28 research outputs found
Visualisierung des y-H2AX-Epitops durch eine innovative Methode zur DNA-Doppelstrangbruch (DSB)-Analyse in der Radiologie.
No full textAbstract 1889: Formation and merger of H69 and H69AR small cell lung cancer (SCLC) tumor cell clusters (TCCs) in vitro
No full textAbstract
A novel manifestation of tumor cell invasiveness, termed mutual cellular pervasion (MCP), was observed in vitro. It involved TCCs formed by SCLC cells.
Suspension cultures of H69 and H69AR cells were grown in flavin-free RPMI 1640 medium with 10% fetal bovine serum as the only source of flavins. Experiments and subcultivations were performed under light with wavelengths above 520 nm. TCC morphology was studied by confocal microscopy and transmission and scanning electron microscopy. Interactions of TCCs were followed by video microscopy in phase contrast and fluorescence modes. Data were analyzed using the Definiens software suite. The interaction of H69AR TCCs with confluent monolayers of human umbilical vascular endothelial cells (HUVEC) was visualized.
Freshly subcultivated, singularized tumor cells rapidly formed aggregates. After two days, solid TCCs prevailed whose surface cells were connected by desmosome-like junctions. Apoptosis but not necrosis became obvious in central parts of clusters with diameters above 100 to 150 μm. TCCs enlarged through both cell proliferation and serial cluster mergers. In the course of TCC merger, four stages were distinguishable: (1) directed cluster movement leading to contact of clusters, (2) formation of a near circular, symmetric structure termed contact disc. It consists of planar surface areas to which both TCCs involved contribute equally. (3) Merger of TCCs took place in the contact disc, and was accompanied by active movement of adjacent cells. (4) Starting from the merger zone, major surface areas of newly formed clusters were mutually pervaded by cells from both participating cell clusters. After numerous merger events, only low numbers of TCCs reaching sizes of up to 2 to 3 mm were present at subcultivation.
No spontaneous bursting, shrinkage, degeneration, or decay of TCCs has been observed. However, when TCCs of H69AR cells were confronted with confluent layers of HUVEC, drastic changes occurred in both formations. First, intense oscillatory cellular movements were triggered on TCC surfaces, preceding the release of cell aggregates which moved away from TCCs to the HUVEC layer. At the same time, HUVEC were disconnected from each other and started moving either randomly or in an oscillatory fashion. Individual tumor cells attached to the much larger HUVEC and spread on their surface, resulting in full covering which secluded HUVEC from the medium.
To our knowledge, neither formation nor merger of malignant TCCs in vitro have been described so far. TCC merger involves mutual cellular penetration of the involved TCC surfaces, i. e. MCP. Contrary to the invasion of tumor cells into non-tumor tissue, pervasion does not lead to destruction but results in TCC growth. However, key features of tumor cell invasion in vivo, such as chemotaxis, spreading and TCC formation as well as disruption of endothelial layers, manifest under conditions in vitro, thus facilitating related experimental studies.
Citation Format: Nico O. Ruprecht, Martin Hungerbuehler, Philipp Kellmann, Nathalie Harder, Günter Schmidt, Carlos Wotzkow, Beat Haenni, Fabian Blank, Hendrik Von Tengg-Kobligk, Christof Granzow. Formation and merger of H69 and H69AR small cell lung cancer (SCLC) tumor cell clusters (TCCs) in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1889. doi:10.1158/1538-7445.AM2017-1889</jats:p
Insight into the dynamic of gadolinium based contrast agent (GBCA) hypersensitivity: Acquisition, persistence and disappearance.
No full textAnaphylaxis against gadolinium based contrast agents (GBCAs) has been reported in all severity grades but is less frequent than hypersensitivity/allergy against iodinated contrast materials. Once a patient acquires such an adverse reaction a life-long premedication is usually the consequence in concert with GBCA-enhanced MR-imaging procedures. The usefulness of this prophylaxis has been questioned, and discussed controversially during the past. The herein presented case may shed some light into the dynamic of contrast-medium-induced hypersensitivity reactions
Improved identification of DNA double strand breaks: γ-H2AX-epitope visualization by confocal microscopy and 3D reconstructed images.
No full textCurrently, in the context of radiology, irradiation-induced and other genotoxic effects are determined by visualizing DSB-induced DNA repair through γ-H2AX immunofluorescence and direct counting of the foci by epifluorescence microscopy. This procedure, however, neglects the 3D nature of the nucleus. The aim of our study was to use confocal microscopy and 3D reconstructed images to improve documentation and analysis of γ-H2AX fluorescence signals after diagnostic examinations. Confluent, non-dividing MRC-5 lung fibroblasts were irradiated in vitro with a Cs-137 source and exposed to radiation doses up to 1000 mGy before fixation and staining with an antibody recognizing the phosphorylated histone variant γ-H2AX. The 3D distribution of γ-H2AX foci was visualized using confocal laser scanning microscopy. 3D reconstruction of the optical slices and γ-H2AX foci counting were performed using Imaris Image Analysis software. In parallel, γ-H2AX foci were counted visually by epifluorescence microscopy. In addition, whole blood was exposed ex vivo to the radiation doses from 200 to 1600 mGy. White blood cells (WBCs) were isolated and stained for γ-H2AX. In fibroblasts, epifluorescence microscopy alone visualized the entirety of fluorescence signals as integral, without correct demarcation of single foci, and at 1000 mGy yielded on average 11.1 foci by manual counting of 2D images in comparison to 36.1 foci with confocal microscopy and 3D reconstruction (p < 0.001). The procedure can also be applied for studies on WBCs. In contrast to epifluorescence microscopy, confocal microscopy and 3D reconstruction enables an improved identification of DSB-induced γ-H2AX foci, allowing for an unbiased, ameliorated quantification
Ex vivo performance comparison of three percutaneous biopsy needle systems.
No full textPURPOSE
The aim of this study is to identify the micro-mechanical characteristics that define biopsy performance in normal ex vivo animal organs.
MATERIALS AND METHODS
Three biopsy systems with differences of needle external diameter, tray height and effective tray length were assessed. Sampling was performed in porcine liver and kidneys with commercially labelled 14G, 16G and 18G, using 2-cm throw needle systems. Five samples were obtained per needle size and per organ, and the experiment was repeated twice for a total of 90 biopsy cores. Samples were analysed and compared in terms of macroscopic aspect, sample length, weight and tissue architecture.
RESULTS
The system with the longest effective needle tray (system A) has shown significant superiority (p < 0.001) versus the other systems regarding the mean weight of tissue obtained for all needle sizes. Furthermore, the 14G needle of system A has shown superiority regarding the number of portal spaces and the 16G regarding the length of kidney fragments obtained.
CONCLUSION
The outcomes obtained with the different biopsy systems were not equal. The micro-mechanical characteristic that appears to influence the quantity and quality of the obtained tissue is the effective needle tray length and not the needle external diameter or the needle tray height. This information should be taken into account in the future design of biopsy needle systems, particularly when potentially used in the assessment of biomarkers and the characterisation of tumour micro-environment.
KEY POINTS
• The amount of obtained tissue mass is not the same among percutaneous biopsy needle systems. • There are different micro-mechanical characteristics that condition the amount of obtained tissue. • The micro-mechanical characteristic that offers more tissue mass for the same calibre is the effective length of the needle tray
Hazard assessment of fluorinated alternatives to long-chain perfluoroalkyl acids (PFAAs) and their precursors : Status quo, ongoing challenges and possible solutions
No full textBecause of concerns over the impact of long-chain perfluoroalkyl acids (PFAAs) on humans and the environment, PFAAs and their precursors are being substituted by alternative substances including fluorinated alternatives that are structurally similar to the substances they replace. Using publicly accessible information, we aimed to identify the status quo of the hazard assessment of identified fluorinated alternatives, to analyze possible systemic shortcomings of the current industrial transition to alternative substances, and to outline possible solutions. Fluorinated alternatives, particularly short-chain PFAAs and perfluoroether carboxylic and sulfonic acids (PFECAs and PFESAs), possess high environmental stability and mobility implying that they have a high global contamination potential. In addition to their potential for causing global exposures, certain fluorinated alternatives have been identified as toxic and are thus likely to pose global risks to humans and the environment. Various factors, particularly the information asymmetry between industry and other stakeholders, have contributed to the current lack of knowledge about the risks posed by fluorinated alternatives. Available cases show that a non-fluorinated substitution strategy (employing either chemical or functionality substitutions) can be a possible long-term, sustainable solution and needs to be further developed and assessed.</p
Using COSMOtherm to predict physicochemical properties of poly- and perfluorinated alkyl substances (PFASs)
No full textRecently, there has been concern about the presence of poly- and perfluorinated alkyl substances (PFASs) in the environment, biota and humans. However, lack of physicochemical data has limited the application of environmental fate models to understand the environmental distribution and ultimate fate of PFASs. We employ the COSMOtherm model to estimate physicochemical properties for 130 individual PFASs, namely perfluoroalkyl acids (including branched isomers for C(4)-C(8) perfluorocarboxylic acids), their precursors and some important intermediates. The estimated physicochemical properties are interpreted using structure-property relationships and rationalised with insight into molecular interactions. Within a homologous series of linear PFASs with the same functional group, both air-water and octanol-water partition coefficient increase with increasing perfluorinated chain length, likely due to increasing molecular volume. For PFASs with the same perfluorinated chain length but different functional groups, the ability of the functional group to form hydrogen bonds strongly influences the chemicals' partitioning behaviour. The partitioning behaviour of all theoretically possible branched isomers can vary considerably; however, the predominant isopropyl and monomethyl branched isomers in technical mixtures have similar properties as their linear counterparts (differences below 0.5 log units). Our property estimates provide a basis for further environmental modelling, but with some caveats and limitations.</p
Monitoring of tumor burden in vivo by optical imaging in a xenograft SCID mouse model: evaluation of two fluorescent proteins of the GFP-superfamily.
No full textBackground Mouse models of human-malignant-melanoma (MM) are important tools to study tumor dynamics. The enhanced green fluorescent protein (EGFP) is widely used in molecular imaging approaches, together with optical scanners, and fluorescence imaging. Purpose Currently, there are no data available as to whether other fluorescent proteins are more suitable. The goal of this preclinical study was to analyze two fluorescent proteins of the GFP superfamily under real-time in vivo conditions using fluorescence reflectance imaging (FRI). Material and Methods The human melanoma cell line MeWo was stable transfected with one plasmid: pEGFP-C1 or pDsRed1-N1. We investigated two severe combined immunodeficiency (SCID)-mice groups: A (solid xenografts) and B (xenografts as metastases). After three weeks, the animals were weekly imaged by FRI. Afterwards the mice were euthanized and metastases were imaged in situ: to quantify the cutis-dependent reduction of emitted light, we compared signal intensities obtained by metastases in vivo with signal intensities obtained by in situ liver parenchyma preparations. Results More than 90% of cells were stable transfected. EGFP-/DsRed-xenograft tumors had identical growth kinetics. In vivo the emitted light by DsRed tumors/metastases was much brighter than by EGFP. DsRed metastases were earlier (3 vs. 5 weeks) and much more sensitive detectable than EGFP metastases. Cutis-dependent reduction of emitted light was greater in EGFP than in DsRed mice (tenfold). Autofluorescence of DsRed was lower than of EGFP. Conclusion We established an in vivo xenograft mouse model (DsRed-MeWo) that is reliable, reproducible, and superior to the EGFP model as a preclinical tool to study innovative therapies by FRI under real-time in vivo conditions
The Dynamic Structure of Gold Supported on Ceria in the Liquid Phase Hydrogenation of Nitrobenzene
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