Unit 9 Slides (1) PDF - Body Health & Immunity

HOW DO YOU STAY HEALTHY? Where are the microbes in a healthy body? BONE sterile! BLOOD sterile! MOUTH NOT sterile! STOMACH *almost* sterile! SMALL INTESTINE *almost* sterile! <104 per ml LARGE INTESTINE MUSCLE sterile! ? BLADDER sterile! SKIN NOT sterile! Images: http://www.scientific-art.c...

HOW DO YOU STAY HEALTHY? Where are the microbes in a healthy body? BONE sterile! BLOOD sterile! MOUTH NOT sterile! STOMACH *almost* sterile! SMALL INTESTINE *almost* sterile! <104 per ml LARGE INTESTINE MUSCLE sterile! ? BLADDER sterile! SKIN NOT sterile! Images: http://www.scientific-art.com/portfolio%20medicine%20pages/minibods.htm Where are the microbes in a healthy body? BONE sterile! BLOOD sterile! MOUTH NOT sterile! STOMACH *almost* sterile! SMALL INTESTINE *almost* sterile! <104 per ml LARGE INTESTINE MUSCLE sterile! ≈1010-11 per ml! BLADDER sterile! SKIN NOT sterile! Images: http://www.scientific-art.com/portfolio%20medicine%20pages/minibods.htm Compartmentalization Microbes are tolerated or even encouraged to grow at some sites. Ø Those exact same microbes can be deadly if introduced to a different site in the body that is meant to be kept sterile. Ø Examples: Ø IMMUNITY INVOLVES OVERLAPPING AND SEQUENTIAL RESPONSE SYSTEMS “innate defenses” “first line” defenses innate immune responses adaptive immune response FIRST LINE DEFENSES “first line” defenses innate immune responses adaptive immune response • First line defenses Always “on” – protects us from vast majority of microbes in the environment. Ø Includes barriers like skin and mucous membranes. Ø Includes antimicrobial substances we produce in our blood, sweat, tears, saliva, and other secretions (stomach acid and bile salts) Ø Includes our commensal microflora Ø INNATE IMMUNE RESPONSES “first line” defenses innate immune responses adaptive immune response • Innate immune responses Rapid response (within minutes to hours) after first-line barriers have been breached Ø Includes phagocytic cells like neutrophils and macrophages Ø Includes antimicrobial proteins in the blood (complement) Ø Involves the recognition of specific microbial molecules and/or tissue damage by receptors on immune cells Ø THE ADAPTIVE IMMUNE RESPONSE “first line” defenses innate immune responses adaptive immune response • Adaptive immune responses Takes several days to weeks to fully develop. Ø Involves “learning” about the microbe to generate antibodies and T-cells that recognize one type of microbe and nothing else. Ø Generates “immunological memory” against a specific microbe so that the host response to a second encounter is rapid and robust. Ø Forms the basis of most vaccines. Ø FUNDAMENTAL CONCEPT • Multiple distinct mechanisms work together to control microbial growth in our tissues Ø Physical barriers Ø Mucous trap and expulsion Ø Antimicrobial peptides Ø Acid Ø Bile Ø Nutrient limitation Ø Oxidative attack (e.g. peroxide) Ø Complement Ø Phagocytes (macrophages and neutrophils) Ø Virus infected cells commit suicide (apoptosis) Ø Antibodies produced by B-cells Ø T-cells kill virally infected cells FIRST-LINE DEFENSES: BODY BORDERS • Body borders serve as first-line defense against invading microbes Some “borders” are thought to be “inside” the body but they are in contact with external environment Ø E.g. Digestive tract – starts at mouth, ends at anus Ø E.g. Respiratory tract – cavity that allows gas exchange Ø Mouth Eye Respiratory tract Digestive tract Urogenital tract Skin Anus Skin Copyright © The McGraw-Hill Companies, Inc. FIRST-LINE DEFENSES: PHYSICAL BARRIERS Skin: • Difficult for microbes to penetrate • All exposed surfaces covered with epithelial cells Ø Tightly packed, rest on fibrous layer • Epidermis: many layers of epithelial cells Outermost cells are dead, filled with keratin Ø Repels water, maintains dry environment Ø Continually slough off along with any attached microbes Ø • Dermis: tightly woven fibrous connective tissue Cells surrounded by collagen and other elastic proteins Ø Cow dermis is main component of leather Ø Nucleus Basement membrane Connective tissue Epidermal layer of the skin • Skin (outer cell layers are embedded with keratin) Copyright © The McGraw-Hill Companies, Inc. FIRST-LINE DEFENSES: PHYSICAL BARRIERS Mucous Membranes: • Line digestive, respiratory, genitourinary tracts • Constantly bathed in secretions (e.g., mucous) – wash microbes away • Peristalsis of intestines, mucociliary escalator of respiratory tract move microbes to areas for elimination Nucleus Basement membrane Connective tissue Stratified epithelium • Skin (outer cell layers are embedded with keratin) • Lining of the mouth, vagina, urethra, and anus Cilia Mucusproducing cell Columnar cell Columnar epithelium • Passages of respiratory system • Various tubes of the reproductive systems Copyright © The McGraw-Hill Companies, Inc. FIRST-LINE DEFENSES: ANTIMICROBIALS Antimicrobial factors in saliva (lysozyme, peroxidase, lactoferrin) • Antimicrobial substances protect skin & mucous membranes: Ø Salt from perspiration on skin Ø Ø Lysozyme degrades peptidoglycan Ø Ø Salt-intolerant organisms most susceptible Tears, saliva, mucus, phagocytic cells, blood Peroxidase enzymes break down hydrogen peroxide to make highly reactive O2 species Saliva, milk, body tissue, phagocytes Ø Catalase-negative organisms most susceptible Ø Lysozyme in tears and other secretions and in phagocytes Removal of inhaled particles Normal microbiota Mucus, cilia Physical barrier of skin, salty residue, fatty acids, normal microbiota Transferrin, calprotectin, Albumen, haptoglobin in blood Rapid pH change from stomach to upper intestine Acid in stomach (low p H) Normal microbiota Flushing of urinary tract pH and normal microbiota of vagina Copyright © The McGraw-Hill Companies, Inc. FIRST-LINE DEFENSES: ANTIMICROBIALS Ø Antimicrobial factors in saliva (lysozyme, peroxidase, lactoferrin) Defensins form pores in microbial membranes Ø Small peptides produced by neutrophils and epithelial cells Acidic stomach, vagina and tears make life inhospitable for most microbes Ø Bile produced by the gall bladder and injected onto stomach contents as they pass into the small intestine is a potent antimicrobial compound Lysozyme in tears and other secretions and in phagocytes Removal of inhaled particles Ø Normal microbiota Mucus, cilia Physical barrier of skin, salty residue, fatty acids, normal microbiota Transferrin, calprotectin, Albumen, haptoglobin in blood Rapid pH change from stomach to upper intestine Acid in stomach (low p H) Normal microbiota Flushing of urinary tract pH and normal microbiota of vagina Copyright © The McGraw-Hill Companies, Inc. FIRST-LINE DEFENSES: NUTRITIONAL IMMUNITY Ø Antimicrobial factors in saliva (lysozyme, peroxidase, lactoferrin) Transferrin Transports iron from stores in the liver to cells that require it for growth Ø Quickly binds free iron to make it unavailable to most microbes Ø Ø Lactoferrin Ø Released at sites of infection to further deplete iron Haptoglobin Ø Bind free hemoglobin released from lysed red blood cells Ø Albumen & calprotectin Ø Bind free zinc to make it unavailable to microbes Ø Lysozyme in tears and other secretions and in phagocytes Removal of inhaled particles Normal microbiota Mucus, cilia Physical barrier of skin, salty residue, fatty acids, normal microbiota Transferrin, calprotectin, Albumen, haptoglobin in blood Rapid pH change from stomach to upper intestine Acid in stomach (low p H) Normal microbiota Flushing of urinary tract pH and normal microbiota of vagina Copyright © The McGraw-Hill Companies, Inc. FIRST-LINE DEFENSES: MICROBIAL HELPERS • Commensal microbiota represent a population of microorganisms that grow on body surfaces of healthy individuals Ø Not part of immune system but provide protection Ø Important for development of immune system in distinguishing harmless microbes from pathogens Antimicrobial factors in saliva (lysozyme, peroxidase, lactoferrin) Lysozyme in tears and other secretions and in phagocytes Removal of inhaled particles Normal microbiota Mucus, cilia Physical barrier of skin, salty residue, fatty acids, normal microbiota Transferrin, calprotectin, Albumen, haptoglobin in blood Rapid pH change from stomach to upper intestine Acid in stomach (low p H) Normal microbiota Flushing of urinary tract pH and normal microbiota of vagina Copyright © The McGraw-Hill Companies, Inc. FIRST-LINE DEFENSES: MICROBIAL HELPERS • Commensal microbes provide protection by: Competitive exclusion of pathogens Ø Cover binding sites, consume available nutrients Ø Produce toxic compounds Ø E. coli may synthesize colicins in intestinal tract Ø Lactobacillus in vagina produce low pH Ø Antimicrobial factors in saliva (lysozyme, peroxidase, lactoferrin) Lysozyme in tears and other secretions and in phagocytes Removal of inhaled particles Normal microbiota Mucus, cilia Physical barrier of skin, salty residue, fatty acids, normal microbiota Transferrin, calprotectin, Albumen, haptoglobin in blood Rapid pH change from stomach to upper intestine Acid in stomach (low p H) Normal microbiota Flushing of urinary tract pH and normal microbiota of vagina Copyright © The McGraw-Hill Companies, Inc. FIRST-LINE DEFENSES: MICROBIAL HELPERS • Disruption of normal microbiota can predispose person to infections • Can occur with antibiotic use: Ø Ø Diarrhea & colitis caused by increased Clostridium difficile growth and toxin production in intestine Excessive growth of Candida albicans in vagina • Patients with inflammatory bowel disease also have effects dysbiosis, and restoring normal flora often helps suppress symptoms Antimicrobial factors in saliva (lysozyme, peroxidase, lactoferrin) Lysozyme in tears and other secretions and in phagocytes Removal of inhaled particles Normal microbiota Mucus, cilia Physical barrier of skin, salty residue, fatty acids, normal microbiota Transferrin, calprotectin, Albumen, haptoglobin in blood Rapid pH change from stomach to upper intestine Acid in stomach (low p H) Normal microbiota Flushing of urinary tract pH and normal microbiota of vagina Copyright © The McGraw-Hill Companies, Inc. INNATE IMMUNE RESPONSE “first line” defenses innate immune responses adaptive immune response • Innate immune response Rapid response (within minutes to hours) after first-line barriers have been breached Ø Involves the recognition of specific microbial molecules and/or tissue damage by immunity factors or receptors on immune cells Ø Includes antimicrobial proteins in the blood (complement) Ø Includes phagocytic cells like neutrophils and macrophages Ø FUNDAMENTAL CONCEPT • Self vs. non-self recognition The immune system must constantly distinguish what is self vs. what is non-self. Ø Some of this is accomplished by immune factors and receptors that recognize molecules made by microbes but that are not found in animals. Ø Peptidoglycan Ø Lipopolysaccharide (LPS) Ø Bacterial flagella Ø Fungal cell wall (e.g. zymosan) Ø double-stranded RNA in cytoplasm (viruses) Ø OVERVIEW OF INNATE DEFENSES • Innate defenses recognize & destroy invaders who breach first-line Pattern-recognition receptors – detect & send warning signals Ø Complement system – remove & destroy invaders; work with adaptive immunity Ø First-line defenses Prevent microbial entry Skin and mucous membranes Microbial invasion Sensor systems Detect microbial invasion Pattern recognition receptors (surfaces, endosomes, and phagosomes of sentinel cells) Innate effector actions Destroy invader Inflammatory response Pattern recognition receptors (cytoplasm of many cell types) Inflammatory response Interferon response Copyright © The McGraw-Hill Companies, Inc. Complement system (blood and tissue fluids) Inflammatory response Opsonization Membrane attack complexes THE COMPLEMENT SYSTEM • Proteins circulating in blood and bathing tissues (in inactive state) • Proteins named in order discovered: C1 through C9 Ø Can split into fragments (e.g. C3 splits to C3a and C3b) Ø Named for their ability to “complement” the function of antibodies • Activated by three pathways that lead to formation of C3 convertase, which splits C3; this is central to all complement functions Ø Classical pathway: complement system is activated by antibodies bound to microbe Ø Alternative pathway: triggered when C3b binds to foreign cell surfaces (C3 unstable, so some C3b always present) Ø Lectin pathway: pattern recognition molecules (mannose-binding lectins, or MBLs) bind to mannose of microbial cells, interact with complement system components EFFECTOR FUNCTIONS OF THE COMPLEMENT SYSTEM In summary - activation produces three major outcomes: • “Opsonization”: Ø C3b binds to bacterial cells and foreign particles, allows phagocytes to engulf more easily • Inflammatory Response: Ø Ø C5a attracts phagocytes to area C3a and C5a increase permeability of blood vessels; also induce mast cells to release cytokines • Lysis of Foreign Cells: Ø Membrane attack complexes (MACs) formed by proteins C5b, C6, C7, C8, and C9 molecules assembling in cell membranes of Gram negatives MEMBRANE ATTACK COMPLEXES FORM HOLES IN CELL MEMBRANES REGULATION OF THE COMPLEMENT SYSTEM • Regulation allows host cells to inhibit inappropriate activation Ø e.g. Molecules in animal/human cell membranes bind regulatory proteins that inactivate C3b, thereby preventing opsonization and/or triggering of alternative pathway a Host cell surface: C3b is quickly inactivated when it attaches to the surface. Complement regulatory protein C3b Other complement proteins C3b attaches to host cell surface; complement regulatory proteins inactivate it. C3 convertase not formed; complement system not activated. Host cell surface b Microbial surface: C3b remains active when it attaches to the surface. Complement regulatory protein Other complement proteins C3b C3b attaches to bacterial surface. Other complement effector proteins attach to C3b bound to the surface, forming a C3 convertase. Complement system activated. Microbial cell surface Copyright © The McGraw-Hill Companies, Inc. LEUKOCYTES (WHITE BLOOD CELLS) • Neutrophils engulf and destroy bacteria and other material Ø Most numerous of leukocytes Ø Also called “polymorphonuclear” cells (or PMNs or ”polys”) Ø Primary component of pus Ø A type of “granulocyte” - contain “granules”, which are membrane bound sacs that hold antimicrobial compounds Ø Important during innate response – have potent antimicrobial activity Photo: Graham Beards https://en.wikipedia.org/wiki/Neutrophil_granulocyte#/media/File:Neutrophils.jpg SPECIALIZED ATTRIBUTES OF NEUTROPHILS • Neutrophils act as rapid response team: move into area and eliminate invaders • Critical role in early stages of inflammation First to be recruited from bloodstream to site of damage Ø More powerful than macrophages, but short life span of 1–2 days in tissues - die once granules used Ø • Methods to kill microbes: Phagocytosis Ø Release enzymatic contents and antimicrobial peptides from granules (degranulation) Ø Neutrophil ”NET” (neutrophil extracellular traps) Ø MONONUCLEAR CELLS Mononuclear Phagocytes Ø Comprise mononuclear phagocyte system (MPS) Ø Includes monocytes (circulate in blood) and cell types that develop from them after leaving the blood stream Ø Differentiate into macrophages and dendritic cells Ø Important sentinel cells Microglial cells in the brain Alveolar macrophages in the lungs Kupffer cells in the liver Resident and recirculating macrophages in the lymph nodes Macrophages and blood monocytes in the spleen Mesangial phagocytes in the kidneys Peritoneal macrophages in the abdominal cavity Copyright © The McGraw-Hill Companies, Inc. Precursors in bone marrow MONONUCLEAR CELLS Macrophage & Dendritic Cells Ø Engulf material in tissues including dead cells, debris, destroy invaders. Ø Always present in tissues; can call in reinforcements Ø Not as potent as killers as neutrophils but live much much longer Ø Bring material to cells of adaptive immune system for “inspection” Ø Communicate with cells of the adaptive immune system to help direct the generation of T-cells and antibodies Monocyte Macrophage Dendritic cell THE PHAGOCYTE RESPONSE 1 Chemotaxis C5a Microbes © Meckes/Ottawa/SPL/Photo Researchers, Inc. C3b 2 Phagocyte Lysosomes Recognition and attachment 6 Pseudopod Phagosome C3b Phagolysosome C3b receptors on phagocyte Digestive enzymes 3 Engulfment 4 Phagosome maturation and phagolysosome formation Copyright © The McGraw-Hill Companies, Inc. 5 Destruction and digestion Exocytosis CYTOKINES HELP COORDINATE IMMUNE SYSTEM RESPONSE • Cytokines allow cells to communicate • Often expressed by damaged or infected cells to guide immune defense • Rarely act alone, often released in combinations • Are varied and have overlapping functions: Chemokines: chemotaxis of immune cells Ø Proinflammatory cytokines: activate immune cells, some nonimmune cells Ø e.g. Interleukin-6 and interferons, which induce cells to upregulate their anti-microbial mechanisms Ø Regulatory cytokines: dampen infection, or stimulate cell division or differentiation Ø PATTERN RECOGNITION RECEPTORS (PRRS) • Allow body to “see” signs of microbial invasion • Detect pathogen-associated molecular patterns (PAMPs) • PAMPs common on all microbes, not just pathogens Cell wall components (lipopolysaccharide, peptidoglycan, lipoteichoic acid, lipoproteins), flagellin subunits, viral RNA molecules Ø May be called MAMPs (for microbe-associated) Ø • Some PRRs recognize DAMPs (for danger-associated), which indicate host cell damage TOLL-LIKE RECEPTORS (TLRS) • 10 different TLRs identified, recognize distinct PAMPs Ø Related to Toll proteins from Drosophila sp. (Fruit flies) Phagosome or endosome • Binding of TLRs to their specific PAMPs initiates signalling cascade Results in expression of immunerelated genes (cytokines, chemokines, antimicrobial genes) Ø Can result in programmed cell death of infected cell Ø Detects lipopolysaccharide (LPS) Detects peptidoglycan Detects flagellin Outside of cell TLRs in cytoplasmic membrane Cytoplasm TLRs in phagosomal or endosomal membrane Lumen of endosome Detects dsRNA Detects bacterial DNA Detects ssRNA Copyright © The McGraw-Hill Companies, Inc. NOD-LIKE RECEPTORS (NLRS) • 23 NLRs identified – all reside in the cytoplasm • Can recognize PAMPs and DAMPs Ø NLR - Detects flagellin RLR - Detects dsRNA Detect microbial factors or cell damage • Also result in expression of immunerelated genes • Defects may be linked with inflammatory disease (e.g. Crohn’s disease) NLR - Detects peptidoglycan RLR - Detects uncapped ssRNA NLR - Detects compounds that indicate cell damage Copyright © The McGraw-Hill Companies, Inc. RIG-LIKE RECEPTORS (RLRS) • Cytoplasmic receptor that detect viral RNA indicating infection NLR - Detects flagellin RLR - Detects dsRNA • Distinguish viral from cellular RNA by 2 characteristics: Often has three phosphates at 5’ end (cytoplasmic RNA hides phosphates) Ø Often double-stranded (cellular RNA single-stranded) Ø NLR - Detects peptidoglycan RLR - Detects uncapped ssRNA NLR - Detects compounds that indicate cell damage Copyright © The McGraw-Hill Companies, Inc. OVERVIEW OF INNATE DEFENSES • Detection of invaders & tissue damage initiate an inflammatory response Local blood vessels undergo changes Ø Phagocytes leave bloodstream & accumulate in tissues Ø First-line defenses Prevent microbial entry Skin and mucous membranes Microbial invasion Sensor systems Detect microbial invasion Pattern recognition receptors (surfaces, endosomes, and phagosomes of sentinel cells) Innate effector actions Destroy invader Inflammatory response Pattern recognition receptors (cytoplasm of many cell types) Inflammatory response Interferon response Copyright © The McGraw-Hill Companies, Inc. Complement system (blood and tissue fluids) Inflammatory response Opsonization Membrane attack complexes THE INFLAMMATORY RESPONSE • Pattern recognition receptor-dependent recognition of microbial products (PAMPs) or tissue damage (DAMPs) can trigger the recruitment of leukocytes into the tissue in attempt to eliminate the microbial invaders and/or deal with the tissue damage • Process is called inflammation because it leads to swelling of the tissues Ø Typically also results in redness, heat, pain, sometimes loss of function • If blood vessels damaged, leads to coagulation and increased permeability THE INFLAMMATORY RESPONSE • Inflammatory process involves cascade of events: Dilation of small blood vessels Ø Greater blood flow into tissues (heat, redness) Ø Slower flow rate to capillaries Ø Leakage of fluids Ø Causes swelling, pain Ø Fluid contains substances to counteract invading microbe Ø Migration of leukocytes from bloodstream to tissues Ø Endothelial cells “grab” phagocytes, slow them down Ø Phagocytes squeeze between cells of vessel Ø Neutrophils, monocytes, lymphocytes arrive at infection site Ø Clotting factors wall off site of infection Ø THE INFLAMMATORY RESPONSE • Dead neutrophils, tissue debris accumulate as pus Ø Large amount of pus = abcess • Extent of inflammation varies Depends on nature of injury Ø Acute inflammation is short term Ø Prevalence of neutrophils as infection brought under control, macrophages clean up damage by ingesting dead cells and debris Ø If acute response fails, chronic inflammation results Ø CHRONIC INFLAMMATION • Chronic inflammation can be dangerous Ø Leads to ongoing recruitment and activation of leukocytes Ø In some cases, macrophages, giant cells accumulate, and granulomas form to ‘wall off ’ the infected tissues • Inflammation can also be dangerous when sensitive tissues affected Ø eg brain swelling with meningitis • Chronic inflammation can occur when particulate debris is too difficult to remove Ø eg asbestos FEVER • Fever is an important host defense mechanism • Strong indicator of infectious disease, especially bacterial • Temperature-regulation center in brain normally holds at 37°C but raises during infection in response to signs of systemic infection Ø Oral temperature >37.8°C is considered a fever • Higher temperatures result from the production of proinflammatory cytokines produced by macrophages following detection of microbial products by TLRs Cytokines & fever-inducing substances are known as pyrogens Ø Endogenous pyrogens produced by host Ø Exogenous pyrogens produced by microbes Ø FEVER • The evolutionary benefit of fevers stems from its effect on the optimal growth rate of microbes • Growth rates of bacteria optimized for 37°C typically drop sharply above optimum, allows more time for defenses • Moderate temperature rise increases rates of enzymes, which influences: Ø Ø Ø Ø Ø Ø Enhances inflammatory response Phagocytic killing by leukocytes Multiplication of lymphocytes Release of attractants for neutrophils Production of interferons and antibodies Release of leukocytes from bone marrow ADAPTIVE IMMUNE RESPONSE • Develops most effective means to eliminate invader • Takes a week or more to build following first exposure Ø Innate immunity must protect during this time, but may not be sufficient to prevent disease Ø Person may not survive long enough for adaptive response to begin • Adaptive immunity has memory Ø More rapid and heightened response upon re-exposure Ø Response has specificity for a single molecular target Ø Vaccination relies upon these properties Ø Some pathogens constantly change to evade adaptive response (e.g. influenza) STRATEGY OF THE ADAPTIVE IMMUNE RESPONSE • First response to the pathogen is the primary response • Adaptive immune system “remembers” mechanisms that proved effective against that specific microbe Ø If encountered again, a stronger secondary response results • Two basic strategies for countering foreign materials: Ø Humoral immunity works to eliminate extracellular threats Ø e.g. Bacteria, toxins, viruses in bloodstream, tissue fluids Ø Cell-mediated immunity (CMI), also called cellular immunity, deals with targets that reside within a host cell Ø e.g. Invading virus infecting cell • Both can damage body’s own tissues if misdirected, so system is tightly regulated to prevent autoimmunity Ø Verification from another immune cell type is typically required (!) THE CELLS OF ADAPTIVE IMMUNITY ARE “LYMPHOCYTES” B cell • B cells BCR Mediate “humoral immunity” Ø Each produce a single type of antibody Ø Each B cell makes a unique receptor (BCR) that binds to only a single type of antigen Ø • T cells T cell TCR Mediate “cellular immunity” Ø Major T cell types include “helper”, “cytotoxic” and “regulatory” Ø Each T cell has a unique receptor (TCR) that only recognizes a single type of antigen Ø WHAT IS AN “ANTIGEN”? • The molecular target of the immune response • Antigen comes from antibody generator • Describes a molecule that reacts specifically with antibody, B-cell receptor, or T-cell receptor • Enormous variety of antigens (e.g. microbes, pollen) • Two general categories: Most are T-dependent antigens: B cell requires confirmation from TH cell (helper T cell) to be activated Ø T-independent antigens: activate B cells without TH cell help; include molecules with repeating subunits such as some carbohydrates Ø HUMORAL IMMUNITY • In humans, B cells develop in bone marrow • When B cell receptor binds an antigen the cell becomes “activated”. It proliferates and differentiate in response to particular extracellular antigens Ø Ø Activation “Naïve” B cell Proliferation and differentiation Plasma Cells: Produce Y-shaped antigenbinding proteins called antibodies Memory B cells: Long-lived cells that are ready to respond quickly if exposed to the same antigen in the future Plasma cells Produce antibodies Effector action and consequence Antibodies Antibodies bind antigen Adaptive immunity (humoral) THE B CELL RECEPTOR • The B-cell receptor (BCR) is membrane-bound version of a B-cell’s specific antibody Ø Binding antigen triggers response Ø Usually needs confirmation from helper T cell • Antibodies./B cell receptors bind to antigens with extremely high degree of specificity • Many different antibodies needed for wide array of antigens, ~3x1011 different specificities are possible in each human individual but each B cell only makes one type of antibody. B cell Plasma membrane Antigen-binding site Antigen B-cell receptor (BCR) CHARACTERISTICS OF PRIMARY RESPONSE • Takes 10–14 days for substantial antibody accumulation Ø Some activated B cells continue dividing, others differentiate to form antibody-secreting plasma cells • Additional exposure to antigen yields much faster secondary response Concentration of antibody • Person may be sick, possibly seriously so, although immune system is actively responding Primary response Days Ag Secondary response Months Days Ag Time after antigen (Ag) injection Copyright © The McGraw-Hill Companies, Inc. Months CHARACTERISTICS OF SECONDARY RESPONSE • Significantly faster, more effective than primary response Ø Pathogens usually eliminated before causing harm Ø Vaccination exploits this natural phenomenon Memory B cells responsible: greater numbers present Ø Antibodies coded by these cells bind antigen effectively Ø When activated, some memory cells quickly become plasma cells, produce antibodies Concentration of antibody • Primary response Days Ag Secondary response Months Days Months Ag Time after antigen (Ag) injection Copyright © The McGraw-Hill Companies, Inc. ANTIBODIES • Also called “immunoglobulins” • Variable region accounts for how different antigens are recognized by different antibodies Antigen-binding site attaches to specific epitope Ø Fit is precise but reversible Ø • Constant region on antibodies allows immune system components to recognize otherwise diverse antibody molecules Antigenbinding site Variable region Light Chain (pink & green) Constant region Heavy Chain (red & blue) Copyright © The McGraw-Hill Companies, Inc. ANTIGENS VS ANTIBODIES • Response to antigens varies depending on type • Proteins generally elicit strong response; carbohydrates and lipids are weaker • Epitope – small region of larger antigen that is actually bound by antibody Also called an ”antigenic determinant” Ø Regions of macromolecules Ø e.g. 10 or so amino acids, may or may not depend on 3D shape Ø Antibodies Epitopes (antigenic determinants) Bacterial cell Epitopes (antigenic determinants) Copyright © The McGraw-Hill Companies, Inc. VARIABLE REGIONS FROM THREE DIFFERENT ANTIBODIES (BLUE) BINDING THREE DIFFERENT EPITOPES ON AN INFLUENZA SURFACE PROTEIN (HEMAGGLUTININ) http://pdb101.rcsb.org/motm/170 THE FUNCTION OF ANTIBODIES Opsonization Bacterium Complement System Activation Neutralization Phagocyte Virus Toxin Complement system protein Bacterium Opsonization by C3b Inflammatory response Lysis of foreign cells Antibody-Dependent Cellular Cytotoxicity (ADCC) Infected “self” cell Immobilization (block motility) and Prevention of Adherence Natural killer cell Cross-Linking Bacterium Bacterium Kills cell Flagellum Copyright © The McGraw-Hill Companies, Inc. OVERVIEW OF CELL-MEDIATED IMMUNITY • T lymphocytes, or T cells • Mature in thymus • Two subsets help eliminate antigens Cytotoxic T (TC) cells and helper T (TH) cells Ø Both have multiple surface copies of T-cell receptor (TCR) Ø Analogous to BCR, but does not recognize free antigen Ø Antigen must be presented by body’s own cells Ø • A third subset is regulatory T (Treg) cells Formerly called T suppressor cells Ø Recently described; current focus of research Ø T CELLS REQUIRE THAT ANTIGENS ARE “PRESENTED” TO THEM BY OTHER CELLS antigen presenting dendritic cell Naïve T cell T cell receptor MHC (antigens can also be displayed by macrophages, Bcells, and infected cells) ANTIGEN PRESENTATION • Antigen presenting cells can be phagocytes like macrophages and dendritic cells. Non phagocytic cells can also present antigens if they are infected. • In the example here a dendritic cell has phagocytosed a bacterial cell and destroyed it. • Small peptide fragments from bacterial proteins are loaded onto MHC molecules and displayed on the surface of the cell. ANTIGEN PRESENTATION • The antigen, bound to the MHC molecule on the surface of the dendritic cell, is a target that can be bound by any T cell that has a matching receptor. • When a correct match is made the naïve T cell becomes activated. ANTIGEN PRESENTATION • Activation also involves other signals between the Tcell and the dendritic cell. • The professional antigen presenting cell can also fine tune the effector T cell response against any given microbe, depending on which pathogen-associated molecular patterns are found in that microbe. AFTER ACTIVATION THE T CELL WILL PROLIFERATE (DIVIDE) TO MAKE MORE OF ITSELF OVERVIEW OF CELL-MEDIATED IMMUNITY • Helper T (TH) cells and cytotoxic T (TC) cells must both be activated before they can multiply Ø Professional phagocytic cells (eg. dendritic cells) are responsible for activation of naïve T cells Ø Ø Confirms antigen signifies danger Once activated, T cells proliferate and differentiate Ø Forms effector TH cells or effector TC cells depending upon activating signal Ø Both types can form memory cells • TC cells respond to intracellular antigens, induce infected cells to undergo cell death (e.g. virally-infected cell) • TH cells help orchestrate humoral and cell-mediated immunity: Ø activate B cells Ø Produces cytokines that direct the function of other classic immune cells and non-immune cells EFFECTOR FUNCTIONS OF TC CELLS Normal cytoplasmic proteins MHC class I molecule All nucleated cells present peptides from cytoplasmic proteins on MHC class I molecules. CD8 T-cell receptor TC cells do not recognize peptides presented by healthy “self” cell. (a) Virus Viral proteins Cytokines Targeted delivery of a “death package” Virally infected “self” cells present viral peptides on MHC class I molecules. (b) TC Cell recognizes viral peptides presented by an infected “self” cell and initiates apoptosis in that target. it also releases cytokines that alert neighboring cells to turn on their antiviral defenses. Tc Cell triggers cell death program in infected target cell, prompting it to undergo apoptosis. Copyright © The McGraw-Hill Companies, Inc. OVERVIEW OF HUMORAL & CELL-MEDIATED IMMUNITY Innate immunity Dendritic cell Adaptive immunity Activates T cells that bind antigens representing “danger” Activation Naive helper T cell Naive B cell Naive cytotoxic T cell Proliferation and differentiation TH cells Plasma cells Deliver cytokines Produce antibodies Tc cells Deliver “death packages” Effector action and consequence Antibodies Antibodies bind antigen Adaptive immunity (humoral) Macrophage that has engulfed invaders Infected Macrophage with increased killing power “self” cell Copyright © The McGraw-Hill Companies, Inc. Adaptive immunity (cell-mediated) Infected “self” cell undergoes apoptosis PREVENTION OF INFECTIONS: VACCINES MGY277 – UNIT 21 – CH. 18.1-18.5 © 2/Jeffrey Hamilton/Ocean/Corbis LECTURE OVERVIEW Principles of Immunization Active & Passive Immunity Vaccines & Immunization Procedures Attenuated/Inactivated Vaccines, Vaccination Strategies, Childhood Immunizations, Current Progress Principles of Immunological Testing Obtaining Antibodies, Quantifying Ag-Ab interactions LECTURE OVERVIEW Principles of Immunization Active & Passive Immunity Vaccines & Immunization Procedures Attenuated/Inactivated Vaccines, Vaccination Strategies, Childhood Immunizations, Current Progress Principles of Immunological Testing Obtaining Antibodies, Quantifying Ag-Ab interactions IMMUNIZATION • Immunization is the process of inducing immunity by purposely introducing antigens from a pathogenic microbe or a tumor cell Has had greatest impact on human health of any medical procedures ➢ Example of how knowledge is power with respect to fighting disease ➢ http://www.behance.net/gallery/2878481/Vaccine-Infographic IMMUNIZATION STRATEGIES ACTIVE IMMUNITY NATURAL ACTIVE IMMUNITY Immunity that results from an immune response in an individual after exposure to an infectious agent. ARTIFICIAL ACTIVE IMMUNITY Immunity that results from an immune response in an individual after vaccination. PASSIVE IMMUNITY NATURAL PASSIVE IMMUNITY Immunity that results when antibodies from a woman are transferred to her developing fetus during pregnancy or to an infant during breast feeding. ARTIFICIAL PASSIVE IMMUNITY Immunity that results when antibodies contained in the serum of other people or animals are injected into an individual. Copyright © The McGraw-Hill Companies, Inc. (top left): © SPL/Photo Researchers; (top right): © Dag Sundberg/Photographer's Choice/Getty Images; (bottom left): © BSIP/Phototake; (bottom right): © SPL/Photo Researchers; PRINCIPLES OF IMMUNIZATION Active Immunity: follows antigen exposure • Natural (infection) or artificial (immunization) Passive Immunity: antibodies from another • No memory; protection is lost once antibodies degrade • Natural: during pregnancy, mother’s IgG antibodies cross placenta; breast milk contains secretory IgA • Artificial: injection of antiserum (contains antibodies) ➢ ➢ ➢ ➢ ➢ Can prevent disease before or soon after exposure Can be used therapeutically to limit duration of certain diseases May block action of microbial toxins using antiserum (antitoxin) Immune globulin (IgG fraction from many donors; variety) Hyperimmune globulin (antibodies to specific microbes) LECTURE OVERVIEW Principles of Immunization Active & Passive Immunity Vaccines & Immunization Procedures Attenuated/Inactivated Vaccines, Vaccination Strategies, Childhood Immunizations, Current Progress Principles of Immunological Testing Obtaining Antibodies, Quantifying Ag-Ab interactions VACCINES & IMMUNIZATION PROCEDURES • Vaccines are preparations from a pathogen or its products used to induce active immunity • Effective vaccines must be safe and have no significant side effects ➢ ➢ Give long-lasting protection Ideally low in cost, stable shelf life, easy to administer • Consideration: Protect the individual versus preventing spread in population? ➢ ➢ Some vaccines protect against disease but do not prevent colonization by the pathogenic microbe Vaccines that protect against colonization cause ‘sterilizing immunity’, which means that the immunized individual can no longer be a carrier ➢ Herd immunity develops when a critical portion of population is immune to disease; infectious agent unable to spread due to insufficient susceptible hosts ➢ Responsible for dramatic declines in childhood diseases HERD IMMUNITY VACCINE CATEGORIES Attenuated Vaccine Inactivated Vaccine ATTENUATED VACCINES • Weakened form of pathogen (i.e. less pathogenic) Replicates in recipient; disease undetectable or mild ➢ Naturally mutated or genetically manipulated to replace normal genes ➢ e.g. Measles, mumps, rubella, chickenpox, yellow fever, polio ➢ • Advantages: single dose usually induces long-lasting immunity due to microbe multiplying in body ➢ Can also inadvertently immunize others by spreading • Disadvantages: sometimes cause disease in immunosuppressed Can occasionally revert or mutate, become pathogenic ➢ Generally not recommended for pregnant women ➢ Logistically, they usually require refrigeration to keep active ➢ INACTIVATED VACCINES • Not viable, so unable to replicate, but retain immunogenicity of pathogen or toxin ➢ Some include whole organism, others only fraction of pathogen • Advantage: cannot cause infections or revert to pathogenic forms ➢ Subunit vaccines allow targeting of immune response to key molecular targets • Disadvantage: no replication, so no amplification in vivo; immune response is limited ➢ ➢ Several booster doses usually needed to compensate for lack of persistence Purified antigens (toxoids/subunit vaccines) are poorly immunogenic – lack “danger signal” to activate dendritic cells ➢ Necessitates use of adjuvants which mimic the danger signal to stimulate an immune response and/or allow slow but constant release of the antigen ➢ e.g. alum, PAMPs, lipid-water emulsions TYPES OF INACTIVATED VACCINES Types Description Notes Example Inactivated–whole agent Killed microbe or inactivated virus Immunogenic but cannot Influenza, rabies, reproduce polio (Salk vaccine) Toxoids Inactivated toxins Antigenic part retained but toxic part destroyed Diphtheria, tetanus Subunit Key protein antigens or antigen fragments from pathogen Avoids parts with possible side effects Acellular pertusis Recombinant Produced by genetically engineered microorganisms VLP Empty capsid Produced by genetically engineered organism HPV Polysaccharide Polysaccharide capsule Not effective in young children - T-dep antigen Pneumococcus (for adults) Conjugate Polysaccharides linked to proteins Converts polysaccharide to T-dep antigen Haemophilus influenzae b, S. pneumoniae Hepatits B (yeast – viral capsid protein) COMPARISON BETWEEN ATTENUATED & INACTIVATED VACCINES Copyright © The McGraw-Hill Companies, Inc. VACCINATION STRATEGY EXAMPLE: CAMPAIGN TO ELIMINATE POLIOMYELITIS • In 1952, over 58,000 polio in the US, leading to 3145 deaths and over 21,000 children with paralysis http://amhistory.si.edu/polio/americanepi/medical.htm VACCINATION STRATEGY EXAMPLE: CAMPAIGN TO ELIMINATE POLIOMYELITIS • National Foundation for Infantile Paralysis, founded by President Theodore Roosevelt, started the “March of Dimes” campaign, which funded poliovirus research leading to vaccine development and, ultimately, delivery. • Three types of poliovirus – entry via the mouth, infects throat and intestinal tract, invades blood, then nerve cells, cause disease www.virology.net VACCINATION STRATEGY EXAMPLE: CAMPAIGN TO ELIMINATE POLIOMYELITIS • William Hammon prepared immunoglobulin-containing fractions from blood of polio survivors, and administered it to those who had been exposed and/or were showing early signs of infection – In 1950, a large clinical trial showed that this strategy was about 80% effective in preventing the development of paralytic poliomyelitis – impractical for widespread use due to the limited supply of immune blood plasma – the success of this passive immunization extremely important proof of concept for immune-mediated control of polio VACCINATION STRATEGY EXAMPLE: CAMPAIGN TO ELIMINATE POLIOMYELITIS • Salk vaccine (mid-1950s) contains inactivated viruses of all three types ➢ Dramatically lowered rate of disease but required series of injections for maximum protection • Sabin attenuated vaccine available in 1961 ➢ Cheaper oral vaccination, although still three doses ➢ Induced better mucosal immunity (secretory IgA response), so better herd immunity ➢ Attenuated viruses can mutate; ~1 out of 2.4 million doses results in poliomyelitis CHILDHOOD IMMUNIZATIONS: RISK / BENEFIT • While most vaccines have negligible risk, some have a detectable risk Measles: ➢ ➢ ➢ ➢ The illness is presented by high fever, generalized rash, and cough, runny nose and/or conjunctivitis (red eyes), with pneumonia as secondary outcome in 1:10. Complications of measles infection include lifelong disabilities such as brain damage, blindness and deafness. Measles infection kills more children than any other vaccine-preventable disease. Before the widespread use of vaccine, 90% of children had contracted measles by the age of 10 years. Today, deaths are due to lack of immunization. The Odds: ➢ Child with measles has 1:1,000 chance of serious brain inflammation (encephalitis) that can lead to brain damage and 1:1000 chance of death from respiratory or neurologic complications ➢ 1:1,000,000 chance of allergic reaction to the measles vaccine SAFE AND EFFECTIVE: http://www.vaccines.com/vaccine-allergic-reaction-odds.cfm Recall: Measles infection causes pneumonia in 1 in 10 infected children and life threatening brain inflammation in 1 in 1000 infected children. CHILDHOOD IMMUNIZATIONS: RISK / BENEFIT Measles: ➢ Victories: ➢ WHO’s Eastern Mediterranean Region implemented widespread measles immunization in 1997; reduced deaths from 96,000 to 7,000 in 10 years ➢ Current measles vaccine coverage 84%; 15.6 million lives saved since 2000 ➢ Setbacks: ➢ Between 1989–1991, immunization rates in US dropped 10%, leading to reemergence of measles and outbreak of 55,000 cases that resulted in 120 deaths THE LIES OF ANDREW WAKEFIELD: INVENTING A LINK BETWEEN MMR VACCINE AND AUTISM Table Taken From: How the case against the MMR vaccine was fixed By Brian Deer (Journalist) British Medical Journal 2011; 342 doi: http://dx.doi.org/10.1136/bmj.c5347 Wakefield was being paid to support a lawsuit claiming the presence of a previously unrecognized ‘bowel-brain syndrome’, which he attributed to the vaccine. He was paid ~$550,000 over 2 years to support this case. For this study, he recruited from anti-MMR campaigners. Much of the data was falsified for the report, clearly disagreeing with the hospital records. CHILDHOOD IMMUNIZATIONS: RISK / REWARD Pertussis: • Routine pertussis (whooping cough) immunization yielded significant decrease in incidence, saved many lives Pertussis involves uncontrollable cough that can lead to vomiting, pneumonia, febrile convulsions, encephalopathy, rib frature, hernia, subconjuctival hemorrhage and occasional death ➢ But because of some adverse reactions to killed whole cell vaccine (fever, aches, very rarely convulsions) caused by inflammatory PAMPs remaining in the vaccine preparation, many parents refused to vaccinate their children ➢ By 1990, highest incidence of pertussis in 20 years, deaths of some children ➢ Safer acellular subunit vaccine is now used ➢ CHILDHOOD IMMUNIZATIONS: RISK / REWARD With most vaccines, there is simply no meaningful risk…. and that’s still not enough to satisfy some folks! (syncope = fainting) VACCINES & IMMUNIZATION PROCEDURES • Some vaccines routinely given, others only under certain circumstances Copyright © The McGraw-Hill Companies, Inc. CURRENT PROGRESS IN IMMUNIZATION • Recent advances yielding safer, more effective vaccines ➢ Conjugate vaccines enlisting T-cell help ➢ New adjuvants being developed ➢ Administering cytokines with vaccine • Novel types being actively studied ➢ Peptide vaccines (key antigenic peptides from pathogens, heat stable) ➢ DNA-based vaccines (inject into muscle tissue, which expresses for a short time) ➢ Therapeutic vaccines (use to cure, rather than to prevent disease) ➢ Drug abuse (antibodies bind elicit drug components in bloodstream to remove ‘high’) Copyright © The McGraw-Hill Companies, Inc. LECTURE OVERVIEW Principles of Immunization Active & Passive Immunity Vaccines & Immunization Procedures Attenuated/Inactivated Vaccines, Vaccination Strategies, Childhood Immunizations, Current Progress Principles of Immunological Testing Obtaining Antibodies, Quantifying Ag-Ab interactions PRINCIPLES OF IMMUNOLOGICAL TESTING • Seronegative: individual not yet exposed to antigen ➢ Has no specific antibodies to that pathogen • Seropositive: individual has been exposed Has produced specific antibodies to pathogen ➢ Seroconversion, process of producing antibodies, takes about 7–10 days; rise in titer is characteristic of infection (even when microbe not detected directly) ➢ Small, steady antibody level indicates previous exposure ➢ • Serology is study of in vitro antibody-antigen interactions Serum is fluid portion of blood after blood clots ➢ Plasma is fluid portion of blood treated to prevent clotting ➢ Other specimens (cerebrospinal fluid, tissues) also tested ➢ OBTAINING ANTIBODIES POLYCLONAL ANTIBODIES • Arise from natural infection or immunization with whole or partial agent; resulting antibodies collected from serum • Yields polyclonal antibodies – multiple naive B cells responded & produced mix of antibodies to variety of epitopes on the antigen More complex antigens will yield more antibodies ➢ Drawback is some may bind closely related organisms (e.g. Shigella and E. coli outer membrane proteins) ➢ • Many applications, including immune detection of antigens in immunoblots, localization of antigens within tissue sections, and immunopurification of proteins from cell lysates ➢ Antibodies to commonly studied antigens often available commercially OBTAINING ANTIBODIES MONOCLONAL ANTIBODIES • Complex, expensive • Antibodies produced have identical constant and variable regions, recognize only single epitope Immunize a mouse with antigen X to activate and induce proliferation of specific B cells. • Can “humanize” by replacing most of antibody allele (i.e. the regions encoding the constant region) with recombinant DNA encoding the analogous DNA from humans ➢ Used as therapeutic drugs since human immune system less likely to destroy B cells from spleen. These are capable of making anti-X antibodies, but die after several generations. B cells die. Myeloma cells. These abnormal plasma cells grow indefinitely, cannot make antibodies, and have a mutation that makes them susceptible to the drug aminopterin. Myeloma cells die. Mix the two cell types along with a chemical that induces their fusion, and then incubate in a medium that contains aminopterin. The B cells and myelomas die, but hybridomas proliferate. Hybridoma cells. These are fusions of B cells and myeloma cells. Select single hybridoma cell that recognizes desired anti-X epitope and maintain it in culture. Harvest antibodies made by the hybridoma cells. Monoclonal antibodies. These all have the same constant and variable regions, and therefore recognize the same epitope and have the same functional characteristics. Copyright © The McGraw-Hill Companies, Inc. LECTURE OVERVIEW Principles of Immunization Active & Passive Immunity Vaccines & Immunization Procedures Attenuated/Inactivated Vaccines, Vaccination Strategies, Childhood Immunizations, Current Progress Principles of Immunological Testing Obtaining Antibodies, Quantifying Ag-Ab interactions