COMPARING INNATE AND ADAPTIVE IMMUNITY

Inflammatory Cytokines and Signaling Mechanisms:

*Cytokine: A family of cell-produced peptides and proteins that mediate pleiotropic pro-inflammatory modulatory effects 1. Monokine: cytokines that were produced by monocytes 2. Lymphokine: cytokines that were produced by lymphocytes 3. Interleukin: cytokine that were produced by leukocytes and acted on leukocytes

Antibody (constant domain) Isotypes

*IgM antibodies 1. First made 2. Pentameric with 10 antigen binding sites 3. Usually low affinity - produced before somatic hypermutation 4. Pentameric structure makes them potent activators of Complement system 5. Large size - confined to blood 6. Critical role - first in dealing with and controlling infections in bloodstream

Mannose Binding Lectin Pathway

1 . A plasma protein belonging to the pattern recognition receptor of innate immunity 2. Binds specifically to mannose residues with defined spacing only found in pathogens 3. Using MASP-1 and MASP-2 as the enzymes to activate C4 and C2 to form Classical C3 Convertase (C4b2a)

Innate Immunity

1. A genetically programmed set of responses that can be mobilized immediately when an infection occurs 2. Because all adaptive immune responses are contingent upon an innate immune response, vaccines must induce both innate and adaptive immune responses.

Summary of inflammatory Cytokines:

1. Activate vascular endothelium and increase vascular permeability: TNF and IL-1 2. Enhance NK cell cytotoxicity: IL-12 3. Induce inflammatory responses and cause tissue injury: IL-1, IL-6, and TNF-alpha 4. Induce migration of leukocytes into inflammatory sites: IL-8 and MCP1 5. Down-regulate inflammatory reactions: IL-10

Terminology:

1. Antisera raised against immunogen contains many antibody molecules that bind to the antigen in slightly different ways 2. Some antibodies in antisera are cross-reactive. A Cross Reaction is defined as the binding of an antibody with closely related molecules in addition to the specific antigen 3. Cross reacting antibodies can cause problems when antisera is used to detect specific antigens 4. Cross reacting antibodies can be removed by absorption with cross reactive antigen, leaving behind only antibodies that react to the specific immunogen

Bone Marrow

1. As the bones develop during the fourth and fifth months of fetal growth, hematopoiesis begins to shift to the bone marrow and by birth this is where practically all hematopoiesis takes place. 2. In adults, hematopoiesis occurs mainly in the bone marrow of the skull, ribs, sternum, vertebral column, pelvis, and femurs. 3. Because blood cells are short-lived, they have to be continually renewed and hematopoiesis is active throughout life.

Antibody Binding characteristics:

1. As the sequence of the CDRs varies from antibody to antibody, so does the shapes and chemical selectivity of the binding pocket created by the CDRs. 2. Antibodies bind ligands whose surfaces and 3-D-chemical properties are complementary to those of the antibody binding pocket.

Major Functions of Complement Activation:

1. Bacterial lysis and killing: forming MAC unit 2. Opsonization for phagocytosis: C3b, C4b 3. Regulation of inflammatory responses: C3a, C4a, and C5a 4. Neutralizing and lytic effect on viral particles: antibody-mediated 5. Immune cell-mediated cytotoxicity: antibody-mediated

Adaptive Immunity

1. Better understood than innate immunity 2. Adaptive immunity is initiated in secondary lymphoid tissues 2. Also called acquired immunity or protective immunity 3. The first time that an adaptive immune response is made to a given pathogen is called the primary immune response. The second and subsequent times that an adaptive immune response is made, and when immunological memory applies, it is called a secondary immune response.

Various functions and roles of complement components:

1. Binding to antigen: antibody complexes and pathogen surfaces 2. Binding to mannose on bacteria 3. Activating enzymes 4. Membrane-binding proteins and opsonins 5. Peptide mediators of inflammation 6. Membrane-attack proteins 7. Complement receptors 8. Complement-regulatory proteins

Structure of Chemokine Classes:

1. C 2. CC 3. CXC 4. CX3C

Components involved in Alternative Pathway:

1. C3: binds to pathogen surface; binds B for cleavage by D; C3bBb is C3 convertase and C3b2Bb is C5 convertase 2. Factor B: Ba - small fragment of B, unknown function Bb - active enzyme of the C3 convertase (C3bBb) and C5 convertase (C3b2Bb) 3. Factor D: plasma serine protease, cleaves B when it is bound to C3b to Ba and Bb 4. Factor P (properdin): plasma protein with affinity for the C3bBb convertase on bacterial cells

Antibody Pocket Formed Determines Antigen It Will Bind:

1. CDRs vary in the sequence of amino acid composition (Meaning - amino acid substitution at each site in the CDR region can vary) 2. There are approximately 20 aa that commonly appear in proteins and each differs slightly in properties with regard to the size, shape and chemical properties of their "side chain" group (R) 3. The linear combination of these aa linked through amide bonds and with their unique side chains determines the shape and chemical binding selectivity of the region. *Acidic amino acids - Glutamic acid, aspartic acid *Basic amino acids - Lysine, arginine, histidine *Small neutral amino acids - Glycine, alanine, serine, cysteine, threonine, glutamine, asparagine *Large neutral amino acids - Phenylalanine, tyrosine, leucine, tryptophan, isoleucine, valine, and methionine

Phagocyte

1. Cells that can specialize in the capture, engulfment, and killing of microorganisms. 2. Neutrophils are the most numerous phagocytes and the most lethal, historically called the microphage 3. Macrophages mean "large phagocyte" and are large, irregularly shaped cells characterized by an extensive cytoplasm with numerous vacuoles, often containing engulfed materials. They are the general scavenger cells of the body, phagocytosing and disposing of dead cells and cell debris as well as invading microorganisms. Unlike neutrophils, macrophages are long-lived and provide warning to other cells and orchestrate the local response to infection. Macrophages resident in the infected tissues are generally the first cell to sense an invading microorganism. They secrete the cytokines that recruit neutrophils and other leukocytes into the infected area. *Monocytes are the mobile progenitors of sedentary tissue cells called macrophages. Monocytes are leukocytes that circulate in the blood. They are distinguished from the granulocytes by being bigger, by having a distinctive indented nucleus, and by looking all the same, hence the name monocyte. Monocytes travel in the blood to tissues, where they mature into macrophages and take up residence there.

Examples of Cytokines:

1. Class I: IL-6, Il-4, IL-2 2. Class II: IFN-alpha, beta, and gamma

Pathogen-Associated Molecular Patterns (PAMPs):

1. Conserved molecular structures on pathogens 2. Produced only by microbes and not by host cells 3. Essential for pathogen metabolism and survival 4. Invariant between microorganisms of a given class

Adjuvant - Mechanism of Action

1. Convert soluble proteins into particulate matter 2. Add a bacterial constituent to boost innate immune system involvement

Dendritic cells

1. Dendritic cells are resident in the body's tissues and have a distinctive star-shaped morphology. Although they have many properties in common with macrophages, their unique function is to act as cellular messengers that are sent to call up an adaptive immune response when it is needed. At such times, dendritic cells that reside in the infected tissue will leave the tissue with a cargo of intact and degraded pathogens and take it to one of several lymphoid organs that specialize in making adaptive immune responses. 2. Dendritic cells in the skin are immature and specialized in the uptake of pathogens and their antigens. They take up antigens at a site of wounding and infection in the skin and carry them to the draining lymph node where they differentiate into mature dendritic cells that are specialized in activating naive T cells.

Immunoglobulin Genes and Rearrangement:

1. Diversity of Ab structure -- Combination of assembly of different V-region gene segments 2. Selective somatic hypermutation of V-region DNA light chain --the V domain is encoded by two segments: * Variable (v) gene segment -- encodes the first 95-103 aa *Joining (J) gene segment -- encodes for up to 13 aa Heavy Chain -- the V domain is encoded by three gene segments: 1. v gene segment 2. J gene segment 3. Diversity (D) gene segment

Antibodies

1. Effector B cells, called plasma cells, secrete soluble forms of immunoglobulins which are known as antibodies 2. Circulate in the blood and can enter infected tissues 3. Antibodies bind to pathogens and cause their inactivation or destruction 4. The most important function of antibodies is to facilitate the engulfment and destruction of extracellular microorganisms and toxins by phagocytes. An antibody specific for one of the antigens on the pathogen surface can coat the entire surface of the antigen. Phagocytic neutrophils and macrophages have cell-surface receptors that bind to the antibody molecule at a site apart from the antigen-binding site that is attached to the pathogen. Thousands of receptors on the phagocyte will then bind to thousands of antibodies on the pathogen. A bacterium coated with antibody thus become more efficiently phagocytosed than an uncoated bacterium. The phenomenon by which a coating of antibody facilitates phagocytes is called opsonization. 4. Immunity due to antibodies and their actions is known as humoral immunity

Antigen-Antibody Binding Forces:

1. Electrostatic forces 2. Hydrogen bonds 3. Van der waals forces 4. Hydrophobic forces - hydrophobic groups interact unfavorably with water and tend to pack together to exclude water molecules. The attraction also involves van der waals forces

Outcomes of Innate Immune Response:

1. Eliminate invading pathogens and maintain normal tissue/organ functions 2. Induce inflammatory response (inflammation) 2. Initiating and directing adaptive immunity

Epitopes for Antibodies Exposed On the Surface of Pathogens

1. Epitope = antigenic determinant 2. Antigen - contains epitope(s) 3. Antibody recognizes and binds to epitope on antigen

Features of Innate Immunity:

1. Evolutionally conserved: Present in all multi-cellular organisms (even plants) 2. Preformed: Genetically inherited (i.e. derived from a preformed genetic code present in the parents) 3. No memory: Not enhanced by prior exposure 4. Limited specificity: one molecule interacts with the same components expressed by various pathogens 5. Regulatory role: Adaptive Immunity

The immune system protects against four classes of pathogens:

1. Extracellular bacteria, parasites, fungi 2. Intracellular bacteria, parasites 3. Viruses (intracellular) 4. Parasitic worms (extracellular)

Human Toll-Like Receptors:

1. First recognized in fruit fly (Drosophila). The human homologues were identified in 1997 2. Discovered in mice in 1998 as the LPS receptor, essential for mice to mount an effective immune response 3. Expressed by myelomonocytic, endothelial, and epithelial cells from various organ systems 4. Located on the cell surface or intracellularly with a common extracellular domain and a conserved cytoplasmic motif 5. Recognize PAMPs with relative specificities 6. Share common activation pathways via TIR signaling domains

Immunoglobulins

1. For B cells, the cell-surface receptors for pathogens are immunoglobulins, whereas those of T cells are known as T-cell receptors. Effector B cells, called plasma cells, secrete soluble forms of these immunoglobulins which are known as antibodies. 2. Immunoglobulins and T-cell receptors are structurally similar molecules. Many millions of different immunoglobulins and T-cell receptors are represented within the population of small lymphocytes in one human being. 3. Any molecule, virus particle, or cell that contains a structure recognized and bound by an immunoglobulin or T-cell receptor is called its corresponding antigen. Surface immunoglobulins and T-cell receptors are thus also referred to as the antigen receptors of lymphocytes. Differences in the amino acid sequences of the variable regions of immunoglobulins and T-cell receptors create a vast variety of binding sites that are specific for different antigens and thus for different pathogens.

Lymphatic System:

1. Frequent sites of infection are the connective tissues, which pathogens penetrate as a result of skin wounds. Intact pathogens, components of pathogens, and pathogen-infected dendritic cells are carried from such sites to the nearest lymph node by the lymphatics. The lymph node receiving the fluid collected at an infected site is called the draining lymph node. 2. The anatomy of the lymph node provides meeting places where lymphocytes coming from the blood encounter pathogens and their products brought from infected connective tissue. Lymphocytes leave the blood through the walls of fine capillaries in secondary lymphoid organs. Arriving lymphocytes segregate to different regions of the lymph node: T cells to the T-cell areas and B cells to B-cell areas known as lymphoid follicles. Pathogens and pathogen-laden dendritic cells from the infected tissue arrive at a lymph node in afferent lymphatic vessels. Several of these unite at the node and then leave it as a single efferent lymphatic vessel. During an infection, pathogen-specific B cells that have bound the pathogen proliferate to form a dense spherical structure called a germinal center in each follicle. A lymph node draining a site of infection increases in size as a result of the proliferation of activated lymphocytes, a phenomenon sometimes referred to as 'swollen glands.' 3. The small fraction of B and T cells bearing receptors that bind to the pathogen or its products will be stimulated to divide and differentiate into effector cells. T cells are activated by dendritic cells, whereupon some of the T cells move to the associated lymphoid follicle where they help activate the B cells to become plasma cells. Other effector T cells and the antibodies secreted by plasma cells are carried by efferent lymph and blood to the infected tissues. There the effector cells and molecules of adaptive immunity work with their counterparts of innate immunity to subdue the infection.

Spleen

1. From the third to the seventh month of fetal life, the spleen is the major site of hematopoiesis. *For a few months in utero the normal human spleen is hematopoietic. By the close of the fifth fetal month, the function has moved permanently into the marrow. Except for some lymphocytes and plasma cells, which continue to be contributed from the white pulp of the spleen and other lymph tissues, the production of blood cells—monocytes, granulocytes, erythrocytes, and megakaryocytes—remains entirely in the marrow. 2. The vast majority of lymphocytes are found in specialized tissues known as lymphoid tissues or lymphoid organs. Spleen is a major lymphoid organ. The other major lymphoid organs are bone marrow, thymus, adenoids, tonsils, appendix, lymph nodes, and Peyer's patches. 3. Lymphoid tissues are functionally divided into two types: Primary or central lymphoid tissues are where lymphocytes develop and mature to the stage at which they are able to respond to a pathogen. The bone marrow and the thymus are the primary lymphoid tissues. All other lymphoid tissues are known as secondary or peripheral lymphoid tissues (including the spleen). They are the sites where mature lymphocytes become stimulated to respond to invading pathogens. 4. The secondary lymphoid organs are dynamic tissues in which lymphocytes are constantly arriving from the blood and departing in the lymph. At any one time, only a very small fraction of lymphocytes are in the blood and lymph; the majority are in lymphoid organs and tissues. 5. The spleen is the lymphoid organ that serves as a filter for the blood, so in that sense it provides adaptive immunity to blood infections. One purpose of the filtration is to remove damaged or senescent red cells; the second function is that of a secondary lymphoid organ that defends the body against blood-borne pathogens. Any micoorganism in the blood is a potential pathogen and source of dangerous systemic infection. Microorganisms and microbial products in the blood are taken up by splenic macrophages and dendritic cells, which then stimulate the B and T cells that arrive in the spleen from the blood. The white pulp in the spleen is where white blood cells gather to provide adaptive immunity. The organization and functions of splenic white pulp are similar to those of the lymph node, the main difference being that both pathogens and lymphocytes enter and leave the spleen in the blood.

Immunogenicity of Proteins

1. Generally only proteins alone can elicit immune responses, usually with help from adjuvants 2. Proteins engage T cells which help B cells make antibodies 3. Proteins engage T cells because T cells recognize antigens as peptide fragments bound to major histocompatibility complex (MHC) molecules 4. Immunological Memory is produced as a result of an intial Primary Immunization (know as "priming") 5. Subsequent immunization (exposure) leads to increasingly stronger response

Clinical Significance:

1. Genetic complement deficiencies, such as C1, C4, or C3 results in increased frequency of SLE (systemic lupus erythematous), immune complex disease, or pyogenic infection 2. Contributing to local tissue damage in many infectious diseases, autoimmune diseases, and immune complex deposition diseases 3. Participated in pathophysiological processes such as septicemia in bacterial infection, shock in DIC (disseminated intravescular coagulation), and ARDS (acute respiratory distress syndrome) 4. Reduction in various diseases such as autoimmune diseases (SLE), chronic hepatitis, and glomerulonephritis, etc.

Receptors for Pathogen Recognition:

1. Goal: To discriminate a large number of potential pathogens from self, with the use of a restricted number of receptors 2. Definition: A family of cell surface molecules or secreted proteins that recognize conserved molecule structures on microbial pathogens (PAMPs) that are not present in higher eukaryotes

Five Features of Inflammation:

1. Heat 2. Redness 3. Swelling 4. Pain 5. Loss of Function

Cytokines

1. Helper T cells secrete cytokines that help other cells of the immune system become fully activated effector cells. 2. As the bacteria begin to divide and set up the infection, cells and proteins in the damaged tissue sense the presence of bacteria, and the cells send out soluble proteins called cytokines that interact with other cells to trigger the innate immune response. The overall effect of the innate immune response is to induce a state of inflammation in the infected tissue. 2. Cytokines induce the local dilation of blood capilarries, which by increasing the blood flow causes the skin to warm and redden. Vasodilation makes the endothelium permeable and increases the leakage of blood plasma into the connective tissue. Expansion of the local fluid volume causes edema or swelling, putting pressure on nerve endings and causing pain. 3. Cytokines also change the adhesive properties of the vascular endothelium, inviting white blood cells to attach to it and move from the blood into the inflamed tissue. Infiltration of cells into the inflamed tissue increases the swelling, and some of the molecules they release contribute to the pain.

Lymphoblast

1. Immature white blood cells that gives rise to B lymphocytes, T lymphocytes, and NK cell lymphocytes 2. A lymphocyte that has gotten larger after being stimulated by an antigen.

Immunogen and Antigen

1. Immunization (with immunogen) leads to specific antibody production 2. Anti-viral antibodies can bind to viral antigen on cell surface An immunogen refers to a molecule that is capable of eliciting an immune response by an organism's immune system, whereas an antigen refers to a molecule that is capable of binding to the product of that immune response. So, an immunogen is necessarily an antigen, but an antigen may not necessarily be an immunogen (e.g. hapten which are not immunogenic). Throughout this site, the term antigen will be used since it refers directly to the molecule that binds to the product of the immune response - the antibody

Basic Properties of Host Receptor:

1. Inherited and essential for host survival 2. Can be secreted, expressed on cell surface, or resident in intracellular component 3. Interaction with microbial components with relative specificity *Important: Receptors with specificity for pathogen molecules recognize patterns of repeated structural motifs 4. Cable of activating pro-inflammatory pathways 5. Related to many human diseases such as bacterial sepsis * Recognition of microbial derived ligands by pattern recognition receptors (PRRs) is a key step in initiating pro-inflammatory signalling pathways. Examples of PRRs linked to the aetiology of sepsis include Toll-like, C-type lectin, RIG-1-like and also Nod-like receptors, which are involved in the formation of the inflammasome, crucial for the maturation of some pro-inflammatory cytokines.

Interfereon: The first line defense against viral infection

1. Interferon (IFNs) are natural cell-signaling proteins produced by the cells of the immune system in response to various challenges, particularly virus with double stranded RNA, a key indicator of viral infection. 2. Interferons assist the immune response by inhibiting viral replication within host cells, activating NK cells and macrophages, increasing antigen presentation to T cells, and increasing the resistance of host cells to viral infection 3. There are 2 known classes of interferon: type I (IFN-alpha, IFN-beta) and type II (IFN-gamma). All are important in fighting viral infections.

Mast cell

1. Is resident in the connective tissue. The activation and degranulation of mast cells at sites of infection make major contributions to inflammation. Has an unknown precursor.

Natural Killer (NK) Cells summary:

1. Killer lymphocytes of the innate immune response: ~ 10% of total lymphocytes (Non-T, Non-B, Large granular lymphocytes) 2. Express CD16 and/or CD56, but not CD3 3. Particularly active state: found in blood, spleen, lung, liver, and GI tract and other tissues and organs 4. Provide innate immunity against intracellular infection 5. Play the vital role in viral infection 6. Move into infected tissues upon pathogen invasion

Chemokines

1. Known as chemotactic cytokines 2. A family of small cytokines secreted by various types of cells 3. Major function: to induce and direct chemotaxis in nearby responsive cells 4. Proteins are classified as chemokines according to shared structural characteristics (small size, approximately 8-10 kDa), and the presence of four cysteine residues in conserved locations that are key to forming their 3-dimensional shape. 5. The receptors are G-protein-coupled receptors with seven transmembrane domains known as chemokine receptors

Common PAMPs:

1. LPS: gram-negative bacteria 2. Peptidoglycan, Lipoteichoic acid: gram-positive bacteria 3. Lipoarabinomannan: mycobacterium 4. Bacterial lipoproteins 5. Bacterial unmethylated DNA 6. Yeast wall mannans 7. Viral double-stranded RNA

Lymph Nodes

1. Lymph nodes are secondary or peripheral lymphoid tissues. They lie at the junctions of an anastomosing network of lymphatic vessels called the lymphatics, which originate in the connective tissues throughout the body and collect the plasma that continually leaks out of blood vessels and forms the extracellular fluid. The lymphatics eventually return this fluid, called lymph, to the blood, chiefly via the thoracic duct, which empties into the left subclavian vein in the neck. Unlike the blood, the lymph is not driven by a dedicated pump and its flow is comparatively sluggish. 2. A unique property of mature B and T cells, which distinguishes them from other blood cells, is that they move through the body in both blood and lymph. Lymphocytes are the only cell type present in lymph in any numbers. When small lymphocytes leave the primary lymphoid tissues in which they have developed, they enter the bloodstream. When they reach the blood capillaries that invest a lymph node, or other secondary lymphoid tissues, small lymphocytes are able to leave the blood and enter the lymph node proper. If a lymphocyte becomes activated by a pathogen it remains in the lymph node; otherwise it will spend some time there and then leave in the efferent lymph and eventually be returned to the blood. This means that the population of lymphocytes within a node is in a continual state of flux, with new lymphocytes entering from the blood while others leave in the efferent lymph. This pattern of movement between blood and lymph is termed lymphocyte recirculation. It allows the lymphocyte population to continually survey the secondary lymphoid organs for evidence of infection. *An exception to this pattern is the spleen, which has no connection to the lymphatic system. Lymphocytes both enter and leave the spleen in the blood. 3. The secondary lymphoid organs such as lymph nodes are dynamic tissues in which lymphocytes are constantly arriving from the blood and departing in the lymph. At any one time, only a very small fraction of lymphocytes are in the blood and lymph; the majority are in lymphoid organs and tissues.

Features of MB-Lectin Pathway:

1. MBL initiates the complement enzymatic reactions 2. MASP-1/2 act as C1 3. MBL is a pattern recognition protein 4. MBL recognizes mannose residues 5. MBL pathway forms classical C3 and C45 convertase 6. Forming membrane attack unit 7. MBL pathway produces C3a, C4a, and C5a 8. MBL binding initiate macrophage phagocytosis (opsonin: complement fragments that coat bacteria and facilitate phagocytosis. The process is known as opsonization)

Karl Landsteiner and modern view of Antibody:

1. Major Findings: * Immunized animals with synthetic compounds that could bind to antibodies * Many of the small synthesized molecules -- such as phenyl arsonates and nitrophenyls -- did not elicit immune responses by themselves. These compounds required coupling to a carrier molecule to generate antibody production 2. The small drugs were termed "haptens" by Landsteiner from the Greek Haptein -- to fasten 3. Anti-hapten antibodies often mediate medically important allergic reactions to drugs, such as penicillin

Neutrophil Granulocytes

1. Most abundant of the granulocytes and of all white blood cells 2. Specialized in the capture, engulfment, and killing of microorganisms. Cells with this function are called phagocytes, of which neutrophils are the most numerous and most lethal. 3. Neutrophils are effector cells of innate immunity that are rapidly mobilized to enter sites of infection and can work in the anaerobic conditions that often prevail in damaged tissue. 4. They are short-lived and die at the site of infection, forming pus, the stuff of pimples and boils *The granulocytes have prominent cytoplasmic granules containing reactive substances that kill microorganisms and enhance inflammation. Because granulocytes have irregularly shaped nuclei with two to five lobes, they are also called polymorphonuclear leukocytes.

Biological Features of Cytokines:

1. Most cytokines are simple polypeptides or glycoproteins produced by various types of cells 2. Production is regulated by inducing stimuli at the level of transcription and translation 3. Production is transient and constitutive production of is uncommon. The action radium is usually short. 4. Cytokines act by binding to high-affinity cell surface receptor (Kd = 10-9 to 10-12 M). Cytokine action: autocrine, paracrine, and endocrine 5. The function of individual cytokines are redundant 6. Cytokines induces the production of additional cytokines, resulting in a cytokine cascade.

Adjuvants Enhance Immunogenicity:

1. Most proteins are poorly immunogenic or non-immunogenic when administered by themselves 2. Strong adaptive responses generally require protein delivered with an adjuvant 3. Adjuvant is a substance that enhances the immunogenicity of molecules

Three enzymes involved in respiratory burst:

1. NADPH oxidase catalyzes formation of the superoxide O2- 2. Superoxide dismutase catalyzes formation of hydrogen peroxide (H2O2) from superoxide 3. Catalase catalyzes the breakdown of hydrogen peroxide to water and oxygen

Plasma cells

1. On encountering the antigen recognized by their antigen receptors, B cells differentiate into antibody-producing plasma cells, and this is their only effector function. Antigen-activated effector T cells, however, undertake a variety of functions within the immune response. One subset of helper T cells remain in the lymph node and stimulate the division and differentiation of pathogen-specific B cells to become antibody-secreting plasma cells. 2. Plasma cells move to the medulla of the lymph node, where they secrete pathogen-specific antibodies, which are taken to the site of infection by the efferent lymph and subsequently the blood. Some plasma cells leave the lymph node and travel via the efferent lymph and the blood to the bone marrow, where they continue to secrete antibodies. 2. Effector T cells are subdivided into cytotoxic T cells and helper T cells. Cytotoxic T cells kill cells that are infected with viruses or with certain bacteria that live inside human cells. They have similar effector functions to NK cells, but NK cells provide these functions during the innate immune response and cytotoxic T cells during the adaptive immune response.

Effector cells

1. Once the pathogen has been recognized, the second part of the innate immune response involves the recruitment of destructive effector mechanisms that kill and eliminate the pathogen. The effector mechanisms are provided by effector cells of various types that engulf bacteria, kill virus-infected cells, or attack protozoan parasites, and a battery of serum proteins called complement that help the effector cells by making pathogens with molecular flags but also attack pathogens in their own right. 3. A typical immune defense (pg. 9): *Bacterial cell surface induces cleavage and activation of complement *One complement fragment covalently bonds to the bacterium, the other fragment attracts an effector cell *The complement receptor on the effector cell binds to the complement fragment on the bacterium *The effector cell engulfs the bacterium by phagocytosis, kills it, and breaks it down

The Relevance of Antibodies to Human Health:

1. Polyclonal Antibody Preparations: *Diagnostic assays *Therapy-Gamma-guard- pooled anti-sera from once infected individuals give short-term protection against various pathogens 2. Monoclonal Antibodies: *Diagnostic assays *Infectious diseases = RSV, Staphylococcus infections, etc. *Cancer- Breast, lymphomas, colon, etc. *Transplantation - tolerance

What is the relevance of Immunology to Pharmacists?

1. Protects the body against infection 2. Immunological responses are important in many diseases: *Infections *AIDS *Autoimmune disorders (e.g. Multiple Sclerosis, Rheumatoid Arthritis) *End-stage organ failure (heart, kidney, liver) due to inflammation *Transplantation *Cancer 3. Many drugs target immune system: *Block inflammation *Inhibit immune system (e.g. transplants) *Boost immune system (e.g. growth factors) 4. Vaccines - Pharmacists can give immunizations 5. Immune system components used in many diagnostic tests

Why do Vaccines Work?

1. Purpose of vaccine is to educate immune system before it encounters pathogen 2. Vaccine generates immune response resulting in production of effector molecules that includes antibodies 3. Antibodies are good because they bind specifically to antigen (pathogen) destroying and removing it from body 4. Higher concentration (titer) of antibody against antigen (pathogen) offer better protection 5. Levels (titers) of antibodies can be increased with subsequent immunizations 6. Antibody affinity for antigen is enhanced by further immunization with same vaccine

Three major functions of Neutrophils:

1. Recognition of bacteria using cell surface receptors 2. Phagocytosis of invading pathogens and dead cells 3. Production of cytokines to initiate inflammation

Three major functions of Macrophages:

1. Recognition of pathogens by PRR receptors 2. Release of soluble mediators (cytokines) 3. Phagocytosis

Summary of Macrophage activities:

1. Reside and recruited to sites of invading microbes and inflammation 2. Recognize pathogens by means of pattern recognition receptor (PRR) and opsonin receptors 3. Phagocytose and kill invading organisms 4. Release soluble mediators (complement products, cytokines, proteases, and prostaglandins) to mediate effector activity 5. Elaborate oxygen and nitrogen radicals 6. Aid wound repair via coagulation, epetheliazation, matrix generation and tissue remodeling 7. Direct the adaptive immune response

Memory

1. Some of the lymphocytes selected during an adaptive immune response persist in the body and provide long-term immunological memory of the pathogen. These memory cells allow subsequent encounters with the same pathogen to elicit a stronger and faster adaptive immune response, which terminates infection with minimal illness. 2. The secondary immune response happens because of immunological memory. That's why the vaccination is done because the purpose is to induce immunological memory to a pathogen so that subsequent infection with the pathogen elicits a strong, fast-acting adaptive response.

Characteristics of the Receptor Molecules of the Innate and Adaptive Immune Systems:

1. Specificity inherited in the genome: Innate - yes, Adaptive - no 2. Expressed by all cells of a particular type (e.g. macrophages): Innate - yes, Adaptive - no 3. Triggers immediate response: Innate - yes, Adaptive - no 4. Recognizes broad classes of pathogens: Innate - yes, Adaptive - no 5. Interacts with a range of molecular structures of a given type: Innate - yes, Adaptive - no 6. Encoded in multiple gene segments: Innate- no, Adaptive - yes 7. Requires gene rearrangement: Innate - no, Adaptive - yes 8. Clonal distribution: Innate - no, Adaptive - yes 9. Able to discriminate between even closely related molecular structures: Innate - no, Adaptive - yes

Binding Specificity of Antibodies

1. Surface Immunoglobulin and secreted antibodies bind to antigens 2. Antigens can be almost anything (proteins, lipids, carbohydrates, nucleic acids, even small organic compounds) 3. Native protein - Antibody binds Denatured protein - Antibody does NOT bind! *Why is this? Specificity!! Because in native, intact molecules, certain determinants may be internally folded and inaccessible to antibody. Denaturation or proteolytic cleavage (e.g. at sites of tissue injury) can conceivably expose previously occult epitopes and allow antibody binding and subsequent effector activity

Biochemical Features:

1. Synthesized mainly by cells in the liver, spleen, lung, bone marrow and macrophages 2. ~10% of serum globulin 3. Sensitive to heat treatment (inactivated by 56 degrees Celsius, 30 minutes) 4. Amounts are relatively stable, not affected by antigen stimulation, adaptive immunity, but by certain diseases 5. Exist in pro-form (inactive)

White Blood Cells

1. The cells of the immune system are principally the white blood cells or leukocytes, and the tissue cells related to them. 2. Along with the other blood cells, they are continually being generated by the body in the developmental process known was hematopoiesis. 3. Leukocytes derive from a common progenitor called the pluripotent hematopoietic stem cell, which also gives rise to red blood cells (erythrocytes) and megakaryocytes. All these cell types, together with their precursor cells, are collectively called hematopoietic cells. 4. Hematopoietic stem cells can divide to give further hematopoietic stem cells, a process called self-renewal. Daughter cells can alternatively become more mature stem cells that commit to one of three cell lineages: the erythroid, myeloid, and lymphoid lineages. 5. The erythroid progenitor gives rise to the erythroid lineage of blood cells, which are the oxygen-carrying erythrocytes and the platelet-producing megakaryocytes. 6. The myeloid progenitor gives rise to the myeloid lineage of cells. The word myeloid means "of the bone marrow." There are at least six cell types produced by the myeloid progenitor cell. These are: the three types of granulocyte (neutrophil, eosinophil, and basophil); the mast cell, which takes up residence in connective and mucosal tissues; the circulating monocyte; which gives rise to the macrophages resident in tissues; and the dendritic cell. 7. The lymphoid progenitor gives rise to the lymphoid lineage of white blood cells. The large granular lymphocytes are effector cells of innate immunity called natural killer cells or NK cells. NK cells are important in the defense against viral infections. They enter infected tissues, where they prevent the spread of infection by killing virus-infected cells and secreting cytokines that impede viral replication in infected cells. The small lymphocytes are the cells responsible for the adaptive immune response. They are small because they circulate in a quiescent and immature form that is functionally inactive. Recognition of a pathogen by small lymphocytes drives a process of lymphocyte selection, growth, and differentiation that after 1-2 weeks produces a powerful response tailored to the invading organism.

Defensins:

1. The defensin molecule is amphipathic in character, meaning that its surface has both hydrophobic and hydrophilic regions. This property allows defensin molecules to penetrate microbial membranes and disrupt their integrity—the mechanism by which they destroy bacteria, fungi, and enveloped viruses. 2. Defensins are constitutively secreted (in constitutive secretion proteins are secreted from a cell continuously, regardless of external factors or signals) at mucosal surfaces, where they protect the epithelium from infection by enteric pathogens and maintain the normal gut flora and microbiota. The α-defensins HD5 and HD6 (also called cryptdins) are secreted by Paneth cells, specialized epithelial cells of the small intestine that are situated at the base of the crypts between the intestinal villi. In addition, Paneth cells secrete other antimicrobial agents, including lysozyme, that contribute to innate immunity. The β-defensins are produced by a broad range of epithelial cells, in particular those of the skin, the respiratory tract, and the urogenital tract. 3. Defensins are also produced by neutrophils, Defensins are also produced by neutrophils, the predominant phagocytes of innate immunity. Neutrophils are recruited to sites of infection throughout the body as a component of the induced innate immune response. The defensins are packaged in the neutrophil's granules and used to kill pathogens that have been phagocytosed by the neutrophil. To prevent defensins from disrupting human cells, they are synthesized as part of longer, inactive polypeptides that are then cleaved to release the active fragment. 4. Individuals differ in their number of copies of a defensin gene: from 2 to 14 copies for α-defensin genes and from 2 to 12 copies for β-defensin genes. The gene copy number determines the amount of protein made, with the result that the arsenal of defensins that a neutrophil carries varies from one person to another. Variation in the amino acid sequences of defensins correlates with their different skills in killing microorganisms. For example, the β-defensin HBD2 specializes in killing Gram-negative bacteria, whereas its relative HBD3 kills Gram-positive and Gram-negative bacteria. Defensins can also differ in the epithelial surfaces they protect: the α-defensin HD5 is secreted in the female urogenital tract, whereas β-defensin HBD1 is secreted in the respiratory tract as well as the urogenital tract.

Lymphocyte

1. The innate immune response works to slow the spread of infection while it calls upon white blood cells called lymphocytes that increase the power and focus of the immune response. Their contribution to defense is the adaptive immune response. 2. While the receptors for innate immunity comprise many different types and each recognize features shared by groups of pathogens and are not specific for a particular pathogen, lymphocytes recognize pathogens by using cell-surface receptors of just one molecular type. These proteins can be made in billions of different versions, each capable of binding a different ligand. This means that the adaptive immune response can be made specific for a particular pathogen by using only those lymphocyte receptors that bind to the infecting pathogen. The lymphocyte receptors are not encoded by conventional genes but by genes that are cut, spliced, and modified during lymphocyte development. In this way, each lymphocyte is programmed to make one variant of the basic receptor type, but among the population of lymphocytes are represented billions of different receptor variants. 3. During infection, only those lymphocytes bearing receptors that recognize the infecting pathogen are selected to participate in the adaptive response. 4. Some of the lymphocytes selected during an adaptive immune response persist in the body and provide long-term immunological memory of the pathogen. These memory cells allow subsequent encounters with the same pathogen to elicit a stronger and faster adaptive immune response, which terminates infection with minimal illness. 5. The lymphoid progenitor gives rise to the lymphoid lineage of white blood cells. The large granular lymphocytes are effector cells of innate immunity called natural killer cells or NK cells. NK cells are important in the defense against viral infections. They enter infected tissues, where they prevent the spread of infection by killing virus-infected cells and secreting cytokines that impede viral replication in infected cells. The small lymphocytes are the cells responsible for the adaptive immune response. They are small because they circulate in a quiescent and immature form that is functionally inactive. Recognition of a pathogen by small lymphocytes drives a process of lymphocyte selection, growth, and differentiation that after 1-2 weeks produces a powerful response tailored to the invading organism. 6. The small lymphocytes, although morphologically indistinguishable from each other, comprise several sub-lineages that are distinguished by their cell-surface receptors and the functions they perform. The most important difference is between B lymphocytes and T lymphocytes, also called B cells and T cells.

Basophil

1. The least abundant granulocyte 2. Also involved in regulating the immune response to parasites, but is so rare that relatively little is known of its contribution to immune defense 3. Granules contain acidic substances that bind basic stains such as hematoxylin

Various Barriers Prevent Pathogens from Crossing Epithelia and Colonizing Tisssues:

1. The outside surface of the human body comprises the skin and the mucosal epithelia that line the digestive, respiratory, and urogenital tracts. These epithelial tissues provide effective physical and chemical barriers that prevent pathogens in the external environment from gaining access to the internal tissues and organs. 2. Further deterring pathogen invasion are the large communities of commensal microorganisms that colonize the skin and mucosal surfaces of healthy individuals. To expand its numbers and become an infection, any aspiring pathogen must compete successfully with the resident commensals for nutrients and space. Pathogenic strains are not always competitive, as exemplified by Clostridium difficile infections, which can only take hold in unhealthy individuals whose populations of commensal bacteria have been decimated by one or more courses of antibiotics.

Inflammation

1. The overall effect of the innate immune response is to induce a state of inflammation in the infected tissue 2. Characterized by: heat, pain, redness, and swelling. These symptoms are not due to the infection itself but to the immune system's response to the pathogen 3. Cytokines induce the local dilation of blood capillaries, which by increasing the blood flow causes the skin to warm and redden. 4. Vascular dilation (vasodilation) introduces gaps between the cells of the endothelium (the thin epithelial layer lining the interior of blood vessels), making the endothelium permeable and increases the leakage of blood plasma into the connective tissue. 5. Expansion of the local fluid volume causes edema or swelling, putting pressure on nerve endings and causing pain. 6. Cytokines also change the adhesive properties of the vascular endothelium, inviting white blood cells to attach to it and move from the blood into the inflamed tissue. WBCs that are usually present in inflamed tissues and release substances that contribute to the inflammation are called inflammatory cells. Infiltration of cells into the inflamed tissue increases the swelling, and some of the molecules they release contribute to the pain.

Structure of Antigen Binding Pocket:

1. The rest of the V(h) and V(l) domains show less variability and the regions between the hypervariable regions are termed "Framework regions" denoted by - FR1, FR2, FR3, and FR4. 2. Framework regions = Beta sheets that provide the structural framework of the domain 3. Hypervariable regions make up the 3 loops at one of the folded molecule which determines the "surface" of protein at that area

Surface Immunoglobulin

1. The surface immunoglobulin is expressed on the surface of B cells 2. The transmembrane region is used to anchor the surface immunoglobulin structure to the B cell surface

Features of NK Cell:

1. Two functions: a) Cell cytotoxicity against virally infected cells and b) Cytokine production 2. Express activating and/or inhibitory receptors for class I MHC antigens 3. Activated by cytokines: IFN-alpha, IFN-Beta 4. Major cytokine produced: IFN-gamma (activating macrophages) 5. Mechanisms of killing: perforin, granzyme, and CD16-mediated cytotoxicity (ADCC) 6. Function is mainly inhibited by IL-10

Pairing of Heavy and Light Chains to form Antigen Binding Pocket:

1. VH and VL domains pair in the antibody molecule bringing the HV loops from each domain together forming a single "HYPERVARIABLE SITE" 2. This site is present at the tip of the antibody molecule and is termed - "Antigen-binding site" or "Antibody-combining site" 3. The hypervariable loops from each of the VH and VL domains at the variable tip of the antibody molecule combine to form a single binding site for antigen. (They do this by forming a POCKET or CAVITY of surface complementarity to the antigen) 4. Hypervariable regions = Complementarity determining regions 5. Abbreviated as CDR1, CDR2, and CDR3

Chemokines

A family of about 40 chemoattractant cytokines. Chemokynes serve to direct the flow of leukocyte traffic throughout the body.

Complement System:

A large group of plasma proteins, upon binding and enzymatic activation, promote microbial destruction, cellular phagocytosis, inflammatory responses, and the cytolytic attack on pathogen and pathogen-infected cells

Hapten

A molecule that reacts with specific antibody but is not immunogenic by itself. They are low molecular weight antigens that cannot activate T cell or B cells due to its inability to bind to MHC proteins. Haptens are immunogenic upon binding covalently to a carrier protein

Antigen

A substance that binds to a specific antibody

Immunogen

A subtance that elicits an immune response. Activates T cells/B cells/other cells

Specialization

Allowing different responses to different types of infections or challenges

Antimicrobial peptides

Antimicrobial peptides are soluble effector molecules of innate immunity that kill a wide range of pathogens. The major family of human antimicrobial peptides is the defensins.

Complement:

As soon as a pathogen penetrates an epithelial barrier and starts to live in a human tissue, the defense mechanisms of innate immunity are brought into play. One of the first weapons to fire is a system of soluble proteins made constitutively by the liver and present in the blood, lymph, and extracellular fluids. These plasma proteins are collectively known as the complement system or just complement. Complement coats the surface of bacteria and extracellular virus particles and makes them more easily phagocytosed Without such a coating, many bacteria resist phagocytosis, especially those that are enclosed in thick polysaccharide capsules. Many complement components are proteolytic enzymes, or proteases, that circulate in functionally inactive forms known as zymogens. Infection triggers complement activation, which proceeds by a series, or cascade, of enzymatic reactions, in which each protease cleaves and activates the next protease in the pathway. Each enzyme is highly specific for the complement component it cleaves, and cleavage is usually at a single site. Although more than 30 proteins make up the complement system, complement component 3 (C3) is by far the most important. People lacking other complement components have relatively minor immunodeficiencies, but people lacking C3 are prone to successive severe infections. When the complement system is activated by infection it leads to the cleavage of C3 into a small C3a fragment and a large C3b fragment. In the process, some of the C3b fragments become covalently bound to the pathogen's surface. This attachment of C3b to pathogen surfaces is the essential function of the complement system; it is called complement fixation because C3b becomes firmly fixed to the pathogen. The bound C3b tags the pathogen for destruction by phagocytes and can also organize the formation of protein complexes that damage the pathogen's membrane. The soluble C3a fragment also contributes to the body's defenses by acting as a chemo-attractant to recruit effector cells, including phagocytes, from the blood to the site of infection.

Common Human PRRs:

CD14 Beta1-integrins CD11/CD18 C-type lectin (Mannose-binding lectin) Scavenger receptors Complement receptors (CR1/CD35) Toll-like receptors (TLR-2, 4, etc.)

Absorption

Cross-reacting antibodies can be removed by absorption with cross reactive antigen, leaving behind only antibodies that react to the specific immunogen

Specificity

Differences in the amino acid sequences of the variable regions of immunoglobulins and T-cell receptors create a vast variety of binding sites that are specific for different antigens and thus for different pathogens. A consequence of this specificity is that adaptive immune response made against one pathogen provides no immunity to another.

Regulation of Inflammation by Complement Cleavage Products (Anaphylatoxins)

During complement activation, C3 and C5 are each cleaved into two fragments, of which the larger (C3b and C5b) continue the pathway of complement activation. The smaller soluble C3a and C5a fragments are also physiologically active, increasing inflammation at the site of complement activation through binding to receptors on several cell types. Inflammation is a major consequence of the innate immune response to infection, which is also called the inflammatory response. In some circumstances, the C3a and C5a fragments induce anaphylactic shock, an acute inflammatory reaction that occurs simultaneously in tissues throughout the body; they are therefore referred to as anaphylatoxins. Of the anaphylatoxins, C5a is more stable and more potent than C3a. Phagocytes, endothelial cells, and mast cells have specific receptors for C5a and C3a. The two receptors are related and of a type that is embedded in the cell membrane and signals through the activation of a guanine-nucleotide-binding protein. The anaphylatoxins induce the contraction of smooth muscle and the degranulation of mast cells and basophils, with the consequent release of histamine and other vasoactive substances that increase capillary permeability. They also have direct vasoactive effects on local blood vessels, increasing blood flow and vascular permeability. These changes make it easier for plasma proteins and cells to pass out of the blood into the site of an infection (Figure 2.15). C5a acts directly on neutrophils and monocytes to increase their adherence to blood vessel walls, and serves as a chemoattractant to direct their migration toward sites of complement fixation. It also increases the phagocytic capacity of these cells, as well as raising the expression of CR1 and CR3 on their surfaces. In these ways, the anaphylatoxins act in concert with other complement components to speed the destruction of pathogens by phagocytes.

Clonal Expansion

During infection, only those lymphocytes bearing receptors that recognize the infecting pathogen are selected to participate in the adaptive response. These then proliferate and differentiate to produce large numbers of effector cells specific for that pathogen. These processes, which select the small subset of pathogen-specific lymphocytes for proliferation and differentiation into effector lymphocytes, are called clonal selection and clonal expansion, respectively. Because these processes take time, the benefit of an adaptive immune response only begins to be felt about a week after the infection has started.

Regulating C1 by C1 inhibitor:

Figure 13.14 C1INH permanently inhibits C1r and C1s. The inactivation of C1r is shown here. C1s is similarly inhibited. C1INH is a member of the serpin family of protease inhibitors. They act as pseudosubstrates. When they are cleaved by the protease, they form a permanent covalent linkage to the protease, which prevents it from releasing the pseudosubstrate and cleaving again. C1INH deficiency causes the syndrome hereditary angioedema.

CD59 protects healthy cells from MAC attacking:

Figure 2.14 CD59 prevents assembly of the membrane attack complex on human cells. Left panel: the formation of a pore by the membrane attack complex (MAC) on a pathogenic microorganism. Right panel: how the human cell-surface protein CD59 prevents pore formation on human cells. By binding to the C5b678 complex, CD59 prevents the polymerization of C9 in the membrane to form a pore.

Alternative Pathway:

Figure 2.3 Complement activation results in covalent attachment of C3b to a pathogen's surface. The key event in complement activation in response to a pathogen is the proteolytic cleavage of complement fragment C3. This cleavage produces a large C3b fragment and a small C3a fragment. C3b is chemically reactive and becomes covalently attached, or fixed, to the pathogen's surface, thereby marking the pathogen as dangerous. C3a recruits phagocytic cells to the site of infection.

Toll-like Receptor -Mediated Inflammatory Cytokine

Figure 3.7 Sensing of LPS by TLR4 on macrophages leads to activation of the transcription factor NFκB and the synthesis of inflammatory cytokines. First panel: LPS is detected by the complex of TLR4, CD14, and MD2 on the macrophage surface. Second panel: the activated receptor binds the adaptor protein MyD88, which binds the protein kinase IRAK4. IRAK4 binds and phosphorylates the adaptor TRAF6, which leads via a kinase cascade to the activation of IKK. Third panel: in the absence of a signal, the transcription factor NFκB is bound by its inhibitor, IκB, which prevents it from entering the nucleus. In the presence of a signal, activated IKK phosphorylates IκB, which induces the release of NFκB from the complex; IκB is degraded. NFκB then enters the nucleus, where it activates genes encoding inflammatory cytokines. Fourth panel: cytokines are synthesized from cytokine mRNA in the cytoplasm and secreted via the endoplasmic reticulum (ER). This MyD88-NFκB pathway is also stimulated by the receptors for cytokines IL-1 and IL-18.

Antibody Structure:

Figure 4.2 The immunoglobulin G (IgG) molecule. As shown in the top panel, each IgG molecule consists of two identical heavy chains (green) and two identical light chains (yellow). Carbohydrate (turquoise) is attached to the heavy chains. The lower panel shows the location of the variable (V) and constant (C) regions in the IgG molecule. The amino-terminal regions (red) of the heavy and light chains are variable in sequence from one IgG molecule to another; the remaining regions are constant in sequence (blue). In IgG a flexible hinge region is located between the two arms and the stem of the Y. Each arm of the Y is made up of a complete light chain paired with the amino-terminal part of a heavy chain, covalently linked by a disulfide bond. The stem of the Y consists of the paired carboxy-terminal portions of the two heavy chains. The two heavy chains are also linked to each other by disulfide bonds. The polypeptide chains of different antibodies vary greatly in amino acid sequence, and the sequence differences are concentrated in the amino-terminal region of each type of chain; this is known as the variable region or V region. This variability is the basis for the great diversity of antigen-binding specificities that antibodies have. Forming the antigen-binding site is a pair of variable regions, one from a heavy and one from a light chain. Every Y-shaped antibody molecule has two identical antigen-binding sites, one at the end of each arm. The remaining parts of the light chain and the heavy chain have limited variation in amino acid sequence between different antibodies and are therefore known as the constant regions or C regions.

Generation of Antibody Diversity

Four main processes of generating Antibody Diversity: 1. There are multiple copies of the different V -gene segments 2. Combinatorial diversity - pairing of different Light and Heavy Chains- This generates a diversity of 2.5 x 106 different antibody molecules 3. Diversity is added at the joints between different segments as a result of recombination process 4. Somatic hypermutation introduces point mutations into rearranged variable region genes

Erythrocytes facilitate the removal of immune complexes from the circulation:

Immune complexes that are covered with C3b fragments can be bound by circulating cells that express the complement receptor CR1. The most numerous of these is the erythrocyte, so that the vast majority of immune complexes become bound to the surface of red blood cells. During their circulation in the blood, erythrocytes pass through areas of the liver and the spleen where tissue macrophages remove and degrade the complexes of complement, antibody, and antigen from the erythrocyte surface while leaving the erythrocyte unscathed (Figure 9.32). Although the vital function of erythrocytes is the transport of gases between lungs and other tissues, they have also acquired functions in the defense and protection of tissues, of which the disposal of immune complexes is a crucial one.

Neutrophils (part i):

In mammalian immune cells, phagocytosis is activated by the microbe-associated molecular pattern or MAMP (commonly known as the pathogen-associated molecular pattern or PAMP). MAMP or PAMP are molecules associated with pathogens and include the bacterial lipopolysaccharide. These molecules are recognized by TLR and other PRRs to activate the innate immune response. The process is assisted by an opsonin, which is any molecule that mark the antigen and enhance the phagocytosis. Opsonin can be an antibody (IgG, IgA), a protein of the complement system (C3b, iC3b), or a mannonse-binding lectin (which initiates the formation of C3b). Opsonin coats an invading antigen thus overriding its negative charge, and also binds to phagocytic cells through specific receptors such as the Fc receptors (which bind to the Fc region on antibody). The antibody coat does not only enhance the binding of the target (antigen) to phagocytic cell, but also increase the expression of complement receptors on neighboring phagocytes.

Respiratory Burst

In the absence of infection, the antimicrobial proteins and peptides in neutrophil granules are kept inactive at low pH. After the granules fuse with the phagosome, the pH within the phagosome is raised through the first two reactions, involving the enzymes NADPH oxidase and superoxide dismutase. Each round of these reactions eliminates a hydrogen ion, thereby reducing the acidity of the phagosome. A product of the two reactions is hydrogen peroxide, which has the potential to damage human cells. (In hair salons and in the manufacture of paper, it is used as a powerful bleach.) The third reaction, involving catalase, the most efficient of all enzymes, promptly gets rid of the hydrogen peroxide produced during the neutrophil's respiratory burst, raising the pH of the phagosome and enabling activation of the antimicrobial peptides and proteins.

Factors that influence the immunogenicity of proteins:

Increase immunogenicity: 1. Large size 2. Intermediate dose (weak immunogenicity if high or low) 3. Subcutaneous > intraperitoneal > intravenous or intragastric 4. Complex 5. Particulate form (weak immunogenicity if Soluble form) 6. Denatured form (weak immunogenicity if Native form) 7. Multiple differences to self protein 8. Slow release adjuvant, bacteria 9. Effective interaction with host MHC

Phagocytosis by Neutrophils

Killing of bacteria by neutrophils involves the fusion of two types of granule and lysosomes with the phagosome. After phagocytosis (first panel), the bacterium is held in a phagosome inside the neutrophil. The neutrophil's azurophilic granules and specific granules fuse with the phagosome, releasing their contents of antimicrobial proteins and peptides (second panel). NAPDH oxidase components contributed by the specific granules enable the respiratory burst to occur, which raises the pH of the phagosome (third panel). Antimicrobial proteins and peptides are activated, and the bacterium is damaged and killed. A subsequent decrease in pH and the fusion of the phagosome with lysosomes containing acid hydrolases results in complete degradation of the bacterium (fourth panel). The neutrophil dies and is phagocytosed by a macrophage (fifth panel).

Summary of MBL:

MASP-2 is able to cleave C4 into C4a and C4b. The C4b binds to adjacent proteins and carbohydrates on the surface of the antigen. C2 then binds to the C4b and C1 cleaves C2 into C2a and C2b. The C4b2a functions as a C3 convertase that can subsequently cleave hundreds of molecules of C3 into C3a and C3b. C3b attaches antigens to phagocytes for opsonization (enhanced attachment) while C3a can promote inflammatory responses that enable body defense cells and defense chemicals to leave the blood and enter the tissues. Some of the C3b combines with the C4b and the C2a. C4b2a3b functions as a C5 convertase that cleaves molecules of C5 into C5a and C5b. C5a is the most potent complement protein triggering inflammation. C5b becomes part of the Membrane Attack Complex (MAC).

Monoclonal Antibodies:

Monoclonal antibodies represent a population of antibodies that recognize a single epitope within an antigen. They are typically produced from a single B cell of an immunized mouse, thereby generating a clonal population of antibodies, identical to one another and all recognizing the same epitope of a specific antigen. Although B cells can be used to harvest antibodies, the disadvantage is these cells have a finite lifespan and will eventually stop producing the antibody. By fusing a specific antibody-producing B cell with a myeloma cell, the limited lifespan of a B cell can be overcome. The resulting immortalized B cell-myeloma hybridoma can provide a constant supply of highly specific monoclonal antibody. Since monoclonal antibodies only recognize one epitope, they generally have low cross reactivity with non-specific antigens. Their epitope specificity, limited cross reactivity, and long term yield make monoclonal antibodies attractive for use in many biological assays and applications.

Classical Pathway:

Once C-reactive protein has bound to a bacterium it can also interact with C1, the first component of the classical pathway of complement activation. C1 has an organization and structure like that of the complex of MBL with MASP-1 and MASP-2 (Figure 3.27). In the C1 molecule, a bouquet of six flowers is formed from 18 C1q polypeptides and 2 molecules each of C1r and C1s, which are inactive serine proteases similar to MASP-1 and MASP-2. C1s becomes an active protease. It cleaves C4, leading to the covalent attachment of C4b to the pathogen surface. It also cleaves C2, leading to the formation of the classical C3 convertase C4bC2a. At this stage the classical and lectin pathways converge. Unique features in the classical pathway of complement fixation are the use of C1q in binding pathogens and the use of C1r and C1s to activate C4 and C2. At the start of infection, complement activation is mainly by the alternative pathway. As the inflammatory response develops and acute-phase proteins are produced, MBL and C-reactive protein provide increased activation of complement via the lectin and classical pathways, respectively. All three pathways contribute to innate immunity, and they work together to produce quantities of C3b fragments and C3 convertases at the pathogen surface.

Innate Immunity (from Dr. Wang):

Our body's natural protective system collectively represents the first line of defense against pathogen infection. The system acts together using various mechanical, biochemical, and biological components to prevent, contain, eliminate pathogens and associate products, and repair tissue damage.

Antibody Definition:

Soluble form of surface immunoglobulin, produced by plasma cells (a terminally differentiated B-cell)

What does specialization/specificity allow for?

Specialization allows for optimal response to different types of microbes.

Cross Reaction

The binding of an antibody with a closely related molecules in addition to the specific antigens

Antibody-Antigen Binding Forces:

The binding of antigens to antibodies is based solely on noncovalent forces—electrostatic forces, hydrogen bonds, van der Waals forces, and hydrophobic interactions. The antigen-binding sites of antibodies are unusually rich in aromatic amino acids, which can participate in many van der Waals and hydrophobic interactions. The better the general fit between the interacting surfaces of antigen and antibody, the stronger are the bonds formed by these short-range forces. Thus, several different antibodies may recognize the same epitope, but the small differences in the shapes and chemical properties of the binding sites give these antibodies different binding strengths, or affinities, for the epitope. Binding caused by van der Waals forces and hydrophobic interactions is complemented by the formation of electrostatic interactions (salt bridges) and hydrogen bonds between particular chemical groups on the antigen and particular amino acid residues of the antibody. In the immune response, the effective antibodies are those that bind tightly to an antigen and do not let go. This behavior contrasts with that of enzymes, which bind a substrate, chemically change the substrate's structure, and then release it

Thymus

The bone marrow and the thymus are the primary lymphoid tissues. B and T lymphocytes both originate from lymphoid precursors in the bone marrow, but B cells complete their maturation in the bone marrow before entering the circulation, whereas T cells leave the bone marrow at an immature stage and migrate in the blood to the thymus where they mature.

Immunization

The deliberate induction of an immune response, generally by injection of an antigen into a test animal or human subject. Immune responses to most antigens elicit the production of both specific antibodies and effector T cells

Somatic Hypermutation of Antibody:

The diversity generated during gene rearrangement is concentrated in the CDR3s of the VH and VL domains. Once a B cell has been activated by antigen, however, further diversification of the whole of the V-domain coding sequence occurs through a process of somatic hypermutation. This introduces single- nucleotide substitutions (point mutations) almost randomly and at a high rate throughout the rearranged V regions of heavy-chain and light-chain genes (Figure 4.25). The immunoglobulin constant regions are not affected, and neither are other B-cell genes. Somatic hypermutation gives rise to B cells bearing mutant immunoglobulin molecules on their surface. Some of these mutant immunoglobulins have substitutions in the antigen-binding site that increase its affinity for the antigen. B cells bearing these mutant high-affinity immunoglobulin receptors compete most effectively for binding to antigen and are preferentially selected to mature into antibody-secreting plasma cells. The changes are concentrated at positions in the heavy-chain and light-chain CDR loops that form the antigen-binding site and directly contact antigen.

Summary of Classical:

The enzyme C1 is able to cleave C4 into C4a and C4b. The C4b binds to adjacent proteins and carbohydrates on the surface of the antigen. C2 then binds to the C4b and C1 cleaves C2 into C2a and C2b. The C4b2a functions as a C3 convertase that can subsequently cleave hundreds of molecules of C3 into C3a and C3b. C3b attaches antigens to phagocytes for opsonization (enhanced attachment) while C3a can promote inflammatory responses that enable body defense cells and defense chemicals to leave the blood and enter the tissues. Some of the C3b combines with the C4b and the C2a. C4b2a3b functions as a C5 convertase that cleaves molecules of C5 into C5a and C5b. C5a is the most potent complement protein triggering inflammation. C5b becomes part of the Membrane Attack Complex (MAC).

Linear and Discontinuous Epitopes:

The epitopes of protein antigens fall into two broad groups (Figure 4.11). Epitopes composed of several successive amino acids in a protein sequence are called linear epitopes (Figure 4.11, left panel). Such epitopes can be formed by accessible loops of a protein antigen. Antibodies that recognize linear epitopes of a native protein often bind to synthetic peptides that contain the amino acid sequence of the epitope. Contrasting with linear epitopes are the discontinuous epitopes (Figure 4.11, right panel).These are formed by two or more parts of the protein antigen that are separated in the amino acid sequence but are brought together in the folded protein. Because discontinuous epitopes are dependent upon three-dimensional structure, antibodies that recognize discontinous epitopes rarely interact with synthetic peptides corresponding to the parts of the protein that form the epitope.

The skin and mucosal surfaces form barriers against infection:

The fixed defenses of skin and mucosa provide well-maintained mechanical, chemical, and microbiological barriers that prevent most pathogens from gaining access to the cells and tissues of the body. When those barriers are breached and pathogens gain entry to the body's soft tissues, the defenses of the innate immune system are brought into play.

Polyclonal Antibodies:

The immune response to an antigen generally involves the activation of multiple B-cells all of which target a specific epitope on that antigen. As a result a large number of antibodies are produced with different specificities and epitope affinities these are known as polyclonal antibodies.

Lymphatic Circulation:

The lymphatic system can be thought of as a drainage system because, as blood circulates through the body, blood plasma leaks into tissues through the thin walls of the capillaries. The portion of blood plasma that escapes is called interstitial or extracellular fluid, and it contains oxygen, glucose, amino acids, and other nutrients needed by tissue cells. Although most of this fluid seeps immediately back into the bloodstream, a percentage of it, along with the particulate matter, is left behind. The lymphatic system removes this fluid and these materials from tissues, returning them via the lymphatic vessels to the bloodstream, and thus prevents a fluid imbalance that would result in the organism's death. The fluid and proteins within the tissues begin their journey back to the bloodstream by passing into tiny lymphatic capillaries that infuse almost every tissue of the body. Only a few regions, including the epidermis of the skin, the mucous membranes, the bone marrow, and the central nervous system, are free of lymphatic capillaries, whereas regions such as the lungs, gut, genitourinary system, and dermis of the skin are densely packed with these vessels. Once within the lymphatic system, the extracellular fluid, which is now called lymph, drains into larger vessels called the lymphatics. These vessels converge to form one of two large vessels called lymphatic trunks, which are connected to veins at the base of the neck.

Immunology

The study of the physiological mechanisms that humans and other animals use to defend their bodies from invasion by all sorts of other organisms

NK Cells:

Three main populations of lymphocytes circulate in the blood: the B cells and T cells of late-acting adaptive immunity, and the natural killer cells (NK cells) of early-acting innate immunity. Reflecting this temporal division in labor, B cells and T cells are small quiescent lymphocytes that require several days of proliferation and differentiation to become useful in resisting infection, whereas NK cells are large and active lymphocytes that are more speedily induced to respond to infection, cancer, or other form of stress. NK cells have two distinctive functions in the innate immune response to viral infection. The first is to kill cells infected with virus. This sacrifice of cells hosting the virus impedes virus replication and thus its spread to neighboring cells. The second function of NK cells is to maintain, and even increase, the state of inflammation in the infected tissue. This is achieved by their secretion of inflammatory cytokines that act mainly on the resident macrophages and increase their capacity to secrete inflammatory cytokines and to phagocytose viral particles and microorganisms in the extracellular environment. NK cells thus contribute to defense against both intracellular and extracellular pathogens. To a rough approximation, the functions performed by NK cells in innate immunity are like those of T cells in adaptive immunity. Because of these functional similarities, many of the effector and cell-surface molecules of NK cells are also found in T cells. Reflecting this overlap, no single marker has been found to define NK cells. In other words, no intracellular protein or cell-surface receptor is exclusively expressed by NK cells and expressed by all of them. For immunologists, the working definition of a human NK cell is a lymphocyte that expresses CD56, a protein of unknown function expressed by all NK cells, and lacks CD3, a cell-surface protein present on all T cells.

Diversity is added to Ends of rearranged V/D/J gene segments:

V-region sequences are constructed from gene segments (V/D/J)

Vasodilation

Vasodilation (vascular dilation) introduces gaps between the cells of the endothelium, making the endothelium permeable and increases the leakage of blood plasma into the connective tissue. Expansion of the local fluid volume causes edema or swelling.

Alternative Pathway

We shall start by describing the alternative pathway of complement activation, which is one of the first responses of the innate immune system, especially to bacterial infection. C3 is made in the liver and secreted into the blood in a conformation that sequesters the thioester bond in an inactive form within the hydrophobic interior of the protein. In the aqueous environment of the blood, the thioester bond becomes active and quickly makes a covalent bond that attaches C3 to another molecule that has either an amino or a hydroxyl group. This usually involves a water molecule, because they are so plentiful, and gives a form of C3 called iC3 or C3(H2O). This hydrolysis is the first step in the alternative pathway of complement activation. The environment near the surface of certain pathogens, particularly bacteria, increases the rate at which C3 is hydrolyzed to give iC3. Also facilitating production of iC3 is the high concentration of C3 in blood. iC3 binds to the inactive complement factor B, making factor B susceptible to cleavage by the protease factor D. This reaction produces a small fragment, Ba, which is released, and a large fragment, Bb, that has protease activity and remains bound to iC3. The iC3bBb complex is a protease that specifically and efficiently cleaves C3 into the C3a and C3b fragments, with exposure of the thioester bond that is in C3b. Proteases that cleave and activate C3 are called C3 convertases, iC3Bb being an example of a soluble C3 convertase. With large numbers of C3 molecules being cleaved and activated, some C3b fragments become covalently attached to amino and hydroxyl groups of the pathogen's outer surface. Like iC3, pathogen-bound C3b binds factor B and facilitates the cleavage of factor B by factor D. This reaction leads to the release of Ba and the formation of a C3bBb complex on the microbial surface. C3bBb is a potent C3 convertase, called the alternative C3 convertase, which works right at the surface of the pathogen. C3bBb binds C3 and cleaves it into C3a and C3b with activation of the thioester bond. Because this convertase is situated at the pathogen's surface and is unable to diffuse away like iC3Bb, a larger proportion of the C3b fragments it produces become fixed to the pathogen. Once some C3 convertase molecules have been assembled, they cleave more C3 and fix more C3b at the microbial surface, leading to the assembly of yet more convertase. This positive-feedback process, in which the C3b product of the enzymatic reaction can assemble more enzyme, is one of progressive amplification of C3 cleavage. From the initial deposition of a few molecules of C3b, the pathogen rapidly becomes coated with C3b.

Complement Activation Regulates Macrophage Phagocytosis

When a pathogen invades a human tissue, the first effector cells of the immune system it encounters are the resident macrophages. Macrophages are the mature forms of circulating monocytes that have left the blood and taken up residence in the tissues. They are prevalent in the connective tissues, the linings of the gastrointestinal and respiratory tracts, the alveoli of the lungs, and in the liver, where they are known as Kupffer cells. Macrophages are long-lived phagocytic cells that participate in both innate and adaptive immunity. Although macrophages are able to phagocytose bacteria and other microorganisms in a nonspecific fashion, the process is made more efficient by receptors on the macrophage surface that bind to specific ligands on microbial surfaces. One such receptor binds to C3b fragments that have been deposited at high density on the surface of a pathogen through activation of the alternative pathway of complement. This receptor is called complement receptor 1 or CR1. The interaction of an array of C3b fragments on a pathogen with an array of CR1 molecules on the macrophage facilitates the engulfment and destruction of the pathogen. Bacteria coated with C3b are more efficiently phagocytosed than uncoated bacteria: the coating of a pathogen with a protein that facilitates phagocytosis is called opsonization

Memory (continued)

With the termination of an infection by the primary immune response, raised levels of high-affinity pathogen-specific antibodies are present throughout the blood, lymph, and tissues, or at every mucosal surface. The antibodies are secreted by plasma cells residing in the bone marrow or in the tissue beneath a mucosal surface, and high levels are sustained for several months after the infection has been cleared. During this time, these antibodies provide protective immunity, ensuring that a subsequent invasion by the pathogen does not cause disease.