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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
Investigation of a Potent Microbiostatic Substance
Secreted by Brain Derived Microphages
by
Victoria A. Roberts
2
Acknowledgements:
Karen Aguirre
Tera M. Fillion
Melissa J. Sargent
Mike
Terry
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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
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.
2.
3.
4.
5.
6.
7.
Neuron
Oligodendrocyte
Capillary
Axon
Astrocyte
Ependymal cell
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
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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 (?)
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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
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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 ……………………………………………………………….
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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,
brown.edu). 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
relapses. The extracellular killing
Figure 2: Cryptococcal Brain Lesions
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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
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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 TCell 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
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Figure 3: Acapsular Cryptococcus
neoformans as utilized at the
Clarkson University Lab
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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
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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,
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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)
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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.
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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
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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?
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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
Microbial Inhibition of Wash
Fractions
Number of Yeast
Colonies
300
Legend
200
100
0
0.0 1.0 5.010.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
Wash Fractions
*(0.0=Yeast control, 1.0=Stimulated
CM control)
unstimulated medium.
Figure 2: Wash fractions show similar inhibition of yeast growth in the experiment which
methods were used as a springboard for summer 2003 research
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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
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.\
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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
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Chapter 6: Conclusion
Future plans of investigation
NMR
Genome
Antimicrobial justification
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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:
http://www.emedicine.com/radio/byname/cryptococcosis-cns.htm April 10th
4) Cryptococcosis:
http://www.brown.edu/Courses/Digital_Path/Lungs/cryptococcosis.htm 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:
http://www.vet.purdue.edu/bms/courses/bms514/chmrx/intmichd.htm#top
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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
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
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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).
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