Medical Microbiology Lecture Notes Test 1.docx

Medical Microbiology Lecture Notes Test 1 8/19 lecture: Human Microbiome in Health and Disease - Microbial flora is in a continual state of change - Microbial flora: collective bacteria and other microorganisms in a host - Nutrition, hormonal, and health changes affect the...

Medical Microbiology Lecture Notes Test 1 8/19 lecture: Human Microbiome in Health and Disease - Microbial flora is in a continual state of change - Microbial flora: collective bacteria and other microorganisms in a host - Nutrition, hormonal, and health changes affect the human microbiota - The human microbiome: each person has a unique set of microbes - Each area of the body has different microbes with different environments - Biggest is the gut microbiome - The Human Microbiome Project: a 5-year multinational study that analyzed the genetic composition of the microbial populations that live in and on healthy adults - A collection of samples from the nose, mouth, skin, gut, and vagina from healthy adult volunteers - The microbes were identified by sequencing targeted regions of the 16S ribosomal RNA gene - 16S ribosomal RNA gene: made up of proteins and RNA's, 2 subunits - It aligns with the Shine Dalgarno sequence to start the translation - It is unique to each species - It is a good genetic marker - They were able to identify the microbes because the 16S ribosomal RNA gene is unique to each species - 18S is for eukaryotes - Operational Taxonomic unit (OUT): used to classify groups of closely related individuals - Core Microbiome: the species that are present at a specific site in 95% or more of individuals - There is a tremendous variation of species among individuals - Different environments on the body have different populations - Ex: different temperature exposures, pH, water availability, etc. - But there is less variation in the functional composition at each site - The taxonomic diversity of a population is great, but the functional properties are highly conserved in microbiomes associated with health - Functional redundancy: a characteristic of species within an ecosystem where certain species contribute in equivalent ways to an ecosystem function such that one species may substitute for another - Microbiome exists in a symbiotic relationship with its host and provides metabolic functions, stimulates innate immunity, and prevents colonization with unwanted pathogens - Normal Flora: consists of a core and secondary microbiota that evolved through a symbiosis with the host and competition with other species - The composition of the microbiota is influenced by personal hygiene, diet, water source, medicines (especially antibiotics), and exposure to environmental toxins - Nutrients play a major role in the relationship between the human host and microbes - Bacteria in the human gut: metabolize complex carbohydrates to provide small-chain fatty acids that can be readily transported and used by the cells of our body - These acids also limit the growth of undesirable bacteria - Bacteria graze on the mucins that line the epithelium, or the oils released in our sweat - Ex: Bacteroidetes and Firmicutes are efficient at breaking down complex carbohydrates and host-derived carbohydrates, including those attached to the mucins or chondroitin sulfates of the protective mucous layer of the intestine - Increases in the ratio of these bacteria in the gut microbiome can lead to a higher efficiency in the storage of the metabolic by-products. - This can be a benefit for malnourished populations or patients with debilitating diseases such as cancer or can lead to obesity in well-nourished populations - Role of the microbiome in diseases - Microbiome research proposes that disease can be caused by a community of organisms rather than a single species of bacteria - Can also influence immunologic and metabolic disorders such as inflammatory bowel disease, obesity, type 2 diabetes, and celiac disease - We are now at the forefront of a new era of redefining the concept of infectious diseases - Dysbiosis: disruption of the normal microflora - Can lead to disease by the elimination of needed organisms or allowing the growth of inappropriate bacteria - Understanding the influence of dysbiosis can lead to both advanced diagnostic tests and novel therapies - Best example: C. difficile disease - A clinical disease preceded by a depletion of the normal flora because of antibiotic use - The effects of microbiome alterations have also been described for the pathogenesis of inflammatory bowel disease and colorectal cancer - Proliferation of bacteria such as Akkermansia muciniphila that produce mucin-degrading sulfatases is responsible for the degradation of the intestinal wall lining - Additionally, an increase in members of the anaerobic family Prevotellaceae leads to upregulation of chemokine-mediated inflammation - Alterations of the microbiome may not be characterized by the presence or absence of a specific microbe, because more than one organism may provide the needed function - It is likely that future diagnostics will measure the presence or absence of a specific gene product (proteomics) or a metabolic function (metabolomics) - Proteomics: the study of the structure and function of proteins, including the way they work and interact with each other inside cells - Metabolomics: the comprehensive measurement of all metabolites and low-molecular-weight molecules in a biological specimen - Probiotics: mixtures of bacteria or yeast that when ingested colonize and proliferate, even temporarily, in the intestine - Commonly gram-positive bacteria: Bifidobacterium, Lactobacillus - And yeast: Saccharomyces - Many are found in ingestible capsules and as food supplements: yogurt, Keifer - They have been used to treat C. difficile: associated with diarrhea and inflammatory bowel disease - Also to provide protection from Salmonella and Helicobacter pylori disease - The species, mixture of species, dose, and viability of the probiotic organisms within a probiotic formulation influence its potency, efficacy, and therapeutic potential - What is clear is that carefully designed "smart probiotics" will likely be an important adjunct to medical therapy in the future - Microbiota of the Skin - Gram-positive, salt-tolerant bacteria - Why? Sweat - Ex: staphylococci, micrococci, and diphtheroids - These grow on oils and use it as a carbon source instead of glucose - Aerobes are on the surface - Ex: staphylococcus ssp. - Anaerobes are deeper in the pores like hair follicles - Ex: Propionibacterium acnes - Yeast is in the moist regions such as underarms, groin, etc. - Ex: Malassezia furfur, and Candida spp. - Respiratory Tract Microbiota - Mouth and Nasopharynx: Upper - Anaerobic: peptostreptococcus, Veillonella, Actynomyces, Fusarium spp. - Aerobic: streptococcus, Haemophilus, Neisseria, Staphylococcus spp. - Opportunists: S. pyogenes, S. aureus, S. pneumonia, N. meningitis, and H. influenza - The larynx, tracheae, bronchioles, and lower airways: lower - Maintained practically sterile, but transient colonization with organisms from the upper respiratory system can colonize - Ex: streptococcus, staphylococcus, Enterobacteriaceae spp. - These microbes suppress pathogens by competitive inhibition in the upper respiratory system - Gastrointestinal Tract Microbiota: biggest and most diverse in the body - General information - It is colonized at birth (if delivered vaginally) - Millions of bacteria per ml of saliva - Large numbers in the large intestine - 100 billion bacteria per gram of feces - Stomach: acid-tolerant organisms - Ex: Lactobacillus, Streptococcus, Helicobacter pylori - Small intestine: bacteria, fungi and parasites - Anaerobes: Peptostreptococcus, Porphyromonas, Prevotella - Gastroenteritis: species that may colonize in small numbers proliferate - Ex: Salmonella, Campyobacter spp - Large intestine: 10^11^ per gram of feces - Anaerobes: Bifidobacterium, Eubacterium, Bacteroides, Enterococcus, Enterobacteriaceae - Population can be altered rapidly by diet, and antibiotic treatment, leading to disease - Contains all nutrients you haven't digested - Genitourinary System - The anterior urethra and the vagina are permanently colonized - Urethra: Enterococcus, Enterobacteriaceae, Candida - Pathogens: N. gonorrheaea, Chlamydia trachomatis - Vagina: influenced by hormonal changes - Lactobacilli, Staphylococcus, Strepococcus, Enterococcus, Enterobacteriaceae, Mycoplasma - Pathogens: Gardnerella, Candida spp., Trichomononas - Bladder: can be transiently colonized, but is cleared by - Acidity of urine and mechanical flushing Online Lecture: General Principles of Laboratory Diagnosis - Microscopy: used for 2 basic purposes - The initial detection of microbes - The preliminary or definitive identification of microbes - The microscopic examination of clinical specimens is used to detect bacterial cells, fungal elements, parasites, and clumps of viruses present in infected cells - Characteristic morphologies can be used for the preliminary identification of most bacteria, and are used for the identification of many fungi and parasites - The microscopic detection of organisms stained with antibodies labeled with fluorescent dyes or other markers has proved to be very useful for the specific identification of many organisms - Bright Field Light Microscopy - Basic components: - light source used to illuminate the specimen - a condenser used to focus the light on the specimen - two lens systems (objective and ocular) used to magnify the image of the specimen - limitations: the resolution of the image (the ability to distinguish that two objects are separate and not one - Dark Field Microscopy: the same objective and ocular lenses are used however a special condenser is used - Advantage: the resolving power of darkfield microscopy is significantly improved compared with that of brightfield microscopy - This makes it possible to detect extremely thin bacteria such as Treponema pallidum - Examination Methods - Direct Examination - The sample can be suspended in water or saline (wet mount) - Can be mixed with alkali to dissolve background material (potassium hydroxide \[KOH\] method) - Can also be mixed with a contrasting dye (lactophenol cotton blue, iodine) - Differential Stains - Gram Stain: best known and most widely used stain - Forms the basis for the phenotypic classification of bacteria - Iron hematoxylin and trichrome stains: invaluable for identifying protozoan parasites - Wright-Giemsa stain: used to identify blood parasites and other selected organisms - Acid-fast stains - Ziehl-Neelsen: requires heating the specimen during the staining procedure - Cold acid-fast stain: Kinyoun Method - Fluorochrome stain: auramine-rhodamine method - In Vitro Culture - Success of culture methods is defined by - The biology of the organism - The site of infection - The patient\'s immune response to the infection - The quality of the culture media - In many infections the organism responsible for the infection will be present among many other organisms that are part of the normal microbial population at the site of infection - Ex: gastroenteritis, pharyngitis, and urethritis - Many media have been developed that suppress the normally present microbes and allow easier detection of clinically important organisms - The patients innate and adaptive immunity may suppress the pathogen, so highly sensitive culture techniques are frequently required - Likewise, some infections are characterized by the presence of relatively few organisms - Culture media purposes - Selective media: selects for the growth of a desired organism - Differential media: used to distinguish between bacterial cultures based on their biochemical properties - Enrichment media: media that allows only the growth of a particular type of microorganism - These are traditional microbiological methods - Pro: very good for metabolic characterization - Con: requires growth in vitro. What if you cannot culture an organism in vitro? - Detection of Microbial Genetic Material - Non-amplified DNA detection: detected with small, labeled DNA or RNA oligonucleotides - Amplified DNA detection: polymerase chain reaction and transcription-mediated amplification - Nucleic Acid analysis: sequencing and restriction fragment length polymorphism - Pros: can detect organisms without culturing them - Little DNA/RNA is needed in most cases - Very sensitive and specific - Cons: what if you cannot isolate DNA/RNA from the site of infection? - Serological Tests - Direct tests detect antigens (from patient samples) - Indirect tests detect antibodies (in patients serum) - Serological Tests to detect antigen - Precipitation and immunodiffusion techniques (soluble antigens) - Immunoassays for cell-associated antigens (immunohistology) - Immunoassays for antibody and soluble antigen - Complement fixation: RBCs are an indicator - ELISA: an enzyme linked to an antibody is the indicator - Western Blot: antigen is immobilized onto a membrane - Monoclonal Antibodies (Mabs): lab-made proteins that mimic the immune systems antibodies and are used to treat a variety of diseases lab-made proteins that mimic the immune systems antibodies and are used to treat a variety of diseases - Hybridoma: "immortal" cancerous B cell fused with an antibody-producing normal B cell - Produces monoclonal antibodies - Kohler and Milstein's 1975 Nature paper: solved the problem of how to generate clones of continuously dividing cells that make antibodies of a known specificity - This ability paved the way to clinical advances - They injected mice with sheep red blood cells and isolated spleen cells, including those that produce antibodies - The antibody-producing spleen cells and myeloma cells fused to create a hybridoma cell - Adalimumab: anti-TNF-α, Rheumatoid arthritis, Crohn's disease, Plaque Psoriasis, Psoriatic Arthritis, Ankylosing Spondylitis, Juvenile Idiopathic Arthritis, Hemolytic disease of the newborn - Etanercept: anti-TNF-α, plaque psoriasis, psoriatic arthritis, rheumatoid arthritis - Abatacept: extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) linked to the modified Fc portion of human immunoglobulin G1 (IgG1), rheumatoid arthritis - Denosumab: anti-RANKL, osteoporosis - Rituximab: anti-CD2-, lymphomas, leukemias - Trastuzumab: anti HER2/neu, breast cancer - The use of monoclonal antibodies in a home pregnancy test - Free monoclonal antibody specific for hCG, a hormone produced during pregnancy - Capture monoclonal antibody bound to substrate - Sandwich formed by the combination of capture antibody and free antibody when hCG is present, creating a color change - Pros of Monoclonal antibodies - Source of sample is the patient - Can sample over time - Cons of Monoclonal antibodies - Antibody presence does not guarantee current infection - Some antibodies show cross-reactivity 8/21 Lecture: Elements of Host Protective Responses - The immune system can have 2 systems: Innate and Adaptive - Innate Responses: you inherit them from your parents - Not pathogen-specific: no memory - Cytokines: regulator proteins secreted by leukocytes - These are soluble molecules - Function: signaling molecules - Hormone-like proteins that act on cells to activate and regulate the innate and immune response - Chemokines: small proteins associated with inflammation - These are soluble molecules - Function: diffuse easily, attract specific cells to sites of inflammation and other immunologically important sites - Neutrophils, basophils, natural killer (NK cells), monocytes, and T cells express receptors and can be activated by specific chemokines - These and other proteins are chemotactic factors that establish a chemical path to attract white blood cells to the site of infection - Interferons: proteins that promote antiviral responses - Subclass of cytokines - Neutrophils: short-lived, primary phagocytosis for extracellular pathogens - Promote the production of other proteins that block infection - Macrophages: long-lived phagocytes - Neutrophils and macrophages are the cell\'s first line of defense and eat what enters - Complement: activated proteins serve as a chemoattractant during inflammatory responses - This is a molecule - Made in the liver and circulates in the blood and tissues - They tag pathogens for destruction - Innate and immune cells communicate by cell-cell interactions and with soluble molecules, including complement cleavage products, cytokines, interferons (IFNs), and chemokines - IFNs: cytokines that are produced in response to viral and other infections or on activation of the immune response - Promote antiviral and antitumor responses and stimulate immune responses - The white blood cells can be distinguished based on - Morphology - Histologic staining - Immunologic functions, and - Intracellular and cell-surface markers (types of receptors and markers on the surface) - B cell receptor and T cell receptor lymphocytes can be distinguished by expression of antigen receptors on their surfaces - You can know if it is a B or T cell receptor because of the specific receptors on their surface - Antigen receptors on B cells: immunoglobulin - Antigen receptors of T cells: T-cell receptors (TCRs) - Other cell-surface proteins distinguish subsets of cells - These marker proteins are defined within clusters of differentiation (CD4), and the markers indicated by "CD" numbers - MHC 1 antigen: all nucleated cells express class 1 major histocompatibility complex antigens - Any cell with a nucleus can express MHC 1 - Proteins are on the surface of the cell - MHC 2 antigen: a special class of cells that are antigen-presenting cells (APCs) express class 2 MHC antigens - These present a piece of the pathogen to neighboring cells - Cells that present antigenic peptides to T cells include dendritic cells, macrophage family cells, B lymphocytes, and a limited number of other cell types - Self-renewing stem cell: found in bone marrow - It produces a pluripotent stem cell - The pluripotent cell can then produce either a Myeloid progenitor cell or a Lymphoid progenitor cell - Myeloid progenitor cell: gives rise to cells like Basophils, Neutrophils, and Monocytes, Dendritic cells, and Macrophages - Lymphoid progenitor cell: gives rise to B and T lymphocytes and Natural Killer cells - Blood cells: all derived from pluripotent stem cells - Stem cells reside in the bone marrow - Stem cells are constantly making tons of cells that will differentiate into T cells, etc. - T cells: differentiated in the thymus - Primary organs: bone marrow and thymus - These are essential to the development of the immune system - Secondary organs: lymph nodes, tonsils, adenoids, spleen, MALT - Primary vs. secondary lymphoid organs - Primary: where cells develop - Bone marrow and thymus - These sites of initial lymphocyte differentiation are essential to the development of the immune system - Thymus: essential at birth for T-cell development but shrinks with aging, and other tissues adopt its function later in life - Secondary: encounters the antigens - DCs and other phage sites bring the antigen here - Lymph nodes, spleen, skin, and mucosa-associated lymphoid tissue (MALT) - These sites are where DCs, innate lymphoid cells (ILCs), B and T lymphocytes, and other cells reside and respond to antigenic challenges - The primary and secondary lymphoid organs produce chemokines and express cell-surface adhesion molecules (addressins) that interact with homing receptors (cell adhesion molecules) to attract and retain these cells - Secondary Organs: Lymph nodes - The lymph node is constructed to optimize the meeting of the innate (DCs and macrophages) and the immune response (B and T) cells to initiate and expand specific immune responses - Lymph nodes and encapsulated and have three layers: cortex, paracortex, and medulla - Cortex: outer layer - Contains mainly B cells, some T cells, follicular DCs, and macrophages arranged in structures called follicles and, if activated, in germinal centers - Primary follicle: B cells develop here - Paracortex: contains T cells and DCs and the DCs present antigens to the T cells to initiate immune responses - Medulla: contains B and T cells and antibody-producing plasma cells, as well as channels for the lymph fluid - Secondary Lymphoid Organs: Spleen - The spleen acts like a lymph node, and also filters antigens, pathogens, and old blood cells - It is less organized than a lymph node but functions about the same - White pulp: consists of arterioles surrounded by lymphoid cells (periarteriolar lymphoid sheath) in which the T cells surround the central arteriole - B cells are organized into primary unstimulated or secondary stimulated follicles that have a germinal center - The germinal center contains memory cells, macrophages, and follicular DCs - Red pulp: storage site for blood cells and the site of turnover of aged platelets and erythrocytes - Secondary Lymphoid Organs: Skin, MALT - A less structured lymphoid tissue - Epidermis of skin: contains keratinocytes and Langerhans cells - Dermis: contains DCs, B and T lymphocytes, macrophages, and mast cells - Large numbers of memory T cells continuously circulate into these two layers of the skin - Peyer patches are in the intestine - MALT: contains less structured aggregates of lymphoid cells - Peyer patches: they are located along the intestinal wall and have special cells in the epithelium (M cells) that deliver antigens from the lumen into this mini lymph node-like structure containing DCs and lymphocytes in defined regions - T cells: interfollicular - B cells: germinal - DCs, T cells, and B cells: reside in the lamina propria layer just under the epithelium - Tonsils: an important part of the MALT - These lymphoepithelial organs sample the microbes in the oral and nasal area - Contains a large number of mature and memory B cells that use their antibodies to sense specific pathogens with DCs and T cells, can initiate immune responses - Swelling of the tonsils may be caused by infection or a response to infection - Polymorphonuclear Leukocytes: AKA neutrophils, or PMNs - These are white blood cells that have a nucleus that can be many different shapes - They are short-lived, 50-70% of circulating leukocytes - They are primary phagocytes: no need to activate them, they are ready to go when they come out of bone marrow - Play a major role during inflammation - First line of defense and eats what enters the bloodstream - Granules contain: myeloperoxidase, β-glucuronidase, elastase, cathepsin G, lysozyme, and lactoferrin - These are all designed to destroy bacteria - Summarized definition: granulocytes with a short life span, multilobed nucleus, and granules; phagocytose and kill bacteria - Millions of these, the most abundance white blood cells in the blood - Eosinophiles: important against parasitic infection, phagocytic - They are activated during an allergic response - Not many in the bloodstream, but can activate when needed - Heavily granulated: phosphatases, peroxidases, basic proteins - Basophils: important during allergic responses, non-phagocytic - Granules contain histamine - Not many in the bloodstream, but can activate when needed - Mononuclear Phagocytes: - Myeloid cells derived from monocytes - Monocytes: follow neutrophils in inflammation - 3-8% blood leukocytes - Horseshoe-shaped nucleus - Has lysosome granules - A precursor to macrophage lineage and dendritic cells - Macrophages: exist in tissue, spleen, lymph nodes, other organs, lung, liver and connective tissue - These have multiple functions: - \#1 function: phagocytosis; through Ig Fc, C3b, and Toll-like and other PAMP receptors - APCs: present antigen on MHC 2 - Secrete cytokines to stimulate inflammation: IF-1, IL-6, TNF-α, IL-12 - Phagocytosis: a huge part of innate immunity - PAMP: recognized by receptors because we do not have these - Ex: pili, flagella, capsule, peptidoglycan, lipopolysaccharide - TLR: receptors that recognize PAMP - Steps: - Chemotaxis and adherence of microbe to phagocyte (bacteria enter) - Ingestion of microbe by phagocyte (it knows it's a pathogen because of the receptors - Formation of a phagosome - Fusion of the phagosome with a lysosome to form a phagolysosome (lysosome is full of enzymes that breakdown proteins) - Digestion of ingested microbe by enzymes - Formation of residual body containing indigestible material - Discharge of waste materials (either released as waste or is loaded onto a receptor that moves outside the cell) - Dendritic cells: professional APCs (they present antigen to T cells) - Myeloid and lymphoid origin - Found in tissues and blood: Langerhans (skin), dermal interstitial cells, splenic marginal cells, liver, thymus, and lymph nodes - Found everywhere under the skin - Plasmacytoid DCs produce INFα in response to viral infections - Follicular DCs in lymph nodes and spleen present antigens to B cells - DCs capture and present antigen on MHC 1 and MHC 2 to T cells - Most potent APC, initiates and determines nature of T-cell response - T cells cannot activate without this reaction - Lymphocytes: B and T cells - B and T cells originate in the bone marrow but also differentiate in other sites - B (bone marrow) cells: make antibodies and also internalize and present antigens - Identified by MHC 2 and receptors for C3b, C3d and complement CR1 and CR2 proteins - Differentiate into memory or plasma cells - T (thymus) cells: control immune responses, also kill virus-infected cells - Express TCR - 2 major classes of T cells: - CD4 T cells: produce cytokines to initiate immune responses and activate macrophages - Subdivided into: TH0, TH1, TH2, TH17, and Treg - Important in B cell development - CD8 T cells: recognize and kill virus-infected cells - NK cells: antibody-dependent killing of virus-infected cells - Compare and contrast innate and adaptive immune systems: - Innate: these cells are ready to go immediately - Macrophages: The number one function is phagocytosis (cleaners) - Present antigen to MHC 2 - Sense signals: secrete interleukins and trigger inflammation - Neutrophils: the first line of defense - The cleanup crew: phagocytosis - Have granules: proteases release oxidative compounds - Adaptive: these cells need a lot of processing until they are ready - B cells: make immunoglobulins - T cells: kill or help 8/26 Lecture: Innate host responses - Barriers to Infection: skin, mucous membranes - Antimicrobial peptides and enzymes found in mucosa: defensins, lysozyme, lactoferrin, IgA, etc. - Defensins: disturb membranes of bacteria - Lysozyme: in our sweat - Acidic environment of the stomach, bladder, and kidneys; bile in the intestine - All full of enzymes that destroy bacteria - Bladder and kidneys have to have a specific pH - Acidity will kill most bacteria - Soluble components of the innate immune system: - Antimicrobial peptides and chelators - Defensins, bactericidal/permeability-increasing peptides (BPIs), and cathelicidins - All peptides produced by neutrophils and epithelial cells - They disrupt microbial membranes and are toxic to bacteria and fungi - Defensins: small cationic peptides - Cathelicidins and BPIs: larger and cleaved to produce microbiocidal peptides - These are secretions - Metal Ion: binding proteins that bind iron or bind zinc and manganese sequester these essential ions to prevent the growth of bacteria and yeast - Complement system: activated directly by fungal and bacterial surfaces and bacterial products (alternate or properdin pathway) by lectin binding to sugars on the bacterial or fungal cell surface (mannose-binding protein), or by complexes of antibody and antigen (classical pathway) - In the bloodstream - Interferons - Activation of the complement system: this system is in your blood inactivated until they become activated - releases multiple factors that are important for opsonization and chemotaxis of inflammatory cells - Composed of: serum proteins activated in a cascade - The complement system is activated directly by fungal and bacterial surfaces and bacterial products (alternate or properdin pathway), by lectin binding to sugars on the bacterial or fungal cell surface (mannose-binding protein), or by complexes of antibi and antigen (classical pathway) - All three pathways will merge to become C3 - The three activation pathways of complement coalesce at a common junction point, which is the activation of the C3 component - Activation by either pathway initiates a cascade of proteolytic events that cleave the proteins into "a," "b," and other subunits - Activated products: once activated, several things will happen - Chemotactic factors will be released: C5a - Chemotactic: the directed movement of cells in a gradient of chemoattractant - Anaphlyotoxins will be released: C3a and C5a - Opsonins will be deposited on pathogens: C3b - Molecules that bind on the surface of a pathogen making it more tasty so others will eat it - B-cell activator will become activated: C3d - Alternative pathway of complement activation: not antibody-dependent - Activated directly by bacterial surfaces - Proteins that are part of the complement system will deposit onto the surface of a microbe - This activates C3 to be cleaved into C3b and C3a - This pathway can be activated before the establishment of an immune response to the infecting bacteria - C3 is spontaneously cleaved in serum and can covalently bind to bacterial surfaces - Properdin factor B binds to the C3b and properdin factor D splits factor B In the complex to yield the Bb active fragment that remains linked to C3b - The complement cascade then continues in a manner analogous to the classical pathway - Summary: C3 combines with factors B, D, and P on the surface of a microbe - This causes C3 to split into fragments C3a and C3b - What activates this pathway? Blood factors that deposit onto C3 - C3b: bid and serves as oposonant - C3a: little and can fuse and serve as a chemoattractant - Classical pathway of complement activation: requires 3 main antibodies that will recognize the microbe, and these antibodies need to be present - To activate: need antibodies already, meaning you have to have been exposed to the pathogen already - The classical complement cascade is initiated by the binding of the first component, C1, to the Fc portion of the antibody when it is bound to cell-surface antigens or to an immune complex with soluble antigens - Activated C1 splits C2 into C2a and C2b, and C4 into C4a and C4b - C2a and C4b combine and activate C3, splitting it into C3a and C3b - The union of C2a and C4b produced C4b2a which is known as C3 convertase - What activates this pathway? antibodies - Lectin pathway of complement activation: a bacterial and fungal defense mechanism independent of antibody - Lectin: a protein that binds to polysaccharides - Activated by sugars present on the microbes themselves - As long as sugars are recognized by lectins present in our blood, then those lectins can activate C2, and C4 will then form the convertase that will cleave C3, and then it will be activated - Mannose-binding protein: a large serum protein that binds to nonreduced mannose, fucose, and glucosamine on bacterial, fungal, and other cell surfaces - Mannose-binding protein resembles and replaces the C1q component of the classical pathway and activates the cleavage of the mannose-binding protein-associated serine protease - What activates this pathway? Lectin - Biological activities of complement components: - C3b causes opsonization - C3a + C5a causes inflammation - C5b + C6 + C7 + C8 + C9 cause the assembly of the membrane attack complex (MAC) that can lyse Gram-negative bacteria (leads to bacteria death) - MAC kills bacteria - Some bacteria evade complement - Capsules prevent C activation and position of opsonization - Opsonins cannot stick to bacteria with thick capsules - Surface lipid-carbohydrates prevent membrane attack complex (MAC) - Enzymatic digestion of C5a - Many more - Interferons (IFNs): small cytokine-like proteins made by lymphocytes in response to viral infection and also in response to inflammation - Can interfere with viral replication but also have systemic effects - 2 large groups: - IFN-α and IFN-β: early antiviral response triggered by double-stranded RNA (viral infection) - DCs and fibroblasts can make IFN-α and IFN-β - Promote transcription of antiviral proteins - Vital for viral infections - Gamma IFN: - Produced by NK and T cells - Activates macrophages and myeloid cells - Activates macrophages so they can efficiently phagocytose - Steps: - Viral RNA from an infecting virus enters the cell - The infecting virus replicates into new viruses - The infecting virus also induces the host cell to produce interferon mRNA - This is translated into alpha and beta interferons - Interferons released by the virus-infected host cell bind to plasma membrane or nuclear membrane receptors on uninfected neighboring host cells - This induces them to synthesize antiviral proteins - These include oligoadenylate synthetase and protein kinase - New viruses released by virus-infected host cell infect neighboring host cells - AVPs degrade viral mRNA and inhibit protein synthesis and thus interfere with viral replication - Myeloid: from bone marrow - Lymphoid: from lymphatic tissue - Innate responses: driven by neutrophils and granule sites - Neutrophils: first line of defense - Have receptors for complement system - Major role in antibacterial and antifungal protection - Receptors bind to microbes and promote phagocytosis - Receptors: C3b, C-type lectin, and opsonin receptors for the Fc portion of antibodies - Have granules that contain antimicrobial proteins and oxidative substances - Terminally differentiated, live less than 3 days, a major component in pus (short half-life) - Mast cells, Basophils, and Eosinophils: granule sites, pro inflammatory - Have a large number of cytoplasmic granules containing antimicrobial substances and mediators of inflammation - Mast cells: present in skin, mucoepithelial tissue, and the lining of small blood vessels and nerves - Basophils: are like mast cells but circulate in the blood, and their granules stain with basic dyes - Mast cells and basophils bind IgE, complement, and microbial products and release histamine and cytokines as part of allergic and inflammatory responses - They both circulate at low levels when healthy - Can bind to IgE: when this happens they are super damaging - Eosinophils: circulate in the blood, their granules stain with acidic dyes, and they are important in antiparasitic responses - Monocyte-Macrophage lineage: these are major phagocytes - Macrophages differentiate from blood monocytes - Macrophages: found in tissue and are more restricted in movement - Like neutrophils, have several receptors that promote phagocytosis - Are able to express MHC2 for antigen presentation: 2 types - M1 macrophages: activated by IFN-Ɣ produced by NK and T cells (TH1 response) - Produce: antibacterial enzymes, cytokines and chemokines - These attract DCs, NK cells and T cells to the site of infection, killing phagocytosed microbes, virally infected cells and tumor cells - M2 macrophages: activated by IL-4 and IL-13 (TH2 response) - Involved in antiparasitic responses, tissue remodeling, wound repair - Myeloid and Plasmacytoid Dendritic cells: whatever the DC cells do will determine what the T cells will do - Dendritic cells are the bridge between the innate and adaptive response - The cytokines they express determine the T cell response - Monocyte myeloid precursors differentiate into immature DCs in tissue and lymphoid organs - Mature DCs are the ultimate antigen-presenting cell (APC) - Initiate antigen-specific T-cell responses - Express receptors that can detect tissue trauma and infection (TLRs) - 3 types of APCs: - Langerhans cells: a type of iDC that remains in the epidermis of the skin - Plasmacytoid DCs: in the blood and generate large amounts of type 1 IFN and cytokines in response to viral and other infections - Follicular DC: in the B-cell-rich follicles of lymph nodes and spleen - Antigens stick to the surface and are displayed to B cells - Natural Killer cells: respond specifically to viral infection - Early response to viral infection and tumor cells - Important in antibody-dependent cellular cytotoxicity (ADCC) - Activated by: - IFN-α and IFN-β (in response to viral and other infections) - TNF-α - IL-12, IL-15, IL-18 (produced by DCs and activated macrophages) - IL-2 (produced by CD4 TH1 cells) - Surface markers: IgG Fc receptor, complement receptors, many T cell-like receptors - When activated: release perforin and granzymes, inducing apoptosis in the target cell - NKT and Ɣ/δ cells reside in tissue and blood - Sense non-peptide antigens (glycolipids from mycobacteria) - Produce IFN-Ɣ, triggering a TH1-type of response - How do they kill? When activated they release perforin and granzymes and it will punch holes (apoptosis) in the cell - Activation of innate cellular responses - Activation of response: bacterial and viral components activate innate and inflammatory responses---toll-like receptors on the cells or inside the cells - Toll-like receptors on the cell: will respond to extracellular pathogens - Toll-like receptors inside the cell: will respond to intracellular pathogens - Toll-like receptors (TLR): responsible for the recognition of pathogen-associated molecular patterns (PAMPs) - Over 13 TLRs have been reported in humans - They activate the NF-kB pathway: this regulates cytokine expression through several adaptor molecules, including MyD88, TIRAP/Mal an dTRIF - Activation of the NF-kB pathway links innate and adaptive immune responses by production of inflammatory cytokines such as IL-1, IL-6, IL-8, TNF alpha, IL-12, chemokines and induction of co-stimulatory molecules such as CD80, CD86, and CD40 - In addition to induction of the cytokine network, MyD88 binds FADD and triggers apoptosis through the caspase cascade - Hence, activation of the apoptosis pathway via TLRs appears to contribute to the repertoire of defense mechanisms utilized by the innate immune response - Toll-like receptors are expressed at a constitutive level but can be upregulated - TLR3, TLR7, TLR8, and TLR9 are localized mainly in endosomal/lysosomal compartments - Human TLR3 is detected in human fibroblast cell lines and TLR9 in in vitro-derived DCs - Specific TLRs will engage different PAMPs from different microbes - Local inflammation: promoted by the inflammasome - Inflammasome: complex present in DCs, macrophages, epithelial and other cells - Formation of these will drive production of specific cytokines and will drive even more inflammation - Whether the adaptive or innate response is needed will depend on the type of trauma that is present to the cell in each case - Chemotaxis and Leukocyte Migration: - Chemotactic factors in response to infection: - C3a, C5a, f-met-leu-phe, chemokines - C3a and C5a: come from complement activation, they are important to attract specific cells to the site of infection - Chemokines and TNF-α cause the endothelial cells to express adhesion in molecules - Leukocytes in circulation slow down, stick, and extravasate to the site of infection - This will drive the neutrophile through the endothelial cells to the tissue where the infection is - When macrophages detect a pathogen they release cytokines and chemokines - This allows the neutrophil to attach and squeeze between cells - Phagocytosis: driven by interactions with the microbe through receptors by recognition of PAMPs - Bacterial attachment mediated by - Opsonins (C3b, Igs, mannose-binding proteins) - Lectins - Fn-binding integrins (gram-positives) - Phagosome fuses with the lysosome - Bactericidal actions: - oxidative burst: myeloperoxidase, nitric oxide, hydrogen peroxide, etc. is released - Cationic proteins, lysozyme, lactoferrin, proteases, etc. degrade the microbe - Steps: - Microbe recognizes the bacteria by its receptors - Bacteria gets engulfed into the microbe - Phagosome fuses with lysosome - It destroys the bacteria - Normal Flora-Associated responses: - The skin and mucosa-associated lymphoid tissue (MALT) of the nares, oral region, and urogenital and gastrointestinal tracts are constantly monitoring and being stimulated by the adjacent normal flora. - DCs continuously probe the intestine and sense the LPS, LTA, flagella, and other components of the bacteria within the lumen. - M2 macrophages, DCs, ILCs, and T cells regulate the response to the flora - Do this by: instructing the epithelial cells to produce mucus and appropriate antimicrobial peptides and by preventing excess inflammation. - An equilibrium is maintained between inflammatory and immune regulatory responses to the microbial stimuli. - Disruption of the equilibrium can result in gastroenteritis, inflammatory bowel disease, or autoimmune diseases. - Acute Inflammation: the process that drives vasodilation, increased vascular permeability, and neutrophil recruitment - Acute-phase proteins are activated: - Vasodilation: causing redness - Increased vascular permeability: swelling or edema - Neutrophil recruitment - Inflammation mediators are beneficial but can cause severe tissue damage if not controlled - Cytokine-induced responses: driven by the presence of LPS and other PAMPs - LPS and other PAMPs stimulate dendritic cells and macrophages to secrete pyrogens - Acute-phase cytokines: IL-1, IL-6, TNF-α and chemokines - TNF-α: triggers the systemic effects of infection: - Stimulates endothelial cells to express adhesion factors and chemokines to attract and activate neutrophils and macrophages. - Promotes fever, metabolic changes and production of Il-1, IL-12 and acute-phase proteins (C-reactive proteins, complement, LPS-binding proteins, protease inhibitors, adherence proteins). - IL-1: produced by activated macrophages, neutrophils, epithelial and endothelial cells. Promotes systemic and local inflammation. - IL-6: produced by many cell types, induces the production of acute-phase proteins in the liver. - Acute-phase response: - Triggered by infection, tissue injury, prostaglandin, interferons, acute-phase cytokines, and inflammation - Promotes fever, anorexia, sleepiness, metabolic changes - IL-1 and TNF-α are pyrogens, acute-phase proteins include C-reactive protein, complement components, LPS-binding proteins, coagulation proteins, etc. - Acute-phase proteins reinforce the innate defenses, but their excessive production can cause shock - Sepsis and cytokine storm: - Generated by an unregulated release of cytokines in response of bacterial components, toxic shock syndrome and certain viremias. - Bacteremia: C5a promotes vascular leakage, neutrophils activation coagulation activation. - DCs produce large amounts of inflammatory cytokines. - Abnormal stimulation of T-cells and APCs by superantigen toxins - Viremia: DCs and T cells produce large amounts of IFN-α. - Bridge to Antigen-specific immune responses: - DCs are key to the transition and determine the nature of the subsequent response. - iDCs are constantly sampling the environment. - If stimulated by PAMPs during infection, they differentiate into DCs, and their role changes; - DCs lose their ability to phagocytize, moves to a lymph node and serves as an antigen presenting cell (APC) to local T cells. - This is how they bridge the response from the innate to the adaptive immune system - Lysozyme: catalyzes hydrolysis of bacterial peptidoglycan - Lactoferrin: bind iron and compete with microorganisms for it - Lactoperoxidase: may be inhibitory to many microorganisms - Beta-lysin: is effective mainly against gram-positive bacteria - Chemotactic factors: induce directed migration of PMNs, monocytes, and other cells - Properdin: activates complement in the absence of antibody-antigen complex - Lectins: bind to microbial carbohydrates to promote phagocytosis - Cationic peptides: disrupt membranes, block cell transport activities 8/28 Lecture: Antigen-specific Immune Responses - Antigen: A protein or carbohydrate that is recognized and sufficient to initiate an immune response. - An antigen is a molecule that is recognized by specific antibodies or the TCR on T cells. - An epitope (antigenic determinant) is the actual molecular structure that interacts with a single antibody molecule or TCR. - Very small, a few amino acids - An antigen can have multiple epitopes - Haptens (incomplete immunogens) can be small molecules and too small to immunize (i.e., initiate a response) an individual but can be recognized by antibodies. Haptens can be made immunogenic by attachment to a carrier molecule, such as a protein. - Adjuvants: chemicals that mimic PAMPs, prolong the presence of the antigen in artificial immunizations. - Usually present in vaccines - T-cell-independent antigens: don't need T-cells to help - The type of immune response initiated by an immunogen depends on its molecular structure. - A primitive but rapid antibody response can be initiated toward bacterial polysaccharides (capsule), peptidoglycan, or flagellin. - T-independent antigens have a large repetitive structure that is sufficient to bind many surface antibody molecules and activate B cells directly without the participation of T-cell help. - Ex: sugar, polysaccharides, carbohydrates - In these cases, the response is limited to production of IgM antibody and plasma cells but memory cells are not generated and anamnestic (booster) responses cannot occur. - T-cell-dependent antigens: do need T-cells to help - Proteins that generate all five classes of immunoglobulins and can elicit memory and an anamnestic response - They need to be processed, broken down, and attached/recognized by either an antigen-presenting cell or a cell that will phagocytose and internalize them - Once they are on MHC 2 then, they can interact with T cells - Process: antigens are processed and presented to B cell - The B cell endocytoses the receptor and places it on MHC2 - Once the receptor is on the MHC2 it can interact with T cells - T cells and B cells engage, communicate, and make cytokines - This interaction makes a plasma cell, which goes on to secrete antibodies - T cells and Cellular immunity: - T cells mature in the thymus - Thymic selection eliminates many immature T cells - T cells respond to Ag by T-cell receptors (TCRs) - T cells require antigen-presenting cells (APCs) - Dendritic cells, macrophages, and B cells - Types of T cells: - CTL: cytotoxic - Kills infected cells - Interacts with CD8 - TH1, TH2, TH7: T-helper cells - Help B cells differentiate - Interacts with CD4 - Treg: T regulatory cell - Regulation of inflammatory processes - Interacts with CD4 - All of these need to see the antigen presented on MHC2 for it to respond - Cell-surface receptors of T cells: made out of 2 polypeptides, an alpha subunit and a beta subunit - The α/β TCR is expressed on most T cells, and these cells are primarily responsible for antigen-activated immune responses. - Classical T cells with the α/β TCR are distinguished further by the expression of either a CD4 or a CD8 molecule. - T cells expressing the γ/δ TCR are present primarily in the mucosa and blood. - They are important for stimulating innate and mucosal immunity. - These cells make up 5% of circulating lymphocytes but expand to between 20% and 60% of T cells during certain bacterial and other types of infections. - The γ/δ TCR senses unusual microbial metabolites and initiates cytokine-mediated immune responses. - The CD4 and CD8 proteins are co-receptors for the TCR, they facilitate the interaction of the TCR with the antigen-presenting MHC molecule and can enhance the activation response. - CD4 binds to class II MHC molecules on the surface of APCs. - CD8 binds to class I MHC molecules on the surface of nucleated cells, including APCs. - The cytoplasmic tails of CD4 and CD8 associate with a protein tyrosine kinase (Lck), which enhances the TCR-induced activation of the cell on binding to the APC or target cell. - Accessory and Adhesion molecules are protein receptors that interact with their protein ligands on APCs and lead to activation of the T cell, promotion of tighter interactions between the cells, or facilitation of the killing of the target cell. - T cells express receptors for many cytokines that activate and regulate T-cell function. - Chemokine receptors distinguish the different T cells and guide the cell to where it will reside in the body. - Summary: - Class 1 restricted interaction - CD8 binds to MHC1 and can interact with MHC1 - CD8 positive T-cells and is a coreceptor - Any cell that has a nucleus can express MHC1 - Class 2 restricted interaction - CD4 binds to MHC2 and can interact with MHC2 - CD4 positive T-cells and is a coreceptor - Macrophages, dendritic, and B cells can express MHC2 - They can also express MHC1 because they all have a nucleus - Peptide presentation by MHC 2: - Exogenous pathway: extracellular loaded onto MHC2 and presented to CD4 T cells - Class 2 MHC are found on antigen-presenting cells and are the major determinant of "invaders" - The MHC 2 presents antigenic peptides from exogenous sources - The MHC 2 presents antigenic peptides to CD4+ T cells - Process: - A phagosome with antigen from outside the cell gets loaded onto MHC2 - This has antigenic peptides - Where did the MHC2 come from? The endoplasmic reticulum, which makes all proteins (and all receptors), makes a class 2 MHC - An invariant chain (a blocker that blocks their binding site) is placed on the class 2 MHC and blocks their binding site - This MHC2 and chain is placed in a vesicle and sent on - Once the MHC2 gets to the phagosome, the chain is broken down - Then an antigenic peptide binds to the Class 2 MHC which then carries it out of the cell - Peptide presentation by MHC 1: - Endogenous pathway: intracellular loaded onto MHC1 and presented to CD8 T cells - Class 1 MHC are found on all nucleated cells and are the major determinant of "self" - The MHC 1 presents antigenic peptides from within the cell - The MHC 1 presents antigenic peptides to CD8+ T cells - Process: - A bad/infected/old protein inside the cell is broken down by a proteasome (machinery for degrading old proteins) into peptides - These peptides move into the endoplasmic reticulum - The ER makes all proteins (and all receptors) and makes MHC1 - MHC1 doesn't get a blockage like MHC2 does, so it is readily available for binding - The peptides that moved into the ER are then bound to MHC1 and moved through the cell - It then goes all the way to the outside of the cell where it will be presented to CD8 T cells and killed - What does CD8 bind to? MHC1 - CD8 function: to kill, not help - What does CD4 bind to? MHC2 - CD4 function: to help - Initiation of CD4+ T-cell responses - Activation through processed Ag loaded on the MHC2 of APCs - MHC 2 presents peptides from proteins degraded in the lysosome - Effects on B and T cells - T cytotoxic cells: - displays CD8+ or T~c~ cells: they recognize when something has been infected, etc. - Target cells are self-carrying endogenous antigens - Activated into cytotoxic T lymphocytes (CTLs) - CTLs recognize Ag + MHC 1 - Induce apoptosis in target cell - To kill a cell: CTL releases perforin and granzymes, which punch holes in the cells - Initiation of CD8+ T-cell responses - Activation through processed Ag loaded on the MHC 1 of DCs - MHC 1 is present in all nucleated cells: "self" - MHC 1 presents peptides from proteins that have been degraded by the proteosome: - Misfolded, viral, tumor - If a macrophage detects a viral infection, what happens? The infection is loaded onto MHC1 - What happens to the macrophage once it loads it? It is killed by CD8 - What if another macrophage does phagocytosis or endocytosis on an antigen? It is loaded onto MHC2 - What happens next? T cell will recognize the antigen (CD4 T cell) and it becomes activated - It is a helper cell, so it will differentiate into a plasma cell, which secretes immunoglobulins - Activation of B cells: antibodies are made up of B cells - B cells are activated by clonal selection and clonal deletion - Clonal selection: B cells differentiate into - Antibody-producing plasma cells - Memory cells - Clonal deletion: at an early differentiation stage, eliminates B cells that recognize host epitopes (self, from our own tissues) - When cells are selected, they will be deleted - Clonal selection: - If a B cell recognizes self-antigen, then it'll be deleted - This happens in the bone marrow - If a B cell recognizes an external antigen, then it'll be kept - This happens in the lymph nodes - Antibodies: proteins that will fold into globules - Globular proteins called immunoglobulins - The number of antigen-binding sites determines valence - Antibodies bind to antigens through antigen-binding sites - Made out of 4 chains - Red and purple: heavy chains - Yellow and green: light chains - They are all crosslinked with disulfide bridges - V section: variable - C section: Constant - Fc (stem) region: a region that interacts with cells - Immunoglobulin (Ig) classes in humans: Ig G, Ig A, Ig M, Ig E, Ig D - IgG antibodies: monomer - Constitutes 80% of serum Abs - Most abundant in blood - Fix complement - In blood, lymph, and intestines - Cross placenta: when someone is pregnant, these antibodies go from the bloodstream to the baby and gives temporary protection - Enhances phagocytosis (serves as an opsonin); neutralize toxins and viruses; protects fetus and newborn - Half-life = 23 days fairly stable - IgM antibodies: first antibody to be secreted by B cells - Pentamer: 10 sites for antigen binding (5 monomers) - constitutes 5-10% of serum Abs - Fix complement in blood, lymph, and on B cells - Good at agglutination of microbes; first Ab produced in response to infection - Half-life = 5 days - IgA antibodies: present in mucosal surfaces - Dimer - Constitutes 10-15% of serum Abs - In secretions - Mucosal protection: this is its main function, even more than IgG - Half-life = 6 days - IgD antibodies: present early in B cell differentiation - Monomer - Constitutes 0.2% of serum Abs - In blood, in lymph, and on B cells - On B cells, initiate immune response - Half-life = 3 days - IgE antibodies: secreted in allergic reactions - Monomer - Constitutes 0.002% of serum Abs very low levels if healthy - On mast cells, on basophils, and in blood - Allergic reactions; lysis of parasitic worms - Degranulates cells - Half-life = 2 days - Time course of the antibody response - This concept is what drives vaccination research - When you are first exposed to an antigen, you see IgM first and then IgG - This is the primary response - Once the infection is resolved, both IgM and IgG go down - Once you see the antigen again, IgG goes way up - Memory cells go into action and secrete more B cells than before