Prostaglandin E Positively Modulates Endothelial Progenitor Cell Homeostasis: An Advanced Treatment Modality for Autologous Cell Therapy

Aims: The mobilization of endothelial progenitor cells (EPC) and their functioning in postnatal neovascularization are tightly regulated. To identify new modulators of EPC homeostasis, we screened biologically active prostaglandin E compounds for their effects on EPC production, trafficking and function. Methods and Results: We found that EPC are a rich source for prostaglandin E 2 (PGE 2), stimulating their number and function in an auto- and paracrine manner. In vivo blockade of PGE 2 production by selective cyclooxygenase-2 inhibition virtually abrogated ischemia-induced EPC mobilization demonstrating its crucial role in EPC homeostasis following tissue ischemia. Conversely, ex vivo treatment of isolated EPC with the clinically approved PGE 1 analogue alprostadil enhanced EPC number and function. These effects were mediated by increased expression of the chemokine receptor CXCR4 and were dependent on nitric oxide synthase activity. Most importantly, ex vivo PGE 1 pretreatment of isolated EPC significantly enhanced their neovascularization capacity in a murine model of hind limb ischemia as assessed by laser Doppler analysis, exercise stress test and immunohistochemistry. Conclusions: The conserved role for PGE in the regulation of EPC homeostasis suggests that ex vivo modulation of the prostaglandin pathway in isolated progenitor cells may represent a novel and safe strategy to facilitate cell-based therapies. Copyright (C) 2009 S. Karger AG, Base

Frequency response of an atomic force microscope in liquids and air: Magnetic versus acoustic excitation

We discuss the dynamics of an amplitude modulation atomic force microscope in different environments such as water and air. Experiments, analytical expressions, and numerical simulations show that the resonance curves depend on the excitation method used to drive the cantilever, either mechanical or magnetic. This dependence is magnified for small force constants and quality factors, i.e., below 1 N/m and 10, respectively. We show that the equation for the observable, the cantilever deflection, depends on the excitation method. Under mechanical excitation, the deflection involves the base and tip displacements, while in magnetic excitation, the cantilever deflection and tip displacement coincideThis work was financially supported by the European Commission (FORCETOOL) and the Ministerio de Educación y Ciencia (MAT2006-03833). We do thank stimulating discussions with J.R. Lozano, S. Patil and N.F. Martinez.Peer reviewe

Patient-centred ambulatory healthcare for people aged 80 and over

People aged 80 and over are the fastest-growing age group in most industrialised countries. On average, this life phase is characterised by a significantly higher burden of morbidity, limitations in daily activities, medical and dental treatment needs and phenomena such as multimorbidity and frailty. However, individual ageing and health trajectories are highly heterogenous. This challenges current healthcare systems that are still primarily organised around acute care occasions. Ambulatory healthcare is in particular demand as the sector closest to people’s lives and the guarantor to enable ageing in place. By now, ambulatory healthcare providers already face considerable work burdens and are the first to encounter the challenges of this demographic change, especially due to lacking adaptations on the health system level. So far, care models for the improvement of ambulatory healthcare for older people have mainly been developed without their participation. These models primarily focused on structural elements such as coordination to manage the complexity of conditions, with mixed results. A more recent approach to redesigning healthcare is the concept of patient-centred care, which puts the patients with their individual goals, expectations and living realities at the centre of healthcare design. Patient-centred care has gained widespread recognition and can now be considered an overall goal for healthcare. However, few studies have systematically incorporated older people’s views to design patient-centred care. In particular, the group of the oldest old, aged 80 and over, were seldom of interest, despite their rapid growth and special healthcare needs. Moreover, the topic of their oral health and healthcare was rarely included in researching health services. Additionally, the investigation of the perspectives of their healthcare providers is needed to understand the practical reality and to advance the support of an appropriate health workforce for an ageing population. Consequently, this dissertation aimed at investigating what matters in developing patientcentred ambulatory healthcare for people aged 80 and over. Three dissertation projects (DPs) were conducted to examine the views of community-dwelling people aged 80 and over and their healthcare providers regarding ambulatory healthcare comprehensively as well as indepth. In DP1, a systematic review of qualitative studies on the views and experiences of people aged 80 and over regarding ambulatory healthcare was conducted. A meta-synthesis of the 22 included primary studies resulted in the development of three core motives that older people have regarding healthcare: feeling safe, feeling like a meaningful human being, and maintaining control and independence. Parallel to that, a meta-summary of the same set of studies was conducted, resulting in 23 specific desirable features of ambulatory healthcare that were systematically appraised on their confidence in the evidence using the tool GRADE CERQual. In DP2, the findings from DP1 were used to further investigate desirable features of ambulatory healthcare from the perspective of community-dwelling people aged 80 and over in Cologne, Germany. In qualitative interviews using a semi-structured interview guide, 22 participants were asked about their perspectives on general ambulatory healthcare and oral healthcare. The interview transcripts were analysed thematically and resulted in a framework of 16 characteristics of good healthcare for the very old, incorporating oral healthcare equally. The study also revealed that older people particularly value and wish for trustful care relationships, that they are rarely aware of their oral health matters, and that they frequently encounter negative stereotypes of older age in the context of healthcare. In DP3, physicians and dentists providing ambulatory healthcare in the state of North-Rhine Westphalia, Germany, were researched. Using a qualitative survey design in the mode of online data collection, they were asked about their perceptions and views on their routine work and interactions with patients aged 80 and over. The results from 77 cases analysed with the approach of structuring qualitative content analysis showed that the healthcare providers found working with the very old particularly challenging due to their medical complexity and nonmedical demands, such as psychosocial matters. The results from all three DPs were taken together to describe and explain what is relevant in the design of patient-centred ambulatory healthcare for the very old. Apart from features of such healthcare, the dissertation discusses the broader implications in referring to the understanding of health, ageing and the role of healthcare, the further development of patientcentred care and the building of a healthcare workforce for the ageing population

Controlling Dynamic Stability and Active Compliance to Improve Quadrupedal Walking

Summary. It is widespread the idea that animal legged locomotion improves wheeled locomotion on very rough terrain. However, the use of legs as locomotion system for vehicles and robots is still far away from competing with wheels and trucks even on natural ground. Walking robots feature two main disadvantages. One is the lack of reacting capabilities from external disturbances, and the other is the very slow walking motion. Both obstacles prevent walking mechanisms from being introduced in industrial processes and from being part of service and assistance robotics. This paper is aimed at solving the two above obstacles by combining a dynamic stability margin that quantifies the impact energy that a robot can withstand, and either controlling a dynamic walk by means of active compliance, which helps the robot react to disturbances. Experiments performed on the SILO4 quadruped robot show a relevant improvement on the walking gait.This work has been partially funded by CICYT (Spain) through Grant DPI2004-05824. The first author is supported by a postdoctoral CSIC-I3P contract granted by the European Social Fund.Peer reviewe

The SARS-coronavirus-host interactome

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock

Analyse der Funktion der NS-Proteine von klinischen HRSV-Isolaten

HRSV ist eine häufige und weltweit verbreitete Ursache von Infektionen des Respirationstraktes. Es führt zu einer entzündlichen Erkrankung der respiratorischen Schleimhäute mit Mukosaödem, Hypersekretion und Bronchospasmus. Die Übertragung des viralen Erregers erfolgt durch Tröpfcheninfektion oder Kontakt mit kontaminierten Gegenständen. HRSV-Infektionen zeigen die höchste Inzidenz bei Säuglingen, vor allem in den ersten zwei bis sechs Lebensmonaten. Bei 25% bis 40% dieser Säuglinge nimmt die Erkrankung einen schweren Verlauf mit Befall des unteren Respirationstraktes in Form einer HRSV-Bronchiolitis oder -Pneumonie. Bei 0,5% bis 2,0% ist eine stationäre Behandlung im Krankenhaus erforderlich. Die Inzidenz nimmt wegen des zunehmend effektiveren Immunsystems mit dem Alter ab. Erwachsene und ältere Kinder zeigen meist keine Symptome bzw. Symptome einer leichten Erkältung. Reinfektionen im Laufe des Lebens sind häufig. Eine effektive kausale Therapie bei HRSV-Infektionen steht derzeit nicht zur Verfügung. Bei Patienten mit leichtem Krankheitsverlauf ist keine spezielle Behandlung erforderlich, therapiert wird symptomatisch. Aktuell ist keine spezifische Prävention in Form einer aktiven Impfung oder als effektive antivirale Therapie etabliert. Angesichts der hohen Inzidenz von HRSV-Infektionen und -Reinfektionen sowie der enormen gesundheitlichen und wirtschaftlichen Auswirkungen ist ein effektiver Impfstoff gegen HRSV als Forschungsziel vorrangig. Das Genom von HRSV, das zur Ordnung der Mononegavirales gehört, besteht aus einem negativ-orientierten RNA-Einzelstrang mit einer Länge von 15 222 Nukleotiden (beim A2-Stamm) und kodiert für zehn subgenomische mRNAs in der Reihenfolge 3’-leader, NS1, NS2, N, P, M, SH, G, F, M2(1+2), L, trailer-5’, die zur Expression von elf viralen Proteinen führen: fünf RNP-assoziierte Proteine, das sind das Nukleoprotein N, das Phosphoprotein P, die große katalytische Untereinheit L der RNA-Polymerase und der Transkriptionselongationsfaktor M2-1 sowie das nicht essentielle M2-2-Protein; vier Hüllproteine, dazu zählen das nicht-glykosylierte Matrixprotein M und drei Oberflächenproteine, im Einzelnen das Fusionsprotein F, das Anheftungsprotein G und das kleine hydrophobe Protein SH; zwei Nicht-Strukturproteine NS1 und NS2. NS1 und NS2 zeichnen die Pneumoviren vor allen anderen Viren der Ordnung der Mononegavirales aus. Beide NS-Proteine sind im Virion nur in Spuren nachweisbar, während sie in infizierten Zellen akkumulieren. Die beiden für die Proteine NS1 und NS2 kodierenden, nichtüberlappenden Gene liegen am 3‘-Ende des Genoms direkt im Anschluss an die leader-Region. NS1 und NS2 stimmen in den vier carboxyterminalen Aminosäuren überein, ansonsten weisen sie keine Sequenzähnlichkeiten auf. Das NS1-Gen hat eine Länge von 552 nt und kodiert für ein leicht saures Protein von 139 AS und 15,7 kD. Das NS2-Gen ist 503 nt lang und kodiert für ein basisches Protein von 124 AS und 14,7 kD. Die für die Ordnung der Mononegavirales charakteristische progressive Attenuation der Transkription sowie die Genlokalisation von NS1 und NS2 am 3‘-Ende lassen auf die höchste Transkriptionsrate für NS1- und NS2-mRNA unter den zehn HSRV-mRNA schließen, was auf eine bedeutende Rolle der NS1- und NS2-Proteine in infizierten Zellen hindeutet. NS1 und NS2 antagonisieren im Zusammenwirken die durch alpha-IFN und beta-IFN induzierte antivirale Antwort des Wirtsorganismus. Hierfür ist eine Koexpression beider NS-Proteine unbedingt erforderlich, ein NS-Protein allein zeigt keine derartige Aktivität. Der Mechanismus, mit dem HRSV die IFN-Antwort des Wirtsorganismus umgeht, ist unklar. In dieser Arbeit wurde die Funktion der NS-Proteine von klinischen HRSV-Isolaten aus fünf bis fünfzehn Monate alten Kindern untersucht. Durch die Anzucht der klinischen HRSV-Isolate in HEp-2-Zellkultur unter identischen Bedingungen wurden zunächst patientenabhängige Faktoren ausgeschaltet und damit die Grundlage für die Vergleichbarkeit der Wachstumseigenschaften der Isolate geschaffen. In den daraufhin erstellten Wachstumskurven konnten deutlich voneinander abweichende Wachstumverhalten der Isolate aufgezeigt werden. Der Befund, dass 3/4 der Bronchiolitis hervorrufenden HRS-Viren hohe infektiöse Titer (>106 infektiöse Viruspartikel/ ml an Tag 3) erreichten, während dies nur bei 1/3 der Bronchitis verursachenden Viren zu beobachten war, könnte auf eine Korrelation zwischen Wachstum in vitro und Pathogenität in vivo hindeuten. Um dies zu belegen, müsste eine größere Zahl von klinischen Isolaten analysiert werden. Die beiden Nicht-Strukturproteine versetzen HRSV in die Lage, die antivirale IFN-Antwort der Wirtszelle zu umgehen. Durch Behandlung von Virus-infizierten Zellkulturen mit IFN ließ sich nachweisen, dass alle klinischen HRSV-Isolate die Eigenschaft der IFN-Resistenz gleichermaßen besitzen und erst durch unphysiologisch hohe IFN Dosen eine wesentliche Inhibierung der Virusreplikation erreicht werden kann. Die in gleicher Weise ausgeprägte α-IFN-Resistenz bei den in Virulenz und Wachstumsgeschwindigkeit unterschiedlichen Viren deutete bereits darauf hin, dass diese Resistenz essentiell für alle klinischen RSV-Isolate ist, und dass zusätzliche Faktoren für das Maß der Aggressivität der Erreger verantwortlich sind. Mittels Nukleotid- und Aminosäuresequenzanalysen von NS1 und NS2 konnte dies weitgehend bestätigt werden. Anhand von RNA aus den HRSV-Isolaten wurde mit Hilfe des Enzyms Reverse Transkriptase cDNA von NS1 und NS2 synthetisiert, die nach dem Prinzip der PCR in vitro amplifiziert wurde. In anschließenden Klonierungsarbeiten wurden aus dem Vektor pBluescript II SK (–) und NS1-DNA bzw. NS1+NS2-DNA als Insert Plasmide konstruiert, in denen die Gensequenzen von NS1 und NS2 ermittelt und rechnergestützt in die entsprechenden Aminosäuresequenzen translatiert wurden. Die Analyse der NS-Sequenzen zeigte eine überraschend hohe Konservierung. Die Isolate waren einschließlich des Long-Stamms diesbezüglich untereinander sehr ähnlich. Diese Beobachtung stimmt mit der IFN-Resistenz überein und zeigt die Bedeutung der NS-Proteine. Die Ergebnisse dieser Arbeit deuten darauf hin, dass Abweichungen in den Sequenzen der übrigen Gene sowie patientenbezogene Faktoren wie Abwehrlage und anatomische Beschaffenheit des Respirationstraktes als Grund für die Unterschiede im Schweregrad der HRSV-Infektion eine Rolle spielen. Angesichts der stabilen Koexpression beider Nicht-Strukturproteine und des dadurch bedingten effektiven IFN-Escape sichern die Gene NS1 und NS2 die Überlebensfähigkeit von HRSV in vivo und stellen ebenso geeignete wie interessante Angriffspunkte in der Entwicklung eines attenuierten Lebendimpfstoffs dar

The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock

Human rights and climate risks for future generations: How moral obligations and the non-discrimination principle can be applied

From an ethical point of view, preventing the development of conditions that threaten the existence of future generations is a necessity; but to what extent can this argument be made using the language of human rights? I contend in this article that this language can provide us with arguments for extending greater consideration to the risks we may be imposing on future generations and the need for institutional representation of these generations’ interests. The application of a human rights perspective to issues of future concern enables us to formulate obligations to upcoming generations on the part of current ones. Further, I consider how the point in time in which a person is born represents a (morally wrong) ground for discrimination