Authors: Geraldina Ortiz, Dr. Bryan and Dr. Dadachova Affiliations: Albert Einstein College of Medicine of Yeshiva University

Introduction

Cryptococcus is a pathogen that affects people with deficient immune system. One of its main virulence factors is its capsule. Studies have shown that DMSO and radiation are able to disrupt this capsule. Radioactive antibodies have also been shown to kill Cryptococcus. The question that arises is how does radiation kill Cryptococcus? We believe that radiation produces hydrogen peroxide.

Introduction cont.

We also did a biodistribution in which we wanted:

1) to evaluate the ability of radioactive antibodies (Re) to find their target; IgG or IgM.

2) We wanted to have an idea of how long they remain in the body.

3) To find out what other organs in the body they accumulate in.

Materials

Flask Cryptococcus

(Cryptococcus) Media (SAB Broth) Reagents to induce

Apoptosis Antibodies Slides Plates Hemocytometer Hydrogen peroxide Centrifuge Incubator

Vortexer Pipette PBS DMSO Counter Gamma ray Counter Pre weight tubes Monoclonial antibodies A/JCr mice Flow Cytometry FLICA kit

Methods

We culture 24067 cells. Counted them on the hemocytometer to see how

concentrated they were. Then we made flasks with SAB(Sabruraoud Broth)

and 24067, in different flask we had the control and different amounts of H2O2 to see the difference in the control and experimental.

We saw that H2O2 kills 24067 when we plated them out.

Then we added Fluorochrome Inhibitor of Caspase (FLICA) to see if they were becoming apoptotic.

We looked at the cells in the fluorescence microscope and by the flow cytometry, to see if the cells took the red fluorescence stain.

Apoptosis Diagram

Methods cont.

In the biodistribution we infected the A/JCr mice, then treated them and finally sacrifice them. We then dissected them and took out several organs.

The organs were placed in labeled tubes, which were then put in the gamma ray counter.

Results

24067 Killing Curve

1

10

100

0m

icro

lite

r

25

mic

rolit

er

37

mic

rolit

er

50

mic

rolit

er

63

mic

rolit

er

10

0m

icro

lite

r

H2O2

CF

Results in fluorescence microscope in 3mM hydrogen peroxide.

Visible (left) and under fluorescence light (right) Demonstrating which cells are becoming apoptotic.

Results cont.

treatment with radioactive antibodies leads to apoptosis

0

2

4

6

8

10

12

0 5 10 15

micrograms of 18B7/tube

% F

LIC

A p

ositiv

e c

ells

3hr hot

3hr cold

17 hr hot

17 hr cold

Results cont.

Biodistribution 13F1+TEPC (4 Hrs%ID/g)

01020304050

Organs

%ID

/g

13Fl

TEPC

Biodistribution 13F1+TEPC ( 24HrsID/g)

02468

Organ

%ID

/g

13FlTEPC

Biodistribution 13F1+TEPC (48Hrs %ID/g)

01234

Organ

%ID

/g

13Fl

TEPC

Discussion

In other experiments not described here, we found that the IgM 13F1was less efficient both in binding and in killing C neoformans in test tubes. In our biodistribution studies, we found that even though IgG molecules are smaller than IgM molecules, the IgG molecules are able to go to the organ more efficiently. The IgM 13F1 doesn't bind as well to the capsule so even though the IgM is bigger it is much less efficient than the IgG in treating infection.

Future work

In the future we want to figure out how radiation kills Cryptococcus and hopefully create a treatment/cure.

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Acknowledgements

Dr.Dadachova Dr.Bryan Xianchun Casadevall lab Dr. Sat Bhattacharya Harlem Children Society Memorial Sloan-Kettering Cancer

Center