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- Forslund, E., et al.
(författare)
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Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations
- 2012
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 3:OCT, s. 300-
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Tidskriftsartikel (refereegranskat)abstract
- Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended periods of time. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells.This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g., conjugation, immune synapse formation, and cytotoxic events.The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at the population level.
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| 2. |
- Hosseinzadeh, Ava, et al.
(författare)
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Stable Redox-Cycling Nitroxide Tempol Inhibits NET Formation
- 2012
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A. - 1664-3224. ; 3
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Tidskriftsartikel (refereegranskat)abstract
- To prevent the spread of pathogens neutrophils as the first line of defense are able to release Neutrophil Extracellular Traps (NETs), a recently discovered form of immune response. Reactive oxygen species (ROS) have been shown to be essential for many different induction routes of NET formation. Therefore, pharmacological inhibition of ROS generation has implications for research and medicine related to NETs. The application of diphenylene iodonium (DPI), an inhibitor of NADPH oxidase activity, is limited due to its toxicity to host cells as well as microbes. Therefore, we investigated the effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) a membrane-permeable radical scavenger on NET formation triggered by phorbol esters and Candida albicans. We quantified the amount of NETs with two complementary methods, using a microscopic analysis and an online fluorescence-based assay. In line with removal of ROS, Tempol reduced the amount of NET formation by neutrophils challenged with those stimuli significantly. Since Tempol efficiently blocks NET formation in vitro, it might be promising to test the effect of Tempol in experimental models of disorders in which NETs probably have hazardous effects.
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| 5. |
- Lycke, Nils Y, 1954, et al.
(författare)
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The role of Peyer's patches in synchronizing gut IgA responses.
- 2012
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Ingår i: Frontiers in immunology. - : Frontiers Media SA. - 1664-3224. ; 3
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Forskningsöversikt (refereegranskat)abstract
- Because Peyer's patches (PP) are the main inductive sites for gut IgA responses we have focused this review on what we know about the function of PP germinal centers (GC). The vast majority of IgA gene sequences in the gut lamina propria (LP) are heavily mutated arguing for an origin in GC. Because PP GC formation is dependent on the presence of CD4 T cells, we speculate that all IgA responses in the normal gut are directly or indirectly T cell-dependent (TD). We hypothesize that the CD4 T cell involvement in gut IgA responses against the microbiota is different from that in systemic responses since cognate T-B cell interactions appear not to be required. In the absence of cognate interactions the function of CD4 follicular helper T cells (Tfh) in PP GC is unclear. However, production of IL-21 and IL-6 is more pronounced than in peripheral lymph nodes. Importantly, we discuss how multiple PP are involved in generating specific IgA responses to TD antigens given orally. Recently we found that oral immunization with NP-hapten conjugated to cholera toxin (NP-CT) stimulated a strong highly synchronized, oligoclonal and affinity matured IgA response. This was achieved through re-utilization of GC in multiple PP as GC IgA B cells emigrated into already established GC. Clonally related B cells were present in both inductive and effector lymphoid tissues in the gut and clonal trees involving multiple PP could be constructed in individual mice. Through adoptive transfer of B1-8(hi) NP-specific B cells we demonstrated that GL7(+) PP B cells could enter into pre-existing GC in PP, a process that was antigen-dependent but did not to require cognate Tfh interactions. Finally, we discuss the role of PP GC for the generation of memory B cells and long-lived plasma cells in the light of contrasting findings regarding IgA memory development to colonizing commensal bacteria versus that to oral immunization with enteropathogens or TD antigens.
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