March 14 Presentation to the new MS Bio students


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On March 14 I presented the history of my research activities and proposals for MS Biology thesis work for the students entering the program at National University,

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March 14 Presentation to the new MS Bio students

  1. 1. The continuously changing scienceFrom infection to cancer and back 2012 Ana Maria Barral
  2. 2. What is Science?“We shall not cease from exploration. And the end of all our exploring will be to arrive where we started and know the place for the first time.” T. S. Eliot
  3. 3. This talk A bit of personal history Cancer and inflammation through the history of Tumor Necrosis Factor alpha (TNFa) Philosophy of science (guaranteed brief) “What we’ve got here is a failure to communicate:” cancer biologists and immunologists Projects:  Ca-pterin  Science course
  4. 4. The dry facts B.Sc of Biochemistry, University of Havana, Cuba  Thesis: protease inhibitors from sea anemones National Institute of Oncology and Radiobiology  Development and characterization of mAbs against malignant melanoma University of Linkoping, Sweden  Ph.D. thesis: Melanoma, thioredoxin, and cytokine regulation La Jolla Institute for Allergy and Immunology, San Diego  Postdoc projects: Exosomes, cytokines and chemokines in Type 1 diabetes Nereus Pharmaceuticals  Characterization of proteasome inhibitors with anti-tumor effect from marine microorganisms
  5. 5. Malignant melanoma Tumor derived from the pigment producing cells (melanocytes) from the skin Least abundant but most lethal skin cancer Presence of tumor antigens and often strong immune response
  6. 6. TNF and receptors Tumor necrosis factor alpha 26 kD protein cleaved to the 17 kD mature TNF (soluble & intracellular) 2 receptors, TNF-R1 and TNF-R2 Currently a whole superfamily of conserved factors and receptors
  7. 7. TNF superfamily
  8. 8. TNF as a link between cancer and infection (inflammation) Original observation: bacterial extracts could provoke tumor necrosis This was caused by a factor released by the host cells in response to bacterial endotoxin TNF= cachexin, circulating factor in parasite infected animals TNF is an important mediator of the inflammatory response, needs to be controlled (septic shock) Tumor cells can also produce TNF
  9. 9. The devil is in the details… There are 2 receptors Human TNF only acts on mTNFR1, mouse TNF on both Some pathways are the same, but the default is for growth Response will depend on context TNF tumor toxicity was mainly observed with concurrent metabolic inhibition
  10. 10. TNFa signaling pathways
  11. 11. Tumor-promoting effect of TNFaBalkwill, Nat Rev Cancr VOLUME 9 | MAY 2009 | 361
  12. 12. TNF and melanoma Previous study: TNF+ primary melanomas present less CD3+ T cell infiltration (Sander & Boeryd, 1996) Goal : how is TNF expressed in melanoma cells and how does it affect the resistance against cytotoxic attack Multiple approaches: cell lines and patient samples, detection of intracellular and secreted TNFa, transfection of cell lines with tagged pro-TNF
  13. 13. TNFa expression protects melanoma cells
  14. 14. TNF is present in Golgi but not in melanosomesTNF and WGA TNF and HMB-45 (melanosome marker)
  15. 15. Immunohistochemical studies of TNF in melanoma patientsTNF staining in primary melanomas
  16. 16. Patients with TNFa+ tumors had a significantly better survival than those with TNFa- tumors Hazard Rate P Confidence intervalTumorThickness 1.555 0.010* 1.113-2.171 TNFa : an independentClark level 2.462 0.247 0.536-11.309 prognostic factor?Mitotic index 1.227 0.039* 1.010-1.490Age 0.996 0.834 0.960-1.034TNFa 0.113 0.046* 0.013-0.966
  17. 17. Study of the intracellular dynamics of TNF in melanoma cells N- -C Pro-TNF Mature TNF GFPN- -C FLAG Pro-TNF Mature TNF GFP
  18. 18. TNF is correctly cleaved inmelanoma cell lines by TACE Uncleaved TNF (yellow), mature TNF (green), Pro-TNF (red)
  19. 19. TNF is transported to the dendritesand transferred to neighboring cells after PMA stimulation
  20. 20. TNF and TNF-receptors are released in exosomesColocalization of TNF and TNFR1 after TNF and TNF-receptors arePMA stimulation in the dendrites present in exosomes
  21. 21. TNF in exosomes provokehigher levels of ROS in T-cells
  22. 22. Conclusions TNF-melanoma project TNF is correctly cleaved in melanoma cell lines Upon stimulation it is transported to the dendrites and transferred to neighboring cells or Released via exosomes Possible local or systemic functional role? CANCER-INFLAMMATION CONNECTION REDUX Who was first, the hen or the egg?
  23. 23. What are Exosomes?Small (60-90 nm) vesicles of endocytic originSecreted by APCs, B-cells, tumor cellsCapable to prime against tumor antigens: immunotherapy“Trojan exosome” hypothesis: HIV uses exosome pathway for budding?Some cytokines can be released via exosomes
  24. 24. Possible role of exosomes during viral infection Exosomes and viruses share similar budding pathways in certain cells Exosomes from antigen-presenting cells present MHC class I-II antigens and costimulatory molecules Could exosomes be released by virus-infected cells and “amplify” the antiviral response?
  25. 25. LCMV lymphocytic choriomeningitis virus + NP: nucleoprotein (np396) CD8 GP: glycoprotein (gp33) CD8+ (gp61) CD4+Viral infection: viremia peaks day 3 CTL response/viral clearance day 6-8Intracranial infection: mice die because of CNS damage byCTLs
  26. 26. RIP-LCMV Mouse Model for Type 1 Diabetes LCMV 1 2 3 4 No Diabetes No Diabetes No Diabetes Overt Diabetes (Day 10-14) T Cell PoolAntigen-specificprecursor T-cells LCMV GP GP GP GP GP GP b b b GP GP GP GP GP GP GP GP GP Inflammation Virus elimination b-Cell Destruction Tolerance Cellular attraction (antigen specific (antigen specific Ignorance (non-specific / innate) adaptive) adaptive)
  27. 27. Exosomes were obtained fromLCMV-infected cells and mice EM of serum exosomes from LCMV-infected mice Western blot of exosomes from splenic DCs of infected mice
  28. 28. Exosomes derived from LCMV-infected DCs andserum express CD11c, some B7.2 and low FasL Exosomes isolated with CD11b-Dynabeads Iad+ PKH62exo Added 2 hrs to splenic Exosomes isolated with Dynabeads GFP-DCs coupled to MHC-II (Ia d) from PKH26 (red) labeled DCs PKH26 exosomes
  29. 29. Exosomes asvaccines…nope.
  30. 30. Cancer causes inflammation
  31. 31. “The Dialectical Biologist”An organism does not compute itself from its DNA.The organism is the consequence of a historicalprocess that goes on from the moment ofconception until the moment of death; at everymoment gene, environment, chance, and theorganism as a whole are all participating. . ..Natural selection is not a consequence of howwell the organism solves a set of fixed problemsposed by the environment; on the contrary, theenvironment and the organism activelycodetermine each other. (Levins and Lewontin,1985)
  32. 32. My philosophy of science Forest and trees Avoid mechanical reductionist thinking ALTHOUGH scientists HAVE to apply a reductionist approach Go back as often as you can to the big picture but avoid superorganic holism Historical approach (lots can be learned from reading the MatMet sections of the old articles) Do not be afraid to question established paradigms: there is no such as absolute truth Exploration of our world is a dynamic process Cross-pollinate
  33. 33. Lost in Translation Tumor biologists focus on the tumor cell, especially on its genes Tumor immunologists study the immune mechanisms reacting (or not) to the tumor cell Few instances of dialogue
  34. 34. Cytokines involved in beta-cell deathIFNg: direct apoptosis (with TNFa) upregulation of MHC class-1TNFa: direct apoptosisIL1: induction of iNOS, NOproductionalso mediates dsRNA mediateddamage (viral infection)SOCS: negative regulator of JAK-STAT responsesInhibits signaling of IL1, IFNg IL2,IL3, IL4 and others
  35. 35. Transgenic mice with cytokine signaling defects in beta-cellsMice used in this study(T. Kay and E. Thomas, Australia)RIP-GP+/SOCS-1+ (SOCS: suppressor of cytokinesignaling)RIP-GP+/IFNgRtg+RIP-GP+/IL1RkoMice were infected with LCMV, and diabetes incidenceand immunological parameters were followed
  36. 36. Diabetes incidence Diabetes incidence 90 80 GP+SOCS- (n=17)Percent diabetes 70 IL1Rko (n=9) 60 IFNgRtg (n=42) 50 40 GP+SOCS+ (n=12) 30 20 10 0 0 25 50 75 100 days
  37. 37. SOCS mice do not present lymphocytic infiltration
  38. 38. SOCS mice do not presentupregulation of the chemokine IP10 (CXCL-10) after viral infection SOCS - SOCS+
  39. 39. SOCS+ islets are more resistant to killing by effector cells or cytokines SOCS- islet 40 40 SOCS + islet SOCS- islet% Cytotoxicity % Cytotocixity 30 SOCS + islet 30 20 20 10 10 0 0 SOCS- SOCS+ TNF TNF+IL1
  40. 40. SOCS+ mice do not upregulate Fas and MHC-class I expression after viral infection Mean Fluorescent Intensity 3500 200Mean Fluorescent Intensity 3000 2500 2000 100 1500 1000 500 0 0 B6 RIP-GP ILR1ko IFNgRtg SOCS+ B6 RIP-GP ILR1ko IFNgRtg SOCS+ Fas MHC class I
  41. 41. SOCS+ local effectors are less activated A C day 5 GP33 day 7 *Mean Fluorescent Intensity 70 8 spleen SOCS+ 60 spleen SOCS- % IFN+ cells 50 6 PLN SOCS+ 40 * PLN SOCS- 4 30 20 2 B 10 D 0 0 RIP-GP ILR1ko IFNgRtg SOCS+ * 2 spleen SOCS+ spleen SOCS- % IFN+ cells GP61 PLN SOCS+ * PLN SOCS- 1 0
  42. 42. SOCS project
  43. 43. Other stuff…
  44. 44. To make things morecomplicated: effect of microbiota
  45. 45. Gut microbiota modulates the immune system Presence of microbes help training of immune system in newborns children C-section vs vaginal birth: colonization by different microbes Parasites can be helpful Autoimmune diseases due to “wrong” microbes Autism? Supraorganisms (animal plus microbes and everything in- between Keep your mind open for more paradigm changes!
  46. 46. An interesting substance…
  47. 47. Calcium pterin as immune modulator SanRx pharmaceuticals (SD startup) has developed DCP: calcium pterin Pterins as natural heterocyclic compounds involved in many biochemical reactions Folates are conjugated pterins Metabolism of amino acids Aromatic compounds, NO Common in vertebrates and also bacteria
  48. 48. DCP storyline Anti-tumoral in vivo effect in several mouse models This effect seems to be related to modulation of IDO (Indoleamine-pyrrole 2,3-dioxygenase) activity IDO degrades tryptophan to kyrunenine Cytokine pattern is also altered- seem to affect Th1 cytokines (IL6 and IFNg especially) Recent results: enhances intracellular killing of Mycobacteria
  49. 49. Possible mechanisms? Mechanism of action: direct effect on IDO? Immunomodulatory effect via cytokines? Direct DNA binding? DCP as supplement (ongoing) Indirect effect through the gut microbiota? There are several bacterial enzymes that require pterin (molybdopterin, cyanopterin)
  50. 50. “Back where you started” Anti-cancer drug development Nereus Pharmaceuticals Emphasis: proteasome inhibitors (modulate NFkB) and angiogenesis inhibitors
  51. 51. Angiogenesis inhibitors
  52. 52. Thesis projects
  53. 53. Project: DCP (Ca-pterin) Currently in the process of gain FDA-approval Regulatory requirements: formulation strategies Combination of folate and Ca-compounds? To explore formulation alternatives based on sound biochemical/pharmacological principles (review process) Analysis of HPLC products of different formulations
  54. 54. Research: the inverted STEM classroom Learning for life: active and interactive, project & inquiry- based Minimal lecturing, use class room time for discussions, projects, activities Use of technology: recorded lectures (podcasts), mobile learning (see Khan academy, iTunes University, Eric Mazur) NU ideal: high proportion of engaged, focused, professional students who value real-life learning, in their own time NU strategic plan is online (competition with state universities & for-profits)
  55. 55. Voice-thread for discussions
  56. 56. Project: online science course In collaboration with School of Education Development of a fully online, interactive science course (Intro to Bio) Pedagogy: development of learning objectives and content/assignment/artifacts to fulfill them Relevance: up to date content, 21st century tools and digital media
  57. 57. Thank you!