What is the immune system? Types of Immunity. Pasteur and rabies vaccine. Historical Role of smallpox. Recognition Response

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1 Recognition Response Effector memory What is the immune system? Types of Immunity Innate Adaptive Anergy: : no response Harmful response: Autoimmunity Historical Role of smallpox Pasteur and rabies vaccine Chinese (variolation( variolation) Lady Montagu (1718) Jenner: : cows (1798) Pasteur: chickens; cholera (1880) Sheep; anthrax 1

2 Transfer of immunity Von Behring and Kitisato Serum Gamma-globulins Antibodies (immunoglobulins( immunoglobulins) Activities gammaglobulins Serum s components 2

3 Types of antibody mediated immunity Active immunity Passive immunity Nobel Prizes Cell-mediated immuity T lymphocytes and macrophage Metchnikoff Phagocytes Chase (1940s): TB in guinea pigs 3

4 2 types of immunity Humoral Cell- mediated (CMI) What is an antigen? How does immune system respond? Role of antibodies Specificity Selective: specificity pre-existing existing Instructional: specifity via Ag template Two theories 4

5 Infection and immunity Viruses: Rotovirus Bacteria: Pseudomonas Yeast: Candida 5

6 Filaria: : parasitic worm Components of immune system Innate Adaptive Specificity Timing Non-specific: innate Skin structures 6

7 Skin section E. coli urinary tract commensals Cilia and mucus lacrimation 7

8 Fever: temperature Physiological barriers Complement Toll-like like receptors TLR s On macrophages G- LPS Produce cytokines Discuss in chapter 3 8

9 factors Phagocytic barriers ph Interferons lysozyme Mac attacking E. coli Macrophage 9

10 Phagocyte Phagocytosis Endocytosis Phagocytosis Receptor-mediated endocytosis pinocytosis 10

11 Phagocytes Inflammation Blood monocytes Neutrophils (PMN s) Tissue macrophages Heat Pain Redness Swelling Collaboration between innate and adaptive Cytokines: soluble mediates that send signals chemokines Signaling Soluble molecule (ligands( ligands) ) and and cell- membrane bound molcule (receptor) 11

12 Defenses Leukocytes 12

13 Adaptive Immunity Antigenic specificity Diversity Immunologic memory Self/non-self recognition Two major groups of cells Lymphocytes Antigen-presenting cells Lymphatic system Lymphocytes: B and T cells 13

14 14

15 Ag-presenting cell and B lymphocyte Immune cells B-cells Bursa Stem cells PMNs: neutrophils Monocytes/ macrophages Lymphocytes B-lymphocytes antigen Spleen, Lymph nodes Phagocytosis: Short-lived Phagocytosis: Long-lived thymus Bursa Plasma cells B-lymphoblasts Memory cells 15

16 Antibody Antibody structure Membrane molecules T-Cells thymus T-lymphocytes Spleen, lymph nodes antigen T- lymphoblasts T-helper T-cytotoxic 16

17 T- lymphocytes Membrane molecules TCR MHC-bound antigens Major Histocompatibility Complex Polymorphic glycoproteins found on all nucleated cells Class I MHC found on all nucleated cells Class II MHC found on antigen-presenting cells Antigen recognition 17

18 Antigen presenting cells Ag-presenting mac and T cell Macrophages B-lymphocytes Dendritic cells Dendritic cell T helper CD4 Ag presentation: Class II MHC Effectors: cytokines Stimulates B-cellsB 18

19 T-cells and mac with Class II MHC T cytotoxic CD8 Ag presentation by class I MHC CTL Virus-infected infected cells Tumor cells Foreign tissue graft Tc activity Antigens and determinants 19

20 How is diversity generated? Genetic rearrangement TCR B-cell antibodies (antigenically( committed) Self-nonself recognition Membrane-bound bound molecules involved in Ag recognition Mem-bound Abs on B cells T cell receptors Class I MHC molecules Class II MHC molecules Major Histocompatibility Complex Polymorphic glycoproteins found on all nucleated cells Class I MHC found on all nucleated cells Class II MHC found on antigen-presenting cells Antigen recognition 20

21 Overview Exogenous Ag: MHC Class II Clonal selection: antigenic commitment Endogenous Ag: MHC Class I 21

22 Primary vs. secondary Graft response Innate vs. Adaptive Immune dysfunction Allergies and asthma Graft rejection and graft vs. host disease Autoimmune disease immunodeficiency 22

23 Overview Allergy ANTIGEN PREPARATION QUESTIONS 1. Define antigen. 2. Define antigenic determinant 3. What is E. coli? 4. How are the cells washed? 5. Why are the cells washed? 6. What is sonication? 7. Why are cells sonicated? 8. How do you determine if sonication is effective? 9. Why do you plate the antigen preparation? ANTIGEN PREPARATION I. Purpose: : To harvest cells and to make a sonicated cell preparation for use as an antigen in immunological procedures. II. Cell Growth: Cells were grown in nutrient broth under aerobic conditions overnight at 37 degrees C. III. Preparation of Cells1 1. Centrifuge cells in sterile tubes at 8000 x g for 10 minutes. (Follow directions for centrifuge using handouts distributed by instructor) 2. Remove supernatant and discard. Add ml of buffer, resuspend cells. 3. Pool cells from large tubes into one small tube and centrifuge e again using table top centrifuge. 4. Remove supernatant, add ml of 0.15 MNaCl, resuspend and centrifuge again if necessary (see instructor). 5. Discard supernatant. 6. Resuspend cells in 0.01 M phosphate buffer in 0.15 MNaCl.. Add enough buffer to give a 10% cell suspension (approximately 9 ml.). IV. Sonication The cell suspension will be sonicated while immersed in an ice bath in order to avoid heat denaturation of the antigenic determinants during sonication. 1. Check the cells microscopically before and after sonication to assure complete disruption. Set the Ultrasonic Dismembrator as directed by instructor. With the probe held stationary on a clamp, raise the cells to the e probe for second bursts. Keep the ice bath around the cells to keep them cool. Centrifuge sonicate as above. Save the supernatant and label as "soluble Ag." Resuspend the pellet in buffer and label as "solid Ag." Store the sonicated preparations in a screw-capped capped tube in the freezer. V. Plate sonicated cells on nutrient agar and incubate at room temperature until next n week. 23