Antibiotika in Wirtschaftsdüngern, Klärschlämmen und Gärresten und deren Interaktion mit Pflanzen

Wirtschaftsdünger, Gärreste und Klärschlämme fallen in großen Mengen an und werden wegen ihres Nährstoffgehaltes für die Düngung in der Landwirtschaft genutzt, wodurch sie Schadstoffe wie Schwermetalle und Antibiotika in landwirtschaftliche Böden transportieren. Ziel der vorliegenden Arbeit war es, die methodischen Grundlagen der chemischen Analyse dieser Stoffe in Wirtschaftsdüngern, Gärresten und Klärschlämmen zu evaluieren und insbesondere für Antibiotika ein geeignetes Analyseverfahren zu entwickeln. In Gefäß- und Modellversuchen wurden zudem direkte toxische und kombinatorische Effekte von Antibiotika und Schwermetallen auf Modellorganismen geprüft und deren ökologische Relevanz abgeschätzt. Die Arbeit erbrachte folgende Ergebnisse:Die CAL-Extraktion von P erwies sich im Vergleich zur Wasser-Extraktion als weniger matrixabhängig und genauer bei der Konzentrationsbestimmung. Die Ergebnisse der CAL-Extraktion entsprachen denen der NAC-Extraktion. Für die eigentliche P-Bestimmung erwies sich die ICP-OES-Messung als genauer im Vergleich zur Kolorimetrie. Tendenziell fanden sich in den Endprodukten der anaeroben Fermentation niedrigere Median- und Maximalkonzentrationen von Antibiotika als in den Eingangssubstraten, die Größenordnung war jedoch vergleichbar. Die Mediankonzentrationen von Wirtschaftsdüngern und Klärschlämmen lagen häufig im Bereich von 100 μg bis wenige mg kg-1 Trockenmasse, der Maximalwert eines Gärrests bei 29,8 mg kg-1 Tetracyclin. Bei vielen Proben wurde eine Kontamination mit mehreren Antibiotika beobachtet. In Modellversuchen (Toxkits) wurde festgestellt, dass Antibiotika in hohen Konzentrationen toxisch auf Daphnia magna und Sinapis alba wirken, und zwar in Form einer Immobilisierung von Daphnia magna und einer Reduzierung der Wurzellänge von Sinapis alba. Die Antibiotika Enrofloxacin, Sulfadiazin und Tetracyclin wiesen in ihrer binären Kombination in Toxkits synergistische toxische Effekte auf. Enrofloxacin konnte im Gefäßversuch nicht in der Bodenlösung detektiert werden, was als Hinweis auf eine Adsorption an die Bodenmatrix gewertet werden kann. Ab einer Enrofloxacin-Konzentration von 3,6 mg kg-1 Sandboden trat eine Reduktion der Biomasseentwicklung von Sinapis alba auf und Enrofloxacin konnte in geringen Konzentrationen im Pflanzenmaterial nachgewiesen werden. Die berechnete Antibiotika-Fracht durch Düngung mit der am stärksten kontaminierten Gärrestprobe betrug 22 μg Tetracyclin kg-1 Boden, was deutlich unter den schädlichen Effektkonzentrationen für Sinapis alba liegt. Auf Grundlage dieser Daten ist derzeit nicht mit einer direkten toxischen Wirkung auf Pflanzen zu rechnen, wenn Wirtschaftsdünger oder Klärschlämme zur Düngung eingesetzt werden. Andere schädliche Effekte in der Umwelt, welche in dieser Studie nicht betrachtet wurden, können jedoch nicht ausgeschlossen werden.Manure, digestates and sewage sludge are produced in large amounts and are used in agriculture because of their nutrient concentration. Through application they can carry pollutants like heavy metals and antibiotics into agricultural soils. The aim of this work was to evaluate the methodological basics of analysis for the phosphorus (P) content in manure, digestates and sewage sludge and the development of a suitable analytical method especially for antibiotics in these complex matrices. Acute and combinatory toxic effects of heavy metals and antibiotics were investigated in a pot trial and in model experiments to estimate their ecological relevance. This work provided the following results:The extraction of P by CAL delivered more reasonable results than a water extraction because CAL extraction was less affected by the matrix. CAL and NAC extraction delivered comparable results so both can be used to determine the P content in digestates. It was shown that the P determination by ICP-OES was more suitable to determine the P content in comparison to the colorimetric measurement by a spectral photometer as P contents were partly overestimated. The median and maximum concentrations of antibiotics tend to be lower in digestates than in the substrates of the anaerobic fermentation. However, the contaminations were in the same order of magnitude. Median antibiotic concentrations in manure, digestate and sewage sludge samples were in the range of 100 μg to a few mg kg-1 on a dry matter basis. The maximum concentration determined in a digestate was 29.8 mg tetracycline kg-1 DW. In small-scale tests (toxkits) it was proven that antibiotics in high concentrations are toxic to Daphnia magna and Sinapis alba. Daphnia magna was immobilised by high antibiotic concentrations and in case of Sinapis alba the root length was reduced by high concentrations of antibiotics. In the toxkits experiments the antibiotics enrofloxacine, sulfadiazine and tetracycline revealed synergistic toxic effects in combination. In a pot experiment, where the combinatory effect of enrofloxacin and copper on growth of Sinapis alba was investigated, it was shown that no enrofloxacin could be detected in the soil solution. This indicates a high adsorption of enrofloxacin to the soil-matrix. In concentrations of 3.6 mg kg-1 soil enrofloxacin caused a reduction in biomass development of Sinapis alba and could be detected in the plant material. In a worst-case scenario it was calculated that up to 22 μg tetracycline kg-1 soil could be transferred to the field as a result of fertilization. This concentration is significantly lower than the toxic effect concentration which was determined for Sinapis alba. Based on the data obtained in the present work, it can be concluded, that currently no acute toxic effects on plant growth should be expected due to fertilization with manure, digestates and sewage sludge. Nevertheless, other toxic effects which were not evaluated in this work, could pose a risk for the environment

Antibiotics in manure, sewage sludge and digestates and their interaction with plants

Wirtschaftsdünger, Gärreste und Klärschlämme fallen in großen Mengen an und werden wegen ihres Nährstoffgehaltes für die Düngung in der Landwirtschaft genutzt, wodurch sie Schadstoffe wie Schwermetalle und Antibiotika in landwirtschaftliche Böden transportieren. Ziel dieser Arbeit war es, die methodischen Grundlagen der Analyse dieser Stoffe in Wirtschaftsdüngern, Gärresten und Klärschlämmen zu evaluieren und insbesondere für Antibiotika ein geeignetes Analyseverfahren zu entwickeln. In Gefäß- und Modellversuchen wurden zudem direkte toxische und kombinatorische Effekte von Antibiotika und Schwermetallen auf Modellorganismen geprüft und deren ökologische Relevanz abgeschätzt. Ergebnisse: Die CAL-Extraktion von P erwies sich im Vergleich zur Wasser-Extraktion als weniger matrixabhängig und genauer bei der Konzentrationsbestimmung. Die Ergebnisse der CAL-Extraktion entsprachen denen der NAC-Extraktion. Für die P-Bestimmung erwies sich die ICP-OES-Messung als genauer im Vergleich zur Kolorimetrie. Tendenziell fanden sich in den Endprodukten der anaeroben Fermentation niedrigere Median- und Maximalkonzentrationen von Antibiotika als in den Eingangssubstraten, die Größenordnung war jedoch vergleichbar. Die Mediankonzentrationen von Wirtschaftsdüngern und Klärschlämmen lagen häufig im Bereich von 100 µg bis wenige mg/kg Trockenmasse, der Maximalwert eines Gärrests bei 29,8 mg/kg Tetracyclin. Bei vielen Proben wurde eine Kontamination mit mehreren Antibiotika beobachtet. In Modellversuchen wurde festgestellt, dass Antibiotika in hohen Konzentrationen toxisch auf Daphnia magna und Sinapis alba wirken. Die Antibiotika Enrofloxacin, Sulfadiazin und Tetracyclin wiesen in ihrer binären Kombination synergistische toxische Effekte auf. Ab einer Enrofloxacin-Konzentration von 3,6 mg/kg Sandboden trat eine Reduktion der Biomasseentwicklung von Sinapis alba auf und Enrofloxacin konnte in geringen Konzentrationen im Pflanzenmaterial nachgewiesen werden. Die berechnete Antibiotika-Fracht durch Düngung mit der am stärksten kontaminierten Gärrestprobe betrug 22 µg Tetracyclin/kg Boden, was deutlich unter den schädlichen Effektkonzentrationen für Sinapis alba liegt. Auf Grundlage dieser Daten ist derzeit nicht mit einer direkten toxischen Wirkung auf Pflanzen zu rechnen, wenn Wirtschaftsdünger oder Klärschlämme zur Düngung eingesetzt werden. Andere schädliche Effekte in der Umwelt, welche in dieser Studie nicht betrachtet wurden, können jedoch nicht ausgeschlossen werden.Manure, digestates and sewage sludge are produced in large amounts and are used in agriculture because of their nutrient concentration. Through application they can carry pollutants like heavy metals and antibiotics into agricultural soils. The aim of this work was to evaluate the methodological basics of analysis for the phosphorus (P) content in manure, digestates and sewage sludge and the development of a suitable analytical method especially for antibiotics in these complex matrices. Acute and combinatory toxic effects of heavy metals and antibiotics were investigated in a pot trial and in model experiments to estimate their ecological relevance. Results: The extraction of P by CAL delivered more reasonable results than a water extraction because CAL extraction was less affected by the matrix. CAL and NAC extraction of digestates delivered comparable results. It was shown that the P determination by ICP-OES was more suitable to determine the P content in comparison to the colorimetric measurement by a spectral photometer. The median and maximum concentrations of antibiotics tend to be lower in digestates than in the substrates of the anaerobic fermentation. However, the contaminations were in the same order of magnitude. Median antibiotic concentrations in manure, digestate and sewage sludge samples were in the range of 100 µg to a few mg/kg on a dry matter basis. The maximum concentration determined in a digestate was 29.8 mg tetracycline/kg DW. In small-scale tests it was proven that antibiotics in high concentrations are toxic to Daphnia magna and Sinapis alba. In the toxkits experiments the antibiotics enrofloxacine, sulfadiazine and tetracycline revealed synergistic toxic effects in combination In concentrations of 3.6 mg/kg soil enrofloxacin caused a reduction in biomass development of Sinapis alba and could be detected in the plant material. In a worst-case scenario it was calculated that up to 22 µg tetracycline/kg soil could be transferred to the field as a result of fertilization. This concentration is significantly lower than the toxic effect concentration which was determined for Sinapis alba. Based on the data obtained in the present work, it can be concluded, that currently no acute toxic effects on plant growth should be expected due to fertilization with manure, digestates and sewage sludge. Nevertheless, other toxic effects which were not evaluated in this work, could pose a risk for the environment

Validation of three-dimensional hydrodynamic models of the Gulf of Finland

A model-intercomparison study was conducted, the first of its kind for the Baltic Sea, whose aim was to systematically simulate the basic three-dimensional hydrographic properties of a realistic, complex basin. Simulations of the hydrographic features of the Gulf of Finland for the summer–autumn of 1996 by six three-dimensional hydrodynamic models were compared. Validation was undertaken using more than 300 vertical hydrographic profiles of salinity and temperature. The analysis of model performance, including averaging of the ensemble results, was undertaken with a view to assessing the potential suitability of the models in reproducing the physics of the Baltic Sea accurately enough to serve as a basis for accurate simulations of biogeochemistry once ecosystem models are incorporated. The performance of the models was generally satisfactory. Nevertheless, all the models had some difficulties in correctly simulating vertical profiles of temperature and salinity, and hence mixed layer dynamics, particularly in the eastern Gulf of Finland. Results emphasized the need for high resolution in both vertical and horizontal directions in order to resolve the complex dynamics and bathymetry of the Baltic Sea. Future work needs to consider the choice of mixing and advection schemes, moving to higher resolution, high-frequency forcing, and the accurate representation of river discharges and boundary conditions

TRAF6 controls excitatory spinogenesis and excitation-inhibition balance though binding neuroplastin

Cell-autonomous mechanisms of early synaptogenesis and their impact on the excitatory-inhibitory brain balance are poorly understood. By analyzing binding motifs in cytoplasmic regions of synaptogenic cell adhesion molecules, we identified a tumor necrosis factor receptor-associated factor 6 (TRAF6) binding motif in neuroplastin. Three-dimensional molecular modelling and biochemical approaches identified amino acids in neuroplastin binding the TRAF-C of TRAF6 with micromolar affinity. TRAF6 is required for spinogenesis and its association with neuroplastin fostered formation of new postsynapses in young hippocampal neurons. Also, TRAF6 is strictly necessary to restore failed spinogenesis in neuroplastin-deficient neurons via neuroplastin expression. These features are independent from neuroplastin extracellular adhesive properties or its known interaction with plasma-membrane Ca2+ ATPases. Furthermore, TRAF6-mediated neuroplastin-dependent spinogenesis determinates the excitatory synapse density and in turn the balance of E-I synapses in mature neurons. These findings provide a highly specific cell-encoded mechanism for early synaptogenesis crucial for neuronal connectivity

TRAF6 controls excitatory spinogenesis and excitation-inhibition balance though binding neuroplastin

Cell-autonomous mechanisms of early synaptogenesis and their impact on the excitatory-inhibitory brain balance are poorly understood. By analyzing binding motifs in cytoplasmic regions of synaptogenic cell adhesion molecules, we identified a tumor necrosis factor receptor-associated factor 6 (TRAF6) binding motif in neuroplastin. Three-dimensional molecular modelling and biochemical approaches identified amino acids in neuroplastin binding the TRAF-C of TRAF6 with micromolar affinity. TRAF6 is required for spinogenesis and its association with neuroplastin fostered formation of new postsynapses in young hippocampal neurons. Also, TRAF6 is strictly necessary to restore failed spinogenesis in neuroplastin-deficient neurons via neuroplastin expression. These features are independent from neuroplastin extracellular adhesive properties or its known interaction with plasma-membrane Ca2+ ATPases. Furthermore, TRAF6-mediated neuroplastin-dependent spinogenesis determinates the excitatory synapse density and in turn the balance of E-I synapses in mature neurons. These findings provide a highly specific cell-encoded mechanism for early synaptogenesis crucial for neuronal connectivity

Severe Asthma Standard-of-Care Background Medication Reduction With Benralizumab: ANDHI in Practice Substudy

Background: The phase IIIb, randomized, parallel-group, placebo-controlled ANDHI double-blind (DB) study extended understanding of the efficacy of benralizumab for patients with severe eosinophilic asthma. Patients from ANDHI DB could join the 56-week ANDHI in Practice (IP) single-arm, open-label extension substudy. Objective: Assess potential for standard-of-care background medication reductions while maintaining asthma control with benralizumab. Methods: Following ANDHI DB completion, eligible adults were enrolled in ANDHI IP. After an 8-week run-in with benralizumab, there were 5 visits to potentially reduce background asthma medications for patients achieving and maintaining protocol-defined asthma control with benralizumab. Main outcome measures for non-oral corticosteroid (OCS)-dependent patients were the proportions with at least 1 background medication reduction (ie, lower inhaled corticosteroid dose, background medication discontinuation) and the number of adapted Global Initiative for Asthma (GINA) step reductions at end of treatment (EOT). Main outcomes for OCS-dependent patients were reductions in daily OCS dosage and proportion achieving OCS dosage of 5 mg or lower at EOT. Results: For non-OCS-dependent patients, 53.3% (n = 208 of 390) achieved at least 1 background medication reduction, increasing to 72.6% (n = 130 of 179) for patients who maintained protocol-defined asthma control at EOT. A total of 41.9% (n = 163 of 389) achieved at least 1 adapted GINA step reduction, increasing to 61.8% (n = 110 of 178) for patients with protocol-defined EOT asthma control. At ANDHI IP baseline, OCS dosages were 5 mg or lower for 40.4% (n = 40 of 99) of OCS-dependent patients. Of OCS-dependent patients, 50.5% (n = 50 of 99) eliminated OCS and 74.7% (n = 74 of 99) achieved dosages of 5 mg or lower at EOT. Conclusions: These findings demonstrate benralizumab's ability to improve asthma control, thereby allowing background medication reduction

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Toxic Effects of Single Antibiotics and Antibiotics in Combination on Germination and Growth of Sinapis alba L.

Antibiotics enter agro-ecosystems via the application of farmyard manure, sewage sludge, animal by-products, or digestates. There are many open questions regarding the behavior of such compounds in the soil like their adsorption, degradation, half-life, and their effects on soil organisms and plants. The impact of antibiotics on the development of antibiotic resistance genes in the environment is regarded as the most important effect that endangers the environment as well as human health. Nevertheless, direct plant toxicity, especially of different antibiotics and heavy metals at the same time, can be of importance as well. In the current study, commercially available phytotoxkits were tested with regard to the toxicity of single antibiotics and antibiotics in combination with the root growth of Sinapis alba L. Additionally, a pot trial was conducted to study the transfer of the observed phytotoxkits results in more complex systems. The phytotoxkits revealed direct toxicity of antibiotics on root development only at high concentrations. The highest toxicity was determined for sulfadiazine, followed by tetracycline and enrofloxacin, showing the least toxicity. When two antibiotics were tested at the same time in the phytotoxkit, synergistic effects were detected. The pot trial indicated lower effect concentrations for enrofloxacin than determined in the phytotoxkit and, therefore, to higher toxicity on plant growth