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- Abu-Humaidan, Anas H.A., et al.
(författare)
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EGFR modulates complement activation in head and neck squamous cell carcinoma
- 2020
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 20:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The epidermal growth factor receptor (EGFR) is pivotal for growth of epithelial cells and is overexpressed in several epithelial cancers like head and neck squamous cell carcinoma (HNSCC). EGFR signalling is also involved in diverse innate immune functions in epithelia. We previously found a role for EGFR in modulating the complement system in skin, this prompted an investigation into EGFR role in complement modulation in HNSCC. Methods: We used patient derived HNSCC cell lines with varying sensitivities to EGFR inhibitors, and generated EGFR inhibition resistant cell lines to study the role of EGFR in modulating complement in HNSCC. Results: We found that HNSCC cell lines activate the complement system when incubated with human serum. This complement activation was increased in cell lines sensitive to EGFR inhibition following the use of the tyrosine kinase inhibitor Iressa. Sensitive cell line made resistant to EGFR-inhibitors displayed complement activation and a decrease in complement regulatory proteins even in the absence of EGFR-inhibitors. Complement activation did not cause lysis of HNSCC cells, and rather led to increased extracellular signal-regulated kinase (ERK) phosphorylation in one cell line. Conclusion: These data indicate that EGFR has a complement modulatory role in HNSCC, and that a prolonged EGFR-inhibition treatment in sensitive cancer cells increases complement activation. This has implications in understanding the response to EGFR inhibitors, in which resistance and inflammatory skin lesions are two major causes for treatment cessation.
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| 2. |
- Abu-Humaidan, Anas H., et al.
(författare)
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Persistent intracellular Staphylococcus aureus in keratinocytes lead to activation of the complement system with subsequent reduction in the intracellular bacterial load
- 2018
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9:MAR
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Tidskriftsartikel (refereegranskat)abstract
- The complement system is an ancient part of the innate immune system important for both tissue homeostasis and host defense. However, bacteria like Staphylococcus aureus (SA) possess elaborative mechanisms for evading both the complement system and other parts of the immune system. One of these evasive mechanisms-important in causing chronic and therapy resistant infections-is the intracellular persistence in non-immune cells. The objective of our study was to investigate whether persistent intracellular SA infection of epidermal keratinocytes resulted in complement activation. Using fluorescence microscopy, we found that persistent SA, surviving intracellularly in keratinocytes, caused activation of the complement system with formation of the terminal complement complex (TCC) at the cell surface. Skin samples from atopic dermatitis patients analyzed by bacterial culture and microscopy, demonstrated that SA colonization was associated with the presence of intracellular bacteria and deposition of the TCC in epidermis in vivo. Complement activation on keratinocytes with persistent intracellular bacteria was found with sera deficient/depleted of the complement components C1q, Mannan-binding lectin, or complement factor B, demonstrating involvement of more than one complement activation pathway. Viable bacterial counts showed that complement activation at the cell surface initiated cellular responses that significantly reduced the intracellular bacterial burden. The use of an inhibitor of the extracellular signal-regulated kinase (ERK) abrogated the complement-induced reduction in intracellular bacterial load. These data bridge the roles of the complement system in tissue homeostasis and innate immunity and illustrate a novel mechanism by which the complement system combats persistent intracellular bacteria in epithelial cells.
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| 3. |
- Fisher, Jane, et al.
(författare)
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Using chemiluminescence imaging of cells (CLIC) for relative protein quantification
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Cell physiology and cellular responses to external stimuli are partly controlled through protein binding, localization, and expression level. Thus, quantification of these processes is pivotal in understanding cellular biology and disease pathophysiology. However, it can be methodologically challenging. Immunofluorescence is a powerful technique, yet quantification by this method can be hampered by auto-fluorescence. Here we describe a simple, sensitive and robust chemiluminescence-based immunoassay (chemiluminescence imaging of cells; CLIC) for relative quantification of proteins. We first employed this method to quantify complement activation in cultured mammalian cells, and to quantify membrane protein expression, shedding, binding and internalization. Moreover, through specific membrane permeabilization we were able to quantify both cytosolic and nuclear proteins, and their translocation. We validated the CLIC quantification method by performing parallel experiments with other quantification methods like ELISA, qPCR, and immunofluorescence microscopy. The workflow of the immunoassay was found to be advantageous in certain instances when compared to these quantification methods. Since the reagents used for CLIC are common to other immunoassays with no need for specialized equipment, and due to the good linearity, dynamic range and signal stability inherent to chemiluminescence, we suggest that this assay is suitable for both small scale and high throughput relative protein quantification studies in whole cells.
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