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Critical Regulation of Thymic Epithelial Cell Function and Thymus Development by Transforming Growth Factor-Beta Signali

Critical Regulation of Thymic Epithelial Cell Function and Thymus Development by Transforming Growth Factor-Beta Signaling. Michael Blazar 2006. Thymus. Blood vessels. Medulla. Cortex. Membrane. http://www.becomehealthynow.com/popups/thymus.htm. Hollander presentation.

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Critical Regulation of Thymic Epithelial Cell Function and Thymus Development by Transforming Growth Factor-Beta Signali

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  1. Critical Regulation of Thymic Epithelial Cell Function and Thymus Development by Transforming Growth Factor-Beta Signaling Michael Blazar 2006

  2. Thymus Blood vessels Medulla Cortex Membrane http://www.becomehealthynow.com/popups/thymus.htm Hollander presentation

  3. Thymocytes and Thymic Epithelial Cells (TEC) (Scanning Electron Microscopy) Hollander presentation TEC Thymocyte

  4. Thymocyte Development Thymic Epithelial Cells (TEC) CD4-CD8- Cortex immature Thymus CD4+CD8+ intermediate Corticomedullary Junction Stem Cell Medulla CD4- CD8+ CD4+ CD8- mature mature Hollander presentation

  5. Previous Studies • Massagué et al. (1998, 2000) • -Mutations in signaling of TGF-β cause developmental disorders and cancer • TGF-β1 inhibits mitosis in epithelial cells • TGF β1-3 causes cell-cycle arrest in epithelial and hematopoietic cells • Kim et al. (2005) • TGF-β stimulates formation of fibrous matrix • TGF-β impacts wound healing, the extracellular matrix, and immunosuppression • Balciunaite et al. (2002) • -Wnt glycoproteins stimulate in vivo growth of TEC in mice

  6. Thymocytes Produce Growth Factors to Stimulate TEC T Cell Thymocytes TGF-β Receptors (RI and RII) Growth Factor TEC Hollander presentation TEC

  7. Cre- and Cre+ Genotypes

  8. Goals of the First Objective Effect of TGF-βRII signaling -Thymus structure -Thymocyte development -T-cell and B-cell populations in the spleen

  9. Hypotheses • Deletion of both TGF-βRII alleles -abnormal thymus structure and development • Deletion of one TGF-βRII allele -normal thymus structure and development

  10. Methods Fluorescent Activated Cell-Sorting (FACS) Staining Immunofluorescence Staining Confocal Microscopy Analysis FACS Calibur Analysis

  11. Immunofluorescence Staining Cre- lox/lox Cre- lox/lox Cre- lox/lox Cre- lox/lox Cre- lox/del Cre+ lox/lox Cre+ lox/lox Cre+ lox/lox Cre+ lox/lox Cre+ lox/del Tears Cysts Abnormal blood vessels Photos taken by author

  12. Results: Percentages of thymocyte populations Cre-: lox/lox to lox/del p=0.76 lox/del: Cre- to Cre+ p=0.27

  13. Results: Percentages of thymocyte populations Cre+: lox/lox to lox/del p=0.46

  14. Results: Percent of T-cells and B-cells in the Spleen Cre+: lox/lox to lox/del p=0.34

  15. Goals of the Second Objective • Effect of adding TGF-β1 -Cell growth of TEC cultures -Programmed cell death of TEC cultures

  16. TEC cultures • Hypothesis • TGF-β will inhibit proliferation in TEC cultures proportional to expression level of TGF-β receptors. Hollander presentation Line TGF- Receptors’ Expression level 1.2 low 2.3 high C6 medium C9 medium

  17. Methods Measure programmed cell death after 24 and 48 hours in culture with/without TGF-β Measure cell growth after 24 and 48 hours in culture with/without TGF-β FACS Calibur Analysis FACS Calibur Analysis

  18. Results: Percent cell growth with/without TGF-β

  19. Results: Percent programmed cell death with/without TGF-β

  20. Results: Percent programmed cell death with/without TGF-β

  21. Conclusions First Objective 1. One TGF-β Receptor II allele was required for normal thymus structure 2. TGF-β Receptor II did not affect thymocyte development and T-cell and B-cell populations in the spleen

  22. Conclusions Second Objective 1. TGF-β addition did not affect cell growth in the TEC culture with the lowest receptor expression level of TGF-β 2. TGF-β addition caused a consistent increase in cell growth in the TEC culture with the highest receptor expression level of TGF-β 3. The effect of TGF-β on cell growth in TEC cultures was lower after 48 hours of culture as compared to 24 hours

  23. Conclusions Second Objective 4. TGF-β addition increased programmed cell death proportional to the receptor expression level of TGF-β 5. When baseline programmed cell death was large (>10%), addition of TGF-β reduced cell death

  24. Future Studies 1. Ideal time of incubation with TGF-β in intact and damaged TEC 2. Effects of TGF-β on cell growth and death of injured TEC in vivo 3. Optimal TGF-β signaling to inhibit cell growth in vivo before chemotherapy and radiation therapy 4. Prevention and repair of injured TEC to aid immune system deficiency and recovery during chemotherapy and radiation therapy

  25. Acknowledgements • Dr. Georg A. Hollander • Mathias Hauri • Jason Gill • Annick Peter • Ms. Fruen • Advanced Science Research team

  26. Critical Regulation of Thymic Epithelial Cell Function and Thymus Development by Transforming Growth Factor-Beta Signaling Michael Blazar 2005/06

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