Infection and Immunity Research Forum

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					            The 2009 Infection and Immunity Research Forum at The University of Western Ontario

                                                                 The Fourth Annual
                                                                 Infection and Immunity Research
                                                                 Forum
                                                                 November 20th, 2009

                                             Mission Statement
!        The Infection and Immunity Research Forum is an initiative organized by the graduate students of the
Department of Microbiology and Immunology at the University of Western Ontario. Prominent scientists will be
invited to present their research within the scope of infection and immunity.  This research forum will also
provide graduate students and post-doctoral fellows with a platform to showcase their own research, by means
of oral and poster presentations, as it pertains to the theme of infection and immunity. The objective of the
Infection and Immunity Research Forum is to allow interaction between both graduate students and post-
doctoral fellows and a world-renowned investigator in the field of infection and immunity. It is anticipated that
this event will foster intellectually stimulating dialogue between participants, and will contribute to the rewarding
academic experience at the University of Western Ontario. The forum will also provide participants with the
unique opportunity to speak with outstanding scientists and colleagues, thus establishing a basis for future
career opportunities and collaborations. Moreover, this endeavour will promote the scientific activities conducted
at the University of Western Ontario to both the local scientific community and the scientific community at large.

                                                    Agenda

                  Time                                Event                              Location

                                                                              In front of Patios 2&3,
                 8:30 AM                 Registration Begins
                                                                              University Hospital

                                                                              Auditorium A, University
                 9:15 AM                 Opening Remarks
                                                                              Hospital

                                         First Oral Presentation Session,     Auditorium A, University
             9:30 - 10:30 AM
                                         Sponsored by Pall Life Sciences      Hospital

                                                      Sara Ramos Romero, “Early activation of PI3K/Akt
                  9:30 - 9:45 AM
                                                                pathway by epicatechin.”

                                                      Enayat Nikoopour, “Th17 polarized from NOD mice
                  9:45 - 10:00 AM                   following mycobacterial adjuvant immunotherapy delay
                                                                      type I diabetes”

                                                  Matthew Miller, “CRAP is a new protein expressed by
                 10:00 - 10:15 AM                 human Cytomegalovirus at early times post-infection and
                                                  localizing to the nuclear lamina”

                                                  Gillian Hewson, “Modulatory Effects of Ginseng Extracts
                 10:15 - 10:30 AM
                                                  on Human Immune Responses”

                                         Coffee Break, Sponsored by
            10:30 - 10:45 AM                                                  Adjacent to Auditorium A
                                         Sarstedt

                                                                              Auditorium A, University
            10:45 - 11:00 AM             Poland Award Presentation
                                                                              Hospital
The 2009 Infection and Immunity Research Forum at The University of Western Ontario



     Time                             Event                            Location

                         Second Oral Presentation
                                                            Auditorium A, University
11:00 - 12:00 PM         Session, Sponsored by
                                                            Hospital
                         Cedarlane

                                   Boram Ham, “Cathepsin B-mediated autophagy flux and
    11:00 - 11:15 AM
                                         autophagic cell death: Another way to die”

                                     Danielle Visschedyk, “Photox: Illuminating the actin-
    11:15 - 11:30 AM
                                      targeting family of mono-ADP-ribosyltransferases”

                                  Samar Sayedyahossein, “The Type of Primary Antigen-
                                  Presenting Cells Determines the Immunomodulatory
    11:30 - 11:45 AM
                                  Capacity of Staphylococcal Peptidoglycan-embedded
                                  Molecules”

                                  Jailal Ablack, “Negative Regulators at the Reigns: The
                                  Role of Cellular Repressors in E1A Conserved Region
    11:45 - 12:00 PM
                                  Three Dependent Activation of Transcription Across
                                  Human Adenovirus Subgroups”

                         Lunch (Requires Registration),     Auditorium A, University
12:00 - 12:45 PM
                         Poster Setup                       Hospital

                         Pall Life Sciences and
                                                            Auditorium A, University
12:45 - 1:00 PM          Cedarlane Oral Presentation
                                                            Hospital
                         Awards Ceremony

                         Keynote Address: Dr. Victor
                         Nizet, “Exploring the Pathogen-    Auditorium A, University
 1:00 - 2:00 PM
                         Host Interface for Novel           Hospital
                         Therapeutic Strategies”

                         Coffee Break, Sponsored by
 2:00 - 2:10 PM                                             Adjacent to Auditorium A
                         Invitrogen

                         CIHR Institute of Infection and
 2:10 - 4:00 PM                                             Patios 2&3, University Hospital
                         Immunity Poster Session

                                                            Auditorium A, University
 4:00 - 5:00 PM          Career Development Talk
                                                            Hospital

                         Reception and Poster Award         The Wave, 2nd Floor UCC
   5:00 PM -             Ceremony (Requires                 Building, The University of
                         Registration)                      Western Ontario
              The 2009 Infection and Immunity Research Forum at The University of Western Ontario

                                                Oral presentations
1. Early activation of PI3K/Akt pathway by epicatechin.         2. Th17 polarized from NOD mice following
                                                                mycobacterial adjuvant immunotherapy delay type I
Sara Ramos-Romero, Department of Physiology, Faculty            diabetes
of Pharmacy, University of Barcelona, Spain; and The
FOCIS Centre for Clinical Immunology and                        Enayat Nikoopour, Jordan Schwartz, Katrina Huszarik,
Immunotherapeutics, Robarts Research Institute, and             Christian Sandrock, Bhagirath Singh, Department of
Departments of Microbiology and Immunology, and                 Microbiology and Immunology, University of Western
Medicine, The University of Western Ontario, London,            Ontario
Ontario, Canada.
Francisco José Pérez-Cano, Àngels Franch, Margarida             IL-17 producing T cells are regarded as potential
Castell, Department of Physiology, Faculty of Pharmacy,         pathogenic T cells in the induction of autoimmune
University of Barcelona, Spain                                  diseases. Previously we have shown that injection of
Joaquim Madrenas, The FOCIS Centre for Clinical                 Mycobacteria containing products such as Bacille
Immunology and Immunotherapeutics, Robarts Research             Calmette-Guerin (BCG) or Complete Freund Adjuvant
Institute, and Departments of Microbiology and                  (CFA), can prevent type I diabetes (T1D) in nonobese
Immunology, and Medicine, The University of Western             diabetic (NOD) mice. We injected NOD mice with
Ontario, London, Ontario, Canada                                mycobacterial products subcutaneously and analyzed the
                                                                IL-17 producing cells from the draining lymph nodes and
Flavonoids are diphenylpropane polyphenols that provide         spleen by restimulating whole cell populations in vitro with
color and protection against UV light to the plants.            or without IL-17 polarizing cytokines. Mice receiving CFA
Epicatechin is a flavanol present in many vegetable              had a concomitant rise in the level of IL-17, IL-22, IL-10
products including tea, apples, grapes, and cocoa. A            and IFN-g in the draining lymph node and spleen. Adoptive
flavanol-enriched diet has been linked to many health            transfer of splenocytes from CFA- injected NOD mice
benefits, but the molecular target of epicatechin remain         polarized with TGF-b plus IL-6 or IL-23 delayed the
unknown. The goal of this project was to determine the          development of diabetes in recipient mice. IL-17 producing
effect of epicatechin on intracellular signaling. First, we     cells induced by CFA maintained their IL-17 producing
studied the in vivo effect of an epicatechin-enriched diet on   ability in the recipient mice. Injection of CFA also changed
inflammatory mediators produced by peritoneal                    the cytokine profile of cells in pancreatic tissue by
macrophages isolated from Wistar rats fed cocoa during 7        increasing IL-17, IL-10 and IFN-g cytokine gene
days. Thereafter, monocytes isolated from human                 expression. Compared to wild type mice, injection of CFA
peripheral blood were incubated with epicatechin at             into MyD88-/- mice drastically reduced but did not abolish
different concentrations and for different times, with or       the level of IL-17 in the lymphoid organs and pancreas
without peptidoglycan. Activation of the ERK-1/-2, PI3K/        indicating that the MyD88 pathway is a major pathway
Akt, and canonical NF- B pathways were determined by            through which Mycobacteria exerts its effect. We suggest
western blotting. We found that in vivo administration of       that the rise in the level of IL-17 after adjuvant therapy in
cocoa correlated with lower production of NO, and               NOD mice has a protective effect on T1D development.
proinflammatory cytokines from macrophages. We also
found that epicatechin, by itself, induced sequential and
rapid phosphorylation of the regulatory subunit of              3. CRAP is a new protein expressed by human
PI3kinase (P85), an event associated with activation of this    Cytomegalovirus at early times post-infection and
enzyme, followed by phosphorylation (i.e., activation) of       localizing to the nuclear lamina
Akt. Such an effect enhanced the activation of this pathway
by peptidoglycan. Activation of these enzymes was also          Matthew S. Miller, Department of Microbiology and
rapidly reversed, and this correlated with an increase in the   Immunology, Schulich School of Medicine and Dentistry,
level of PTEN, a negative regulator of the PI3K/Akt             The University of Western Ontario
pathway. Based on these results, we conclude that the           Leesa Pennell, Department of Immunology, Faculty of
biological effects of epicatechin, and perhaps cocoa            Medicine, University of Toronto
extracts containing this flavonoid, are induced by selective     Marc Geadah, Laura Hertel, Department of Microbiology
and rapid activation of the PI3K/Akt pathway in monocytes.      and Immunology, Schulich School of Medicine and
                                                                Dentistry, The University of Western Ontario
             The 2009 Infection and Immunity Research Forum at The University of Western Ontario


4. Modulatory Effects of Ginseng Extracts on Human            process is prevented, a Rip-1 (receptor interacting
Immune Responses                                              protein-1)-dependent autophagic cell death becomes
                                                              prominent. To date, how autophagy, which is generally
Hewson G.L., Toth J.M., Chau L.A., Madrenas J.                known as a cell survival process, induces cell death is
The FOCIS Centre for Clinical Immunology and                  unknown. We found that cathepsin B, a lysosomal
Immunotherapeutics, Robarts Research Institute, and the       protease, plays a key role in autophagy flux. Cathepsin B-
Departments of Microbiology and Immunology, and               deficient macrophages or inhibition of cathepsin B by the
Medicine, The University of Western Ontario, London, ON,      chemical inhibitor CA-074-ME caused an accumulation of
Canada                                                        autolysosomes and LC3-II (microtubule associated protein
                                                              1 light chain 3). These cells were more susceptible to
Despite being used as an herbal remedy for thousands of       autophagic cell death induced by serum starvation or the
years, the immunomodulatory effects of ginseng remain         treatment of the pan-caspase inhibitor zVAD-fmk than wild-
uncertain. This is because most of the studies to date have   type or non- treated macrophages. The RIP-1-specific
used neoplastic cell lines whose results cannot be            inhibitor Necrostatin-1 inhibited the autolysosome
extrapolated to normal human immune cells. Therefore,         accumulation and autophagic cell death. These results
the goal of our project is to characterize the effects of     suggest that cathepsin B is a key player in autophagy flux
North-American ginseng (Panax quinquefolius) extracts on      and the blockage of autophagy flux leads to autophagic
the innate and adaptive immune responses of normal            cell death. Our study identified a novel role of cathepsin B
human peripheral blood mononuclear cells (PBMCs)              in autophagy flux and autophagic cell death, which can be
(monocytes, B cells, T cells).      Our hypothesis is that    a potential therapeutic strategy for inflammatory diseases
ginseng has immunomodulatory effects by itself and on the     such as sepsis and septic shock.
profile of cytokine production in response to pathogen-
associated molecular patterns [endotoxin (LPS)] and toxins
[staphylococcal superantigens (SAgs)]. To test this           6. Photox: Illuminating the actin-targeting family of
hypothesis we measured the production of cytokines and        mono-ADP-ribosyltransferases
reactive oxygen species (ROS) by PBMCs in response to
ethanol, aqueous and polysaccharide extracts of ginseng       Danielle Visschedyk, Alex Perieteanu, Zachari Turgeon,
alone or in combination with LPS or SAgs. We found that       Robert Fieldhouse, A. Rod Merrill, University of Guelph
ginseng extracts by themselves induced production of
IL-6, TNF-alpha and IL-10 by PBMCs with the aqueous           Photorhabdus luminescens is a pathogenic bacterium that
fraction being more potent than the crude polysaccharide      produces many toxic proteins. Previously primarily known
fraction and the ethanol fraction. In addition, the aqueous   to target insects, Photorhabdus has been studied for its
fraction triggered IL-1beta production. Such a response       potential use in agriculture and the control of pests.
was similar for aqueous extracts from 4 out of 5 different    However, Photorhabdus infections of humans are now
sources.      Induction of cytokines by ginseng extracts      beginning to be seen in the United States and
enhanced the proinflammatory innate response to LPS.           Australia.The mono-ADP-ribosyltransferases (mARTs) are
However, IL-2 production to SAgs (an adaptive response)       an enzyme class produced by numerous pathogenic
was downregulated.          In addition, ginseng aqueous      bacteria and participate in disease in plants and animals,
fractions induced the production of ROS by monocytes.         including humans. We have discovered and characterized
Based on these results, we conclude that North-American       a novel mART from P. luminescens which has been named
ginseng, and in particular its aqueous fraction, has          Photox. This 46 kDa toxin shows high homology to other
immunomodulatory properties, enhancing innate immune          actin-targeting mARTs in key catalytic regions and a similar
responses while downregulating adaptive immune                core catalytic fold. Furthermore, Photox shows in vivo
responses. Key words: Ginseng, Human immune cells,            cytotoxic activity against yeast, and in vitro, enzymatic
Innate Immunity, Adaptive Immunity, Cytokines                 activity is quite high (kcat, 2235 +/- 270 min-1).
                                                              Substitutions of hallmark catalytic residues results in
                                                              drastic decreases in mART activity. This toxin specifically
5. Cathepsin B-mediated autophagy flux and                     ADP- ribosylates actin at Arg177, targeting each of alpha-,
autophagic cell death: Another way to die                     beta-, and gamma-actin isoforms, and inhibiting regular
                                                              polymerization of actin filaments. By epifluorescent
Boram Ham, Soon-Duck Ha, Sung O. Kim, UWO                     microscopy, Photox has been seen to associate with actin
                                                              within yeast cells. After nearly a decade since the last
Macrophages are key innate immune cells, releasing large      addition to this enzyme family, Photox is the newest actin-
quantities of inflammatory cytokines upon activation, and      targeting ADP-ribosyltransferase.
cell death of activated macrophages can be a crucial
process in preventing overwhelming inflammation. Our
previous studies showed that activated macrophages
undergo apoptotic cell death, however, when apoptosis
             The 2009 Infection and Immunity Research Forum at The University of Western Ontario


7. The Type of Primary Antigen-Presenting Cells               8. Negative Regulators at the Reigns: The Role of
Determines the Immunomodulatory Capacity of                   Cellular Repressors in E1A Conserved Region Three
Staphylococcal Peptidoglycan-embedded Molecules               Dependent Activation of Transcription Across Human
                                                              Adenovirus Subgroups
Samar Sayedyahossein, Department of Microbiology and
Immunology, The University of Western Ontario, London,        Jailal Ablack, Department of Microbiology & Immunology
Ontario, Canada.                                              UWO
Thu A. Chau, The FOCIS Centre for clinical Immunology         Peter Pelka, Department of Oncology, UWO
and Immunotherapeutics, Robarts Research Institute,           Joe Mymryk, Departments of Oncology and Microbiology &
London, Ontario, Canada.                                      Immunology
David E. Heinrichs, Department of Microbiology and
Immunology, The University of Western Ontario, London,        Viruses are obligate intracellular parasites, and over a
Ontario, Canada.                                              nearly infinite number of selections have evolved efficient
Joaquín Madrenas, The FOCIS Centre for clinical               and potent means to hijack the cellular environment. In the
Immunology and Immunotherapeutics, Robarts Research           case of DNA tumor viruses, a critical step in the viral
Institute, Department of Microbiology and Immunology, The     replication is the expression of early genes that are
University of Western Ontario, Department of Medicine,        responsible for making the intracellular milieu conducive to
The University of Western Ontario, London, Ontario,           viral replication and initiating the viral replication cycle. In
Canada.                                                       Human Adenovirus (hAd), the expression of early genes is
                                                              mediated by the E1A oncoprotein--a paradigm of viral
S. aureus is a common human-commensal Gram-positive           transactivation.      The largest E1A isoform contains a C4
bacterium. S. aureus is also considered the most important    Zinc finger domain within Conserved Region 3 (CR3) that
cause of serious and fatal infections in the Western          is responsible for this function. hAd Serotype 5 (hAd5)
hemisphere. Staphylococcal superantigens (SAgs) are           E1A CR3 interacts with key cellular factors in order to
pyrogenic exotoxins that have potent activating properties    target itself to viral promoters and activate transcription.
on human T lymphocytes, and play a pathophysiological         We have expanded this model to include representative
role in many S. aureus-induced diseases including Toxic       members of each hAd subgroup and examined the
Shock Syndrome (TSS).            Recent studies from our      function and interaction with cellular transcriptional
laboratory have shown that staphylococcal peptidoglycan-      regulatory proteins for each CR3. There are dramatic
embedded molecules (PGN), a component of Gram-                differences in how representative CR3s activate
positive bacterial cell wall, have an immunomodulatory        transcription as Gal4 DNA-binding domain fusions.
effect on human T cell activation by binding to TLR2 on       Surprisingly, these differences are not due to the role of the
antigen-presenting cells (APCs) and signaling through the     established core targets TBP and MED23, as there are no
canonical NF-κB pathway to induce IL-10 production and        differences in the interaction of all six representative CR3s
inhibition of the T cell responses to SAgs. The nature of     with these factors.       Furthermore, all six CR3s fail to
PGN-mediated immunomodulation is unknown.               We    activate transcription in MED23-/- MEFs, thus MED23 is
hypothesized that the capacity of PGN to modulate human       absolutely required for CR3 function, but does not account
T cell responses is dependent on the type and stage of        for the observed differences. Examination of the interaction
differentiation of APCs and their profile of cytokine          of these representative CR3s with the remaining known
production in response to PGN. We found that monocytes        cellular targets revealed some dramatic differences.
and macrophages as APC were able to mediate significant        Specifically, hAd3 CR3 binds strongly to the cellular
down-regulation of IL-2 production in response to SAg in      repressor BS69 which may explain its relatively weak
the presence of PGN (P < 0.05).               In contrast,    ability to activate transcription. Similarly, Ad9 CR3-- the
immunomodulation was not significant when monocyte-            weakest activator tested-- has a relatively strong
derived dendritic cells were used as APC. These results       interaction with the cellular repressor CtBP. I recently
were not due to differences in SEE presentation by these      identified the cellular histone acetyl-transferase GCN5 as a
APCs. The immunomodulatory effect correlated with the         new target of CR3. All six CR3s interact with GCN5 and
ability to produce IL-10 as well as IL-6 and TNF-α, but not   GCN5 knockdown increased transcriptional activation by
with IL-12 or IL-1β production. Intracellular staining of     all CR3s. Therefore, GCN5 is a newly identified and
peripheral blood mononuclear cells responding to PGN          universal negative regulator of CR3 function.             Taken
showed that IL-10 is mostly produced by non-CD3+              together, these results expand the current paradigm of viral
populations (monocytes, and perhaps B cells). Based on        transactivation to include negative regulators of
these results, we propose that modulation of SAg-induced      transcription as key components of the viral transcriptional
T cell responses by PGN depends on the type of primary        activation complex.          This work suggests that viral
APC. This provides a starting point to assess the different   transactivation is highly regulated by balancing interactions
outcomes associated with S. aureus colonization vs. S.        with activating and repressing factors, and that this
aureus-induced sepsis and TSS.                                balance differs between hAd serotypes that have evolved
                                                              to target different tissues.