Bi1_2009_Lecture19_full

Bi1_2009_Lecture19_full - Shingles* • Caused by...

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Unformatted text preview: Shingles* • Caused by reactivation of Herpes Zoster Virus • Infection of the nerve roots • After recover from chicken pox, virus is dormant in nerve roots -- usually dormant forever • Virus can be reactivated by stress, aging, or a weakened immune system • Reactivated virus causes shingles, not chicken pox Response to a question in class on Friday May 8 Example of reactivation by a herpes virus -Herpes Simplex virus and cold sores •A virus in a latent state is not being replicated. Figure 11-4 • Can’t be detected by the immune system (not producing viral proteins that can be displayed as peptides on MHC proteins). • Infection by herpes simplex virus is cleared by the immune system, but a residual infection persists in sensory neurons that innervate the lips. • Neurons express very low levels of class I MHC molecules, so hard for CD8 T cells to recognize and attack infected neurons. • If virus is reactivated (stress or alteration in immune status), area served by the infected nerve is reinfected and you get a new cold sore. Antibodies -- serum proteins that combat pathogens ~10 nm (100 Å) Immunoglobulin G (IgG) • Typical mammal (e.g., you, a mouse) has the capacity to make >1016 different types of antibodies. Arrangement of CDRs in Ab combining sites: CDR3s always in center, CDR1 and CDR2 always on sides CDR2 (L) CDR3 (H) CDR1 (H) CDR2 (H) CDR1 (L) CDR3 (L) Branden and Tooze, Fig. 15.13 Janeway et al. (2005) Immunobiology: The Immune system in health and disease Garland Publishing, 6th edition, figure 3-8 Enyzme-linked immunosorbent assay (ELISA) to detect anti-HIV-1 p24 antibodies Microtitre well coated with p24 Serum added Anti-human antibody labeled with enzyme added Enzyme substrate added Note: False positives can result from cross-reactions. See Figure 49.15 in Freeman 3/E Western blot to detect anti-HIV-1 antibodies Lyse (break open) laboratory strain of HIV-1 in detergent Separate HIV proteins by SDS-PAGE* Transfer proteins to membrane (blot) Cut membrane and incubate in blood sample from individual being tested 6 -gp160 -gp120 -p66 -p55 -pg41 -p32 -p24 -p17 Detect bound antibodies Days after HIV-1 infection 30 SDS is a detergent that binds to and unfolds proteins; PAGE: polyacrylamide gel electrophoresis Clicker question In SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins migrate towards the positive electrode because: 1) 2) 3) 4) The polypeptide backbone is negatively charged. The sidechains are negatively charged. SDS is negatively charged. Migration is random; ~half of proteins migrate towards the positive electrode. What antibodies do • Don’t directly kill anything • Can block entry of a virus into a host cell or prevent virus from replicating (neutralizing antibodies) gp41 gp120 IgG CD4 CCR5 What antibodies do • Can tag invaders for destruction (first of three ways) – By complement -- binding of IgM or IgG to repeated epitope on invader surface triggers “classical” pathway (movie in extra material at end of Antibodies Lecture 1) Electron micrographs of ~100 Å diameter membrane attack complex channels that are one of the end results of complement activation. What antibodies do • Can tag invaders for destruction (2nd of three ways) – By macrophages: antibodies opsinize (decorate the surface of) invaders -Fc receptors on macrophages bind to exposed Fcs to increase phagocytosis What antibodies do • Can tag invaders for destruction (3rd of three ways) – By Natural Killer (NK) cells: Fc receptors on NK cells bind to exposed Fcs to activate Antibody-Dependent Cellular Cytotoxicity (ADCC) • • NK cells bear surface Fc receptors (CD16). If Fc regions of IgG are clustered or aggregated by antigen on a target cell, they bind to CD16. Binding to CD16 causes contents of granules inside cells to be released --> lysis of target cells. Antibodies can bind to different epitopes on the same Protein -antigen The immune response to any antigen is polyclonal antigens Note that these three Fabs bind to different regions of the model antigen lysozyme Figure courtesy of Ian Wilson, Scripps Institute Haptens What if you want a single, chemically-homogeneous antibody against an antigen? Answer: you make a monoclonal antibody -- see next slide and link to Köhler and Milstein’s 1984 Nobel Prize lecture on Bi1 website. B cell hybridomas secrete monoclonal antibodies Polyclonal B cells secreting antibodies against antigen A cannot be grown in tissue culture, so can’t produce a clone secreting a single type of antibody. Fuse B cells with myeloma (malignant tumor) cells. These cells have been immortalized (can be grown in tissue culture). Use myeloma cells that lack the enzyme hypoxanthine:guanine phosphoribosyl transferase (HGPRT) enzyme. Resulting hybrid cells (hybridomas) secrete antibody and can be grown in tissue culture. Make clones of single hybridomas. Figure A-14 part 1 of 2 Monoclonal antibodies have many uses in biology, biotechnology, medicine • Used to detect presence and/or quantity of an antigen; e.g., Western blot, ELISA, immunofluorescence microscopy, immunoelectron microscopy, flow cytometry. • Used to purify antigens; e.g., immunoprecipitation (e.g., CHiP), immunoaffinity chromatograpy. • Used for medical applications, especially for the treatment of cancer. 160 different monoclonal antibodies in clinical trials or awaiting FDA approval (August 2006). Examples: Therapeutic uses of monoclonal antibodies •Rituximab (Genentech) -- against CD20 antigen on surface of normal and malignant B cells. Used to treat non-Hodgkin’s lymphomas (B cell lymphomas). •Herceptin (Genentech) -- against HER2 antigen. Given to patients with metastatic breast cancer whose tumors overexpress the HER2 protein (growth factor receptor). (HER2-positive breast cancers are more aggressive than HER2-negative breast cancers.) Different ways monoclonal antibodies are used to eliminate tumors Figure 14-17 e.g., ricin or Pseudomonas toxin Can also link antibody to a chemotherapy drug (e.g., adriamycin) (ADCC) Labeling techniques for immunofluorescence microscopy and flow cytometry Kuby, Kindt, Goldsby, Osborne Immunology Textbook Flow cytometry -Fluorescence Activated Cell Sorting (FACS) Acells A+ cells A two-color FACS analysis Single color FACS analysis (e.g., using anti-A antibody) -- note this is a log scale Kuby, Kindt, Goldsby, Osborne Immunology Textbook Stanford used flow cytometry to screen blood before HIV tests were available • Reduced ratio of CD4+ to CD8+ T cells in AIDS patients • July 1983 to June 1985, Stanford Blood Center used flow cytometry to test donated units for CD4:CD8 ratio • Did not transfuse blood from donors with CD4:CD8 ratio < 0.85 • Most other blood banks did no screening • ~10,000 cases of transfusion-transmitted AIDS in US before HIV test available in 1985 Galel et al., 1995, “Prevention of AIDS transmission through screening of the blood supply” Annu. Rev. Immunol. 201-227 Laboratory use of complement: (Low tech version of FACS) Example: Have mixture CD4 T cells and CD8 T cells Want only CD8 T cells Add anti-CD4 antibody to mixture of T cells. It binds. Now add complement, and CD4 T cells will be killed, leaving you with CD8 T cells only. Another way to sort cells Kuby, Kindt, Goldsby, Osborne Immunology Textbook In vitro selection to produce human monoclonal antibodies or increase affinity of existing monoclonal antibody Figure A-15 Generate library of heavy and light chain variable regions using spleen DNA. Or introduce random mutations into variable regions genes of a specific antibody. Clone into a phage so Multiply phage that each phage display library in expresses one bacteria, bind phage VH-VL surface fusion to surface coated protein. with antigen. Wash away unbound phage. Repeat procedure (multiply recovered phage, bind to antigen, wash away unbound phage) for several cycles. Recover specific high-affinity antigen binding VH-VL regions. Clicker question Epitope : CDR 1) HV region: Ag 2) Ab : Ig 3) Fab : Fc 4) Ag : Ab Clicker question After an egg is fertilized, the DNA in the egg is copied. Copies are passed to daughter cells, copied again, passed to new daughter cells, etc. etc. With the exception of errors arising during copying (mutations), all somatic* cells end up with same DNA as the fertilized egg. *somatic = not a germline cell; i.e., not sperm or egg 1) True? 2) False? Clicker question If you sequenced the receptors and proteins of the innate immune system from identical twins, they would be identical, regardless of differences in immunological experience. If you sequenced the receptors and proteins of the adaptive immune system from identical twins, they would be identical, regardless of differences in immunological experience. 1) 2) 3) 4) True, True False, False True, False False, True Extra slides for reference The diversity of antigen receptors in both B and T cells is generated by rearrangements of gene segments Antibodies (and T cell receptors) are encoded by sets of gene segments. During development of a B (or T) cell, gene segments are joined randomly by DNA recombination (irreversible). Juxtaposed gene segments encode variable part of the antibody (or T cell receptor). Different cells join gene segments differently, so receptors are unique. Each B cell bears many copies of its unique receptor (membrane-bound antibody). Each T cell bears many copies of its unique receptor (T cell receptor; TCR). Figure 1-18 Variability within antibody V domains clusters in three regions Wu and Kabat index of variability: # aa that occur at that position / frequency of most common aa at that position 3 hypervariable (HV) regions (CDRs) Light Heavy 24-34 31-35 50-56 50-65 89-97 95-102 Janeway et al. Immunobiology Figure 3.6 V regions encoded by >1 gene segment (light chains) Important point: Rearrangement for antibody genes is at the DNA level -- different from RNA splicing, which occurs in many genes Kuby Immunology textbook V regions encoded by >1 gene segment (heavy chains) Kuby Immunology textbook ...
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This note was uploaded on 09/25/2010 for the course BIO 1 taught by Professor Bakorman during the Spring '09 term at Caltech.

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