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

Investigation of a Potent Microbiostatic Substance Secreted by Brain Derived Microphages

byVictoria A. Roberts

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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.

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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

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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1. Neuron2. Oligodendrocyte3. Capillary4. Axon5. Astrocyte6. Ependymal cell7. Microglial cell

Figure 1: Physiological composition of brain

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

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Figure 2: Cryptococcal Brain Lesions

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

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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 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

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Figure 3: Acapsular Cryptococcus neoformans as utilized at the Clarkson University Lab

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

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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Microbial Inhibition of WashFractions

0.01.05.010.015.020.025.030.035.040.045.050.055.060.00

100

200

300Legend

Wash Fractions*(0.0=Yeast control, 1.0=Stimulated

CM control)

Nu

mb

er

of

Yeast

Co

lon

ies

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.

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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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