Immunology. A broad field encompassing both basic and clinical applications, deals with antigens, antibodies and cell mediated host defense functions, specially as they relate to immunity to disease, hypersensitivity biological reactions, allergies and rejection of foreign tissues. Immunity
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A broad field encompassing both basic and clinical applications, deals with antigens, antibodies and cell mediated host defense functions, specially as they relate to immunity to disease, hypersensitivity biological reactions, allergies and rejection of foreign tissues.
Body defense against exogenous(microbes) and endogenous(tumor cells) agents. It is:
1- Innate(natural, nonadaptive, or nonspecific) immunity.
2- Adaptive(acquired, or specific) immunity. Occurs after exposure to antigen. It is mediated by either antibodies(Humeral immunity) or lymphoid cells(Cellular immunity). It can be:
1- Physiological barriers at the portal of entrey
A- The Skin
B- Mucous Membranes
2- Nonadaptive Immunologic Mechanisms
A- Reticuloendothelial system:Mononuclear phagocytic cells in blood, lymphoid tissue, liver, spleen, bone marrow, lung and other tissues that are efficient in uptake and removal of particulate mater from lymph channels and the blood stream.
B- Alternative pathway of complement
C3 parasites, endotoxines, microbial surface C5, C6, C7, C8, C9(0psonization, lysis of bacteria).
C- Phagocytosis: Engulfment and killing of microbes by phagocytic cells which include microphage (granuolocytes,polymorphs) and macrophage. The process of killing includes both nonoxidative and oxidative mechanisms.
D- Inflammatory response
E- Fever: Endotoxines and Interleukin 1
F- Interferon: Virus induced protein that have antiviral activity after 48 hours of viral infection.
IFNa, IFNb, INFy.
G- NK cells: lyse tumor cells and virus infected cells.
This type of immunity occurs in response to infection called ADAPTIVE as the immune system must adapt itself to previously unseen molecules. Following recovery from certain infections with a particular micro-organism, individuals will never again develop infection with the same organism, but can become infected with other micro-organisms, i.e. he/she is protected against one micro-organism. This form of protection is called IMMUNITY and an individual is said to be IMMUNISED against that organism. The induction of immunity by infection or with a vaccine is called ACTIVE IMMUNITY.
Acquired Immunity (adaptive Immunity/specific Immunity)
A. Hypervariable (HVR) or complementarity determining regions (CDR)
Comparisons of the amino acid sequences of the variable regions of Ig's show that most of the variability resides in three regions called the hypervariable regions or the complementarity determining regions as illustrated in Figure 3. Antibodies with different specificities (i.e. different combining sites) have different CDR's while antibodies of the exact same specificity have identical CDR's (i.e. CDR --> Ab Combing site). CDR's are found in both the H and the L chains.
B. Framework regions
The regions between the CDR's in the variable region are called the framework regions (FR) (Figure 3). Based on similarities and differences in the framework regions the immunoglobulin heavy and light chain variable regions can be divided into groups and subgroups. These represent the products of different variable region genes.
1. Effector functions - The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment (See Figure 5). Normally the ability of an antibody to carry out an effector function requires the prior binding of an antigen. However, there are exceptions to this rule.
C. F(ab')2 - Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites (Figure 6). This fragment was called F(ab')2 because it was divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab')2 binds antigen but it does not mediate the effector functions of antibodies
V. IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS
Immunoglobulin fragments produced by proteolytic digestion have proven very useful in elucidating structure/function relationships in immunoglobulins.
A.Fab - Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 4. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.
1. Antigen binding - These fragments were called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL. An antibody is able to bind a particular antigenic determinant because it has a particular combination of VH and VL. Different combinations of a VH and VL result in antibodies that can bind a different antigenic determinants.
B. Fc - Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.
A. Immunoglobulin classes - The immunoglobulins can be divided into 5 different classes based on differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a given class will have very similar heavy chain constant regions. These differences can be detected by sequence studies or more commonly by serological means (i.e. by the use of antibodies directed to these differences).
1. IgG - Gamma heavy chains
2. IgM - Mu heavy chains
3. IgA - Alpha heavy chains
4. IgD - Delta heavy chains
5. IgE - Epsilon heavy chains
B. Immunoglobulin Subclasses - The classes of immunoglobulins can de divided into subclasses based on small differences in the amino acid sequences in the constant region of the heavy chains. All immunoglobulins within a subclass will have very similar heavy chain constant region amino acid sequences. Again these differences are most commonly detected by serological means.
1. IgG Subclasses
a) IgG1 - Gamma 1 heavy chains
b) IgG2 - Gamma 2 heavy chains
c) IgG3 - Gamma 3 heavy chains
d) IgG4 - Gamma 4 heavy chains
a) IgA1 - Alpha 1 heavy chains
b) IgA2 - Alpha 2 heavy chains
C. Immunoglobulin Types - Immunoglobulins can also be classified by the type of light chain that they have. Light chain types are based on differences in the amino acid sequence in the constant region of the light chain. These differences are detected by serological means.
1. Kappa light chains
2. Lambda light chains
D. Immunoglobulin Subtypes - The light chains can also be divided into subtypes based on differences in the amino acid sequences in the constant region of the light chain.
1. Lambda subtypes
a) Lambda 1
b) Lambda 2
c) Lambda 3
d) Lambda 4
E. Nomenclature - Immunoglobulins are named based on the class, or subclass of the heavy chain and type or subtype of light chain. Unless it is stated precisely you are to assume that all subclass, types and subtypes are present. IgG means that all subclasses and types are present.
F. Heterogeneity - Immunoglobulins considered as a population of molecules are normally very heterogeneous because they are composed of different classes and subclasses each of which has different types and subtypes of light chains. In addition, different immunoglobulin molecules can have different antigen binding properties because of different VH and VL regions.
1. Structure - The structures of the IgG subclasses are presented in Figure 7. All IgG's are monomers (7S immunoglobulin). The subclasses differ in the number of disulfide bonds and length of the hinge region.
2. Properties - Most versatile immunoglobulin because it is capable of carrying out all of the functions of immunoglobulin molecules.
a) IgG is the major Ig in serum - 75% of serum Ig is IgG
b) IgG is the major Ig in extra vascular spaces
c) Placental transfer - IgG is the only class of Ig that crosses the placenta. Transfer is mediated by receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally; IgG2 does not cross well.
d) Fixes complement - Not all subclasses fix equally well; IgG4 does not fix complement
e) Binding to cells - Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc receptors on PMN's, monocytes and macrophages is that the cell can now internalize the antigen better. The antibody has prepared the antigen for eating by the phagocytic cells. The term opsonin is used to describe substances that enhance phagocytosis. IgG is a good opsonin. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions.
1. Structure - Serum IgA is a monomer but IgA found in secretions is a dimer as presented in Figure 11. When IgA exits as a dimer, a J chain is associated with it.
When IgA is found in secretions is also has another protein associated with it called the secretory piece or T piece; sIgA is sometimes referred to as 11S immunoglobulin. Unlike the remainder of the IgA which is made in the plasma cell, the secretory piece is made in epithelial cells and is added to the IgA as it passes into the secretions (Figure 12). The secretory piece helps IgA to be transported across mucosa and also protects it from degradation in the secretions.
a) IgA is the 2nd most common serum Ig.
b) IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. Since it is found in secretions secretory IgA is important in local (mucosal) immunity.
c) Normally IgA does not fix complement, unless aggregated.
d) IgA can binding to some cells - PMN's and some lymphocytes.
1. Structure - The structure of IgD is presented in the Figure 13. IgD exists only as a monomer.
a) IgD is found in low levels in serum; its role in serum uncertain.
b) IgD is primarily found on B cell surfaces where it functions as a receptor for antigen. IgD on the surface of B cells has extra amino acids at C-terminal end for anchoring to the membrane. It also associates with the Ig-alpha and Ig-beta chains.
c) IgD does not bind complement.
1. Structure - The structure of IgM is presented in Figure 8. IgM normally exists as a pentamer (19S immunoglobulin) but it can also exist as a monomer. In the pentameric form all heavy chains are identical and all light chains are identical. Thus, the valence is theoretically 10. IgM has an extra domain on the mu chain (CH4) and it has another protein covalently bound via a S-S bond called the J chain. This chain functions in polymerization of the molecule into a pentamer.
a) IgM is the 3rd most common serum Ig.
b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen.
c) As a consequence of its pentameric structure, IgM is a good complement fixing Ig. Thus, IgM antibodies are very efficient in leading to the lysis of microorganisms.
d) As a consequence of its structure, IgM is also a good agglutinating Ig . Thus, IgM antibodies are very good in clumping microorganisms for eventual elimination from the body.
e) IgM binds to some cells via Fc receptors.
f) B cell surface Ig - Surface IgM exists as a monomer and lacks J chain but it has an extra 20 amino acids at the C-terminal end to anchor it into the membrane (Figure 9). Cell surface IgM functions as a receptor for antigen on B cells. Surface IgM is noncovalently associated with two additional proteins in the membrane of the B cell called Ig-alpha and Ig-beta as indicated in Figure 10. These additional proteins act as signal transducing molecules since the cytoplasmic tail of the Ig molecule itself is too short to transduce a signal. Contact between surface immunoglobulin and an antigen is required before a signal can be transduced by the Ig-alpha and Ig-beta chains. In the case of T-independent antigens, contact between the antigen and surface immunoglobulin is sufficient to activate B cells to differentiate into antibody secreting plasma cells. However, for T-dependent antigens, a second signal provided by helper T cells is required before B cells are activated.
1. Structure - The structure of IgE is presented in Figure 14. IgE exists as a monomer and has an extra domain in the constant region.
a) IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen.
b) Involved in allergic reactions - As a consequence of its binding to basophils an mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms.
c) IgE also plays a role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite.
d) IgE does not fix complement.
Figure 1Electrophoretic separation of serumproteins
A. Immunoglobulins (Ig) - Glycoprotein molecules which are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that when antibody-containing serum is place in an electrical field the antibodies, which were responsible for immunity, migrated with the globular proteins (Figure 1).
II. GENERAL FUNCTIONS OF IMMUNOGLOBULINS
A. Ag binding - Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host.
1. Valency - The valency of antibody refers to the number of antigenic determinants that an individual antibody molecule can bind. The valency of all antibodies is at least two and in some instances more.
B. Effector Functions - Often the binding of an antibody to an antigen has no direct biological effect. Rather, the significant biological effects are a consequence of secondary "effector functions" of antibodies. The immunoglobulins mediate a variety of these effector functions. Usually the ability to carry out a particular effector function requires that the antibody bind to its antigen. Not every immunoglobulin will mediate all effector functions.
1. Fixation of complement - lysis of cells, release of biologically active molecules
2. Binding to various cell types - phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins and the binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts. The binding results in transfer of the immunoglobulin across the placenta and the transferred maternal antibodies provide immunity to the fetus and newborn
IMMUNOGLOBULINS - STRUCTURE AND FUNCTION
a) opening up of junctions between endothelial cells in post-capillary venule to allow plasma proteins to escapeb) adhesion of leukocytes to endothelial cells of post-capillary venule, followed by emigration of phagocytes into tissues.
Inflammation is localised to area of infection/tissue injury by release of substances from micro-organisms or chemicals (chemical mediators) released from cells in tissues, e.g. histamine from MAST CELLS. Once organisms destroyed inflammation settles down (RESOLVES).
The scanning electron micrograph above, shows a human macrophage (gray) approaching a chain of Streptococcus pyogenes (yellow). Riding atop the macrophage is a spherical lymphocyte. Both macrophages and lymphocytes can be found near an infection, and the interaction between these cells is important in eliminating infection.
1. Antigen Processing. When the macrophage eats bacteria, proteins (antigens) from the bacteria are broken down into short peptide chains and those peptides are then "displayed" on the macrophage surface attached to special molecules called MHC II (for Major Histocompatibility Complex Class II). Bacterial peptides are similarly processed and displayed on MHC II molecules on the surface of B lymphocytes.
2. Helper T Cell Stimulating B Cell. When a T lymphocyte "sees" the same peptide on the macrophage and on the B cell, the T cell stimulates the B cell to turn on antibody production.
3. Antibody Production. The stimulated B cell undergoes repeated cell divisions, enlargement and differentiation to form a clone of antibody secreting plasma cells. Hence. through specific antigen recognition of the invader, clonal expansion and B cell differentiation you acquire an effective number of plasma cells all secreting the same needed antibody. That antibody then binds to the bacteria making them easier to ingest by white cells. Antibody combined with a plasma component called "complement" may also kill the bacteria directly.
Cellular Basis Of Immune Response
Strategy Two: Elimination of micro-organisms which normally survive for long periods in macrophages.
Even when antibody and complement opsonise certain bacteria and phagocytosis occurs, they are not killed but survive and multiply in macrophages, e.g. Mycobacterium tuberculosis. Elimination occurs by use of a subpopulation of cells calledHELPER T cells. These cells recognise macrophages containing intracellular bacteria by means of T cells antigen receptor, which is not an antibody. They help macrophages to kill bacteria by synthesising soluble molecules (CYTOKINES) which stimulate bacterial killing mechanisms of macrophages.
Strategy Three - Elimination of micro-organisms which infect cells without an endogenous antimicrobial defence system.
Viruses are obligate intracellular pathogens, can infect any type of cells, and most cells do not possess antimicrobial mechanisms. During intracellular replication virus proteins appear on the surface of the infected cell. A second subset of T cells - cytotoxic T cells - recognises these viruses (foreign) antigens and secretes cytotoxic molecules which kill the infected cells.
The surface of each T cell also displays thousands of identical T cell receptors (TCRs).
The best understood CD8+ T cells are cytotoxic T lymphocytes (CTLs). They secrete molecules that destroy the cell to which they have bound. This is a very useful function if the target cell is infected with a virus because the cell is usually destroyed before it can release a fresh crop of viruses able to infect other cells.
An example will show the beauty and biological efficiency of this mechanism.
Every time you get a virus infection, say influenza (flu), the virus invades certain cells of your body (in this case cells of the respiratory passages). Once inside, the virus subverts the metabolism of the cell to make more virus. This involves synthesizing a number of different macromolecules encoded by the viral genome.
In due course, these are assembled into a fresh crop of virus particles that leave the cell (often killing it in the process) and spread to new target cells.
Except while in transit from their old homes to their new, the viruses work inside of your cells safe from any antibodies that might be present in blood, lymph, and secretions.
But early in the process, infected cells display fragments of the viral proteins in their surface class I molecules. CTLs specific for that antigen will be able to bind to the infected cell and often will be able to destroy it before it can release a fresh crop of viruses.
In general, the role of the CD8+ T cells is to monitor all the cells of the body, ready to destroy any that express foreign antigen fragments in their class I molecules.
CD8 T Cells
Are substances that can induce antibody formation and react with their specific antibodies. Are called immunogenes.
The features that determine immunogenicity are:
2- Molecular size.
3- Chemical and structural complexity.
4- Molecular weight > 1oooo.
5- Dosage, route and timing of Ag administration.
Incomplete Ag which can be conjugated with a carrier protein to form a complete Ag.
Substances that stimulate the immune response.
It includes initiation of the infection process & the mechanism that lead to the development of signs & symptoms of disease. The following are some definitions:
The most frequent portals of entry of pathogenic bacteria into the body are are the sites where m. m. meet with the skin – Respiratory, Gastrointestinal, Genital, & Urinary Tract.
Transmission of Infection
Bacteria enter the body adhere to host cells, usually epithelial cells
Multiply Spread directly through tissues or via lymphatic system to the blood stream(transient or persistent). Through blood bacteria spread widely in the body to reach tissues particularly suitable for their multiplication.
1- Adherence Factors
2- Invasion of host cells and tissues – Enzyme production, capsule.
3- Toxin Production – Exotoxins, Endotoxins, Enteroxins.
4- Intracellular Pathogenicity.
Bacterial Virulence Factors
The complement system plays an essential role in host defence against infectious agents and in the inflammatory process. It consists of about twenty plasma proteins that function either as enzymes or as binding proteins. In addition to these plasma proteins, the complement system includes multiple distinct cell-surface receptors that exhibit specificity for the physiological fragments of complement proteins and that occur on inflammatory cells and cells of the immune system. There are also several regulatory membrane proteins that function to prevent autologous complement activation and protect host cells from accidental complement attack (1).
The role of complement in host defence has been established through genetic deficiencies of certain complement components, which may result in life-threatening recurrent bacterial infections or immune complex diseases. Such deficiencies shall be discussed later.
The role of complement in inflammation and tissue injury has become apparent through clinical investigations and discoveries that the pathogenesis of certain experimental inflammatory diseases is complement-dependent.
The complement system can be activated by two different pathways: the classical complement pathway and the alternative complement pathway.
The classical pathway is activated by the binding of antibody molecules (specifically IgM and IgG1, 2 and 3) to a foreign particle. This pathway is antibody-dependent.
The alternative pathway seems to be of major importance in host defence against bacterial infection because, unlike the classical pathway, it is activated by invading micro-organisms and does not require antibody. This pathway is antibody-independent.
The alternative pathway constitutes a humoral component of natural defence against infections, which can operate without antibodies. The six proteins C3, B, D, H, I, and P together perform the functions of initiation, recognition and activation of this pathway which results in the formation of activator-bound C3/C5 convertase.
The classical pathway functions to mediate the specific antibody response. It is as elaborately controlled as the alternative pathway, although it does lack the spontaneous initiation ability; i.e. the antibody independent recognition function, and the feedback amplification mechanism. Both activation pathways contain an initial enzyme that catalyses the formation of the target cell bound C3 convertase which in turn generates the C5 convertase. This results in the cleavage and activation of C5 and, therefore, in the assembly of the membrane attack complex (MAC). The MAC is assembled from five hydrophilic precursor proteins: C5, C6, C7, C8, and C9. Activation of the MAC is a consequence of the activity of either the classical or the alternative pathway on the surface of a cell. Through its metastable membrane binding site, the forming MAC binds firmly to target membranes owing to hydrophobic interactions with the lipid bilayer. The final events are the unfolding, the oligomerisation, or the polymerisation of C9, which causes the weakening of membrane structure, and the formation of transmembrane channels thus leading to osmotic lysis of the cell. MAC assembly is regulated by the S protein of plasma, and by homologous restriction factors of host cell membranes. Complement mediated lysis occurs in many kinds of cells: erythrocytes, platelets, bacteria, viruses possessing a lipoprotein envelope, and lymphocytes.
Complement is a complex system containing more than 30 various glycoproteins present in serum in the form of components, factors, or other regulators and/or on the surface of different cells in the form of receptors. These are present in the blood serum in an inactive state and are activated by immune complexes (the classical pathway), by carbohydrates (the lectin pathway), or by other substances, mainly of bacterial origin (the alternative pathway)
Figure 1.2: Different activation pathways of the complement system (MBP: mannan-binding protein; MASP: MBP-associated serine protease; B: factor B; D: factor D; P: properdin; MAC: membrane attack complex)