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Radioactive treatment of C. neoformans infection.

Authors: Geraldina Ortiz, Dr. Bryan and Dr. Dadachova Affiliations: Albert Einstein College of Medicine of Yeshiva University. Radioactive treatment of C. neoformans infection. Introduction. Cryptococcus is a pathogen that affects people with deficient immune system.

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Radioactive treatment of C. neoformans infection.

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  1. Authors: Geraldina Ortiz, Dr. Bryan and Dr. Dadachova Affiliations: Albert Einstein College of Medicine of Yeshiva University Radioactive treatment of C. neoformans infection.

  2. 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.

  3. 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.

  4. 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

  5. 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 H2O2to 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.

  6. Apoptosis Diagram

  7. 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.

  8. Results

  9. Results in fluorescence microscope in 3mM hydrogen peroxide. Visible (left) and under fluorescence light (right) Demonstrating which cells are becoming apoptotic.

  10. Results cont.

  11. Results cont.

  12. 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.

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

  14. Reference • Aberg JA Cryptococcosis and HIV [Online] Available • http://hivinsite.ucsf.edu/insite?page=kb-05-02-05 • Apoptosis Celular [Online Image] Available • http://www.dialogica.com.ar/medline/archives/001772.php July 14, 2004 • Bertout, S., Renaud, F., Swinne, D., Mallié, M., Bastide, J.-M. (1999). Genetic Multilocus Studies of Different Strains of Cryptococcus neoformans: Taxonomy and Genetic Structure. J. Clin. Microbiol. 37: 715-720 • Bryan R, Zaragoza O, Zhang T, Ortiz G, Casadevall A, Dadachova E. "Radiological Studies Reveal Radial Difference in the Architecture of The Polysaccharide Capsule of Cryptococcus neoformans" Eukaryotic Cell 4.2 (2004) P465-475 • Casadevall, A. and Perfect, R.J. Cryptococcus neoformans (1998) American Society for Microbiology U.S.A • Chaturvedi V, B Wong, and SL Newman. Oxidative Killing of Cryptococcus neoformans by human Neutrophils. Evidence that fungal mannitol protects by scavenging reactive oxygen intermediates. J. Immununol. 1996; 156:L • How do you want to die? [Online] Available • http://healthy-living.org/html/what_a_choice_.html August 22, 2006 • Lopez, A. The lancet, May 27, 2006, Vol.367 Pp. 1747-1757. New release • Milenic DE, Brady ED, Brechbiel MW. Antibody Targeted Radiation Cancer Therapy. Nature Rev. Drug Disc. 3: 488-498, 2004 [Journal] • Pringle, J.R., Preston, R.A., Adams, A.E., Stearns, T., Drubin, D.G., Haarer, B.K., Jones, E.W.  (1989).  Fluorescence Microscopy Methods for Yeast.  Meth Cell Biol 31: 357-435. • Pringle, J.R., Adams, A.E.M., Drubin, D.G., and Haarer, B.K. (1991). Immunofluorescene methods for yeast. Methods Enzymol.194, 565-602 • üFröhlich, K.-U. & Madeo, F. (2000) Apoptosis in yeast—a monocellular organism exhibits altruistic behavior. FEBS Lett. 473, 6–9.10.1016/s0014-5793(00)01474-5 • 4ü • Wentworth P Jr, Jones LH, Wentworth AD, Zhu x, Larsen NA, Wilson IA, Xu X, Goddard Lerner RA. “Antibody Catalysis of the Oxidation of Water.” Science 293, 1806-1811 (2001) PN5290. • Wentworth, P. Jr. et al. 2002 Evidence for antibody-catalyzed ozone formation in bacterial killing and inflammation. Science. 298: 2195-2199

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

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