Antibodies PDF

Antibodies Dr Sherko Ali Omer Dept. of Basic Medical Sciences Learning objectives By the end of this session, students should be able to : • Define the natures and structure of antibodies • Identify different Immunoglobulin classes and their function • Explain antigenic determinants of immunoglobu...

Antibodies Dr Sherko Ali Omer Dept. of Basic Medical Sciences Learning objectives By the end of this session, students should be able to : • Define the natures and structure of antibodies • Identify different Immunoglobulin classes and their function • Explain antigenic determinants of immunoglobulins • Define the monoclonal antibodies mAb, explain their production and identify their application in different medical fields • Identify the genetic behind immunoglobulins diversity and mechanism to produce different antibody isotypes 2 Antibody or Immunoglobulin Specialized glycoprotein, produced from activated B cells (plasma cells) in response to an antigen. Immunoglobulin (Ig) are capable of combining with the antigen that triggered their production. Arne Tiselius in 1939 subjected the serum to electrophoresis, the serum proteins are separated into four fragments- albumin, globulin α, β and γ. Antibodies are located in the γ-globulin fraction; because they immunologically react with the antigen; they were given the name as immunoglobulin. 3 Antibody or Immunoglobulin 4 Antibody or Immunoglobulin Both the terms, immunoglobulin (Ig) and antibody are used interchangeably; representing the physiological & functional properties of same molecule respectively. Immunoglobulin constitutes 20-25 per cent of total serum proteins. There are five classes (or isotypes) of immunoglobulins: 0000 IgG, IgA, IgM, IgD and IgE 5 Structure of antibody An antibody molecule is a ‘Y-shaped’ heterodimer; composed of four polypeptide chains: f • Two identical light (L) chains, of molecular weight 25,000 Da each. • Two identical heavy (H) chains each having molecular weight 50,000 Da or more. 6 Structure of antibody All four H and L chains are bound to each other by disulfide bonds, and by noncovalent f interactions such as salt linkages, hydrogen bonds, and hydrophobic bonds. All the chains have two ends- an amino terminal end (NH3) and a carboxyl terminal end (COOH). 7 Antibody classes, Heavy chain There are five classes of H chains and two classes of light chains. The five classes of H chains are structurally and antigenically distinct; each is designated by the Greek letters γ, α, μ, δ and ε and is present only in a particular class of lg. The five classes of immunoglobulins (lgG, lgA, lgM, lgD and lgE) are classified based on the amino acid sequences of the heavy chains. 8 Antibody classes There are five classes of H chains: Immunoglobulin class Heavy chain type IgG IgA IgM IgD IgE γ (gamma) α (alpha) µ (mu) δ (delta) ε (epsilon) 9 Antibody structure, Light chain There are of two types of L chain: • Kappa (κ) • Lambda (λ) 01 These were named after Korngold and Lapari who originally described them. In humans, 60% of the light chains are kappa and 40% are lambda type (ratio 3:2). Both the light chains of an antibody molecule should be of same type, either κ or λ, but never both. L chains are composed of 214 amino acids; whereas the number of amino acids in the heavy chain varies ranging from 446 (in α chain) to 576 (in μ chain). 8 2 O 0 10 Antibody structure, Variable and Constant Regions Each H and L chain comprises of two regions: variable (V) and constant (C) region, depending upon whether the amino acid sequences of the regions show variable or uniform pattern among different antibodies. ruler V region represents the antigen binding site of the antibody. Of 0ns 11 Antibody structure, Variable and Constant Regions Within the variable region, there are some zones (hot spots) that show relatively higher variability in the amino acid sequences called as hypervariable regions or complementarity determining regions (CDRs). These form the antigen-binding site. There are three hot spots in the L and four in the H chain respectively. 12 Antibody structure, Variable and Constant Regions The amino acid sequence of constant region shows uniform pattern. Constant region constitutes the remaining part of an Ig molecule other than that of variable region. Length of the constant regions is approximately 104 amino acids for light chain, 330 amino acids for γ, α and δ heavy chains and 440 amino acids for µ and ε heavy chains. 13 Antibody structure, Paratope βParatope isItthe site on the hypervariable to regions that make actual contact with the epitope of an antigen. body 14 Antibody structure, Domains Light chain contains one variable domain (VL) and one constant domain (CL). T Heavy chains possess one variable domain (VH) and 3 or 4 numbers of constant domains (CH). Heavy chain of γ, α and δ have three constant domains-CH1, CH2 and CH3. Heavy chains µ and ε have four constant domains- CH1 to CH 4. mqqi.be 15 Antibody structure, Hinge Region Hinge region is rich in proline and cysteine. It is quite flexible, allowing the Ig molecule to assume different positions, thus helps the antibody in reaching towards the antigen. at 0 Hinge region is sensitive to various enzymatic digestions. 16 Antibody Enzymatic Digestion When an immunoglobulin molecule is subjected to enzymatic digestion, it generates various fragments. Types of digestion: i Papain digestion Pepsin digestion Mercaptoethanol reduction 17 Antibody Enzymatic Digestion, Papain Digestion Papain digestion of Ig results in three fragments each having a sedimentation coefficient (s) of 3.5: • Two Fab fragments • Fc fragment 18 Antibody Enzymatic Digestion, Pepsin Digestion Pepsin digestion of Ig results in: • One F (ab')2 fragment • Many smaller fragments of Fc 19 Antibody Enzymatic Digestion, Mercaptoethanol Reduction Mercaptoethanol reduction of Ig molecule- generates four fragments (two H and two L chains) as it cleaves only disulphide bonds sparing the peptide bonds. 20 Functions of Immunoglobulins Antigen binding (Fab region) Through fragment antigen binding, Ig will protect the host. It also Interact with the antigen. Valency of an antibody refers to the number of Fab regions it possesses. Thus, a simple monomeric antibody molecule has a valency of two. 21 Functions of Immunoglobulins Effector or biological functions (by Fc region) Fixation of complement: Ig coating the target cell binds to complement through its Fc receptor which leads to complement mediated lysis of the target cell. Binding to various cell types: Phagocytic cells, lymphocytes, platelets, mast cells, NK cell, eosinophils and basophils bear Fc receptors (FcR) that bind to Fc region of Ig. Binding can activate the cells to perform some biological functions. Some Ig (e.g. IgG) also bind to receptors on placental trophoblasts, which results in transfer of the Ig across the placenta. 22 Immunoglobulin Classes, Properties and Function Five classes of Ig: lgG, IgA, IgM, IgD and IgE, Each with different property and function. A G D E M 23 Immunoglobulin G (IgG) IgG constitutes about 70-80% of total Igs 0 e of the body. IgG has maximum daily production, highest serum concentration and longest half-life of 23 days. 24 Immunoglobulin G (IgG) 0000 IgG has four subclasses- IgG1, IgG2, IgG3 and IgG4; all differ from each other in the amino acid sequences of the constant region of their γ-heavy chain. e Subclasses vary in their biological functions, length of hinge region and number of disulphide bridges. IgG3 has longest hinge region with 11 inter-chain disulphide bonds. 25 Functions of IgG IgG can cross placenta- hence provide immunity to the fetus and new born. Among subclasses, IgG2 has the poorest ability to cross placenta. Complement fixing: Complement fixing ability of subclasses varies - IgG3> IgG1> IgG2. IgG4 does not fix complements. Phagocytosis: Aid phagocytosis as it bind to antigen and has receptors on phagocytes. 26 Functions of IgG IgG mediates precipitation and neutralization reactions. IgG plays a major role in neutralization of toxins as it can easily diffuse into extravascular space. IgG is raised after long time following infection and represents chronic or past infection (recovery). 27 Immunoglobulin M (IgM) Among all Igs, IgM has highest molecular weight, and maximum sedimentation coefficient (19 S). IgM is present only in intravascular compartment, not in body fluids or secretions. IT_ IgM exists in both monomeric and pentameric forms where five units are joined by J chain, but it present as membrane-bound antibody on B cells as monomeric form. When present in secreted form, it is pentameric in nature. 28 Functions of IgM Form in acute infection Complement fixing Antigen receptor Acts as an opsonin E Fetal immunity? Protection against intravascular organisms Mediate agglutination 29 Immunoglobulin A (IgA) IgA is the second most abundant class of Ig next to IgG, constituting about 10-15% of total serum Ig. IgA exists in both monomeric and dimeric forms. lgA in serum is predominantly in monomeric form. Serum lgA interacts with the Fc receptors expressed on immune effector cells, to initiate various functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), degranulation of immune cells, etc. 30 Secretory IgA Dimeric in nature; two IgA monomeric units joined by a J chain. Dimeril fory Secretory component: Location-Predominant antibody found in body secretions like milk, saliva, tears, intestinal & respiratory tract mucosal secretions. e Secretory component is derived from poly Ig receptor present on the serosal surfaces of the epithelial cells. 31 Formation of secretory IgA Dimeric sIgA is synthesised by plasma cells situated near mucosal epithelium, J chain is also produced in the same cell. Dimeric sIgA binds to poly Ig receptor on the basolateral surface of mucosal epithelium Receptor- IgA complex is endocytosed into mucosal epithelial cells Receptor is partially cleaved leaving behind a part of it (secretory component) Subsequently sIgA (complex of dimeric IgA with J chain and secretory component) is released into the mucosal secretions. 32 Function of secretory IgA Secretory IgA (sIgA) play role in: • Local or mucosal immunity • Effective against bacteria like Salmonella,Vibrio, Neisseria, and viruses like polio and influenza • Breast milk is rich in sIgA and provides good protection to the immunologically immature infant gut • Secretory component protects IgA from denaturation by bacterial proteases produced by intestinal flora. 33 Subclasses of IgA Depending upon the amino acid sequences in the constant region of heavy chain, IgA exists in two isotypes or subclasses: • IgA1 • IgA2 34 Immunoglobulin E (IgE) Minimum daily production, lowest serum concentration, and shortest half life. IgE is the only heat labile antibody (inactivated at 56°C in one hour). IgE has affinity for the surface of tissue cells (mainly mast cells) of the same species (homocytotropism). E IgE is the mediator of type I hypersensitivity (allergy) reactions and it is elevated in helminthic infections. 35 Immunoglobulin D (IgD) IgD is found as membrane Ig on the surface of B cells and acts as a B cell receptor along with IgM. Has the highest carbohydrate content among all the Igs. No other function is known for IgD so far. O O C 36 Properties and functions of Immunoglobulins Properties of various immunoglobulins Property IgG IgA IgM IgD IgE Usual form Monomer Monomer,dimer Monomer Monomer Valency 2 2 or 4 Monomer,Penta mer 2 or 10 2 2 Other chains None J chain None None Subclasses G1, G2, G3, G4 J chain, secretory component A1, A2 None None None Molecular weight (kDa) 150 150-600 900 150 190 Serum level mg/mL 9.5–12.5 IgA1- 3.0 IgA2 - 0.5 1.5 0.03 0.0003 % of total serum Ig 75–85% 10–15% 5–10% 0.3% 0.019% Half-life, days 23* 6 5 3 2.5 Daily production mg/kg 34 24 3.3 0.4 0.0023 37 Properties and functions of Immunoglobulins Properties of various immunoglobulins Property Intravascular distribution (%) Sedimentation coefficient Complement activation Classical Alternative Binds to Fc receptors of phagocytes Placental transfer Mediates coagglutination Mucosal transport Mast cell degranulation Marker for B cells Heat stability IgG 45% IgA 42% IgM 80% IgD 75% IgE 50% 7 7 19 7 8 ++ (IgG3>1>2) ++ – + - +++ ? ** – - – - Yes (except IgG2) Yes (except IgG3) + Yes + + + + + yes 38 Antigenic Determinants of Immunoglobulins The entire Ig molecule is not immunogenic, but it contains some antigenic determinants at specific sites. Based on the location of antigenic determinants, the Ig molecules are divided into: • Isotypes • Allotypes • Idiotypes 39 Ig Isotypes The five classes of Igs and their subclasses are called as isotypes. Vary from each other in the amino acid sequences of the constant region of their heavy chains. Such variation is called as isotypic variation. 40 Ig Allotypes Immunoglobulin allotypes are genetically determined antigenic . differences in Immunoglobulins that varies in different members of the same species. These differences are located in C region so that a particular isotype may have several alternative allelic structure. 41 Ig Allotypes To date, three systems of allotypic markers have been characterized for humans: Kappa light chain (Km system). γ heavy chain (Gm system. α heavy chain (Am system). Antibody to allotype determinants can be produced by injecting Igcontaining these determinants from one member to another within a given species. Anti allotype specific antibodies may also be developed following blood transfusion or by maternal passage of IgG into the fetus. 42 Ig Idiotypes Unique amino acid sequence present in paratope region (in VH and VL regions) of one member of a species acts as antigenic determinant to other members of the same species. Such antigenic determinants are called as idiotopes and the sum total of idiotopes on an Ig molecule constitutes its idiotypes. 43 Ig Idiotypes Idiotypes in an individual arise continuously from mutations (somatic hypermutations) in the genes of variable region. Idiotypes may act as foreign to the host itself; however do not evoke autoimmune response because they are present in small numbers. 44 Abnormal Immunoglobulins Abnormal Ig Explanation Bence Jones proteins Produced in a neoplastic condition of plasma cells called multiple myeloma (light chain disease) Waldenstrom’s macroglobulinemia Lymphoma affecting B cells producing excess IgM Heavy chain diseases Characterized by an excessive production of heavy chains that are short and truncated Cryoglobulinemia Condition where the blood contains cryoglobulins; a type of Ig that becomes insoluble (precipitate) at low temperatures but redissolves again if the blood is heated 8 45 Polyclonal vs. Monoclonal Nature of Antibody Naturally antigen having multiple epitopes enters the body where each epitope may stimulate one clone of B cells producing one type of antibody. So the serum contains mixture of antibodies (polyclonal antibody) derived from different clones of B cells. When only one clone of B cell is stimulated by a single epitope of an antigen and then is allowed to proliferate and produce antibodies; such antibodies are referred to as monoclonal antibodies (mAb). 46 Monoclonal Antibody Antibodies derived from a single clone of plasma cell; all having the same antigen specificity- i.e. produced against a single epitope of an antigen. mAb (Hybridoma technique) was developed by Georges Kohler and Cesar Milstein in 1975 ; for which they were awarded Nobel Prize in 1984. 47 mAb, Principles of Hybridoma Technique Hybridoma technique fuse B cells from an immunized animal (typically a mouse) with a myeloma cell line and growing the cells under conditions in which the unfused normal and tumor cells cannot survive. The resultant fused cells that grow out are called hybridomas; each hybridoma makes only one Ig, i.e. mAb. 48 mAb, Principles of Hybridoma Technique • Mouse is injected with an antigen, after an interval, the mouse splenic B cells are obtained. • Myeloma (cancerous immortal plasma) cells Used as a source of immortal cell. myeloma cells are genetically modified with two mutation so that they lose the ability to produce their own antibody but retain immortal property. • Fusion between B and myeloma cells occur in polyethylene glycol broth. 49 mAb, Principles of Hybridoma Technique • Following the fusion three types of cells will result Unfused myeloma cells Unfused mouse splenic B cells Fused hybridoma cells • Sub culturing the cells on HAT (hypoxanthine, aminopterin and thymidine) media, only fused hybridoma cell survive • Due to HAT media, HGPRT (hypoxanthine guanine phosphoribosyl transferase) and thymidine kinase are needed for cell survival, so any cell (e.g. myeloma cell) that lacks HGPRT cannot grow on HAT medium 50 mAb, Principles of Hybridoma Technique • Unfused splenic B cells can grow but do not survive long as they are not immortal • Unfused myeloma cells- Cannot grow as they lack HGPRT enzyme to perform the salvage pathway of purine synthesis • Hybridoma cells can grow and survive long • cells is dispensed into multi-well plates to such an extent that each well contains only one cell • Hybridoma cells producing the desired mAb by RIA or ELISA techniques using the specific antigen fragments, and are selectively proliferated. 51 mAb, Principles of Hybridoma Technique • The selected hybridoma cells can be maintained in two ways: • Hybridoma cell is cultured to generate a clone of identical cells; producing pure form of mAb. • Desired hybridoma cell is injected into the peritoneal cavity of mouse where it can multiply and produce mAb in ascitic fluid. 52 Modifications in mAb by recombination • Mouse mAb - 100% mouse derived proteins • Chimeric mAb is prepared by recombination of 34% mouse proteins (variable region) and 66% human proteins (constant region). • Humanized mAb- Only the antigen binding site (i.e. CDRcomplementarity determining regions) is mouse derived (10%) and the remaining part of mAb is human derived. • Human mAb- 100% of amino acids are human derived. It is the best accepted mAb in humans. 53 Types of monoclonal antibodies Mouse mAb can induce immune response in humans producing human anti-mouse antibody (HAMA); It is eliminated faster from the body. Hence mouse derived monoclonal antibodies are not the best for human use humanuse. 54 Applications of monoclonal antibodies Diagnostic reagents- mAb used widely for detection of antigen using different techniques • Detection of infections, such as hepatitis B, serogrouping of streptococci, etc. • Pregnancy detection test—by using monoclonal antibody against human chorionic gonadotropin • Blood grouping can be done by using anti-A and anti-B monoclonal antibodies • Tumor detection and imaging: By using mAb specific for tumor antigens secreted by tumor cells (e.g. prostate specific antigen) • Tissue typing for transplantation can be done by using anti-HLA monoclonal antibodies. 55 Applications of monoclonal antibodies Isolation and purification- mAb can be used to purify individual molecule from a mixture even when they are present in low concentration. e.g. interferon and coagulation factor VIII. Identification of cells and clones- TH and TC cells are identified by using anti-CD4 and anti-CD8 mAb using flocytometry. Monitoring proteins and drug level in serum. As a passive immunity Ig - as in post exposure prophylaxis against various infections, mAb targeting specific antigens of the infecting organism can be administered, examples include- immunoglobulins against hepatitis B, rabies, and tetanus. 56 Applications of monoclonal antibodies Therapeutic use- Treatment of various inflammatory & allergic diseases and cancer. The mechanisms by which monoclonal antibodies work as therapeutic: • Suppress immune system • Kill or inhibit malignant cells • Inhibit angiogenesis Monoclonal Targeted against antibody Suppress immune system Adalimumab and TNF-α Infliximab Used in treatment of Omalizumab Daclizumab Muromonab Anticancer Trastuzumab Rituximab Inhibit angiogenesis Bevacizumab IgE IL-2 receptor CD3 Asthma Rejection of kidney transplants HER-2 CD20 Breast cancer Lymphoma VEGF(Vascular endothelial growth factor) Platelet receptor GpIIb/IIIa Colorectal cancers Abciximab Rheumatoid arthritis Crohn's disease Coronary artery 57 disease Applications of monoclonal antibodies 1 Used as immunotoxin- mAb conjugated with bacterial/chemical toxins (e.g. diphtheria toxin) can be used to kill the target cells such as cancer cells. mAb against surface receptors helps in binding to the target cells and the toxin helps in target cell killing. Used as enzymes- Abzyme is a monoclonal antibody with catalytic activity. 58 Genetics of Antibody Production The mechanism of organization of Ig gene is unique and different from the classical ‘one gene-one polypeptide’ genetic model. In contrast, Ig polypeptide chains are coded by more than one gene. 59 Multigene organization of Immunoglobulin There are three basic principles of this model which are summarized below. 1. Ig molecule is not coded by a single gene 2. Heavy chain is coded by four gene segments- V (variable), D (diversity) and J (joining) and C (constant) gene segments. Light chains are coded by three genes V, J and C gene segments. 3. Ig genes are encoded in different chromosomes: • H chain gene family is located on chromosome 14 • Kappa light chain gene family is located on chromosome 2 • Lambda light chain gene family is located on chromosome 22 60 Multigene organization of Immunoglobulin Multiple genes exist for each genetic segment of Ig chain. Ig molecule is produced by recombination between various gene segments. Gene rearrangement occurs at: Rearrangement at DNA level Rearrangement at RNA level 61 Multigene organization of Immunoglobulin 62 Rearrangement at DNA level There is rearrangement and splicing between the DNA segments of variable region of both H and L chains. H chain gene region undergoes rearrangement first followed by L chain gene region. H chain, D-J joining occurs first followed by V-DJ joining whereas in L chain, only V -J joining takes place. • Recombination of gene segments occurs at the time of joining which is mediated by special recombinase enzymes, encoded by RAG (recombination activation genes). • C gene segments of both H & L chains are not joined at DNA level; but remain separate. 63 Rearrangement at RNA level V, D, J and C gene segments are transcribed to generate primary RNA transcript. C region RNA transcripts combine with variable region RNA transcripts to generate complete H and L chains. Differential Ig RNA processing- It is an important event which occurs at post translational level which is responsible for• Directing the synthesis of immunoglobulin as membrane bound Ig or secretory Ig. • Simultaneous expression of membrane Ig (IgM and IgD) on surface of mature B cells. 64 Antibody Diversity Human immune system is capable of producing vast number of antibodies (108 or even more) corresponding to various epitopes of different antigens. Gene segment V D J C Number of genes H chain κ L chain λ L chain 51 40 30 27 0 0 6 5 4 9 1 4 Type of joining Possible combinations V-D-J combinations in heavy chain V-J combinations in κ chain 51VH X 27DH X 6JH genes = 8262 40Vκ X 5Jκ genes = 200 V-J combinations in λ chain Combinations of H & L chains 30Vλ X 4Jλ genes = 120 8262 X 200 X 120 = 2.64 X 106 65 Mechanism of antibody diversity • Multiple genes for each segment coding for Ig chain • Many possible combinations of joining of variable region gene segments • Junctional flexibility :V-DJ, D-J and V-J joining can take place at any level of several nucleotides present at the ends of V, D, J segments. • Junctional diversity: The V/D/J joining is a highly inaccurate process that results in the addition or subtraction of variable number of nucleotides and, thus, generates junctional diversity. • Somatic hypermutation: following antigenic stimulus to B cells,V region genes undergo point mutations (resulting from nucleotide substitutions) at a higher frequency, hence named as hypermutation. This helps in affinity maturation of B cells. 66 Class switching or isotype switching Class switching allows any given VH domain to associate with the constant region of any isotype. This enables antibody specificity to remain constant while the biological effector activities of the molecule vary. 67 Learning Objectives By the end of this session, students should be able to: 1. Summarize events in the process of opsonization. 2. Interpret the role played by eosinophils in host defense during multicellular parasitic infection via ADCC, and the function of eosinophils in regulation of type I hypersensitivity reactions. 3. Compare both the live attenuated oral (Sabin) poliovirus vaccine with the inactivated poliovirus (Salk) vaccine, in terms of mucosal immunity and secretory IgA production. 4. Infer the process of isotype switching in the primary and secondary immune responses. Case Study 1: The immune response observed in an apparently healthy 12-year-old Caucasian male after recurrent exposure to a bacterial antigen is characterized by rapid increase in serum lgG level. Some immunoglobulin molecules are attached to the surface of macrophages, neutrophils and B lymphocytes. Which of the following is the cell attachment site for the immunoglobulin molecule shown below? Explain and discuss your answer. A. A B. B C. C D. D E. E Answer: E. E The basic lgG immunoglobulin structure is pictured above, and the question asks for the identification of the part of the immunoglobulin that binds receptors on macrophages, neutrophils and B-lymphocytes. These cell types express cell surface proteins known as Fc receptors (FcR) that bind specifically to the Fc portion of lgG molecules. This binding is essential for the process of opsonization. Opsonization refers to the promotion of phagocytosis of tagged material by phagocytic cells such as neutrophils and macrophages. lgG acts as an opsonin by binding antigens (i.e. bacterial surface proteins, etc.) at its Fab sites and subsequently binding a phagocyte at its Fc site. This signals for the phagocytosis of the Fab bound antigen by the phagocyte. The Fc region of the immunoglobulin molecule near the carboxy terminal is the attachment site to Fc receptors. A similar process occurs with lgE antibody in type I hypersensitivity reactions. lgE binds allergenic antigen at its Fab sites and binds Fc receptors on mast cells and basophils. Once multiple lgE molecules bind antigen and the Fc receptor on the mast cell or basophil and subsequently cross-link with each other, these cells will degranulate thereby releasing multiple vasoactive substances into the local milieu. IF Educational Objective: The carboxy terminal of the Fc portion of the heavy immunoglobulin chains represents the site that binds to the Fc receptors on neutrophils and macrophages. Antibody bound to antigen is able to signal for the phagocytosis of that antigen by a conformational change of the Fc region allowing binding to the Fc receptor on phagocytes. This leads to subsequent phagocytosis of the organism / antibody complex and subsequent destruction of the organism. 0 8 bindstoantigen cells presenting bindsto usually maste O O 00 o Case Study 2: A 23-year-old man comes to the physician with dysuria and increased urinary frequency. He is an active duty member of the military and recently returned from sub-Saharan Africa, where he had been stationed for the last year. The patient's symptoms have persisted for several months and have failed to resolve following antibiotic treatment. His blood eosinophil count is elevated. Urine microscopy shows schistosome eggs. He is started on praziquantel and experiences improvement in his symptoms. The elevated eosinophils in this patient contribute to the host defense against schistosomiasis through which of the following mechanisms? Explain and discuss your answer. A. Antibody-dependent cell-mediated cytotoxicity B. B lymphocyte chemotaxis C. Complement activation D. Immediate hypersensitivity E. MHC class I antigen processing Answer: A. Antibody-dependent cell-mediated cytotoxicity Eosinophils perform the following functions: 1. Parasitic defense: Eosinophil proliferation and activation during multicellular parasitic infection is stimulated by IL-5 produced by TH2 and mast cells (not to be confused with IL-4, which stimulates lgE production). When a parasite invades the mucosa or enters the bloodstream, it is coated by lgG and lgA antibodies that bind the Fc receptors located on the eosinophil cell surface. This triggers eosinophil degranulation and release of cytotoxic proteins (e.g., major basic protein) and reactive oxygen intermediates, substances that damage and destroy antibody-bound parasites. This mechanism is an example of antibody-dependent cellmediated cytotoxicity (ADCC), which is also used by macrophages, neutrophils, and natural killer cells. 2. Type I hypersensitivity reactions: Eosinophils also synthesize prostaglandins, leukotrienes, and cytokines that contribute to the inflammation seen in late-phase type I hypersensitivity and chronic allergic reactions. Educational Objective: Eosinophils play a role in host defense during multicellular parasitic infection. When stimulated by antibodies bound to a parasitic organism, they destroy the parasite via antibody-dependent cell-mediated It cytotoxicity with enzymes from their cytoplasmic granules. Another Is function of eosinophils is regulation of type I hypersensitivity reactions. e Case Study 3: f infectious viral disease a ecting the central nervous system that causes paralysis Preventive disease specialists working in a developing country are investigating vaccination options to limit the spread of poliomyelitis. As part of the study, 2 patients are vaccinated against poliomyelitis. One patient receives an intramuscular inactivated vaccine and the other patient receives a live attenuated oral vaccine. One month after vaccination, the levels of which of the following poliovirus antibodies will differ the most between these 2 patients? Explain and discuss your answer. A. Cerebrospinal fluid lgG B. Duodenal luminal lgA C. Serum lgA D. Serum lgG E. Serum lgM Answer: B. Duodenal luminal lgA Secretory lgA is the major antibody associated with mucosa l immunity. Upon intestinal exposure to a novel antigen, B cells found in mesenteric lymph nodes and Peyer's patches become activated and preferentially migrate to the lamina propria underlying the intestinal mucosa. There, they become fully differentiated plasma cells that begin to synthesize lgA dimers (linked by J chain). These lgA dimers then bind to the polymeric immunoglobulin receptor (plgR) found on the basolateral surface of intestinal epithelial cells and undergo transcytosis. As the linked lgA dimer is released into the intestinal lumen, a portion of plgR remains attached to the antibody (secretory component), forming the complete secretory lgA molecule. Stimulation of local secretory lgA production is best promoted when the corresponding mucosal surfaces are directly stimulated by the antigen. In addition, live attenuated vaccines generally produce stronger immune responses than killed vaccines by acting as a persistent stimulus that better activates helper and cytotoxic T cells. As a result, the live attenuated oral (Sabin) poliovirus vaccine generates a much more robust oropharyngeal and intestinal mucosal lgA response than the inactivated poliovirus (Salk) vaccine. Educational Objective: The live attenuated oral (Sabin) poliovirus vaccine produces a stronger mucosal secretory lgA immune response than does the inactivated poliovirus (Salk) vaccine. This increase in mucosal lgA offers immune protection at the site of viral entry by inhibiting attachment to intestinal epithelial cells. O Case Study 4: An 8-year-old boy is brought to the pediatrician with fever, runny nose, and malaise. After examining the child, the pediatrician determines that he has a viral infection and does not require any specific treatment. The mother asks why an antibiotic is not necessary, and the physician explains the differences between bacterial and viral infections. The immune system is composed of both cellular and humeral components, which are able to mount an effective response against many types of viral and bacterial infections. A section of a normal lymph node is shown in the image below. The structures indicated by the arrows are most likely to contain cells undergoing which of the following processes? Explain and discuss your answer. is thymus ti tdti 1 A. lsotype switching B. Negative selection C. Tolerance development D. VDJ recombination E. VJ recombination Answer: A. lsotype switching B-cell precursors proliferate and mature in the bone marrow. Mature Bcells then leave the bone marrow and migrate to lymphoid organs and peripheral tissues, where they are exposed to antigens. On first exposure to a new antigen, a clone of B-cells becomes activated. Some activated B-cells differentiate into short-lived plasma cells that release antigen-specific lgM through a T-cell independent process. However, most activated B-cells migrate to lymphoid follicles located in the lymph node cortex where they form germinal centers (arrows) that are the site of B-cell proliferation during the immune response. A portion of these activated B-cells form long-lived memory cells that remain dormant in the lymph node until the next encounter with the same antigen, but the majority transform into antibody-secreting plasma cells. lsotype switching (from lgM to other types of immunoglobulins) also occurs in the germinal centers late in the primary response, providing activated Bcells the ability to produce antigen-specific antibodies of differing isotypes . Heavy chain constant regions are isotype-specific and distinguish the 5 isotypes (lgM, lgG, lgA, lgE, and lgD), while the variable regions are antigenspecific. Light chains are antigen-specific and do not determine isotype. lsotype switching first requires interaction of the CD40 receptor on activated B-cells with the CD40 ligand (CD154) expressed by activated Tcells. Afterward, isotype switching can occur through genetic rearrangement of the heavy chain constant regions. This process is modulated by T-cell cytokines such as lL-2, IL-4, lL-5, lL-6, and IFN-γ. After the primary immune response, subsequent encounters with the same antigen generate a predominantly lgG response (or lgA in the case of a mucosa! response). Educational Objective: The primary immune response to a new antigen initially results in plasma cells that only produce IgM. Isotype switching later occurs in the germinal centers of lymph nodes and requires interaction of CD40 receptor on B-cells with the CD40 ligand (CD 154) expressed by activated T cells. IgG is the main serum immunoglobulin of the secondary response.