Vivo Y1s 2015 Scatter File BEST Download

[Yufei Labbe]

<div>Hepatocyte growth factor/scatter factor (HGF/SF) is a potent mitogen, motogen, and morphogen for epithelial cells expressing its tyrosine kinase receptor, the c-met proto-oncogene product, and is required for normal development in the mouse. Inappropriate stimulation of Met signal transduction induces aberrant morphogenesis and oncogenesis in mice and has been implicated in human cancer. NK1 is a naturally occurring HGF/SF splice variant composed of only the amino terminus and first kringle domain. While the biological activities of NK1 have been controversial, in vitro data suggest that it may have therapeutic value as an HGF/SF antagonist. Here, we directly test this hypothesis in vivo by expressing mouse NK1 in transgenic mice and comparing the consequent effects with those observed for mice carrying an HGF/SF transgene. Despite robust expression, NK1 did not behave as an HGF/SF antagonist in vivo. Instead, NK1-transgenic mice displayed most of the phenotypic characteristics associated with HGF/SF-transgenic mice, including enlarged livers, ectopic skeletal-muscle formation, progressive renal disease, aberrant pigment cell localization, precocious mammary lobuloalveolar development, and the appearance of mammary, hepatocellular, and melanocytic tumors. And like HGF/SF-transgenic livers, NK1 livers had higher levels of tyrosine-phosphorylated complexes associated with Met, suggesting that the mechanistic basis for the effects of NK1 overexpression in vivo was autocrine activation of Met. We conclude that NK1 acts in vivo as a partial agonist. As such, the efficacy of NK1 as a therapeutic HGF/SF antagonist must be seriously questioned.</div><div></div><div></div><div></div><div></div><div></div><div>vivo y1s 2015 scatter file download</div><div></div><div>Download: https://t.co/HjPTjb3Hfl </div><div></div><div></div><div>A novel technique, backscatter scanning electron microscopy (BSE) was used to study frustrated phagocytosis since it provides high-contrast detection of nanowires, allowing clear discrimination between the nanofibres and other cellular features. A human monocyte-derived macrophage cell line THP-1 was used to investigate cell-nanowire interaction in vitro and the parietal pleura, the site of fibre retention after inhalation exposure was chosen to visualise the cell- fibre interaction in vivo after direct pleural installation of AgNWs.</div><div></div><div></div><div>Whilst we evoked frustrated phagocytosis in the pro-inflammatory effects, we also noted that intermediate 5 μm length fibres, were fully enclosed by macrophages but were pro-inflammatory, as previously recorded[14]. Due to restrictions on space we could not fully explore the morphological basis of this new effect and here we extend these findings to fully characterise fibre length effects particularly frustrated phagocytosis in vitro and in vivo. We utilised the technique of backscatter electron microscopy in particular to investigate this phenomenon. We also describe the retention of fibres in the parietal mesothelial layer, an anatomical region not hitherto identified a retention compartment for fibres.</div><div></div><div></div><div>Backscatter scanning electron microscopy examination was carried out on the macrophage accumulations on the parietal pleura samples from mice injected with AgNW3, AgNW5 and AgNW10 at 24 hour post intrapleural instillation in order to examine the development of interaction between the mesothelial cell layer/inflammatory cells on the surface. The dose of 5 μg per mouse used in this study lead to a significant increase in inflammation after treatment with AgNW5, AgNW10, AgNW14 and AgNW28 as previously reported[14]. A dose response was performed by increasing the dose of AgNW3 up to 10 μg which resulted in no significant increase in inflammation. The dose of AgNW5 was decreased to 1 μg and 2.5 μg whereby 2.5 μg continuously showed significant inflammation[14]. AgNW3 treatment was chosen to investigate if any short fibre were retained in the pleural space after intrapleural injection even though no inflammatory response was observed. AgNW5 and AgNW10 treatments were chosen since both treatments lead to a significant increase in pleural inflammation however differ in their interactions with pleural macrophages[14]. No AgNW3 could be observed on the parietal pleura surface 24 hour post treatments confirming that short fibres are readily cleared from the pleural space. In contrast to AgNW3, both AgNW5 and AgNW10 lead to an aggregation of inflammatory cells on the surface of the parietal pleura with accumulation of nanowires within the lesion area (Figure7A,C). Most AgNW5 were fully phagocytosed by pleural macrophages (Figure7A, B and insert, stars). In comparison, AgNW10 showed a number of fibres only partly phagocytosed and therefore leading to frustrated phagocytosis (Figure7C,D white arrow). Some AgNW5 and AgNW10 were not taken up by macrophages as indicated by the yellow arrows (Figure7A,C). By looking at the surface with higher magnification unclosed membrane could be visualised in pleural macrophages phagocytosing AgNW10 (Figure7D and insert, black arrows).</div><div></div><div></div><div>So far, we hypothesised that after a prolonged exposure to fibres in general up to 7 days, the instilled fibres in the pleural space would either be cleared from the pleural space and enter the lymphatic drainage system or be accumulated in a granulomatous lesion on the parietal pleura at stomata[15]. By using backscatter imaging, for the first time it was possible to visualise nanowires in the mesothelial cell layer of the parietal pleura. Both treatments, AgNW5 and AgNW10 (but not AgNW3 which were not observed as they had been cleared) showed nanofibres either on the surface of the mesothelial cells (black arrow Figure9B) or taken up by these cells (white arrow Figure9A,B). This is especially surprising for AgNW5, since pleural macrophages are able to phagocytose these fibres completely as shown in Figure7B. By comparing the structure/shape of the nanowires associated with the mesothelial layer (Figure9) and within macrophages (Figure8B,E), it can quite clearly be seen that the nanowires are still intact after 1 week in the mesothelial layer whereas within the phago-lysosome of a macrophage the fibres start to degrade and lose integrity. These images show evidence that not all fibres longer than 5 μm are internalised by macrophages during the early inflammatory reaction, and that some are compartmentalised into the mesothelial layer which could lead to direct effects on the mesothelial cells and pleural diseases in a later stage if they are biopersistent.</div><div></div><div></div><div>The use of a novel technique, backscatter electron microscopy allowed us to distinguish between membrane bound fibres and unphagocytosed fibres and to visualise the interaction of inflammatory cells with different length of nanofibres on the parietal pleura, the site of fibre retention in vivo.</div><div></div><div></div><div></div><div></div><div></div><div></div><div>An in vivo study performed by Oberdorster et al. assessed the clearance of small (3 μm in diameter) and large (10 μm in diameter) polystyrene microspheres in rat and identified minimal clearance of larger spheres after a 200 days post position period[18]. This was considered to be due to impaired clearance of particles when the macrophage particle load is 60% of its normal volume.</div><div></div><div></div><div>Frustrated phagocytosis has been implicated to play a major role in the development of an inflammatory milieu after exposure to fibres in vitro[19, 20]. We recently demonstrated that frustrated phagocytosis is a major factor in the genesis of inflammation in the pleural space after deposition of long fibres whereby macrophages undergoing frustrated phagocytosis of the long fibres release factors that promote a potent pro-inflammatory cytokine response from adjacent mesothelial cells[20]. In addition we have demonstrated that 5 μm is the threshold for pro-inflammatory effects of fibre in the pleural space for a wide range of fibers[14]. However, so far frustrated phagocytosis has not been fully visualised in vivo in the pleura and the fibre length threshold for frustrated phagocytosis in vivo is unknown. Here we showed that the length cut off value at which long fibres can be fully phagocytosed by macrophages differs in vitro and in vivo. Whereas in vitro fibres of 10 μm could be completely phagocytosed, in vivo inflammatory cells on the parietal pleura showed incomplete uptake and frustrated phagocytosis of 10 μm fibres. There are a number of potential explanations for this anomalous difference between in vivo and in vitro length-dependent effect. The in vivo model is a pleural granuloma in mice and this is very different from the in vitro situation. Firstly the cells used in vitro are human cells, secondly they are a cell line and thirdly they are not in the granulomatous milieu but are spread out singly on plastic, submerged in medium with 10% serum, a very abnormal environment. We conclude that investigations on frustrated phagocytosis and its relation to the pathological effects of fibres need to be carried out in vivo although future work could be focussed on trying different cell lines and culture conditions with the aim of finding a better in vitro model that mimics the in vivo findings.</div><div></div><div></div><div>Interestingly, pleural injection of AgNW5 lead to significant inflammation in the pleural space[14] but was not associated with frustrated phagocytosis in vivo. These results show that frustrated phagocytosis is not solely responsible for the onset of an inflammatory reaction in the pleural space after exposure to fibrous particles and that fibres around 5 μm can be fully phagocytosed but nonetheless cause sufficient cellular stress to induce pro-inflammatory effects (Figure10).</div><div></div><div></div><div>Length categories of frustrated phagocytosis and pleural inflammation and diagrammatic representation of frustrated phagocytosis. A) Summary of frustrated phagocytosis and pleural inflammation in vitro and in vivo. B) Diagrammatic representations of the length-dependent effects seen at the level of the macrophage in granulomas on the parietal pleura after instillation of fibres. These suggest that fibres 3 μm and shorter can be phagocytosed with no inflammation and cleared from the pleural space. Fibres 5 μm long can be entirely enclosed but are associated with inflammation whilst 10 μm fibres clearly cause frustrated phagocytosis and inflammation.</div><div></div><div> ffe2fad269</div>