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Project name: Release of Neutrophil Extracellular Traps (NETs) in response to inflammatory stimuli Instructor: Slava Berger, Department of Medicine Abstract/introduction: Neutrophils are the major antimicrobial phagocytes of the innate immune system. They are one of the first effector cells to arrive at the site of infection and play critical roles in pathogen clearance, recruitment, as well as in activation of other immune cells. To combat microbes, neutrophils employ three major strategies: the well-known (1) phagocytosis and (2) degranulation, which are beyond the scope of this research; (3) A novel host defense structure named neutrophil extracellular traps (NETs). NETs are networks of DNA and antimicrobial factors that are released by neutrophils to trap and kill pathogens. They can trap and kill various bacterial, fungal and protozoan pathogens, and their release is one of the first lines of defense against pathogens. NETs may act serving as effective antimicrobial defenses, but also as putative sources of molecules that induce inflammation and may promote tissue damage. Deficient NET formation predisposes humans to severe infection, while uncontrolled NET formation contributes to inflammation, vascular injury and tissue damage. Making too many or not disposing of NETs at the right time and in the right place is pathogenic. Therefore, regulating NET formation is important to prevent many pathological conditions. Thus the present study will determine the effect of various inflammatory and pathogenic stimuli on the release of NETs. In this project we will isolate human neutrophils from the blood of healthy volunteers. We will grow the cells in culture and activate them with various inflammatory and pathogenic stimuli. These stimuli will include pro-inflammatory molecules, membrane components from bacterial and fungal origin. After the stimulation the morphological changes of the cells will be followed by light microscopy. NET formation will be visualized by staining with specific antibodies and fluorescent dyes, and will be quantified. The expected results will contribute to further research of NET formation phenomenon in various pathologies and health condition. Figure 1. Bacteria caught in NETs. Scanning electron microscopy of human neutrophils incubated with Salmonella, a bacterium that causes typhoid fever and gastroenteritis. The bacteria are trapped in NETs. Bar, 1 μm (Brinkmann et al. J Cell Biol. 2012, 198:773-83). Figure 2. Visualizing NETs using neutrophil antimicrobial antibodies or DNA-intercalating dyes by confocal microscopy. Image was taken in our laboratory. Student mission / Objective: 1. Acquiring knowledge and being exposed to the work with human blood and leukocyte isolation using Ficoll-gradient. 2. Flow Cytometry analysis of various leukocyte populations using specific antibodies. 3. Working with neutrophil primary cell cultures. 4. Studying the NET formation by using fluorescent staining of the cells and analysis with flourometer and confocal microscopy. 5. Studying proteins which mediate NET formation using confocal microscopy and Western blotting analysis. 6. Observation of NET formation using live-cell imaging Time-Lapse microscopy. Requirements: 1. Basic background on the inflammatory/immune system 2. Basic knowledge of human blood and its structure. 3. The willingness to work with human blood and cells Please read the attached paper entitled: “Neutrophil extracellular traps: Is immunity the second function of chromatin?” and answer the following questions: 1. Write a brief summary about the innate immune system and its function. 2. Name the blood cells and write the main role of each cell. 3. Write a brief summary about the role of NET formation in one of the diseases mentioned in the attached paper. Recommended reading material: In order to answer these questions, you are free to use scientific papers and scientific websites including Wikipedia. If you have any questions or trouble understanding the requirements, feel free to contact me : [email protected]