Copy of Post MT Notes (1).pdf

Lecture 17 – Microbial infection and pathogenesis - Infection: a microorganism is established in a host, whether or not a host is harmed - Disease: actual damage or injury that impairs host function - Pathogens: microbial parasites that cause disease or tissue damage in a host - Pathogenicity: the a...

Lecture 17 – Microbial infection and pathogenesis - Infection: a microorganism is established in a host, whether or not a host is harmed - Disease: actual damage or injury that impairs host function - Pathogens: microbial parasites that cause disease or tissue damage in a host - Pathogenicity: the ability of a parasite to inflict damage on the host - Adherence: enhanced ability of microbes to attach to host tissues - Necessary but not sufficient to start disease - Pathogens typically adhere to epithelial cells through interactions between molecules on the pathogen and host tissues - Pathogens can form biofilm and adhere to the host via the biofilm - Pathogens gain access to host tissues by way of a portal of entry: - mucous membranes, skin surface, puncture wounds, cuts, insect bites, or other abrasions - critical in establishing an infection - Adhesins: receptors (made up of glycoprotein or lipoprotein) on pathogen’s surface that enable it to bind to host cells - Specificity - Host receptors include: extracellular matrix, cell surface glycoproteins, membrane lipids - Ex. Influenza virus uses the surface hemagglutinin to specifically interact with mucosal cells of the upper respiratory tract - Capsules in adherence and protection - The bacterial capsule forms a thick coating outside the plasma membrane and cell wall and serves two important functions in bacterial pathogenicity - The capsule is both sticky and contains specific receptors to facilitate attachment to host tissues - Capsules can evade the host defense system - Fimbriae, pili, and flagella as adherence structures - Fimbriae are structures uniformly distributed on bacterial cell surface - Implicated in specific adherence in infections by enteric bacteria (Escherichia, Salmonella, and Shigella) as well as Neisseria gonorrhoeae - Pili are involved in the attachment to urogenital epithelia by Neisseria gonorrhoeae - Flagella may also facilitate adherence to host cells - Colonization: growth of microorganisms after they have gained access to host tissues - Process begins at birth when exposure to a suite of harmless bacteria and viruses to establish the initial normal microbiota - Colonization usually starts in the mucous membranes - - - - - Tightly packed epithelial cells that line the surface of urogenital, respiratory and digestive tracts secrete mucous, a thick secretion of water-soluble glycoproteins - Mucous retains moisture and naturally inhibits microbial attachment through physical processes; e.g., sneezing and swallowing - Some microbes, pathogens and nonpathogens, adhere and colonize - Process: (a) Loose association upon initial exposure (b) Penetration of mucus leading to adhesion and colonization (c) Further colonization leading to biofilm formation Growth of microbial community and tooth decay - Saliva contains acid glycoproteins that forms a film on tooth surface providing an attachment site - First colonizers are Streptococcus sobrinus and S mutans - Sucrose (table sugar) triggers the formation of capsules and dextran to secure attachment to the tooth and gum surface - Dental plaque: thick biofilm caused by extensive bacterial growth - Besides streptococci, many other bacteria and a few archaea are found in dental plaques - Streptococcus sobrinus and S mutans are lactic acid producers - High local concentrations of lactic acid decalcifies tooth enamel, resulting in cavities Invasion and systemic infection - Invasion: ability of a pathogen to grow in host tissue, spread and cause disease - Some pathogens remain localized after initial entry multiplying and invading a single site; - Bacteremia: presence of bacteria in the bloodstream; usually asymptomatic because the immune system will remove them - Septicemia: bacteria multiplying in the bloodstream and spread systematically from an initial point and produce toxins - Usually begins at a specific organ such as intestine, kidney and lung and spread rapidly throughout the body - May lead to massive inflammation, septic shock (blood pressure drops to dangerously low level), and death Virulence: the relative ability of a pathogen to cause disease Virulent factors: toxic or destructive compounds produced by the pathogen that directly or indirectly enhance invasiveness and host damage by facilitating and promoting infection Quantification of virulence - mortality, illness, or pathological lesions - For example, mortality can be estimated experimentally to determine the amount needed to kill 50% of the test animals LD50 (lethal dose50) - High virulent pathogens require a few infectious agents to cause disease; - Example of low virulent pathogen: Vibrio cholera requiring a large inoculum to initiate disease Virulence attenuation: the decrease or loss of virulence - Attenuated strains of pathogens can be developed by culturing in the laboratory through passages rather than isolated from diseased individuals - Presumably mutated strains of pathogens (less virulent or loss of virulence) can grow faster than pathogenic strains when selective pressure is absent Virulence attenuation in vaccine development - Attenuated strains of pathogens are valuable because they are used in vaccine production - First rabies vaccine developed by Louis Pasteur - Greater efficacy and generate stronger immune response than killed microbes Genetics of virulence - The outcome of an infectious disease is the net result of genetic and physiological features of both the pathogen and host - Virulence of a pathogen maybe firmly encoded by chromosomal genes or by mobile elements - Example: virulence in Salmonella - Genes associated with pathogenicity are clustered in pathogenicity islands on the chromosome - Some virulence factors are carried by plasmids, which can spread rapidly in the population The compromised host - The pathogen-host interaction is dependent on both the host and the pathogen - Opportunistic infections: those caused by organisms that do not cause disease in heathy hosts - Compromised hosts: individuals in whom one or more mechanisms to disease is inactive - Infection with viruses such as HIV weaken the immune system - Many hospital patients with noninfectious diseases are compromised hosts - Nosocomial infections: healthcare associated infections - Main causes; e.g., surgery, biopsy, catheterization, hypodermic injection - Affect two millions each year in the United States with ~5% mortality rate Enzymes and toxins of pathogenesis - Infectious bacteria release enzymes that breakdown the host’s tissues - The polysaccharide hyaluronic acid is an extracellular matrix component that holds the cells together - Streptococcus pyogenes produces hyaluronidase that digests hyaluronic acid to get to the deeper tissues - Clostridia that cause gangrene produce collagenase (collagen is the major protein in the connective tissues) to get to the deeper tissues with anoxic environment Coagulase and streptokinase as virulence factors - manipulate blood clotting - Coagulase promotes blood clotting , blocking access to the bacteria by the immune system - May account for the localized nature of infections such as boils and pimples - Streptokinase dissolves blood clot formed by the host to isolate the pathogen - Pathogens that produce streptokinase can invade deeper tissues - Streptokinase is used as a pharmaceutical to minimize blood clots following heart attacks and other treatments Toxins - Toxicity: the ability of an organism to cause disease by means of a toxin that inhibits host cell function or kills host cells - Exotoxins: toxic proteins secreted by the pathogen - Based on their mechanisms, exotoxins are grouped into three classes: - AB toxins - two subunits: Subunit B facilitates Subunit A to cross the cytoplasmic membrane of the host; Subunit A causes damage to the host cell - Examples: toxins that cause diphtheria, tetanus, botulism, and cholera - Cytolytic toxins - Superantigen toxins - Enterotoxins: exotoxins whose site of action is the small intestine, causing secretion of fluids into the intestinal lumen, resulting in vomiting and diarrhea Diphtheria exotoxin: blockage of protein synthesis - Diphtheria toxin: an AB exotoxin produced by the bacterium Corynebacterium diphtheriae - The gene encoding the toxin is encoded by a prophage - The B subunit binds to the cytoplasmic membrane and cleaves itself to release the A subunit - The A subunit catalyzes ADP-ribosylation of the elongation factor EF-2 - The EF-2-ADP is unable to transfer amino acids to the growing protein chains, thereby shutting down protein synthesis - The Shiga toxin uses the same mechanism Neurological exotoxin: botulinum toxin - Botulinum toxin is produced by the anaerobic Grampositive bacterium Clostridium botulinum - consuming contaminated canned food - Muscle contraction is the result of muscle receptor interacting with the neurotransmitter acetylcholine - The botulinum toxin cleaves the proteins involved in coordinating the release of acetylcholine - Without the excitatory acetylcholine signal, muscle cannot contract - Can lead to death by suffocation if the diaphragm muscles are severely affected - Chronic pain is often caused by stress-induced muscle tension; local application of low doses of botulinum toxin can provide relief Neurological exotoxin: tetanus toxin - Tetanus toxin is produced by Clostridium tetanus, an obligate anaerobe - The bacteria grow in the body in deep wounds that become anoxic - On contact with the nervous system, the toxin is transported through the motor neurons to the spinal cord - The toxin binds to the inhibitory interneurons, preventing the release of the inhibitory neurotransmitter glycine (normally required for blocking the release of acetylcholine) - The toxin results in the flooding of the neurotransmitter acetylcholine in the neuromuscular junctions, leading to muscle contraction Cholera enterotoxin, and AB-type exotoxin - Enterotoxins: toxic activity affecting the small intestine - Generally cause massive secretion of fluid into the intestinal lumen, resulting in vomiting and diarrhea - Cholera starts by intake of food or water contaminated with Vibrio cholera - The bacteria travel to, and colonize, the small intestine - Subunit B of the toxin binds to the epithelial cells - Subunit A enters the cell and activates the enzyme adenylyl cyclase, converting ATP to cyclic AMP - Cyclic AMP regulates multiple cellular processes including ionic balance - Blocks the uptake of Na+ , resulting in the efflux of chloride and bicarbonate and the massive secretion of water - Can be reversed by replenishing loss fluids with clean rehydration solution Cytolytic exotoxins: work by degrading cytoplasmic membrane, causing cell lysis and cell death - Many are phospholipases, enzymes that degrade membrane phospholipid - Some form pore to release cell contents Endotoxins: NOT proteins, but are part of the lipopolysaccharide component of the outer membrane of Gram-negative bacteria - Released in toxic amounts only when the bacterial cells lyse and the toxin is solubilized - Generally less toxic than exotoxins Innate immunity - Immunity: the ability of an organism to resist infection - Two systems: innate immunity and adaptive immunity - Innate immunity: nonspecific immunity - Built-in capability of multicellular organisms to target pathogens that are seeking to colonize the host - Does not require previous exposure to a pathogen of its products - Phagocytes are primary effector cells - Rapid response within hours - Adaptive immunity: Acquired ability to recognize and destroy a particular pathogen or its products - Lymphocytes are primary effector cells - Response several days - Dependent on previous exposure to the pathogen or its products (specificity) - Directed toward an individual molecular component of the pathogen (antigen) - Antibodies from plasma cells and cytotoxic T cells clear specific infection - B and T lymphocytes have focused attack on pathogen, get post exposure immunity - Natural host resistance - Normal microbiota helps host resist pathogens(skin and gut) - The ability of microbes to cause disease varies between species, each species has varying sensitivity - Different pathogens invade different tissues - Ex. AIDS on T-helper lymphocytes vs Botulism on Botulism Motor neuron end plate - Routes of infection are crucial; e.g., tetanus is in deep wounds while Salmonella is ingested - Competitive exclusion: pathogens do not easily infect tissues because the harmless microbes limit available nutrients and sites for infection Physical and chemical barriers to infection - tight junctions between epithelial cells that line the body tissues inhibit invasion and infection - Mucosal membranes are coated with a thick layer of mucous to trap microorganisms, pollen, and other foreign agents - Epithelial cells underlying the mucous layer have cilia on their surface that carry out coordinated movements to expel microorganisms - Stomach acid inhibits bacterial growth - Skin is salty and acidic, limiting bacterial growth - Lysozyme in tears, saliva, milk, and other secretions lyse bacteria Cells and organs of the immune system - Blood and lymph circulation - Cells and proteins of the immune system are transported to various tissues and organs of the body via blood and lymph - Blood and lymph are fluids from separate circulation systems in the human body - Blood is pumped through arteries and capillaries and returns from the body through veins - Lymph drains from extravascular tissues into lymph capillaries and lymph ducts, then into lymph nodes throughout the lymphatic system - Lymph nodes contain high concentration of immune system cells Connections between the blood and lymphatic systems - In capillary beds, leukocytes and solutes pass from blood in the lymphatic system - Lymph containing antibodies and immune cells empties into the blood circulatory system via the thoracic lymph duct Blood constituents - Whole blood comprises plasma and cells - Plasma: contains proteins and other solutes where blood cells are suspended - Outside the body, insoluble proteins in the plasma trap the cells in insoluble mass (blood clot) - Serum: portion of blood that is not cells or clotting proteins - Major cell types of blood - Erythrocytes (red blood cells) – 4.2-6.2 X 10^9 /mL - Leukocytes – all nucleated cells – 4.5-11 X 10^6 /mL - Lymphocytes, granulocytes, and monocytes(see slides for pic) Differentiation of leukocytes (nucleated white blood cells) - Hematopoietic stem cells: precursor blood cells found in bone marrow and the gut - - - - Hematopoiesis: differentiation of blood stem cells into different blood cells influenced by soluble cytokines and chemokines, proteins that direct immune cell production, function, and movement Following differentiation, the cells enter the circulation and migrate to other parts of the body for further maturation Myeloid cells: derived from a myeloid precursor cell; can be divided into two categories: - Antigen-presenting cells: engulf, process, and present antigens to T-lymphocytes, - T-lymphocytes: then initiate an adaptive immune response - Monocytes, macrophages, and dendritic cells - Granulocytes: contain toxins or enzymes that are released to kill target cells - Cytoplasmic granules contain histamine and other mediators that function to initiate an inflammatory response when released - Neutrophils, basophils, eosinophils Lymphocytes - Lymphoid stem cells produce T cells, B cells and natural killer cells - Natural killer cells function primarily in innate immunity; - remove viruses and tumour cells by recognizing specific cell-surface molecules - Lymphocytes: specialized leukocytes involved exclusively in adaptive immune response; two types: - - - - B cells: originate and mature in bone marrow - T cells: originate in bone marrow, but mature in thymus - Bone marrow and thymus are primary lymphoid organs - Each lymphocyte produces a unique protein that interacts with a single antigenic determinant - T cells: T cell receptors - B cells: antibodies or immunoglobulins Phagocytes and microbial invasion - Innate immunity is primarily driven by the activities of phagocytes - Phagocytes recognize common structural features found on pathogens - Respond within minutes - Phagocyte-pathogen interactions: result in activation of genes and translation of proteins that eventually lead to the destruction of the pathogen - Not always effective in controlling infection - Certain phagocytes can activate adaptive immunity by processing and presenting antigens to receptors on T lymphocytes - Microbial invasion is the ability of a pathogen to enter host cells or tissues, multiply, spread, and cause disease Recruitment of phagocytes - Tissue damage, such as caused by puncture wound, can lead by invasion by microorganisms, - Resident leukocytes and damaged cells release cytokines and chemokines - a subclass of cytokines that attract circulating immune cells to the site of injury - Phagocytes, in response to the cytokine-chemokine gradient, are recruited to the site of injury and squeezed out of the blood capillaries, - The invading phagocytes are cleared by the phagocytes Pathogen recognition - Phagocytes must be able to recognize, capture, and destroy pathogens to clear infection and restore body tissue to a healthy state - Pathogen-associated molecular pattern: pathogens have structures and molecules not found in or on host cells (e.g., peptidoglycan, flagella, dsRNA (virus)) - Lipopolysaccharide of outer membranes of Gram negative bacteria is a common pathogen-associated molecular pattern - Pattern recognition receptors: leukocytes have membrane bound or soluble proteins that recognize pathogen-associated molecular pattern - Binding of pattern-associated molecular pattern by pattern-recognition receptors stimulates the phagocytes to engulf and destroy the pathogen - Toll-like receptors: a class of pattern recognition receptors - Each toll-like receptor on a human phagocyte recognizes and interacts with a specific pathogen-associate molecular pattern - Upon encountering a pathogen-associate molecular pattern, the toll-like receptor sends a signal to the nucleus Phagocyte signal transduction - Signal transduction in phagocyte: upon activation of toll-like receptors, a leukocyte will start a phosphorylation cascade to transmit the activation signal to the nucleus, - which will activate transcription factors to turn on genes in response to the activation signal - NFκB (nuclear factor kappa-light-chain-enhancer) is a key transcription factor that is activated in many different pathways Phagocytosis and phagolysosomes - Upon recognition of pathogen-associated molecular patterns by their toll-like receptors, phagocytes engulf the pathogens - Phagosome: the membrane-bound vesicle that surrounds the bacterium - The phagosome fuses a lysosome to form a phagolysosome - Phagocyte produces toxic reactive oxygen intermediates to kill the bacteria within a phagolysosome; - thus preventing damage to the phagocyte itself Phagocyte inhibition - Some pathogens can survive the phagolysosome - Mycobacteria tuberculosis: produces carotenoids to neutralize singlet oxygen and has a waxy cell wall that absorbs free radicals. This pathogen lives and divides within phagocytes - Some pathogens such as Streptococcus pyrogenes: produce leukocidins, which kill white blood cells. Dead white blood cells are found in pus - Some pathogens contain a capsule, which makes it difficult for the phagocyte to engulf them Inflammation: a nonspecific reaction to noxious stimuli (e.g., physical injury, toxins, and pathogens) - Local infection leads to inflammation in a small part of the body, followed by healing - Cytokines and chemokines released by injured cells and phagocytes draw white blood cells to a site of inflammation - Leukocytes at the site of infection release proinflammatory cytokines including interleukin-1 (IL-1) - The cytokines increase vascular permeability causing swelling, pain, and heat localized at site of infection - The pressure associated with swelling force fluids away from the blood vessels and into the lymphatic system - Strengthen immune response - Prevent the spread of pathogens to the bloodstream - Effective inflammatory response isolates and limits tissue damage, destroying damaged cells and pathogens Systemic inflammation and septic shock - Systemic inflammation: occurs when the inflammatory response fails to localize the pathogens and the reaction spreads throughout the body (leads to inflammation and disease throughout the body) - Inflammatory cells and mediators spread throughout the entire circulatory and lymphatic systems - Can lead to septic shock - SS: when increased vascular permeability resulting in a decrease of blood pressure, which can cause damage to multiple organs at the same time - Gram-negative bacteria: contain LPS, which triggers a proinflammatory cytokine response from leukocytes as their Toll-like receptors are activated - leads to a cytokine storm, which can be fatal. - Example: Salmonella species or Escherichia coli, which can be introduced into the peritoneal cavity or the bloodstream by a ruptured or leaking bowel Fever - Inflammatory response can induce fever because of released Cytokines, such as interleukin-1 and interleukin-6, - Fever: a condition of elevated body temperature - These cytokines stimulate the hypothalamus, the temperature control centre of the brain, to produce prostaglandins - Prostaglandins are chemical signals that raise the body temperature - Benefits of fever - Increases circulation rate, expedites leukocytes to get to the site of infection - Reduce the growth of pathogens because they do not tolerate the higher temperature - Increase in transferrins, which bind and sequester iron, thereby depriving pathogens of this important nutrient The complement system - Complement (C’) or the complement system: a set of circulating, inactive proteins that are sequentially activated in response to a pathogen - Functions to boost the efficiency of both the innate and adaptive immune responses for the destruction of pathogens - Three pathways: - a) The sequence, orientation, and activity of the components of the classical complement pathway. - b) The mannose-binding lectin pathway. - c) The alternative pathway - 3 OUTCOMES - Complement C3b coating the target making it easier for phagocytes to engulf it - Complement C3a diffuses to the surrounding area serving as a chemoattractant - Complement C5a binding to the target forming the membrane attack complex, resulting in cell lysis Natural killer cells: cytotoxic lymphocytes - Major Histocompatibility Complex I (MHC1 proteins): All nucleated cells have on their surface - Many virus-infected cells and tumour cells do not have MHC I proteins and produce a stress protein genes - When encountering a cell with stress proteins and without MHC I proteins, a natural killer cell will activate and destroy the target by producing: - Granzyme: an enzyme that induces apoptosis (programmed cell death) - Perforin: pokes holes in the target membrane Interferons: small cytokine proteins produced by virally infected cells - serve as a warning system and prevent viral replication by stimulating the production of antiviral proteins in uninfected cells once they receive the interferon signal from infected cells Lecture 19 – Adaptive immunity: highly specific host defense What is part of our cells n helps vs is acc immunity. - innate immunity: broadly targeted responses triggered by general features of microorganisms - Adaptive immunity: directed toward specific molecular components of the microbes mediated by a special class of antigen-reactive leukocytes called lymphocytes - B lymphocytes: from bone marrow and mature in bone marrow, produce antibodies (soluble in blood stream) that interact and protect against extracellular antigen - Conferring antibody-mediated immunity to the host - T lymphocytes (T cells): from bone marrow, mature in thymus. display antigen-specific receptors on their surface that defend against intracellular pathogens, such as viruses and some bacteria - Conferring cellular immunity to the host - Not based on soluble proteins Specificity: dependent on lymphocyte receptors interacting with individual antigen (from pathogen) - T lymphocytes (T cells) display antigen-specific receptors call T cell receptors - Each receptor specific to one antigen or limited type of antigen - All the receptors are identical on the lymphocyte - B lymphocytes display membrane-bound immunoglobulins on their surface - Same specificity to one time but also are antibodies (immunoglobulins) Memory: the first antigen exposure induces multiplication of antigen-reactive cells, resulting in multiple clones. Subsequent exposures to the same antigen result in rapid production of large quantities of antigen-reactive T cells or antibodies 1. Multiply like craz 2. All the cells exposed to same antigen, the memory cells interact with antigens = multiply = react Primary immune response: first exposure to an antigen in which antigen recognition by specific B or T lymphocytes leads to B and T cell activation, proliferation, and differentiation - Exposure = multiply & differentiate (primary response few days) - continuous exposure does not result in secondary response bc need refractory period, the cells need to become memory cells - Primary response not good enough for immunization, booster stimulates secondary response (strong response takes approx 3 months for humans) Secondary immune response: subsequent exposure to the same antigen activates clones from the primary immune response to generate stronger and faster response - Vaccination with killed or weakened pathogens, or their products, is a means of conferring immunity Tolerance: the acquired ability to make an adaptive response to discriminate between host (self) and foreign (nonself) antigens - How to distinguish self vs foreign - Failure to develop tolerance may result in reactions against self, called autoimmunity - Precursor T cells travel from the bone marrow to the thymus, they mature, and are put under both positive and negative selective pressure - How to know which cells to keep? - First: Positive selection – T cells that recognize MHC peptides are retained - Must recognize Major Histocompatibility Complex proteins on surface - If not, they don't multiply - Second: Negative selection – T cells that pass the positive selection and strongly bind to self-antigens are selected against - If they react too strongly with MHC, they don’t pass - Clonal deletions – more than 99 percent of T cells that enter the thymus do not survive the selection process; remaining T cells react strongly with foreign antigens B cell selection and tolerance ONLY when they see an antigen do they go thru selec. - Enormous diversity of antigen-reactive B cells - Positive B cell selection occurs when the B cell receptors encounter an antigen that they recognize: - Proliferate, make more copies (with the help of T helper cells) - Differentiate into antibody-producing plasma cells (many) and memory cells (few) - Negative B cell selection occurs in the bone marrow, where self-reactive B cells are deleted (clonal deletion), or silenced because they lack a T cell help signal Antigens: substances that react with antibodies or T cell receptors - Not all antigens are immunogenic Immunogens: substances that elicit an immune response Intrinsic factors that determine immunogenicity include: - Size: haptens, which are small molecules, are not immunogens but they may induce an immune response if attached to a larger carrier molecule - Complexity: complex proteins and carbohydrates are good immunogens, while molecules with simple repeating units (e.g., DNA, mRNA: too similar to make unique structure to be recognized bc they r simply af) are poor immunogens - Vaccines often attached to protein (immunogenic) - Physical form: insoluble molecules or aggregates are usually excellent immunogens Extrinsic factors that determine immunogenicity include - Dose: micrograms to a gram - Route: injection is more effective than oral exposure - A large, oral dose of an immunogen may induce tolerance rather than immunity Antibodies do not interact with an entire antigen, but only with a distinct portion of the molecule called an antigenic determinant or epitope - Where they can bind: antigenic determinant or epitope - Antigens typically contain several different epitopes, each capable of reacting with a different antibody - May include sugars, short peptides of four to six amino acids, and other organic molecules that are components of a larger immunogen - T cell receptors recognize epitope only after the antigen has been processed (partially degraded) - example: antigen-presenting cells such as Macrophages, B lymphocytes etc. - They chew up antigens and put them on the surface as a couple of proteins? Active vs Passive immunity B lymphocyte (B cell): each B cell has ~100,000 identical antibodies on its surface called B cell receptors - B cells interact with antigen and T helper (Th) cells to produce antibodies - B cell receptors bind to antigen (pathogen), internalize, and digest it - The pathogen-derived peptides are affixed to Major Histocompatibility Complex II proteins and display on the surface of the B cell (antigen-presenting cell) - The antigen-presenting B cell interacts with an antigen-specific T cell, T helper cell - T helper cell secretes cytokines to activate the B cell to produce clones that differentiate into plasma cells (producing antibodies) and memory cells Antibodies (or immunoglobulins, Igs): either soluble or cell surface antigen receptors - bind to toxins or viruses to neutralize them - Toxin bound by antitoxin antibody is neutralized and doesn’t bind bind to foreign cells and make them easier to engulf by phagocytes Structure and function of immunoglobulin G (IgG) - Five major classes of antibodies: IgG, IgA, IgM, IgD, and IgE - differences in amino acid sequence of their heavy chain - different structural features, expression patterns, and functional roles - IgG is the most common antibody circulating in the body - Four polypeptide chains: two heavy and two light chains - The two heavy and two light chains are held together covalently by disulfide bond. - One heavy and one light chain interact to form an antigen-binding unit. - each IgG molecule has two antigen-binding sites. - The constant domain is identical for all IgG - The Fc stem binds receptors on phagocytes to facilitate phagocytosis - The variable domains bind antigen igG used in soluble secrtion, part of secondary Primary and secondary antibody responses in serum - Primary antibody response: produces short-lived plasma cells that live for less than a week; mostly IgM - Low concentration - Secondary antibody responses (subsequent exposures): response quicker - Memory cells do not need T cell help - Produces 10-100 times more antibodies, mostly IgG Antigen binding by immunoglobulin - Binding depends on shape of pocket - Binding is a function of the folding pattern of the heavy and light polypeptide chain - Variable domains of different antibodies are different from one another, especially in complementarity-determining regions (CDRs) - The three CRDs provide most of the molecular contacts with antigen (see slide) - The antigen-binding site of an antibody is large enough to accommodate the binding of an epitope with both the heavy and light chain variable regions - Different antibodies bind their epitopes with different strengths, called binding affinities Generating antibody diversity Multiple unusual mechanisms at play: - Somatic recombination - The gene encoding each immunoglobulin is constructed from several immunoglobulin gene segment - As each B cell matures, immunoglobulin gene segments undergo random rearrangements by recombination and deletion of intervening segments - Once one antibody rearrangement is successful, the process stops - Allelic exclusion: only the rearranged allele is expressed so that each B cell produces only one antibody - The C are for the dif Ig# The recombination of the gene segments between V-D and D-J is imprecise - Can vary by several nucleotides and change the amino acid sequence thus generating diversity - Random heavy and light chain reassortment - Two different light chains: kappa and lambda - The five heavy chains (five classes of immunoglobulins) can join with either the kappa or lambda light chain to form antibody - From number of gene segments, gene rearrangement and reassortment can generate >3.3 million possible antibodies - Coding for joint diversity - Somatic Hypermutation: mutation rate of B cell immunoglobulin genes is higher than other genes - Occurs only in the V regions of heavy and light chains, creating slightly altered Ig cell surface receptors with changing binding affinities for the antigen - Affinity maturation: B cells with receptors displaying higher affinity for the antigen are selected (those who bind tighter r maintained, highly specific) - The strengthening of antibody binding is responsible for the dramatically stronger secondary immune response MHC Class I proteins: found on surface of all nucleated cells - Present internal antigens to T-cytotoxic cells - Internal (cytoplasmic) antigens can originate from viral proteins or cancer proteins - If the peptide presented by MHC Class I is recognized by the T cell receptor of a T-cytotoxic cell, the antigen-containing cell is destroyed - MHC Class I proteins are the major antigen barriers for tissue transplant MHC Class II proteins: found only on the surface of antigen-presenting cells (B lymphocytes, macrophages, and dendritic cells) - Present antigens to T-helper cells - Stimulate cytokine production and lead to antibody-mediated immunity or inflammatory responses Based on the peptides (self or foreign) presented by MHC proteins, the immune system distinguishes cells with foreign antigens from cells with self antigens Antigen presentation by MHC I proteins - - Protein antigens degraded by the proteasome in the cytoplasm = fragments Peptide fragments are transported into the endoplasmic reticulum through a pore formed by the TAP (transporter associated with antigen processing) proteins) MHC I proteins in the endoplasmic reticulum are stabilized by chaperonins until peptide fragments are bound peptide fragments are bound by MHC I, the complex is transported to the cell surface The MHC I-peptide complex interacts with T cell receptors (CD8) on the surface of T-cytotoxic cells The T-cytotoxic cell is activated by the binding events, causing it to release cytokines and cytolytic toxins and kill the target cell T cell-mediated immunity : depend on MHC 1 OR 2 - Antigen-presenting cells, such as the phagocytes in innate immunity, ingest, degrade, and process antigens - then present antigens to T cells that secrete protein cytokines that activate the adaptive immune response - T-helper cells produce and release cytokine that induce inflammation - T-cytotoxic cells produce and release perforin and granzyme for target cell lysis - T cell receptors of a given T cell bind only to MHC molecules having foreign antigens embedded in the MHC protein Diversity of T cell receptors is generated by some of the same mechanisms involved in producing diversity of antibodies - Somatic recombination, Random chain reassortment, Coding for joint diversity Immunoglobulin gene superfamily: immunoglobulins, T cell receptors and MHC proteins - Consists of two nonidentical polypeptide chains - Constant and variable regions - Share protein domains - Similar mechanisms of generating diversity for immunoglobulins and T cell receptors T-cytotoxic cells - Directly kill cells that display surface foreign antigens - Contact between T-cytotoxic cell and target cell is required for cell death - On contact, granules in T cytotoxic cell migrate to contact site - Degranulation releases granzymes (causing apoptosis) and perforin (causing pores formation in target cell) T-helper cells - T-helper -1 subset activates macrophages - T-helper-2 subset plays a crucial role in B lymphocyte activation and antibody production Lecture 20 Disorders of the immune system: - Hypersensitivity: - Allergies/Immediate hypersensitivity/Type I - Cytotoxic/Type II - immune complex/Type III - Delayed-type hypersensitivity/Type IV - Autoimmunity: Condition when immune system attack self-antigens. - Autoantibodies or Cell-mediated autoimmune disorders Superantigens - Superantigens bring together APCs and T-cells, but bypass the antigen-TCR complementarity. - They do this by targeting conserved regions outside the antigen-binding sites. - This activates a large fraction of T cells in the body: up to 25%. Causes superantigen shock. Vaccines: disease prevention - Inoculate person with an inactivated pathogen or pathogenic epitope. - Injected, memory cells deployed - This causes the production of memory cells which can mount a swift and strong response if actually infected - mRNA: Antigen produced inside body instead of getting it - Protein - Inactivated: Heat/chemical treated pathogens (e.g. Vibrio cholerae, Influenza) - Attenuated: avirulent strains of a pathogen (e.g. Tuberculosis, measles, Rubella) - Subunit: component of pathogens like toxoids, isolated virulence antigens (e.g. Tetanus toxoid, meningitis caused by Neisseria meningitidis) - Conjugate: genetically engineered antigens (often polysaccharides) coupled with large harmless proteins that elicits an effective immune response (pneumococcal vaccines, meningitis caused by Haemophilus influenzae) - Nucleic acid vaccines: - DNA vaccines: antigens delivered to cells directly in plasmids. Host cells transcribe and translate the antigen to produce the antigen (e.g. Hepatitis A, B) - mRNA vaccines: antigens delivered as pre-processed mRNA for translation in the host cells (e.g. Pfizer’s COVID-19 vaccine). - Advantages: virtually zero risk of infection from the vaccine itself. mRNA vaccines also have a short half life, difficult to transport for temperature requirements. - Disadvantages: vaccine delivery and host cell update; mRNA vaccines can be unstable - Plant Based: Antigen delivered into plants using Agrobacterium tumefaciens. - extracted from plant cells in the form of virus-like particles (VLP): - VLP: antigens embedded in phospholipid vesicles. - Elicits a strong immune response, but lacks pathogen DNA, hence cannot cause disease. - Large farm for vaccines: sustainable, instead of factors - No vaccines exist Immunotherapy - Anticancer vaccines - Prophylactic: preventive; targets oncogenic pathogens - EX. HPV leads to cervical cancers, so we use vaccine to lower HPV which lowers our chances - Therapeutic: Immune cells can be sensitized to tumor antigens to mount a response against cancer cells. - Anticancer therapies: Checkpoint inhibitors - cancer cells overexpress immune-suppressing checkpoint proteins - PD1: programmed cell death protein 1 - Checkpoint inhibitors block the activity of these proteins to prevent neutralization of cytotoxic T cells - pembrolizumab; binds to PD-1 receptors of T-cells - We can block checkpoints to stop immune system from recognizing - Adoptive T-cell transfer - Tumor infiltrating T cells (TILs): - Extract T-cells that have natural anticancer ability from within tumors. - Propagate in the lab. - infuse back into the patient. - In use to treat melanoma. - Chimeric antigen receptor (CAR) T cells: (more advanced version) - T cells with engineered receptors that can recognize tumor antigens even without their presentation on MHC complexes. - T cells extracted from patients. - Engineered using viral vectors to give cells enhanced receptors. - Infuse back into patients. - In use to treat melanoma. - T cells even more sensitive than natural cancer T cells - Challenges: Overcoming immunosuppressive tumor microenvironments. Checkpoint inhibitors often used in combination to improve this. - Side effects: - Neurologic toxicity - inflammation (MORE t cells = higher chance inflammation) - Variation in treatment outcome from person to person. Gut microbiome - People who respond to PD1 more have dif gut microbiome than those who don't - Patients that seem to respond better to anticancer treatments show higher amounts of Bifidobacterium in their feces. - Beneficial gut bacteria like Bifidobacterium are thought to induce release of immune-stimulating cytokines - enhance tumor clearance in mice - Why feces transplant instead of purified bacteria? We don't know exact composition of bacteria in microbiome, don't know exactly what heals. Antibacterial drugs - Target: Cell wall synthesis - Β-lactams: inhibits transpeptidation reaction (catalyzed by penicillin binding proteins (ex.transpeptidase)) during cross-linking of peptidoglycan - Most widely used - B-lactam ring stays consistent, other parts change for effectivity - Bc we don't have cell walls - More in + bacteria - Isoniazid: analog of nicotinamide, a vitamin required for mycolic acid essential for mycobacterium cell wall synthesis. - Used to treat tuberculosis. - Vancomycin: akin to β-lactams, but binds to the two D-ala residues on the end of the peptide chains, preventing cross-linking of peptidoglycan. - Only effective in Gram positive bacteria (bc outer membrane) - Does Not inhibit enzyme, binds to peptides so cross linking cant attach - Protein synthesis - Inhibits 70S prokaryotic ribosomes (50S+30S). (not our ribsomes - Aminoglycosides: Inhibits the 30S subunit. Broad spectrum. - Side effects like nephrotoxicity limit their use. - Example: gentamicin. - Tetracyclines: Inhibits the 30S subunit. Broad spectrum. - Both aminoglycosides and tetracyclines are produced by several species of streptomyces - Example: tigecycline. - Macrolides: Inhibits the 50S subunit. - Example: azithromycin. - For humans, same effect bacteria cant produce proteins - Nucleic acid synthesis: - Quinolones: Inhibits DNA gyrase, preventing DNA packaging and unpacking in bacteria. - Broad spectrum - Fluoroquinolones are the most predominantly used. - Rifamycin: Inhibits RNA polymerase. - Can fuck us up too a lil - Actinomycin: Blocks RNA elongation by binding to DNA. - Folic acid synthesis inhibitors: - Sulfonamides (sulfa drugs): growth factor analogs - Sulfanilamide, for example, mimics p-aminobenzoic acid (PABA) required to produce folic acid - Folic acid is essential in nucleic acid synthesis. - Commonly used with trimethoprim, which inhibits another step of folic acid synthesis. - Membrane disruptors: - Daptomycin: binds to cytoplasmic membrane of bacteria and forms pores - Polymyxins: disrupts the outer membrane and forms pores in the cytoplasmic membrane of bacteria. - Generally not used, can dissolve our cell membrane but some selectivity bc of LPS - Lipid biosynthesis - Platensimycin: inhibits fatty acid synthesis. - We dont do fatty acid synthesis Antiviral drugs - Reverse transcriptase inhibitors (RTIs): blocks reverse transcription that converts viral RNA genomes to DNA in retroviruses (e.g. HIV). - Virus use our machinery so targeting them targets us - Nucleoside RTIs (NRTIs): dideoxy analogs of nucleosides for chain termination I THINK - Sanger sequencing methods - Has toxic side-effects. Example: Zidovudine. - Non-nucleoside RTIs (NNRTIs): non-competitive inhibitor of reverse transcriptase. (we dont have this so its chill on us) - Protease inhibitors: inhibits viral protease required for viral protein processing - Fusion inhibitors: binds to viral membrane proteins required for docking and host-cell entry. Neuraminidase inhibitors: blocks release influenza particles from host cell surface. Interferons: IFNs, produced by host cells against viruses Antifungal Anti-protozoan drugs - Quinine and derivatives: blocks hemoglobin metabolism in species of Plasmodium which cause malaria - Artemisinin: antimalarial drug derived from Artemisia plants. - Unclear mechanism of action, but possibly produces free-radicals upon haem metabolism. - Metronidazole: blocks nucleic acid synthesis in anaerobic organisms. - Used to treat infections caused by Giardia intestinalis, Trichomonas vaginalis, Entamoeba histolytica - Mebendazole: inhibits synthesis of microtubules. Popularly used to treat helminth infections. Antibiotic resistance: antibiotic-resistant pathogens. - Widespread (mis)use of antibiotics. - Unnecessary prescriptions - Self-prescriptions, often in LMICs. - Patients not completing treatment regimens. - Use in animals in agriculture. - Proper use of antibiotics - Limiting use in agriculture - incentives to discover and commercialize new antibiotics - Antibiotics that inhibit novel targets: platensimycin inhibits lipid biosynthesis, a target largely not targeted in bacteria. - Antibiotics that inhibit hard-to-evolve targets: teixobactin inhibits a small lipid molecule involved in bacterial cell wall synthesis, and is difficult to evolve resistance against. - Enzyme inhibitors: β-lactamase inhibitors e.g. clavulanic acid - Drug combinations