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MDSC 321 Final Exam GRADED A
What is MHC? - answer-✔Major histocompatibility complex What was MHC first recognized for? - answer-✔Self/non-self discrimination in transplant rejection What is MHC absolutely essential for? - answer-✔Displaying an antigen to T-cells What is the only way T-cells can see antigens? - answer-✔In the context of self MHC How many classes of MHC are there? Define them and their function. - answer-✔I
- presentations of antigens inside the cell to killer T-cells II - presentation of extracellular antigens to helper T-cells III - secreted proteins not involved in antigen presentation Where is MHC II expressed? - answer-✔Only in membranes of antigen presenting cells (macrophages and dendritic cells and B-cells) What are the names of the 3 genes MHC II has? - answer-✔HLA-DP, HLA-DQ, HLA-DR How many chains does each MHC II gene have? - answer-✔2 chains - alpha and beta
Where is MHC I NOT expressed? - answer-✔Red blood cells How many chains do MHC I genes have? - answer-✔1 chain - alpha What are the names of the genes MHC I has? - answer-✔HLA-A, HLA-B, HLA-C What does polygenic mean? How many genes does MHC have? - answer-✔more than 1 gene; MHC has 6 True or False: not all MHC molecules within an individual are the same. - answer- ✔False. MHC molecules differ between individuals, but are the same within one. What is MHC a result of? - answer-✔Polymorphism What is a polymorphism? How do they arise? - answer-✔multiples alleles in a gene loci; arise due to mutations How many MHC are expressed on an antigen presenting cell? Explain where they come from. - answer-✔(3 MHC I + 3 MHC II from mom) + (3 MHC I + 3 MHC II from dad) = 12 Where does the diversity in an MHC molecule occur? - answer-✔Peptide binding cleft What is/are the difference(s) between the peptide binding cleft of MHC I and II? What do these differences mean? - answer-✔Class I has cleft that is closed at the end - peptide must be completely contained
- Transport whole complex to surface within vesicles How many chains does a TCR have? - answer-✔ 2 What are the chains of a TCR held together by? - answer-✔Disulfide linkage What is TCR antigen recognition mediated by? - answer-✔V domain of both peptide chains How many hypervariable loops contact the antigen in a TCR? Name them. - answer-✔ 1 - CDR Is there anything else that contributes to antigen recognition? - answer-✔An extended looping structure called HV True or False: TCR contacts both MHC and the antigen simultaneously. - answer- ✔True Which hypervariable domain does most of the antigen recognition? - answer- ✔CDR What do the remaining hypervariable domains recognize? Name them. - answer- ✔CDR1 and CDR2 recognize the MHC What is self-restriction? - answer-✔TCR repertoire is generated through the positive/negative selection of immature T-cells in the thymus - if it doesn't recognize MHC, it is killed
What are the 2 co-receptors that engage the MHC-antigen complex? What do they bind to? - answer-✔CD8 (killer) - binds to MHC I CD4 (helper) - binds to MHC II What does the binding of co-receptors to MHC result in? - answer-✔Lowers activation threshold of T-cells - T-cells can recognize much smaller infections What is CD3? - answer-✔Signalling molecule True or False: the cytoplasmic tail of the TCR can mediate intracellular signalling on its own. - answer-✔False. Does all signalling with ITAM motifs Explain the twist on the 12/23 rule that occurs with TCR. - answer-✔With B-cells, it is impossible to have 2 D's join together. T-cells have alternative joining of D segments (can have VJ, VDJ, VDDJ) How is additional variation created within the T-cell repertoire? - answer- ✔Junctional diversity - P and N nucleotides added between each TCD segments Which hypervariable loops do NOT have diversity? Why? - answer-✔CDR1 and CDR2 - must recognize MHC every time Does allelic exclusion occur in TCR? Is it as strict? - answer-✔Yes and no
Describe the competitive ELISA process. What would more antigen in the sample mean? - answer-✔1. Incubate antibody with antigen to be measured.
- Add this mixture to the antigen coated well.
- Add enzyme conjugated secondary antibody
- Add substrate and measure color. More antigen = less free antibodies = stronger color. What are the advantages of the ELISA processes? (Name 1) - answer-✔Not radioactive Highly sensitive Lots of samples can be measured What is Fluorescent Activated Cell Sorter? - answer-✔The quantification and identification of cell populations based on surface antigens labelled with fluorescent antibodies Explain the process of cell sorting using magnetic bead/antibody conjugates. - answer-✔1. Add specific antibody to the cell extract containing the unknown antigen.
- Add a secondary antibody coupled to magnetic beads
- Apply a magnet and rinse to remove unbound material Explain the process of affinity chromatography. - answer-✔1. Add specific antibody bound to insoluble beads to beaker
- Add solution containing a mixture of antigens
- Wash away the unbound antigens
- Elute unknown antigen.
What is papain digestion? What are the fragments generated? - answer-✔Digesting the antibody into 3 pieces. 2 FAB (fragment of antigen binding) 1 Fc (fragment of crystallisation) What is pepsin digestion? What are the fragments generated? - answer-✔Digesting the antibody into 2 pieces. FAB2 (in tact) and Fc. What is the advantage of digesting the antibody? - answer-✔Certain experimental setups where you have to use fragmented antibody. Example: other leukocytes could bind to the Fc part of the antibody and mess up results. What is immunohistochemistry? What's an example? - answer-✔Using antibodies as a tool to highlight the immune system. Using gold to label antibodies. What is the direct method of seeing antibodies with fluorochrome? - answer- ✔Primary antibody binds to cells with membrane antigen. What is the indirect method of seeing antibodies with fluorochrome? - answer- ✔Label an anti-isotype antibody --> this will bind to the primary antibody attached to the membrane antigen. What is the advantage of modifying the variable domain of an antibody? - answer- ✔To take advantage of the abilities of binding domains of antibodies from other species, without causing the reaction that would normally occur.
What does a non-identity in a DID tell us? - answer-✔The antibodies in each well are different to one another, but each of them match an antigen in the antigen well. Explain the direct Coombs test. - answer-✔1. Blood sample taken.
- Blood tested to see which antibodies attach to the surface of the RBC
- Washed RBC's are incubated with antihuman antibodies
- RBC agglutinates
- Antihuman antibodies form links between RBC's by binding to human antibodies attached to the RBC's Explain the indirect Coomb's test. - answer-✔1. Obtain serum containing antibodies.
- Donor's blood sample is added to the tube with the serum.
- Recipient's antibodies that target the donor's RBC's form antibody-antigen complexes.
- Antihuman antibodies added to the solution
- Agglutination of RBC's occur because human antibodies are attached to RBC's. How can agglutination assays be modified for other purposes? - answer- ✔Influenza can cause RBC agglutination by the hemagglutin on the virus binding the RBC. This property can be used to screen for protective immunity. What is complement fixation/RBC lysis? - answer-✔The recognition of an antibody leading to consumption from the sample. Later addition of washed RBC and anti-RBC antibodies result in no lysis. If there is no initial recognition, the complement is not consumed and is available to lyse RBC added later.
True or False: monoclonal antibody production is a preparation where antibodies come from different B-cells. - answer-✔False. All antibodies produced in this way come from the SAME B-cell. Where are the B-cells harvested from during monoclonal antibody production? - answer-✔The spleen. True or False: the spleen cells produce antibodies solely for the antigen the mouse was challenged with during monoclonal antibody production. - answer-✔False. The mouse makes a mixture of antigen specific and antigen non-specific B-cells. What is the role of the B-cell cancer in monoclonal antibody production? - answer- ✔When mixed with the antigen-specific/non-specific antibodies, they produce hybrids that will live forever in culture. What is the name of the B-cell cancer and antigen-specific/non-specific B-cell fusions? - answer-✔Hybridomas How do we isolate the antigen-specific hybridomas from the sample? - answer- ✔Culture in HAT medium. What is HAT medium? How does it isolate the hybridomas we want? - answer- ✔Hypoxanthine-aminopterin-thymidine medium. Contains inhibitors of de novo nucleic acid synthesis pathway (this kills the B-cell cancer). Spleen-spleen fusions die after a period of time because they do not live forever in culture. Why must the monoclonal antibodies be improved after harvesting them? (Name 2 reasons) - answer-✔- Too antigenic
- Too short/too long half life
What can you achieve with a bi or trispecific antibody? - answer-✔Make 1 drug instead of 2 or 3 that achieve the same things. What are some disadvantages of monoclonal antibody production? - answer-✔- Limited use in other species due to lack of suitable myeloma cells and poor cell fusion
- Very slow
- Not all desired targets will achieve a good antibody response. What is a voluntold? - answer-✔A person in a different class/societal position who was used as experiments for early immunology. What is the basic process of using cell culture as an experimental model? - answer- ✔Give cells in a Petri dish a stimuli and observe the response. What is a flow chamber? What does it tell us? - answer-✔Blood enters a chamber and then can be coated with anything we want. Shows us how cells behave. What is the advantage of using 3D tissue cultures? - answer-✔Get cells to grow into 3D structures. Allow us to study tissue invasion. What are the advantanges of using cell cultures as an experimental model? Name
- Disadvantages? Name 2. - answer-✔Advantages:
- No ethics
- Simple system
- Lots of reagents
- Easy to image
- High throughput Disadvantages:
- no adaptive immunity
- no tissue microenvironment
- Hard to study processed beyond cellular level What are the advantages and disadvantages of using flies/worms as an experimental model? - answer-✔Advantages:
- cheap
- no ethics
- rapid breeding
- easy genetic manipulation
- innate immunity
- high throughput Disadvantages:
- no adaptive immmunity
- small, hard to do surgery
- limited reagents What are the 2 most popular models for basic immunity? - answer-✔Zebrafish and rodents What are the advantages and disadvantages of using zebrafish as an experimental model? - answer-✔Advantages:
- cheap
What are the 3 R's required for approval for use of animals as experimental models? - answer-✔Reduce - use as few animals as possible Replace - with lower organisms (such as mice) Refine - get more data out of each animal/improve technique to minimize discomfort What are the advantages of using prisoners to complete clinical trials? - answer-✔- Provide an opportunity to repay debt to society
- Higher than average prevelance of disease
- Access to advanced health care prisoners wouldn't normally get
- Ability to do followup studies
- Over-representation of minority populations What are the arguments against using prisoners to complete clinical trials? - answer-✔Freedom Informed consent Privacy Incentive What are the two types of human studies? - answer-✔Observational and interventional How are observational studies carried out? - answer-✔Don't affect normal course of treatment, simply watch and observe what happens. How are interventional studies carried out? - answer-✔Test a new treatment by intervening on the patient's health in a way that may or may not help them.
What are the four stages of interventional studies? - answer-✔Phase 1 - make sure the drug is safe Phase 2 - does it work? Phase 3 - large scale; what are the side effects? does it work? Phase 4 - follow up (what side effects come up over a longer time that did not show up earlier?) How can you ethically test treatments/immunizations for potentially deadly disease? - answer-✔Take advantages of situations that already exist in the world (ie: find an area with a higher prevalence for that disease) What are the two types of immunodeficieny? Define them. - answer-✔Primary - congenital Secondary - acquired What is SCID? What type of immunodeficiency is this? - answer-✔Severe Combined Immunodeficiency - collection of conditions resulting in failure of mature B-cell and T-cell development. PRIMARY What is the result of SCID? - answer-✔Severe recurrent infections early in life How is SCID treated? - answer-✔Bone marrow transplant What is ADA deficiency? - answer-✔Mutation in adenosine deaminase. Ultimately inhibits DNA replication.
True or False: T-cell immunodeficiency is more common than B-cell deficiency. - answer-✔False. T-cell immunodeficiency is rare because most also result in B-cell defects. What is bare lymphocyte syndrome? What does it result in? - answer-✔Lack of MHC II expression. Results in lack of helper T-cells available to assist B-cells What is the treatment for bare lymphocyte syndrome? - answer-✔Bone marrow transplant What TAP-deficiency syndrome? What does it result in? - answer-✔Lack of MHC I expression. Results in lack of killer T-cells and therefore no killing of infected targets. What is the treatment for TAP-deficiency syndrome? - answer-✔No treatment What is DiGeorge syndrome? What does it result in? - answer-✔No thymus. Results in generalized immune deficiency (T-cells don't develop and B-cells do not produce antibodies) What is the treatment for DiGeorge syndrome? - answer-✔Thymic transplant What is CGD? What does it result in? What kind of immunodeficiency is this? - answer-✔Chronic granulomatous disease. Results in inability to kill internalized targets due to inability to make ROS. Consequently results in granuloma formation. Example of phagocyte immunodeficiency.
What is LAD? What does it result in? - answer-✔Leukocyte adhesion deficiency. Results in lack of integrins and failure of the leukocyte recruitment cascade. Also results in deficiency of complement receptors. What might secondary immunodeficiency arise from? - answer-✔Medical treatments or infections. What is AIDS? What does it result from? - answer-✔Acquired Immunodeficiency Syndrome. Results from infection with Human Immunodeficiency Virus. How is HIV able to infect the immune system? - answer-✔Virus is picked up by the dendritic cells which transport it to the lymph nodes. Virus is then transferred to helper T-cells and cannot be removed. True or False: HIV caused 26,000,000 deaths between its discovery in 1984 and
- answer-✔False. HIV doesn't kill humans, AIDS does (and was responsible for all those deaths) What is the advantage to using combination therapy to combat HIV? - answer- ✔More drugs = more mutations for resistance needed for the virus to survive. Why do combined therapy drugs work? - answer-✔HIV uses completely different method for cell division. Making drugs that combat the proteins and enzymes required for HIV division does not harm to our own cells. What is autoimmunity? - answer-✔A breakdown in immune tolerance leading to inappropriate immune response directed against self components.
