Structural basis for inflammation-driven shedding of CD163 ectodomain and tumor necrosis factor-α in macrophages

The haptoglobin-hemoglobin receptor CD163 and proTNF-α are transmembrane macrophage proteins subjected to cleavage by the inflammation-responsive protease ADAM17. This leads to release of soluble CD163 (sCD163) and bioactive TNF-α. Sequence comparison of the juxtamembrane region identified similar palindromic sequences in human CD163 ((1044)Arg-Ser-Ser-Arg) and proTNF-α ((78)Arg-Ser-Ser-Ser-Arg). In proTNF-α the Arg-Ser-Ser-Ser-Arg sequence is situated next to the previously established ADAM17 cleavage site. Site-directed mutagenesis revealed that the sequences harbor essential information for efficient cleavage of the two proteins upon ADAM17 stimulation. This was further evidenced by analysis of mouse CD163 that, like CD163 in other non-primates, does not contain the palindromic CD163 sequence in the juxtamembrane region. Mouse CD163 resisted endotoxin- and phorbol ester-induced shedding, and ex vivo analysis of knock-in of the Arg-Ser-Ser-Arg sequence in mouse CD163 revealed a receptor shedding comparable with that of human CD163. In conclusion, we have identified an essential substrate motif for ADAM17-mediated CD163 and proTNF-α cleavage in macrophages. In addition, the present data indicate that CD163, by incorporation of this motif in late evolution, underwent a modification that allows for an instant down-regulation of surface CD163 expression and inhibition of hemoglobin uptake. This regulatory modality seems to have coincided with the evolution of an enhanced hemoglobin-protecting role of the haptoglobin-CD163 system in primate

Apoptose induziert die proteolytische Prozessierung des IL-6 Rezeptors und trägt zum proinflammatorischen Trans-Signalling von Neutrophilen bei

Interleukin-6 (IL-6) trans-signalling has emerged as a prominent regulator of immune responses during both innate and acquired immunity. Regulation of IL-6 trans-signalling is reliant upon the release of soluble IL-6R (sIL-6R), which binds IL-6 to create an agonistic IL-6/sIL-6R complex capable of activating cell types that would normally not respond to IL-6 alone. Soluble IL-6R is generated by proteolytic cleavage of the membrane-anchored precursor (shedding) or from alternative spliced mRNA. Several inducers of IL-6R shedding such as bacterial pore forming toxins, bacterial metalloproteases, the phorbol ester PMA, cholesterol depleting agents and C-reactive protein have been identified in previous studies. IL-6R shedding induced by PMA and cholesterol depletion is dependent on the metalloprotease ADAM17 (A Disintegrin And Metalloprotease), and to a lesser extent on the related sheddase ADAM10. In this study apoptosis was identified as a natural stimulus of IL-6R shedding. Apoptosis, or programmed cell death, plays an important role in regulating growth, tissue homeostasis, development and immune responses. The induction of apoptosis is mediated by extrinsic and intrinsic pathways which require the cooperation of a series of molecules including signal molecules and receptors, enzymes and gene regulating proteins. Among them, the caspase-cascade signalling system is crucial for induction, transduction and amplification of intracellular apoptotic signals. This study demonstrates that intrinsic and extrinsic apoptotic stimulation of cells by DNA-damage, cytokine deprivation, UV irradiation and Fas ligation promotes shedding of the IL-6R from the cell surface thereby generating sIL-6R. Apoptosis-induced shedding of the IL-6R was caspase dependent but PKC, MAPK and ROS independent. Inhibition of ADAM17 during apoptosis by small pharmacological inhibitors or a dominant-negative ADAM17 variant resulted in prevention of IL-6R shedding. The sIL-6R generated during apoptosis was biological active and formed complexes with IL-6 thereby facilitating IL-6 trans-signalling in nonapoptotic cells. Neutrophils have a short life span and die rapidly via apoptosis in vivo and in vitro. In this study neutrophils were used as a cellular model. Induction of apoptosis by Fas-ligation or UV irradiation of primary human neutrophils promoted IL-6R shedding thereby resulting in a two-fold increase of sIL-6R release. Neutrophils represent the first cell population infiltrating inflamed areas and play a crucial role during inflammatory responses due to their extraordinary microbial capacity. Following phagocytosis of invaded microorganisms neutrophils undergo apoptosis as a mechanism to promote resolution of inflammation. The second cell population which migrates into the inflamed area consists mostly of mononuclear phagocytes (monocytes) which are responsible for the clearance of apoptotic neutrophils thus leading to resolution of the inflammatory reaction. Findings in this study indicate that apoptosis-induced shedding of the IL-6R from the neutrophils surface facilitates formation of an [IL-6/sIL-6R] complex which directs IL-6 trans-signalling presumably on endothelial cells to promote recruitment of mononuclear phagocytic cells involved in the nonphlogisic removal of apoptotic neutrophils. Using a mouse model of acute inflammation, it was demonstrated in this study that blocking IL-6R-mediated signal transduction by neutralizing antibodies or soluble gp130-Fc during acute inflammation resulted in a normal influx of neutrophils but a severely impaired influx of monocytes into the inflamed area. Therefore, IL-6R shedding and subsequent IL-6 trans-signalling represent important steps for the resolution of inflammatory responses by controlling the recruitment of mononuclear phagocytes. Thus, shedding of the IL-6R during neutrophil apoptosis may have profound effects on the outcome of the inflammatory response.Interleukin-6 (IL-6) vermitteltes Trans-signalling ist ein wichtiger regulatorischer Signalweg für die Steuerung der angeborenen und erworbenen Immunität. IL-6 Trans-signalling wird durch die Freisetzung vom löslichen Interleukin-6-Rezeptor (sIL-6R) moduliert; dieser bindet an IL-6, um einen IL-6/sIL-6R Komplex zu bilden. Der Komplex aus IL-6 und sIL-6R kann dann Zellen stimulieren, die lediglich gp130 exprimieren und normallerweise nicht in der Lage sind auf IL-6 zu reagieren, da ihnen die ligandenbindende Untereinheit (IL-6R) fehlt. Die lösliche Form des IL-6Rs kann entweder durch limitierte Proteolyse des membranständigen Rezeptors oder durch alternatives Spleißen der IL-6R mRNA generiert werden. In vorherigen Arbeiten konnte bereits gezeigt werden, dass die limitierte Proteolyse des IL-6Rs (Shedding) durch bakterielle porenformende Toxine, bakterielle Metalloproteasen, den Phorbolester PMA, Cholesterinentzug und C-reaktivem Protein induziert werden kann. Shedding des IL-6Rs durch Phorbolester und Cholesterinentzug wird hauptsächlich durch die Metalloprotease ADAM17 (A Disintegrin And Metalloprotease 17) und zum geringen Teil von der verwandten Protease ADAM10 vermittelt. Im Rahmen dieser Arbeit konnte gezeigt werden, dass apoptotische Zellen vermehrt den IL-6R spalten; demnach stellen apoptotische Prozesse einen physiologischen Stimulus für induzierte IL-6R Proteolyse dar. Apoptose wird auch als programmierter Zelltod bezeichnet und spielt in der Regulation von Zellwachstum, Gewebehomöostase und Immunreaktionen eine entscheidende Rolle. Apoptose kann abhängig von der Art des auslösenden Reizes auf verschiedene Wege eingeleitet werden; zum einen durch Aktivierung von Todesrezeptoren auf der Zellmembran und zum anderen durch mitochondrial vermittelte Signalwege. Hauptmerkmal beider Apoptosewege sind Caspasen (Cysteinproteasen), welche die morphologischen und biochemischen Merkmale der Apoptose durch Spaltung von zellulären Bestandteilen hervorrufen. Im Rahmen dieser Arbeit konnte gezeigt werden, dass sowohl todesrezeptoren- als auch mitochondrialvermittelte Apoptose zu vermehrter Proteolyse des IL-6Rs führt. Dabei wurde zelluläre Apoptose durch verschiedene Wege induziert, wie zum Beispiel durch toxische Substanzen, die zur Schädigung des genetischen Materials führen, UV-Bestrahlung, Aktivierung des Todesrezeptors CD95 (Fas) oder Entzug von Überlebensfaktoren. Das durch Apoptose induzierte IL-6R Shedding war Caspase-abhängig jedoch PKC-, MAPK- und ROS- unabhängig. Des Weiteren führte eine Inhibition von ADAM17 durch kleine pharmakologische Inhibitoren oder durch eine dominant-negative ADAM17 Variante während des programmierten Zelltodes zur kompletten Unterbindung von apoptosevermitteltem IL-6R Shedding. Der lösliche IL-6R, welcher während der Apoptose vermehrt gebildet wurde, war biologisch aktiv und konnte im Komplex mit IL-6 in nicht-apoptotischen gp130 exprimierenden Zellen Trans-signalling auslösen. Polymorphkernige Neutrophile sind kurzlebige Zellen mit einer Lebensdauer von 6-8 Stunden; danach sterben Neutrophile durch Apoptose. Im Rahmen dieser Arbeit wurden Neutrophile als zelluläres System verwendet. CD95 (Fas) Aktivierung und UV-Bestrahlung lösten in primären humanen Neutrophilen Apoptose aus, welche mit erhöhtem Shedding des IL-6Rs einherging. Bei Entzündungsprozessen stellen Neutrophile die erste Verteidigungslinie gegen Mikroorganismen dar. Neutrophile phagozytieren Mikroorganismen und töten diese durch vermehrte Produktion von Sauerstoffradikalen ab. Um die inflammatorische Reaktion zu beenden, müssen die aktivierten Neutrophilen eliminiert werden. Dies passiert durch die Einleitung von Apoptose in aktivierten Neutrophilen und anschließender Phagocytose durch mononukleäre Immunzellen (Monocyten), welche als zweite Zellpopulation in den Entzündungsort einwandern. Ergebnisse in dieser Arbeit zeigen, dass apoptotische Neutrophile verstärkt den IL-6R spalten. Dieser bildet einen Komplex mit IL-6. Der Komplex aus sIL-6R/IL-6 stimuliert daraufhin endotheliale Zellen, Mediatoren auszuschütten, die an der Rekrutierung von Monocyten beteiligt sind. Mit Hilfe eines murinen Krankheitsmodelles (Air-pouch-Modell) konnte gezeigt werden, dass Blockierung von sIL-6R-vermittelten Signalwegen durch neutralisierende Antikörper oder rekombinantem löslichen gp130-Fc zu einer stark verminderten Akkumulation von Monocyten in den Entzündungsherd führt. Demnach wird der sIL-6R von apoptotischen Neutrophilen freigesetzt und beeinflusst bei entzündlichen Prozessen die Einwanderung von Monocyten, welche für das Beenden der inflammatorischen Reaktion verantwortlich sind

Light emission efficiency of GdAlO3:Ce (GAP:Ce) powder screens under X-ray radiography conditions

The aim of this study was to evaluate the performance of GdAlO3:Ce powder scintillator screens for use in medical imaging applications. This powder phosphor, also known as GAP:Ce scintillator, is a non-hygroscopic material, emitting blue light with short decay time and it has never been used in medical imaging. Various scintillator screens with coating thickness, ranging from 14.7 to 121.1 mg/cm2, were prepared in our laboratory from GdAlO3:Ce powder (Phosphor Technology, Ltd) by sedimentation on silica substrates. The Quantum Detection Efficiency (QDE) and Energy Absorption Efficiency (EAE) were evaluated. Absolute luminescence efficiency measurements were performed under exposure conditions employed in diagnostic radiology (50-140 kV, 63 mAs), using an experimental setup based on a photomultiplier tube. The emission spectrum of GAP:Ce, after calculating the matching factor, was found compatible with various existing optical detectors. QDE and EAE of GAP, comparing to YAP has exhibited higher values. Absolute Efficiency values were found to increase with increasing X-ray tube voltage. Although for values higher than 120 kVp a decrease was observe

STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.Lukas Kenner and Jan Pencik are supported by FWF, P26011 and the Genome Research-Austria project “Inflammobiota” grants. Helmut Dolznig is supported by the Herzfelder Family Foundation and the Niederösterr. Forschungs-und Bildungsges.m.b.H (nfb). Richard Moriggl is supported by grant SFB-F2807 and SFB-F4707 from the Austrian Science Fund (FWF), Ali Moazzami is supported by Infrastructure for biosciences-Strategic fund, SciLifeLab and Formas, Zoran Culig is supported by FWF, P24428, Athena Chalaris and Stefan Rose-John are supported by the Deutsche Forschungsgemeinschaft (Grant SFB 877, Project A1and the Cluster of Excellence --“Inflammation at Interfaces”). Work of the Aberger lab was supported by the Austrian Science Fund FWF (Projects P25629 and W1213), the European FP7 Marie-Curie Initial Training Network HEALING and the priority program Biosciences and Health of the Paris-Lodron University of Salzburg. Valeria Poli is supported by the Italian Association for Cancer Research (AIRC, No IG13009). Richard Kennedy and Steven Walker are supported by the McClay Foundation and the Movember Centre of Excellence (PC-UK and Movember). Gerda Egger is supported by FWF, P27616. Tim Malcolm and Suzanne Turner are supported by Leukaemia and Lymphoma Research.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms873

Strategic forecasting of GHG intensity : econometrically enhanced LCA for the Singapore-Rotterdam containership green corridor

Green shipping corridors are networks of ports and shipping lanes designated exclusively for low and zero-carbon alternative fuels. By sharing the expenses and benefits of using these fuels, participants in these corridors are incentivized to do so. The Port of Rotterdam and the Maritime and Port Authority of Singapore (MPA) have signed a Memorandum of Understanding (MoU) to establish the world's longest green and digital corridor, facilitating low- and zero-carbon shipping. Currently, there is a lack of studies and research determining the actual green character of this specific green corridor. Here is a forecast based on econometric parameters (GDP, population, TEU demand) and the integration between LCA and the Holt-Winters additive method, evaluating the GHG intensity of bio-methanol, e-methanol, and MDO utilized by an 8000 TEU containership in this green corridor until 2050. It is reported that e-methanol produced by solar power has the lowest WTW emissions with -24,49 g CO2 Eq./ MJ, a value that aligns with the Fuel EU targets until 2045. Starting with Fuel EU, the total amount of penalties for that specific fuel choice is 80.673.138,5 €, and according to the EU ETS, the highest amount needed to be paid for allowances is 4.352.943,4 € in 2045. This study demonstrates the critical importance of the source of electricity during production and the need for additional investments in renewables in Rotterdam and Singapore, as only e-methanol, as opposed to bio-methanol and MDO, can be considered a promising net-zero pathway for shipping and a cost-effective option in the coming years until 2050

Total Quality Management in Higher Education with Balance Scorecard Technique

Abstrac

Mid-term effects of fire on soil properties of North-East Mediterranean ecosystems

Fire is a fundamental ecological process with a long history on Earth, determining the distribution of vegetation formations across the globe. Fire, however, does not only affect the vegetation but also the soil on which vegetation grows, creating a post-fire environment that differs significantly in terms of soil chemical and physical properties from the pre-fire environment. The duration of these alterations remains largely unknown and depends both on the vegetation condition and the fire characteristics. In the current study, we investigate the effect of fire on some chemical and physical properties 11 years after the event in four plant communities. Two of them constitute typical Mediterranean fire-prone plant communities, dominated by sclerophyllous Mediterranean shrubs, such as Quercus coccifera and Q. ilex, while the other two are not considered fire prone and are dominated by deciduous broadleaved species such as Q. petraea and Castanea sativa, respectively. The results indicate that fire affects the soil properties of the various communities in a different manner. Burned sites in the Q. coccifera community have a significantly lower concentration of organic matter, total nitrogen, and available magnesium. At the same time, they have a significantly higher concentration of sand particles and a lower concentration of clay particles. The effect of fire on the soil properties of the other three communities is less dramatic, with differences only in total phosphorus, organic matter, and total nitrogen. The results are discussed in relation to the site conditions and the post-fire regeneration of plant communities

ADAM17 substrate release in proximal tubule drives kidney fibrosis

Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism, we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of a disintegrin and metalloprotease 17 (ADAM17); of its cleavage-activated proligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG); and of the substrates\u27 receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of proinflammatory and profibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorphic mice, specific ADAM17 inhibitor-treated WT mice, or mice with inducible KO of ADAM17 in proximal tubule (Slc34a1-Cre) were significantly protected against these effects. In vitro, in proximal tubule cells, we show that AREG has unique profibrotic actions that are potentiated by TNFα-induced AREG cleavage. In vivo, in acute kidney injury (AKI) and chronic kidney disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a therapeutic target for human kidney fibrosis