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  1. 1. Clarkson University Investigation of a Potent Microbiostatic Substance Secreted by Brain Derived Microphages A Thesis by Victoria. A. Roberts Department of Biology Submitted in partial fulfillment of the requirements for a Bachelor of Science Degree with University Honors April 2004 Accepted by the Honors Program ____________________________ Advisor Date ____________________________ Honors Reader Date ____________________________ Honors Director Date
  2. 2. Investigation of a Potent Microbiostatic Substance Secreted by Brain Derived Microphages by Victoria A. Roberts 2
  3. 3. Acknowledgements: Karen Aguirre Tera M. Fillion Melissa J. Sargent Mike Terry 3
  4. 4. Outcome Objectives: • Problem Identification • Framing the Problem Environment/ Problem Scope • Formulating a Hypothesis • Conducting an Investigation • Analysis • Supporting a Conclusion • Presentation of Results In place of another proposal, as I have already completed one for summer research last year, I have worked on completing a draft of my thesis on which I shall continue work this summer. This rough draft and the extensive literature search that I have completed represent my progress on my thesis in addition to the conclusion of my necessary thesis research. 4
  5. 5. Executive Summary: It has long been believed that infectious pathogens are unable to penetrate the tight junctions of endothelial cells that line the capillaries that oxygenate the brain. This seemingly impenetrable blood brain barrier seemed the brains primary line of defense against invading fungi among other pathogens. Within recent years the second line of immunological defense beyond this endothelial wall has focused on the activities of a brain derived macrophage. Microglial cells are normally bone marrow derived cells that 1. Neuron 2. Oligodendrocyte 3. Capillary 4. Axon 5. Astrocyte 6. Ependymal cell 7. Microglial cell Figure 1: Physiological composition of brain provide nutrients to neurons; however, when stimulated by interferon gamma, which signals an infection, they quickly become immunologically active to protect the host. Most often the microglial cells phagocitize the intrusive antigen. However, very little is known about the extracellular methods employed. Previous investigation in professor Aguirre’s lab has shown that properly stimulated cells of a retrovirally-transformed murine microglial cell line (BV2) inhibit the proliferation of the fungus Cryptococcus neoformans. The growth of the avirlent reporter strain (cap67) was inhibited by a specific 5
  6. 6. and novel potent microbiostatic substance secreted by IFN-Y stimulated brain derived microphages. Cryptococcus neoformans is responsible for cryptococcal pneumonia and cryptococcal menigoencephalitis. It is the third most common central nervous system syndrome associated with AIDS and if left untreated is lethal. If initial infection is survived lifelong maintenance on anti-fungal medications is necessary. Identification of a new method of treatment would be optimal. Therefore the novel microbiostatic substance is a source of hope for new drug therapies. Purification and peripheral examination of the basic properties of the compound are the basis of this thesis. Primary interests include the molecular weight, ability to diffuse, try sin sensitivity, heat sensitivity and target range against a broad spectrum of pathogens. An unsuccessful attempt was made to visualize the band corresponding to molecular weight standards from an electrophoreased gel. Instead a general high molecular weight was determined by the range of activity shown off the collected fractions of a BioRad column. The antimyotic substance was shown to be trypsin sensitive and heat stable. Examination of the target range showed significant activity against similar yeasts such as Saccharomyces cerevisiae and Candida albicans . No significant inhibition was shown against bacteria such as E. coli, the slime mold Dictyostelium discoideum, or the amoeba Niglarei Fowlarri (?) 6
  7. 7. Table of Contents: 1. Title Page 2. Acknowledgements 3. Abstract/ Executive Summary 4. Table of Contents 5. List of Figures 6. Introduction 7. Background, literature review and definition of terms 8. Methodology 9. Results 10. Discussion 11. Conclusions 12. Bibliography 13. Appendix 7
  8. 8. List of Figures: Page 1. Physiological Composition of Brain- Microglial Cells ……...……… 5 2. Brain lesions caused by Cryptococcus neoformans …..………… 9 3. Acapsular Cryptococcus neoformans ………………...…………… 4. Column Experimental Set-up………………………………………. 5. Yeast-Column Graph 1 ……………………………………………. 6. Yeast –Column Graph 2 …………………………………………… 7. D. discoideum: Experiment 1 ……………………………………... 8. D. discoideum Experiment 2 …………………………………….. 9. Gel 1 ………………………………………………………………. 10. Gel 2 ………………………………………………………………. 11. Gel 3 ………………………………………………………………. 8
  9. 9. Chapter 1: Introduction The opportunistic fungal pathogen Cryptococcus neoformans becomes deadly when it reaches the central nervous system by hematogenous dissemination from the lungs (Med Pix). Common clinical signs are fever, headache, nausea, vomiting, confusion and blurred vision, skin sores and if the disease is in the lung then pneumonia. This ubiquitous yeast like fungi infected over 1,200 people in New York City alone in 1990. It was the most common infection of the central nervous system within the city. C. neoformans is especially pronounced in immuniosupressed individuals. The incidence of infection is 2-4 cases per thousand immunocompromised patients (Crytococcosis, Five to ten percent of individuals with AIDS develop the infection which either results in death or life long antifungal therapy to prevent a relapse of infection (A. Casadevall, Cryptococcus neoformans). The fungi are most commonly contracted by airborne spores in the vicinity of bird droppings. In the majority of cases the encapsulated fungi remains in the lung. However, in the event that it is able to pass the blood brain barrier where it is able to cause cryptococcal brain lesions microglial function as immune effector cells and destroy the intrusive fungi using an arsenal of different methods. To fight C. neoformans as the ethologic agent of Cryptoccocal meningoencephalitis and Cryptococcal pneumonia it is necessary to develop more effective therapies to prevent future Figure 2: Cryptococcal Brain Lesions 9
  10. 10. relapses. The extracellular killing mechanisms of the microglial cell line may provide a spring board of new drug therapies I plan to include information from the following articles to enrich the introduction • IFN gamma: Efficacy of Recombinant Gamma Interferon for Treatment of Systemic Cryptococcosis in SCID Mice • Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans • Enhancement of antifungal chemotherapy by interferon-gamma in experimental systemic cryptococcosis 10
  11. 11. Chapter 2: Background • Jason Crowe’s Graph- preceding Mike this Graph shows the BV2 yeast killing assay which can be used to show the differences in the stimulated and unstimulated yeast killing ability of microglial cells. Also his BV2 yeast killing assay because of the continued use of Cap 67 in my own experiments • Data from Janel Smith’s thesis that shows that the interferon gamma stimulated BV2 cells are effective at killing cap 67. Shows the importance of the particular type of stimulation • Other studies on the problem: Mike’s Graph showing how the experiments started with the use of the mw column. • Other Articles to be discussed that led to this point in our lab: o Basic Immunological methods of antigen recognition and methods of antigen killing o Potential Mechanisms of Neurologic Disease in HIV Infection o Anticryptococcal Resistance in the Mouse Brain: Beneficial Effects of o Local Administration of Heat-Inactivated Yeast Cells o Interdependency of Interleukin-10 and Interleukin-12 in Regulation of T- Cell Differentiation and Effector Function of Monocytes in Response to Stimulation with Cryptococcus neoformans • Previous research showing that the myotic substance is only shown in the supernatant • Not Nitric Oxide • Crude Prep fractionation 11
  12. 12. Figure 3: Acapsular Cryptococcus neoformans as utilized at the Clarkson University Lab 12
  13. 13. Chapter 3: Methodology Nitrocellulose Paper Experiments: (plan to include a picture of the experiment when retrieved) Initial analysis of the antiyotic activity of C. neoformans used agar (?) plates and small pieces of nitrocellulose paper. The plates were plated with a concentration of 1 x 10 -4 (?) of the C. neoformans strain Cap 67. Each piece of nitrocellulose paper was used in triplicate. After thoroughly soaking each cn2 piece of nitrocellulose paper in either conditioned medium, unconditioned medium or a phosphate buffered saline (PBS) control the paper was patted dry on a sterile paper towel and then gently place on the agar plate. It was expected that areas of yeast growth would be inhibited around the nitrocellulose paper soaked in conditioned medium because of the presence of activated BV2 microglial cells. An alternative experiment which controlled for the minimal differences in moisture of the nitrocellulose paper placed the paper squares under the agar which solidified above the papers. Native Gel Electrophoresis: (plan to include a picture of the gel running apparatus) It was expected that the proteins were not heat killed therefore a native gel electrophoresis was used to attempt to visualize the molecular weight of the unknown compound. The first gel was loaded with 20 ul of conditioned medium and 4 ml of sample buffer with two lanes of standard molecular weight markers on each side. The second gel used the same setup but used unconditioned medium. After each gel was electrophoresed down the gel it was then placed over night at 30 volts to transfer onto nitrocellulose paper. After the completion of the transfer the pieces of trimmed 13
  14. 14. nitrocellulose paper was then placed face down on C. neoformans plated agar plates. It was hoped that the conditioned medium would inhibit the growth of the yeast only along a certain line of the gel. If the concentration of the conditioned BV2 supernatant was high enough then the yeast inhibition would show at a line corresponding with a molecular weight marker. This would give an approximate molecular weight estimate. BioRad Column Experiment: As an alternative method for determining the molecular weight of the antimyotic component a Bio Rad column composed of an organic filamentous packing substance was used. The 25 in long column was oriented precisely at a 90 degree angle to the lab bench and made uniform by a two hour thawing period in a sterile environment. Then sterile PBS was run over the column to make sure the column was uniformly distributed after unthawing. While the experimenter is careful not to disrupt the surface of the column the initial PBS is drained until a moist layer of the gel was exposed. Then 1 ml of the BioRad Gel standards was loaded onto the gel and allowed to absorb. Then it was pushed through the column by 25 ml increments of additional PBS. The first collection of 15 ml of void volume was collected, followed by forty 1 ml increments and a final 15 ml wash volume. In between collections the stopcock at the base of the column was turned perpendicular to the length of the column to stop the flow. To analyze the collected fractions each was run over a non native gel. Twenty microleters of every odd fraction, 14
  15. 15. including the wash and void volumes were run over the gel at 70 volts. The same procedure was used for 1 ml of the unstimulated and stimulated medium. By correlating the fractions with the most antimyotic activity with the corresponding molecular weight fractions it would be possible to determine a rough estimate of the molecular weight of the compound. Trypsin Experiment: Trypsin is a serine protease. It binds to substrates specifically based on the positively charged amino acid side groups lysine and arginine. (Worthington). If the myotic component of the microglial supernatant is a protein with a positively charged side chain its bonds will be disrupted and the protein will be rendered useless. Standard procedures for a trypsin assay require that the test sample be covered in a few mills of trypsin for approximately three minutes while left on the shaker. Then the trypsin is deactivated by a protein rich serum.. Then the remaining supernatant is plated with Cap67 and its inhibitive properties are measured in comparison to the non stimulated BV2 supernatant similarly plated with Cap67. (General description, will be expounded upon) Heat Stable Experiment: If the unknown compound is a protein it will denature when exposed to heat. To test the ability of heat to denature the effective fungi killing factor within the microglial cell supernatant various samples were heated for duration of (?) and then plated with the Cap 67 strain. (General, will be expounded upon) Experiments Testing Antimyotic Target Range: Bakers Yeast and Thrush Yeast: (methods to be added) 15
  16. 16. Conditioned Medium Yeast Experiments: 1 ml fractions harvested from the BioRad columns were concentrated using microcentrifuge concentrators and the fractions were tested in quadruple against yeast plated at a concentration of 2 x 10 -4 on agar plates. Initially the void, wash, conditioned medium, unconditioned medium and a control of only growth medium were tested on a 96 well plate. After a two day incubation period all wells were plated at 10 -2 and 10 -3 (units) concentrations. After two days they were refrigerated to slow additional growth and counted as soon as possible using a hemocetometer. E. coli Experiments: (Methods to be added) Dictyostelium discoideum Experiments: D. discoideum was ordered from California Biological Supply and arrived ready to plate. It was plated North to South across a Petri dish over a West to East smear of E. coli which served as the slime mold’s food source. After a thick culture of D. discoideum appeared cloudy white across the Petri dish it was removed with a rubber policeman and put into solution using a LPS solvent. The D. discoideum was then colored with a neutral red stain to enhance visual contrast of the slime mold against the agar plates. Following the staining protocol twelve dish plates were filled with 1 ml of Dictyostelium agar. The plate was divided into three conditions: 0.2 ml conditioned medium, 0.2 ml unconditioned medium and 0.2 ml PBS. Then following the distribution of those volumes 100 ul of D. discoideum was put into each well. The plate is covered and incubation is unnecessary. Pictures were taken at a maximum of two days following the experiment. 16
  17. 17. Chapter 4: Results Results of classification Mw --- not a defensin : Microbial Inhibition of Wash Fractions Graphs from yeast experiments Pictures of Dictyostelium Get pictures of spot test exp Figure ?: Wash fractions show similar inhibition of yeast growth in the experiment which methods were used as a springboard for summer 2003 research 17
  18. 18. Figure ?: The collected wash fractions of stimulated medium over the column showed specific regions of C. neoformans growth inhibition verified by the lower numbers of yeast colonies (shown in logs). So what kind of substance do we think we’re dealing with? 18
  19. 19. Figure ?: Experiment with D. discoideum shows no statistically significant difference in the slime mold growth when suspended in distilled water [left] or LPS buffer [right]. Figure ?: Final experiment with D. discoideum shows that there is no significant difference in the inhibition of slime mold growth in the presence of stimulated or unstimulated medium. Microbial Inhibition of Wash Fractions Number of Yeast 300 Legend Colonies 200 100 0 0.0 1.0 5.010.0 20.0 30.0 40.0 50.0 60.0 15.0 25.0 35.0 45.0 55.0 W ash Fractions *(0.0=Yeast control, 1.0=Stimulated CM control) Figure 2: Wash fractions show similar inhibition of yeast growth in the experiment which methods were used as a springboard for summer 2003 research 19
  20. 20. Figure ?: Both Silver stain (top) and coomassie blue (bottom) stains were used to verify the molecular weight band location of the antimyotic agent. Every odd fraction was run 1-39 including the void and wash. Figure ?: Stimulated and unstimulated mediums from the BV2 cells were run in series with a molecular weight marker flanking the outmost columns. Died only with Coomassie blue stain. The bottom cropped gel was run with stimulated medium. 20
  21. 21. Figure ?: Stronger molecular weight markers were constructed by finding the maximum concentration of various proteins to produce optimal band visibility when run over the column. Molecular weight marker is shown on the far right preceded by various concentrations of proteins. 21
  22. 22. Chapter 5: Discussion Do we think that this could be a drug? What would the steps to becoming a drug be? What kinds of things are used for drugs to kill fungal pathogens? Possible mechanisms for killing. Ring compound and function of shape and method of killing 22
  23. 23. Chapter 6: Conclusion Future plans of investigation NMR Genome Antimicrobial justification 23
  24. 24. Chapter 7: Preliminary Literature Search 1 ) Casadevall, A., and Perfect, J., Cryptococcus neoformans, Washington, D.C. Albert Einstein College of Medicine of Yeshiva University 2) MedPix Contributer Hervey D. Segall, MD Cryptococal meningencephalitis Factoid 1470 created 2001-03-23 3) Emedicine: Excerpt from Cryptococcosis, CNS: April 10th 4) Cryptococcosis: April 11th. 5) Physiology Diagram: Shier,Butler, Lewis. Student Online Learning Center: Hole’s Essentials of Human Anatomy and Physiology. McGraw Hill, 2000. 6) Krause, K. H., Professional Phagocytes: Predators and Prey of Microorganisms. Schwez Med Wochenschr 2000; 130; 97-100. 7) Diamond R. D. et al., Factors influencing killing of Cryptococcus neoformans by human leukocytes in vitro. Public Health Report. 1996 May-June; 111(3):226-35. 8) Kaplan, J. E. et al., Preventing opportunistic infections in human immunodeficiency virus-infected persons: implications for the developing world. Tropical Medical Hygene. 1996 Jul; 55(1):1-11. 9) Ganz, T. et al., Defensins: Natural peptide antibiotics of human neutrophils. Clinical Investigation. 1985 Oct; (4):1427-35. 10) Introduction to Antimicrobial Drug – excerpt from book from offline 11) Antimicrobial Drug Development Outline: 24
  25. 25. 12) Smith, Janel L., Investigation of Interactions Between Murine Brain-Derived Macrophages and T Lymphocytes in an Experimental Infection with Cryptococcus neoformans. Honors Thesis May 2003. 13) Crowe, Jason J., Mechanism of Fungistasis of Cryptococcus neoformans Cells by Brain- Derived Macrophages in a Murine Model. Honors Thesis May 2003. 14) Aguirre, K. et al. MHC Class II-Positive Perivascular Microglial Cells Mediate Resistance to Cryptococcus neoformans Brain Infection, GLIA 39:184-188 (2002). 15) Benjamini, Eli. et al. Immunology: A Short Course. Fourth Ed. Wiley- Liss, NY: 2000. 16) Worthington- biochem supply : Trypsin reference April 14, 2004. 17) IFN gamma: Efficacy of Recombinant Gamma Interferon for Treatment of Systemic 18) Cryptococcosis in SCID Mice 19) Fungicidal activity of IFN-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans 20) Enhancement of antifungal chemotherapy by interferon-gamma in experimental systemic cryptococcosis 21) Potential Mechanisms of Neurologic Disease in HIV Infection 22) Anticryptococcal Resistance in the Mouse Brain: Beneficial Effects of Local Administration of Heat-Inactivated Yeast Cells 25
  26. 26. Interdependency of Interleukin-10 and Interleukin-12 in Regulation of T-Cell Differentiation and Effector Function of Monocytes in Response to Stimulation with Cryptococcus neoformans 26
  27. 27. Chapter 8: Appendix Pathology: Cryptococcal pneumonia: Cell Stain showing fluid collection Cat scan and chest X ray are diagnostic tools used to visualize the fluid cavities caused by cryptoccal infection Cryptococcal meningoencephalitis: Capsular polysaccharide stains bright red Cat scans diagnostic of cryptococcal menigoencephalitis will show hydrocephalus, atrophy, leptomenigeal enhancement and abscess formation (Med Pix). 27