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Unit 3 Immunology and Complement

Unit 3 Immunology and Complement. Part 2 Terry Kotrla, MS, MT(ASCP)BB. Overview of Immunity. Immunoglobulins. Humans produce specific proteins or immunoglobulins which can be differentiated on the basis of: Size Biologic function Biochemical properties Serological activity.

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Unit 3 Immunology and Complement

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  1. Unit 3 Immunology and Complement Part 2 Terry Kotrla, MS, MT(ASCP)BB

  2. Overview of Immunity

  3. Immunoglobulins • Humans produce specific proteins or immunoglobulins which can be differentiated on the basis of: • Size • Biologic function • Biochemical properties • Serological activity

  4. Basic Structure of Immunoglobulins • An antibody digested by papain yields three fragments • Two Fab which consist of antigen binding site, can sensitize. • One Fc, whic is the region that determines biological properties of the Ig.

  5. Basic Structure of Immunoglobulins • An antibody digested by pepsin yields two fragments: • One Fab2 which consist of 2 antigen binding sites joined together, able to agglutinate. • One Fc,the region that determines biological properties of the Ig.

  6. Immunoglobulin Classes

  7. IgMClass

  8. IgM • Largest of all the antibody molecules, consists of five of the basic units (pentamer) mu heavy chains joined together by a structure known as J-chain. • Accounts for about 5-10% of the immunoglobulin pool. • Restricted almost entirely to the intravascular space due to its large size. • Fixes complement, much more efficient than IgG in the activation of complement and agglutination. • First antibody to be produced and is of greatest importance in the first few days of a primary immune response to an infecting organism. • Does not cross the placenta. • Many blood group antibodies that are capable of agglutinating antigen positive RBCs suspended in saline in tests performed at 22 C are IgM causing visible agglutination, ie, ABO antibodies. • IgM antibodies are potent agglutinators that activate complement very efficiently.

  9. IgG • Most abundant of the immunoglobulins in the plasma • One basic structural unit, i.e. Y-shaped molecule having 2 light chains and 2 Gamma heavy chains. • Produced in response to a wide variety of antigens, including bacteria, viruses and RBC and WBC allo-antigens. • Coats organisms to enhance phagocytosis by neutrophils and macrophages. • Through its ability to cross the placenta, maternal IgG provides the major line of defense against infection for the first few weeks of a baby's life. • It is the predominant antibody produced in the secondary response. • The serologic behavior and characteristics of IgG antibodies make them one of the most clinically significant in blood banking. • Most blood group antigens capable of eliciting an immune response result in the production of IgG antibodies. • These antibodies are detected by serologic test procedures based on their behavior characteristics, such as reactivity at 37 C, complement activation, indirect agglutination and hemolysis. • Much of routine blood banking involves serologic test procedures designed to detect and identify IgG antibodies. • Four subclasses which differ in their heavy chain composition and in some of their characteristics such as biologic activities. IgG1, IgG2, IgG3 and IgG4.

  10. IgA • Found in saliva, tears, colostrum breast milk and in nasal, bronchial and intestinal secretions. • IgA present in large quantities in colostrum and breast milk, is transferred across the gut mucosa in the neonate and plays an important role in protecting the neonate from infection. • Produced in high concentrations by lymphoid tissues lining the gastrointestinal, respiratory and genitourinary tracts. • Plays an important role in protection against respiratory, urinary tract and bowel infections and preventing absorption of potential antigens in the food we eat. • Represents 10 to 15% of the total circulatory immunoglobulin pool. • In plasma IgA may exist as a single basic structural unit or as two or three basic units joined together. • The IgA present in secretions exists as two basic units (a dimer) attached to another molecule know as secretory component. • 1) This substance is produced by the cells lining the mucous membranes. • 2) It is thought to protect the IgA in secretions from destruction by digestive enzymes. • IgA does not cross the placenta and does not bind complement. • For blood banking, an IgA deficient individual may produce anti-IgA which can cause severe, life-threatening anaphylactic reactions during transfusion. Once identified these individuals must be transfused with blood and components which lack IgA.

  11. IgA Structure • The dimeric IgA molecule. • 1 H-chain, • 2 L-chain, • 3 J-chain, • 4 secretory component

  12. IgE • Trace plasma protein (only about 0.004%) in the plasma of non-parasitized individuals. • Major importance mediating some types of allergic reactions and is generally responsible for an individual's immunity to invading parasites. • Fc region binds strongly to a receptor on mast cells and basophils and, when antigen is bound it causes the basophil (or mast cell) to release histamines and heparin from these cells, resulting in allergic symptoms. • Clinical effects of IgE mediated reactions include increased vascular permeability, skin rashes, respiratory tract constriction (wheezing), and increased secretions from epithelium (watery eyes, runny nose). • Not much else is known about its biologic role. • IgE does not fix complement and does not cross the placenta. • No blood group antibodies have been reported to belong to this class.

  13. IgE

  14. IgD • Accounts for less than 1% of the total immunoglobulin pool. • This is primarily a cell membrane immunoglobulin found on the surface of B lymphocytes. • IgD does not fix complement and does not cross the placenta. • Little is known about the function of this class of antibody. • No blood group antibodies have been reported to belong to this class.

  15. Clinical Significance of Blood Group Antibodies • A blood group antibody is considered clinically significant if it has been associated with the following: • Has caused hemolytic transfusion reactions (destruction of transfused red cells) or • Implicated in Hemolytic disease of the fetus and newborn (HDFN) (destruction of fetal cells)

  16. Blood Group Antigens • At least 30 blood groups with over 600 antigens. • Individuals may produce antibodies to blood group antigens they do not possess when exposed to blood through transfusion or pregnancy. • Second exposure may result in immune hemolysis of red blood cells.

  17. Transfusion Reaction • Term used to describe an unfavorable response by a recipient to the infusion of blood or blood products and include the following: • In-vivo hemolysis (either immediate or delayed) • Decreased survival of transfused cells • Anaphylaxis • Graft-versus-host disease • Post-transfusion purpura • Alloimmunization • Sepsis due to bacterial contaminated components, • Disease transmission. • Will be discussed in detail later

  18. Severity • Depends on a number of factors, including the characteristics of the antibody class involved. • Antibodies to the ABO system antigens are predominantly IgM, cause complement activation andintravascular hemolysis. • Other RBC antigens induce formation of IgG classantibodies which may cause accelerated RBC destruction extravascularly. • Symptoms of response to incompatible ABO transfusion may include fever, low back pain, nausea and vomiting, circulatory shock, anemia, jaundice, and kidney failure which may ultimately result in death. • Primary immune response may be asymptomatic due to slow destruction of RBCs. • Secondary response symptomatic due to memory B cells and rapid antibody production.

  19. Antibody Mediated Hemolysis • Hemolysis can be intravascular or extravascular. • INTRAVASCULAR: Antibodies destroy the red cells IN THE CIRCULATION. Due to of IgM and activation of complement with destruction of RBCs, VERY BAD, will see RED serum/plasma. • EXTRAVASCULAR: hemolysis is due to RBCs being coated with IgG and destroyed OUTSIDE the circulation in the RES system. If it occurs slowly may not be detectable.

  20. Transfusion Reactions • Screening donor blood for disease markers significantly decreased transfusion transmitted diseases. • Reactions to donor WBCs and platelets relatively common but usually not severe. • ABO reactions severe and PREVENTABLE by following protocols. • “Other” blood group antibodies may or may not be detectable. • It is YOUR duty to provide serologically compatible blood and blood components for transfusion.

  21. Complement • Spend quality time on your notes from Serology.

  22. Complement • Integral part of the immune system. • Three pathways • Classical • Alternative or properdin • Lectin • Three primary functions: • Lysis of antibody coated cells, such as bacteria and RBCs. • Mediation of opsonization, preparation of foreign cells for phagocytosis. • Generation of peptide fragments that regulate features of the inflammatory and immune response.

  23. Importance in Blood Banking • Two major areas: • Some antigen-antibody complexes cause sufficient quantities of complement to be bound to RBCs to complete activation cycle, causing hemolysis. • Antigen-antibody complexes initiate complement binding in such a way that allows demonstration of the existence of such complexes by the use of serologic techniques. • Fresh serum necessary to detect complement mediated in-vitro reactions.

  24. The Classic Pathway • Eleven components involved, numbered C1 to C9. • Complement cascade requires presence of cations, both calcium and magnesium. • Activation of the classic pathway almost always initiated by immunoglobulin. • Requires only 1 molecule of IgM (has 5 Fc). • Requires 2 molecules of IgG (has 1 Fc).

  25. Two IgG, One IgM

  26. The Classic Pathway • Recognition Phase - Recognition unit: C1q,C1r,C1s. • Activation Phase -Activation Unit: C4b,C2b,C3b,C5b • Attack Phase – Attack Unit:C5b,C6,C7,C8 and C9 • Classic pathway: C1,C4,C2,C3,C5,C6,C7,C8,C9 • Must go to completion for hemolysis to occur. The next two slides are to assist you in your studies.

  27. Classical Pathway

  28. Alternative (Properdin) Pathway • Proteins in the alternative pathway perform activities similar to those in the classic pathway but are usually non-antibody triggered. • Any one of a variety of substances can initiate complement activation including: • bacterial polysaccharides and lipopolysaccharides, • endotoxins, • cobra venom, • trypsin like enzymes, • aggregates of IgA and IgG4 that do not activate C1. • C1, C4 and C2 do not participate. • Alternative pathway: C3,C5,C6,C7,C8,C9

  29. Alternative Pathway

  30. Lectin Pathway • Activation begins when mannan-binding protein (MBP) binds to the mannose groups of microbial carbohydrates. • Two more lectin pathway proteins called MASP1 and MASP2 (equivalent to C1r and C1s of the classical pathway) now bind to the MBP. • This forms an enzyme similar to C1 of the classical complement pathway that is able to cleave C4 and C2 to form C4bC2a, the C3 convertase capable of enzymatically splitting hundreds of molecules of C3 into C3a and C3b. • The beneficial results are the same as in the classical complement pathway above: • trigger inflammation (C5a>C3a>c4a); • chemotactically attract phagocytes to the infection site (C5a); • promote the attachment of antigens to phagocytes via enhanced attachment or opsonization (C3b>C4b); • serves as a second signal for the activation of naive B-lymphocytes (C3d); • cause lysis of gram-negative bacteria and human cells displaying foreign epitopes (MAC); • and remove harmful immune complexes from the body (C3b>C4b).

  31. Lectin Pathway - FYI • Overview of the lectin complement pathway. In humans, MBL and ficolin that are lectins form complexes with MASPs (MASP-1,MASP-2 and MASP-3) and sMAP. Note that MBL consists of several sizes of oligomers and that the composition of MASPs and sMAP of each MBL oligomer has not been fully elucidated. Once the complexes bind to carbohydrates on the surfaces of microbes, activated MASPs cleave C4, C2 and C3.

  32. Activation of Pathways

  33. Order of Activation of 3 Pathways

  34. Regulation of Complement • Activation of complement cascade results in complex series of molecular event with potent biologic consequences. • Modulating mechanisms are necessary to regulate complement activation and control production of biologically active split products. • First mechanism is spontaneous decay of activated components. • Second mechanism involves specific control proteins that modulate the activity of certain complement components at critical activation steps. • C1 inhibitor blocks activities of C1r and C1s. • Other factors inhibit activation of other complement components. • A number of proteins act to control the membrane attack unit. • Bottom line, gotta turn it off!

  35. References • http://en.wikipedia.org/wiki/Antibody • Complement: http://www.medicine.uiowa.edu/martinlab/complement.html

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