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,

1. The continuously
changing science
From infection to cancer
and back
2012
Ana Maria Barral

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. 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. 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. 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. 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. TNF superfamily

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. 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. TNFa signaling pathways

11. Tumor-promoting effect of TNFa
Balkwill, Nat Rev Cancr VOLUME 9 | MAY 2009 | 361

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. TNFa expression protects
melanoma cells

14. TNF is present in Golgi but not in
melanosomes
TNF and WGA TNF and HMB-45 (melanosome marker)

15. Immunohistochemical studies of
TNF in melanoma patients
TNF staining in primary melanomas

16. Patients with TNFa+ tumors
had a significantly better
survival than those with TNFa-
tumors
Hazard Rate P Confidence interval
Tumor
Thickness 1.555 0.010* 1.113-2.171 TNFa : an independent
Clark level 2.462 0.247 0.536-11.309 prognostic factor?
Mitotic index 1.227 0.039* 1.010-1.490
Age 0.996 0.834 0.960-1.034
TNFa 0.113 0.046* 0.013-0.966

17. Study of the intracellular dynamics
of TNF in melanoma cells
N- -C
Pro-TNF Mature TNF GFP
N- -C
FLAG Pro-TNF Mature TNF GFP

18. TNF is correctly cleaved in
melanoma cell lines by TACE
Uncleaved TNF (yellow), mature TNF (green),
Pro-TNF (red)

19. TNF is transported to the dendrites
and transferred to neighboring cells
after PMA stimulation

20. TNF and TNF-receptors are
released in exosomes
Colocalization of TNF and TNFR1 after TNF and TNF-receptors are
PMA stimulation in the dendrites present in exosomes

21. TNF in exosomes provoke
higher levels of ROS in T-cells

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. What are Exosomes?
Small (60-90 nm) vesicles of endocytic origin
Secreted by APCs, B-cells, tumor cells
Capable to prime against tumor antigens: immunotherapy
“Trojan exosome” hypothesis: HIV uses exosome pathway for budding?
Some cytokines can be released via exosomes

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. 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 6-8
Intracranial infection: mice die because of CNS damage by
CTLs

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
Pool
Antigen-specific
precursor 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. Exosomes were obtained from
LCMV-infected cells and mice
EM of serum exosomes from LCMV-infected
mice
Western blot of exosomes from splenic DCs
of infected mice

28. Exosomes derived from LCMV-infected DCs and
serum 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. Exosomes as
vaccines…nope.

30. Cancer causes inflammation

31. “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)

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. 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. Cytokines involved in beta-cell
death
IFNg: direct apoptosis (with TNFa)
upregulation of MHC class-1
TNFa: direct apoptosis
IL1: induction of iNOS, NO
production
also mediates dsRNA mediated
damage (viral infection)
SOCS: negative regulator of JAK-
STAT responses
Inhibits signaling of IL1, IFNg IL2,
IL3, IL4 and others

35. 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+/IFNgRtg+
RIP-GP+/IL1Rko
Mice were infected with LCMV, and diabetes incidence
and immunological parameters were followed

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. SOCS mice do not present
lymphocytic infiltration

38. SOCS mice do not present
upregulation of the chemokine IP10
(CXCL-10) after viral infection
SOCS - SOCS+

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. SOCS+ mice do not upregulate Fas
and MHC-class I expression after
viral infection
Mean Fluorescent Intensity
3500 200
Mean 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. 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. SOCS project

43. Other stuff…

44. To make things more
complicated: effect of
microbiota

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. An interesting substance…

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. 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. 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. “Back where you started”
 Anti-cancer drug development
 Nereus Pharmaceuticals
 Emphasis: proteasome inhibitors (modulate NFkB) and
angiogenesis inhibitors

52. Angiogenesis inhibitors

53. Thesis projects

54. 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

55. 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)

56. Voice-thread for discussions

57. 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

58. Thank you!