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Innate Immune Response
Infection by C. diphtheriae is most commonly localized to the upper respiratory tract, while the circulating exotoxin causes widespread and potentially lethal damage. The innate immune system is the body's first lines of defense, including the epithelial barriers and the non-specific killing of pathogens by macrophages and neutrophils. To establish an infection, C. diphtheriae must first breach the mucosal epithelial barriers in the throat or airway and colonize the epithelial surface (Janeway, Jr. et al., 2005). In clinical diphtheria, an infection manifests itself by forming a pseudomembrane on the tonsils, uvula, soft palate, pharynx, and nasopharynx. Satellite infections can spread into trachea, bronchi, esophagus and stomach. However, the site of infection remains localized on the epithelial surfaces (Hadfield et al., 2000).
As the bacteria multiply, the cells of the innate immune system recognize the invading bacteria by their non-self surface molecules. The macrophages quickly recognize the bacteria and destroy the pathogens through phagocytosis. Macrophages also recruit neutrophils to the site where they too can assist in phagocytosis and removal of the bacteria. In addition, macrophages release cytokines and chemokines in order to cause inflammation. The inflammatory response attracts more effector cells to help fight the infection, helps stop the spread of the pathogen and promotes healing of the damaged tissue. Finally, the innate immune response will initiate the adaptive immune response if it is not enough to control the infection (Janeway, Jr. et al., 2005). The first signs of the diphtheria infection are edema and hyperemia of the infected areas, which indicate an innate inflammatory response is occurring. As the infection progresses, the underlying epithelium dies and a thick secretion of fibrin, pus, sloughed-off epithelial cells, host immune cells and bacteria forms. This fibrous network forms the pseudomembrane, which is often visible as a gray, green or black membrane in the back of the throat. If the infection is not checked, necrosis will extend deeper into the underlying tissue. These more severe lesions are more vascularized with greater numbers of neutrophils, representing the increasing innate immune response. The exotoxin secreted by the bacteria can enter the bloodstream through these vascularized patches to cause more severe damage elsewhere in the body (Hadfield et al., 2000).
Cellular Immune Response
The effector functions of the cellular immune response are essential for protection against diphtheria toxin. There are three classes of effector T cells, including CD8+ cytotoxic T cells and two types of CD4+ T helper cells. The usual function CD8 cytotoxic T cells is to recognize and kill cells that are displaying viral peptides on their cell surfaces, indicating that they are infected. Clinical diphtheria is caused by the exotoxin entering cells and stopping all protein synthesis. Once the exotoxin fragment A has entered the host cell, the cell is almost certainly going to die anyway so attack by a cytotoxic T cell would not necessarily stop the spread of the disease. In addition, if protein synthesis in the cell had stopped because of the fragment A interference, then the display of foreign peptides on the cell surface would also be compromised. The function of TH1 and TH2 cells is important in the response against the initial bacterial infection and the circulating exotoxin. Armed TH1 cells activate macrophages. When these cells are activated in turn, they increase their microbial activity and their ability to activate other T cells to further the immune response. This response is essential for the removal of the initial bacterial infection. Armed TH2 cells help initiate the humoral immune response by inducing B cells to differentiate and produce immunoglobulins (antibodies). The effector function of TH2 cells is very important in the defense against the toxin itself. Since the toxin is the disease causing agent, neutralization of the toxin can prevent the clinical disease associated with infection by a toxigenic strain (Janeway, Jr. et al., 2005).
Humoral Immune Response
The most effective way to prevent the toxic effects of diphtheria toxin is to simply prevent it from entering the host cell. Organisms that are highly susceptible to diphtheria toxin have high numbers of the HB-EFG precursor fragment B cell surface receptor. Mice and rats are not susceptible to the toxin because they do not have the specific membrane receptors that are required for fragment B binding. Without these specific receptors, the toxin proenzyme can not cleave to release the toxic fragment A into the cytosol to interfere with protein synthesis (Boquet and Pappenheimer, Jr., 1976; Holmes, 2000). In humans and other susceptible species, the most effective way to prevent the clinical disease is to neutralize the toxin by antibody binding so that the toxin can not adhere to and enter the host cell.
The clinical disease associated with C. diphtheriae is caused by the circulating exotoxin, so antibody recognition of the toxin itself is essential for humoral immunity. Fragments A and B of the diphtheria toxin are immunologically distinct. It has been found that antibodies against the C-terminal region of fragment B are more effective at preventing internalization than antibodies against the N-terminal region, which is the fragment A region. The function of fragment B is primarily adhesion to the host cell and internalization of the toxin, so logically an antibody blocking the functionality of this region would prevent internalization of the toxin. Anti-fragment B antibodies have been found to be primarily responsible for the neutralization of the toxin (Collier, 1975).
There are various explanations for why anti-fragment A antibodies are not as effective as inhibiting the enzymatic activity of the toxin. Fragment A is not activated until the toxin has been cleaved and fragment A has entered the host cell. Many immunogenic determinants on fragment A are not exposed until the toxin has been cleaved as fragment A passes across the plasma membrane. Clearly, if the antigenic sites on fragment A are not exposed until this point in the infection it may be too late for the anti-fragment A antibodies to have any significant neutralizing effects (Cryz, Welkos and Holmes, 1980). Alternatively, a high-affinity anti-fragment A antibody may bind to an exposed site on fragment A before it is cleaved from fragment B. This antibody may become detached when the toxin is cleaved or because of conformational changes when fragment B adheres to the host cell (Collier, 1975). Diphtheria toxin primarily induces an IgG antibody response, with a smaller response in IgA antibodies (Lue et al., 1990).
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Collier, R. John. “Diphtheria Toxin: Mode of Action and Structure.” Bacteriological Reviews 39.1 (1975): 54-85.
Cryz, Stanley J., Susan L. Welkos and Randall K. Holmes. "Immunochemical Studies of Diphtherial Toxin and Related Nontoxic Mutant Proteins." Infection and Immunity 30.3 (1980): 835-846.
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Holmes, Ryan K. “Biology and Molecular Epidemiology of Diphtheria Toxin and the tox Gene.” The Journal of Infectious Diseases 181 (Suppl 1) (2000): S156-67.
Janeway, Jr., Charles A., Paul Travers, Mark Walport and Mark J. Shlomik. Immunobiology: The Immune System in Health and Disease. 6th ed. New York: Garland Science, 2005. pp. 37-100, 319-361.
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