MCB 150 problem set 2

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Biology

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Feb 20, 2024

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Problem Set 2 Name:Nathan Perez MCB150, Spring 2024 Student ID: 3037236398 Prof. Barton Your GSI: Mari Due: Feb 2 at 5pm Please feel to discuss these problems with other students but write your answers independently and in your own words. 1. Predict which pattern recognition receptor(s) is (are) likely to be important for the sensing of the following microbes. a) E. coli (a gram-negative bacterium that replicates extracellularly): The most important for sensing an E. coli would be TLR-4. b) Influenza (a single-stranded RNA virus): The most important PRR would be TLR-7 for influenza c) Listeria (a gram-positive bacterium that escapes from phagosomes): The most important PRR would be TLR-2. d) Herpes simplex virus 1 (a DNA virus): TLR-9 would be the most important PRR for herpes because it recognized unmethylated CpG. 2. You are studying how macrophages respond to infection with a bacterial pathogen that uses a secretion system to inject proteins into the cell. The proteins prevent phagocytosis by blocking activation of Rho GTPases. You suspect that an inflammasome may be activated in the infected cells. a ) Describe 2 consequences of infection that you could measure to test your hypothesis and how you would measure them. - The production and secretion of Proinflammatory cytokines: Cytokines like IL-1beta and IL-18 which play a major role in the mediation of recurring other immune cells to the site of infection. I would measure this by using ELisa for IL1beta and IL-18 to measure the concentrations of the cytokines in the supernatant of the infected macrophage cultures, - The induction of Pyroptosis : Pyroptosis is mediated by the activation of gasdermin D by caspase-1 and we would measure this by using flow cytometry to detect certain cells viability through using stains to see the comparison. b) Assuming you had access to macrophages from any knockout mouse you want, what gene would you knock out to test your hypothesis. - I would knock out either caspase-1 or pyrin to test my hypothesis. 3. TRIF is an adaptor in TLR signaling that leads to IRF3 activation, yet mice lacking IRF3 are more susceptible to many viral infections than mice lacking TRIF. Explain. - IRF3 is a much stronger signal to fight infections compared to TRIF. TRIF is more of a soldier in the fight of infections while IRF3 is more of a boss and has the ability to bring in other strong helpers in fighting the infection. 4. Describe 3 events that occur upon microbe recognition in a tissue and lead to recruitment of immune cells to the site of infection. 1. Detecting signals: Immune cells use special sensors like PRR rto detect the microbes by the body’s innate immune system. These receptors are able to identify PAMPs which are unique to

the molecules found on the microbe. When PAMPS and PRRs bind they trigger immune cells to be produced to release an immune response. 2. Release signaling molecules: The immune cells release molecules like cytokine and chemokines which are signaling proteins that stimulate immune response and promote inflammation and fevers. They alert and recruit other immune cells to the site of interest. 3. Cytokines make blood vessels near infections more permeable and cause cells to display sticky molecules. This lets immune cells leave the bloodstream and enter the infected tissue more easily, sticking to vessel walls and moving into the tissue to fight the infection. 5. Predict the relative severity to infection of humans with mutations in the following genes. Provide a brief explanation of your reasoning. a) E-selectin: Increase severity of infections because E-selectin helps with white blood cells to move to the infection sites. If it wasn’t working then the body would not be able to fight off infection. b) ICAM-1: More severe infections because ICAM-1 helps white blood cells stick to the blood vessel walls and move into the infected tissue. c) TNF: this depends on the mutation on how severe it could become. It is important for inflammation and fighting infections. Having too many TNF could also cause damage or if there is a mutation. 6. One of the common measures that clinicians use to assess a patient who they suspect may have an infection is levels of C Reactive Protein in their blood. Why? - Clinicians use CRP levels to assess a patient’s blood to figure out if they have an infection because it is an indicator of inflammation, when CRP are high that usually means they have an infection and when the infection is brought down CRP levels decrease as well. 7. Some cytokines induced by innate immune activation can have profound systemic effects (both positive and negative). Describe the mechanisms in place to limit systemic levels of these cytokines. - Cytokine Inhibitors: Certain molecules act as sponges or blockers for cytokines, effectively neutralizing them before they can trigger further inflammation. - Regulatory Immune Cells: Some immune cells specialize in calming down the immune response, reducing cytokine output and keeping the response from getting out of hand. - 8. What cytokines would you predict dendritic cells to produce during a) a viral infection? - IFN alpha and beta, IL-18, and IFN gamma b) an extracellular bacterial infection? - IL-1beta, TNF-alpha, chemokines 9. You are performing experiments with dendritic cells isolated from TLR2+/- (heterozygote) or TLR2-/- (knockout) mice. You analyze a few different parameters after stimulating the cells with lipopeptides (a ligand for TLR2/1). Draw the expected results for the following a) western blot for IkBa on lysates of the stimulated dendritic cells b) surface expression of CD80 (a costimulatory molecule) on the dendritic cells, as measured by flow cytometry

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