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Datasets for 'Generation of functional hepatocyte 3D discoids in an acoustofluidic bioreactor'
Full text linkThis dataset supports the publication: 'Generation of functional hepatocyte 3D discoids in an acoustofluidic bioreactor' By: Khedr, Mogib; Messaoudi, Walid, Mohamed; Jonnalagadda, Umesh; Abdelmotelb, Ahmed; Glynne-Jones, Peter; Hill, Martyn; Khakoo, Salim I; Abu Hilal, Mohammad. In: Biomicrofluidics</span
The roles of beta and alpha tryptases in asthma: genetic and immunopharmacological studies
No full textTryptases, the dominant secretory granular proteins from human mast cells, are emerging as important mediators in asthma and allergy. The β- and α- tryptases have highly similar nucleotide sequences and located on the same locus. While the entire population expresses β-tryptase, the α-tryptase gene exhibits copy number variation (CNV). We have studied the association of expression of these allelic variants with asthma or allergic diseases. We have investigated also the potential actions of β- and α-tryptases in vitro and in vivo. We have found that the one alpha tryptase copy allele was significantly associated with lower total serum IgE levels (Z= -2.39, p=0.01) and a tri-allelic architecture with alleles carrying no, one or two copies of the α-tryptase gene was postulated. The addition of βtryptase to epithelial cells induced upregulation of mRNA for IL-8, IL-6 and TNF-α, while α-tryptase on the other hand was without effect in this model. Injection of β-tryptase into the mouse peritoneum induced great accumulation of neutrophils but accumulation of other cell types was less marked. Under the same conditions, injection of α- tryptase induced less neutrophilia but eosinophils, macrophages and mast cells numbers were significantly increased. The actions of β-tryptase seemed be independent of PAR-2 receptors but not the case for α-tryptase, where PAR-2 pathway might take the leads. In conclusion, recombinant α-tryptase may be a stimulus for the recruitment of inflammatory cells and altered cytokine gene expression with effects distinct from those of β-tryptase
Pro-inflammatory actions of the exodomain shed from Protease Activated Receptor 2 (PAR-2)
No full textRationale:Activation of PAR-2 by proteases has been implicated as a key mechanism in allergic and other inflammatory conditions. Proteolytic cleavage can lead to exposure of a tethered ligand whose binding to the receptor stimulates cell signalling, and the release from PAR-2 of an exodomain fragment (PAR-2E). Potential functions of PAR-2E have been little studied.Methods:PAR-2E was synthesised and its stability investigated in human biological fluids, cell supernatants and with selected proteases. The ability of PAR-2E to stimulate calcium flux in cultures of human umbilical vein endothelial cells was examined using a fluorescence based microplate procedure, and altered RNA expression by whole genome microarray analysis and quantitative PCR (qPCR) for selected genes. Adhesion molecule expression was investigated by flow cytometry. In parallel studies, nucleated cells were enumerated and levels of matrix metalloproteases (MMP) determined by gelatin zymography in peritoneal lavage fluid from C57BL/6 mice injected intraperitoneally with PAR-2E.Results:PAR-2E was highly susceptible to degradation by proteases, but its addition to cells resulted in calcium flux, and increased expression of genes for various adhesion molecules (including ICAM1, EPCAM and ITGAL), and cytokines (TNFAIP3 and IL1B). PAR-2E-induced upregulation of ICAM1, VCAM-1, EPCAM and ITGAL was observed on flow cytometry. Injection of PAR-2E into mice was associated with eosinophilia and raised levels of MMP2 in peritoneal lavage fluid.Conclusions:PAR-2E may act as a stimulus for increased expression of adhesion molecules, cytokine release and eosinophilia, and deserves consideration as a mediator of inflammation following PAR-2 activation
Vasoactive intestinal peptide (VIP) induces proliferation of human hepatocytes
No full textBackground: VIP is a gastrointestinal peptide hormone which regulates cell proliferation and differentiation in many cell types. In vitro, hepatocytes have limited viability and proliferative capacity. Although several growth factors such as epidermal growth factor (EGF) have been studied, role of VIP is still unclear. We have investigated the effect of VIP on proliferation of human hepatocytes.METHODS: Human hepatocytes were isolated by two-steps collagenase protocol from liver specimens obtained from patients undergoing liver surgery. Cells were grown in Williams’ E media on collagen coated culture plate. Following 4 to 6 hours of cell plating, treatment with VIP or EGF was started and continued for 3 or 5 days. DNA replication was investigated by measuring Bromodeoxyuridine, BrdU incorporation using immunoflorescent staining. In parallel experiments; 1, 3 or 5 days total RNA was isolated and RT–PCR was performed. In the cell culture supernatant, urea and albumin concentrations were detected.RESULTS: VIP was able to increase total number of hepatocytes and number of proliferating cells in a dose dependent manner markedly at day 3 of treatment. Treatment with VIP was associated with an increase in mRNA expression of ki-67 and H3 genes in a dose dependent process. Although mRNA expression of albumin gene was increased significantly with EGF, no marked alteration was found with VIP treatment. With increasing time, addition of VIP was associated with a decrease in albumin and urea secretion from liver cells.CONCLUSIONS: VIP was able to induce proliferation of human hepatocytes but with little effects on hepatocytes differentiation.<br/
Alpha tryptase: Potential roles in inflammation distinct from those of b-tryptase
No full textRATIONALE: Tryptases are among the most abundant products of the humanmast cell. Beta-tryptase has emerged as an important mediator of allergicinflammation. The copy number of a-tryptase has recently beenfound to be associated with the asthma phenotype, but the function ofthis allelic variant to b-tryptase is unknown.We have investigated potentialactions of a-tryptase.METHODS: C57BL/6 mice were injected intra-peritoneally with recombinanta or b-tryptases (0.005 or 0.5 ug/mouse; 12 mice per group). After6, 12 or 24 h, mice were killed and peritoneal lavage performed.Inflammatory cells were enumerated and levels of albumin and total proteindetermined. Gelatine zymography was applied to examine the activityof matrix metalloprotease (MMP)-2 and MMP-9. In separate experiments,cells of the human bronchial epithelial line 16HBE were incubated withtryptases and expression of mRNA for IL-8, IL-6 and TNF-a examinedby quantitative PCR.RESULTS: Injection of a-tryptase induced the accumulation of neutrophils,eosinophils, macrophages and mast cells (p<0.01) but not lymphocytes.Under the same conditions, b-tryptase stimulated greaterneutrophil recruitment, but the accumulation of other cell types was lessmarked. MMP-9 activity in lavage fluid was unaffected in a-tryptase injectedmice, but it was increased in those injected with b-tryptase.Following addition of a-tryptase to epithelial cells, there was down-regulationof mRNA for IL-6 and TNF-a (p<0.05) whereas b-tryptase inducedstrong up-regulation of the cytokines investigated.CONCLUSION: Recombinant a-tryptase may be a stimulus for the recruitmentof inflammatory cells and altered cytokine gene expressionwith effects different from those of b-tryptase
Generation of functional hepatocyte 3D discoids in an acoustofluidic bioreactor
No full textUltrasonic standing wave systems have previously been used for the generation of 3D constructs for a range of cell types. In the present study, we cultured cells from the human hepatoma Huh7 cell line in a Bulk Acoustic Wave (BAW) field and studied their viability, their functions and their response to the anti-cancer drug, 5 Fluorouracil (5FU). We found that cells grown in the acoustofluidic bioreactor (AFB) expressed no reduction in viability up to 6 h of exposure compared to those cultured in a conventional 2D system. In addition, constructs created in the AFB and subsequently cultured outside of it had improved functionality including higher albumin and urea production than 2D or pellet cultures. The viability of Huh7 cells grown in the ultrasound field to 5FU anti-cancer drug was comparable to that of cells cultured in the 2D system, showing rapid diffusion into the aggregate core. We have shown that AFB formed 3D cell constructs have improved functionality over conventional 2D monolayer and could be a promising model for anti-cancer drug testing
Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: lack of a role of protease activated receptor 2 (PAR2)
No full textBackground: tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2.Objectives: to investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model.Methods: we have injected recombinant human βII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours mice were euthanized, peritoneal lavage performed and inflammatory changes investigated.Results: tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat-inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 h after tryptase injection there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice.Conclusions: our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Though these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2. This article is protected by copyright. All rights reserved.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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