The TH1 Response to The Attenuated Bovine Mycobacterium Teberculosis Vaccine for The Prevention of Asthma in Mus Musculus

Janine Low, Mahie Lie, and Annie Yang

Writer’s comment: Thanks to Don Meisenheimer and ENL 104E, we spent countless hours drumming our fingers on little white cubes while staring lifelessly into a monitor at a rhythmically blinking cursor to compose the grant proposal that lies ahead. But also thanks to Don and “Writing in the Sciences,” we have gained a better understanding of research papers as well as a method to write them clearly, succinctly, and technically. Specifically, we learned to focus each section of the proposal to a different audience. The experiment that we proposed was relevant not only to finding a more permanent treatment for asthmatics, but also to how it could decrease the medical expenses for such an individual. We chose to write about asthma because it is a well-known health concern which affects a large population. Ironically, none of the authors has asthma.
—Janine Low, Mahie Lie, and Annie Yang

Instructor’s comment: Janine, Mahie, and Annie produced this grant proposal in a week and a half, basing it on Janine’s previous eight weeks of research and writing on the topic, including her literature review and IMRAD-style paper, and the interview she conducted with a campus professor. The proposal arises from discrepancies, in fact, that the three of them identified between printed research findings and the more up-to-date information garnered from the interview. What impresses me about the proposal is its success in terms of both technical depth and technique, presenting sophisticated scientific thoughts with an elegance in writing style rare in undergraduate composition courses.
—Don Meisenheimer, English Department

The Th1 Response to the Attenuated Bovine Mycobacterium tuberculosis Vaccine for the Prevention of Asthma in Mus Musculus

         Asthma is an autoimmune disorder affecting over 17 million Americans. Current methods of treatment using corticosteroid inhalers have long-term deleterious side effects. In a study of Japanese school children, Taro Shirakawa et al. found that the attenuated bovine Mycobacterium tuberculosis vaccine (BCG) not only prevented tuberculosis, but also decreased the incidence of asthma by enhancing the production of Th1 helper cells and IFN-g. Tuberculosis also remains an epidemic worldwide; the estimated cost of treating tuberculosis in the U.S. alone is $700 million per year. In a similar study researching the Th1/Th2 response in mice, Gesine Hansen concluded that Th1 enhancement might actually aggravate asthma. Yet Hansen’s experiment is flawed in that the test mice received direct application of Th1 and Th2 cells rather than naturally eliciting the immune response via BCG injection. Additionally, the age and sample size of the murine model did not correspond to the human children tested in Shirakawa’s research. Furthermore, neither study provided information on long-term effects. We propose to improve upon the methods employed by Shirakawa and Hansen by injecting BCG into Mus Musculus to stimulate a natural Th1 response, as well as correlating murine age to human age using an appropriately large sample size. Cytokine analysis from blood serum drawn at 6 and 26 weeks of age will determine allergen response. Furthermore, we seek to examine long-term effects of administering BCG vaccinations by examining lung morphology and immune cell composition of the airways. Our aim is to provide evidence for the efficacy and safety of the BCG vaccine in mice. In doing so, we hope to provide a sound basis for further studies leading to future applications of BCG as a preventative measure for both tuberculosis and asthma in the United States.

Starting Date: June 1, 2010
Dates of Proposed Period of Support: June 1, 2010 - September 30, 2011
Submission Date: December 11, 2000
Total Funds Requested for Initial Budget Period: $ 9,350
Principal Investigators: Janine Low Ph.D., Mahie Lie DVM, Annie Yang Ph.D.

Introduction
The University of California at San Francisco Asthma Clinical Research Center, the University of California at Davis School of Veterinary Medicine, and the Carson Edward Biochemistry Laboratory collectively request $9, 350 from the Donald K. Meisenheimer Humanitarian Fund for a clinical study of the tuberculosis vaccine as a possible prevention for asthma. The prevalence of asthma has increased 75% from the years 1980 to 1994, currently affecting an estimated 17 million Americans.1 More than 5,300 deaths from asthma occur annually.2
         Asthma is an allergic disorder in which the airways in the lungs are easily inflamed, causing difficulty in breathing. A multitude of factors can trigger an asthma attack, especially the inhalation of airborne particles, such as second-hand smoke, dust, and pet dander. To protect the body, immune cells attack the invading particles and release chemicals called histamines that cause bronchial smooth muscles to contract, thereby decreasing the diameters of the airways. In a nonasthmatic individual, this inflammatory response is minimal and therefore does not affect one’s ability to breathe. In contrast, an asthmatic has a hyperactive histamine response, resulting in severely constricted airways and compromised breathing ability. Though there are no known permanent treatments for asthma, inhalers containing corticosteroids (chemicals which reduce inflammation) temporarily relax the bronchial muscles and allow restoration of normal breathing.3 Although corticosteroids have been invaluable in managing asthma attacks, bone loss and other deleterious side effects have been recorded from long-term use.4
         One proposed method to combat the development of asthma and allergic diseases is the administration of childhood vaccinations for the bacterial infection tuberculosis (TB).5 Not only does TB remain one of the largest causes of death in Third-World countries, it affects the U.S as well; the estimated cost of treating tuberculosis in the U.S. alone is $700 million per year.6 Yet current U.S. medical protocol excludes use of the tuberculosis vaccine, primarily because its employment may complicate diagnosis of the disease.5 Since its discovery in 1908, however, the TB vaccine has been used extensively in Japan and other countries.6 The Japanese Ministry of Health and Welfare requires TB vaccinations for children at 3 months, 6 years, and 12 years of age.5 Retrospective studies of these children reveal that in addition to preventing tuberculosis, the TB vaccine also reduces the inflammatory response associated with asthma.5 Still, these compelling studies only focused on young children who have not yet reached adulthood and therefore its long-term effects remain unknown.
         Our objective is to study the long-term effects of such inoculations for preventing asthma. The TB vaccine has great potential for relieving the distress of those afflicted with asthma and other allergic disorders. Not only would the employment of the tuberculosis vaccine potentially alleviate the suffering of millions of Americans affected by asthma, it would also prevent needless discomfort from the symptoms associated with the highly contagious disease tuberculosis, while conserving medical resources and the tremendous time and costs of treatment.
         The following section provides background information on past studies and how we will integrate elements from existing research and will seek to improve upon their methods. Thereafter, the description of proposed research will demonstrate how our study is relevant to the field of medicine and asthma. The reference, key personnel, and budget sections follow, providing details and justification of the personnel, supplies, and costs involved.

Background
         The mammalian immune system is comprised of T lymphocytes, cells that help fight off infection. One type of T lymphocyte is the T helper, which is categorized as either a Th1 anti-inflammatory cell or a Th2 pro-inflammatory cell.9 A healthy immune system modulates the balance of Th1 and Th2 production.10 Taro Shirakawa’s research team devised a retrospective study of the attenuated bovine Mycobacterium tuberculosis vaccine, or Bacillus Calmette Guerin (BCG), which stimulates the Th1 response, or antibody formation, early in a child’s life. He found that these children were less likely to suffer from asthma because the introduction of tuberculosis in small amounts triggers a large formation of Th1 cells. Th1 cells prevent the overproduction of Th2 cells, which causes lung inflammation.5 On the one hand, the Th2 inflammatory response prevents more allergic particles from entering the lungs. On the other hand, Th1 cells recognize new foreign particles, fight off the invaders, and “remember” what the enemies look like for future reference. In learning about the enemy, Th1 memory cells also make interferon gamma (IFN-g), which inhibits formation of interleukin-4 (IL-4), a cytokine responsible for the asthmatic response. Cytokines such as IFN-g and IL-4 are cells which signal proteins that elicit necessary immune responses from other cells.
         In response to Shirakawa’s compelling study, Gesine Hansen et al. studied a similar Th1/Th2 reaction using BALB/c mouse models. Since Th1 cells oppose Th2 function, Hansen believed Th1 enhancement in an immune system free animal would prevent Th2 expression and ultimately asthmatic responses. Hansen concluded that increasing the Th1 levels might actually cause serious inflammation of the airways. Hansen discovered the reverse effect of what Shirakawa found, which was a benefit from increased Th1 production.7
         However, Hansen’s study is nonrealistic for three reasons. First of all, Hansen’s methods are artificial. By directly injecting Th1 and Th2 cells into the mice, Hansen creates a replica of a passive immune system, instead of eliciting a natural immune response. In contrast, the subjects in Shirakawa’s study actively launch an immune response by reacting to the BCG vaccine. Hansen does not know the correct ratio of Th1 to Th2 cell secretions in response to vaccinations or other stimuli, whereas the body regulates this ratio. As Stephen Hauser, professor at UCSF and the Chair of Neurology, points out, “a simple autoimmunity, characterized by ‘bad’ T-cells that secrete inflammatory cytokines and ‘good’ T-cells that secrete anti-inflammatory cytokines, is untenable.”10 As the immune system is comprised of a complex and delicate combination of checks and balances which are not fully understood, attempting to replicate an artificial immune system is difficult. Furthermore, a possibility remains that Th1 and Th2 formation produces beneficial or necessary intermediates in associated biochemical pathways, as opposed to passively receiving direct administration of Th1 or Th2 cells.
         Second, the age of the mice used in Hansen’s research does not correlate to the age of the humans studied by Shirakawa. Hansen studied sexually mature8 6-10 week old mice, whereas Shirakawa’s results and conclusions of the effects of the BCG vaccine were based on children. Therefore, Hansen’s results cannot effectively compare to Shirakawa’s conclusions as immune system function may be affected by age.
         Finally, Hansen’s study did not provide the sample size for the experiment. Though this is a minor oversight, sample size is an important aspect of this type of study. Since the number of animals tested is not given, it is unknown whether the results are statistically significant.
         In addition, neither Hansen nor Shirakawa tested the long-term effects of manipulating Th1 and Th2 levels in the immune system. Changing the ratios of the immune cells in both experiments may lead to serious, unforeseen problems, as inflammation is part of the healing process.
         Our research team will address these unresolved issues by incorporating elements of Shirakawa’s and Hansen’s research and making the necessary adjustments to improve upon their work’s deficiencies. We propose to inoculate the BCG vaccine into mice with a known genetic history in order to induce an immune response rather than create an artificial balance of Th1 and Th2 cells. Additionally, we will correlate the age of mice to the age of human children in Shirakawa’s study. We will use an appropriately large sample size (30), as well as study any long-term effects the BCG vaccine may induce. Since the trial duration is one year, the long-term effects of BCG will be determined by examination of geriatric mice at the end of our study.

Description of Proposed Research
         Our aim is to duplicate Shirakawa’s study using a mouse model in order to examine any possible long-term deleterious effects resulting from manipulation of the immune system via the Bacillus Calmette Guerin (BCG) vaccine. We seek to combine the methods of both Shirakawa’s and Hansen’s experiments to more accurately describe the Th1 response as it applies to asthma. Our objective is to provide evidence in support of BCG vaccines as a safe and effective preventative measure against asthma by measuring the Th1 response in inoculated Mus Musculus. Such evidence will provide a basis for further research that will serve as stepping stones for future approval of the widespread use of the BCG vaccine in the U.S.

Rationale for Using a Murine Model
         Murine lung development is an acceptable model for studying human lung morphology. Since Mus Musculus have a lifespan of 1-2 years,8 we will be able to study long-term effects on their developmental and geriatric physiology within a reasonable timeframe, whereas this is more difficult in human subjects. In order to correlate the murine model to Shirakawa’s human study, we propose to use 2-week old mice with a known genetic and disease-free history. All animal protocols used will follow approved standards of the University of California at Davis Committee on Animal Welfare.

Administration of the BCG Vaccine
         We will use a sample size of 30 animals. The control group will consist of 15 mice, which will receive placebo injections of phosphate-buffered saline (PBS). The test group will consist of 15 mice, which will receive the attenuated bovine Mycobacterium tuberculosis (BCG) vaccine. Either PBS or the BCG vaccine will be administered to the mice, at 2, 4, and 6 weeks of age, which roughly corresponds to the Japanese vaccine protocol of administering BCG to children at 3 months, 6 years, and 12 year of age. One-tenth of a cubic centimeter (0.1 cc) of PBS or BCG will be administered intradermally in the interscapular region of the mouse.

Cytokine Analysis
         Blood samples will be drawn 24 hours after the 2nd and 6th week injection and used for cytokine analysis. ELISAs will be performed on the blood serum to detect cytokines for Th1 and Th2, IFN-g and IL-4, respectively. Biotinylated detection of IFN-g will be determined by color change using the detection chemicals R4-6A2 and XMG1.2. Likewise, detection of IL-4 will be determined by color change using 11B11 and BVD6-24G2. This process will be repeated at 20 weeks after the last injection of BCG to see if the immune response has been sustained through adulthood. The cytokine levels of the control group and the BCG treated group will be compared for statistically significant differences. An unpaired student’s t-test will be used to quantify statistical significance based on a 95% confidence interval (p 0.05).

Necropsy Analysis
         Necropsies will be performed on euthanized animals at one year of age in order to study long-term effects of the BCG vaccine on geriatric murine physiology. Specifically, the lung and liver tissues as well as cell composition in the lungs by lavage and histology analysis will be examined. The cause of death will be determined for those specimens that expire during the course of the experiment.

Long-Range Goal of Study
         The long-range goal of this study is to provide a solid starting point in assessing the efficacy of the attenuated bovine Mycobacterium tuberculosis vaccine for the prevention of asthma, and the long-term health effects associated with administration of BCG. We hope to provide a sound experimental basis for further research that will eventually lead to the approval of the BCG vaccine for application in the U.S. for the prevention of both tuberculosis and asthma.

References

1. United States Centers for Disease Control. Forecasted State-Specific Estimates of Self-Reported Asthma Prevalence—1998. Morbidity and Mortality 47:1022-1025 (December 4, 1998).

2. United States Centers for Disease Control. National Center for Health Statistics. National Vital Statistics Report. 47 (4) (1997).

3. http://www.aaaai.org/public/fastfacts/glossary.stm

4. Journal of Allergy and Clinical Immunology 101: 445-450 (1998) at http://pharminfo.com/pubs/pnn/pnn25_6.html

5. Shirakawa T, Enomoto T, Shimazu S, Hopkin J. The Inverse Association Between Tuberculin Responses and Atopic Disorder. Science 275 (5296): 41 (January 1997).

6. Report from a workshop conducted by the U.S. Department of Health and Human Services Advisory Council for Elimination of Tuberculosis, the U.S. National Vaccine Program Office and the National Institute of Allergy and Infectious Diseases of the US National Institutes for Health, March 5-6, 1998, from the National Institute of Allergy and Infectious Diseases at www.niaid.nih.gov/publications/blueprint/page1.htm.

7. Hansen G, Berry G, Dekruff R, Umetau D. Allergen-Specific Th1 Fails to Counterbalance Th2 Cell-Induced Airway Hyperreactivity but Cause Severe Airway Inflammation. The Journal of Clinical Investigation 103 (2):175-183.

8.Animaldiversity.ummz.umich.edu/accounts/mus/m._musculus$nar-rative.html

9. UCSF Researchers Advise Caution in Testing Treatments for MS. Doctors’ Guide: Global Edition December 19, 1996.

10. Rook G, Educating the Immune System. Science and Medicine July/August: 54-63.

Other References

Castro M, Chaplin D, Walter M, Holtzman M. Could Asthma Be Worsened by Stimulating the T-Helper Type 1 Immune Response? American Journal of Respiratory, Cell and Molecular Biology 22 (2); 143-146 (February 2000).

Cookson W, Moffatt. Asthma—An Epidemic in the Absence of Infection? Science 275 (5296): 41 (January 1997).

Pinkerton K, Personal Interview. (November 9, 2000).

http://www.rodentia.com/wmc/index.html

Vogel G. Why the Rise in Asthma Cases? Science 276 (5319): 1645.

Von Mutius E, Martinez F, Fritzsch C, Nicolai T, Reitmeir P, Thiemann H. Skin Test Reactivity and Number of Siblings. British Medical Journal 308: 692-695 (March 12, 1994).

Key Personnel

Janine Low Ph.D will perform necropsies and morphological analyses of lung and liver tissues. Mahie Lie DVM will oversee animal care and health. Annie Yang Ph.D will supervise and coordinate the cytokine analysis. The principal investigator and the two collaborators will collate and publish the findings. Biographical sketches of the key investigators are provided on the following three pages.
         Research technicians are responsible for the animals’ daily care and maintenance, in addition to assisting with vaccine administration, cytokine analysis, and necropsy. Undergraduate animal technicians will aid research technicians with animal maintenance. Finally, Maury Smovich will compute all statistical information.

Budget Justification
         The total requested funds from the Donald K. Meisenheimer Humanitarian Fund is $9,350. The costs for this experiment, totaling $193,060, are summarized in the following table. Included in the budget are fees for supplies and expertise provided by the personnel for testing and data analysis. Travel reimbursements are included for Janine Low who will commute from San Francisco to Davis in order to conference with the other key personnel and assist with the research. The costs are significantly reduced, as the research facilities and necessary equipment will be provided by the UCD Carson Edward Biochemistry Laboratory, supervised by Annie Yang Ph.D. Additionally, the laboratory technicians have assisted the key investigators in the past, which will eliminate the time and costs involved in advertising for and interviewing prospective candidates.