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Example research essay topic: Immune Response Infectious Diseases - 2,154 words

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Fetal Immune Response To Infection With Toxoplasmosa gondi Tom Repici Immunology 4200 11 - 21 - 98 ABSTRACT I will be addressing the possible immunological barriers that may be involved with challenge to infection of Toxoplasmosa gondii, the protozoan that causes toxoplasmosis. It is widely known that toxoplasmosis is a devastating disease, with often drastic consequences upon infection. In pregnant mothers, these consequences can be very horrid. Such effects are more felt by the unborn fetus than by the mother. These effects include abortion, premature birth, and severe growth retardation (Creamy et. al. , 1994).

Falkner et. al. have shown that the human fetus is capable of producing antibodies as early as ten weeks gestation time. We also know that antibodies to toxoplasmosis exist, which are of Ig G, Ig M, and, recently demonstrated, although in low levels, Ig E isotype's. These facts give rise to some questions regarding the bizarre reactions of the fetus, which should be more than protected enough from infection. Why is the fetus damaged, or even aborted from such a challenge, when it has the ability to produce antibodies?

Are any of the antibodies produced specific for toxoplasmosis? Are the maternal antibodies that may exist to toxoplasmosis found in fetal serum (due to the passive immunity from mother to child), or are they blocked by some selection mechanism in the placenta? My objectives are to find out 1) if the placenta does in fact serve as an antibody sponge (Creamy et. al. ), 2) if the fetus produces antibodies specific for toxoplasmosis, 3) if fetal antibodies specific for toxoplasmosis are found, what isotype they are (to determine if they might be of maternal origin, since Ig G readily crosses the placenta), 4) if the antibodies found have a high affinity for toxoplasmosis immunogen and are capable of eliciting an effective immune response. BACKGROUND AND SIGNIFICANCE I will be addressing the fetal response to challenge with toxoplasmosis infection. We do know that there is immune response activity in the fetus at the age of four months gestation, thus they are capable of producing antibodies along with the ones that they receive via the placenta in passive immunity.

We also know that there are antibodies specific for toxoplasmosis. Lastly, we know that the disease is treatable, only after infection is acquired, by which time irreversible damage to the fetus may have been done. Toxoplasmosis is the common name given to the disease caused by the protozoan Toxoplasmosa gondii. The disease is usually found in animals, and has found to be transmitted to pregnant mothers by poor handling of cats (which are the only carriers of the protist?

s oocytes or eggs), and by ingesting uncooked meat. The protists enter the body in the form of trophozoites, which can reside in tissues in the form of tissue cysts. It is this form that can possibly reside in the placenta, and thus release its contents into the blood and then cross the barrier to the fetus. We still need to know why the disease is so devastating to the fetus. We need to know if a healthy, pregnant mother? s antibodies to toxoplasmosis can cross the placenta to the unborn fetus, or if the placenta blocks them by some mechanism.

We also need to know if the fetus produces antibodies specific for toxoplasmosis. To answer these questions, a series of experiments have to be devised. Such experiments must be carried out in vivo most of the time. This would call for subjects who would be willing to give to experimental conditions. These conditions would include placental tissue samplings, and blood samplings as well. Some experiments can be conducted in vitro.

These would be various assays needed to test the blood for presence of antibodies, antigen, etc. EXPERIMENTAL DESIGN The first experiment to carry out would involve pregnant mice, preferably in the late gestational period, around 17 - 19 days. It is in his time that the fetus? s immune system should be able to function properly enough to provide the data needed (Rush, 1968). The first experiment is to determine if the placenta acts in a selective manner, by trapping maternal antibodies that may be specific for toxoplasmosis. A pregnant mouse within the gestational range shown above would be parent ally introduced to toxoplasmosis antigen labeled with a fluorescent dye or radioactive chromium.

A period of 7 - 10 days time for antigen uptake would be required, although monitoring the blood sample for any immune activity would be needed. Upon satisfactory completion of infection, blood samples from the fetus and mother would be needed. The samples from the mother and fetus would be centrifuged and separated. If any tagged antigen is found in the fetal serum bound to Ig, it is assumable that the fetus does display some immune activity toward toxoplasmosis.

The fetal Ig with the toxoplasmosis would then be examined for any similarity to Ig bound to antigen in the mother. The testing for similarity of Ig molecules is of importance, because it could answer the question of whether or not the placenta is a filter for maternal antibody. The two Ig molecules would be broken down via Papain digestion to obtain the Fab fragments (four total; 2 maternal, 2 fetal) and 2 Fc fragments (one maternal, one fetal). The Fc fragments are of interest, because it is this segment that holds the key to answering if the 2 are of the same origin.

Determining the isotype is done by breaking down the fragments and subjecting them to Southern Blotting, and the results of the blot will tell if the fragments are the same. If the fetus shows isotype other than that of Ig G, it is safe to say that it is capable of producing its own antibodies to toxoplasmosis. This is because Ig G is the main isotype of antibody that is capable of crossing the placental barrier. If Ig G is found in the fetal serum, a test to determine if the Fab fragments of both the fetal and maternal Ig molecules are of same amino acid sequence must be done.

This is necessary to prove if the Ig G found in the fetal serum is of maternal origin. The same test would be done to the Fab fragment as to the Fc fragment; ie subjecting them to Southern Blot. If they match, then it is safe to say that the maternal antibody to toxoplasmosis does in fact cross the placenta, and that the placenta does not filter out toxoplasmosis antibodies. After establishing the presence of antibodies to toxoplasmosis in the fetus, assays to determine the functioning of the system would be needed. This would call for a wide range of assays to show Ig affinity to antigen, the ability of the c omelet to elicit a response, etc.

The first assay conducted would involve isolated Ig from fetal serum, regardless of isotype, but the Ig must be specific for toxoplasmosis. This should be easy to come by in an infection situation, since antibodies to toxoplasmosis are produced in an infection situation to that specific antigen. These isolated Ig molecules would then be subjected to equilibrium dialysis. This assay would involve Ig specific for toxoplasmosis and toxoplasmosis antigen labeled with chromium 51. The results of this assay would prove if the Ig specific for toxoplasmosis does in fact have a high enough affinity for antigen, which in an indirect sense, could tell how well the Ig-Ag interaction would be at eliminating the antigen by the complement system. In other words, the higher the affinity, the better the chance of getting the antigen coated and ready for destruction by the complement system.

This leads to the next test, which would determine the ability of the Ag-Ig complex to activate the complement system. This assay would call for the fetus to be infected with labeled toxoplasmosis directly, and samples of blood to be drawn from the fetus after sufficient time has been allowed for any Ig-Ag reactions to occur. The samples would then be mixed with complement, and then the mixture would be checked for any lysed toxoplasmosis antigen by the increased presence of label present in solution. Any signs of released label would indicate that the Ig is able to produce an ideal humoral response. Another test to perform to observe immune reaction would be to observe the antibody? s ability of antibody-dependent cell-mediated cytotoxicity (ADCC).

This involves labeled antibody, along with eosinophils, NK (natural killer) cells, and neutrophils, all from fetal serum and labeled with a marker. A cell culture infected with toxoplasmosis would be mixed with antibody, and then incubated at 37 degrees Fahrenheit for several hours to ensure good binding of antibody to the target cell. After this period of time, the labeled phagocytic cells would be added. If label is found in solution, it is safe to say that the antibodies, when in contact with antigen, can elicit a cell-mediated response. The last assay will measure the ability of antigen to ensure a cellular response by making memory B cells. This would involve the direct hemolytic plaque assay, in which a SRBC (sheep red blood cell) would be conjugated with toxoplasmosis antigen.

After conjugation, the antigen bearing Specs would be injected into a fetal mouse and allowed time to infect; about 4 days time should be sufficient. After 4 days, the spleen of the fetus would be removed, and put in culture in agar medium, and complement would be added. The mature would be incubated at 37 degrees Celsius for 1 hour, after which the dish would be checked for plaques indicating plasma cell activity. It should be noted that the cells forming the plaque will be of Ig M isotype. To further observe if a secondary response could be established, the indirect hemolytic assay could be implemented. Positive results from the indirect method would indicate Ig G isotype, indicating a secondary response and cell memory.

Positive results from both assays indicate a cellular response to antigen. DISCUSSION The data from the previous assays could give insight on how the human fetus could react to toxoplasmosis infection. By finding out if the fetus produces antibodies to toxoplasmosis, we know that the fetus is capable of at least eliciting a humoral response of some nature, no matter how small. The assays to measure affinity to antigen also amplify the possibility of antibody molecules to create a response, mainly by the complement system.

By finding out if the fetus can produce plasma cells tells us if it can actually give off a secondary response, or if memory is achieved, if a second encounter with toxoplasmosis happens. By finding out if the maternal antibody is caught up in the placenta also yields information on the possibility of antibody selection, which may occur in it. The benefits of finding out if the fetus can respond to toxoplasmosis will enable us to find ways to assist the unborn in its fight for life upon infection. By seeing that it can produce antibodies tells us that a drug may be formed to assist in binding of antigen to antibody, or by creating a drug that could possibly assist in binding of antigen to complement. The formation of plasma cells tells us that the fetus can form memory to such an antigenic challenge, which is of importance if future encounter occurs.

The total benefit of these assays is to gain an overall knowledge of how the fetus really does or how it could respond to antigen challenge, mainly of such a devastating disease such as toxoplasmosis. If we gain insight on how the fetus responds, it will open the door to the next chapter of research to find controls to stop fetal infections such as toxoplasmosis from being fatal as they are. WORKS CITED 1) Creamy, Robert K. , Resnik, Robert. Maternal-Fetal Medicine. W.

B. Saunders Co. , Philadelphia. 1994. 2) Rush, Robert. The Mouse. Burgess Publishing Co. , Minneapolis. 196 REFERENCES 1) Creamy, Robert K. , Resnik, Robert. Maternal-Fetal Medicine. W.

B. Saunders Co. , Philadelphia. 1994. 2) Cooper, Max et. al. B Lymphocytes In The Immune Response. Elsevier/North-Holland, New York. 1978. 3) Falkner, Frank, Tanner, JM. Human Growth: A Comprehensive Treatise, Vol. 1.

Plenum Press, NY. 1986. 4) Golub, Edward S. The Cellular Basis of the Immune Response. Singer Assoc. , Mass. 1977. 5) Innes, EA. ? Toxoplasmosis: comparative species susceptibility and host response. ? Comparative Immunology of Microbiological Infectious Diseases. February 1997. (recv?

d via Pub Med) 6) Kovarik, Jiri, Sie grist, Claire-Anne. ? Immunity in early life. ? Immunology Today. Vol. 18, no 4. April 1998. 7) Mandell, Michael et. al.

Principles and Practices of Infectious Diseases. Churchill Livingstone, NY. 1990. 8) Nahamias AJ, Kourtis AP. ? The great balancing acts. The pregnant woman, placenta, fetus, and infectious agents. ? Clinical Perinatology.

June 1997. (recv? d via Pub Med) 9) Rush, Rober. The Mouse. Burgess Publishing, Minneapolis. 1968.


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