Lecture 1 Flashcards
Four Main Classes of Pathogens
- Extracellular bacteria / parasites / fungi
- Intracellular bacteria / parasites
- Viruses (intracellular)
- Parasitic worms (extracellular)
Most cells are better equipped to fight EXTRACELLULAR pathogens.
Define the following terms:
- Commensal bacteria
- Pathogen
- Antigen
- Leukocyte
- Lymphocyte
Commensal bacteria: complex community of microorganisms that live in the mucosal surface of our digestive tracts; 10x more numerous than our own cells; protects us by maintaining the barriers to outcompeting pathogenic bacteria
Pathogen: microbe that can cause disease (= symptoms)
Antigen: material (from pathogen) that induces an immune response
Leukocyte: white blood cells (incl lymphocytes and myeloid cells)
Lymphocyte: specialized white blood cells that mediate adaptive immunity
Compare Innate and Adaptive Immunity on the following:
- Response time
- Response to repeat infection
- Found in (what?)
- Ligands / inducers
- Receptors that mediate pathogen recognition
- Receptor diversity
Response time: (i) hours vs (a) days
Response to repeat infection: (i) identical to primary; no memory vs (a) stronger response upon second exposure; memory
Found in: (i) all multicellular orgs vs (a) vertebrates only; came later in evolution
Ligands / inducers: (i) pathogen associated molecular patterns vs (a) virtually any component of pathogen
Receptors that mediate pathogen recognition: (i) pattern recognition receptors, toll like receptors vs (a) antibodies and t cell antigen receptors
Receptor diversity: (i) limited bc fixed in germline vs (a) unlimited bc generated by V(D)J recombination
What are the 5 types of lymphocytes?
T helper cells: regulate other immune cells → THE GENERALS
T cytotoxic (killer) cells: kill infected cells → THE SOLDIERS
B cells: produce antibodies (immunoglobulin); can also produce cytokines → THE MEDICS
Dendritic cells and macrophage: can directly kill microbes by phagocytosis and other mechs BUT also help to activate T cells (connection btwn innate and adaptive imm)
NK cells: lymphocytes that have characteristics of innate and adaptive immunity; kills virus-infected cells by lysing them; involved with antibody-dependent cell-mediated cytotoxicity (ADCC)
Provide two theories of immune recognition.
Pattern Recognition theory: pathogens contain molecular patterns that differ from host cells “pathogen-associated molecular patterns) (aka PAMPs) ;; recognized by immune cells using specific receptors → FOREIGN
- Pathway: TLR4 → NFkB → inflammatory cytokines
- Eg. lipopolysaccharide LPS in gram negative bacteria
Danger theory: Pathogens cause damage to tissues which leaders to the release of alarm signals that trigger immune responses, sometimes called “damage associated molecular patterns” (aka DAMPs) → NATIVE; released by dying cells / tissues
- Pathway: NALP3 inflammation → pyroptosis
- Eg. uric acid release from dying cells
Define the following:
- Innate Immune System
- Adaptive Immune System
- Immunological Memory
Innate: rapid and non specific; initiated for every injury / infection that occurs to the body; use toll like receptors (TLRs) to mediate response → FIRST LINE OF DEFENSE
Adaptive: develops over time but very specific; initiated only for pathogens; retains memory for faster response in case of future infections; use antibodies and T cell receptors (TCRs) to mediate response → SUIT UP FOR WAR
Immunological memory: core part of adaptive immune system; develops over time but can be a lifelong response ;; usually in regards to the maintenance of memory B and T cells and high serum or mucosal antibody levels that are intended to protect against reinfection
What are the 2 fluid systems. Describe the connection.
Lymph and blood.
Lymphatic system collects lymph (extracellular fluid) that drains from tissues into LYMPH NODES, and eventually returns to blood via thoracic duct.
Naive lymphocytes enter lymph nodes from the blood. Pathogens and their antigens are transported from tissues via lymphatic vessels to the lymph nodes, where they encounter immune cells and activate them.
Contrast primary vs secondary lymphoid organs. Provide examples of each.
Primary: where immune cells develop → creation of naive cells
- Thymus: primary lymphoid organ for T Cell Development
- Bone Marrow: … B cell development
Secondary: where immune cells encounter antigens → activation of naive cells
- Lymph nodes: collect antigens from tissues
- Spleen: … blood stream
Define leukocytes. Which of them belong to the innate v adaptive immune system? What’s the special case?
Leukocytes: white blood cells that derive from hematopoietic stem cells
MYELOID LINEAGE = INNATE → white blood cells other than lymphocytes BUT incl phagocytes
Ie. Monocyte / macrophage, neutrophil, eosinophil, basophil
LYMPHOID LINEAGE = ADAPTIVE → aka lymphocytes; mostly small, relatively inactive cells BUT can be activated / proliferate when triggered by an immune response
Ie. Natural Killer cell, T cells (helper and cytotoxic), B cells
SPECIAL CASE = DENDRITIC CELLS → can arise from either myeloid or lymphoid progenitors
Define the following:
- Bactericidal Mechanisms
- Effector Cells
Bactericidal mechanisms: any mechanisms which eradicates or kills bacteria
Effector cells: perform functions of an immune response, such as cell killing / activation, that can clear the infectious agent from the body
What are the functions of the five myeloid cells?
Macrophage: phagocytosis and activation of bactericidal mechanisms; can present antigens to naive cells to trigger an immune response
Neutrophil: most common white blood cell; phagocytosis of extracellular pathogens; activation of bactericidal mechanisms
Eosinophil: combat parasitic infections via opsonization; effector cell in allergic reactions
Mast cell: releases of granules containing histamine and active agents; has Fc Receptors
Basophil: has Fc receptors → textbook says similar function as mast cell but slide says “unknown activated function”
What are the functions of dendritic cells?
Important bridge btwn innate and adaptive immunity
Myeloid function: antigen uptake in peripheral sites for presentation → early responders to infection
Lymphoid function: antigen presentation to naive lymphocytes for activation → turns on adaptive immunity
Explain how adaptive immunity works in terms of “clonal selection”.
Each lymphocyte bears a single type of receptor w a unique specificity → over time, diversity of these receptors ensures that the immune system can respond to any pathogen
(1) High affinity binding btwn a foreign molecule and lymphocyte receptor leads to lymphocyte activation
(2) This specific lymphocyte with its receptor is now “selected” for “clonal expansion”, referring to the antigen-driven proliferation of mature naive clone of the chosen lymphocyte that can target the foreign antigen
(3) Activation of these clonal cells can lead to eradication of the antigen.
Define the following:
- PAMPs
- DAMPs
- PRRs
PAMPs: aka pathogen associated molecular pathways; molecules specifically associated with groups of pathogens that are recognized by cells of the innate system
DAMPs: aka damage associated molecular pathways → ie efflux of potassium detected in the cytosol indicates cell damage
PRRs: aka pattern recognition receptors; receptors of the innate immune system that recognize PAMPs
Define the following:
- Complements
- Cytokines
Complement: set of plasma / serum proteins that act together against pathogens in extracellular settings; generated in the liver but circulate in the blood as inactive forms; can coat pathogen to facilitate removal or directly kill certain pathogens
Cytokines: aka interleukins; small secreted peptides used for intracellular communication btwn cells in order to influence cell behavior (ie turn on / off immune responses); requires a receptor in order to induce new activity in a cell
What events can arise once a PRR has bound a PAMP?
Increased secretion of antimicrobial products
Increased microbial killing in phagosomes
Production of inflammatory mediators
Increased ability to turn-on T cells
What is inflammation and how does it happen?
Complex series of events induced by infection or tissue damage; one possible end of the complement cascade
Symptoms: redness, pain, swelling, heat
Steps
- Bacteria triggers macrophages and mast cells to release cytokines and chemokines (ie histamine)
- Vasodilation occurs, leading to increased vascular permeability and blood flow to affected area → causes redness, heat, and swelling
- Inflammatory cells migrate into tissue via chemotaxis, releasing inflammatory mediators that cause pain
Chemotaxis: directed migration of cells from blood stream to site of injury via concentration gradient of extracellular signals (in this case, signals are chemokines OR the structurally similar C3A and C5A anaphylatoxins)
What mechanisms make up cell-mediated immunity vs humoral immunity?
Cell-mediated:
- Phagocytosis (cellular eating)
- Cytotoxicity (cellular killing)
Humoral:
- Complements: circulating proteins that can kill pathogens in various ways
- Antibodies: proteins secreted by B cells that assist in more efficient destruction by indirect means
What are the steps of phagocytosis and which cells perform it? What is the function of phagocytosis? What is the relationship between PRRs and phagocytosis?
Macrophages, neutrophils, dendritic cells (also eosinophils) – Provides protection against extracellular pathogens
- Bacterium attaches to membrane
- Bacterium is ingested, forming phagosome
- Phagosome fuses with lysosome
- Lysosomal enzymes digest the bacteria
- Digested material is released from the cell
Many phagocytic receptors are also PRRs involved in innate immune destruction
Encounters with pathogens via PRRs activate the phagocyte by triggering release of inflammatory mediators and increasing phagocytic activity (ie the ability to kill pathogens)
Define these terms. What do they have in common? What are their differences?
- Necrosis
- Apoptosis
- Pyroptosis
All forms of cell suicide, which is used to disrupt the spread of intracellular pathogens and help protect the host by causing an “early death” of the hijacked cell
Apoptosis considered “tidy” while Necrosis and Pyroptosis are “messy” as they rupture the cell membrane and release the foreign bodies
Necrosis: cell organelles and DNA swell and clump until the cell eventually bursts, releasing the foreign bodies → triggers inflammation
Apoptosis: aka “programmed” or “immunologically silent” cell death; broken down from the inside by nucleases and proteases, leading to nuclear fragmentation, proteolysis, blebbing, then death by phagocytosis
Pyroptosis: aka “fiery” death; mediated by catalytic activity of caspase-1 within inflammasome (which can be activated in response to PAMPs or DAMPs) → triggers inflammation and involved with microbial response
Define the following:
- Antigen
- Adjuvant
Antigen: something that generates an immune response by binding specifically to an antibody or by generating peptide fragments that are recognized by a T cell receptor (ie antigen presenting cell)
Adjuvant: any substance that enhances the immune response to an antigen with which it is mixed
What are three components of bacterial cell walls and why are they important to how our immune system functions?
Lipopolysaccharide, aka LPS
Peptidoglycan
Lipoprotein, made of lipopeptides
Are not found in mammalian cells → thus they are PAMPs that can trigger the immune system and can act as good adjuvants
What are TLRs and their functions?
Toll Like Receptors (TLRs) are subsets of PRRs; some can reside on the cell surface while others reside in intracellular spaces
TLRs on plasma membrane recognize bacterial components ;; TLRs on intracellular vacuoles respond to nucleic acid sensing from viruses or bacteria, and are found in endosomes
Ligand induced dimerization of Leucine Rich Repeat (LRR) domains leads to dimerization of the intracellular signaling Toll Interleukin 1 Receptor (TIR) domain
- LRR domains in extracellular domain form a horse-shoe like structure that can bind ligand to the inner or outer groove
- Genes containing LLRs and TIR domains separated by a transmembrane segment are VERY LIKELY to be TLRs
Provide the location, name, and specificity for 9 TLRs given in class. (Some of these TLRs may join together to form complexes.)
Found on PLASMA MEMBRANE:
- TLR-2 / 6 complex recognizes diacyl lipopeptides
- TLR-2 / 1 complex triacyl lipopeptides)
- TLR 4 / MD-2 complex recognizes LPS
- TLR 5 recognizes flagella, which is a bacterial component for movement (not part of cell wall)
Found on INTRACELLULAR VACUOLES:
- TLR 3 responds to dsRNA (produced during viral replication)
- TLR 7 responds to ssRNA (found within viral genomes)
- TLR 9 responds to CpG DNA (unmethylated bacterial DNA)
Describe the TLR Signalling Pathway.
OVERVIEW: TLR binds PAMP > creation of TIR complex > Kinase cascade degrades IkB > NKfB binds to promoters > release of inflammatory cytokines
- TIR domain of TLRs helps to transduce an intracellular signal by interacting with MyD88 (an essential signaling adaptor for many TLR signalling events; also has its own TIR domain)
- homotypic interactions of the two TIR regions lead to creation of a complex
Complex will cause a kinase cascade that results in degradation of IkB, which inhibits NFkB - Once no longer inhibited, NFkB will bind to promoters and turn on gene expression, leading to release of inflammatory cytokines
Compare the TLR Signalling Pathway of Mammals against that of Flies.
Both have innate immunity BUT flies also have embryonic development
Mammals have TLR1-12 while Flies only have Tolls
Mammals have adaptor MyD88 while Flies have dMyD88
Mammals have inhibitor IkB while Flies have cactus
Mammals have transcription factor NFkB (p60/p65) while Flies have Dif/Dorsal
The target genes of mammals are inflammatory cytokines, antimicrobial products, etc ;; while those of flies are antimicrobial products, etc
What are interferons and their functions?
type of cytokine that is specialized to fight viruses; named for their ability to INTERFERE with viral replication in vitro
Functions:
- Makes the host an inhospitable place for the virus to replicate by blocking protein synthesis of the host cell → now virus can’t hijack machinery for replication bc machinery is no longer working
- Increase MHC class 1 expression and antigen presentation in all cells
- Activate dendritic cells and macrophages
- Activate NK cells to kill virus-infected cells
What kinds of PAMPs trigger Type 1 Interferons?
Viral infections induce strong expression of Type 1 interferons aka IFN-alpha and IFN-beta, which promote potent antiviral effector mechs
Vacuolar TLRs (involved in nucleic acid sensing) often respond to viral PAMPs and trigger production of type 1 interferons
TLR3 can link recognition of viral PAMPs to activation of the IRF and induction of antiviral cytokines (Type 1 interferons, alpha and beta) via the transcription factor IRF3
- IRF transcription factors are inactive, cytosolic in resting cells
- TLR3 signaling thru TRIF leads to phosphorylation of IRF3, leading to nuclear transport and activation of the target gene, IFN-beta (a type 1 interferon)
What are the benefits and hazards of TLR signaling / activation in regards to shock? (Experiment with mice.)
TLR helps to initiate inflammation in response to local infection
Systemic TLR activation can have disastrous consequences (esp w gram negative bacteria, ie LPS), such as release of inflammatory cytokines into the entire body’s bloodstream → leads to vascular permeability throughout the body, leading to decreased blood volume overall and subsequent collapse of vessels, eventually leading to organ failure and death
Mice lacking TLR4 are resistant to LPS-induced shock
Mice lacking TLR4 are also more sensitive to localized infection with gram negative bacteria
Describe NOD-like receptors.
large family of mammalian cytosolic innate immune sensors that contain nucleotide oligomerization domain (NOD) domain, aka nucleotide binding (NBD) domain
NOD1 and NOD2 (aka NLR with card domain, NLRC) mediate cytosolic sensing of bacterial PAMPs
Functions
- LRR domains involved in sensing PAMPs / DAMPs
- Some activate via NFkB transcription factors
- Others activate a multi protein complex called inflammasome (contains enzyme caspase, which can be activated to process cytokine proproteins)
Discuss NLRPs or NALPs.
Defined as NLRs that activate inflammasomes have PYRIN domains, aka NLRP / NALP
Senses eflux of potassium out of the cell (indicating infection or cellular damage) via cytosolic sensors
Can be triggered by crystalized molecules
- Uric acid crystals from Gout
- Aluminum hydroxide crystals from Alum adjuvant
- Cholesterol crystals from artherosclerosis
NLRP3 activation is associated w inflammatory response via association with adaptor protein ASC (aka PYCARD) and cleaving of Caspase 1
(1) Potassium efflux induces dissociation of chaperones that keep NLRP3 in an inactive conformation
(2) NLRP3 forms oligomers with ASC causing proteolytic cleavage of pro-caspase 1
(3) Caspase 1 releases mature inflammatory cytokines from their proproteins
Describe the connections between adaptive and innate immunity.
When innate immune signaling is insufficient to clear a pathogen, the adaptive immune system kicks in → thus, innate immune signaling turns ON the adaptive immune response (innate imm response overlaps in time with the initial triggering of the adaptive immune response)
Stimulation of dendritic cells by PAMPs promotes their ability to turn on immune cells (ie T cells) by:
- Inducing DC migration from sites of infection to lymph nodes
- Up-regulation of molecules on DC involved in APC function
Discuss active versus passive immunity.
Behring and Kitasato Experiment: immunize rabbits with tetanus bacteria (treated)→ isolate serum from immunized rabbits and inject into naive rabbit (create treated naives); have control group of untreated naives → challenge all three groups with lethal dose of live tetanus bacteria → found that only treated and treated naives survived
Active immunity: animal that was directly injected with the killed pathogen; has immunity for life
Passive immunity: short lived and taken from an actively immunized organism; very specific
Active substance in the “immune serum” that provides protection is ANTIBODIES.
Describe the various protein structures and the interactions within them.
Made of non covalent bonds that are relatively long and low energy (compared to a covalent bond) → less energy needed to break non covalent bonds BUT because there’s a lot of interactions btwn non covalents, it can lead to a very stable structure overall
Primary structure: amino acid sequence of polypeptide chain
Secondary: alpha helix and beta pleated; more interactions
Tertiary: more of a 3D structure that has coalesced due to the interactions btwn them
- Domain: portion of a protein that can form a compact 3D structure
- Can be covalently linked to other domains via flexible region of peptides; all part of the same peptide chain tho
Quaternary: two (or more) folded polypeptides that come together to interact, leading to a dimeric protein molecule
What is the Tiselius and Kabat Experiment? Be able to draw the graph.
(1) immunized rabbits with the purified protein chicken ovalbumin
(2) serum taken from immunized rabbit contains a substance with the capacity to bind to ovalbumin, creating a precipitate
(3) isolate serum into two parts for comparison, one via electrophoresis for identification of different protein fractions (aka starting serum; found albumin, alpha, beta, and gamma peaks) and the other for removal of ovalbumin-binding materials (aka depleted serum)
(4) compared starting v depleted serum, found that gamma had most absorbance / binding activity, THUS must contain antibody (termed it “immunoglobulin”)
Describe the structure of antibodies. Be able to draw and identify certain areas.
Comprised of two types of polypeptide chains: 2 Heavy Chains + 2 Light Chains = 4 chains total
Chains are held together by interchain disulfide bonds and non-covalent interactions
Ig Domains are structures made up of repeating structural units of ~110 amino acids. Each antibody block makes up one Ig domain, thus there are multiple Ig domains in a chain.
Heavy has 4 domains, Light Chain has 8.
C terminal Ig domains are constant and encoded by the germline, thus involved with effector functions. Variations here are limited BUT are responsible for different antibody isotypes and whether they are membrane bound or secreted.
N terminal Ig domain is variable and generated by somatic DNA rearrangements, thus acts as the antigen binding domain bc have essentially unlimited diversity.
Antibodies are BIVALENT, thus have two identical antigen binding sites. More binding sites induce cooperative binding, leading to stronger binding relationships and less dissociation (compared to monovalent)
Hinge region allows flexible movement of antigen binding arms
Define immunoglobulin fold. What is it associated with?
Commonly used structural motif amongst cell surface protein; used throughout the body to maintain good protein-protein interactions
All Ig domains have a similar 3D structure → 2 beta pleated sheets come together to form a sandwich, held together by disulfide bond and hydrophobic interactions, yielding three flexible loops at the end (aka hypervariable regions 1-3)
HV1-3 (aka complementary determining regions, CDR 1-3) is located at each N terminal tip, thus there are 6 CDRs per chain → high variability means that each antibody molecule has a unique antigen binding site with its own dimensions and complementarity (aka specificity)
Intervening framework regions (FR1-4), aka non hypervariable regions, will make up the rest of the structure (ie region of the C terminal)
