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Non-adaptive immunity is a combination of several plasma proteins and cellular systems. These include complement system, fibrinolytic and fibrinolytic systems. Cellular systems include polymorphonuclear and platelet systems (PMN), mast cells endothelial and platelet cells (thrombocytes), macrophages, dendritic and NK cells. These are collectively called “mediators for inflammation”. These molecules can be preformed or made from scratch on request. Although these molecules can cause inflammation, the ultimate goal of their creation is defense, not inflammation. Inflammation is a temporary state that makes it easier for infectious agents to be combated. These molecules have many similarities in their functions. Evolutionary pressure appears to have favoured organisms with backup systems to backup system for backup systems. It’s not rocket science but it works in a similar way. It is possible to reduce inflammation but not completely, but it is difficult to prevent it. Cellular subsystems that contribute to defense/inflammatory mediators: Preformed molecules are stored within granules, and then released as needed: Histamine, serotonin and lysosomal protein Newly synthesized molecules: protaglandins (PAF), reactive oxygen species (ROS), NO cytokines I interferons (1.1 COMPLEMENT) The primary purpose of the complement system is to combat bacterial infections. It functions at multiple levels. It can recognize many bacteria and alert and recruit them, increase visibility to phagocytes, and even lyse them. At least three pathways can activate the complement system. Two evolutionary older pathways, the “alternative” or lectin pathways, are the two. Both pathways are activated on many bacteria surfaces and contribute to innate immunity. Although the third pathway is mainly antibody-activated, it was discovered first. It is sometimes called the “classical path” in an unfair way. It is the alternative pathway to complement activation that involves the spontaneous hydroysis a plasma complement component C3 thioester bond, resulting in C3(H2O). C3(H2O), which has a changed conformation, allows for the binding of plasma protein factor B. This results in C3(H2O). C3b can bind covalently to cellular or bacterial surfaces if it is generated close to them. The same process is repeated on the membrane. Factor B attaches to the surface, which will be cleaved using factor D. One of many protective proteins can stop C3b from binding to the membrane of a cell. Inhibitors are complemented by protease factor I (letter I as in Iris), which cleaves C3b into enzymatically active products (iC3b). These inhibitors are not present on bacterial surfaces, which allows the complement cascades to continue. The bacterial surface facilitates factor P (properdine), which stabilizes the membrane-bound complex C3bBb. This complex, which is the C3 convertase for the alternative pathway, then works as an amplifier tool by rapidly cleaving hundreds more C3 molecules. Soluble C3a diffuses in the surrounding, attracting phagocytes to the area of infection via chemotaxis. C3b fragments, and their cleavage products C3dg, C3dg, and C3bi, are deposited on the bacteria’s surface in increasing numbers. They are recognized by specific complement receptors (CR1 to CR4) that are found on the membranes of phagocytes. Optonization is a function that makes the bacterium “delicious” for phagocytes. It’s also known as “delicacy”. The complement cascade doesn’t stop here: activation of C5 through C9 eventually results in membrane pores, which sometimes lyse bacterium. C3a-C4a and C5a are sometimes called “anaphylatoxins”. They have additional functions: they attract phagocytes and cause mast cell degranulation. This allows for easier access to plasma proteins and leukocytes at the site of infection. The lectin pathway for complement activation takes advantage of the fact that many bacteria surfaces have mannose sugar molecules with a specific spacing. The oligomeric plasma protein-mannan-binding lecin (MBL) binds with this pattern of mannose moieties and activates proteases MASP-1 (MASP=MBL activated protease, which is similar in structure as C1r or C1s). This cleaves C4 and C2 to create a second type C3 convertase, consisting of C4b & C2b. The resulting events are identical to the ones in the alternative pathway. C2a was the original name for the large C2 fragment. It was later renamed C2b to unify all large fragments of “b”. Unfortunately, nomenclature has been inconsistent since then. 4 The classical pathway typically begins with antigen-bound antibodies recruiting C1q, then binding and sequential activation C1r/C1s serine proteases. The C1s cleaves the C4 and the C2, and the C4b-C2b forms the C3 convertase in the classical pathway. This pathway can be activated even if there are no antibodies. CRP (Creactive Protein) binds to bacteria surfaces and activates C1q. Pharmacology cross-reference: Humanized monoclonal antibody Eculizumab binds with complement component C5, interfering with its cleavage, and stopping activation of the lytic pathways. This is useful when complement activation causes hemolysis. Eculizumab treatment may increase the chance of meningococcal infection due to its importance in fighting them. This can be prevented with previous vaccination. 1.2 COAGULATION/FIBRINOLYSIS SYSTEM AND KININ SYSTEM Frequently, coagulation (more about that in cardiovascular pathophysiology) and kinin systems are activated simultaneously by a process called contact activation. This process, as the name suggests, is initiated by three plasma proteins coming into contact with negatively charged surfaces. These surfaces could be basal membranes, collagen, or aggregated plateslets for a laceration. Or bacterial surfaces for an infection. These plasma proteins are Hageman-factor, high molecular weight-kininogen HMWK and prekallikrein. Contact with a negatively charged surface activates factor XII, initiating the entire coagulation process. Factor XII also cleaves prekallikrein, which releases the active protease Kallikrein. This in turn releases HMWK’s non-apeptide bradykinin. Bradykinin is known to increase small vessel permeability and dilate small vessels indirectly through the endothelium. It also favors smooth muscle contraction and is the strongest pain mediator. Bradykinin, like other kinins, has a short half-life and is inactivated by peptidases such as angiotensin converting enzyme. Pharmacology cross-reference: ACE inhibitors are often used to lower blood pressure. However, they can also cause coughing. It is thought that this happens due to an increase of bradykinin activity. These plasma protein cascades lead to an inflammatory reaction and blockage of small vessels by coagulation. This is helpful in preventing the spread of infection via blood. Blood pressure drives plasma to be filtered out of vessels with enhanced permeability and forms tissue lymph. The plasma is then diverted to regional lymph nodes where phagocytes, and other leukocytes, are waiting to initiate additional defense measures. The activation of plasma protein cascades is, in many ways, a prerequisite for the next step: activation of cellular system at the infection site. How are the participating cells activated 5 1.3 ACTIVATION OF CELLS, PATTERN RECOGNITION RECEPTORS Neutrophil granulocytes Neutrophil granulocytes (frequently designated PMN, for polymorphonuclear leukocytes) are able to directly recognize and phagocytize many bacteria, but not the most crucial polysaccharide-capsulated pathogens. These agents can only be recognized and phagocytized after opsonization by complement via complement receptors on neutrophils. How does neutrophils get from the bloodstream to their intended site of action? A host of molecules are released from the site of infection and eventually reach endothelial cells in nearby vessels. LPS (lipopolysaccharide), derived from bacteria, C3a C4a, and C5a, as well as signaling molecules from first macrophages, e.g. the chemokine IL-8 and TNFa. These signals are quickly reacted to by endothelial cells, which expose new proteins like ICAM-1 and 2, on their membranes. They are then tightly bound to cell-cell contact proteins of neutrophils or other leukocytes. Neutrophils are normally rolling along the endothelium by dynamic contacts between their sialyl-Lewis-x-carbohydrates and selectin proteins on the endothelial plasma membrane. The neutrophil stops abruptly when the PMN-integrins bind to the ICAMs. It squeezes between two endothelial cell and approaches the focal infection. Here, neutrophils kill and phagocytize bacteria. Neutrophil extracellular traps = net are another way neutrophils can kill pathogens. They do this by ejecting the DNA (or chromatin) of the target cells. Either the cells die during the NET-forming process or they eject their nucleus and enzymes, while continuing to phagocytize. As bacteria can also be killed in harsh environments, neutrophils die quickly once activated. Macrophages pick up their remains. Mast cells Mast cell activation involves activating mast cells to release histamine through a wide range of stimuli, including heat and cold, scratching and laceration, and complement activation. Mast cells can degranulate later, as a result of an adaptive immune response. We will be looking at activation of endothelial and thrombocytes in cardiocascular pathophysiology to avoid redundancy. Pattern recognition receptors activate macrophages/dendritic cell activation. To detect the presence of pathogens macrophages/dendritic cells express a wider range of receptors than neutrophils. These pattern recognition receptors (PRRs), which recognize pathogen-associated molecular structures (PAMPS), are conserved in large classes of pathogens because of their functional importance. Many of these receptors are located at plasma membranes. 6 C-type lectins is one group that recognizes certain sugar units, which are often found at the terminal position on carbohydrate chains. The C-type lectins also include the mannose receptor and DC-SIGN, which are typical of dendritic cell. Langerin is another example. In parallel to the mannan binding protein, the “mannose receptor”, recognizes terminal mannose and N-acetyglucosamin. There are many PAMP receptors in the large group of Toll like receptors (TLRs, fruit fly Drosophila Toll is the first to be identified from this family). TLR4 can be activated by bacteria lipopolysaccharide. TLR1/TLR2 is activated by TLR2/TLR6 and TLR2/TLR6 are activated by bacterial peptides and Glycan. TLR5 binds flagellin. A group of polynucleotiderecognizing TLRs checks the content of endosomes: TLR9 recognizes bacterial DNA without the CpG methylations typical for human DNA, TLR3 virus-typical double-stranded RNA, TLR7 and -8 single-stranded RNA. There are three other types of receptors that sense PAMPS intracellularly in the cytoplasm. These receptors are not only expressed by macrophages or dendritic cell, but also many other types cells. For example, NOD-like receptors, NOD1 (or NOD2), sense components of the peptidogycan in the bacterial cell walls. Other NLRs, upon activation, form a large complex cytoplasmic, the inflammasome. The inflammasome is essential for cell activation. It helps to cleave IL-1b from inactive precursors and also assists in cell activation. Some NLRs recognize not only PAMPs but also patterns that indicate damaged or dying cells. These may include a decrease in intracellular K+ concentration, or the appearance uric acid crystals that are a result of the removal of DNA purine bases. Some NLRs can also be used as receptors for cells that are “danger threatening”. RIG-like receptors, (RLHs),: The cytoplasmic RNA helicase RIG-1 and related proteins such as MDA-5 are virus receptors. RIG I recognizes single stranded viralRNA by a free triphosphate located at the 5′ end – this is without the typical cap structure of our mRNA. MDA 5 recognizes double stranded DNA, which occurs in the replication cycle for many viruses, but is not usually found in our cells. Cyclic GMPAMP synthase cGAS: Double-stranded DNA is “belongs to” the nucleus or to mitochondria. Something is wrong as soon as DNA appears within the cytosol. Either a virus is present in the cell or the cell’s organization is in disarray. The enzyme cGAS recognises cytosolicDNA and creates the cyclic nucleotide cGAMP (from GTP and ATP). This activates the protein STING (stimulator for interferon gene), which activates interferon genes and other emergency program. These receptors can recognize PAMPs as well as molecular patterns that are typical of cells in distress, as we’ve seen. K+ can leak out of cells if they are damaged. DNA is found in the cytosol if the nucleus and mitochondria are damaged. These patterns, analogous to PAMPS (danger associated molecular pattern), are known as DAMPs (danger-associated molecular patterns). The first time PRRs were discovered was early in the evolution. They have been an important tool in multicellular organisms’ battle with bacteria for long periods of time. For example, the sea urchin genome contains over 200 receptors for Toll-like and NOD-like receptors. C3-derivatives that are deposited on pathogens activate complement receptors. 7 The activation of these macrophage receptors results in phagocytosis, which is the killing or breakdown of ingested bacteria. A profound change in gene expression of macrophages results in the release of a variety of cytokines, including IL-1b and IL-6. These cytokines attract and activate other cells within the defense system. These cytokines reach the liver via the bloodstream and launch another tool for non-specific defense: the production of acute phase protein. Macrophages and the dendritic cell also produce certain membrane-associated proteins upon activation. These proteins (CD80, CD86) are necessary to trigger an adaptive immune response. What is the difference between macrophages, dendritic and dendritic cell? Macrophages tend to be more non-adaptive in defense. They can phagocytize large quantities of particulate matter and are called “heavy earth moving machinery”. The adaptive side of defense is where dendritic cells operate: they gather antigenic materials and take them to the lymph nodes to be passed to T cells. They can phagocytize but not do the heavy lifting. Macropinocytosis, which is a process of taking up large amounts of fluids and all soluble antigens in the surrounding environment, can be used to take up many antigens. Infected with viruses is another way that dendritic cell can take up antigens. This is essential to initiate an adaptive antiviral immune reaction. Our dendritic cells can live for a long time, as they are derived from hematopoietic cells found in the yolk sac wall or the fetal liver. Later, dendritic cell production also occurs in bone marrow. There are two stages in the life of dendritic cells. While functionally young and immature they roam the peripheral, collecting material but not having the tools to activate T cell. The chemokine receptors determine where they go. They follow the chemokine trails into peripheral tissues with these receptors. Some Langerhans cells, for example, may remain in target tissues for many years. Others, such as Langerhans cells, might only stay there for a few days. However, any “traumatic” infection that is triggered by TLR signaling causes them to grow and rush to the lymph node. This happens because chemokines are now recognized by CCR7 (chemokine receptor 7). Although mature dendritic cells are unable to absorb antigen, they still have all the necessary resources for productive relations with T cells, including lots of MHC molecules and B7 molecules. These battle-hardened and worldly-wise dendritic cell populations secrete chemokine CCL18 which is attractive to young, inexperienced T cells. The implications of this are only revealed later (section 2.2). Innate lymphoid cell Our innate defense system includes cells that appear exactly like T or B lymphocytes under the microscope but do not express receptors for either T or B cells. These cells are called innate lymphoid. These cells can be activated by cytokines from macrophages and dendritic cell cytokines, which may contribute to non-adaptive defense. This is a partial list of the cell types. We will not be focusing on natural killer cells as part of this group. 1.4 VASOACTIVE MINES: HISTAMINE and SEROTONIN Histamine, which is released from mast cells granules causes vascular dilatation, which results in an increase in permeability. It is made by the decarboxylation and synthesis of the amino acid histidine. There are four types histamine receptors. They all belong to the G protein-coupled 7TM Family. The H1 and H4 receptors mediate the proinflammatory 8 functions of histamine. These receptors can be blocked by drugs. They are often used to treat allergies, unwelcome aspects of inflammation (runny nose, stuffed nose), and motion sickness. To reduce gastric acid production, H2 receptor blockers can be used. Histamine is released via the H1 receptors to increase small vessel size and permeability. It also recruits other leukocytes via the H4 receptors. Serotonin is mostly released from activated, aggregated thrombocytes. It activates platelets and increases their ability to bind to clotting factors. Tryptophan is the basis of serotonin. 1.5 LYSOSOMAL ENEZYMES Proteases include acid hydrolases, collagenase and cathepsins. The bactericidic proteins (lysozyme and defensin), kill and degrade phagocytized bacterial cells. Tissue destruction is a common side effect. Proteases are also released from cells. TNFa appears to be the most important driver of protease production among all cytokines. 1.6 PROSTAGLANDINS and LEUKOTRIENES Prostaglandins are made from arachidonic, which is a polyunsaturated fat acid component of phospholipids. Arachidonic acid can be mobilized from the membranes using phospholipases, and then metabolized by either cyclooxygenases to prostaglandins or lipoxygenase to leukotrienes. There are two cyclooxygenase enzyme isoenzymes that are expressed and controlled in a different way. Many tissues contain COX1. It plays a vital role in many tissues, including protecting the mucosa and renal perfusion. When the natural immune system activates, COX2 is produced. Prostaglandins have a very short half-life and only affect the cells within a few hundred meters of their source. Prostaglandins have many functions in different tissues. Their pro-inflammatory functions make up only a small portion of their entire spectrum. Prostaglandins are not able to be described in general terms. Their functions depend on the type of tissue and the specific prostaglandin molecules that are present. Pro-inflammatory effects can be seen in isolation. PGE2 or PGD2 prodilatation is promoted by prostaglandins (the “2”, in prostaglandin names, refers to the number of double bonds within the molecule). Not by itself, PGE2 causes pain by stimulating the effects of pain-causing stimuli like bradykinin or elevated extracellular potassium. Prostaglandins also have opposite effects on blood coagulation. One prostaglandin, thromboxane (produced by thrombocytes), promotes coagulation while the other, prostacyclin (released by endothelial cell), inhibits it. PGE2 plays a key role in the onset of fever in the hypothalamus. Endothelial cells in the organum vasculosum terminalis of the front wall, third ventricle produce PGE2 in response to IL-1b and IL-6 from activated macrophages. This mechanism raises the temperature set in the hypothalamus. Because many pathogens have enzymes that are optimized for normal body temperatures, fever reduces their proliferation rate. Some steps necessary for adaptive immune response (antigen presentations) are also accelerated at 9 From an evolutionary point of view, fever is an old trick in fighting infections: if possible, poikilothermic fish swim to warmer waters upon experimental Klebsiella-infection, which increases survival rates. It is not rational to reduce fever by pharmacologic means. The bronchial constriction caused by leukotrienes D4, E4 and E4 is exacerbated by vascular permeability. They are key players in bronchial asthma. Leukotriene C4 is chemotactic, activates PMN. Cross reference in Pharmacology: Prostaglandins/leukotrienes have a wide range of effects. There are many ways to interfere pharmacologically with them, and there are equally good options for undesirable side effects. Cortisol, and other glucocorticosteroids, inhibit the phospholipase that releases arachidonic acids from phospholipids. This inhibits the synthesis of prostaglandins as well as leukotrienes. Glucocorticoids are strong anti-inflammatory agents. Acetylsalicylic Acid (Aspirin(r),) and other NSAIDs, non-steroidal anti-inflammatory drug (NSAIDs), act as an anti-inflammatory, anti-inflammatory and fever-reducing agent by inhibiting cyclooxygenases. Conventional COX inhibitors block both isoenzymes and can cause side effects such as gastritis, ulceration, and intestinal bleeding. It was clear that blocking COX2 (a cyclooxygenase enzyme) would be sufficient to produce anti-inflammatory effects. COX2-specific drugs were created promising reduced side effects. Celecoxib was one of the first to show this principle. The withdrawal of COX2-inhibitor Rofecoxib from the market was due to an increase in the risk of stroke and myocardial damage. To reduce the chance of thromboembolic complications, low doses are used for acetylsalicylic acids. We will examine the mechanism of platelet inhibition within “cardiovascular pathophysiology”. Hyperactivity in one pathway may be caused by the main bifurcation of arachidonic acid metabolic. This mechanism can cause bronchial asthma in sensitive people by blocking COX with NSAIDs. This sensitivity is likely to have a genetic basis. People with this condition may also develop symptoms when they eat foods containing salicylates such as bell peppers, oranges and curry. The use of leukotriene receptor blocking drugs (e.g. Montelukast) and lipoxygenase inhibitors, which are not yet approved in Austria, Germany, or Switzerland, can pharmacologically reduce the effects of leukotriene. These medications are mostly used in asthma therapy. 1.7 PLATELET ACTIVATING FACTOR (PAF) PAF is a phospholipid released by thrombocytes, basophils/mast cells, neutrophils, monocytes/macrophages and endothelial cells. It is known to have many pro-inflammatory properties, including activating 10 platelets, increasing vascular permeability and bronchial constriction, and activating neutrophil chemotaxis. 1.8 REACTIVE OXYGEN SECTIONS After phagocytosis, stimulation by mediators such as PAF, neutrophils or macrophages activate their NADPH-oxidase enzyme complex and produce chemically extremely aggressive oxygen derived reactants such as superoxide-anion, hydrogen peroxide, singlet oxygen (1O2), or hydroxyl radicals(.OH). This is known as oxidative burst or respiratory. Myeloperoxidase is another enzyme that produces hypochlorite anion. These reactive oxygen species, or ROS, are very toxic and chemically modify all types of bacterial macromolecules. Although this is a great way to kill phagocytized organisms, it also kills the phagocyte and often damages the surrounding tissues. Chronic inflammation can lead to malignancies and increase the likelihood of them occurring over time. This is because oxidative damage to the bases of DNA causes a higher rate of mutations. Endothelial cells and macrophages produce 1.9NO Nitrogen oxide (NO) which has two functions. It dilates blood vessels, and contributes to the death of phagocytized microorganisms. Endothelial cells that sense mediators of inflammation activate endothelial NOS (eNOS) and produce large amounts of NO to relax nearby smooth muscle cells. Although macrophages do not express NO synthase in their own cells, they can be stimulated with cytokines such as IFNg or TNFa to activate the enzyme. Pathogen killing can be enhanced by iNOS, which is cytokine-inducible NOS. 1.10 CYTOKINES and CHEMOKINES The meaning of “cytokine” is a bit fuzzy. It is a signaling molecule that contains a number of polypeptides. There are many cytokines with very different functions and targets cells. Their names are often confusing. A few examples: interleukins, TNFa (tumor necrosis factor-a), lymphotoxin, IFNg (interferong), G-CSF (granulocyte-colony stimulating factor), GM-CSF (granulocyte/macrophage-colony stimulating factor), c-kit-Ligand, TGF-b (transforming growth factor-b). Chemotaxis is mediated by a large number of cytokines. These proteins are called chemokines and have a small (8-10 kDa), conserved structure consisting of three b sheets and a Cterminal a-helix. Based on their tertiary structures, they can be divided into four subfamilies, CC, CXC or CXXXC. Chemokines are named after their subfamily. An “L” stands for ligand, and a number is used: CCL2,CXCLn. Receptors get an “R” instead, e. g., CCR5, CXCRn. Receptors, too, have a common structure: all of them are 11 7-transmembrane-helix (7TM) receptors, which are G protein-coupled. All immune cells can reach the correct place at the right moment thanks to the guiding system of chemokine gradient fields and chemokine receivers. Let’s look at the cytokine mix released by macrophages upon activation of their pattern recognition receptors. After recognizing and phagocytosing the bacteria, macrophages release cytokines TNFa and IL-1b. IL-12 activates NK (natural killer) and ILC1 cells (innate lymphoid-1), and aids in the differentiation and maturation a specific subset of T cells (these cell types will be explained later in sections 1.13 to 2.13 respectively). IL-8, a chemokine, has the systematic name CXCL8. It recruits neutrophils via CXCR1 or -2 receptors. TNFa, IL-1b, and IL-6 are all part of a team that has many overlapping functions. They can have both local and distant effects. We will be looking at TNFa to illustrate the complex biological effects that a single cytokine can have. In the next section, we will first look at the strategy and then the implementation. Cross reference in Pharmacology: Many cytokines can be produced as recombinant protein and used as drugs. For example, G-CSF is used to stimulate neutrophil formation. Inhibiting immune reactions can be made easier by counteracting certain cytokines. Cortisol and other corticoids in higher than physiologic levels are immunosuppressive. It has a suppressive effect that reduces the expression of many cytokines (e. g. TNFa, IL-1b and IL-2), in large part. You can use recombinant proteins to counteract specific cytokines to suppress a limited aspect of an immune reaction, without risking generalized immune suppression. The anti-TNFa treatment is used to treat Crohn’s disease, rheumatoid and severe forms psoriasis. 1.11 TNFa AND AN ACUTE PHASE REACTION Although many types of cells can produce TNFa, the majority is produced by activated macrophages. Almost all cells have TNFa receptors. The activation of receptors results in the expression of genes that contribute to the defense of the organism against infection. The molecule’s purpose is to coordinate a non-adaptive defense response on both a local level and at the systemic level. First, we will look at abstract strategies and then move on to practical mechanisms. Strategy: Local: If an epithelial barrier has been breached, it’s essential to limit the ensuing bacteria to that area. Sepsis, which is a potentially life-threatening condition, would be the most dangerous. You can prevent this by increasing the permeability in the small blood vessels, and by closing the draining veins with clotting. This is caused by increased blood pressure which is localized by 12 vasodilatation. It causes a slow movement in tissue lymph towards the regional lymph node and some pathogens. With its many phagocytes, the lymph node acts as a filter to stop further spread. Leukocytes from the blood are also recruited to the area of primary infection. Endothelial cells are then instructed to assist them. Experimental evidence shows that if pathogenic bacteria is administered to a rabbit’s paw, it will usually contain the infection. The bacteria can spread through the blood to all other organs if the rabbit is also injected with antibodies against TNFa. These TNFa effects are a double-edged knife. Sometimes they occur too late and the bacteria has already spread. TNFa can be a problem in this situation, leading to sepsis and septic shock. The bacteria spreads to macrophages all over the body. The TNFa is released by macrophages in the liver, spleen and lung. This causes plasma volume to plummet (vascular leakage syndrome). The coagulation cascade kicks off everywhere in the body. It is joined by the fibrinolytic cacade. This consumes all available clotting factors (disseminated inner coagulation), and causes profuse bleeding. These processes can be very difficult to stop once they have started. This condition results in most patients being permanently disabled. Systemic level: TNFa is released in small amounts from inflamed areas. This allows for the spread of the infection to other parts. This can cause fever and the feeling of being sick. It also causes energy conservation, but it mobilizes energy to make more defense equipment, plasma proteins, and neutrophils. Implementation: At the local level: TNFa activates endothelial cell walls nearby, which then express adhesion proteins that allow leukocytes dock. They can also retract slightly to increase permeability and let leukocytes through. activates thrombocytes, causing them to attach to the endothelium. This causes the vessel to close its draining arm and cause coagulation to begin. These two effects allow IgG and complement components to reach the source of infection. They facilitate extravasation of leukocytes, and increase flow to local lymph nodes. Tissue lymph flow transports pathogen antigens, which are packaged in phagocytes. This helps to activate an adaptive immune response. Induces iNOS in macrophages. NO contributes to the killing of pathogens as well as vascular dilatation. Induces cyclooxygenase (lipoxygenase) and protaglandin synthesis. This induces tissue destruction and fibroblast proliferation. They are also related to relatives that were later discovered, and they both bind to the same receptor. These molecules have been grouped under the “type I-interferons” heading. Type-I interferons are signaling molecules that virus-infected cells secrete to slow or inhibit virus replication in nearby cells. This allows for a faster and more adaptive immune response. When viruses are replicated in human cells, many of them produce intermediates that contain long double-stranded DNA. This type of RNA is not normally found in human cells. They only have RNA-molecules that are very short and double-stranded between loops. The appearance of long stretches double-strandedRNA is an indicator of potential viral infection and stimulates the expression and secretion type I-interferons. TLR3 recognizes double-stranded DNA in endosomes. The RIG-like receptor protein MDA-5 detects double-stranded transcripts in the cytosol. Interferon responses can also be triggered by single-stranded viralRNA. RNA that contains no 5′-triphosphate ends in cytosol (i.e. Without the typical cap structure of our mRNA, RNA without RIG-like receptors (RIG-I) is recognized. Almost all cells have RIGlike receptors. Another warning sign of possible virus attack is the presence of DNA in the cell cytosol. The cytosolic enzyme Cyclic GMP AMP Synthase (cGAS), which is activated by binding DNA, is a cytosolic one. It generates the cyclic nucleotide (cGAMP) by binding to DNA. This activates the adapter protein, STING (STimulator for INterferon Genes). STING coordinates a protein compound that eventually leads to activation of transcription factors like IRF3 (interferon regulator factor 3), which in turn stimulates expression of interferon genes. The virus-infected cells are not able to benefit from Type I interferons. They warn nearby cells to prevent virus replication. Jak/STAT signal transduction allows activation of the type I interferon receptor in neighboring cells. This results in the induction specific genes, which makes intracellular conditions favorable for virus replication. Protein kinase R is one of the induced proteins. It is activated via double stranded RNA. It inhibits ribosomal mRNA transcription by phosphorylating eukaryotic Translation initiation Factor eIF2. As it depends on the host cell machinery for virus proteins to be produced, this severely limits replication possibilities. This harsh measure can also negatively impact the functioning of host cells. Induction of the Oligoadenylate Synthase enzyme activates a second antiviral mechanism. This enzyme oligomerizes ATP through the formation of unusual 2′-5’ bonds (normally, nucleotide connection are 3′-5’). These 2′-5’A activate RNase L which is an inactive form RNase and breaks down both viral and cellular RNA. Type I-interferons induce additional anitviral protein as well as proteins that promote adaptive immune responses with the ultimate goal of eliminating the virus. These include MHC-class I molecules (see section 2.8) and components of the protease that are important for antigen processing. NK cells are also protected by enhanced MHC-I expression in non-infected cells. 15 Type I-interferons, which activate, are similar to IL-12 and NK cells. Cross reference in Pharmacology: As therapeutics, recombinant type 1 interferons can be injected. Although viral infections seem like a logical indication, interferons can be expensive and can have severe side effects (e.g., anemia, depression, flu-like symptoms upon injection) and are very costly. IFNa was not recommended for life-threatening viral diseases such as hepatitis C. However, specific virus replication inhibitors can be used to treat hepatitis C with fewer side effects. Other applications, which are not related to viral infections but are logical consequences of interferons’ effects, are also available. IFNb is used for reducing relapses in multiple sclerosis relapsing-remitting forms. 1.13 NK CELLS The appearance and function of natural killer (NK) cells is similar to cytotoxic T lymphocytes. However, they lack the T cell receptor that T cells use to identify virus-infected cells. They are included among the innate lymphoid lymphoid cells. How do they identify cells that need to be killed? The catch phrase missing or modified self may be one of the cellular properties that activates NK cells. The NK cells are essential in the initial phases of defense against viruses but also against other infectious agents. They can also be used to eliminate rogue cells and prevent the formation of tumors. Two types of receptors are expressed by NK cells: inhibiting and activating. The inhibiting receptors detect normal MHC-I molecules in cells probed by NK cells. Normal MHC-I cells will be left alone. A cell that lacks MHC-1 or expressing an altered MHC-1 (missing or altered self) will be left alone. However, activating NK receptors can only recognize the cell and cause apoptosis. Many viruses, including herpes viruses infected cells, suppress MHC-I expression. This trick gives viruses a distinct advantage in the future, since these cells can’t be identified by cytotoxic T cell cells. However, they are vulnerable to NK cell attack. Alternate mechanisms may also be used to activate NK cells. Many cells produce proteins such as MICA (MHCI-chain-related A), that act as ligands to activate the NK receptor NKG2D (natural killing group 2, member D) under cellular stress. This can happen in some cells as a result of oncogenic mutations. High levels of MICA in NK cells cause them to axe questionable cells. It’s better to be safe than sorry. NK cells can be activated by cytokines (especially IL-12), but not by direct contact with cell-cells. In turn, NK cell responses by secreting cytokines (primarily IFNg), which act as a stimulant on macrophages. This mechanism was crucial in the defense against Leishmania, a protozoon spread by sandflies. Leishmania species can be taken into macrophages but are able to inactivate them. Dendritic cells also recognize Leishmania and activate NK cells via IL-12. NK cells are then activated by IFNg to stimulate macrophages to eliminate intracellular parasites. 16 Although NK cells make up part of the adaptive immune system, they are also capable of being directed by antibodies to specific structures in a process called antibody-dependent cellular cytotoxicity. 2. THE ADAPTIVE IMMUNE REPONSE. There are many ways to combat pathogens. Antibodies are a great tool to fight extracellular pathogens. 2.1 ANTIBODIES A antibody (=immunoglobulin), is made up of two heavy and two lighter chains that are joined by disulfide bond. There are five alternative types of heavy chains (m,g,d,a,e- all encoded on chromosome 14,) which give rise to IgM, IgG and IgD as well as IgA and IgE. Type k and l light chains can be encoded on chromosomes 22 and 2. IgM is always composed of five immunoglobulin units joined together, while IgA may contain two. A few terms are used in immunology. An antibody can have a variable or constant region. The constant region of the genome encodes the protein. As such, it is determinable like all other proteins. However, the variable area is created by rearrangement. This involves cutting and pasting DNA. The variable region of immunoglobulin binds to antigen. Antigens are anything that can trigger an adaptive immune response. Its chemical makeup is not important. It can contain polypeptides and carbohydrates, as well as fats, carbs, and nucleic acid. It is necessary to have a minimum size. When coupled with larger carriers, very small molecules can only be used as antigens (or haptens). Antibodies can recognize large, three-dimensional surface structures. This contact can be established by any non-covalent binding force, including hydrogen bonds, electrostatic attraction, Van der Waals-, and hydrophobic forces. Antigen binding can be reversed. Most biological macromolecules contain multiple structures that can elicit an immune response. These are known as antigenic determinants and epitopes. Cross-reaction is a phenomenon where two different macromolecules may be bound by the exact same antibody. These statements are about antigens that have been bound to antibodies. T-lymphocytes recognize 17 antigens. These are more restricted. T-lymphocytes detect epitopes that range from 8 to 20 amino acid. An antoantibody is an antibody that has a high affinity for a specific macromolecule in its organism. The corresponding macromolecule is known as an autoantigen. Histocompatibility antibodies are antigens that can cause rejection after organ transplantation. Their most important group is encoded on a small portion of the short arm chromosome 6. This is the major histocompatibility complicated (MHC). The minor histocompatibility gene group includes all other genes that have variants that could cause rejection. Three fragments are formed when a specific protease is used for the digestion of the Y-formed antigen. Two identical fragments are called Fab (fractional antibody binding) and one fragment represents the other end. It contains a large portion of the constant area. This fraction was crystallized in early experiments and is known as Fc (fraction crystallizable). This is the “back end” of an antibody. Many cells in the immune system have receptors that can bind to it. These receptors are called Fc-receptors. They are named after the heavy chain they recognize. Fcg-R, Fca-R, Fca/u/R (for IgA), Fca/u/R (for IgA, IgM), Fce–R (for IgE). Most of these receptors have an affinity too low to bind single antibodies for extended periods of time. Antigen-binding results in larger immune complexes. Cooperative binding between multiple Fc ends with their receptors allows for rapid internalization via phagocytosis. This provides a mechanism to speed up antigen clearance. An exception to this rule are mast cells and eosinophils, which also bind free (meaning non-antigen-complexed) IgE via their high-affinity Fc-e-receptors. 2.2 HOW DO ANTIBODIES IMPACT ON DEFENSE Antigenic bacteria, viruses, and parasites are all common. B-lymphocyte-derived plasma cell cells will then produce specific antibodies after a lag phase lasting at least five days. This is a period that we must endure with the help of our innate immunity. These antibodies then bind with the pathogens. What’s the point? What does this mean? Depending on the pathogen, antibodies may help in at least five ways: neutralizing viruses, targeting toxins, and enhancing complement-lysis bacteria opsonizing (“yummifying”) cells ADCC (antibody dependent cellular cytotoxicity). NK cells can sense cells that have bound antibodies via their Fc receptors. These cells then go on to be killed. These could be virus-infected cells that have exposed their cell membrane to viral envelope proteins. To neutralize viruses or toxins, you must stun them in all directions with antibodies so they can no longer make contact with their receptors. Each virus contacts a specific protein to enter a cell. This is called its receptor. The protein is not intended to be a receptor for viruses; it serves a different physiological function. HIV (human immunodeficiency viruses) uses the lymphocyte transmembrane proteins CD4 as its receptor. We will examine CD4 in section 2.9. It is possible to infuse neutralizing antibodies with some viruses, but not HIV. HBV vaccination is a good example. The vaccine is recombinant and contains the HBs-antigen recombinant protein. It induces neutralizing antibodies. HBV is instantly studded with antibodies if it later enters the body. It is unable to enter the liver cells and remains completely innocuous. Tetanus and diphtheria aren’t caused by bacteria, but by the toxins they produce. These bacterial toxins can also be used to direct cells to behave in the best interests of the bacteria by binding and misusing cellular protein. Anti-toxin antibodies can be neutralized by immunizing babies with inactivated versions. The disease-causing toxins are not able to bind to the receptors of their receptors, so if a child is later infected, they will not be noticed. Complement-activation via the classical pathway: IgM and two of the four subclasses of IgG activate complement. These antibodies’ Fc part binds to complement component C1q. Further steps are described in section 1. This can only be achieved after antibodies have bound the antigen, creating an immune complex — modifying their conformation. Complement cannot be activated by free soluble antibodies. This is important because complement can also be activated via alternative pathways and lectin pathways. The process is made more efficient by antibodies: there are more opsonizing cells that can be deposited with C3b, which makes it much quicker and more efficient. With a greater chance of bacterial destruction, there are more complement pores. Immunoglobulins can also opsonize via Fc receptors on phagocytes. Complement receptors are important for managing immune complex-waste. CR1 can be found on both leukocytes and red blood cells. It binds to C3b, which has been deposited on immune complicatedes. The immune complexes are then transported to the liver and spleen by erythrocytes. There, they will be picked up by phagocytes. When this transport system becomes overwhelmed, soluble immune compounds can build up at the sites of filtration (e.g., renalglomerula) and cause disease. IgM (2.3 IMMUNOGLOBULIN CATEGORIES (ISOTYPES). IgM is a pentamer composed of five Y-formed cells arranged in a circular pattern. It is the first to respond to infection and gradually declines thereafter. It can be used to distinguish between a new infection and an older one. An acutely infected patient will show specific IgM but not IgG. A patient who has been infected for a long time will only show IgG. IgM’s ability to activate complement is so strong, that one bound IgM “crab” acts as a landing pad for C1q. This is unlike IgG where IgG requires at least two IgG molecules to be bound at a certain distance. C1q can then travel between them. IgM is small enough to be confined to blood plasma. It is too large to fit between endothelial cell walls. IgG, the standard model antibody that appears later in an immune response than IgM, is IgG. There are four subclasses IgG1-IgG4, of which IgG1 & IgG3 effectively activate complement. 19 IgG is a class of antibodies that are transported through the placenta. It provides antibodies for newborn children for 2-3 months against pathogens “seen” in the mother’s eyes. IgG has a half-life of approximately 21 days in blood, which is about twice that of IgM. IgG can reach plasma concentrations above a certain threshold, which is necessary for the effective neutralization or elimination of viruses and toxins. IgA is a monomer found in blood. Its main function is to protect the epithelial surface. There are two subclasses of IgA (IgA1 & IgA2). It must be synthesized in the submucosa, as a dimer that is joined to a J-chain. A submucosal cell, such as an epithelial one in the intestine, or a salivary gland can bind the dimer through a poly-Ig receptor and transcytotically transport it in a vessel to the apical membrane. It is then released through the cleavage and release of the receptor. The secretory component (SC) of the receptor is still attached to the IgA dimer. It is now called sIgA (secretory IgA). SC protects the sIgA against proteolytic digestion in intestinal tract. Strong glycosylation focuses sIgA within the epithelium’s thin mucus layer. By keeping viruses, bacteria, and toxins near the surface, immune exclusion is achieved. IgE was developed to combat parasites (worms, protozoa). It is not present in plasma in large quantities like other isotypes. Most of it is tightly bound to the high-affinity Fce receptor of mast cells. These receptors are located in connective tissue beneath outer and inner surfaces (e.g. skin, gut, and bronchi). When a worm enters the epithelial barrier it binds and crosslinks specific IgE. This results in mast cell degranulation. Additional IgE can bind to the parasite. Mast cells produce histamine and other molecules that attract eosinophils. The H1 receptors induce an inflammatory response that facilitates movement of eosinophils. They are guided in their chemotaxis via H4 receptors. Eosinophil granulocytes also express Fce-receptor and attack the parasite by secretion high-toxic basic proteins from large eosinophil granules. Parasite infections are becoming less common in developed countries. The immune system can confuse harmless entities like grass pollen or tree pollen with dangerous parasites. This is a problem. IgE that is normally useful can then become a problem, causing hay fever and bronchial asthma. IgD can be found on the cell membranes of newly formed B lymphocytes and in small amounts in plasma. It is unlikely that soluble IgD plays a role in defense. 2.4 IMMUNOGLOBULIN DIAGNOSIS It is possible to measure the concentrations of an entire class of immunoglobulins (e.g. IgE in blood) or antigen-specific immunoglobulins. Antigen-specific antibody concentrations used to be expressed in “titer” form. The titer is the final step in a serial dilution giving positive results in qualitative tests. A classic example of such a vintage test is the complement fixation test. This test uses erythrocytes to test whether they lyse after adding a serum dilution or complement. The patient’s serum was dilute 1:10 – 1:120 – 1:140 -1:80, 1:160 – 1:1320. The titer of the antibody was 1:160 if the test comes back positive at 1:10 through 1:160, but not at 1:1320. It was often expressed reciprocally, with a “titer of 160”. We’ll be looking at three different methods to measure antibody concentrations: Western blot, immunofluorescence and ELISA. Monoclonal antibodies will be required for all three. 20 In the beginning, monoclonal antibodies were required to detect biomolecules. A rabbit, a laboratory animal, was given the purified molecule and its serum was used for immunologic testing. However, this antiserum (in lab jargon, “polyclonal antibodies”) is not a precise tool. It has a wide range of antibodies that can be used against any antigens that the lab animal came in contact with. These side specificities can cause test results to be distorted. Monoclonal antibodies Monoclonal antibodies eliminate the specificity problem because they are amplified copies of one antibody that is produced by a single cell. It is tedious and time-consuming to create a monoclonal antibodies. The usual procedure involves repeatedly immunizing a mouse with the antigen of concern, in this case, human IgM. After several weeks of receiving human IgM injections, antibodies will be produced against human IgM. The majority of the antibodies produced by B cells will be found in the mouse’s liver. These cells must be removed from the animal to obtain them. It would be easy to simply take these cells and harvest the desired antibody. However, the cells will stop growing and cease proliferating very quickly. They are fused with a mouse tumor cell strain to give them unlimited survival and proliferation potential. The tumor cells also have a biochemical Achilles heel, which can be used later to eliminate unwanted cells. A simple laboratory procedure with polyethylene glycol can perform cell fusion. The fusion reaction will not only produce the desired B/tumor cells fusions but also many non-fused cells. The next step is to ensure that only the desired fusion cells survive. They will die after only a few days if they are not fused or unfused. Fused tumor cells or cells that are not fused are dangerous as they can quickly outgrow the cells. They can be killed using a trick. The tumor cell line is deficient in an enzyme important to recycle purine nucleotides, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The tumor cells need tetrahydrofolic acids to continue to grow and produce new purine bases. It is possible to stop the growth of tetrahydrofolic acids by adding aminopterin, its antagonist, to the culture. After fusion, cells are cultivated in HAT media, which is named after hypoxanthine, aminopterin, and thymidine, which can also be made without tetrahydrofolic acids. What happens next? What happens? B cells die anyway. Only desired B cells/tumor cells fusions survive. They can proliferate because they use an intact copy of HGPRT from the mouse B-cell to recycle purines. These cells are called hybridoma cells after a time in culture. This is a fusion cell which grows like lymphoma. These are all the types of B cells that were originally found in the mouse’s spleen. Many of these cells will not make any antibodies, some will produce antibodies that are unrelated to our antigen and few will produce high affinity antibodies to human IgM. How do you find them? Next, limit dilution is used. Hybridoma cells in medium are diluted and then distributed across hundreds to thousands of microtiter wells. It is important that only one hybridoma cell is present in each well. Monoclonal means that the hybridoma cells growing up will be monoclonal. The antibody produced by hybridoma cells is secreted into the culture supernatant or medium. There is still the challenge of finding the two, three, or five cell clones that produce antibody against our antigen. This is in addition to the thousands of other clones or none at all. An immunological assay (usually ELISA) is used to detect antibodies binding our antigen. Once the hybridoma cell-clone is found, it can be grown and maintained indefinitely. Monoclonal antibodies can also be isolated from the culture medium. Monoclonal antibodies that are monoclonal against the most important diagnostic macromolecules of today are readily available. Monoclonal antibodies are also increasingly being used in anti-TNFa therapy. They are mainly made from mouse anti-mouse antibody (HAMA) and would therefore elicit an immune reaction in humans. Monoclonals that have been “humanized” are used. This means that all of the parts of the mouse antibodies not required for binding antigen are replaced with human counterparts. ELISA The concentration of antibodies in sera from patients can be measured using many methods, but the most popular is ELISA (enzyme linked immunosorbent assay). A test for IgM could be used to determine if there has been a recent virus infection. The microtiter plates are coated with virus protein or virus. Next, incubate the wells with diluted patient serum. If antibodies are present, they will bind the plastic-bound viruses proteins. Monoclonal mouse antibody against the human IgM is then added after thorough washing. This antibody is identical to the one we made in the previous section but has been linked with an enzyme like horse radish peroxidase. The enzyme-linked antibody will also bind if there is anti-virus IgM present in the patient’s serum. The enzyme-linked antibody that was not bind to the serum will be removed if it does not contain anti-virus IgM. Then, a colorless substrate molecular is added. This is then metabolized by horseradish peroxidase to produce a bright color pigment. Photometrically, the amount of color is determined by dividing the anti-virus IgM level in the patient’s serum. Photometrically, the color indicates that the patient has IgM for the virus. A negative color signifies that there is no anti-virus IgM. A parallel test could be performed using another monoclonal antibody to human IgG. This would allow the patient to verify if they were infected with the virus longer ago. Western blot (immunoblot). Western blots can be used to confirm HIV infection. The HIV proteins are separated using a polyacrylamide gel, and then denatured with the detergent SDS. The ELISA uses the same steps to blot the contaminated virus proteins. First, the membrane is treated with diluted patient serum. Next, it is treated with an enzyme-linked monoclonal anti human antibody. Finally, it is coated with substrate. There are washing steps in between. If the patient has antibodies against HIV, this will show in the form of colored bands on the membrane.Immunofluorescence Sometimes, for instance in autoimmune disease, it is important to test whether a patient has antibodies against certain tissue structures, without knowing the exact molecule the antibody might recognize. A droplet of diluted anti-nuclear antibody is used to determine if a patient has antinuclear antibodies. Cells or a section of tissue are placed on a glass slide, and then incubated with the droplet. If there are antibodies that bind to a nuclear structure, these can be detected again using a mouse monoclonal antibody against human antibody coupled with fluorescent dye. The fluorescence microscope will show brightly visible nuclei if the patient has anti-nuclear antibodies. In the absence of ANA they will remain dark. Anti-nuclear antibodies don’t bind to the cell nuclei in vivo, and should therefore not be present. They can bind to autoantigens in the cell nucleus (DNA or histones) when they are released from necrotic cell–cell death via apoptosis prevents this problem. These immune complexes can then lead to diseases like systemic lupus. Immunoelectrophoresis For an overview whether normal amounts of IgM, IgG and IgA are present in human serum, immunoelectrophoresis is informative. First, serum proteins must be separated electrophoretically using a gel. Next, the rabbit anti-human serum is applied to a groove parallel to the axis for separation. The gel is then agitated to allow the rabbit anti-human serum to diffuse through it towards the separated proteins. The formation of precipitation arcs where serum proteins meet antibody allows for the identification of three distinct arcs for IgM/IgG and IgA. This arc would not be present if IgA is deficient. 2.5 THE GENERATOR FOR ANTIBODY DIVERSITY What is the secret to our ability to create antibodies against almost any antigen in the world? The polypeptide chains that make up antibodies are genetically encoded. However, the human genome is only about 25,000. Even if all of them were able to encode antibodies, it wouldn’t be enough. This conundrum is solved by the unique molecular random generator, rearrangement (somatic-recombination). Parts of the heavy and light chains form the variable region of an immunoglobulin. The variable portion in the heavy chain is not encoded in a linear fashion in the genome. Instead, it is split into separate gene segments of three types: V, D, and J (variable diversity and joining). Each of these segments can be found in different ways. For the heavy chain, there are 40 (V), 23 (20 D) and 6 (16 J). The complete variable region of the heavy chain is assembled randomly by adding one V, one D, and one J segment to the DNA. An enzyme complex that contains RAG-proteins, (recombination activating genes), excises interfering DNA by recognizing so called recombination signals sequences (RSS). Normal DNA repair proteins then rejoin the segments. There are 40x23x6 methods to recombine segments. This results in 5520 possible heavy chains just by arranging the building blocks. This is just one example. The rejoining process can be messy. Nucleotides can be lost, or more often, added by the enzyme terminaldeoxynucleotidyltransferase (TdT), which can cause enormous variability. This is known as junctional diversity, or imprecise linking. Individually, light chain genes are made along the same lines. However, they don’t have D segments. They only have V and J segments. There are 320 possible ways to build a light-chain using the k locus as well as the l locus. Randomly generated heavy chains can be combined with randomly generated light ones to increase the level of variability. You can generate different antibodies by simply rearranging the building blocks without imprecise joining. Immature bone marrow B cells precursors are used for somatic recombination. Specific quality control mechanisms are used to monitor the maintenance of a productive reading frame. A specific kinase called BTK (Bruton’s tyrosine kinase) signals the successful assembly of a heavy-chain. The absence of a BTK signal means that both the heavy chain gene and the B cell are affected by frame shifts. This causes the B cell to become ineffective and go into apoptosis. After an antibody is assembled successfully, it can be expressed as a transmembrane proteins in the form a B-cell receptor. There is a difference between secreted and B cell receptor antibodies. This is due to a transmembrane region encoded by an exon that can be added or removed by alternative splicing. Cross reference in Pharmacology: Ibrutinib is an inhibitor of Bruton’s tyrosine kinase. It is used to treat CLL, and other B-cell non Hodgkin lymphomas. Somatic hypermutation is an additional mechanism that can increase variability and allow for the development of high-affinity antibodies. This happens especially when the antigen cannot quickly be eliminated. The rate of somatic mutation occurs at approximately a thousandfold higher rates in B cells that are rapidly multiplying in lymphoid centers. These regions that determine complementarity are also known as hypervariable areas. How does this mutation rate occur? The most common form of DNA damage in cells is spontaneous hydrolytic desamination of Cytosine. This causes uracil. This process is only possible in B cells. The enzyme AID (activation induced cytidine desaminase) is activated to speed it up. AID can only be activated in genomic regions with high levels of transcription, since the DNA strands must be separated to allow the enzyme to function. Deamination is the equivalent of a point mutation. While cytosine pairs up with guanine in a cytosine pair, uracil forms two hydrogen bond with adenine in a uracil. Secondary repair processes, which uracil is prohibited in DNA, can lead to additional exchange possibilities. These mutations can increase antibody affinity. The respective B cells will then be able to retain antigen longer and receive a stronger stimuli to proliferate. Over time, somatic hypermutation favors the shift to antibodies with higher affinity. There are four mechanisms that contribute to antibody diversity. This seems like an absurdity. Isn’t DNA supposed to transmit genetic information as accurately as possible? This rigid system is how it is possible for a random generator to develop. Comparing different species we see that all vertebrates use a RAG-based random generator in order to increase defense against infections. All vertebrates? Not quite! Some primeval jawless fish species, such as lamprey or hagfish, do not have this ability. Our genome does not contain a minimalistic, high-tech machine. It is more like a confused accumulation of ancient sediments. It contains multiple copies of “molecular non-sensical machines”, such as retroviruses or transposons, many of which are inactivated by mutations. What does “molecular-nonsense machines” mean? Imagine a contraption that can only make copies of itself. If there were enough resources, this would result in a flood of machines. Viruses are, in principle, nothing more than that. A unit of DNA that contains the information necessary to make enzymes, with the ability to extract the unit from the surrounding DNA and implant it elsewhere, is another type of nonsense machine. This is called a transposon. The following genetic accident occurred in the Silurian, approximately 440-420 million years ago: An active transposon was inserted into a transmembrane gene. It was fatal! It could be reexcised by the transposon, which would make it possible to heal. This structure was the nucleus for our antibody-and T cell receptor-loci. It evolved through multiple locus doublings, followed by mutational drift. The original transmembrane proteins and the RAG proteins correspond to the transposon nucleases. The original transposon’s left and right delimitations for excision were usually all that was left. These base sequences, which we now call recombination signals sequneces (RSS), are short base sequences. Only one transposon copy, the one on chromosome 11, has active nucleases. The two small RAG genes, which are very close together, do not contain introns. This is a rare feature in humen gene transcriptions. This genetic “accident” occurred in a Silurian fish, which allowed the random generator to the adaptive immune system. This “invention” gave rise to a huge selective advantage in the fight against infective threats. The offspring of this fish drove all other vertebrates into extinction with the exception of some lampreys and hagfish. Rearrangement can create a variable region that can be passed from one isotype of the organism to another. This can be done again by cutting and pasting DNA. RAG proteins are not involved in this process. Exons that encode the constant regions for all classes of antibodies are located on chromosome 14. They are clustered with u (for IgM), plus d (which will not be considered) being the closest to variable region segments. Next, g (IgG), an (IgA), and e (IgE). The production of IgM is achieved by using the closest constant region, which is u after a successful VDJ rearrangement. The segment that encodes u and d in the first B cell is removed. This positions the exons that code the g constant area adjacent to the VDJ. After undergoing class switch, these cells now produce IgG. The variable region remains the same. The antibody binds to the same antigen with identical affinity but it is now IgG. An analogous situation is when an immune reaction occurs, there are two possible classes of class switch: a results in IgA and e results in IgE. The cytokines produced by T-lymphocytes, other cells, can influence the probability and type of class switching. In the germinal centers secondary follicles, class switch takes place spatially and time-synchronistically with somatic hypermutation. Both are initiated by the same enzyme, AID. Switch regions in gene segments that are part of heavy chain constant regions can easily form single-chain DNA loops. These temporary loops are where AID deaminates cytosine and leads to uracil. This is actually a targeted, accelerated version of spontaneous deamination through hydrolysis which occurs regularly in our cells. Uracil is a “wrong base” in DNA that can be quickly removed by a dedicated repair program. Uracil is removed by UNG (uracil DNA-glycosylase), followed by removal of deoxyribose by APE1 (apurinic/apyrimidinic endonuclease 1), generating a single strand break as part of the normal repair process. A double strand break is possible if the same thing happens on the opposite strand, just a few nucleotides down. This form of DNA cleavage is possible in class switch recombination. It occurs simultaneously at two different locations. Interrupting DNA with heavy chain segments u or d is removed and the far ends are joined using the non-homologous ending joining (NHEJ), double strand break repair method. VDR segments are placed next to exons that encode the g heavy chains (or less often the a and e chains), which results in class switching from IgM (or IgA) to IgG (or IgA or IgE). 2.6 HOW DO YOU DISTINGUISH ENTIRELY DANGEROUS, USELESS, AND USEFUL ANTIBODIES Is it not dangerous to have random antibodies? Depending on the infection, one would expect to find some useful antibodies. However, more antibodies will likely be ineffective and even harmful. They could cause autoimmune disease by binding to our bodies. Safeguards exist. Clones of B cells that have rearranged antibodies to recognize ubiquitous selfantigens are prone to apoptosis (clonal deletion), or they can become frozen and cannot be activated (central anergy). These protective mechanisms don’t always work well and sometimes autoantibodies can be made. Infecting pathogens is a way to distinguish between useful and non-useful antibodies. Infected pathogens are constantly changing the structure of new antibodies in bone marrow cells. They migrate to the peripheral lymphatic tissues once it becomes clear that they don’t recognize self-antigens. Many die after waiting in vain. An infection will result in a wide range of antibodies being produced by the invading pathogen. These antibodies act as “B cells receptors” and are found on resting lymph nodes and other lymphoid tissues. One out of every million B cell receptors that matches an antigen of the pathogen is activated to cause a specific B cell to become proliferative. All other B cells are not affected. This is known as “clonal selection”. It is the antigen that selects cells that can react to it. Thus, it determines which antibodies are useful. An activated cell produces many daughter cells, a clone. These cells differentiate and secrete large quantities of antibody. There is a transmembrane region at the terminus of their heavy chain that can be included or excluded by alternative transcription. This is what makes the difference between secreted and B cell receptor. Our immune system is always fighting subliminal infections so there are many proliferating “useful” B cells. The random nature of antibody production means that the percentage of useful cells is higher than one would expect. 2.7 T Cell Help Antibodies can be dangerous tools that could cause autoimmune damage. If a single contact between a B cell receptor and an antigen was sufficient to trigger large-scale antibody production, it would be very dangerous. As a safety measure, a “safety trap” must be released before a B-cell can activate. This is analogous to a gun. This complex process is called “T cell help” and it’s accomplished through a complex process. The exception to this rule is the so-called “T cell independent antigens”. These antigens are often linear antigens that have repetitve epitopes and are capable of crosslinking multiple B-cell receptors or other pattern recognition receptors. This activation results in the production of IgM with a low affinity. Without T cell support, neither affinity maturation nor class switching is possible. Understanding how T cells interact with other cells and lymphoid tissues is essential in order to understand their functions. 2.8 LYMPHATIC SYSTEM MARROW and THYMUS are the primary lymphatic organs. These are the places where new, “naive”, B- and/or T cells are generated and rearrange their receptors. The bone marrow is where hematopoietic stem cell give rise to lymphoid precursor cells. These B cells are differentiated in the bone marrow. However, the name B cell comes from the gut-associated organ of birds, the bursa Fabricii. This organ doesn’t exist for humans. The thymus, which is located on top of your heart, houses lymphoid progenitors. Here they differentiate and go through complex quality control procedures. Only a fraction of these mature naive thymocytes can leave the thymus. (Explained in section 2.2.1). The thymus is an embryonic organ that develops from the 3rd and 4th pharyngeal pouches. It is of endodermal origin, with two diverticula which unite in the middle. They then migrate caudally to the developing organ. A deletion of 30-50 genes can cause the development to be disrupted in the most severe case of complex DiGeorge syndrome. The parathyroid glands, which develop from the same area, are often missing in this case. This can lead to T-cell defects and hypocalcemia. The thymus in children and adolescents is densely filled with epithelial and thymocyte cells. Adults are seeing fatty tissue replace larger areas. It isn’t as bad as you might think. Adults have been through multiple infections so they are well-equipped with memory cells. This reduces the need for T cells. 27 Mature, naive B- and T cells, as well as precursors of APC (antigen presenting cells, including monocytes/macrophages and dendritic cells) from the bone marrow emigrate from the central lymphatic organs. The bloodstream is the main route lymphocytes use to travel. APC travel through the bloodstream to freely roam tissues. All types of cells eventually meet at the peripheral lymphatic organs, which include lymph nodes and GALT/Peyer plaques, tonsils, BALT, and spleen. LYMPH nodes appear static under the microscope but can be better compared to a large international airport with a lot of cells constantly arriving and departing. There are several outlets and inlets for lymph nodes. The interstitial fluid from the blood capillaries is transported by the afferent lymphatic vessels that reach the most peripheral lymph nodes. The lymph flow causes dendritic cells laden with ingested material to drift to the lymph nodes. The lymph flow can increase dramatically in the event of infection. It carries with it pathogens as well as their antigenic molecules. This is both outside and inside activated macrophages, dendritic, and dendritic cell. A lymph node acts as a command center that provides real-time information about the antigenic situation at the periphery. Through specialized high-endothelial veins, lymphocytes continuously enter the lymph node from the blood. If activated, B cells move to the cortex and form germinal centers in follicles. Specialized “follicular Dendritic” cells are able to immobilize immune complexes and their Fc- or complement receptors. This makes the antigens “visible” for proliferating cells. T cells can wander to nearby paracortical regions. In the lymph node’s middle, there are activated B cells which have already differentiated into plasma cells and more macrophages. Each lymph node is connected to the next lymphode via an efferent vessel. Finally, the lymph node can be reached by the thoracic drain to the blood. Warning: Dendritic and follicular cells are two completely different types of cells that have been given similar names (dendritic = trees-like). Specialized APC that ingest antigen in their periphery and present processed antigen on MHC2 to T cells, dendritic cells. Follicular dendritic cell sits in germinal centers. They use complement receptors as well as Fc receptors to fix antigen containing immune complexes onto their outer surfaces for B cells to view. GALT (gut associated lymphoid tissue), includes Peyer’s patches within the small intestine and lymph follicles distributed along the entire intestinal wall. Peyer’s patches consist of functional units made up of specialized epithelium with M-cells (microfolded, multifenestrated), that transport small amounts antigen through the epithelial barrier via transcytosis. The underlying lymphatic tissue contains dendritic cells and B-cell follicles, as well as peripheral T-helper area. The GALT-activated lymphocytes’ clonal descendants travel via blood and lymphatics to recirculate back into the GALT and other mucosa-associated lymphoid tissue. Most plasma cells derived mostly from activated B cells make dimeric IgA. This is then transported back into the lumen. These mechanisms not only protect our mucosal surfaces, but also allow breastfeeding mothers to protect their baby by secreting IgA against the same oral pathogens that her immune system detects. The double-edged sword of transcytotic uptake from the gut via M cells is transcytotic.