When stimulated by an appropriate antigen, T-lymphocytes respond by dividing and differentiating into cytotoxic T cells (also called Killer T cells). Various other T cells release biologically active soluble factors that mediate the response of other cells involved in the immune response. The soluble factors collectively known as lymphokines are effective in mediating the responses of monocytes and macrophages. Like B-lymphocytes, the T-lymphocytes have surface receptors that can react with antigens, triggering the cell-mediated response. These surface factors however are not immune-globulins, as they are in B-lymphocytes (humoral immunity).
Although manufactured in small concentrations, the lymphokines are extremely active. One lymphokine, called the chemotactic factor (CF), draws phagocytes to the antigen site. Another, the migration inhibition factor (MIF) prevents macrophages from moving away. The third, the macrophage aggregation factor (MAF), causes phagocytes to clump together at the site. A fourth, the macrophage activating factor (also MAF), appears to increase the mobility of phagocytes and the number of lysosomal enzymes in each. The overall effect is to increase the efficiency of phagocytosis of antigens and bring about a specific response to the disease.
The four types of lymphokines represent over 50 lymphokines that have been described so far. In 1979, the term interleukin was coined for substances produced by white blood cells (-leuko) that have an effect on other white blood cells linter.). One lymphokine, called interleukin l, is a T-lymphocyte protein that is believed to stimulate the maturation of T-lymphocytes. Another lymphokine, interleukin 2, is also a T-lymphocyte protein, but its function is to activate T-lymphocytes to rapidly grow and divide. Interleukin 2 has found practical use in the treatment of tumors. (Microbiology Resistance to Disease Notes Study Material)
Lymphokines disappear rapidly once the antigen has been eliminated. However, a person will remain immune to future effects of the antigen because a colony (or clone) of identical T-lymphocytes remains in the tissues. These cells are called memory T-lymphocytes. If the antigens reappear in the tissues, the memory cells will rapidly revert to lymphoblasts that secrete lymphokines to eliminate the antigens. This is one reason for long-term immunity to disease. (Microbiology Resistance to Disease Notes Study Material)
Cellular immunity is a chief means of resistance to bacterial diseases like leprosy and tuberculosis, fungal diseases like candidiasis and cryptococcosis, and many pathogenic protozoa and helminthic parasites. The process is also active in many viral and rickettsial diseases because these organisms multiply within cells where antibodies are ineffective. Scientists believe that certain viruses, Tickettsiae, and fungi induce antigens to form on the surface of infected cells, and t the antigens stimulate a type of T-lymphocyte called a killer T-lymphocyte (or killer T-cell) to lyse the infected cell after contacting them. Killer T-lymphocytes also appear to be a factor in the destruction of cancer cells.
There are also two other members of the so-called T-cell family. One, helper T-lymphocytes, present in some immune responses, bind to antigens and assist the response by B-lymphocytes to the antigens. The collaboration is therefore essential to the immune response controlled by B-lymphocytes. Another suppressor T-lymphocytes apparently interfere with the function of B-lymphocytes and prevent and exaggregate immune response. They also are thought to help prevent an immunological response to oneself.
In victims of AIDS, an abnormally low number of helper T-lymphocytes exists in the immune system together with an unusually high number of suppressor T-lymphocytes. These factors lead to the suppression of the immune system that characterizes the disease. Suppressor and helper T-lymphocytes are often called regulator cells since they regulate B-lymphocyte function. By contrast, other T-lymphocytes are called effector cells as they affect an immune response directly. (Microbiology Resistance to Disease Notes Study Material)
[II] Specific resistance and antibodies
(Humoral immunity)
Humoral immunity begins as antigenic determinants from macrophages stimulate B-lymphocytes. Helper T-lymphocytes assist the stimulation. The B-lymphocytes do not enter the circulation. Rather, they remain in the lymphoid tissue and multiply to form a clone of cells called plasma cells. Plasma cells are about two to three times the size of B-lymphocytes and have elaborate internal details. Their principal products are protein molecules called antibodies.
Antibodies are synthesized according to directions in the Ir genes and are released into circulation at a rate of several thousand molecules per second. The term humoral immunity is used because the interaction between antibodies and antigens occurs in the bloodstream (“humor” refers to blood). (Microbiology Resistance to Disease Notes Study Material)
Plasma cells continue to produce antibodies for two to three days or until the antigenic stimulation comes to an end. At this point, the plasma cells die off and are replaced by a second clone of B-lymphocytes called memory B-lymphocytes. These memory cells remain in the lymphoid tissue for many years and become active when the antigen reappears. Rapid production of antibodies by memory cells usually ensures that a second episode of the disease does not occur. (Microbiology Resistance to Disease Notes Study Material)
Immunoglobulin (Ig)
Antibody molecules represent about 17 percent of the total protein in the blood serum. An enormous variety of antibodies should be evident from only a single fact that a single bacterium may have a thousand different antigens, and that each antigen might elicit its own highly specific antibody. The term immunoglobulin (Ig) is used synonymously with antibodies because antibody molecules exhibit the characteristics of the globulin group of proteins. (Microbiology Resistance to Disease Notes Study Material)
Structure and Types of Antibodies
It was only during the late 1950s and early 1960s that details about the nature of antibodies began to emerge. The research focused on the Bence Jones proteins identified a century earlier by Henry Bence Jones. Enough information and several theories were available about the origin and functions of these proteins, but little was known about their detailed structure.
It was presumed that an understanding of the structure of these proteins (now known to be antibodies) would provide clues to their unique specificity for antigens. The answers were provided by Gerald M: Edelman and Rodney M. Porter who described the chemical composition and structure of proteins and received the 1972 Nobel Prize in Physiology or Medicine. (Resistance to Disease Notes Study Material)
The basic antibody molecule consists of four polypeptide chains: two identical “heavy” (H) chains and two identical “light” (L) chains. These chains are joined together by sulphur to sulphur (disulphide) linkages to form a Y-shaped structure. Each heavy chain consists of about 400 amino acids, while each light chain has about 200 amino acids. (Microbiology Resistance to Disease Notes Study Material)
Within each polypeptide chain there exist constant and variable regions. The amino acids in the constant region of both light and heavy chains are virtually identical among antibodies. However, the amino acids of the variable region vary among the hundreds of different antibodies. Thus the variable regions of light and heavy chains combine to form a highly specific, three-dimensional structure somewhat analogous to the active site of an enzyme. This portion of the antibody molecule combines with the antigenic determinant.
Moreover, the “arms” of the antibody are identical so that a single antibody molecule may combine with two antigen molecules. This combination may lead to complex antibody and antigen molecules. (Microbiology Resistance to Disease Notes Study Material)
When an antibody molecule is sectioned with papain, an enzyme from papaya fruit, the molecule separates at the hinge point, and two functionally different segments are isolated: (i) the Fab fragment, for “fragment-antigen-binding”, is the portion that will combine with the antigenic determinant, (ii) the Fc fragment, for “fragment able to be crystallized”, has various functions: it is the part of the antibody molecule that combines with phagocytes in opsonization; it appears to neutralize viral receptor sites; it attaches to certain cells in allergic reactions, and it activates the complement system in resistance mechanisms.
At present time, five types of antibodies have been identified, based on differences in the heavy (H) chains. The five classes of antibodies (immunoglobulins, abbreviated as Ig), are designated as IgM, IgG, IgA, IgE, and IgD. The structures of these antibodies are shown in Figure.
IgM consists of a pentamer (five subunits), whose segments are connected by a glycoprotein called a joining or J chain. IgA is a dimer (two subunits), with the segments also connected by a J chain. In addition, there is a secretory component that enables the molecule to leave secretory epithelial cells. The remaining three antibodies are monomers (one subunit).
IgM is the first antibody to appear in the circulation after stimulation of B-lymphocytes. It is the principal component of the primary antibody response and the largest antibody molecule. Owing to its size, most LG. remains in circulation. IgM is formed during fetal cases of rubella on toxoplasmosis, indicating that a certain immunological competence exists in the fetus. About 5 to 10 percent of the antibody component of normal serum consists of this antibody. (Microbiology Resistance to Disease Notes Study Material)
IgG is the classical gamma globulin and is the major circulating antibody, comprising about 80 percent of the total antibody component in normal serum. IgG appears about 24 to 48 hours after antigenic stimulation and continues antigen-antibody interaction begun by IgM. It is thus the antibody of secondary antibody response. In addition, it provides long-term resistance to disease as a product of the memory B-lymphocytes. injections of a vaccine considerably raise the level of this antibody in the serum. lgG is also the maternal antibody that crosses the placenta and renders immunity to the fetus until the child is able to make antibodies at about six months of age.
IgA is approximately 10 percent of the total antibody component of normal serum. One form of this antibody, called serum IgG, exists in the serum and is similar to IgA. A second form accumulates in body secretions and is referred to as secretory IgA. This antibody provides resistance in the respiratory and gastrointestinal tracts, possibly by inhibiting the attachment of the parasites to tissues. It is also located in tears and saliva, and in the colostrum, the first milk secreted by a nursing mother. When consumed by a child, the antibodies may lend resistance to gastrointestinal disorders. (Microbiology Resistance to Disease Notes Study Material)
IgE plays a major role in allergic reactions by sensitizing cells to certain antigens. This process will be discussed later. The functions and significance of IgD are not known at present. Perhaps this antibody may stimulate B-lymphocytes to secrete other antibodies.
Origin (Diversity) of Antibodies – Clonal selection theory
For decades, immunologists were puzzled by how the limited number of lymphocytes in the lymphoid tissue could produce an enormous variety of antibodies. Several theories have been put forward from time to time to explain the origin of antibodies. As early as 1900 Ehrlich had provided details of the selective theory, according to which the antigen-stimulated synthesis of an antibody by cells that already made the antibody at a low level in advance of immunization i.e. all the necessary genetic information was present before the cell encounters the antigen. This view was, however, abandoned in the 1920s. (Microbiology Resistance to Disease Notes Study Material)
From among early theories, one called the template theory (also instructive theory) suggested that antigens modified the lymphocytes, which then provided a design, or template, against which the antibody was formed. The antigen was thought to direct the folding of the antibody molecule in its active form. The main supporters of this theory were Breinl and Haurowitz Pauling in the 1940s, and 1950s. This theory was later abandoned when it became evident that the shape of a protein (i.e. its activity) is determined by its amino acid sequence.
Most immunologists currently favor a process called the clonal selection hypothesis, the main idea put by Jerne, Talmage, and Burnet in 1959 or so. According to this hypothesis, the immune system contains a cadre of different B-lymphocytes able to produce every conceivable antibody. Even without antigenic stimulation, the lymphocytes produce a small number of antibody molecules that localize on the lymphocyte surface. The antibodies then serve as receptor sites for antigenic determinants.
When antigenic determinants later enter the lymphoid tissue, they interact only with complementary receptor sites. Helper T-lymphocytes appear to assist the interaction. This union “selects out” certain B-lymphocytes and stimulates them to undergo transformation leading to clones of plasma cells. An antigenic determinant is thus an extracellular signal that triggers cell division and synchronizes the development of a particular cell type. (Microbiology Resistance to Disease Notes Study Material)
How can one avoid synthesizing antibodies that react with their own antigens? To explain this, it is hypothesized that interactions between B-lymphocytes and antigens during fetal development are quite different from those that occur after birth. The clonal selection theory depends on the development and differentiation of a large population of B-lymphocytes during fetal development.
To explain the development of tolerance to self-antigens, this theory says that during fetal development, surface receptors of certain B cells react with self-antigens. The lymphocytes that have reacted with self-antigens are unable to divide to form a clone of cells; rather this leads to the destruction of those lymphocytes. (Microbiology Resistance to Disease Notes Study Material)
Briefly one may say that the clonal selection hypothesis is the process by which antigenic determinants select B-lymphocytes having complementary receptor sites and stimulate them to form clones of plasma cells. Does this hypothesis imply that an individual is born with all the different B-lymphocytes ever needed? However, current research indicated otherwise. It appears that a person originally possesses a limited number of B-lymphocytes, which differentiate to form the final cadre of cells.
During differentiation, a great deal of gene shuffling takes place within the DNA that eventually makes up the immune response (Ir) genes. Gene segments, acting as transposons, appear to migrate from one chromosome location to another, thereby producing a variety of genetic codes. The genes that code for antibody molecules are also subject to an exceptionally high frequency of local mutations. These recombinations and mutations thus yield an immense number of Ir gene combinations from a limited number of original genes. The result is a huge repertoire of different antibodies that B-lymphocytes can produce at their receptor sites.
Antigen-Antibody interactions
(Antibody in specific resistance)
In order for specific resistance to develop, antibodies must interact with antigens in such a way that the antigen is altered. This alteration may result in the death of a microbe that possesses the antigen, inactivation of the antigen, or increased susceptibility of the antigen to other body defenses (maybe to phagocytosis). The following are the ways by which the antigen may be altered, rendering resistance to the body.
1. Neutralisation. Certain antibodies called neutralizing antibodies, react with antigens (say viral capsids), and prevent the viruses to attach to host cells. Influenza viruses are inhibited by neuraminidase antibodies in this way. Neutralizing antibodies also bind viruses together in clumps thereby encouraging phagocytosis. Moreover, neutralization represents a vital defense mechanism against toxins. The antibodies, called antitoxins, alter the toxin molecules near their active sites and mask their toxicity. Neutralization of toxin molecules also increases their size, thus encouraging phagocytosis. (Microbiology Resistance to Disease Notes Study Material)
2. Other reactions. Some antibodies, called agglutinins react with antigens on the surface of organisms such as bacteria. This action causes clumping, or agglutination of the organisms, and enhances phagocytosis. Movement is inhibited if antibodies react with antigens on the flagella, and the organisms may be clumped together by their flagella. (Microbiology Resistance to Disease Notes Study Material)
There are antibodies, called precipitins. They react with dissolved antigens and convert them to solid precipitates. In this form, the antigens are usually inactive and move easily phagocytized. (Microbiology Resistance to Disease Notes Study Material)
There are also opsonins, the antibodies that stimulate phagocytosis by direct intervention. The antigen attaches to the Fab fragment of the antibody while the Fc portion inserts to a receptor site on the phagocyte. Example – inactivation of Streptococcus pneumoniae. Normally the microbe resists phagocytosis but when antibodies react with the M protein in the bacterial capsule phagocytosis occurs quickly.
3. Complement system. This is an example of an antigen-antibody reaction in which a group of proteins, called the complement system is involved. This system originally described in 1895 by Jules Bordet, a Belgian working with Metchnikoff at the Pasteur Institute, is said to involve a series of 11 proteins that function in a cascading series of reactions. The system exists in all normal sera and is activated by IgM or IgG. The complement works with antibodies to cause opsonization, chemotaxis, and lysis of bacterial cells.
The pathway, also referred to as the classical pathway for activation of the complement is set into motion by the interaction of antigen and antibody molecules. Usually, the interaction takes place on the surface of a cell such as a bacterium. As a result of a number of reactions (attachment, binding, etc. of units of the system to antibody) there is formed a series of substances.
One of these is an attack complex (C5b, C6, C7, C8, C9). This complex increases cell membrane permeability and induces the cell to undergo lysis through the leakage of its cytoplasm. Another substance in the cascade (C5a) attracts phagocytes through chemotaxis, while another substance (C2, C3, C4b) facilitates phagocytosis by binding the cell to the phagocyte. Still, other fragments (C3a and C5a) are anaphylatoxins. These react with tissue cells called mast cells and induce the release of histamine, which contracts smooth muscles and thereby increases the movement of phagocytes out of blood vessels to the infection site.
Louis Pillemer and his associates 1954 proposed an alternative pathway or properdin system for activation of the complement system. A serum protein, properdin was shown to function in the activation of the system. Properdin functioned in a way different from the classical one, and hence the name alternative pathway. In this system, several complement components are bypassed, and an antigen-antibody reaction is not required for stimulation. This pathway is stimulated by endotoxins as well as by capsular polysaccharides in streptococci and by certain fungi. (Microbiology Resistance to Disease Notes Study Material)
The complement system is particularly useful against Gram-negative bacteria as it alters their cell walls and makes them susceptible to lysozyme. A complement component encourages the release of lysozyme from local macrophages to react with bacteria. In Gram-positive bacteria, the peptidoglycan of the cell wall resists the attack complex, but lysozyme from macrophages degrades the cell wall.
Monoclonal Antibodies
(Hybridoma Technology)
An antibody-secreting cell, like any other cell, can become cancerous. Unchecked, the cell proliferates to become a mass of cells called myeloma. Since myeloma begins as a single cell, all of its progeny constitute a clone with identical genetic characteristics. The remarkable feature of this clone is that the cells produce only a single type of antibody. Thus the serum of an animal with myeloma contains enough amounts of one antibody, and the tissue culture of myeloma produces only one antibody.
Georges J.F. Kohler (W. Germany) and Cesar Milstein (Argentina) 1975 developed a method for the production of antibodies from a single clone of myeloma cells in the laboratory. Antibodies from this clone were named monoclonal antibodies. The method of their formation is shown in Figure.
The antigens are injected into mice. The plasma cells from the spleen of immunized mice are extracted. These immunized plasma cells are then fused with unstimulated myeloma cells from other mice to form a clone of hybrid cells. This fusion results in a hybridoma (hybrid myeloma), which is immortal and is programmed to produce a single antibody against the original antigen. The plasma cells supply the programme for the antibody, and the myeloma cells supply immortality. A hybridoma is thus a cancerous (proliferating) mass of cells all of which synthesize the same kind of antibody molecule. (Microbiology Resistance to Disease Notes Study Material)
The tumor develops from a hybrid cell (and hence the name hybridoma) formed by the fusion of a cancerous lymphoid cell (myeloma) and a spleen cell from an animal that has been provoked (immunized) with a specific antigen. A cell from the spleen of an animal sensitized into synthesizing one type of antibody (monoclonal) when fused with a myeloma cell that has a property of uncontrolled proliferation results in a hybrid cell that divides to form a tumor. Monoclonals can now be made to order for any given antigen. In 1984, Kohler and Milstein shared the Nobel Prize in Physiology or Medicine for the development of the monoclonal antibody technique.
Monoclonal antibodies and hybridoma technology have been important breakthroughs in biomedicine. As compared with ordinary antibodies monoclonal ones are more uniform and pure. They have been used to pinpoint the antigens on the surfaces of parasites, that have been utilized in vaccine production By conjugating a fluorescent dye to a monoclonal antibody we can scan a tissue. a cell, or part of a cell for the presence and/or site of a specific protein. This method was used to locate Z-DNA in chromosomes of Drosophila.
Monoclonal antibodies are important in the treatment of tumors. Tumor cells are removed from the patient and injected into the mouse, whereupon the spleen of the mouse begins to produce tumor antibodies. Spleen cells are then fused with myeloma cells to produce a hybridoma that produces antibodies for that specific tumor. When the antibodies are injected back into the patient, they react specifically with the tumor cells without destroying other tissue cells. It is very likely that monoclonal antibodies could also be used to carry drugs (antibody tagged with the drug) to the tumor and destroy its cells.
In 1983, diagnostic tests for gonorrhea and chlamydial urethritis, called Gonozyme and Chlamydiazyme tests respectively were developed by use of monoclonal antibodies. Such antibodies are also used for cleansing bone marrow before transplant, in treating disorders of the immune system, etc. (Microbiology Resistance to Disease Notes Study Material)
Microbiology Resistance to Disease Notes Study Material
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