Immunology and Immunotechnology note.pptx

Lecture note

1. Immunotechnology BIOT 531 Haramaya University 1 Dr. Zekeria Yusuf

2. Topics to be covered IMMUNE SYSTEM AND IMMUNE RESPONSEs ANTIGENS AND ANTIBODIES MAJOR HISTOCOMPATIBILITY COMPLEX AND TRANSPLANTATION HYPERSENTITVITY REACTIONS: Immunological techniques ATOIMMUNITY AND IMMUNOMODULATION : immunization Dr. Zekeria Yusuf 2

3. Immunology Immunology is the study of our protection from foreign macromolecules or invading organisms and our responses to them. •Host – e.g. human,… •Foreign macromolecule, antigen – e.g. virus protein, worm, parasite (Everything that should not be in my body) Dr. Zekeria Yusuf 3

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5. Immunology has its origins in the study of how the body protects itself against infectious diseases caused by microorganisms, such as bacteria, viruses, protozoa, and fungi, and also parasitic organisms, such as helminth worms. Important initial barriers to infection are physical (e.g. the skin ), enhanced by substances secreted by the body, such as saliva and tears, that contain molecules that can neutralise bacteria. The internal mucosal tissues (e.g. lungs & airways , and the gut ) are coated with mucus that is able to trap potential infectants . In the airways, mobile ciliate hairs work together to transport contaminants away from vulnerable areas. Tissues such as the skin, mucosal surfaces and airways also contain populations of immune cells that can respond to infectants that breach these physical defences. Both systems work in close cooperation and, to an important extent, the adaptive immune system relies upon the innate immune system to alert it to potential targets, and shape its response to them. 5 Dr. Zekeria Yusuf

6. Immune Sysyem Immune system = cells, tissues, and molecules that mediate resistance to infections Immunology = study of structure and function of the immune system Immunity = resistance of a host to pathogens and their toxic effects Immune response = collective and coordinated response to the introduction of foreign substances in an individual mediated by the cells and molecules of the immune system 6 Dr. Zekeria Yusuf

7. Immune tissues All immune cells originate in the bone marrow , deriving from haematopoietic stem cells , but an important set of immune cells (T lymphocytes) undergo maturation in an organ known as the thymus . The thymus and bone marrow are known as primary lymphoid tissues . Secondary lymphoid tissues , namely the lymph nodes , spleen and mucosa-associated lymphoid tissues ( MALT ) are important sites for generating adaptive immune responses and contain the lymphocytes (key adaptive cells). The lymphatic system is a system of vessels draining fluid (derived from blood plasma) from body tissues. Lymph nodes, that house lymphocytes, are positioned along draining lymph vessels, and monitor the lymph for signs of infection. MALT tissues are important in mucosal immune responses, and reflect the particular importance of the gut and airways in immune defence. The spleen essentially serves as a ‘lymph node’ for the blood. 7 Dr. Zekeria Yusuf

8. Microbes are ubiquitous in nature, extraordinarily diverse, rapidly evolve to exploit opportunities to infect hosts and to evade their immune systems. Microbes: why they are formidable foes? 8 Dr. Zekeria Yusuf

9. 8 hours = 280 trillion bacteria!!!! Exponential growth Number of bacteria Time (hrs) Many pathogens can expand rapidly in the nutrient-rich environment of the host. 9 Dr. Zekeria Yusuf

10. Pathogens rapidly evolve to avoid the host immune response Virally encoded decoy receptors 10 Dr. Zekeria Yusuf

11. Gross anatomy of the immune system Some key definitions: Pathogen : microbe that causes disease Antigen : material (from a pathogen) that induces an immune response Innate (natural) immunity : rapid, non specific immune response Adaptive (acquired) immunity : slower, specific immune response Leukocytes : blood cells Lymphocytes : specialized blood cells that mediate adaptive immunity (e.g. T and B cells) 11 Dr. Zekeria Yusuf

12. The cells of the immune system circulate through the body via lymph and blood. Pathogens and their antigens are transported from tissues via lymphatic vessels to the lymph nodes where they encounter immune cells. 12 Dr. Zekeria Yusuf

13. The cells of the immune system spend much of their time in lymphoid organs. They develop (arise) in primary lymphoid organs , and they interact with antigens in secondary lymphoid organs . Thymus : primary lymphoid organ for T cell development Bone marrow : primary lymphoid organ for B cell development Lymph nodes : collect antigens from tissues Spleen : collects antigens from blood stream 13 Dr. Zekeria Yusuf

14. PRINCIPAL FUNCTION OF THE IMMUNE SYSTEM • To protect humans from pathogenic microorganisms • Pathogenic microorganisms (Pathogens) – Microorganisms capable of causing infection and/or disease • Infection : – Ability of pathogen to enter host, multiply and stimulate an immune response • Disease: – Clinical manifestations associated with infection Dr. Zekeria Yusuf 14

15. Role of the immune system Defense against microbes Defense against the growth of tumor cells kills the growth of tumor cells Homeostasis destruction of abnormal or dead cells (e.g. dead red or white blood cells, antigen-antibody complex) 15 Dr. Zekeria Yusuf

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17. Immune System Organs Cells Molecules 17 Dr. Zekeria Yusuf

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23. Immune system…. Innate Immunity Humoral Immunity B cells, antibody, complement Phagocytes, Toll-like receptors Cellular Immunity T cells, TCR, development, selection, tolerance, NK cells Immune Responses inflammation, allergy, autoimmunity, infection 23 Dr. Zekeria Yusuf

24. Immune System: (1) organs Tonsils and adenoids Thymus Lymph nodes Spleen Payer’s patches Appendix Lymphatic vessels Bone marrow Primary Lymphoid Organs –Bone Marrow and Thymus –Maturation Site • Secondary Lymphoid Organs –Spleen, lymph nodes, –MALT (mucosal associated lymph tissue) –GALT (gut associated lymph tissue) –Trap antigen, APC , Lymphocyte Proliferation

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27. Immune system: (2) cells Lymphocytes T-lymphocytes B-Lymphocytes, plasma cells natural killer lymphocytes Monocytes , Macrophage Granulocytes neutrophils eosinophils basophils

28. Immune system: (3) molecules Antibodies Complement Cytokines Interleukines Interferons 28 Dr. Zekeria Yusuf

29. The Nature of Disease • Pathogenic Organisms • Genetic Disorders • Toxic Chemicals • Other Environmental Factors • Physical Damage to Organs • Nutritional Disorders Types of Pathogenic Organisms • Viruses • Bacteria • Protozoan • Fungi • Animal • Parasites Dr. Zekeria Yusuf 29 Immunity is body's ability to resist or eliminate potentially harmful foreign materials (antigens) or abnormal cells and pathogens.

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35. History: what imparts Immunity? Emil von Behring and Kitasato (1890) Serum from vaccinated animals was protective ( diptheria ) Metchinkoff (1880) Cell based Immunity Merrill Chase (1940)- Transfer of WBC (immunity to tuberculosis) Both serum and cells contribute to immunity 35 Dr. Zekeria Yusuf

36. The immune system A functional system – NOT an organ system: Complex system – includes Skin – physical barrier Lining of mucus membranes – physical barrier Secretions – tears, mucus etc - antimicrobial Blood cells and vasculature – WBCs Bone marrow Liver – makes compl e ment proteins Lymphatic system and lymphoid organs Most tissues – have resident immune cells 36 Dr. Zekeria Yusuf

37. Why study the immune system? Importance of the immune system in human health Provides model systems for studies of: gene regulation molecular recognition signal transduction etc, etc Provides powerful techniques for use in medicine and science 37 Dr. Zekeria Yusuf

38. Immune dysfunction Important pathologies may result from immune dysfunction. Inborn (‘congenital’) immunodeficiencies , with a genetic basis, can disable all, or part, of the immune response (both innate and adaptive) – resulting in vulnerabilities to infection or cancers. Examples include severe combined immunodeficiency ( SCID ) and common variable immunodeficiency ( CVID ). In addition, autoimmunity occurs when the immune system mistakenly targets self tissues, resulting in chronic inflammatory conditions and tissue destruction. Examples include: type 1 diabetes , rheumatoid arthritis , and multiple sclerosis . 38 Dr. Zekeria Yusuf

39. Transplantation science Identification of the important role of the MHC in allowing the body to discriminate between self/non-self tissues has greatly enhanced the success of tissue and organ transplantation , by allowing tissue matching . This has been aided by the development of immunosuppressive drugs that are becoming increasingly sophisticated as we identify more specific elements within the immune system to target. 39 Dr. Zekeria Yusuf

40. Vaccines Vaccines can utilise harmless elements from particular pathogens to prime the immune system, so that if the pathogen is actually encountered, it is met with a stronger secondary (‘memory’) response and dealt with more quickly. Alternatively, vaccines may also utilise live, but attenuated, variants of the pathogen to induce a protective immune response. The role of vaccines remains central to the importance of immunology as a healthcare science– with keystone contributions in the disease areas of smallpox , polio , tuberculosis , measles , mumps , rubella and papillomavirus , amongst many others. However, success can depend on the target pathogen, and effective vaccines for HIV , hepatitis C and malaria remain elusive, in large part due to the mutability of these organisms as targets for the immune system. 40 Dr. Zekeria Yusuf

41. Diseases associated with immune system dysfunction Autoimmunity (SLE, Arthritis, Myasthenia gravis, Graves disease) Immunodeficiency (inherited, acquired) Allergy (environment, drugs) Cancer (Leukemia, Lymphoma) 41 Dr. Zekeria Yusuf

42. Myasthenia gravis 42 Dr. Zekeria Yusuf

43. Asthma & Allergy 43 Dr. Zekeria Yusuf

44. Therapeutics Vaccination Adoptive immunotherapy Transplantation 44 Dr. Zekeria Yusuf

45. Treating cancer 45 Dr. Zekeria Yusuf

46. Powerful methods for detecting and quantitating proteins and cells based on the highly specific binding of antibodies ( immunoglobulins ) ELISA: e nzyme- l inked i mmuno s orbant a ssay (measures small amounts of hormones, drugs, microbes in body fluids) Western blot (detects disease associated proteins) Flow cytometry (quantifies various cell types in mixed population-- HIV patients) 46 Dr. Zekeria Yusuf

47. Western Blot Separate proteins by size using PAGE gel Transfer gel to blotting membrane Probe membrane with antibody specific for protein of interest Detect bound antibody by chemiluminesence 47 Dr. Zekeria Yusuf

48. Immunofluoresence Immunofluoresence 48 Dr. Zekeria Yusuf

49. Flow Cytometry: Quantitative Single-cell Immunofluoresence 49 Dr. Zekeria Yusuf

50. Immunity Immunity ( immunis - Latin-exempt, state of protection from infectious diseases) Immunity is body's ability to resist or eliminate potentially harmful foreign materials or abnormal cells consists of following activities: Defense against invading pathogens (viruses & bacteria) Removal of 'worn-out' cells (e.g., old RBCs) & tissue debris (e.g., from injury or disease) Identification & destruction of abnormal or mutant cells (primary defense against cancer) Rejection of 'foreign' cells (e.g., organ transplant) Inappropriate responses: Allergies - response to normally harmless substances Autoimmune diseases 50 Dr. Zekeria Yusuf

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57. Two types of immunity Innate (non-adaptive) first line of immune response relies on mechanisms that exist before infection Acquired (adaptive) Second line of response (if innate fails) relies on mechanisms that adapt after infection handled by T - and B - lymphocytes o ne cell determines one antigenic determinant 57 Dr. Zekeria Yusuf

58. A typical immune response INNATE IMMUNITY Rapid responses to a broad range of microbes ACQUIRED IMMUNITY Slower responses to specific microbes External defenses Internal defenses Skin Mucous membranes Secretions Phagocytic cells Antimicrobial proteins Inflammatory response Natural killer cells Humoral response (antibodies) Cell-mediated response (cytotoxic lymphocytes) Invading microbes (pathogens) Complement 58 Dr. Zekeria Yusuf

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61. Innate immunity Mast cells and basophils are innate cell types that, when activated, secrete histamine, which can be an important inflammatory mediator produced in response to initial tissue damage as a result of infection. Mast cells are tissue resident (e.g. in mucosal tissues) whilst basophils are found in the blood. In particular, they play a key role in the so-called allergic response . Innate immunity comprises both cellular and humoral (‘in solution’) elements. The cellular elements are represented notably by phagocytes (specifically neutrophils and macrophages ) that can respond to signs of infection (i.e. inflammation) in the tissues and home-in on infective bacteria before neutralising and engulfing them (‘ phagocytosis ’). Recognition of microorganisms by the innate system occurs via characteristic pathogen-associated molecular patterns ( PAMPs ) on microbial surfaces, and an important family of innate receptors called pattern-recognition receptors ( PRRs ) are responsible for this (notably including Toll-like receptors [ TLRs ]). The natural killer ( NK ) cell is another important innate cell that is able to detect and target intracellular infection of body cells by viruses. A further specialised innate cell is the eosinophil that plays a particular role in targeting larger infective organisms, such as parasitic worms. 61 Dr. Zekeria Yusuf

62. Found in: Substances that trigger: Receptors: all multicellular organisms A limited number of pathogen-associated molecular patterns (PAMPs) A limited number of Pattern Recognition Receptors (PRRs) expressed on many cells types Vertebrates only Virtually any component of pathogens Highly variable receptors of 2 types: antibody made by B cells and TCR made by T cells 62 Dr. Zekeria Yusuf

63. Comparison of the adaptive and innate immune responses innate adaptive Response time hours days Response to identical to primary stronger response upon repeat infection second exposure Receptors that pattern recognition receptors antibodies and T cell antigen receptors (TCR) Mediate pathogen Toll-like receptors (TLR) Recognition limited diversity, unlimited diversity fixed in germline generated by V(D)J recombination Ligands Pathogen associated molecular virtually any component of pathogen patterns (PAMPs) 63 Dr. Zekeria Yusuf

64. Innate immunity vs Adaptive Immunity No memory No time lag Not antigen specific A lag period Antigen specific Development of memory Innate Immunity (first line of defense) Adaptive Immunity (second line of defense) 64 Dr. Zekeria Yusuf

65. Innate immunity Based on genetic make-up Relies on already formed components Rapid response: within minutes of infection Not specific same molecules / cells respond to a range of pathogens Has no memory same response after repeated exposure Does not lead to clonal expansion 65 Dr. Zekeria Yusuf

66. Physical & Biochemical barriers 66 Dr. Zekeria Yusuf

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68. Defensins Originally isolated from frog skin based on their ability to kill bacteria Small polypeptides (<10kDa) secreted at mucosal surfaces Direct bacteriocidal properties Insertion into biological membranes leading to target cell lysis Inhibited by cholesterol (specificity) 68 Dr. Zekeria Yusuf

69. Phagocytosis “cellular eating” 3. Phagosome fuses with lysosome. 1. Bacterium attaches to membrane 2. Bacterium is ingested, forming phagosome, 4. Lysosomal enzymes digest the bacteria. 5. Digested material is released from cell. Phagocytes: macrophage, neutrophils, dendritic cells 69 Dr. Zekeria Yusuf

70. Phagocytes use a variety of methods to destroy ingested microbes. (household bleach) 70 Dr. Zekeria Yusuf

71. Macrophage fight microbes by phagocytosis and production of toxic molecules A macrophage produces reactive oxygen species to aid in destruction of the microbe Reactive oxygen is revealed by the blue dye, NBT 71 Dr. Zekeria Yusuf

72. The innate i mmune System Interactions between the two systems 72 Dr. Zekeria Yusuf

73. White blood cells (WBCs) Macrophages B-lymphocytes T-lymphocytes Natural killer(NK) cells Mast cells 73 Dr. Zekeria Yusuf

74. Many different types of blood cells participate in the immune response to microbes: Innate immune cells: phagocytes ” macrophage, neutrophils , dendritic cells Adaptive immune cells: “ lymphocytes” T cells, B cells Cells of the immune system 74 Dr. Zekeria Yusuf

75. Most blood cells act to fight infection. Adaptive immunity Innate immunity Blood cells lineages. 75 Dr. Zekeria Yusuf

76. Lymphocytes of the adaptive immune system T helper cells: regulate other immune cells T cytotoxic (killer) cells: kill infected cells B cells: produce antibodies (immunoglobulin) Dendritic cells and macrophage: directly kill microbes by phagocytosis and other mechanisms. They also help to activate T cells (connection between innate and adaptive immunity) NK cells are lymphocytes that have characteristics of innate and adaptive immunity. 76 Dr. Zekeria Yusuf

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78. Some microbes hijack cellular machinery to replicate and spread. Intracellular pathogens include viruses (influenza, HIV) and intracellular bacteria (listeria) and intracellular parasites (malaria, toxoplasma). cell-autonomous defense: cell produces an immune response that acts on itself 78 Dr. Zekeria Yusuf

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82. CELLS OF INNATE AND ADAPTIVE IMMUNITY I. Myeloid Lineage: 1. Neutrophil : principal phagocytic cell of innate immunity 2. Eosinophil : principal defender against parasites 3. Basophil : functions similar to Eosinophils and mast cells. referred to as polymorph nuclear leukocytes ( PMN’s ), nuclei are multilobed (2 to 5). Granulocytes: cytoplasmic granules Dr. Zekeria Yusuf 82

83. Myeloid lineage – Monocytes : •Leukocytes with bean shaped or brain-like convoluted nuclei •Circulate in blood with half life of 8 hours •Precursors of tissue macrophages –Macrophages: •Mononuclear phagocytic cells in tissue •Derive from blood monocytes •Participate in innate and adaptive immunity Dr. Zekeria Yusuf 83

84. Myeloid lineage… – Dendritic cells: •Cells with dendriform (star shaped) morphology • Interdigitating reticular cells (synonym) •Capture and present antigens to T lymphocytes -Mast cells: •Located in mucous membrane and connective tissue throughout body •Major effector cell in allergy •Modulation of initial immune response Dr. Zekeria Yusuf 84

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92. CELLS OF INNATE AND ADAPTIVE IMMUNITY.. II. Lymphoid Lineage –Large lymphocytes (large granular lymphocytes): •Natural killer ( NK ) cells ( CD16 , CD56 ) •Innate immunity to viruses and other intracellular pathogens •Participate in antibody-dependent cell-mediated cytotoxicity ( ADCC ) – Small lymphocytes: •B cells ( CD19 ) •T cells ( CD3 , CD4 or CD8 ) • role in adaptive immunity Dr. Zekeria Yusuf 92

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96. FUNCTION OF B LYMPHOCYTES: 1) interact with antigenic epitopes , using their immunoglobulin receptors. 2) subsequently develop into plasma cells, secreting large amounts of specific antibody. 3) circulate as memory cells. 4) present antigenic peptides to T cells, consequent upon interiorization and processing of the original antigen. Dr. Zekeria Yusuf 96

97. CLONAL SELECTION OF B LYMPHOCYTES: clonal selection, accounts for antibody formation. Each individual has a large pool of B lymphocytes (about 107). Each immunologically responsive B cell bears a surface receptor (either IgM or IgD ) that can react with one antigen (or closely related group of antigens); i.e., there are about 10 7 different specificities. An antigen interacts with the B lymphocyte that shows the best "fit" with its immunoglobulin surface receptor. After the antigen binds, the B cell is stimulated to proliferate and form a clone of cells . These selected B cells soon become plasma cells and secrete antibody specific for the antigen. Dr. Zekeria Yusuf 97

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99. MEMORY T cells : Memory T cells, as the name implies, endow our host defenses with the ability to respond rapidly and vigorously for many years after the initial exposure to a microbe or other foreign material. This memory response to a specific antigen is due to several features: (1) many memory cells are produced, so that the secondary response is greater than the primary response, in which very few cells respond. (2) memory cells live for many years or have the capacity to reproduce themselves. (3) memory cells are activated by smaller amounts of antigen and require less costimulation than do naïve, unactivated T cells. (4) activated memory cells produce greater amounts of interleukins than do naïve T cells when they are first activated. Dr. Zekeria Yusuf 99

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105. STRUCTURE AND FUNCTIONS OF IMMUNE CELLS – T CELLS T lymphocytes also arise in the bone marrow. Unlike B cells, which mature within the bone marrow, T cells migrate to the thymus gland to mature. During its maturation within the thymus, the T cell comes to express a unique antigen-binding molecule, called the T-cell receptor, on its membrane.Unlike membrane-bound antibodies on B cells, which can recognize antigen alone, T-cell receptors can recognize only antigen that is bound to cell-membrane proteins called major histocompatibility complex ( MHC ) molecules. MHC molecules that function in this recognition event, which is termed “antigen presentation,” are polymorphic (genetically diverse) glycoproteins found on cell membranes. Dr. Zekeria Yusuf 105

106. MHC … There are two major types of MHC molecules: Class I MHC molecules, which are expressed by nearly all nucleated cells of vertebrate species, consist of a heavy chain linked to a small invariant protein called _2- microglobulin . Class II MHC molecules, which consist of an alpha and a beta glycoprotein chain, are expressed only by antigenpresenting cells. When a naive T cell encounters antigen combined with a MHC molecule on a cell, the T cell proliferates and differentiates into memory T cells and various effector T cells. There are two well-defined subpopulations of T cells: T helper (TH) and T cytotoxic ( TC ) cells.Although a third type of T cell, called a T suppressor (TS) cell, has been postulated, recent evidence suggests that it may not be distinct from TH and TC subpopulations. T-helper and T cytotoxic cells can be distinguished from one another by the presence of either CD4 or CD8 membrane glycoproteins on their surfaces. Dr. Zekeria Yusuf 106

107. THE CLUSTER OF DIFFERENTIATION (CD) • A protocol for identification and investigation of cell surface molecules • CD number assigned on basis of 1 cell surface molecule recognized by 2 specific monoclonal antibodies • CD nomenclature established in 1982 – 1st International Workshop and Conference on Human Leukocyte Differentiation Antigens ( HLDA ) Dr. Zekeria Yusuf 107

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114. Effector mechanisms: how the immune system protects Immune Effector Mechanisms : Cell-mediated immunity : Phagocytosis (cellular eating) cytotoxcity (cellular killing) Humoral immunity : complement: group of serum proteins that can directly kill pathogens. antibodies: (also called immunoglobulin) proteins secreted by B cells that bind directly and specifically to pathogens. Antibodies target pathogens by marking them for destruction by other components of the immune system. Macrophage fight microbes by engulfing and digesting them ( phagocytosis or cellullar eating). 114 Dr. Zekeria Yusuf

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117. Innate immunity: mechanisms Mechanical barriers / surface secretion skin, acidic pH in stomach, cilia Humoral mechanisms lysozymes, basic proteins, complement, interferons Cellular defense mechanisms natural killer cells neutrophils, macrophages,, mast cells, basophils, eosinophils Neutrophil NK Cell Monocyte Macrophage Basophils & Mast cells Eosinophils 117 Dr. Zekeria Yusuf

118. Neutrophils in innate immune response Most abundant WBCs (~50-60%) Efficient phagocytes Most important cells of the innate immune system 118 Dr. Zekeria Yusuf

119. Cellular and Inflammatory Components of Innate Immunity •Cellular – Phagocytic cells •Inflammatory – Vasodilation –Capillary permeability Phago = to eat Cyte = cell WBCs ( eg . Neutrophils ) – find, eat and digest microbes ! Neutrophils (a type of white blood cell) are attracted to bacterial products. Here they are moving toward a gradient of the bacterial peptide fMLP . 119 Dr. Zekeria Yusuf

120. How do neutrophils eat and digest microbes ? Granules 120 Dr. Zekeria Yusuf

121. What’s in the granules ? Lysozyme – digests bacterial cell wall; other antimicrobial proteins 121 Dr. Zekeria Yusuf

122. Additional role of neutrophils Triggers inflammatory response 122 Dr. Zekeria Yusuf

123. Monocytes Monocytes (~5% of WBCs) Migrate into the tissues and become Macrophages Lung Bone Liver Brain intestine 123 Dr. Zekeria Yusuf

124. Macrophages “Big eaters” Phagocytosis of microbes in tissue ( neutrophils are present only in blood) Antigen presentation 124 Dr. Zekeria Yusuf

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126. Natural killer cells Not B-lymphocytes / T-lymphocytes Important part of the innate immune system Kill virus /bacteria infected cells (Intracellular pathogens) Kills cancer cells 126 Dr. Zekeria Yusuf

127. NK cells differentiate choose cells to kill ? Uninfected cell / Normal cell Microbe infected cell / cancer cell Some cell surface proteins are missing 127 Dr. Zekeria Yusuf

128. How does the killer kill ? Kills both host cells and microbes Release of granules with perforins and proteases 128 Dr. Zekeria Yusuf

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131. Inflammation Complex biological process by which body responds to pathogens and irritants Associated with swelling of tissue Key player in innate immune response 131 Dr. Zekeria Yusuf

132. All roads lead to inflammation Inflammation Neutrophils Monocytes /macrophages NK cells TLRs Cytokines /IFN C` proteins Coagulation proteins Cellular Extracellular 132 Dr. Zekeria Yusuf

133. Inflammation and vascular changes Vasodilatation Increased capillary permeability Normal blood vessel Dilated blood vessel Normal blood vessel Leaky blood vessel 133 Dr. Zekeria Yusuf

134. Signs of inflammation Vascular changes Vasodilatation Capillary permeability Heat / redness Temporary loss of function Fever Swelling Pain 134 Dr. Zekeria Yusuf

135. Inflammation: a complex series of events induced by tissue damage (described in medical literature in 1500s) Redness (rubor) Pain (dolor) Swelling (tumor) Heat (calor) 135 Dr. Zekeria Yusuf

136. Signs of inflammation 136 Dr. Zekeria Yusuf

137. Infection can induce inflammation, but even sterile injuries can be sufficient to induce inflammation. Inflammation occurs when injured tissues release mediators that promote vasodialation (increased blood flow) and chemotaxis (directed migration) of leukocytes. 137 Dr. Zekeria Yusuf

138. Inflammation causes blood cells to move from blood stream to site of injury 138 Dr. Zekeria Yusuf

139. Inflammation and innate immunity Mast cells – similar to basophils in blood; mast cells are present in tissues and release histamines in response to wound / infection /irritant Histamine Pathogen removal Adaptive immune response + + + 139 Dr. Zekeria Yusuf

140. Blood cells (leukocytes) travel from the blood stream into tissues by a process known as extravasation Blood cells can also be attracted to sites of infection by products produced by pathogens, as well as by chemoattractants made by host ( chemokines , inflammatory meditators ). 140 Dr. Zekeria Yusuf

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143. Summary: role of Inflammation in innate immunity Initiation of phagocytosis – killing of pathogen Limiting the spread of infection Stimulate adaptive immune response Initiate tissue repair 143 Dr. Zekeria Yusuf

144. The good and bad about inflammation Acute /short-term -Good chronic /long-term - Bad 144 Dr. Zekeria Yusuf

145. Chronic inflammation = tissue damage Chronic inflammation - macrophages in the injured tissue. Macrophages release toxins (including reactive oxygen species or ROS ) that injure tissues chronic inflammation is almost always accompanied by tissue destruction. Normal tissue Tissue : chronic inflammation 145 Dr. Zekeria Yusuf

146. Chronic inflammation and tissue damage Reduced tissue function Tissue damage Chronic inflammation Activation of immune cells Killing of host cells 146 Dr. Zekeria Yusuf

147. Reactive oxygen species  ( ROS ) are chemically reactive  molecules containing oxygen. Examples include peroxides, superoxide, hydroxyl radical. Thus , the selective oxidation of critical signal transduction proteins probably represents a regulatory mechanism that functions to minimize the generation of ROS through respiratory control mechanisms . The reduction of the rate of ROS generation, in turn, will promote cellular survival under conditions of oxidative stress, when reactive oxygen and nitrogen species overwhelm cellular antioxidant defense systems, by minimizing the non-selective oxidation of a range of biomolecules . Since protein aggregation occurs if protein repair and degradative systems are unable to act upon oxidized proteins and restore cellular function, the reduction of the oxidative load on the cell by the down-regulation of the electron transport chain functions to minimize protein aggregation.  OXIDATIVE damage to cellular proteins 147 Dr. Zekeria Yusuf

148. Chronic inflammation and Cancer ROS O 2 - OH - DNA Mutation Cancer 148 Dr. Zekeria Yusuf

149. 1. Anatomical Barriers - Mechanical Factors Skin Flushing action of saliva, tears, urine Mucociliary escalator Innate immune system- External defenses 149 Dr. Zekeria Yusuf

150. Anatomical Barriers – Chemical factors Antimicrobial Peptides in sweat Lysozyme in tears /saliva HCl in stomach 150 Dr. Zekeria Yusuf

151. Anatomical Barriers – Biological fac tors Normal flora – microbes in many parts of the body Normal flora – competes with pathogens for nutrients and space Normal flora – > 1000 species of bacteria 151 Dr. Zekeria Yusuf

152. 1. Innate immune system: components of Blood   Compl e ment proteins Coagulation proteins Cytokines WBCs Extracellular Innate immune system- internal defenses 152 Dr. Zekeria Yusuf

153. innate response – internal defenses – Cellular (WBCs) Come into play when the external defenses are breached Neutrophils Monocytes /macrophages NK cells TLRs 153 Dr. Zekeria Yusuf

154. Toll-like receptors (TLRs) Transmembrane proteins Present on macrophages / few other cells Conserved across vertebrates Important part of innate immune system They look out for microbes (or their components) They bind to the microbes (or their components) They trigger a cascade of events to kill or protect against pathogens THEY ARE INNATE IMMUNE SENSORS 154 Dr. Zekeria Yusuf

155. Which microbial components are recognized by TLRs ? 155 Dr. Zekeria Yusuf

156. What happens when a TLR bind to a microbe ? TLR binding to microbe Inflammation Secretion of Cytokines / Interferon Phagocytosis of infected cell Apoptosis of infected cell Enhanced immune response Killing of infected cell 156 Dr. Zekeria Yusuf

157. Apoptosis: Cellular Suicide Nuclear fragmentation Proteolysis Blebbing Death Remnants undergo phagocytosis 157 Dr. Zekeria Yusuf

158. Apoptosis versus Necrosis Tidy: contents of cells degraded from within, producing small cellular “blebs” Programmed from inside the cell Messy: contents of cell released. Induced by external insult 158 Dr. Zekeria Yusuf

159. Cell death by necrosis is more likely to produce inflammation. 159 Dr. Zekeria Yusuf

160. Interferons are cytokines that are produced in response to viral infection. Produce an “anti-viral state” in target cells. Acts on cell that produces it, as well as neighboring cells. Together with dsRNA, act to triggering the Protein Kinase R (PKR) pathway. Shuts down protein synthesis machinery of cells, thus preventing viral replication. Cells can avoid being hijacked by viruses by activating the Protein Kinase R (PKR) pathway. PKR is triggered by dsRNA and interferon. 160 Dr. Zekeria Yusuf

161. Protein Kinase R: Interfering with Infection RNA- binding domain Kinase domain dsRNA or interferon PKR eIF2a Phosphorylated PKR (active) Phosphorylated eIF2a (inactive) 161 Dr. Zekeria Yusuf

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164. Cytokines and chemokines Cytokines form an important family of proteins that function as immune mediators and have important roles during immune responses – they can serve to both stimulate or inhibit the differentiation, proliferation or activity of immune cells. A subset of cytokines, chemokines , play an important role in guiding immune cells to sites of infection by forming a chemical ‘trail’. 164 Dr. Zekeria Yusuf

165. Cytokines: (also called interleukins) Small secreted peptides that used for intracellular communication between cells of the immune system Can turn on or off immune responses Mediate inflammation Chemokines: subset of cytokines that specialize in regulating cell motility 165 Dr. Zekeria Yusuf

166. Cytokines Small proteins – secreted by cells of the immune system Affect the behaviour of other cells signalling molecules Key players in innate and acquired immunity 166 Dr. Zekeria Yusuf

167. Which cells release cytokines ? Cells of the immune system: Neutrophils – when they encounter a pathogen Macrophages – when they encounter a pathogen TLRs – bind to microbe / components of a microbe NK cells – on encountering a microbe infected cell /tumour cell Lymphocytes – when they are activated 167 Dr. Zekeria Yusuf

168. Examples of cytokines Interferons Interleukins Tumour necrosis factor (TNF) 168 Dr. Zekeria Yusuf

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171. Interferons (IFN) Signalling proteins produced by by virus infected monocytes and lymphocytes Secreted proteins – Key anti-viral proteins “Interfere” with virus replication Warn the neighbouring cells that a virus is around... If we did not have IFNs – most of us may die of influenza virus infection 171 Dr. Zekeria Yusuf

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173. 173 How does IFN warn the neighbouring cells ? Dr. Zekeria Yusuf

174. 174 The infected cells release IFN antiviral state antiviral state antiviral state antiviral state Dr. Zekeria Yusuf

175. 175 Virus infects the neighbouring cells antiviral state antiviral state antiviral state antiviral state Dr. Zekeria Yusuf

176. 176 Prewarned cells are able to quickly inhibit the virus antiviral state antiviral state antiviral state antiviral state Dr. Zekeria Yusuf

177. How do interferons inhibit viruses ? Host protein Induction Cascade of events Virus ds -RNA Activation Inactive host protein Active host protein Inhibition of host protein synthesis Virus cannot replicate 177 Dr. Zekeria Yusuf

178. Interleukins Interleukins – 1-37 Not stored inside cells Quickly synthesized and secreted in response to infection Key modulators of behaviour of immune cells Mostly secreted by T-lymphocytes & macrophages 178 Dr. Zekeria Yusuf

179. What to interleukins do ? Interleukins Proliferation of immune cells Activation of immune cells Increase antibody production Inflammation 179 Dr. Zekeria Yusuf

180. Tumour necrosis factor (TNF) TNF Killing of cancer Fever Inflammation 180 Dr. Zekeria Yusuf

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182. complement system The complement system represents the humoral arm of innate immunity, and consists of a number of proteins (found in solution in the blood) that can interact directly, or indirectly, with infective bacteria (through different activation pathways). Inflammation, as a result of infection, allows plasma, containing complement proteins, to enter infected tissues. Once activated, the member proteins assemble to form complexes on the surface of microbes that punch holes in the membrane. The complement activation pathways are termed: the classical pathway , the alternative pathway , and the mannose-binding lectin pathway . 182 Dr. Zekeria Yusuf

183. Compl e ment (C`)  a large number of distinct plasma proteins that react with one another (C1 thro’ C9) Complement can bind to microbes and coat the microbes Essential part of innate immune response Enhances adaptive immune resposne (taught later) 183 Dr. Zekeria Yusuf

184. Complement proteins: role in innate immune system C`proteins Inflammation Facilitates phagocytosis Direct lysis of pathogens 184 Dr. Zekeria Yusuf

185. How do C` proteins facilitate phagocystosis ? Bacteria coated with C` Neutrophils have C` receptors Initiation of phagocytosis 185 Dr. Zekeria Yusuf

186. How do C` proteins lyse pathogens? Membrane attack complex formed by c` proteins 186 Dr. Zekeria Yusuf

187. Coagulation proteins Coagulation: mechanism to stop bleeding after injury to blood vessels Complex pathway involves Platelets Coagulation factors Vitamin K 187 Dr. Zekeria Yusuf

188. How does blood clot ? 188 Dr. Zekeria Yusuf

189. Coagulation: Delicate balance Coagulation proteins Anticoagulants Blood clotting Inflammation Apoptosis ( prog . Cell death) Prevent blood clotting Inhibit inflammation Inhibit apoptosis Too much of clotting – Problem Too little clotting - Problem Maintenance of a balance 189 Dr. Zekeria Yusuf

190. Coagulation and innate immunity Coagulation proteins Anticoagulants Pathogens and cytokines Increased inflammation and increased apoptosis of infected cells 190 Dr. Zekeria Yusuf

191. Summary: what happens when external defenses fail ? INNATE IMMUNITY Rapid responses to a broad range of microbes ACQUIRED IMMUNITY Slower responses to specific microbes External defenses Internal defenses Skin Mucous membranes Secretions Phagocytic cells Antimicrobial proteins Inflammatory response Natural killer cells Humoral response (antibodies) Cell-mediated response (cytotoxic lymphocytes) Invading microbes (pathogens) Complement 191 Dr. Zekeria Yusuf

192. Summary: innate response – internal defenses Cellular Neutrophils Monocytes /macrophages NK cells TLRs Extracellular C ytokines C omplement C oagulation 192 Dr. Zekeria Yusuf

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215. Mechanism of antibody production Dr. Zekeria Yusuf 215

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219. Antibody production… Dr. Zekeria Yusuf 219

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221. Technique for the production of monoclonal antibodies Dr. Zekeria Yusuf 221

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223. Applications of monoclonal antibodies Dr. Zekeria Yusuf 223

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231. T – CELL CLONING The analysis of specificity and effector function in T cells depends heavily on the study of monoclonal populations of T lymphocytes. These can be obtained in four distinct ways. as for B-cell hybridomas , normal T cells proliferating in response to specific antigen can be fused to malignant T-cell lymphoma lines to generate T-cell hybrids. The hybrids express the receptor of the normal T cell, but proliferate indefinitely owing to the cancerous state of the lymphoma parent. T-cell hybrids can be cloned to yield a population of cells all having the same T-cell receptor. When stimulated by their specific antigen these cells release cytokines such as the T-cell growth factor interleukin-2 (IL-2) and the production of cytokines is used as an assay to assess the antigen specificity of the T-cell hybrid. Dr. Zekeria Yusuf 231

232. T-cell cloning… T-cell hybrids are excellent tools for the analysis of T-cell specificity, as they grow readily in suspension culture. However, they cannot be used to analyze the regulation of specific T-cell proliferation in response to antigen because they are continually dividing. T-cell hybrids also cannot be transferred into an animal to test for function in vivo because they would give rise to tumors. Functional analysis of T-cell hybrids is also confounded by the fact that the malignant partner cell affects their behavior in functional assays. Therefore, the regulation of T-cell growth and the effector functions of T cells must be studied using T-cell clones. Dr. Zekeria Yusuf 232

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235. Immunogens and antigens Immunogen / antigen: a substance that elicits an immune response [i.e. a humoral (antibody response) or cell-mediated immune response] Immune response gen erator Though the two terms are used interchangeably – there are differences between the two Antigen: any substance that binds to an antibody (or a T-cell receptor) – but some antigens cannot elicit an immune response. All immunogens are antigens but not all antigens are immunogens 235 Dr. Zekeria Yusuf

236. Epitope Epitope: the portion of an antigen that is recognized and bound by an antibody ( Ab ) or a T-cell receptor (TCR) epitope = antigenic determinant Two types: B-cell Epitopes – recognized by B-cells T-cell Epitopes – recognized by T cells 236 Dr. Zekeria Yusuf

237. Epitopes Epitope: the portion of an antigen that is recognized and bound by an Ab or a T Cell receptor One protein may have multiple antigenic determinant 237 Dr. Zekeria Yusuf

238. Immunogenicity Immunogencity : is the ability to induce a humoral (antibody) and/or cell-mediated immune response. W eak immunogens Strong immunogens 238 Dr. Zekeria Yusuf

239. What determines immunogenicity ? Foreignness: essential for immunogenicity (self-responsive immune cells are eliminated during lymphocyte development) Size: Bigger>Smaller Chemical composition: Proteins > nucleic acids / polysaccharides / lipids Structure: Primary /secondary /tertiary structures play a role Physical form: Particulate> Soluble 239 Dr. Zekeria Yusuf

240. Host factors affecting immunogencity Difference across species (interspecies) Differences within a species ( intraspecies ) - Responders / non-responders to vaccine - differences in disease severity in epidemics Genetics Age 240 Dr. Zekeria Yusuf

241. Isoantigens Isoantigens : Antigens present in some but not all members of a species Blood group antigens – basis of blood grouping MHC (major histocompatibility complex)- cell surface glycoproteins 241 Dr. Zekeria Yusuf

242. Autoantigens Autoantigens are substances capable of immunizing the host from which they are obtained. Self antigens are ordinarily non-antigenic Modifications of self-antigens are capable of eliciting an immune response 242 Dr. Zekeria Yusuf

243. Haptens Haptens are small molecules which are non-immunogenic , thus could never induce an immune response by themselves. 243 Dr. Zekeria Yusuf

244. Adaptive immunity Key to the adaptive immune response is the lymphocyte . There are several subtypes, however these fall under two broad designations: T lymphocytes and B lymphocytes (commonly known as T cells and B cells ). Although both originate in the bone marrow, T cells mature in the t hymus, whilst B cells mature in the b one marrow. During an organism’s early development a large number of B- and T cells are produced, each of which has the ability to recognise a specific, and essentially unique, molecular target. An important aspect of this maturation process is that, for both of these cell types, cells that recognise targets within the body (‘self’ tissue) are identified and weeded-out. An additional aspect of the maturation process for T cells is that further distinct subsets are produced – helper T cells (also called CD4 + T cells ) and cytotoxic T cells (also called CD8 + T cells ). The individual specificity of lymphocytes is key to the generation of adaptive responses. Adaptive immunity utilises many kinds of receptor to coordinate its activities. T cells carry T-cell receptors ( TCR ), whilst B cells carry B-cell receptors ( BCR ), and variations in the fine structure of these receptors account for the individual specificity described above. In addition, another set of receptors, encoded by the major histocompatibility complex ( MHC ), play an important role in adaptive immunity. MHC class I receptors are displayed on a majority of body cells, whilst MHC class II receptors are restricted to antigen-presenting cells ( APCs ). Both of these receptor types interact with TCRs . 244 Dr. Zekeria Yusuf

245. A daptive immune response The adaptive immune response consists of two branches, a cellular adaptive response (effected by cytotoxic T cells) and a humoral adaptive response (effected by B cells). The former is directed especially towards pathogens that have colonised body cells or body cells that have become malignant (as in cancer). The latter generally targets pathogens or molecules (antigens) that are free in the bloodstream or present at mucosal surfaces. As suggested by its name, the helper T cell plays a central role in both of these responses since, once activated, it can shape the subsequent immune response through the particular molecules that it secretes – in particular, controlling the activation of other cell types – as such it is an important ‘gatekeeper’. Two subtypes of helper T cells ( Th1 and Th2 ) have been identified as being responsible for guiding adaptive responses towards either a cellular profile ( Th1 ) or a humoral profile ( Th2 ). Th17 cells have recently been identified and are thought to play a further specialised role. Effective regulation of immune responses is also vital to ensure that they don’t themselves cause unnecessary tissue damage, and regulatory T cells ( Tregs ) are a subset of T cell that play an important role in this process. 245 Dr. Zekeria Yusuf

246. Initiation of adaptive immunity Antigen-presenting cells are functionally-defined cells that are able too initiate adaptive immune responses by presenting antigen to T cells. Major APCs are dendritic cells ( DCs ), which are found throughout the body – however macrophages and B cells may also serve as APCs , with the former providing an important link from innate immunity. Dendritic cells continuously monitor the bodily environment by absorbing protein fragments that they acquire from their surroundings, and presenting them on the their cell surface in association with MHC receptors. DCs may be activated by local innate immune signals (induced by infection) causing them to migrate through the lymph (or blood) to lymph nodes where they present antigen to T cells. If a protein fragment is recognised by a particular cytotoxic T cell this will suggest that it is of foreign origin (due to elimination of cells recognising ‘’self’’) leading to a cellular adaptive response. Similarly, B cells in the lymph node may encounter free antigen carried in the lymph, leading to a humoral adaptive response. In both cases, concurrent activation of helper T cells is usually necessary to ensure an effective overall response. 246 Dr. Zekeria Yusuf

247. The cellular adaptive response Body cells are continuously processing protein derived from the internal cellular environment and presenting it in association with MHC class I receptors. This will typically be ‘self’ antigen (that is ignored by the immune system), but can also be peptides derived from infecting viruses or bacteria, or aberrant cancer peptides. Activated cytotoxic T cells of a given specificity proliferate in the lymph and then migrate to sites of infection where they monitor body cells for signs of intracellular infection or aberrant self proteins associated with cancer-presented on MHC class I molecules – using their TCRs . If they encounter antigen that they recognise, this indicates infection or malignancy, and they are then able to induce apoptosis ( autodestruction ) of targeted body cells. This constitutes the cellular adaptive response. 247 Dr. Zekeria Yusuf

248. The humoral adaptive response As already stated, B cells can recognise antigen through direct recognition of antigen via their BCRs , without the need for prior processing or presentation via a receptor – so they are key to identifying extracellular pathogens (e.g. bacteria in the lymph). Once activated, B cells differentiate into plasma cells that are capable of secreting antibody molecules into the circulation (small molecules that match the individual specificity of the parent cell) that are then able to find their targets elsewhere in the body. Once bound to a target, antibody molecules can activate the classical pathway of the complement system , thereby directing it to neutralise its targets with great specificity. Binding of antibody also enhances phagocytosis . 248 Dr. Zekeria Yusuf

249. Immune memory It is important to note that an effective primary adaptive response (e.g. relating to a pathogen that hasn’t previously been encountered) takes some time to develop, since only small numbers of target-specific B- and T cells are present initially and, once activated, they must first proliferate through a process known as clonal selection , to form effector cells . A proportion of these effector cells go on to form a stock of long-lived memory cells ensuring that if a particular pathogen is encountered again, any subsequent secondary adaptive response (or ‘memory response’) develops more quickly and is thus more effective. 249 Dr. Zekeria Yusuf

250. Adaptive immunity: second line of response Based upon resistance acquired during life Relies on genetic events and cellular growth Responds more slowly, over few days Is specific each cell responds to a single epitope on an antigen Has anamnestic memory repeated exposure leads to faster, stronger response Leads to clonal expansion 250 Dr. Zekeria Yusuf

251. Adaptive Immunity: active and passive Active Immunity Passive Immunity Natural clinical, sub-clinical infection via breast milk, placenta Artificial Vaccination: Live, killed, purified antigen vaccine immune serum, immune cells

252. Adaptive immunity: mechanisms Cell-mediated immune response (CMIR) T-lymphocytes eliminate intracellular microbes that survive within phagocytes or other infected cells Humoral immune response (HIR) B-lymphocytes mediated by antibodies eliminate extra-cellular microbes and their toxins Plasma cell (Derived from B-lymphocyte, produces antibodies) 252 Dr. Zekeria Yusuf

253. Cell-mediated immune response T-cell recognizes peptide antigen on macrophage in association with major histo-compatibility complex (MHC) class identifies molecules on cell surfaces helps body distinguish self from non-self T-cell goes into effectors cells stage that is able to kill infected cells

254. T lymphocytes 2 types helper T- lymphocytes (CD4+) CD4+ T cells activate phagocytes to kill microbes cytolytic T-lymphocyte (CD8+) CD8+ T cells destroy infected cells containing microbes or microbial proteins 254 Dr. Zekeria Yusuf

255. Cell mediated immune response Primary response production of specific clones of effector T cells and memory clones develops in several days does not limit the infection Secondary response more pronounced, faster more effective at limiting the infection Example - cytotoxic reactions against intracellular parasites, delayed hypersensitivity (e.g., Tuberculin test) and allograft rejection 255 Dr. Zekeria Yusuf

256. Humoral immune response B lymphocytes recognize specific antigens proliferate and differentiate into antibody-secreting plasma cells Antibodies bind to specific antigens on microbes; destroy microbes via specific mechanisms Some B lymphocytes evolve into the resting state - memory cells

257. Antibodies (immunoglobulins) Belong to the gamma-globulin fraction of serum proteins Y-shaped or T-shaped polypeptides 2 identical heavy chains 2 identical light chains All immunoglobulins are not antibodies Five kinds of antibodies IgG, IgM, IgA, IgD, IgE

258. IMMUNOLOGICAL TECHNIQUES ( Immunotechnology ) Agglutination, precipitation , immune – fluorescence, immunoelectrophoresis , immunoblotting , ELISA, RIA , Flow cytometry . Production and purification of antibodies, determination of antibody titre by RIDand EID , production of hybridoma . T – cell cloning: mechanism of antigen recognition by T and B – lymphocytes, Importance of antigen and MHC class II molecules in T – cell cloning. Antigen specific and alloreactive T – cell cloning – immunologically relevant antigens and T– cell subtypes and Applications in vaccine development. Dr. Zekeria Yusuf 258

259. What is an antibody? Produced by Plasma cell (B-lymphocytes producing Ab ) Essential part of adaptive immunity: S pecifically bind a unique antigenic epitope (also called an antigenic determinant) Possesses antigen binding sites Members of the class of proteins called immunoglobulins , Antibodies are defined as blood protein produced in response to and counteracting a specific antigen. • Chemically the antibodies are globulins and are called as immunoglobins . 259 Dr. Zekeria Yusuf

260. ANTIGEN: – A toxin or other foreign substance which induces an immune response in the body, especially the production of antibodies. ANTIBODY: – A blood protein produced in response to and counteracting a specific antigen. Dr. Zekeria Yusuf 260

261. TYPES OF ANTIGEN • Complete antigen or immunogen : – These are the antigens which are able to induce antibody formation by themselves. • In complete antigen or hapten : – These are the chemical substances which are not able to induce antibody formation by themselves. Dr. Zekeria Yusuf 261

262. Properties of antigen 1. FOREIGNNESS: – Immune system has the ability to distinguish between self and non-self antigens. – Only antigens which are foreign to the individual induce an immune response. 2. SIZE: – When the size of the antigen is more means it can cause severe infection. Dr. Zekeria Yusuf 262

263. Properties of antigen…. 3. CHEMICAL NATURE: – The proteins present in the antigen will stimulate the immune response. 4. SUSCEPTIBILITY TO TISSUE ENZYME: – Only those substance which can be metabolized and can be converted to soluble form by the action of tissue enzyme act as good antigens. Dr. Zekeria Yusuf 263

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268. Antibodies (immunoglobulins) Belong to the gamma-globulin fraction of serum proteins Y-shaped or T-shaped polypeptides 2 identical heavy chains 2 identical light chains All immunoglobulins are not antibodies Five kinds of antibodies IgG, IgM, IgA, IgD, IgE

269. What does an antibody look like ? 2 identical heavy chains 2 identical light chains Each heavy chain – has a constant and a variable region Each light chain has a constant and a variable region H H L L Constant region Variable region 269 Dr. Zekeria Yusuf

270. Antibody: structure and function Fab – fragment antigen binding Fc - Fragment constant 270 Dr. Zekeria Yusuf

271. Antibodies can also be divided into two regions based on their functions. The tip of the “Y”, consisting of one constant and one variable domain from each heavy and light chain of the antibody, contains the region that binds the antigen. It is for this reason that we call it the Fab (fragment, antigen binding) region . The base of the “Y”, composed of two heavy chains, plays a a role in modulating immune cell activity. This region is called the Fc (Fragment, crystallizable ) region . The Fc region can bind to cell receptors, complement proteins, as well as to other immune molecules, thereby initiating different physiological effects including opsonization , cell lysis , and degranulation . Dr. Zekeria Yusuf 271

272. Antibody: Fab Fab region Variable region of the antibody Tip of the antibody Binds the antigen Specificity of antigen binding determined by V H and V L 272 Dr. Zekeria Yusuf

273. Antibody: Fc Fc region Constant region Base of the antibody Can bind cell receptors and complement proteins 273 Dr. Zekeria Yusuf

274. Antibodies occur in 2 forms Soluble Ag: secreted in blood and tissue Membrane-bound Ag : found on surface of B-cell, also known as a B-cell receptor (BCR) Antibodies exist in two forms 274 Dr. Zekeria Yusuf

275. CLASSES OF IMMUNOGLOBULIN Immunoglobulin G (IgG) or Gamma globulin • IgG is the major serum globulin constituting approximately 75% of total immunoglobulins with normal serum concentration of 15- 16mg /ml. • its molecular weight is 1,50000 Daltons. • it has a half life of 21 days.it is distributed equally between intravascular and extravascular compartments. IgG appears later in infection(2 weeks after infection) but persist for longer period of time. Dr. Zekeria Yusuf 275

276. IgG 70-75% of total immuniglobulin Secreted in high quantities in secondary exposures Cross the placenta Major functions / applications neutralize microbes and toxins opsonize antigens for phagocytosis activate the complement protect the newborn 4-fold rise or fall indicates active infection A single positive sample indicates past exposure 276 Dr. Zekeria Yusuf

277. CLASSES OF IMMUNOGLOBULIN … Immunoglobulin M ( IgM ) or Mu ( μ) globulin or macro globulin • constitute about 10% of total immunoglobulins with normal serum concentration of 1.2mg /ml. • its molecular weight is 9,00000 Daltons. • It is usually the first immunoglobulin that appears early (7-8 days) following stimulation by an antigen but persists for short duration (4- 6wks ). its presence in serum indicates recent infection. Dr. Zekeria Yusuf 277

278. IgM Secreted initially during primary infection Cannot cross the placenta Major functions / applications secreted first during primary exposure activates the complement used as a marker of recent infection Presence in newborn means infection Single positive sample in serum or CSF indicates recent or active infection Used to detect early phase of infection 278 Dr. Zekeria Yusuf

279. CLASSES OF IMMUNOGLOBULIN … Immunoglobulin A ( IgA ) or Alpha globulin or α globulin • It is the second most abundant immunoglobulin constituting approximately 15% of total immunoglobulin with normal serum concentration of 0 .6-4.2 mg/ml. • It is a fast moving globulin found in high concentration in colostrums, saliva, tears, nasal fluid, sweat, milk and in secretions of respiratory, intestinal and genital tracts. Dr. Zekeria Yusuf 279

280. IgA Monomeric in serum Dimeric with secretory component in the lumen of the gastro-intestinal tract and in the respiratory tract Major function / application neutralizes microbes and toxins Sero-diagnosis of tuberculosis Synthicial respiratory virus tests 280 Dr. Zekeria Yusuf

281. CLASSES OF IMMUNOGLOBULIN … Immunoglobulin D ( IgD ) or globulin • It is present in concentration of 0.03 mg/ml. • It has a molecular weight of 1,80,000 Dalton. • IgD is present on the surface of B lymphocytes. Dr. Zekeria Yusuf 281

282. IgD Monomeric Major functions / applications present on the surface of B lymphocytes functions as membrane receptor role unclear has a role in antigen stimulated lymphocyte differentiation 282 Dr. Zekeria Yusuf

283. CLASSES OF IMMUNOGLOBULIN … Immunoglobulin E ( IgE ) or Epsilon globulin • It is mostly distributed extravascularly and is produced by a very small proportion of plasma cells. • It lives in intestinal and respiratory tract. The antigen antibody reaction is a molecular association similar to an enzyme-substrate interaction, with an important distinction: it does not lead to an irreversible chemical alteration in either the antibody or the antigen. The association between an antibody and an antigen involves various noncovalent interactions between the antigenic determinant, or epitope , of the antigen and the variable-region ( VH / VL ) domain of the antibody molecule, particularly the hypervariable regions, or complementarity determining regions ( CDRs ). Dr. Zekeria Yusuf 283

284. Serodiagnosis of infectious and non infectious allergies (e.g., allergic bronchopulmonary aspergillosis, parasitic diseases) IgE Mediates type I hypersensitivity Monomeric Major functions / applications associated with anaphylaxis plays a role in immunity to helminthic parasites 284 Dr. Zekeria Yusuf

285. Sequential IgM-IgG humoral response IgM produced as a first response to many antigens levels remain high transiently IgG produced after IgM higher levels persist in small amounts throughout life produced in large amounts during secondary response persistence of antigen sensitive ‘memory cells’ after primary response

286. IgM – IgG sequential response First stimulus Time Second stimulus Antibody titer IgM IgG Anamnestic response 286 Dr. Zekeria Yusuf

287. Antibody-antigen Binding The binding of antibody with its corresponding Ag may result in agglutination, precipitation, complement fixation, greater susceptibility to ingestion and destruction by phagocytes of neutralization. • Such reactions are useful in laboratory diagnosis of various diseases Dr. Zekeria Yusuf 287

288. Antigen-antibody specificity The exquisite specificity of antigen antibody interactions has led to the development of a variety of immunologic assays, which can be used to detect the presence of either antibody or antigen. • Immunoassays have played vital roles in diagnosing diseases, monitoring the level of the humoral immune response, and identifying molecules of biological or medical interest. The noncovalent interactions that form the basis of antigen antibody (Ag- Ab ) binding include hydrogen bonds, ionic bonds, ydrophobic interactions, and van der Waals interactions. Dr. Zekeria Yusuf 288

289. Antigen-antibody specificity… Because these interactions are individually weak (compared with a covalent bond), a large number of such interactions are required to form a strong Ag- Ab interaction. • Furthermore, each of these noncovalent interactions operates over a very short distance, generally about 1 angstrom, Å; consequently, a strong Ag- Ab interaction depends on a very close fit between the antigen and antibody. • Such fits require a high degree of complementarity between antigen and antibody, a requirement that underlies the exquisite specificity that characterizes antigen-antibody interactions. Dr. Zekeria Yusuf 289

290. Antibody Affinity Is a Quantitative Measure of Binding Strength • The combined strength of the noncovalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope . • Low-affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high-affinity antibodies bind antigen more tightly and remain bound longer. The combined strength of the noncovalent interactions between a single antigen-binding site on an antibody and a single epitope is the affinity of the antibody for that epitope . • Low-affinity antibodies bind antigen weakly and tend to dissociate readily, whereas high affinity antibodies bind antigen more tightly and remain bound longer. Dr. Zekeria Yusuf 290

291. Antibody Avidity Incorporates Affinity of Multiple Binding Sites • The affinity at one binding site does not always reflect the true strength of the antibody-antigen interaction. • When complex antigens containing multiple, repeating antigenic determinants are mixed with antibodies containing multiple binding sites, the interaction of an antibody molecule with an antigen molecule at one site will increase the probability of reaction between those two molecules at a second site. • The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. • The avidity of an antibody is a better measure of its binding capacity within biological systems (e.g., the reaction of an antibody with antigenic determinants on a virus or bacterial cell) than the affinity of its individual binding sites. Dr. Zekeria Yusuf 291

292. Antibody Avidity… • High avidity can compensate for low affinity. • For example, secreted pentameric IgM often has a lower affinity than IgG , but the high avidity of IgM , resulting from its higher valence, enables it to bind antigen effectively. The valence of an antibody or antigen is the number of different molecules that it can combine with at one time. Dr. Zekeria Yusuf 292

293. Cross-Reactivity • Although Ag- Ab reactions are highly specific, in some cases antibody elicited by one antigen can cross-react with an unrelated antigen. • Such cross-reactivity occurs if two different antigens share an identical or very similar epitope . • In the latter case, the antibody’s affinity for the cross-reacting epitope is usually less than that for the original epitope . Antigen: antibody complexes are noncovalently associated groups of antigen and antibody molecules that can vary in size from small soluble complexes to large insoluble complexes that precipitate out of solution; they are also known as immune complexes. Dr. Zekeria Yusuf 293

294. Cross-reactivity… • Cross-reactivity is often observed among polysaccharide antigens that contain similar oligosaccharide residues. • The ABO blood-group antigens, for example, are glycoproteins expressed on red blood cells. • Subtle differences in the terminal residues of the sugars attached to these surface proteins distinguish the A and B blood-group antigens. • An individual lacking one or both of these antigens will have serum antibodies to the missing antigen(s). • The antibodies are induced not by exposure to red blood cell antigens but by exposure to cross-reacting microbial antigens present on common intestinal bacteria. These microbial antigens induce the formation of antibodies in individuals lacking the similar blood-group antigens on their red blood cells. • (In individuals possessing these antigens, complementary antibodies would be eliminated during the developmental stage in which antibodies that recognize self epitopes are weeded out.) Dr. Zekeria Yusuf 294

295. Cross-reactivity… • The blood-group antibodies, although elicited by microbial antigens, will cross react with similar oligosaccharides on foreign red blood cells, providing the basis for blood typing tests and accounting for the necessity of compatible blood types during blood transfusions. • A type A individual has anti-B antibodies; a type B individual has anti-A; and a type O individual thus has anti-A and anti-B. The bacterium Streptococcus pyogenes , for example, expresses cell-wall proteins called M antigens. • Antibodies produced to streptococcal M antigens have been shown to cross-react with several myocardial and skeletal muscle proteins and have been implicated in heart and kidney damage following streptococcal infections. Dr. Zekeria Yusuf 295

296. Structure of antigens • Formation of an Ag- Ab lattice depends on the valency of both the antibody and antigen: • The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments. • The antigen must be either bivalent or polyvalent; that is, it must have at least two copies of the same epitope , or have different epitopes that react with different antibodies present in polyclonal antisera . Dr. Zekeria Yusuf 296

297. Structure of antigens… Different antibodies bind to distinct epitopes on an antigen molecule. The surface of an antigen possesses many potential antigenic determinants or epitopes , distinct sites to which an antibody can bind. The number of antibody molecules that can bind to a molecule of antigen at one time defines the antigen‘s valence. Steric considerations can limit the number of different antibodies that bind to the surface of an antigen at any one time (center and bottom panels) so that the number of epitopes on an antigen is always greater than or equal to its valence. Dr. Zekeria Yusuf 297

298. • Experiments with myoglobin illustrate the requirement that protein antigens be bivalent or polyvalent for a precipitin reaction to occur. • Myoglobin precipitates well with specific polyclonal antisera but fails to precipitate with a specific monoclonal antibody because it contains multiple, distinct epitopes but only a single copy of each epitope . • Myoglobin thus can form a crosslinked lattice structure with polyclonal antisera but not with monoclonal antisera . • The principles that underlie precipitation reactions are presented because they are essential for an understanding of commonly used immunological assays. • Although various modifications of the precipitation reaction were at one time the major types of assay used in immunology, they have been largely replaced by methods that are faster and, because they are far more sensitive, require only very small quantities of antigen or antibody. Dr. Zekeria Yusuf 298

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300. PRECIPITATION • When a soluble antigen combines with its corresponding Ab in presence of electrolytes ( NaCl ) at a suitable temperature ( 370c ) and pH 7.5, the Ag- Ab complex forms an insoluble precipitate, a reaction called precipitation. Dr. Zekeria Yusuf 300

301. Precipitation Reactions • Antibody and soluble antigen interacting in aqueous solution form a lattice that eventually develops into a visible precipitate. • Antibodies that aggregate soluble antigens are called precipitins. • Although formation of the soluble Ag- Ab complex occurs within minutes, formation of the visible precipitate occurs more slowly and often takes a day or two to reach completion. Dr. Zekeria Yusuf 301

302. A. Simple precipitation test 1. Ring test: – The test is very simple for detection of Ag. In a test tube (narrow test tube) serum is taken, over it antigen is layered or poured. – A white zone is formed at the junction where the Ag- Ab reactions take place eg : Ascoli’s test in anthrax Dr. Zekeria Yusuf 302

303. 2. Slide test: – A drop of antigen and antibody is placed on a clean slide and mixed by shaking; floccules appear indicating positive test eg : VDRL 3. Tube test: – Kahn test for syphilis is tube flocculation test . – Ag is added to the tube containing antibody; if the Ag- Ab reaction takes place, floccules appear indicating positive test. Dr. Zekeria Yusuf 303

304. 4. Gel diffusion test: – Single diffusion in one dimension: – Double diffusion in one dimension – Single diffusion in two dimension (Radial immunodiffusion ) – Double diffusion in two dimension ( Ouchterlony technique) Dr. Zekeria Yusuf 304

305. B. IMMUNODIFFUSION TESTS 1) Immunoelectrophoresis 2) Rocket electrophoresis 3) counter Immunoelectrophoresis Dr. Zekeria Yusuf 305

306. IMMUNODIFFUSION TESTS.. Precipitation Reactions in Gels Yield Visible Precipitin Lines • Immune precipitates can form not only in solution but also in an agar matrix. When antigen and antibody diffuse toward one another in agar, or when antibody is incorporated into the agar and antigen diffuses into the antibody-containing matrix, a visible line of precipitation will form. • As in a precipitation reaction in fluid, visible precipitation occurs in the region of equivalence, whereas no visible precipitate forms in regions of antibody or antigen excess. Dr. Zekeria Yusuf 306

307. IMMUNODIFFUSION TESTS… • Two types of immunodiffusion reactions can be used to determine relative concentrations of antibodies or antigens, to compare antigens, or to determine the relative purity of an antigen preparation. • They are radial immunodiffusion (the Mancini method) and double immunodiffusion (the Ouchterlony method); both are carried out in a semisolid medium such as agar. • In radial immunodiffusion , an antigen sample is placed in a well and allowed to diffuse into agar containing a suitable dilution of an antiserum. Dr. Zekeria Yusuf 307

308. IMMUNODIFFUSION TESTS… • As the antigen diffuses into the agar, the region of equivalence is established and a ring of precipitation, a precipitin ring, forms round the well . • The area of the precipitin ring is proportional to the concentration of antigen. • By comparing the area of the precipitin ring with a standard curve (obtained by measuring the precipitin areas of known concentrations of the antigen), the concentration of the antigen sample can be determined. • In the Ouchterlony method, both antigen and antibody diffuse radially from wells toward each other, thereby establishing a concentration gradient. • As equivalence is reached, a visible line of precipitation, a precipitin line, forms Dr. Zekeria Yusuf 308

309. Immunoelectrophoresis Combines Electrophoresis and Double Immunodiffusion • In immunoelectrophoresis , the antigen mixture is first electrophoresed to separate its components by charge. • Troughs are then cut into the agar gel parallel to the direction of the electric field, and antiserum is added to the troughs. • Antibody and antigen then diffuse toward each other and produce lines of precipitation where they meet in appropriate proportions. • Immunoelectrophoresis is used in clinical laboratories to detect the presence or absence of proteins in the serum. • A sample of serum is electrophoresed , and the individual serum components are identified with antisera specific for a given protein or immunoglobulin class. • This technique is useful in determining whether a patient produces abnormally low amounts of one or more isotypes , characteristic of certain immunodeficiency diseases. • It can also show whether a patient overproduces some serum protein, such as albumin, immunoglobulin, or transferrin . Dr. Zekeria Yusuf 309

310. • The immunoelectrophoretic pattern of serum from patients with multiple myeloma, for example, shows a heavy distorted arc caused by the large amount of myeloma protein, which is monoclonal Ig & therefore uniformly charged. • Because immunoelectrophoresis is a strictly qualitative technique that only detects relatively high antibody concentrations (greater than several hundred g/ml), it utility is limited to the detection of quantitative abnormalities only when the departure from normal is striking, as in immunodeficiency states an immunoproliferative disorders. Dr. Zekeria Yusuf 310

311. Uses of precipitation reaction • Used for identification of bacteria • Used for identification of antigenic components of bacteria eg : Ascoli’s test • Used for the standardization of toxins and antitoxins • Used for the demonstration of unknown Ab in serum eg : VDRL • Used for detection of human blood, semen etc • Testing for food adultration . Dr. Zekeria Yusuf 311

312. VENEREAL DISEASE RESEARCH LABORATORY TEST • It is the most widely used precipitation test used in diagnosis of venereal disease. • Procedure: – in this test .05 ml of inactivated serum ( 560c for 30 minutes) of patients is taken in a special slide with rings or depressions of 14/ 16mm diameter each and depth of 1.75mm . – one drop of Ag ( cardiolipin ) is added with a insulin syringe the Ag- Ab reaction is studied under low power objective of microscope. Results: – presence of clumps indicate positive test where as uniformly distributed crystals indicate negative test. Dr. Zekeria Yusuf 312

313. AGGLUTINATION • When a particulate antigen (bacteria, RBC) is mixed with its corresponding Ab in the resence of electrolytes ( NaCl ) at a suitable temperature ( 370c ) and pH 7.4, the particles are clumbed or agglutinated. • IgM Ab’s agglutinate better than IgG Ab’s Dr. Zekeria Yusuf 313

314. Agglutination Reactions • The interaction between antibody and a particulate antigen results in visible clumping called agglutination. • Antibodies that produce such reactions are called agglutinins. • Agglutination reactions are similar in principle to precipitation reactions; they depend on the crosslinking of polyvalent antigens. Dr. Zekeria Yusuf 314

315. Agglutination Reactions… Techniques • Slide agglutination test (micro agglutination): it is widely used method to detect unknown Ag (bacteria, RBC in blood grouping etc). on a clean glass slide a drop of Ag is mixed with a drop of antiserum. • The reaction occurs immediately. Clumps are formed in few minutes. • Tube agglutination test (macro agglutination): it is quantitative test used to determine or estimate the titre of Ab and to confirm the result of slide agglutination test. • It is routinely used for serological diagnosis of typhoid, paratyphoid, brucellosis, and typhyoid fever. Dr. Zekeria Yusuf 315

316. 1. WIDAL TEST • Widal test is an agglutination test employed in the serological diagnosis of enteric fever caused by S typhi and S parathyphi A and B. •Principle: The serum of patient is tested for O and H antibodies by using antigenic suspension S typhi O (somatic Ag) and Styphi H ( flagellar Ag) respectively. For paratyphoid testing , the Ag suspension used are S. paratyphi AH and S. paratyphi BH . Dr. Zekeria Yusuf 316

317. • widal test may be conducted in two ways – WIDAL ( slide agglutination/ micro agglutination) test – WIDAL (tube agglutination/ macro agglutination) test Dr. Zekeria Yusuf 317

318. WIDAL (slide agglutination/ micro agglutination) test • In each four rings of the slide , labeled as O,H,AH and BH add one drop of serum. • Add corresponding Ag drop in each ring of the glass plate • Mix the Ag suspension and the diluted serum drop in each ring by using separate applicator stick • Slowly rock and tilt the glass plate and observe for 3 minutes • Record the degree of agglutination as 4+, 3+,2+,1+ or no agglutination Dr. Zekeria Yusuf 318

319. Interpretation • Agglutination appears by the end of the first week. The titer increase during second, third and fourth week after which it gradually decreases. • Single test is not of much value. Demonstration of rising titre of Ab by testing two or more serum specimen is more meaningful than single test. • Patient already tested with antibiotics may not show any rise in titre , instead there may be fall in titre . Dr. Zekeria Yusuf 319

320. Patient who has received vaccine against salmonella may give false positive reaction. • Individuals who had suffered from enteric fever in past sometimes develop antisalmonella Ab during an unrelated or closely related infection. This is called anamnestic response with temporary rise in H titer only. • Ag suspension with fimbrial Ag may sometimes give false positive reactions. Dr. Zekeria Yusuf 320

321. COMPLIMENT FIXATION TEST ( eg Wasserman reaction): • it is a name given to series of some nonspecific protein that occur in normal serum which are activated by Ag- Ab reactions & subsequently mediate a number of biologically important reactions. Dr. Zekeria Yusuf 321

322. Procedure • The reaction consists of two steps – Test system – Indicator system or hemolytic system Dr. Zekeria Yusuf 322

323. Test system: • Inactivated patients serum is mixed with Ag ( cardiolipin ). • A measured amount of complement( 2units of guinea pig serum) is added to this mixture. • The mixture is incubated for one hour at 370c in water bath. • If Ag and Ab match, the compliment will be fixed. Dr. Zekeria Yusuf 323

324. Indicator system or hemolytic system: • It consist of sensitized sheep’s RBC (sheep red cell coated with haemolysin 4- MHD ). • The indicator system is added to the test mixture and incubated again for 30 min at 370c in water bath. Dr. Zekeria Yusuf 324

325. Observation and interpretations • Absence of hemolysis : – if hemolysis does not occur it indicates that the compliment has been utilized/ fixed in the primary reaction between Ag and Ab and hence there is no hemolysis . – This is a positive test and confirms the presence of particular Ab in patients serum and a particular disease. Dr. Zekeria Yusuf 325

326. Presence of hemolysis : – if hemolysis takes place, it indicates that compliment has not utilized in primary reaction but utilized by indicator system resulting in lysis of red cells. – This is a negative test and confirms the absence of particular Ab in patients serum and hence no disease. Dr. Zekeria Yusuf 326

327. Bacterial Agglutination Is Used To Diagnose Infection • A bacterial infection often elicits the production of serum antibodies specific for surface antigens on the bacterial cells. • The presence of such antibodies can be detected by bacterial agglutination reactions. • Serum from a patient thought to be infected with a given bacterium is serially diluted in an array of tubes to which the bacteria is added. • The last tube showing visible agglutination will reflect the serum antibody titer of the patient. • The agglutinin titer is defined as the reciprocal of the greatest serum dilution that elicits a positive agglutination reaction. Dr. Zekeria Yusuf 327

328. • For example, if serial twofold dilutions of serum are prepared and if the dilution of 1/640 shows agglutination but the dilution of 1/1280 does not, then the agglutination titer of the patient’s serum is 640. • In some cases serum can be diluted up to 1/50,000 and still show agglutination of bacteria. • The agglutinin titer of an antiserum can be used to diagnose a bacterial infection. Dr. Zekeria Yusuf 328

329. • Patients with typhoid fever, for example, show a significant rise in the agglutination titer to Salmonella typhi . • Agglutination reactions also provide a way to type bacteria. • For instance, different species of the bacterium Salmonella can be distinguished by agglutination reactions with a panel of typing antisera . Dr. Zekeria Yusuf 329

330. Hemagglutination Is Used in Blood Typing • Agglutination reactions are routinely performed to type red blood cells ( RBCs ). • In typing for the ABO antigens, RBCs are mixed on a slide with antisera to the A or B blood-group antigens. If the antigen is present on the cells, they agglutinate, forming a visible clump on the slide. • Determination of which antigens are present on donor and recipient RBCs is the basis for matching blood types for transfusions. Dr. Zekeria Yusuf 330

331. In Agglutination Inhibition, Absence of Agglutination is diagnostic of Antigen • A modification of the agglutination reaction, called agglutination inhibition, provides a highly sensitive assay for small quantities of an antigen. • For example, one of the early types of home pregnancy test kits included latex particles coated with human chorionic gonadotropin ( HCG ) and antibody to HCG . • The addition of urine from a pregnant woman, which contained HCG , inhibited agglutination of the latex particles when the anti- HCG antibody was added; thus the absence of agglutination indicated pregnancy. Dr. Zekeria Yusuf 331

332. • Agglutination inhibition assays can also be used to determine whether an individual is using certain types of illegal drugs, such as cocaine or heroin. • A urine or blood sample is first incubated with antibody specific for the suspected drug. • Then red blood cells (or other particles) coated with the drug are added. • If the red blood cells are not agglutinated by the antibody, it indicates the sample contained an antigen recognized by the antibody, suggesting that the individual was using the illicit drug. Dr. Zekeria Yusuf 332

333. ENZYME LINKED IMMUNOSORBANT ASSAY (ELISA) • it is a recently developed method for measuring or detecting Ag- Ab reaction and has been now used in the detection of variety of Ab sand Ag s such as hormones, toxins, virus etc. • There are two methods of ELISA – Direct method used for detection of Ag – indirect method used for detection of Ab Dr. Zekeria Yusuf 333

334. DIRECT METHOD (DOUBLE ANTIBODY SANDWICH ELISA) • The wells of micro- ELISA plates are coated with antibodies which adhere to the surface of each well covalently. The walls are emptied and washed so that any free or excess antibodies are removed. • The solution to be tested for Ag is placed on the Ab coated well and incubated for two hours at 370c . the Ag if specific to Ab , will bind to it forming Ag- Ab complex. • The wells are again washed thoroughly with buffer to remove any unbound or free Ag. Dr. Zekeria Yusuf 334

335. Now enzyme linked Ab (second Ab ) is added to the wells and incubated at 370c for 1hr . The Ag is thus sandwiched between the first Ab (that coated to well) and enzyme linked second Ab. • Again through washing is done with buffer solution to remove excess amount of enzyme linked second antibody which have not bound to the Ag molecule. • Now specific substrate is added to the wells and left at room temperature till the development of specific colour . The enzyme phosphatase splits the substrate to yellow compound and this colour in the reaction may be read visually or estimated colourimetrically . • The intensity of the colour is directly proportional to the amount of enzyme that is specifically bound to the well; which in turn is proportional to concentration of Ag present in solution or specimen. Dr. Zekeria Yusuf 335

336. INDIRECT METHOD • The wells of micro- ELISA plate are coated with antigen which adsorb to the surface of well. The wells are emptied and washed to remove free and excess antigen. • The test serum is added to the Ag coated wells and plate incubated for 2 hrs at 370c . The antigen if specific to Ab , will bind with it forming AG- Ab complex. • The wells are again washed thoroughly with buffer to remove any unbound/ free Ab. • Enzyme linked second antibody is added to wells and incubated at 370c for 1 hr • Again through washing is done to remove any unbound enzyme linked second antibody. Dr. Zekeria Yusuf 336

337. • Substrate is added and well left at room temperature. The development of colour indicates presence of antibodies in serum. The intensity of colour is measured by spectrophotometer. • The intensity of colour is associated with amount of enzyme linked second antibody which in turn is proportional to concentration of Ab’s present in the test serum. Dr. Zekeria Yusuf 337

338. Failure of immune response Immune response helps individuals defend against microbes some cancers Immune response can fail hypersensitivity reactions immunodeficiency 338 Dr. Zekeria Yusuf

339. HYPERSENSITIVITY REACTIONS An immune response mobilizes a battery of effector molecules that act to remove antigen by various mechanisms. Generally, these effector molecules induce a localized inflammatory response that eliminates antigen without extensively damaging the host’s tissue. Under certain circumstances, however, this inflammatory response can have deleterious effects, resulting in significant tissue damage or even death. This inappropriate immune response is termed hypersensitivity or allergy. Although the word hypersensitivity implies an increased response, the response is not always heightened but may, instead, be an inappropriate immune response to an antigen. Hypersensitive reactions may develop in the course of either humoral or cell mediated responses. Dr. Zekeria Yusuf 339

340. Hypersensitivity reactions Cause cell damage through excessive immune response to antigens Hypersensitivity overreaction to infectious agents Allergy overreaction to environmental substances Autoimmunity overreaction to self 340 Dr. Zekeria Yusuf

341. Immunodeficiency Loss or inadequate function of various components of the immune system Can occur in any part or state of the immune system physical barrier, phagocytes, B lymphocytes, T lymphocytes, complement, natural killer cells The immuno -compromised host has an impaired function of immune system is at high risk of infection 341 Dr. Zekeria Yusuf

342. Immunodeficiency Congenital (primary) immunodeficiency genetic abnormality defect in lymphocyte maturation Acquired (secondary) immunodeficiency results from infections, nutritional deficiencies or treatments AIDS, chronic leukemia 342 Dr. Zekeria Yusuf

343. Altered immunity: immuno-compromised Disorder Compromised function Altered anatomic barrier Mucus membrane Reduction in IgA Microbe binding Gastro-intestinal tract Elevated pH Bacteria killing Change in flora Colonization resistance Immune system Innate immunity Reduction of complement Activates phagocytosis Opsonization of bacteria Membrane attack complex Neutropenia Monocytopenia Phagocytosis Bacteria killing Adaptive immunity Reduction of T cells Activation of macrophages Activation of B lymphocytes Hypo-gammaglobulinemia Neutralizes pathogens and toxins, opsonization, complement activation

344. Summary (1) Innate immunity relies on mechanisms already existing before microbe infects host is the first line of defense has no memory for subsequent exposure relies on non specific mechanisms 344 Dr. Zekeria Yusuf

345. Summary (2) Adaptive immunity develops following entry of microbe into the host comes into action after innate immunity fails to get rid of microbe has memory to deal with subsequent exposure happens through specific cells T cells (cell mediated) B cells (antibody mediated) 345 Dr. Zekeria Yusuf

346. Summary (3) Primary immune response short lasting smaller in magnitude Secondary immune response longer in duration larger in magnitude develop ‘memory cells’ following primary response Failure of immune response can result in: hypersensitivity immunodeficiency 346 Dr. Zekeria Yusuf

347. STRUCTURE AND PROPERTIES OF ANTIGENS – ISO AND ALLOANTIGENS Substances that be recognized by the immunoglobulin receptor of B cells, or by the Tcell receptor when complexed with MHC , are called antigens. Fundamental differences in the way B and T lymphocytes recognize antigen determine which molecular features of an antigen are recognized by each branch of the immune system. Immunogenicity and antigenicity are related but distinct immunologic properties that sometimes are confused. Immunogenicity is the ability to induce a humoral and/or cellmediated immune response. Dr. Zekeria Yusuf 347

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349. Immunogenicity Although a substance that induces a specific immune response is usually called an antigen, it is more appropriately called an immunogen . Antigenicity is the ability to combine specifically with the final products of the above responses (i.e., antibodies and/or cell-surface receptors). Although all molecules that have the property of immunogenicity also have the property of antigenicity , the reverse is not true. Some small molecules, called haptens , are antigenic but incapable, by themselves, of inducing a specific immune response. In other words, they lack immunogenicity. Dr. Zekeria Yusuf 349

350. ANTIGEN SPECIFICITY The antigenic specificity of the immune system permits it to distinguish subtle differences among antigens. Antibodies can distinguish between two protein molecules that differ in only a single amino acid. The immune system is capable of generating tremendous diversity in its recognition molecules, allowing it to recognize billions of unique structures on foreign antigens. Once the immune system has recognized and responded to an antigen, it exhibits immunologic memory; that is, a second encounter with the same antigen induces a heightened state of immune reactivity. Because of this attribute, the immune system can confer life-long immunity to many infectious agents after an initial encounter. Finally, the immune system normally responds only to foreign antigens, indicating that it is capable of self/ nonself recognition. The ability of the immune system to distinguish self from nonself and respond only to nonself molecules is essential, since the outcome of an inappropriate response to self molecules can be fatal. Dr. Zekeria Yusuf 350

351. STRUCTURE AND FUNCTIONS OF MHC AND HLA SYSTEMS Every mamaliaan species studied till date possesses a tightly linked cluster of genes, the major histocompatibility complex ( MHC ), whose products play roles in intercellular recognition and in discrimination between self and nonself . The MHC participates in the development of both humoral and cellmediated immune responses. While antibodies may react with antigens alone, most T cells recognize antigen only when it is combined with an MHC molecule. Furthermore, because MHC molecules act as antigen-presenting structures, the particular set of MHC molecules expressed by an individual influences the repertoire of antigens to which that individual’s TH and TC cells can respond. For this reason, the MHC partly determines the response of an individual to antigens of infectious organisms, and it has therefore been implicated in the susceptibility to disease and in the development of autoimmunity. The recent understanding that natural killer cells express receptors for MHC class I antigens and the fact that the receptor– MHC interaction may lead to inhibition or activation expands the known role of this gene family. Dr. Zekeria Yusuf 351

352. Functions of MHC COMPLEX: The MHC molecules act as cell surface markers which enable infected cells to signal cytotoxic and helper Tcells . There is no doubt that this role in immune responsiveness is immensely important, and in this respect the rich polymorphism of the MHC region represents a species response to maximize protection against diverse microorganisms. The major histocompatibility complex ( MHC ) is a large genetic complex with multiple loci. The MHC loci encode two major classes of membrane-bound glycoproteins : class I and class II MHC molecules. TH cells generally recognize antigen combined with class II molecules, whereas TC cells generally recognize antigen combined with class I molecules. Dr. Zekeria Yusuf 352

353. TISSUE TRANSPLANTATION Transplantation the term in immunology, refers to the act of transferring cells, tissues, or organs from one site to another. The desire to accomplish transplants stems from the realization that many diseases can be cured by implantation of a healthy organ, tissue, or cells (a graft) from one individual (the donor) to another in need of the transplant (the recipient or host). The development of surgical techniques that allow the facile reimplantation of organs has removed one barrier to successful transplantation, but others remain. One is the lack of organs for transplantation. Although a supply of organs is provided by accident victims and, in some cases, living donors, there are more patients in need of transplants than there are organs available. Dr. Zekeria Yusuf 353

354. TISSUE TYPING METHODS FOR TISSUE AND ORGAN TRANSPLANTATIONS Since differences in blood group and major histocompatibility antigens are responsible for the most intense graft-rejection reactions, various tissue-typing procedures to identify these antigens have been developed to screen potential donor and recipient cells. Initially, donor and recipient are screened for ABO blood-group compatibility. The blood-group antigens are expressed on RBCs , epithelial cells, and endothelial cells. Antibodies produced in the recipient to any of these antigens that are present on transplanted tissue will induce antibody mediated complement lysis of the incompatible donor cells. HLA typing of potential donors and a recipient can be accomplished with a microcytotoxicity test. Dr. Zekeria Yusuf 354

355. Autoimmunity Basically means immunity to self •A condition that occurs when the immune system mistakenly attacks and destroys healthy body tissue Autoimmunity is the failure of an organism in recognizing its own constituent parts as non self, which allows an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Autoimmunity is often caused by a lack of germ development of a target body and as such the immune response acts against its own cells and tissues. Dr. Zekeria Yusuf 355

356. Autoimmunity… Autoimmunity results from a failure or breakdown of the mechanisms normally responsible for maintaining self-tolerance in B cells, T cells, or both. • The major factors that contribute to the development of autoimmunity are genetic susceptibility and environmental triggers, such as infections. • Autoimmune diseases may be either systemic or organ specific. • Various effector mechanisms are responsible for tissue injury in different autoimmune diseases. • Epitope spreading: Autoimmune reactions initiated against one self antigen that injure tissues may result in the release and alterations of other tissue antigens, activation of lymphocytes specific for these other antigens, and exacerbation of the disease. Dr. Zekeria Yusuf 356

357. AUTOIMMUNITY Dr. Zekeria Yusuf 357

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361. AUTOIMMUNE DISEASES •A Group of 60 to 80 chronic inflammatory diseases with genetic predisposition and environmental modulation •Prevalence of 5% to 8% in US •Prevalence is greater for females than males 75% of cases •4th largest disease class in women •Autoimmunity is a destructive immune response against self antigens (how does this compare to hypersensitivities?) •Once started, autoimmune diseases are hard to stop Severity ranges from minor to lethal. Dr. Zekeria Yusuf 361

362. AUTOIMMUNE DISEASES •Autoimmunity can be caused by immunological, genetic, viral, drug-induced, and hormonal factors. •There are 4 immunological mechanisms of autoimmunity. •All mechanisms cause abnormal B or T cell activation. •Centrality of the Ternary Complex •Most instances of autoimmune diseases occur with multiple mechanisms, which makes treatment difficult. Dr. Zekeria Yusuf 362

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366. APPLICATIONS OF MEDICAL BIOTECHNOLOGY 1. PHARMACOLOGY 2. GENE THERAPY 3. STEM CELLS 4. TISSUE ENGINEERING Dr. Zekeria Yusuf 366

367. 1. Pharmacology 1. INSULIN PRODUCTION Production of genetically engineered human insulin was one of the first breakthroughs of biotechnology in the pharmaceutical industry. Insulin was first produced in Escherichia coli through recombinant DNA technology in 1978. Mass production of human proteins, vaccines, etc. by genetically modifying bacteria or viruses. The human gene for insulin is placed into bacteria, are cultured and allowed to produce insulin which is collected, purified and sold to diabetics worldwide. Grow bacteria that make the insulin protein (fermentation) Isolate the protein from all the other stuff that was in the fermentation tank (purification) Convert the insulin to its active form (processing) Dr. Zekeria Yusuf 367

368. 2. Human growth hormone Production of human growth hormone was first done in 1979 using recombinant DNA technology. scientists produced human growth hormone by inserting DNA coding for human growth hormone into a plasmid that was implanted in Escherichia coli bacteria. This gene that was inserted into the plasmid was created by reverse transcription of the mRNA found in pituitary glands to complementary DNA. Prior to this development, human growth hormone was extracted from the pituitary glands of cadavers, as animal growth hormones have no therapeutic value in humans. Dr. Zekeria Yusuf 368

369. 3.HUMAN BLOOD CLOTTING FACTOR Production of human clotting factors was enhanced through recombinant DNA technology. Human clotting factor ix was the first to be produced through recombinant DNA technology using transgenic Chinese hamster ovary cells in 1986. Plasmids containing the factor IX gene, along with plasmids with a gene that codes for resistance to methotrexate , were inserted into Chinese hamster ovary cells via transfection . Dr. Zekeria Yusuf 369

370. 4.GENE PILL 1. Gene pill delivers DNA to Intestine 2. DNA is absorbed by gut cells 3. Protein drug is synthesized inside the cells 4. Protein drug is secreted into the blood Dr. Zekeria Yusuf 370

371. 5. MONOCLONAL ANTIBODIES ( MAB ) They are so called because they are clones of an individual parent cell. Remember, antibodies are specific proteins that target pathogens invading our body. Steps in making them: 1. Human antibody genes are put into a mouse. 2. Mouse is infected causing it to make human antibody producing cells (B-cells). 3. These cells are removed from the mouse and fused with a tumour cell. 4. Now we have a tumour cell that is constantly producing antibodies and more cells like itself. Dr. Zekeria Yusuf 371

372. Monoclonal antibodies… This technology is used primarily to fight off cancer cells as these monoclonal antibodies can be “trained” to target markers that show up on cancer cells. The mAbs will then destroy the cancer cell and go looking for more. Dr. Zekeria Yusuf 372

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374. Gene therapy Gene therapy is the use of DNA as a pharmaceutical agent to treat disease. It derives its name from the idea that DNA can be used to supplement or alter genes within an individual's cells as a therapy to treat disease The most common form of gene therapy involves using DNA that encodes a functional, therapeutic gene to replace a mutated gene. Gene therapy is of two types , somatic gene therapy and germ line gene therapy. Dr. Zekeria Yusuf 374

375. GENE THERAPY FOR DISEASES Gene Therapy has made important medical advances in less than two decades. Within this short time span, it has moved from the conceptual stage to technology development and laboratory research to clinical translational trials for a variety of deadly diseases. Dr. Zekeria Yusuf 375

376. GENE THERAPY FOR GENETIC DISORDERS SEVERE COMBINED IMMUNE DEFICIENCY (ADA- SCID ) ADA- SCID is also known as the bubble boy disease. Affected children are born without an effective immune system and will succumb to infections outside of the bubble without bone marrow transplantation from matched donors. The therapeutic gene called ADA was introduced into the bone marrow cells of such patients in the laboratory, followed by transplantation of the genetically corrected cells back to the same patients. The immune system was reconstituted in all six treated patients without noticeable side effects, who now live normal lives with their families without the need for further treatment. Dr. Zekeria Yusuf 376

377. CHRONIC GRANULOMATUS DISORDER ( CGD ) CGD is a genetic disease in the immune system that leads to the patients' inability to fight off bacterial and fungal infections that can be fatal. Using similar technologies as in the ADA- SCID trial, investigators in Germany treated two patients with this disease, whose reconstituted immune systems have since been able to provide them with full protection against microbial infections for at least two years. Dr. Zekeria Yusuf 377

378. HEMOPHILIA Patients born with Hemophilia are not able to induce blood clots and suffer from external and internal bleeding that can be life threatening. The therapeutic gene was introduced into the liver of patients, who then acquired the ability to have normal blood clotting time. Dr. Zekeria Yusuf 378

379. GENE THERAPY FOR ACQUIRED DISEASES Multiple gene therapy strategies have been developed to treat a wide variety of acquired diseases like: Cancer Parkinson's Disease Huntington's Disease Influenza HIV Hepatitis Dr. Zekeria Yusuf 379

380. STEM CELLS A stem cell is a cell that has the potential to become any cell type in the human body. The easiest place to get stem cells is from an embryo. Stem cells are introduced into a damaged area of the body where, under the right conditions, will replace the damaged area. Dr. Zekeria Yusuf 380

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382. Stem cells are currently being tested to treat everything from Crohn’s disease to baldness! The main areas where stem cells have proven their worth is in bone marrow transplants, replacing damaged heart tissue after a heart attack and replacing damaged nerve tissue which gives hope to anyone who has had a spinal cord injury. Dr. Zekeria Yusuf 382

383. STEM CELLS (SOURCES) Embryonic stem cells Infant and adult stem cells Present in small numbers in Bone marrow Peripheral blood Skin epithelium Umbilical cord blood Dental pulp of infant’s teeth May be obtained by reprogramming somatic cells Introduction of retroviruses carrying reprogramming genes into fibroblasts Dr. Zekeria Yusuf 383

384. TISSUE ENGINEERING A form of regenerative medicine, tissue engineering is the creation of human tissue outside the body for later replacement. Usually occurs on a tissue scaffold, but can be grown on/in other organisms. Dr. Zekeria Yusuf 384

385. The technique to grow an ear follows the steps 1) taking a tiny piece of cartilage tissue, 2) dissolving away the white springy tissue to collect the actual cells inside (the cells are microscopic & trapped inside the white tissue called matrix) 3) expanding the number of cells by various methods in the lab 4) placing that increased volume of cells on or in mould that have a shape of an ear 5) implanting the new ear onto the patient. Dr. Zekeria Yusuf 385

386. IMMUNIZATION thru vaccination Vaccines – conventional, peptide vaccines, subunit, DNA vaccines, toxoids , antisera , edible vaccines, plantibodies , ISCOMs , recombinant antibodies . Immune stimulatory complexes, Common immunization programmes – BCG , small pox, DPT , polio, measles, Hepatitits – B. Dr. Zekeria Yusuf 386

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394. TOXOIDS Some bacterial pathogens, including those that cause diphtheria and tetanus, produce exotoxins . These exotoxins produce many of the disease symptoms that result from infection. Diphtheria and tetanus vaccines, for example, can be made by purifying the bacterial exotoxin and then inactivating the toxin with formaldehyde to form a toxoid . Vaccination with the toxoid induces anti- toxoid antibodies, which are also capable of binding to the toxin and neutralizing its effects. The problem of obtaining sufficient quantities of the purified toxins to prepare the vaccines has been overcome by cloning the exotoxin genes and then expressing them in easily grown host cells. In this way, large quantities of the exotoxin can be produced, purified, and subsequently inactivated. Dr. Zekeria Yusuf 394

395. ANTISERA An antigen binding site in an antibody ( paratope ) is a reflection of the three-dimensional structure of part of the antigen ( epitope ). This unique amino acid structure in the antibody is known as the idiotype , which can be considered as a mirror of the epitope in the antigen. Antibodies can be raised against the idiotype by injecting the antibody into another animal. This anti- idiotype antibody mimics part of the three dimensional structure of the antigen. This can be used as a vaccine. When the anti- idiotype antibody is injected into a vaccinee , antibodies ( antianti-idiotype antiobodies ) are formed that recognize a structure similar to part of the virus and might potentially neutralize the virus. Dr. Zekeria Yusuf 395

396. EDIBLE VACCINES Edible vaccines are composed of antigenic proteins and do not contain pathogenic genes (because obviously they use attenuated strains). Thus, they have no way of establishing infection and safety is assured. Oral administration is possible , production is highly efficient and can be easily scaled up. Dr. Zekeria Yusuf 396

397. PLANTIBODIES A plantibody (a portmanteau derived from plant and antibody) is an antibody produced by genetically engineered crops. Antibodies are part of animal immune systems, and are produced in plants by transforming them with antibody genes from animals. This was first done in 1989, with a mouse antibody made by tobacco plants. Although plants do not naturally make antibodies, plantibodies have been shown to function in the same way as normal antibodies. There are two potential major applications for plantibodies . A number of companies in addition to Biolex , including Planet Biotechnology in California, and Medicago in Canada are pursuing the commercial development of plantibodies as therapies for everything from cancer to the common cold. Secondly, the production of plantibodies might be used to make (genetically modified) crops resistant to plant pathogens. Dr. Zekeria Yusuf 397

398. RECOMBINANT ANTIBODIES Theoretically, the gene encoding any immunogenic protein can be cloned and expressed in bacterial, yeast, or mammalian cells using recombinant DNA technology. A number of genes encoding surface antigens from viral, bacterial, and protozoan pathogens have been successfully cloned into bacterial, yeast, insect, or mammalian expression systems, and the expressed antigens used for vaccine development. The first such recombinant antigen vaccine approved for human use is the hepatitis B vaccine. This vaccine was developed by cloning the gene for the major surface antigen of hepatitis B virus ( HBsAg ) and expressing it in yeast cells. The recombinant yeast cells are grown in large fermenters , & HBsAg accumulates in the cells. The yeast cells are harvested and disrupted by high pressure, releasing the recombinant HBsAg , which is then purified by conventional biochemical techniques. This recombinant hepatitis B vaccine has been shown to induce the production of protective antibodies Dr. Zekeria Yusuf 398

399. ISCOM (Immune stimulating complex) Immune stimulating complexes ( ISCOMs ) are spherical open cage-like structures (typically 40 nm in diameter) that are spontaneously formed when mixing together cholesterol, phospholipids and Quillaia saponins under a specific stoichiometry . The complex displays immune stimulating properties and is thus mainly used as a vaccineadjuvant in order to induce a stronger immune response and longer protection. Dr. Zekeria Yusuf 399

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404. Tumor immunity Explain how the immune system of the host responds to the presence of a tumour . Tumors arise from accumulated genetic mutations Carcinogenesis is a multistep process at both the phenotypic and the genetic levels resulting from the accumulation of multiple mutations. Immune system react to antigens that it recognizes as foreign. Tumor cells can be recognized by the immune system as non-self. Recognition and destruction of non-self tumor cells by the immune system (immunological resistance of the host against the development of cancer). Dr. Zekeria Yusuf 404

405. Tumor antigens two categories: based on their patterns of expression: 1. Tumor-specific antigens - present only on tumor cells and not on any normal cells 2. Tumor-associated antigens - present on tumor cells and also on some normal cells Dr. Zekeria Yusuf 405

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