourse related data. The models were designed to identify discipline specific factors influencing the occurrence of clinical events on racecourses in Switzerland. RESULTS: Factors influencing the risk of clinical events during races were different for each discipline. The risk of a clinical event in trot racing was lower for racing on a Porphyre-sand track than on grass tracks. Horses whose driver was also their trainer had an approximately two times higher risk for clinical events. In obstacle races, longer distances (2401-3300 m and 3301-5400 m respectively) had a protective effect compared to racing over shorter distances. In flat racing, five racecourses reported significantly less clinical events. In all three disciplines, finishing 8th place or later was associated with clinical events. CONCLUSIONS: Changes in management that aim to improve the safety and welfare of racehorses, such as racetrack adaptations, need to be individualised for each discipline.