March 14 Presentation to the new MS Bio students
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Transcript of March 14 Presentation to the new MS Bio students
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The continuously changing scienceFrom infection to cancer and back
2012
Ana Maria Barral
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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
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This talkA 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-pterinScience course
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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
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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
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TNF and receptorsTumor 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
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TNF superfamily
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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
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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
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TNFa signaling pathways
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Tumor-promoting effect of TNFa
Balkwill, Nat Rev Cancr VOLUME 9 | MAY 2009 | 361
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TNF and melanomaPrevious 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
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TNFa expression protects melanoma cells
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TNF is present in Golgi but not in melanosomes
TNF and WGA TNF and HMB-45 (melanosome marker)
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Immunohistochemical studies of TNF in melanoma patients
TNF staining in primary melanomas
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Hazard Rate P Confidence intervalTumorThickness 1.555 0.010* 1.113-2.171Clark level 2.462 0.247 0.536-11.309Mitotic 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
Patients with TNFa+ tumorshad a significantly bettersurvival than those with TNFa-tumors
TNFa : an independentprognostic factor?
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Study of the intracellular dynamics of TNF in melanoma
cells
Pro-TNF Mature TNF GFP
FLAG Pro-TNF Mature TNF GFP
N-
N-
-C
-C
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TNF is correctly cleaved in melanoma cell lines by TACE
Uncleaved TNF (yellow), mature TNF (green),Pro-TNF (red)
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TNF is transported to the dendrites and transferred to neighboring cells after PMA
stimulation
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TNF and TNF-receptors are released in exosomes
Colocalization of TNF and TNFR1 afterPMA stimulation in the dendrites
TNF and TNF-receptors arepresent in exosomes
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TNF in exosomes provoke higher levels of ROS in T-
cells
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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?
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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
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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?
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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 by CTLs
LCMV lymphocytic choriomeningitis virus
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RIP-LCMV Mouse Model for Type 1 Diabetes
T CellPool
bGP
GPGP
GP
ToleranceIgnorance
1
Antigen-specificprecursor T-cells
b-Cell Destruction(antigen specific
adaptive)
GP
GPGP
4
Overt Diabetes(Day 10-14)
Virus elimination(antigen specific
adaptive)
3
bGP
GPGP
GP
No DiabetesNo Diabetes
LCMV2
bGP
GPGP
GP
InflammationCellular attraction
(non-specific / innate)
LCMV
No Diabetes
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Exosomes were obtained from LCMV-infected cells and mice
EM of serum exosomes from LCMV-infected miceWestern blot of exosomes from splenic DCsof infected mice
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Exosomes derived from LCMV-infected DCs and serum express CD11c, some B7.2 and low FasL
Iad+ PKH62exo
Exosomes isolated with CD11b-Dynabeads
Exosomes isolated with Dynabeads coupled to MHC-II (Iad) from PKH26 (red) labeled DCs
Added 2 hrs to splenic GFP-DCs
PKH26 exosomes
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Exosomes as vaccines…nope.
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Cancer causes inflammation
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“The Dialectical Biologist”An organism does not compute itself from its DNA. The organism is the consequence of a historical process that goes on from the moment of conception until the moment of death; at every moment gene, environment, chance, and the organism as a whole are all participating. . . .Natural selection is not a consequence of how well the organism solves a set of fixed problems posed by the environment; on the contrary, the environment and the organism actively codetermine each other. (Levins and Lewontin, 1985)
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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
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Lost in TranslationTumor 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
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Cytokines involved in beta-cell death
IFNg: direct apoptosis (with TNFa) upregulation of MHC class-1TNFa: direct apoptosisIL1: induction of iNOS, NO productionalso mediates dsRNA mediated damage (viral infection)
SOCS: negative regulator of JAK-STAT responsesInhibits signaling of IL1, IFNg IL2, IL3, IL4 and others
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Transgenic mice with cytokine signaling defects in beta-cells
Mice used in this study(T. Kay and E. Thomas, Australia)
RIP-GP+/SOCS-1+ (SOCS: suppressor of cytokine signaling)
RIP-GP+/IFNgRtg+
RIP-GP+/IL1Rko
Mice were infected with LCMV, and diabetes incidenceand immunological parameters were followed
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Diabetes incidence
Diabetes incidence
0 25 50 75 1000
10
20
30
40
50
60
70
80
90
GP+SOCS+ (n=12)
GP+SOCS- (n=17)
IFNgRtg (n=42)
IL1Rko (n=9)
days
Per
cen
t d
iab
etes
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SOCS mice do not present lymphocytic infiltration
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SOCS mice do not present upregulation of the chemokine
IP10 (CXCL-10) after viral infection
SOCS - SOCS+
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SOCS+ islets are more resistant to killing by
effector cells or cytokines
SOCS- SOCS+ 0
10
20
30
40SOCS- isletSOCS + islet
% C
ytot
oxic
ity
TNF TNF+IL10
10
20
30
40SOCS- isletSOCS + islet
% C
ytot
ocix
ity
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SOCS+ mice do not upregulate Fas and MHC-class I expression
after viral infection
Fas MHC class I
B6 RIP-GP ILR1ko IFNgRtg SOCS+0
500
1000
1500
2000
2500
3000
3500
Me
an
Flu
ore
sc
en
t In
ten
sit
y
B6 RIP-GP ILR1ko IFNgRtg SOCS+0
100
200
Mea
n F
luo
resc
ent
Inte
nsi
ty
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SOCS+ local effectors are less activated
GP61
0
1
2
*
*spleen SOCS+spleen SOCS-PLN SOCS+PLN SOCS-
% I
FN
+ c
ell
s
A C
B D
GP33
0
2
4
6
8 *
*
spleen SOCS+spleen SOCS-
PLN SOCS+
PLN SOCS-
% IF
N+
cel
lsRIP-GP ILR1ko IFNgRtg SOCS+
0
10
20
30
40
50
60
70 day 7day 5
Mea
n Fl
uore
scen
t In
tens
ity
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SOCS project
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Other stuff…
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To make things more complicated: effect of
microbiota
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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!
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An interesting substance…
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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
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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
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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)
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“Back where you started”Anti-cancer drug development
Nereus Pharmaceuticals
Emphasis: proteasome inhibitors (modulate NFkB) and angiogenesis inhibitors
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Angiogenesis inhibitors
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Thesis projects
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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
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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)
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Voice-thread for discussions
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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
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Thank you!