Oberflächenexpression der AMP-Desaminase 2 auf humanen Immunzellen und ihre Rolle im extrazellulären Purinmetabolismus

Extrazelluläre Purine tragen maßgeblich zur Regulation des inflammatorischen Milieus bei: Während ATP als proinflammatorisches Alarmin wirkt, vermittelt Adenosin entzündungshemmende Effekte. Das Gleichgewicht dieser Adeninmetabolite wird im Wesentlichen durch die Ektonukleotidasen Ektonukleosidtriphosphatdiphosphohydrolase 1 (CD39) und Ekto-5'- Nukleotidase (CD73) vermittelt, die ATP über AMP zu Adenosin verstoffwechseln. AMP-Desaminasen (AMPD) sind Enzyme des intrazellulären Purinstoffwechsels, die die Desaminierung von AMP zu IMP katalysieren. Damit erleichtern sie einerseits den Abbau von ATP und verringern andererseits den AMP-Bestand für die Adenosin-Produktion durch CD73. An der Zelloberfläche humaner Immunzellen ist dieser Mechanismus bisher nicht beschrieben. Ziel dieser Arbeit war die Analyse der Expression von AMPD2 an der Oberfläche humaner Leukozyten sowie deren Regulation unter inflammatorischen Bedingungen. Mittels Immunpräzipitation (IP) aus Membranfraktionen sowie durch Anreicherung des Oberflächenproteins mittels Oberflächenbiotinylierung wiesen wir AMPD2 in der Plasmamembran von HEK293 Zellen, HMEC-1 Zellen, U-937 Zellen und CD14+ Monozyten nach. Der Proteinnachweis erfolgte mittels Western Blot und Massenspektrometrie. Zusätzlich visualisierten wir die Oberflächenexpression immunfluoreszenzmikroskopisch auf U-937 Zellen sowie auf humanen mononukleären Zellen aus dem peripheren Blut (PBMCs). Durchflusszytometrisch zeigten wir eine signifikante Steigerung der Expression der oberflächlichen AMPD2 (eAMPD2) auf humanen Monozyten nach Toll-like-Rezeptor (TLR)-Stimulation (p=0,0078) sowie eine Reduktion dieser durch Inhibition des sekretorischen Wegs (p=0,0078). Durch die zusätzliche Behandlung mit Dexamethason (Dex) ließ sich der Effekt der Immunstimulation teilweise reduzieren. Im Vergleich zu bezüglich Geschlecht und Alter vergleichbaren Kontrollen zeigten PBMCs von Patient*innen mit rheumatoider Arthritis (RA) eine erhöhte Grundexpression von eAMPD2. Eine erhöhte eAMPD2-Expression auf CD14+ Monozyten war nicht mit einer Reduktion der extrazellulären Adenosinkonzentration (eADO) verbunden. Hingegen konnten wir das anti-inflammatorische Potential von extrazellulärem IMP im Sinne einer Reduktion der Sekretion von Tumornekrosefaktor-alpha (TNF-α) durch PBMCs nachweisen. Zusammenfassend identifiziert diese Arbeit erstmals eAMPD2 als Ektoenzym auf humanen Immunzellen als neuen Regulator des extrazellulären Purinstoffwechsels. Durch AMP-Verbrauch könnte die Desaminase den Abbau von proinflammatorischem ATP erleichtern und das bestehende System der Ektonukleotidasen CD39 und CD73 um die Produktion des langlebigen anti-inflammatorischen Moleküls IMP ergänzen.Extracellular purine metabolites are powerful mediators of the inflammatory milieu: ATP acts as a pro-inflammatory alarmin, while adenosine represents a potent anti-inflammatory molecule. The ectonucleotidases ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73) regulate the balance of these metabolites by catalysing the sequential degradation of ATP to adenosine via AMP. Intracellular AMP deaminases (AMPD) mediate AMP deamination to IMP. Thereby, they facilitate degradation of ATP on the one hand and impair adenosine production by reducing AMP supply for CD73 on the other hand. The mechanism of AMP deamination has not yet been recognised on the surface of immune cells. This study examined the surface expression of AMPD2 in human leukocytes and its modification under inflammatory conditions. We performed immunoprecipitation (IP) from membrane fractions as well as enrichment of surface protein by surface biotinylation to detect AMPD2 in the plasma membrane of HEK293 cells, HMEC-1 cells, U-937 cells and CD14+ monocytes. The enzyme was identified by western blot and mass spectrometry. Additionally, surface expression was visualised with the help of immunofluorescence microscopy of U-937 cells and human peripheral blood mononuclear cells (PBMCs). Flow cytometry revealed a significant increase in surface AMPD2 (eAMPD2) expression on human monocytes after toll-like receptor (TLR) stimulation (p=0.0078), while a reduction was observed after Golgi transport inhibition (p=0.0078). Concomitant incubation with dexamethasone (Dex) partly prevented the increase caused by immunostimulation. PBMCs from patients with rheumatoid arthritis (RA) showed an elevated expression of eAMPD2 compared to sex- and age-matched healthy controls. An increase in eAMPD2 expression on CD14+ monocytes was not associated with reduced levels of extracellular adenosine (eADO). On the other hand, we verified the anti-inflammatory potential of extracellular IMP by demonstrating a reduction in tumor necrosis factor-alpha (TNF-α) secretion from PBMCs. In summary, this study shows AMPD2 surface expression on human immune cells for the first time and thereby identifies a novel ectoenzyme in the extracellular purine metabolism. We hypothesise that the deaminase enhances the degradation of pro-inflammatory ATP by removing AMP from the extracellular space and adds the production of long-lived anti-inflammatory IMP to the existing system of ectonucleotidases

The Forgotten Kingdom.: New investigations in the prehistory of Eswatini

The kingdom of Eswatini provides a rich archaeological sequence covering all time periods from the Early Stone Age to the Iron Age. For over 27 years though, no or very little archaeological research was conducted in the country. In the scope of a new project funded by the German Research Foundation (DFG) we aim to re-excavate and re-date Lion Cavern, the potentially oldest ochre mine in the world. In addition, we conduct a largescale geological survey for outcrops of ochre and test their geochemical signatures for comparative studies with archaeological ochre pieces from MSA and LSA assemblages in Eswatini. Here we present a review of the research history of the kingdom and some preliminary results from our ongoing project

Assessment of the quality of mini-HTA

Objectives: Mini-HTA (health technology assessment) is increasingly being applied in Denmark as an input for decisions on the use of health technologies. Mini-HTA is a form or check list with questions concerning the prerequisites for and consequences of health technologies. At the national level, the National Board of Health uses mini-HTA when hospitals apply for permission to introduce new treatments. Mini-HTA is also compulsory in Danish Regions' annual collection of early warnings. At the local level some hospitals have made mini-HTA compulsory when clinical departments apply for funding for new technologies. The objective of this study is to assess the quality of the information included in mini-HTA used at Danish hospitals and to discuss the consequences of this to decision making. Methods: The quality of mini-HTA is assessed by use of an INATHA checklist for HTA reports. Data consists of reviews of the quality in fifty-two mini-HTAs produced by Danish hospitals in 2008. Results: The mini-HTAs generally include descriptions of the assessed technology and the comparator, but information about the selection and interpretation of the clinical literature and other data is often missing. The level of evidence for the clinical effects and the main references are generally included. Only 25 percent of the mini-HTAs include a quantitative estimate of the size of the clinical effects. Organizational consequences inside the clinical department is described in 81percent of the cases and 92 percent includes a cost estimate. Conclusions: The results show that the quality of the information in many cases is insufficient. There is a strong need for quality assurance of mini-HTAs to improve the accuracy of the information, however, without harming the timeliness and the limited use of resources in producing the reports

Status Quo of Progress Testing in Veterinary Medical Education and Lessons Learned

Progress testing is an assessment tool for longitudinal measurement of increase in knowledge of a specific group, e.g., students, which is well-known in medical education. This article gives an overview of progress testing in veterinary education with a focus on the progress test of the German-speaking countries. The "progress test veterinary medicine" (PTT) was developed in 2013 as part of a project by the Competence Centre for E-Learning, Didactics and Educational Research in Veterinary Medicine-a project cooperation of all German-speaking institutes for veterinary medicine in Germany, Austria, and Switzerland. After the end of the project, the PTT was still continued at six locations, at each of the five German schools for veterinary medicine and additionally in Austria. Further changes to the PTT platform and the analysis were carried out to optimize the PTT for continuing to offer the test from 2017 to 2019. The PTT is an interdisciplinary, formative electronic online test. It is taken annually and is composed of 136 multiple-choice single best answer questions. In addition, a "don't know" option is given. The content of the PTT refers to the day 1 competencies described by the European Association of Establishments for Veterinary Education. The platform Q-Exam (R) Institutions (IQuL GmbH, Bergisch Gladbach, Germany) is used for creating and administrating the PTT questions, the review processes and organizing of the online question database. After compiling the test by means of a blueprint, the PTT file is made available at every location. After the last PTT in 2018, the link to an evaluation was sent to the students from four out of these six partner Universities. The 450 analyzed questionnaires showed that the students mainly use the PTT to compare their individual results with those of fellow students in the respective semester. To conclude our study, a checklist with our main findings for implementing progress testing was created

Toll-Like Receptor 7 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus. Toll-like receptor 7 (TLR7) is involved in host innate immunity against pathogens, and its aberrant activation is linked to the development of systemic lupus erythematosus (SLE, also called ‘‘lupus’’). Type I interferons (IFN) are apparently driving forces for lupus pathogenesis. Previously, we found that EBV latent membrane protein 1 (LMP1) primes cells for IFN production. In this report, the relationship among EBV LMP1, TLRs, and IFN production are examined. We find that TLR7 activation increases the expression of EBV LMP1, and IFN regulatory factor 7 (IRF7) is involved in the stimulation process. TLR7 activation did not induce IFNs from EBV-infected cells, but potentiates those cells for IFN production by TLR3 or TLR9 activation. In addition, we find that LMP1 and IFNs are coexpressed in the same cells in some lupus patients. Therefore, the aberrant activation of TLR7 might induce LMP1 expression and LMP1-expression cells may be producing IFNs in lupus patients. These results suggest EBV might be an exacerbating factor in some lupus patients via promoting IFN production

Safety and Efficacy of Risuteganib in Intermediate Non-exudative Age-Related Macular Degeneration

Purpose : Risuteganib is a small synthetic peptide that regulates select integrin functions involved in the pathogenesis of dry age-related macular degeneration (AMD). This study evaluated the safety and efficay of risuteganib for the treatment of dry AMD. Methods : Randomized, double-masked, placebo-controlled Phase 2 study in eyes with intermediate dry AMD presenting with best-corrected visual acuity (BCVA) between 20/40-20/200 was conducted across multiple centers in the United States. Patients were randomized to receive either intravitreal 1.0mg risuteganib or sham injection at baseline. At week 16, patients in the risuteganib group received a second dose and the sham group crossed over and receive a single dose of 1.0mg risuteganib. The primary endpoint was the percentage of population with ≥ 8 letters BCVA gain from baseline to week 28 in 1.0mg risuteganib vs baseline to week 12 for sham. Results : Forty-five patients were enrolled in the study. At baseline, mean patient age was 78.8 and 75.9 years and mean baseline BCVA was 67.1 and 64.4 letters in the sham and risuteganib groups, respectively. The primary endpoint was met; 48% of patients in the risuteganib group at week 28 and 7% of patients in the sham group at week 12 gained > 8 letters from baseline (p=0.013). Of the risuteganib treated patients, 20% gained > 15 letters at week 28; no patients in the sham group at week 12 had this gain. On a post-hoc masked analysis by 2 independent reading centers, greater outer retinal and photoreceptor thickness and volume and smaller ellipsoid zone defect area in the central 1 mm zone at baseline were associated with increased BCVA response to risuteganib. Risuteganib demonstrated a good safety profile in this study. Conclusions : Risuteganib showed significant benefit over sham in patients with dry AMD with respect to proportion of patients gaining > 8 letters of BCVA from baseline. Furthermore, post hoc analysis provides preliminary insights into baseline anatomic features that may help to determine likelihood of BCVA response to risuteganib. These findings will be confirmed in an upcoming larger trial

Age-related increase of mitochondrial content in human memory CD4+ T cells contributes to ROS-mediated increased expression of proinflammatory cytokines

Cellular metabolism modulates effector functions in human CD4+ T (Th) cells by providing energy and building blocks. Conversely, cellular metabolic responses are modulated by various influences, e.g., age. Thus, we hypothesized that metabolic reprogramming in human Th cells during aging modulates effector functions and contributes to “inflammaging”, an aging-related, chronic, sterile, low-grade inflammatory state characterized by specific proinflammatory cytokines. Analyzing the metabolic response of human naive and memory Th cells from young and aged individuals, we observed that memory Th cells exhibit higher glycolytic and mitochondrial fluxes than naive Th cells. In contrast, the metabolism of the latter was not affected by donor age. Memory Th cells from aged donors showed a higher respiratory capacity, mitochondrial content, and intracellular ROS production than those from young donors without altering glucose uptake and cellular ATP levels, which finally resulted in higher secreted amounts of proinflammatory cytokines, e.g., IFN-γ, IP-10 from memory Th cells taken from aged donors after TCR-stimulation which were sensitive to ROS inhibition. These findings suggest that metabolic reprogramming in human memory Th cells during aging results in an increased expression of proinflammatory cytokines through enhanced ROS production, which may contribute to the pathogenesis of inflammaging

Orbital effects of a monochromatic plane gravitational wave with ultra-low frequency incident on a gravitationally bound two-body system

We analytically compute the long-term orbital variations of a test particle orbiting a central body acted upon by an incident monochromatic plane gravitational wave. We assume that the characteristic size of the perturbed two-body system is much smaller than the wavelength of the wave. Moreover, we also suppose that the wave's frequency is much smaller than the particle's orbital one. We make neither a priori assumptions about the direction of the wavevector nor on the orbital geometry of the planet. We find that, while the semi-major axis is left unaffected, the eccentricity, the inclination, the longitude of the ascending node, the longitude of pericenter and the mean anomaly undergo non-vanishing long-term changes. They are not secular trends because of the slow modulation introduced by the tidal matrix coefficients and by the orbital elements themselves. They could be useful to indepenedently constrain the ultra-low frequency waves which may have been indirectly detected in the BICEP2 experiment. Our calculation holds, in general, for any gravitationally bound two-body system whose characteristic frequency is much larger than the frequency of the external wave. It is also valid for a generic perturbation of tidal type with constant coefficients over timescales of the order of the orbital period of the perturbed particle.Comment: LaTex2e, 24 pages, no figures, no tables. Changes suggested by the referees include