By Andrew Kliewer, Jeff Siswanto, and Rony Eshaque CHE 100 LAB Due: November 15, 2004.

1. By Andrew Kliewer, Jeff Siswanto, and Rony Eshaque CHE 100 LAB Due: November 15, 2004.

2. S M A L L P O X V A C C I N E S • Small pox is caused by 2 strains of the virus name Variola, Variola major which is more severe and Variola minor • There is no treatment for this, but vaccines can help protect against the disease. • The earliest form of vaccination was inoculation which was to take fluid from lesions of infected person. This is known to be effective because the symptoms in persons given the inoculation had less sever symptoms than those that didn’t receive the inoculation. • Edward Jenner who invented this method is known as a father of small pox vaccination • The vaccine Dryvax is licensed in U.S.A., although it is not regularly used since small pox is declared as eradicated. The vaccine is however given to people who work with live vaccines is places for disease control. • With the wake of terrorism, there is a strong possibility that it might be used as biological weapon

3. P O L I O V A C C I N E S • In early 1900, vaccine development for polio begun • Polio is caused by three strains of viruses which have RNA. • In 1947, Dr. Jonas Salk began his research on Polio virus • He needed to process the viruses in a way that they will be less infectious • In 1952, he developed a vaccine using the mixtures of three types of viruses that grow in monkey kidney cultures • He invented a chemical called formalin that inactivated the whole virus • The results of the vaccines were dramatic and government granted permission for the vaccines to be used in the country • In the field of recombinant biotechnology, scientists are attempting to alter the gene of Polio virus

4. I N S U L I N • In the late 1900, Banting and Best discovered Insulin • Insulin is hormone produced by the pancreas in the body • Diabetic people can’t produce insulin naturally • Pancreatic islets and the insulin protein were isolated from animals • Bovine made from the cow’s pancreatic cells and porcine made from the pig’s pancreatic cells work very well • In 1980, technology allowed scientists to make human insulin • The human gene which codes for the insulin was copied and then put inside a bacteria • By using this technique, pharmaceutical companies can isolate pure human insulin • The advantage of human insulin is that there is lower chance of inducing an allergic reaction because all human beings have the same insulin

5. M O N O C L O N A L A N T I B O D I E S • What is? • Antibodies are natural defenses that act to fight infectious agents (antigens) such as viruses and disease-causing bacteria entering through the body • Production of antibodies can be stimulated by vaccines • Certain antibodies are “activated” by diseases to prevent future invasion of the same disease • Uses of antibodies • Antibodies are extremely specific, meaning that they bind and destroy certain antigens • Antibodies are used to protect diseases, diagnose illnesses, detect drugs and any blood abnormalities • Antibodies are potentially safer than drugs since they do not attack the body’s own cells, hence the patient experiences no side effects

6. M O N O C L O N A L A N T I B O D I E S • History • Nobel prize winners Köhler and Milstein devised a technique in 1975 which allowed them to produce antibodies • Past Objective • To mass produce antibody of a single specificity • Produce an antibody that can reproduce itself indefinitely • Procedure • Köhler and Milstein used a technique called somatic cell hybridization by fusing myeloma cells (found in tumor of bone marrow) with cells from an immunized mouse • “Hybrid” cells containing one selective drug are produced and cultured in a medium

7. … … … … … … Once a significant amount of antibodies are produced, the spleen of the animal is removed and its cells are cultivated Animal produces antibodies Antigen of choice injected into one animal Drugs are injected into the medium which will kill remaining myolema cells. The remaining spleen cells will die in 1 to 2 weeks. “Hybrid” cells called “hybridomas” that are inherited from cells immune to the antigen survive. Polyethylene glycol is added to promote fusion between spleen and myeloma cells, producing “hybrids” Hybridomas continue to replicate and inherit their characteristics Fused spleen cells Fused myeloma cells Unfused myeloma cells Unfused spleen cells hybridomas Hybridomas are analyzed and determined whether the desired antibody is secreted. Selected hybridomas are cloned and cultivated in great quantities. Cell death Cell death PROLIFERATION Clonal Expansion Flow chart adapted from Johns Hopkins Arthritis [5]

8. M O N O C L O N A L A N T I B O D I E S • Production • The conventional method of injecting an antigen to a number of animals were inefficient because the animals often yield unwanted substances and very little usable antibody (the procedure is referred to as polyclonal antibody technology) • Since monoclonal antibody technology was developed, high demands lead to mass production of natural antibodies • Cultures are used to cultivate cells that produce natural antibodies • Tumor cells are known to be fused with cells to produce “hybridomas” • These particular cells have the ability to endlessly replicate themselves thus it is possible to produce great quantities of antibodies • Advantages of the monoclonal antibody technology • An impure antigen may be injected to one animal since the monoclonal antibody of interest is cultivated by individual selection strategy • Hybridomas are immortal hence there is an unlimited source of antibody

9. C Y T O K I N E S What are they? • Cytokines are proteins that act as communicators between cells. They are created by one cell and act upon another, relaying messages that tell the cell to grow, stop growing, move to a trouble spot, or otherwise somehow modify its function • Some uses include activation of hormones that both promote and discourage growth, begin immunal activities against harmful bacteria and viruses, and promote wound healing Uses • Promote growth and healing: they have possible uses in patients who do not produce sufficient cytokines on their own • Cytokines activate killer T cells, which are vital in fighting against deformed or corrupted cells such as tumors • Cytokines activate hematopoiesis which can be used to treat patients with anemia or decreased blood cell counts

10. C Y T O K I N E S History • Relatively new idea. Originally appeared in the 1980’s when a cytokine therapy was developed in the 1980’s, originating from the need of leukocyte IFN, and cloning of HuIFN(alpha) subtypes. • Medicines such as Insulin are included in this field despite the fact that insulin is not actually a cytokine. It does however promote DNA synthesis and cell proliferation. Recent Uses/Discoveries • Hematopoiesis: boost production of mature blood cells of various lineages by using lineage specific cytokines as replacement therapy (has been used to increase red blood cell counts, neutrophils, eosinophils, and monocytes in people with aids). • One recent cytokine to be licensed is an interferon. In 1993, FDA approved interferon beta (Betaseron) for multiple sclerosis. Studies showed it helped prevent flare-ups of the disease. • Way Produced: • The DNA strand that produces the cytokine desired is removed from a cell. It is then inserted into an E.coli • The E-coli then produce the cytokine in high concentration, and when the • desired amount is reached, inserted into people where it activates (or deactivates) certain cells, and creates the desired effects

11. C Y T O K I N E S : Bone Remodeling Cycle Current Research • Nerve growth for use in Alzheimer's disease and brain-derived neurotrophic factor (BDNF) and for amyotrophic lateral sclerosis • Wound Healing: Insertion of the wound healing cytokines (transforming growth factor beta (TGF- beta) and others) has sped up the healing rate of rats when given the TGF up to 24 hours before wounding Example of Bone Remodeling Cycle Quiescence Mineralization Activation Formation ‘Coupling’ Resorption

12. C Y T O K I N E S Problems with Cytokines • The cytokines have the correct DNA structure, but because they aren’t synthesized by the body itself, they wont be identical in their ‘post-translational modifications’. This could affect how the cytokines react to their target cells when inserted into the body • If mammalian cells are used to create the cytokines (yeast etc) then there is a possibility of other bacteria or viruses being present, therefore chemical cleansing is required, which may introduce some undesired chemicals into the body • Over exposure to cytokines results in a toxicity reaction with symptoms similar to those of the flu, such as headache and malaise • For patients who received greater amounts of cytokines (30 million units), they experience disorientation, confusion and eventually comatose. This means that cytokines, while not lethal, cannot be taken for extended periods of time • The biggest problem with cytokines is that different cytokines may act upon the same cell with the same results, meaning that finding the specific cytokine desired is very difficult

13. W O R K S C I T E D [1] Vaccines. Available Online: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/V/Vaccines.html, [2004,Nov. 09]. [2] Perez, Arely., 2003. Small Pox. Available Online: http://www.goshen.edu/bio/Biol206/Biol206LabProject/smallpox/Smallpox.html, [2004,Nov. 05]. [3] Morganstein, Linda., 2002. Development of Polio Vaccines. Available Online: http://www.accessexcellence.org/AE/AEC/CC/polio.html, [2004,Nov. 05]. [4] Insulin. Available Online: http://www.endocrineweb.com/diabetes/2insulin.html, [2004,Nov. 05]. [5] Soloski, Mark J., 2004. What on Earth is Monoclonal Antibody? Available Online: http://www.hopkins-arthritis.som.jhmi.edu/edu/mono_anti.html [2004, Nov. 7]. [6] Meager, Anthony. 1990.Cytokines. Buckingham: Open University Press

14. W O R K S C I T E D [7] Access Excelence, 2004. Monoclonal Antibody Technology - The Basics. Available Online: http://www.accessexcellence.org/RC/AB/IE/Monoclonal_Antibody.html [2004, Nov. 5]. [8] Kimball, John W., 2004. Monoclonal Antibodies. Available Online: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html [2004, Nov. 8]. [9] Cotner, J.  and T. Johengen. 1999. The Impact of a Recurrent Coastal Plume on Phosphorus Dynamics and Production in Lake Michigan. Available Online: http://www.glerl.noaa.gov/eegle/projects/p09/p09.html [1999, Sept. 15]. [10] Marian Segal 1995 Cytokines: Putting Body Mechanics to Work. Available Online: http://www.fda.gov/bbs/topics/CONSUMER/CON0291f.html [11] Clemens, M.J. 1991. Cytokines. Oxford: BIOS Scientific Publishers Limited