An improved method for high-throughput quantification of autophagy in mammalian cells

Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays

Progressive skin fibrosis is associated with a decline in lung function and worse survival in patients with diffuse cutaneous systemic sclerosis in the European Scleroderma Trials and Research (EUSTAR) cohort.

Objectives To determine whether progressive skin fibrosis is associated with visceral organ progression and mortality during follow-up in patients with diffuse cutaneous systemic sclerosis (dcSSc). Methods We evaluated patients from the European Scleroderma Trials and Research database with dcSSc, baseline modified Rodnan skin score (mRSS) ≥7, valid mRSS at 12±3 months after baseline and ≥1 annual follow-up visit. Progressive skin fibrosis was defined as an increase in mRSS >5 and ≥25% from baseline to 12±3 months. Outcomes were pulmonary, cardiovascular and renal progression, and all-cause death. Associations between skin progression and outcomes were evaluated by Kaplan-Meier survival analysis and multivariable Cox regression. Results Of 1021 included patients, 78 (7.6%) had progressive skin fibrosis (skin progressors). Median follow-up was 3.4 years. Survival analyses indicated that skin progressors had a significantly higher probability of FVC decline ≥10% (53.6% vs 34.4%; p<0.001) and all-cause death (15.4% vs 7.3%; p=0.003) than non-progressors. These significant associations were also found in subgroup analyses of patients with either low baseline mRSS (≤22/51) or short disease duration (≤15 months). In multivariable analyses, skin progression within 1 year was independently associated with FVC decline ≥10% (HR 1.79, 95% CI 1.20 to 2.65) and all-cause death (HR 2.58, 95% CI 1.31 to 5.09). Conclusions Progressive skin fibrosis within 1 year is associated with decline in lung function and worse survival in dcSSc during follow-up. These results confirm mRSS as a surrogate marker in dcSSc, which will be helpful for cohort enrichment in future trials and risk stratification in clinical practice

Racial differences in systemic sclerosis disease presentation: a European Scleroderma Trials and Research group study

Objectives. Racial factors play a significant role in SSc. We evaluated differences in SSc presentations between white patients (WP), Asian patients (AP) and black patients (BP) and analysed the effects of geographical locations.Methods. SSc characteristics of patients from the EUSTAR cohort were cross-sectionally compared across racial groups using survival and multiple logistic regression analyses.Results. The study included 9162 WP, 341 AP and 181 BP. AP developed the first non-RP feature faster than WP but slower than BP. AP were less frequently anti-centromere (ACA; odds ratio (OR) = 0.4, P < 0.001) and more frequently anti-topoisomerase-I autoantibodies (ATA) positive (OR = 1.2, P = 0.068), while BP were less likely to be ACA and ATA positive than were WP [OR(ACA) = 0.3, P < 0.001; OR(ATA) = 0.5, P = 0.020]. AP had less often (OR = 0.7, P = 0.06) and BP more often (OR = 2.7, P < 0.001) diffuse skin involvement than had WP.AP and BP were more likely to have pulmonary hypertension [OR(AP) = 2.6, P < 0.001; OR(BP) = 2.7, P = 0.03 vs WP] and a reduced forced vital capacity [OR(AP) = 2.5, P < 0.001; OR(BP) = 2.4, P < 0.004] than were WP. AP more often had an impaired diffusing capacity of the lung than had BP and WP [OR(AP vs BP) = 1.9, P = 0.038; OR(AP vs WP) = 2.4, P < 0.001]. After RP onset, AP and BP had a higher hazard to die than had WP [hazard ratio (HR) (AP) = 1.6, P = 0.011; HR(BP) = 2.1, P < 0.001].Conclusion. Compared with WP, and mostly independent of geographical location, AP have a faster and earlier disease onset with high prevalences of ATA, pulmonary hypertension and forced vital capacity impairment and higher mortality. BP had the fastest disease onset, a high prevalence of diffuse skin involvement and nominally the highest mortality

Feasibility study for the use of self‐collected nasal swabs to identify pathogens among participants of a population‐based surveillance system for acute respiratory infections (GrippeWeb‐Plus)—Germany, 2016

Background Internet‐based participatory surveillance systems, such as the German GrippeWeb, monitor the frequency of acute respiratory illnesses on population level. In order to interpret syndromic information better, we devised a microbiological feasibility study (GrippeWeb‐Plus) to test whether self‐collection of anterior nasal swabs is operationally possible, acceptable for participants and can yield valid data. Methods We recruited 103 GrippeWeb participants (73 adults and 30 children) and provided them with a kit, instructions and a questionnaire for each sample. In the first half of 2016, participants took an anterior nasal swab and sent it to the Robert Koch Institute whenever an acute respiratory illness occurred. Reporting of illnesses through the GrippeWeb platform continued as usual. We analysed swabs for the presence of human c‐myc‐DNA and 22 viral and bacterial pathogens. After the study, we sent participants an evaluation questionnaire. We analysed timeliness, completeness, acceptability and validity. Results One hundred and two participants submitted 225 analysable swabs. Ninety per cent of swabs were taken within 3 days of symptom onset. Eighty‐nine per cent of swabs had a corresponding reported illness in the GrippeWeb system. Ninety‐nine per cent of adults and 96% of children would be willing to participate in a self‐swabbing scheme for a longer period. All swabs contained c‐myc‐DNA. In 119 swabs, we identified any of 14 viruses but no bacteria. The positivity rate of influenza was similar to that in the German physician sentinel. Conclusion Self‐collection of anterior nasal swabs proofed to be feasible, was well accepted by participants, gave valid results and was an informative adjunct to syndromic data.Peer Reviewe

Industrielle Mikroplastikemissionen - Handlungsempfehlungen

EmiStop ist ein Projekt, welches in der Initiative „Plastik in der Umwelt“ des Bundesministeriums für Bildung und Forschung über drei Jahre die Eintragspfade von Mikroplastik (ausgenommen Fasern und Rezyklate) in der kunststoffproduzierenden und -verarbeitenden Industrie sowie der zwischengeschalteten Logistik mit Fokus auf das Wassermanagement und die (Ab)Wasserinfrastruktur untersucht hat. In enger Zusammenarbeit mit einzelnen Betriebsstandorten und Industrieverbänden sowie durch Betriebsbegehungen, Probenahmen und Laboranalysen wurden angepasste Probenahmestrategien und -methoden, analytische Verfahren für Mikroplastik sowie Maßnahmen zur Vermeidung des Eintrags von Mikroplastik in die Umwelt entwickelt. Ein Projektergebnis stellen diese Handlungsempfehlungen für Akteur:innen aus der Industrie dar. Sie sollen als Leitfaden dienen, um Mikroplastikemissionen in die Umwelt an Betriebsstandorten zu vermeiden oder zu vermindern

Progressive interstitial lung disease in patients with systemic sclerosis-associated interstitial lung disease in the EUSTAR database

Objectives To identify overall disease course, progression patterns and risk factors predictive for progressive interstitial lung disease (ILD) in patients with systemic sclerosis-associated ILD (SSc-ILD), using data from the European Scleroderma Trials And Research (EUSTAR) database over long-term follow-up. Methods Eligible patients with SSc-ILD were registered in the EUSTAR database and had measurements of forced vital capacity (FVC) at baseline and after 12±3 months. Long-term progressive ILD and progression patterns were assessed in patients with multiple FVC measurements. Potential predictors of ILD progression were analysed using multivariable mixed-effect models. Results 826 patients with SSc-ILD were included. Over 12±3 months, 219 (27%) showed progressive ILD: either moderate (FVC decline 5% to 10%) or significant (FVC decline >10%). A total of 535 (65%) patients had multiple FVC measurements available over mean 5-year follow-up. In each 12-month period, 23% to 27% of SSc-ILD patients showed progressive ILD, but only a minority of patients showed progression in consecutive periods. Most patients with progressive ILD (58%) had a pattern of slow lung function decline, with more periods of stability/improvement than decline, whereas only 8% showed rapid, continuously declining FVC; 178 (33%) experienced no episode of FVC decline. The strongest predictive factors for FVC decline over 5 years were male sex, higher modified Rodnan skin score and reflux/dysphagia symptoms. Conclusion SSc-ILD shows a heterogeneous and variable disease course, and thus monitoring all patients closely is important. Novel treatment concepts, with treatment initiation before FVC decline occurs, should aim for prevention of progression to avoid irreversible organ damage