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Lecture 3

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University of Toronto St. George
Dana Philpott

IMM250 – Lecture 3 – 1. Shigella’s portal of entry - the M cell a. M cells – kind of epithelial cells overlies the Peyer’s patches which is between villi. Have very thin cytoplasm. Could be why Shigella uses this to transport itself. b. Shigella doesn’t like to go through microvilli, instead go through M cells. 2. Once the barrier is breached how are pathogens detected and dealt with? a. Innate immune detection (this lecture) b. Cellular and Humoral innate factors (next week) 3. Innate immunity a. • Innate denotes a property of some thing or action which is essential and specific to that thing or action, and which is wholly independent of any other object, action or consequence. Also known as “inborn”. Represents THE immune system is in most multicellular organisms (from plants, sea urchins, flies, hydra, etc) -– innate immunity is in both multi cellular organisms. Plants, insects, hydra…humans. Most of the time this is the efficient system against microorganisms, 4. Innate versus adaptive immunity a. Innate immunity i. Conserved throughout evolution In all multicellular organism – have being evolved throughout evolution ii. Organisms possess a set number of recognition molecules – because it has a modified system it has evolved common systems in species iii. Cells are immediately active – once they detect the organism they can put in a program of defense iv. Has no memory- if a cell sees a Shigella today, it is going to react in the exact same way the next day b. Adaptive immunity - i. Evolved 500 million yrs ago? ii. Unique to vertebrates only in vertebrates iii. Infinite number of recognition molecules (antibodies) iv. Cells require priming –requires the innate system sees it first before this system comes into play v. Provides memory of infection – basic of vaccines 5. Innate and adaptive immunity work together in humans a. l In the absence of innate immunity, infections cannot be controlled. Need innate immunity to initiate adaptive immunity. b. l In the absence of adaptive immunity, infection are first controlled by innate immunity but cannot be cleared. c. In normal individuals, infections are cleared by the innate and adaptive immune responses. 6. The discovery of the innate immune system a. Metchnikoff first described role of phagocytes in invertebrates (star fish) – studies how different cells in the blood of the larvae responded to different organisms through the microscope. Watched how different blood cells move out of the cells rush to the threat. He also noticed phagocytosis. First to describe the system we call phagocytosis. How did the starfish know that something was coming in through the epithelial barrier? 7. How can our cells tell that something is foreign? a. Recognition of exogenous microbial products: “non-self” – Dr. Charles Janeway – recognize things which are brought in during tissue injuring, eg: a thorn picking you and this will bring you microbial products inside. b. Recognition of endogenous danger signals: “modifiedself” – Dr. polly Matzinger – recognizing modified cell. Thorn poking into the hand, and rather than responding to the microbial product you respond to the change in the cell which emits signals. 8. Turns out they are both right! a. Innate immunity depends upon the recognition of: i. Non-self: MAMPs (microbial-associated molecular patterns) / PAMPs (pathogen associated molecular patterns) a. – “Signatures” of microbial infection - pieces of microbes growing when we have some sort of injury or infection ii. Modified-self: DAMPs (danger-associated molecular patterns) a. - Danger signals released from dead or dying cells during injury 9. Overview of innate immunity a. Recognition my MAMP/DAMP by a PRM (Pattern recognition Molecule/ Pattern Recognition Receptors) to induce a Signal Transduction(changes in the cell) 10. What are MAMPs? a. Also known as PAMPs (pathogen-associated molecular patterns) b. Represent structural components of microorganisms unable to be modified – in innate system we have only a set no of receptors. So we evolved to recognize very constrained structures which is available in bacteria, fungi etc. this shows that the microorganism has being unable to modify through evolution. 11. Bacteria can be differentiated into 2 groups based on the “Gram-stain” – represents the cell wall of different types of bacteria. If you isolate bacteria from the blood it is important to find what kind of bacteria it is, because antibiotics can have different effects on the type of bacteria. The way to do is isolate bacteria  fix it to the slide (by going It through a Bunsen burner)  add crystal violet and stain them with purple color  treat the cell with iodide  which helps the retain the purple color in the wall  treat it with alcohol to decolour it if positive it would still retain the purple color,negative, the color would wash away then stain it with saffronin to counter strain it. – will be taken up gram negative. a. Positive - main color is purple color. Doesn’t take up Saffronin. - Staphylococcus b. Negative- quickly takes up the Saffronin color – E coli 12. Bacteria can be differentiated into 2 groups based on the “Gram-stain” 13. Gram-positive cell wall a. Staphylococcus is an example, if you cut a cross section of its cell wall you se that it b. Have a thick layer of peptidoglycan outside the cytoplasmic membrane of the bacteria c. There are other stuff sticking into the peptidoglycan like lipoteichoci acid, wall teichoic acid. d. The reason that the color is retaind is because the crystal violet complex gets stuck into the complex cell wall (peptidoglycan) and cant remove in decolourization 14. Gram-negative cell wall a. Ex : E coli- cross section of it has b. Plasmic membrane, inner membrane. The peptidoglycan is found within two layers, and it is a very thin layer within the outer and inner membrane. The lipopolysaccharide (endotoxins) is the one in the outer membrane. This is the one which makes the outer membrane of gram negative bacteria. When doing gram stain and decolorization the crystal violet washes away because there is very little peptidoglycan to react with. 15. MAMPs include these bacterial cell wall products a. Lipotechoic acid (Gram +) - lipidated carbohydrates b. LPS (Gram -; also called “endotoxin”) lipopolysaccharides c. Peptidoglycan (both; also the target of some antibiotics) - peptide carbohydrates i. * Lipidated carbohydrates and peptide-carbohydrates 16. And also “extensions” a. - base unit making up flagella is called “flagellin” – extension of bacteria used to swim i. Protein 17. MAMPs also include a. Genetic material of bacteria and viruses i. DNA: CpG sequences found in microbial DNA (cytosine and guanine separated by a phosphate group, “p”) 1. Quite unique to microbes – in humans this sequences are highly suppressed in our genome, and if they are present it is being modified and methylate them and all our components are made so it is unable to recognize its own DNA as MAMPs. Unique to microbes is unmethylated long sequences of CpG. 2. These sequences are suppressed and/or modified in mammalian genetic material ii. RNA: double or single stranded 1. genetic material often associated with viruses iii. * Nucleic acid 18. MAMPs associated with fungus a. Zymosan - carbohydrate from the cell wall of yeast b. Beta-glucan - carbohydrate from the cell wall of other fungi i. * carbohydrates 19. What are DAMPs? a. High intracellular levels of reactive oxygen species (ROS) Produced by cells that are “blocked” in phagocytic process (and probably destined to die) b. Release of potassium (K+) i. Released by cells with damaged membranes 20. K+ efflux a. K+ is kept at high concentrations INSIDE the cell b. Leakage means something is wrong - cell is dying 21. What are ROS? a. Highly toxic oxygen-derived molecules b. Generated by process called “oxidative burst” - use of oxygen by cells of the innate immune system to form ROS in order to kill microbes during the phagocytosis process. we have recognition of microbe or other particle. It gets engulfed in the phygasome. once the bacteria is within the phagasome, it send oxidative bursts. It dumps these ROS into the vacuole and kills the bacteria within it. Bacteria get degraded. Very important for the killing of the bacteria. 22. Phagocytosis ROS as a DAMP a. “Frustrated” phagocytosis leads to high amounts of intracellular ROS i. Triggers: asbestos (lung disease), uric acid crystals (gout), aluminium (vaccine adjuvant), amyloid (accumulates in Alzheimer’s disease)- when there is large crystals or large bodies, and when it cannot be digested through phagocytosis, the macrophage sees more in it and phagocyte tries to engulf the huge microbes. And then he cannot because they are huge. This is frustrated phagocytosis. Then cell creates high levels of ROS within the cytoplasm and dumps it on the bacteria. On this process we get a high level product of ROS. And ROS is DAMP. ii. These are triggered by asbestos, uric acid crystals, aluminium, amyloid. These generate high levels of ROS, which the body detects as a DAMP then. 23. MAMPs and DAMPs are the “sensees”, what are the sensors? a. PRR - pattern recognition receptors i. Soluble PRRs 1. Collectins (eg: surfactant, mannose-binding lectin), Ficolins b. Cell signaling PRRs 1. Toll-like receptors (TLR), Nod-like receptors (NLR), RIG-I-like receptors (RLR) 24. Collectins – Soluble PRR a. Assemble into multi-meric structures -proteins b. • Bind microbial carbohydrates c. • Link to the complement system for destruction of microbe d. Proteins which are floating around in the blood stream looking for carbohydrate structures associated with microorganisms in the blood stream. Collectins are monomers, kind of like hexameric structures which can then bind to carbohydrates structures on the surface of microbes. When binding they are recognizing these structures on bacteria and fungus, then they link this to the system called complement system. Which is important for the destruction of the microbes. 25. Cell Signaling Pattern Recognition Receptors a. Membrane Associated i. TLR – Toll Like Receptors- 1. Structure: Have an extra cellular domain which forms coiled cellanoid domains, have a cytoplasmic domain which sticks inside the cell and this is how signals are being transferred. b. Cytoplasmic – within the cytoplasm, no attachment to the membrane. floating around the cell and looking for DAMP and MAMP. i. NLR – Nod Like Receptors ii. RLR – Rig I like Receptors 26. TLRs, NLRs, & RLRs a. History of the discovery of these PRRs b. The MAMPs/DAMPs they recognize c. How they tell the cell that they have recognized a MAMP/DAMP i. Signal transduction and activation of gene expression 27. Christiane Nuesslein-Volhard: discovery of Drosophila Toll a. Identified a protein she called “Toll” meaning “COOL” in German b. Helps the Drosophila embryo to differentiate its top from its bottom (She won the Nobel prize for Medicine for her work on Drosophila development in 1995) c. She found that one of the mutant flies had a bizzare phenotype. They found that the fly was missing a gene called Toll. Toll protein helps the embryo differentiate the top from the bottom. 28. Nicolas Gay: Toll and inner part of the Human IL-1 receptor is similar a. Searching for proteins similar to Toll b. Shows cytoplasmic domain of Toll related to that of hIL-1R (now called a TIR domain for Toll IL-1 Receptor) i. Why does a protein involved in human inflammation look like one involved in fly neural tube development? c. When he looked at the toll protein in the cytoplasmic domain, it looked very similar to the cytoplasmic domain to the one in human proteins. Which was the different fromm one receptor. He questioned why did the flies domain and the humans domain look the same? 29. Bruno Lemaitre and Jules Hoffmann: Drosophila use Toll to defend from infection with fungus a. Infected Toll-deficient adult flies with Aspergillus fumigatus b. All flies died after 2-3 days c. Flies use Toll to defend from fun
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