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MSU MMG 451 - Homework 3.6

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Complete the following learning objectives by reading the introduction of the article and reviewing section 5-12 of the textbook. Figures 5.20 and 5.21 will also be helpful.MMG 451 – Spring 2021Unit 3 – Adaptive Immunity: T cellsHomework 3.6 – Due 04-01-21 before 3:00 PMThe learning objectives listed below relate the research article provided on the course D2L site. Please note that students are NOT expected to read and understand the entire research article. The research article is:Mice expressing human ERAP1 variants associated with ankylosing spondylitis have altered T-cell repertoires and NK cell functions, as well as increased in utero and perinatal mortality. David P. W. Rastall, Fadel S. Alyaquob, Patrick O’Connell, Yuliya Pepelyayeva, Douglas Peters, Sarah Godbehere-Roosa, Cristiane Pereira-Hicks, Yasser A. Aldhamen and Andrea Amalfitano.Complete the following learning objectives by reading the introduction of the article and reviewing section 5-12 of the textbook. Figures 5.20 and 5.21 will also be helpful. - Describe the symptoms of the autoimmune immune disease ankylosing spondylitis (AS).- Pain and stiffness in the lower back and hips, especially in the morning and after periods of inactivity and neck pain and fatigue. Over time, symptoms can worsen, improve, or stop at irregular intervals.-The joint between the base of the spine and the pelvis-The vertebrae in the lower back-The places where tendons and ligaments attach to bones-The cartilage between the breastbone and ribs-Hip and shoulder joints- Identify the two genes that have the strongest association with AS. Rationalize how this relates to the hypothesis about the primary cause of AS.HLA – B27 and HLA-A29- Explain the rational for referring to one ERAP allele as ERAP1-High vs. another as ERAP1-Low.Some MHC class I molecules emerge from the ER carrying a weakly bound peptide that falls out of the peptide-binding groove during transport. When this happens, calreticulin in the vesicle membrane can stabilize the heterodimer of the MHC class I heavy chain and beta-2-microglobulin, and there is a mechanism that can recycle this ‘empty’ MHC class I molecule back to the ER, where it can try again toacquire a tightly binding peptide.- Define the term immunodominant epitope/peptide (you may need to google this).Immunodominant epitopes are from complex antigens that initiate T-cell responses. T-cell epitopes are peptides derived from antigens and recognized by the T-cell receptor (TCR) when bound to MHC molecules displayed on the cell surface of APCs. (a) CD4 T-cells express the CD4 coreceptor, which binds to MHC II, and recognize peptides presented by MHC II molecules.- Draw graphs to demonstrate the rate of peptide trimming and MHC I expression by ERAP1-High and ERAP1-Low proteins as well as secretion of IL-1 by cells expressing ERAP1-High and ERAP1-Low proteins that was demonstrated in previous publications.MHC I secretion rate of Expression of IL-1 peptidetrimming ERAP1- HighERAP1-Low ERAP1- High ERAP1-Low ERAP1- High ERAP1-LowThe videos and questions below are meant as a guide to understand some of the techniques used to gather the data in the research article:Flow cytometry:One of the methods used to perform immunology research is flow cytometry. Here is a link to a YouTube video (12 minutes) that describes this method. https://www.youtube.com/watch?v=sfWWxFBltpQ&t=120sWatch the video and find the answers to the questions below:- About how many cells can be analyzed per second using flow cytometry? Thousands of cells per second- What is the difference between a cell with high forward scatter vs. a cell with low forward scatter?High forward scatter has bigger cell size and forward scatter has smaller cell size- What is the difference between a cell with high side scatter vs. a cell with low side scatter?High side scatter has more granularity and low side scatter has less granularity- What are fluorophores?Fluorescent molecules- What type of molecule are fluorophores often attached to (artificially)? Hint: it’s something B cells makeAntibodies- What is the difference between the molecules: Alexa Fluor 488 and R-PE?Alexa Fluor 488 is green and uses a 530-nanometer band pass filter and R-PE is orange with high quantum yields and uses a 585-nanometer band pass filter.ELISPOT:Another method that is used in the research article above is the ELISPOT. To learn about this method, watch this short animation on YouTube at https://www.youtube.com/watch?v=TXet7c0mLlA- Review ELISAs and identify one similarity between ELISAs and ELISpot and one difference between the two methods.ELISPOT stands for enzyme-linked immunoSPOT and is widely used for monitoring cellular immune responses and has found clinical applications in diagnostics and in the monitoring of graft tolerance or rejectionin transplant patients. The ELISPOT technique has proven to be among the most useful ways to monitor cell-mediated immunity, due to its sensitive and accurate detection of rare antigen-specific T cells (or B cells. ELISAstands for enzyme-linked immunosorbent assay) and is the method to detect an analyte (usually a protein) by means of an antibody. The antibody is then reported back through a secondary antibody coupled to an enzymethat will cause an enzyme-specific substrate to stain the contents of the well.In comparison, an ELISA determines the total concentration of the secreted signaling protein or antibody, whereas an ELISPOT detects individual cytokine or antibody secreting cells answering the question 'what is the frequency of secreting cells?’, although any comparison between these two methods is not very relevant asthey both serve very different


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