Investigation of the biochemistry and function of neutrophil serine protease 4 (NSP4)

Serine proteases in cytoplasmic granules of neutrophils (NSPs), namely neutrophil elastase, cathepsin G (CG) and proteinase 3, have been under intense investigation for several decades. They are mainly known for their role in intracellular killing of pathogens and are also increasingly recognized as key regulators of innate immune responses. In 2009, I identified a fourth serine protease in neutrophils that has been completely overlooked and neglected so far. The aim of this thesis was an in-depth biochemical and functional characterization of this novel serine protease 4 (NSP4) of human neutrophils. Using monoclonal antibodies to NSP4, the distribution of NSP4 in normal human tissues was studied. NSP4 was observed only in neutrophils and neutrophil precursors of the bone marrow. The content of NSP4 in neutrophil lysates was about 20-fold lower compared to CG. Nevertheless, NSP4 was found to be released into the supernatant upon neutrophil activation. NSP4 could be further identified as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions. For the functional analysis, the production and yield of recombinant NSP4 was clearly improved using different expression systems and DNA construct modifications. The proteolytic specificity was analyzed using E. coli peptide libraries, mass spectrometry and several synthetic peptide libraries. All these analyses clearly revealed an arginine specificity for NSP4. Consistent with this, NSP4 was strongly inhibited by heparin-accelerated antithrombin and C1 inhibitor and, with lower efficacy, by α1-proteinase inhibitor (α1PI). The data allowed me to generate an NSP4-specific α1PI variant that was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils. This finding strongly indicated that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. In addition, dipeptidyl peptidase I (DPPI) was identified as the activator of NSP4 in vivo, as DPPI deficiency resulted in complete absence of NSP4 in a Papillon-Lefèvre patient. Analysis of cell-based calcium assays revealed that proteinase-activated receptor-2 may represent a potential natural substrate of NSP4. So far, NSP4-deficient mice did not show an abnormal phenotype under clean housing conditions. Activation of isolated neutrophils by phorbol esters or immune complexes was also not impaired. This study establishes NSP4 as the only arginine-specific pre-activated serine protease stored in azurophil granules of neutrophils that may fullfil a quite distinct, supportive role in neutrophil responses to tissue damage and bacterial infections.Die Granula-assoziierten Serinproteasen der Neutrophilen, genannt Neutrophilen-Elastase, Cathepsin G (CG) und Proteinase 3, werden bereits seit über 30 Jahren intensiv erforscht. Sie sind hauptsächlich für ihre Funktion bei der intrazellulären Degradation von pathogenen Keimen bekannt. Außerdem werden sie zunehmend als wichtige Regulatoren der angeborenen Immunantwort angesehen. Im Jahr 2009 identifizierte ich eine vierte Serinprotease in Neutrophilen, die bis dahin komplett übersehen wurde. Das Ziel dieser Arbeit war eine ausführliche Untersuchung der Biochemie und Funktion dieser neuen Serinprotease 4 (NSP4) in humanen Neutrophilen. NSP4 konnte in immunhistochemischen Analysen normaler Gewebe nur in Neutrophilen und deren Vorläuferzellen im Knochenmark nachgewiesen werden. Die Menge von NSP4 im Gesamtzelllysat von Neutrophilen war 20-fach geringer als die von CG. Dennoch konnte ich nachweisen, dass NSP4 von aktivierten Neutrophilen sezerniert wird. In subzellulären Neutrophilen-Fraktionen war NSP4 in erster Linie mit azurophilen Granula assoziiert. Die Produktion und Ausbeute rekombinanter, aktiver NSP4 wurde mithilfe verschiedener DNA-Konstrukte und Expressionssysteme deutlich verbessert. Für die Bestimmung der proteolytischen Spezifität wurden E. coli Peptidbibliotheken, Massenspektrometrie und synthetische Peptidbibliotheken eingesetzt. Alle Ergebnisse ergaben eindeutig eine Arginin-Spezifität für NSP4. NSP4 wurde sehr gut von Heparin-Antithrombin, C1 Inhibitor und mit geringerer Effizienz von α1-Proteinase-Inhibitor (α1PI) blockiert. Die Ergebnisse ermöglichten die Herstellung einer NSP4-spezifischen α1PI Variante, die nachweislich mit der gesamten NSP4 aus Neutrophilen-Zelllysat und aus Überständen aktivierter Neutrophilen einen kovalenten Komplex bildete. Dies war ein klarer Hinweis für die Speicherung und Sekretion der NSP4 als aktive Endoprotease. Außerdem konnte ich zeigen, dass NSP4 in vivo von Dipeptidylpeptidase I (DPPI) aktiviert wird, da in einem Papillon-Lefèvre Patienten ohne funktionelle DPPI auch NSP4 nicht nachweisbar war. Intrazelluläre Kalziummessungen in HaCaT-Zellen ergaben, dass der Proteinase-aktivierte Rezeptor-2 ein mögliches natürliches Substrat für NSP4 sein könnte. In einer Pathogen-freien Reinraumumgebung zeigten NSP4-defiziente Mäuse bislang noch keinen Phänotyp. Auch die Aktivierung isolierter Neutrophilen durch Phorbolester oder Immunkomplexe war nicht beeinträchtigt. In dieser Arbeit gelang es mir, die erste aktive Arginin-spezifische Serinprotease (NSP4) in azurophilen Granula von Neutrophilen zu identifizieren. Wegen ihres Vorkommens in allen sequenzierten Genomen höherer Vertebraten könnte NSP4 eine wichtige Rolle bei der Steuerung und Verstärkung Neutrophilen-abhängiger Immunantworten zukommen

Machine learning algorithms performed no better than regression models for prognostication in traumatic brain injury

Objective: We aimed to explore the added value of common machine learning (ML) algorithms for prediction of outcome for moderate and severe traumatic brain injury. Study Design and Setting: We performed logistic regression (LR), lasso regression, and ridge regression with key baseline predictors in the IMPACT-II database (15 studies, n = 11,022). ML algorithms included support vector machines, random forests, gradient boosting machines, and artificial neural networks and were trained using the same predictors. To assess generalizability of predictions, we performed internal, internal-external, and external validation on the recent CENTER-TBI study (patients with Glasgow Coma Scale <13, n = 1,554). Both calibration (calibration slope/intercept) and discrimination (area under the curve) was quantified. Results: In the IMPACT-II database, 3,332/11,022 (30%) died and 5,233(48%) had unfavorable outcome (Glasgow Outcome Scale less than 4). In the CENTER-TBI study, 348/1,554(29%) died and 651(54%) had unfavorable outcome. Discrimination and calibration varied widely between the studies and less so between the studied algorithms. The mean area under the curve was 0.82 for mortality and 0.77 for unfavorable outcomes in the CENTER-TBI study. Conclusion: ML algorithms may not outperform traditional regression approaches in a low-dimensional setting for outcome prediction after moderate or severe traumatic brain injury. Similar to regression-based prediction models, ML algorithms should be rigorously validated to ensure applicability to new populations

Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good

Search for stop and higgsino production using diphoton Higgs boson decays

Results are presented of a search for a "natural" supersymmetry scenario with gauge mediated symmetry breaking. It is assumed that only the supersymmetric partners of the top-quark (stop) and the Higgs boson (higgsino) are accessible. Events are examined in which there are two photons forming a Higgs boson candidate, and at least two b-quark jets. In 19.7 inverse femtobarns of proton-proton collision data at sqrt(s) = 8 TeV, recorded in the CMS experiment, no evidence of a signal is found and lower limits at the 95% confidence level are set, excluding the stop mass below 360 to 410 GeV, depending on the higgsino mass

Severe early onset preeclampsia: short and long term clinical, psychosocial and biochemical aspects

Preeclampsia is a pregnancy specific disorder commonly defined as de novo hypertension and proteinuria after 20 weeks gestational age. It occurs in approximately 3-5% of pregnancies and it is still a major cause of both foetal and maternal morbidity and mortality worldwide1. As extensive research has not yet elucidated the aetiology of preeclampsia, there are no rational preventive or therapeutic interventions available. The only rational treatment is delivery, which benefits the mother but is not in the interest of the foetus, if remote from term. Early onset preeclampsia (<32 weeks’ gestational age) occurs in less than 1% of pregnancies. It is, however often associated with maternal morbidity as the risk of progression to severe maternal disease is inversely related with gestational age at onset2. Resulting prematurity is therefore the main cause of neonatal mortality and morbidity in patients with severe preeclampsia3. Although the discussion is ongoing, perinatal survival is suggested to be increased in patients with preterm preeclampsia by expectant, non-interventional management. This temporising treatment option to lengthen pregnancy includes the use of antihypertensive medication to control hypertension, magnesium sulphate to prevent eclampsia and corticosteroids to enhance foetal lung maturity4. With optimal maternal haemodynamic status and reassuring foetal condition this results on average in an extension of 2 weeks. Prolongation of these pregnancies is a great challenge for clinicians to balance between potential maternal risks on one the eve hand and possible foetal benefits on the other. Clinical controversies regarding prolongation of preterm preeclamptic pregnancies still exist – also taking into account that preeclampsia is the leading cause of maternal mortality in the Netherlands5 - a debate which is even more pronounced in very preterm pregnancies with questionable foetal viability6-9. Do maternal risks of prolongation of these very early pregnancies outweigh the chances of neonatal survival? Counselling of women with very early onset preeclampsia not only comprises of knowledge of the outcome of those particular pregnancies, but also knowledge of outcomes of future pregnancies of these women is of major clinical importance. This thesis opens with a review of the literature on identifiable risk factors of preeclampsia

Search for anomalous production of events with three or more leptons in pp collisions at √s = 8TeV

Published by the American Physical Society under the terms of the Creative Commons Attribution 3.0 License. Further distribution of this work must maintain attribution to the author(s) and the published articles title, journal citation, and DOI.A search for physics beyond the standard model in events with at least three leptons is presented. The data sample, corresponding to an integrated luminosity of 19.5fb-1 of proton-proton collisions with center-of-mass energy s=8TeV, was collected by the CMS experiment at the LHC during 2012. The data are divided into exclusive categories based on the number of leptons and their flavor, the presence or absence of an opposite-sign, same-flavor lepton pair (OSSF), the invariant mass of the OSSF pair, the presence or absence of a tagged bottom-quark jet, the number of identified hadronically decaying τ leptons, and the magnitude of the missing transverse energy and of the scalar sum of jet transverse momenta. The numbers of observed events are found to be consistent with the expected numbers from standard model processes, and limits are placed on new-physics scenarios that yield multilepton final states. In particular, scenarios that predict Higgs boson production in the context of supersymmetric decay chains are examined. We also place a 95% confidence level upper limit of 1.3% on the branching fraction for the decay of a top quark to a charm quark and a Higgs boson (t→cH), which translates to a bound on the left- and right-handed top-charm flavor-violating Higgs Yukawa couplings, λtcH and λctH, respectively, of |λtcH|2+|λctH|2<0.21

MUSiC : a model-unspecific search for new physics in proton-proton collisions at root s=13TeV

Results of the Model Unspecific Search in CMS (MUSiC), using proton-proton collision data recorded at the LHC at a centre-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1), are presented. The MUSiC analysis searches for anomalies that could be signatures of physics beyond the standard model. The analysis is based on the comparison of observed data with the standard model prediction, as determined from simulation, in several hundred final states and multiple kinematic distributions. Events containing at least one electron or muon are classified based on their final state topology, and an automated search algorithm surveys the observed data for deviations from the prediction. The sensitivity of the search is validated using multiple methods. No significant deviations from the predictions have been observed. For a wide range of final state topologies, agreement is found between the data and the standard model simulation. This analysis complements dedicated search analyses by significantly expanding the range of final states covered using a model independent approach with the largest data set to date to probe phase space regions beyond the reach of previous general searches.Peer reviewe

Measurement of prompt open-charm production cross sections in proton-proton collisions at root s=13 TeV

The production cross sections for prompt open-charm mesons in proton-proton collisions at a center-of-mass energy of 13TeV are reported. The measurement is performed using a data sample collected by the CMS experiment corresponding to an integrated luminosity of 29 nb(-1). The differential production cross sections of the D*(+/-), D-+/-, and D-0 ((D) over bar (0)) mesons are presented in ranges of transverse momentum and pseudorapidity 4 < p(T) < 100 GeV and vertical bar eta vertical bar < 2.1, respectively. The results are compared to several theoretical calculations and to previous measurements.Peer reviewe