Immunology Field Trip-Background

Your body is equipped with a highly adaptable personal defense system called the immune system. The immune system is responsible for protecting your body against any foreign invaders. Normally, the immune system concentrates on bacteria and viruses, but cancer cells are also targeted by the immune system. Like all complicated systems, the immune system does occasionally malfunction. Sometimes the immune system attacks the body and the result is an autoimmune disease, such as rheumatoid arthritis. More commonly, the immune system overreacts to foreign materials that are not dangerous, such as pollen, resulting in allergies. All foreign materials that elicit an immune response are called antigens.

The immune system mounts two types of defense: specific and non-specific. A non-specific response is immediate and general. Non-specific responses include fevers, and several general chemical responses. The specific immune response is based on a cellular response to a particular antigen. The immune system is complex, highly regulated and extremely effective. This overview is far from complete, but will give a working overview of the cellular response.

The immune system is composed of several cell types and many specific molecules. This general overview includes only the basic cells and molecules. Immune cells, also known as white blood cells, are generated in the bone marrow. The main cell types involved in standard immune response are B cells, T cells, and macrophages. All of these cells are trained to recognize “self” (the host) or “non-self” (foreign material). In some instances the immune system improperly identifies “self” as “foreign” and the immune system attacks the host body. This is referred to as an autoimmune disease and some examples are rheumatoid arthritis, lupus and Crohn’s.

Foreign material can be anything not recognized as self. This means any part of a bacterium such as membrane proteins, membrane sugars, or cell wall will be identified as foreign and elicit an immune response. When a bacterium enters the body, a macrophage (“giant eater”) will recognize it as foreign and engulf it. Once the bacterium has been “eaten” by the macrophage, it is broken up and pieces of it are displayed on the outside of the macrophage. T cells near the macrophage will inspect the antigens displayed by the macrophage. Unlike macrophages, which will “eat” anything, each T cell recognizes only one antigen. Among the T cell population a wide variety of antigens is recognized, but each T cell is responsible for recognizing a particular antigen. When a T cell recognizes an antigen it begins an attack specifically designed to target that antigen. T cells release a series of molecules which activate B cells, excite other components of the immune system and boost the activity of other T cells.

If T cells are the control station for the immune response, B cells are the workhorse of the immune system. Each B cell is capable of producing antibodies to a single antigen. Antibodies are proteins and they are designed to interact with a particular antigen. An activated B cell begins producing large quantities of antibody. The antibody interacts with the antigen, coating the exterior of an invading bacterium with heavy protein flags. Other immune system components respond to the antibody tagged bacterium and destroy the cell. Macrophages will consume cells marked with antibodies, certain T cells will destroy marked cells and chemical poisons called complement will attach to antibodies and destroy marked cells.

On an initial exposure it takes the body 5-10 days to develop a specific immune response. Considering that bacteria reproduce every 20 minutes, this is a very long time. Often a person is exposed to the same bacteria repeatedly. To reduce the response time, the immune system has developed a memory which allows for a much faster response time. When a B cell is activated, it begins dividing to produce more copies of itself and increase the quantity of antibody produced. Some of those copies do not participate in the immune response, but quietly remain in the body long after the infection has ended and the active B cells have died. These remaining B cells are called memory cells. T cells also generate memory cells. The role of the memory cells is to mount a faster immune response to a second exposure to the same antigen.

This immune memory has been used to protect people from common, but deadly, diseases such as whooping cough, measles, mumps, small pox and polio. Most children in industrialized countries are vaccinated against these diseases. A vaccination provides a representative antigen from the pathogen (disease causing organism). The antigen itself is not able to cause the disease, but it is enough to activate the immune system and develop a set of memory cells which will provide lasting protecting against the pathogen. For example, small pox was eradicated from the world in a global vaccination effort, which is why people are no longer vaccinated against it. There are two known government samples of small pox which have been maintained for scientific and medical research. There is a concern that terrorist organizations may have illegally accessed small pox samples, hence the current discussion about small pox vaccinations.

Vaccines can be developed for pathogens that maintain the same antigenic markers. Pathogens that change frequently, such as the flu virus and Human Immunodeficiency Virus (HIV), present a considerable challenge in vaccine development. A new flu vaccine is developed annually to address the most recent changes in the flu virus. HIV mutates so rapidly that no effect vaccines have been developed.

Antibodies (Ab), also referred to as immunoglobins (Ig), are produced by the immune system in response to specific antigens (Ag). The ability of an antibody to recognize and bind an antigen is called specificity. Antibodies are fairly large proteins (~150kD) composed of two heavy and two light chains. Structurally, they look like the letter “Y”.

Antibodies are manufactured by B cells and each B cell produces antibodies capable of recognizing only one specific antigen. Nevertheless, it is estimated that the immune system is capable of producing 100 million to a trillion antibodies of unique specificity. Variation in antibodies is generated by “mix and match” genetic procedures at both the DNA and RNA level. Each light chain is composed of a variable (V_L), joining (J_L) and constant (C_L) region and each heavy chain is composed of a variable (V_H), diversity (D_H), joining (J_H) and constant (C_H) region. There are 300 light chain variable regions, 4 joining regions and two constant regions. There are as many as 1000 heavy chain variable regions, 13 diversity and 4 joining regions. There are eight different heavy chain constant regions, depending on the immunoglobin class.

	Variable Regions	Diversity Regions	Joining Regions	Constant Regions
Light Chain	300	N/A	4	2
Heavy Chain	1000	13	4	8

Immature B cells generate unique light chains by splicing the V_L region to one of the J_L regions. Unique heavy chains are generated by splicing V_H, D_H, and J_H regions together. This splicing occurs at the DNA level and the extra regions are discarded from the B cell genome. This ensures that each B cell produces only one variation of antibody. 3000 different light chains and 5000 heavy chains are possible based solely on combinations of variable, diverse, joining and constant regions. Additional diversity is generated by “sloppy” DNA splicing. RNA transcripts are also spliced to produce unique antibody products in each cell.

There are five classes of immunoglobins in mammals: IgA, IgE, IgD, IgM and IgG. These antibodies differ in the heavy chain constant regions.

IgA is found in mucosal areas such as intestine, vagina, in tears, saliva and breast milk. IgE is responsible for allergic responses. The function of IgD is not clear. IgM is the default form of antibody produced by B cells. After activation, B cells produce a burst of IgMs and then switch to IgG production. IgG is the form most active in immune responses. Birds produce another variant called an IgY.

A critical component of studying proteins or other molecules in the laboratory is the ability to identify and locate them. Proteins can be “labeled” or marked with radioactivity or otherwise modified to allow researchers to track them during an experiment, but it is difficult to label a particular protein in a mix and there is always the chance that the label will change the way the protein behaves.

The specificity of antibodies makes them a valuable tool in the lab. Antibodies can be used to track a protein. The antibody itself is visualized by labeling. The label does not interfere with the function of the antibody. Common labels or tags attached to the end of the antibody include radioactive tags, and chemiluminescent or colorimetric reagents. Common uses of antibodies are affinity chromatography for separating specific proteins from a mix, ELISAs, dot blots and westerns for identification of proteins and immunocytology for highlighting protein activity within cells.

Antibodies to a particular protein are generated by injecting the protein of interest in an animal. Although an isolated protein is not likely to cause the injected animal a problem, the immune system will recognize it as foreign and raise an immune response. After this, the antibodies can be collected in a blood sample and isolated for use in the lab.

Ironically, the healthier a person is, the more easily they are able to maintain their health. The immune system is complex to develop and expensive for the body to maintain. As a result, infants and young children whose immune systems are not fully developed are more susceptible to disease. Older people, whose cellular systems are slowing down, are also susceptible to disease. People who are suffering from malnutrition, chronic disease, exhaustion or other problems which tax their bodies are also at risk. HIV infects and destroys T cells, effectively preventing the cellular response from activating which leads to the development of Acquired Immune Deficiency Syndrome (AIDS). People infected with HIV lose their immune system and die from a variety of common, opportunistic infections.

The rapid strep test is used to determine whether or not streptococcal bacteria are present in the throat. Results can be obtained at the Dr.’s office within 10 minutes. Identification of strep infection is important so that appropriate antibiotic therapy can begin immediately. Long term consequences of strep throat can include inflammation, damage to the nervous system and permanent damage to the heart and kidneys. The traditional test for strep involves growing a bacterial culture from the patient’s throat sample, and can take two to three days for results to be available. When using the traditional test, physicians would often prescribe antibiotics immediately, without results, based on physical symptoms or on a precautionary basis. We all know that overuse or misuse of antibiotics should be avoided, a problem which the rapid strep test helps to alleviate.

The rapid strep test begins with a throat swab (make ‘em gag!) in the clinic. The presence of streptococcal bacteria Group A in the throat swab, will yield a sample that contains a specific streptococcal protein (antigen) that is further extracted with chemicals. At the clinic an antibody solution is added to the extracted patient sample, then a substrate is added. If a color change takes place, or a “+” symbol is indicated on a test kit, then the person has a strep infection. The test is fast, easy and relatively inexpensive – a recent web search found a company advertising a kit that could test 25 samples for $85.00.

It is very important to note that the rapid strep test has limitations. First, the test is designed to detect a protein (antigen) found in streptococcal bacteria Group A – there are other infection causing bacteria, including some strep types, that may not be detected by the test. Second, even if the infection is caused by strep Group A bacteria, the sensitivity of the test is 75%-80%. This means that 1 in 4, or 1 in 5 people infected by strep A will not be accurately diagnosed. Also, a test can appear negative even in the case of strep A infection, if the person took any antibiotics prior to the test. For these reasons, the traditional strep culture test is still used to confirm the diagnosis of a rapid strep test.

The HIV antibody test tests for the presence of antibodies to the human immunodeficiency virus (HIV) in a patient’s blood. Serum is extracted from the blood and then added to a microtiter plate coated with HIV proteins (antigens). A secondary antibody (antihuman Immunoglobulin) along with a reagent is added to the plate, then a substrate is added. If a color change takes place to blue-green, then HIV infection is indicated. This can be followed with an acid wash (turning the color to yellow) prior to spectrophotometry. This enzyme immunoassay (EIA) test is described as “99% accurate at 6 months of infection by HIV strain 1” (CDC AIDs Hotline), since it can take the human body up to 6 months to develop antibodies to HIV and since there are two main strains of HIV. HIV 1 is the strain found primarily in the United States, the test can also indicate infection by HIV 2, but not with the same degree of accuracy. However, HIV 2 is found mainly in West Africa, and so HIV 2 will mainly be of concern to people who have been in contact with blood from this region.

Generally if a first EIA, which takes 90 minutes, is positive, it will be followed by a second EIA and a Western Blot for confirmation. One of the main limitations of this test is that it may take 3-6 months for sufficient quantities of the antibody to be present in the patient’s blood.

There are several other types of test available for HIV, some which yield results more quickly after infection. They include Radioimmunoprecipitation assay (RIPA), which is expensive but can be used with low antibody levels, and the Polymerase Chain Reaction (PCR) which can also be used soon after infection, and to design strain specific drug therapy, but is also costly. Another test, the p24 antigen capture assay, was originally used to screen blood supplies, but not usually used to diagnose individual HIV infection. Two more cost-effective tests in development include the Rapid latex agglutination assay and the Dot-blot immunobinding assay. Recently tests have become available which can detect HIV antibodies in saliva and urine. Two AIDS Hotline recommended websites about HIV testing are: www.cdc.gov and www.ashastd.org.

HIV: health and medical information about HIV and AIDs. www.medicinenet.com. Retrieved 1 May 2003.

To learn more about the immune response, immunity and disease, investigate these web sites:

1) Name a common autoimmune disease. What are the symptoms? What causes the symptoms? What treatments are available? Remember to include references.

2) What would happen if each of the following cell types were removed from the immune system: macrophage, T cells, B cells.

3) It is known that if a person develops cow pox and recovers, and then is exposed to small pox and he or she is less likely to develop small pox. Explain why.

5) List the mandatory vaccines for attending school in Wisconsin and which diseases they provide protection against. What are acceptable reasons not to receive these vaccinations?

6) What are some diseases for which vaccination is not mandatory? Under what circumstances might you wish to be vaccinated against one of these diseases?

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