TY - JOUR A1 - Fehrholz, Markus A1 - Seidenspinner, Silvia A1 - Kunzmann, Steffen T1 - Expression of surfactant protein B is dependent on cell density in H441 lung epithelial cells JF - PLoS ONE N2 - Background Expression of surfactant protein (SP)-B, which assures the structural stability of the pulmonary surfactant film, is influenced by various stimuli, including glucocorticoids; however, the role that cell-cell contact plays in SP-B transcription remains unknown. The aim of the current study was to investigate the impact of cell-cell contact on SP-B mRNA and mature SP-B expression in the lung epithelial cell line H441. Methods Different quantities of H441 cells per growth area were either left untreated or incubated with dexamethasone. The expression of SP-B, SP-B transcription factors, and tight junction proteins were determined by qPCR and immunoblotting. The influence of cell density on SP-B mRNA stability was investigated using the transcription inhibitor actinomycin D. Results SP-B mRNA and mature SP-B expression levels were significantly elevated in untreated and dexamethasone-treated H441 cells with increasing cell density. High cell density as a sole stimulus was found to barely have an impact on SP-B transcription factor and tight junction mRNA levels, while its stimulatory ability on SP-B mRNA expression could be mimicked using SP-B-negative cells. SP-B mRNA stability was significantly increased in high-density cells, but not by dexamethasone alone. Conclusion SP-B expression in H441 cells is dependent on cell-cell contact, which increases mRNA stability and thereby potentiates the glucocorticoid-mediated induction of transcription. Loss of cell integrity might contribute to reduced SP-B secretion in damaged lung cells via downregulation of SP-B transcription. Cell density-mediated effects should thus receive greater attention in future cell culture-based research. KW - messenger RNA KW - surfactants KW - epithelial cells KW - transcription factors KW - gene expression KW - tight junctions KW - adenocarcinoma of the lung KW - immunoblotting Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158291 VL - 12 IS - 9 ER - TY - JOUR A1 - Fehrholz, Markus A1 - Glaser, Kirsten A1 - Seidenspinner, Silvia A1 - Ottensmeier, Barbara A1 - Curstedt, Tore A1 - Speer, Christian P. A1 - Kunzmann, Steffen T1 - Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4\(^+\) Lymphocytes JF - PLoS One N2 - Background Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown. Aim The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4\(^+\) lymphocytes. Methods Purified human CD4\(^+\) T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry. Results Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4\(^+\) lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4\(^+\) lymphocytes. Conclusion For the first time, the immunomodulatory capacity of CHF5633 on CD4\(^+\) lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4\(^+\) cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4+ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance. KW - lymphocytes KW - surfactants KW - flow cytometry KW - monocytes KW - RNA isolation KW - T cells KW - cytokines KW - inflammation Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146419 VL - 11 IS - 4 ER - TY - JOUR A1 - Glaser, Kirsten A1 - Fehrholz, Markus A1 - Curstedt, Tore A1 - Kunzmann, Steffen A1 - Speer, Christian P. T1 - Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14\(^{+}\) Monocytes JF - PLoS ONE N2 - Background Surfactant replacement therapy is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome. New generation synthetic surfactants represent a promising alternative to animal-derived surfactants. CHF5633, a new generation reconstituted synthetic surfactant containing SP-B and SP-C analogs and two synthetic phospholipids has demonstrated biophysical effectiveness in vitro and in vivo. While several surfactant preparations have previously been ascribed immunomodulatory capacities, in vitro data on immunomodulation by CHF5633 are limited, so far. Our study aimed to investigate pro- and anti-inflammatory effects of CHF5633 on native and LPS-stimulated human adult monocytes. Methods Highly purified adult CD14\(^{+}\) cells, either native or simultaneously stimulated with LPS, were exposed to CHF5633, its components, or poractant alfa (Curosurf\(^{®}\)). Subsequent expression of TNF-α, IL-1β, IL-8 and IL-10 mRNA was quantified by real-time quantitative PCR, corresponding intracellular cytokine synthesis was analyzed by flow cytometry. Potential effects on TLR2 and TLR4 mRNA and protein expression were monitored by qPCR and flow cytometry. Results Neither CHF5633 nor any of its components induced inflammation or apoptosis in native adult CD14\(^{+}\) monocytes. Moreover, LPS-induced pro-inflammatory responses were not aggravated by simultaneous exposure of monocytes to CHF5633 or its components. In LPS-stimulated monocytes, exposure to CHF5633 led to a significant decrease in TNF-α mRNA (0.57 ± 0.23-fold, p = 0.043 at 4h; 0.56 ± 0.27-fold, p = 0.042 at 14h). Reduction of LPS-induced IL-1β mRNA expression was not significant (0.73 ± 0.16, p = 0.17 at 4h). LPS-induced IL-8 and IL-10 mRNA and protein expression were unaffected by CHF5633. For all cytokines, the observed CHF5633 effects paralleled a Curosurf®-induced modulation of cytokine response. TLR2 and TLR4 mRNA and protein expression were not affected by CHF5633 and Curosurf®, neither in native nor in LPS-stimulated adult monocytes. Conclusion The new generation reconstituted synthetic surfactant CHF5633 was tested for potential immunomodulation on native and LPS-activated adult human monocytes. Our data confirm that CHF5633 does not exert unintended pro-inflammatory effects in both settings. On the contrary, CHF5633 significantly suppressed TNF-α mRNA expression in LPS-stimulated adult monocytes, indicating potential anti-inflammatory effects. KW - adults KW - monocytes KW - surfactants KW - cytokines KW - protein expression KW - flow cytometry KW - messenger RNA KW - cloning Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180195 VL - 11 IS - 1 ER - TY - JOUR A1 - Paletta, Daniel A1 - Fichtner, Alina Suzann A1 - Starick, Lisa A1 - Porcelli, Steven A. A1 - Savage, Paul B. A1 - Herrmann, Thomas T1 - Species Specific Differences of CD1d Oligomer Loading In Vitro JF - PLoS One N2 - CD1d molecules are MHC class I-like molecules that present glycolipids to iNKT cells. The highly conserved interaction between CD1d:α-Galactosylceramide (αGC) complexes and the iNKT TCR not only defines this population of αβ T cells but can also be used for its direct identification. Therefore, CD1d oligomers are a widely used tool for iNKT cell related investigations. To this end, the lipid chains of the antigen have to be inserted into the hydrophobic pockets of the CD1d binding cleft, often with help of surfactants. In this study, we investigated the influence of different surfactants (Triton X-100, Tween 20, Tyloxapol) on in vitro loading of CD1d molecules derived from four different species (human, mouse, rat and cotton rat) with αGC and derivatives carrying modifications of the acyl-chain (DB01-1, PBS44) and a 6-acetamido-6-deoxy-addition at the galactosyl head group (PBS57). We also compared rat CD1d dimers with tetramers and staining of an iNKT TCR transductant was used as readout for loading efficacy. The results underlined the importance of CD1d loading efficacy for proper analysis of iNKT TCR binding and demonstrated the necessity to adjust loading conditions for each oligomer/glycolipid combination. The efficient usage of surfactants as a tool for CD1d loading was revealed to be species-specific and depending on the origin of the CD1d producing cells. Additional variation of surfactant-dependent loading efficacy between tested glycolipids was influenced by the acyl-chain length and the modification of the galactosyl head group with PBS57 showing the least dependence on surfactants and the lowest degree of species-dependent differences. KW - cell staining KW - major histocompatibility complex KW - binding analysis KW - oligomers KW - glycolipids KW - lipids KW - surfactants KW - T cells Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124879 VL - 10 IS - 11 ER - TY - THES A1 - Forster, Wilhelmina Alison T1 - Mechanisms of cuticular uptake of xenobiotics into living plants T1 - Mechanismen der kutikulären Xenobiotika-Aufnahme in lebende Pflanzen N2 - The objective of this Thesis was to progress the understanding of the mechanisms of cuticular uptake into living plant foliage, thereby enabling uptake of important compounds such as pesticides and pollutants to be modelled. The uptake of three model compounds, applied in the presence and absence of surfactants, into the leaves of three plant species (Chenopodium album L., Hedera helix L. and Stephanotis floribunda Brongn) was determined. The results with 2-deoxy-D-glucose (DOG), 2,4-dichlorophenoxy-acetic acid (2,4-D) and epoxiconazole in the presence of surfactants (the polyethylene glycol monododecyl ethers C12EO3, C12EO6, C12EO10, and a trisiloxane ethoxylate with mean ethylene oxide (EO) content of 7.5, all used at one equimolar concentration) illustrated that the initial dose (nmol mm-2) of xenobiotic applied to plant foliage was a strong positive determinant of uptake. Using this new approach for whole plant uptake, uptake on a per unit area basis was found to be related to initial dose of xenobiotic applied, by an equation of the form: Uptake(nmol mm-2) = a [ID]b at time t = 24 hours, where ID is the initial dose or the mass of xenobiotic applied per unit area (M(nmol xenobiotic applied)/A(droplet spread area)). Total mass uptake can then be calculated from an equation of the form: Total Uptake(nmol) = a [ID]b.A. In order to verify this relationship, further studies determined the uptake of three pesticides, applied as commercial and model formulations in the presence of a wide range of surfactants, into the leaves of three plant species (bentazone into Chenopodium album L. and Sinapis alba L., epoxiconazole and pyraclostrobin into Triticum aestivum L.). The results confirmed that the initial dose (nmol mm-2) of xenobiotic applied to plant foliage is a strong, positive determinant of uptake. In a novel approach, further studies used this relationship (nmol mm-2 uptake versus ID; termed the uptake ratio) to establish the relative importance of species, active ingredient (AI), AI concentration (g L-1) and surfactant to uptake. Species, AI, its concentration, and surfactant all significantly affected the uptake ratio. Overall, 88% of the deviance could be explained. More useful was the analysis of the individual xenobiotics, where the models explained 83%, 85%, and 94% of the variance in uptake ratio for DOG, 2,4-D, and epoxiconazole, respectively. In all cases, species, surfactant, and AI concentration significantly affected the uptake ratio. However, there were differences in the relative importance of these factors among the xenobiotics studied. Concentration of AI increased in importance with increasing lipophilicity of AI, while species was much less important for the most lipophilic compound. Surfactant became less important with increasing AI lipophilicity, although it was always important. The preceding studies considered uptake at only one time interval (24 hours). Total uptake after 24 hours can be the same for a compound formulated with different surfactants, but rates of uptake (and therefore rain-fastness and subsequent translocation to target sites) can be quite different. Therefore, there was a requirement to be able to model uptake over time into whole plants. Hence, the objective of further studies was to determine whether a logistic-kinetic penetration model, developed using isolated plant cuticles, could be applied to whole plant uptake. Uptake over 24 hours was determined for three model compounds, applied in the presence and absence of surfactants, into the leaves of two plant species. Overall, the model fitted the whole plant uptake data well. Using the equations developed, based on initial dose, to calculate uptake at 24 hours, in conjunction with the logistic-kinetic model, has significantly progressed our understanding and ability to model uptake. The advantages of the models and equations described are that few variables are required, and they are simple to measure. N2 - Das Ziel dieser Dissertation war es, das Verständnis der Mechanismen der Aufnahme von Wirkstoffen über die Kutikula in die Blätter einer lebenden Pflanze zu verbessern und es dadurch möglich zu machen, die Aufnahme von wichtigen Verbindungen wie z.B. von Pestiziden und Schadstoffen zu modellieren. Es wurde die Aufnahme von drei Modellverbindungen ermittelt, die in Anwesenheit und Abwesenheit von oberflächenaktiven Stoffen in die Blätter von drei Pflanzenarten (Chenopodium album L., Hedera helix L. und Stephanotis floribunda Brongn.) aufgetragen wurden. Das Ergebnis mit 2-Deoxy-D-Glucose (DOG), 2,4-Dichlorphenoxy-Essigsäure (2,4-D) und Epoxiconazol in Anwesenheit der oberflächenaktiven Stoffe (die Polyethylenglycolmonododecylether C12EO3, C12EO6, C12EO10, und ein Trisiloxanethoxylat mit einem EO-Mittelwert von 7,5; wobei alle in einer äquimolaren Konzentration und daher in verschiedenen prozentualen Konzentrationen verwendet wurden) zeigte, dass die auf die Pflanzenblätter aufgetragene anfängliche Dosis (nmol mm-2) an Xenobiotikum ein starker, positiv bestimmender Faktor für die Aufnahme war. Verwendet man diese neue Beschreibung der Aufnahme von Xenobiotica in ganze Pflanzen, so kann man feststellen, dass die Aufnahme pro Einheitsfläche von der anfänglichen Dosis von aufgetragenem Xenobiotikum abhängig ist, und zwar nach folgender Gleichung: Aufnahme(nmol mm-2) = a [ID]b bei einer Zeit t = 24 Stunden, wobei ID für die anfängliche Dosis oder die Masse an pro Einheitsfläche aufgetragenem Xenobiotikum steht (M(nmol aufgetragenes Xenobiotikum)/A(Tropfenausbreitungsbereich)). Die Gesamtaufnahme der Masse kann dann aus einer Gleichung der Formel: Gesamtaufnahme(nmol) = a [ID]b.A errechnet werden. Um diese Beziehung zu bestätigen, wurde in zusätzlichen Studien die Aufnahme von drei Pestiziden ermittelt, die als gewerbliche und Modellformulierungen in Anwesenheit einer großen Auswahl von oberflächenaktiven Stoffen in die Blätter von drei Pflanzenarten (Bentazon in Chenopodium album L. und Sinapis alba L., Epoxiconazol und Pyraclostrobin in Triticum aestivum L.) aufgetragen wurden. Die Ergebnisse bestätigten die Feststellung, dass die anfängliche, auf die Pflanzenblätter aufgetragene Dosis (nmol mm-2) an Xenobiotikum ein starker, positiv bestimmender Faktor der Aufnahme ist. Bei einem neuartigen Ansatz verwendeten zusätzliche Studien dieses Verhältnis (nmol mm-2 Aufnahme pro ID; genannt Aufnahmeverhältnis), um die relative Bedeutung der Arten, der Wirksubstanz (AI), der AI-Konzentrationen (g L-1) und der oberflächenaktiven Stoffe für die Aufnahme zu ermitteln. Die Art, AI, ihre Konzentration und die oberflächenaktiven Stoffe hatten alle einen erheblichen Einfluss auf das Aufnahmeverhältnis. Insgesamt konnte 88 % der Abweichung erklärt werden. Noch nützlicher war die Analyse der einzelnen Xenobiotika, bei denen die Modelle 83 %, 85 % und 94 % der Varianz im Aufnahmeverhältnis jeweils für DOG, 2,4-D und Epoxiconazol erklärten. In allen Fällen hatten die Arten, der oberflächenaktive Stoff und die AI-Konzentration einen erheblichen Einfluss auf das Aufnahmeverhältnis. Es gab jedoch Unterschiede bei der relativen Bedeutung dieser Faktoren unter den untersuchten Xenobiotika. Die Konzentration von AI gewann größere Bedeutung mit einer erhöhten Fettlöslichkeit von AI, während die Art eine weit geringere Rolle für die meisten lipophilen Verbindungen spielte. Oberflächenaktive Stoffe verloren an Bedeutung mit zunehmender AI-Fettlöslichkeit, obwohl diese stets von Bedeutung waren. Die bisher dargestellten Studien zogen die Aufnahme bei nur einem Uhrzeitintervall (24 Stunden) in Betracht. Die Gesamtaufnahme nach 24 Stunden kann zwar bei einer Verbindung, die mit verschiedenen oberflächenaktiven Stoffen formuliert ist, die gleiche sein, die Aufnahmeraten (und daher die Regenfestigkeit und anschließende Translokation an Zielstellen) können dabei jedoch völlig verschieden sein. Es bestand daher die Notwendigkeit, die Aufnahme in vollständigen Pflanzen im Zeitablauf modellieren zu können. Infolgedessen war es das Ziel zusätzlicher Studien festzustellen, ob ein logistisch-kinetisches Penetrationsmodell, das unter Anwendung isolierter pflanzlicher Kutikeln entwickelt wurde, bei der Gesamtpflanzenaufnahme zum Einsatz kommen könnte. Die Aufnahme über 24 Stunden wurde für drei Modellverbindungen ermittelt, die in Anwesenheit und Abwesenheit von oberflächenaktiven Stoffen in die Blätter von zwei Pflanzenarten aufgetragen wurden. Insgesamt gesehen entsprach das Modell den Pflanzenaufnahmedaten sehr gut. KW - Kutikula KW - Xenobiotikum KW - Modelle KW - kutikuläre Aufnahme KW - Pestizide KW - oberflächenaktive Stoffe KW - lebende Pflanzen KW - models KW - cuticular uptake KW - pesticides KW - surfactants KW - living plants Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-21240 ER -