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The continuously changing scienceFrom infection to cancer and back 2012 Ana Maria Barral
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
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
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
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
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
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
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
Tumor-promoting effect of TNFaBalkwill, Nat Rev Cancr VOLUME 9 | MAY 2009 | 361
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
TNF is present in Golgi but not in melanosomesTNF and WGA TNF and HMB-45 (melanosome marker)
Immunohistochemical studies of TNF in melanoma patientsTNF staining in primary melanomas
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
Study of the intracellular dynamics of TNF in melanoma cells N- -C Pro-TNF Mature TNF GFPN- -C FLAG Pro-TNF Mature TNF GFP
TNF is correctly cleaved inmelanoma cell lines by TACE Uncleaved TNF (yellow), mature TNF (green), Pro-TNF (red)
TNF is transported to the dendritesand transferred to neighboring cells after PMA stimulation
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
TNF in exosomes provokehigher levels of ROS in T-cells
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?
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
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?
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
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)
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
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
“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)
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
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
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
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+/IFNgRtg+RIP-GP+/IL1RkoMice were infected with LCMV, and diabetes incidenceand immunological parameters were followed
To make things morecomplicated: effect of microbiota
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!
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
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
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)
“Back where you started” Anti-cancer drug development Nereus Pharmaceuticals Emphasis: proteasome inhibitors (modulate NFkB) and angiogenesis inhibitors
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
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)
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