"2006 Infection and Immunity Research Forum"
The graduate students of the Department of Microbiology and Immunology, University of Western Ontario present: 2006 Infection and Immunity Research Forum 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. Direct support of the Infection and Immunity Research Forum is proudly provided by the Department of Microbiology and Immunology at the University of Western Ontario. Agenda for November 24, 2006 Time Event Where 8:00 am - 8:45 am Registration begins In front of Auditorium A, University Hospital 8:45 am - 9:00 am Welcome Auditorium A, University Hospital 9:00 am - 9:15 am Kingsley Anukam "HIV/AIDS in Africa, might probiotic help?" 9:15 am - 9:30 am Patrick Paladino "The cellular response to virus particle entry constitutes a primary antiviral strategy that does not require TLRs or RIG-I." 9:30 am - 9:45 am Jörg H. Fritz "Nod1-mediated peptidoglycan recognition controls the onset of adaptive immunity." 9:45 am - 10:00 am Michelle McCully "Up-regulation of receptor interacting protein 2 correlates with rapid resolution of peritoneal dialysis- associated peritonitis." 10:00 am - 10:15 am Dennis Ng "AKT activation is required to overcome the effect of Anthrax lethal toxin in human myeloid cells." 10:15 am - 10:30 am Coffee Break 10:30 am - 11:00 am Presentation of Roche award for best oral presentation and Auditorium A, University Hospital Poland Award 11:00 am - 12:00 pm Dr. Norma W. Andrews "Intracellular pathogens: New turns in Auditorium A, University Hospital the road to lysosomes." 12:00 pm - 1:30 pm Lunch/ Set up Posters Patios 2 & 3, University Hospital 1:45 pm - 3:45 pm Poster Presentations Patios 2 & 3, University Hospital 4:00 pm - 5:00 pm Career Development Auditorium B, University Hospital 5:00 pm - 7:00 pm Reception and presentation of poster prizes The Wave, UCC, 2nd floor Registration tickets are required for the lunch and reception. ***Please note that Photo ID may be required for purchase of alcoholic beverages at the reception.*** http://www.uwo.ca/mni/MNI/IIRF/ 1 2006 Infection and Immunity Research Forum Oral Presentations 9:00 am - 9:15 am 9:15 am - 9:30 am HIV/AIDS IN AFRICA, MIGHT PROBIOTICS HELP? THE CELLULAR RESPONSE TO VIRUS PARTICLE ENTRY CONSITIUTES A PRIMARY ANTIVIRAL STRATAGY THAT Kingsley Anukam Lawson Health Research Institute and Benson DOES NOT REQUIRE TLRS OR RIG-I Idahosa University Nigeria. Patrick Paladino Department of Pathology and Molecular Medicine Gregor Reid Lawson Health Research Institute, and Surgery, UWO Derek T. Cummings Department of Pathology and Molecular Medicine Ryan S. Noyce Department of Biochemistry and Biomedical Introduction: HIV/AIDS is the leading cause of death in Africa today. The epidemic Sciences has surpassed armed conflicts fuelled by multinational oil companies in Africa. In sub- Saharan Africa, around one quarter of females under the age of 30 have HIV and an Department of Pathology and Molecular Medicine, estimated half billion are at risk of acquiring the virus through sexual contact. Efforts Karen L. Mossman and Biochemistry and Biomedical Sciences to provide condoms, develop a vaccine, or anti-retrovirals, have failed to stem the epidemic. The innate immune system responds to pathogen infection by eliciting a non- The risk factors associated with HIV in Africa: Bacterial vaginosis (BV) is a common specific immune response following recognition of various pathogen associated condition characterized by the replacement of lactobacilli by either anaerobic bacteria molecular patterns. Toll-like receptors (TLRs) and the RNA helicases, RIG-I and such as Atopobium, Prevotella, Gardnerella and Mobiluncus or intracellular MDA5, recognize foreign nucleic acid within endosomal and cytoplasmic Mycoplasma or aerobic bacteria such as E. coli and enterococci. The net result is an compartments, respectively, initiating a signaling cascade that involves the induction elevated pH, production of sialidase by colonizing bacteria. BV organisms produce of type I IFN through the transcription factors IRF3 and NF?B. However, a recent multiple virulence factors including toxins and proteases that can degrade sIgA, and paradigm has emerged in which bacterial DNA and double-stranded B-form DNA they have been found to induce inflammation and local damage, with a 19 fold trigger type I IFN production through an uncharacterized TLR- and RIG-I-independent increase in inflammatory cytokines reported, along with elevation in pH. This pathway. We have previously described a response in primary fibroblasts wherein the inflammation is believed to be a factor associated with a significantly increased risk of entry of diverse RNA and DNA enveloped virus particles is sufficient to induce a sexually transmitted diseases (STDs) including HIV. Failure to eradicate BV is subset of ISGs and a complete antiviral response in an IRF3-dependent, IFN- arguably of most concern in sub-Saharan Africa for several reasons. Black women, independent manner. Here, we show that the innate immune response to virus for reasons that are not fully understood, have a particularly high incidence of BV. particle entry is independent of both TLR and RIG-I pathways, confirming the existence of novel innate immune mechanisms that result in the activation of IRF3. The rationale for probiotics: The strong correlation between loss of lactobacilli, Furthermore, we propose a model of innate antiviral immunity in which exposure to dominance of BV causing organisms, inflammation and elevated pH, and increased increasing numbers of virus particles elevates the complexity of the cellular response risk of HIV, lends credence to recolonization of the vagina with probiotic lactobacilli from an intracellular, IFN-independent response to one involving secretion of may reduce the risk of HIV infection in a portion of women. cytokines and activation of infiltrating immune cells. Studies already carried out in Africa: There is tremendous interest in the potentials of probiotics for health maintenance among university students. Both animal and human studies have shown that probiotics can be administered safely without side effects. As BV increases the risk of HIV in women, we have shown that augmentation of metronidazole with probiotic GR-1 and RC-14 can cure BV. Recent published data by our team have reported an effective (90%) cure of symptomatic BV using probiotic lactobacilli. Given the correlation between BV and HIV, and the high risk of the latter in Nigeria, intravaginal use of lactobacilli could provide women with a self-use therapy We have demonstrated that probiotic yogurt can to stop diarrhea and restore a CD4 count in women already with HIV/AIDS. This is the first study to show the benefits of probiotic yogurt on quality of life of women in Nigeria with HIV/AIDS, and suggests that perhaps a simple fermented food can provide some relief in the management of the AIDS epidemic in Africa. More studies and funds are needed to transfer this simple preventive measure to Africa, instead of relying on only anti-retroviral therapy. http://www.uwo.ca/mni/MNI/IIRF/ 2 2006 Infection and Immunity Research Forum Oral Presentations 9:30 am - 9:45 am 9:45 am - 10:00 am Nod1-mediated peptidoglycan recognition controls the Up-regulation of receptor interacting protein 2 correlates onset of adaptive immunity with rapid resolution of peritoneal dialysis-associated peritonitis Jörg H. Fritz Department of Immunology, University of Toronto Michelle McCully Dept. of Microbiology & Immunology, UWO Lionel Le Bourhis Department of Immunology, University of Toronto Stephen E. Girardin Department of Laboratory Medicine and M.L. Baroja Robarts Research Institute Pathobiology, University of Toronto Robarts Research Institute T.A. Chau Dana J. Philpott Department of Immunology, University of Toronto Peter G. Blake Dept. of Medicine, UWO The mammalian NACHT-LRR protein family member Nod1 has been shown to play Joaquin Madrenas Dept. of Microbiology & Immunology and Medicine, an essential role in innate immunity by sensing specific peptidoglycan (PGN) UWO structures. Here we show that Nod1-mediated immune recognition of the PGN- Currently, there is no prognostic factor for peritoneal dialysis (PD)-associated derived muropeptide FK156 elicits priming of antigen-specific T and B cell immunity. peritonitis. Since resolution of PD-associated peritonitis correlates with a cell- Furthermore, we demonstrate that the synergistic activation of Nod1 and Toll-like mediated Th1 response, we hypothesized that up-regulation of receptor interacting receptors by classical adjuvant preparations containing mycobacterial cell wall protein 2 (RIP2) - a putative kinase linked with Th1 responses – could be a useful components is key for priming type 1, type 2 as well as TH17 immune pathways and prognostic parameter in this clinical setting. To test this hypothesis, we analyzed subsequent full-blown antigen-specific antibody responses in vivo. The importance of RIP2 expression in human immune cells under basal conditions and during the these findings is highlighted by the observation that Nod1 is required for optimal course of PD-associated peritonitis. We found that RIP2 expression increased upon generation of immunity after oral infection with Helicobacter pylori. Taken together, cell activation with bacterial toxins (LPS, peptidoglycan, lipoteichoic acid), and that these findings support the importance of PGN recognition by Nod1 for instructing the temporal profile of induction differed for each immune cell lineage depending on adaptive immunity, and thereby contributing to successful therapeutic vaccination its involvement in innate or adaptive phases of the immune response. More approaches as well as host defense against bacterial infection. importantly, we found that RIP2 expression increased in peritoneal immune cells during PD-associated peritonitis, and that this up-regulation correlated with clinical outcome. Specifically, early induction of RIP2 expression in peritoneal CD14+ cells correlated rapid resolution of infection, while minimal induction of RIP2 expression correlated with protracted evolution resulting in catheter loss in 36% of the patients. Patients with protracted evolutions also had lower levels of IL-12p40 and higher levels of IL-8 and MCP-1, consistent with a delayed transition from innate to adaptive immunity. Based on our data, we conclude that the up-regulation of RIP2 expression is a useful tool to monitor the transition from innate to adaptive Th1 responses to PD- associated peritonitis, and to predict the clinical outcome of these infections. 10:00 am - 10:30 am AKT activation is required to overcome the effect of Anthrax lethal toxin in human myeloid cells Dennis Ng Dept. of Microbiology and Immunology, UWO Soon D. Ha Dept. of Microbiology and Immunology, UWO Sung O. Kim Dept. of Microbiology and Immunology, UWO Anthrax lethal toxin (LeTx) is believed to be the fatal factor in Bacillus anthracis infections. This lethal disease often progresses despite extensive antibiotic treatments, and a novel therapeutic strategy is warranted. To facilitate development of effective therapies against Anthrax, insight into the pathogenic effects of LeTx is required. Since LeTx primarily targets myeloid immune cells, compromising both innate and adaptive immunity, we investigated long-term effects of LeTx on human myeloid cells. Brief exposure of LeTx to isolated human peripheral blood mononuclear cells (hPMBC) caused permanent inhibition of mitogen activation kinase kinases (MAPKKs) and immune response in both monocytes and non- myeloids cells. We found that the permanent inhibitory effect of LeTx is due to persistent LeTx protease activity in non-proliferating cells, and cells require proliferating capability to overcome the effect. In proliferating human myeloid cell line THP-1 and U937 cells, short exposure of LeTx completely blocked activation of MAPKKs and tumor necrosis factor-a (TNF-a) production up to 4 days. Interestingly, we also found that LeTx completely inhibited the entry into the G2 phase of the cell cycle, however cell cycle could re-initiate after 3 days. Furthermore, we showed that the release of G2 cell cycle blockage required LeTx-induced phospholipase A2- dependent Akt activation, and this Akt activation also promoted survival of the THP-1 cells. Our study suggests that the effects of LeTx in non-proliferating cells can be permanent, and Akt-dependent restoration of immune cells is required for recovering host immune response. http://www.uwo.ca/mni/MNI/IIRF/ 3 2006 Infecion and Immunity Research Forum Poster Presenters Poster # Presenter Title 1 J.N.G Ablack The Role of the pCAF Histone Acetyltransferase in Human Adenovirus E1A Conserved Region Three Dependent Transcription 2 Kemi Adeyanju The contribution of the HIV-1 Tat protein to adverse drug reactions induced by sulphonamides. 3 Khaled Aly Surface localization of the Trans-Zeatin Synthesizing protein (Tzs) is type IV secretion system dependent. 4 Negin Ashki NITRIC OXIDE INDUCES DOSE-DEPENDENT AND REVERSIBLE CHANGES IN THE ELECTROPHYSIOLOGICAL PROPERTIES OF AXONS IN THE GUINEA PIG SPINAL CORD 5 C. Bauer Cigarette Smoke Impairs Induction of Type I Interferons in Primary Lung Fibroblasts 6 W Brintnell Anti-citrulline immune responses in acute and chronic arthritis in DR4 tg mice. 7 Tracy Chew IRF-3 PROTEIN ABUNDANCE IN VERO CELLS UNDERLIES THEIR DIFFERENTIAL RESPONSE TO VIRUS-DERIVED STIMULI . 8 Laura Copeman Immune Responses to Cellular Xenotransplants are Modified by Sertoli Cells 9 Melinda Demendi ELUCIDATION OF THE BIOCHEMICAL FUNCTION OF CJ1123c, A PUTATIVE ACETYL-TRANSFERASE IN CAMPYLOBACTER JEJUNI 10 Tao Dong Succinate Selection for rpoS Mutations in Verocytotoxin-Producing Escherichia coli 11 Todd Fairhead The Role of RIP2 in Vascularized Allograft Rejection 12 R. S. Flannagan Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo 13 Hamed Ghanei Functional characterization of MsbA from Pseudomonas aeruginosa 14 Stephen Hanson Investigating the consequences of VSV-M interaction with mitochondria 15 Nathan Ho The Biosynthesis of 6-deoxy-heptose sugars in Yersinia pseudotuberculosis. 16 Amber Jarvis-Metcalfe Visualization of Xanthomonas campestris pv. vesicatoria infection of tomato using GFP and confocal laser scanning microscopy 17 Katarina Kaluzny INVESTIGATION OF THE WZY ALPHA AND BETA O-ANTIGEN POLYMERASES IN PSEUDOMONAS AERUGINOSA. 18 Mark G. Kirchhof Stomatin-Like Protein 2 Stabilizes TCR-Dependent Signalling Microclusters In Effector T Cells Abstracts to posters can be found online at http://www.uwo.ca/mni/MNI/IIRF/ 4 Poster # Presenter Title 19 Simon Lam A Dual Reporter System for Simultaneous Monitoring of Bacterial Growth and Viability 20 Julie Lamothe Characterization of the mechanism used by Burkholderia cenocepacia to persist within macrophages 21 Dirk Lange MODIFIED HEPTOSE SYNTHESIS IN THE CAMPYLOBACTER JEJUNI CAPSULE AND THEIR INVOLVEMENT IN VIRULENCE 22 Caitlin D. Lemke Characterization of SLP-2 in lymphocyte signaling and activation in vitro and in vivo 23 Matilde Leon-Ponte Serotonin receptor signaling triggers early T cell activation events 24 Theresa Lindhout Contributions of lipopolysaccharide to the motility of Pseudomonas aeruginosa 25 Slade A. Loutet Mechanisms of antimicrobial peptide resistance in Burkholderia cenocepacia 26 Dalam Ly Protection from Type 1 Diabetes by iNKT Cells May Require Interactions with CD4+CD25+ Regulatory T Cells 27 Maloney, K.E. SEARCHING FOR THE FUNCTION OF MGTC USING IN VITRO AND EX VIVO METHODS 28 Kris Marshall Identification and analysis of non-conventional nuclear import signals using Adenovirus E1. 29 Andrew Martins Probiotic-treated Macrophages Release Cytokines Capable of Influencing the Maturation of Dendritic Cells: A Role for G-CSF 30 Megan McGarry Topological mapping and Functional analysis of the E.coli protein WaaL 31 Dana McIvor Altering Monocyte Responses Through Chemokine Modulation. 32 W. Monty McKillop Characterization of CD11d leukocyte integrin expression 33 Alexandra Merkx-Jacques The role of protein glycosylation in the virulence of the gastric pathogens Helicobacter pylori and Campylobacter jejuni. 34 Kanishka Mohib Inhibition of Indoleamine-2,3 dioxygenase (IDO) Activity in Renal Tubular Cells Prevents Renal Ischemia Reperfusion Injury 35 Enayat Nikoopour The evaluation of pro and anti inflammatory cytokine gene polymorphisms in patients with Behcet’s disease 36 Ryan Noyce Characterization of the IRF3-mediated antiviral response to virus particle entry 37 Ximena Ortega A lipopolysaccharide modification gene cluster encoding the synthesis and transfer of 4-amino-arabinose is essential for survival of Burkholderia cenocepacia 39 Kinnari B. Patel Identifying the regions of WbaP critical for function and interaction with O-antigen biosynthesis proteins in Salmonella enterica 40 Peter Pelka Nek9, a NIMA-related kinase is a novel target for adenovirus E1A proteins Abstracts to posters can be found online at http://www.uwo.ca/mni/MNI/IIRF/ 5 Poster # Presenter Title 41 A. K. M. Nur-ur Rahman MOLECULAR BASIS OF T CELL RECEPTOR SELECTIVITY, CROSS-REACTIVITY AND ALLELIC DISCRIMINATION BY A BACTERIAL SUPERANTIGEN: INTEGRATIVE FUNCTIONAL AND ENERGETIC MAPPING OF THE SpeC-Vbeta2.1 MOLECULAR INTERFACE 42 Brad Shrum Magnetic resonance imaging of innocuously labelled dendritic cells in vivo. 43 Vijayakumar Somalinga Role of HP0366 in the secretion and biosynthesis of virulence factors in Helicobacter pylori 44 Kyle Stephenson Vesicular Stomatitis Virus expressing IL-15 as a potential oncolytic virus 45 Patricia L. Taylor Elucidation of the catalytic mechanism of phosphoheptose isomerase (GmhA), a drug target for combating Gram negative bacterial infection. 46 Wendy Teft Inverse agonist activity of soluble CTLA-4 ligands requires oligomerization of this receptor and CD28 expression 47 Tushar Shakya Finding Novel Inhibitors for the Antibiotic Resistance Enzyme APH(3’)-IIIa 48 Christie L. Vermeiren Heme Recognition by a Staphylococcus aureus NEAT Domain 49 Enrique D. Vinés Glu-Arg salt bridges are critical for an interaction between iron- hydroxamate binding protein FhuD2 and the FhuBGC2 ATP-binding cassette transporter in Staphylococcus 50 Costin Vladau Dendritic cell-mediated immune modulation: Targeted gene silencing using siRNA-bearing immunoliposomes 51 Shuang Wang Preclinical efficacy of CD28 signaling inhibitor 6-thio-GTP in prolongation of cardiac allograft survival 52 R. Alejandra Ward Characteristtics of Three Classes of Immunoglobulins from the Avian Egg 53 Alanna Watson Regulation of growth factor-stimulated chemotaxis and chemokinesis of primary murine keratinocytes 54 Erin Westman Biochemical characterization of WbpI, a putative UDP-2,3- diacetamido-2,3-dideoxy-D-glucuronic acid 2-epimerase from Pseudomonas aeruginosa serotype O5 55 Maryam Yeganegi EFFECT OF LACTOBACILLI ON PLACENTAL COX2 AND PGDH EXPRESSION IN LPS-ACTIVATED TROPHOBLASTS 56 Ahmed F. Yousef Wrestling with SUMO: Human Adenovirus E1A’s interaction with the SUMO conjugase UBC9. 57 Caleb CJ Zavitz Lymphocyte responses in human smokers and model animals Abstracts to posters can be found online at http://www.uwo.ca/mni/MNI/IIRF/ 6 2006 Infection and Immunity Research Forum Poster Presentations POSTER #: 1 POSTER #: 3 The Role of the pCAF Histone Acetyltransferase in Surface localization of the Trans-Zeatin Synthesizing Human Adenovirus E1A Conserved Region Three protein (Tzs) is type IV secretion system dependent. Dependent Transcription Khaled Aly Ph.D. Candidate, McMaster University J.N.G Ablack Department of Microbiology and Immunology, University of Christian Baron McMaster University Western Ontario M. Shuen Department of Microbiology and Immunology, University of Western Ontario J.S. Mymryk Departments of Oncology, Microbiology and Immunology, Many Gram-negative pathogens use type IV secretion systems (T4SSs) as mechanisms of virulence in order to deliver effectors into their target hosts. Protein-protein interaction studies University of Western Ontario and cellular localization analysis of different T4SS machineries and substrate proteins are necessary approaches towards resolving the mechanism of substrate delivery. The action of several plant hormones is associated with the virulence of many phytopathogens. The Adenoviruses make up a family of over 100 members that infect a wide range of hosts Agrobacterium tumefaciens nopaline strain C58 encodes a trans-zeatin synthesizing protein including birds, reptiles and mammals. There are 51 serotypes of human adenoviruses (hAd) (Tzs). Tzs is an isopentenyl transferase that is involved in complex metabolic pathways in that are further divided into six sub-groups based on several criteria including oncogenic plants such as the regulation of shoot formation. Here, we show that Tzs interacts with the potential. E1A is the first gene expressed from the adenovirus genome. The largest E1A T4SS component VirB5. In virB5 deletion strain CB1005, Tzs co-fractionation with proteins in mRNA encodes a 289 residue protein. Alignment of the amino acid (aa) sequence of E1A the range of 230-67 kDa was abolished as shown by size exclusion chromatography. Tzs was 289R protein across serotypes reveals four regions of conservation appropriately named detected in sheared and sedimented A. tumefaciens appendages and blebs. Immuno-EM conserved regions 1-4 (CR1-4). Neither E1A, as a whole, or CR3 have any DNA binding or analysis further revealed that Tzs is a surface localized protein and that its localization is T4SS enzymatic activity. The CR3 portion of the hAd-5 E1A protein functions as a potent dependent. Our results show that the assembly of a wild-type T4SS apparatus is required for transcriptional activator and is required to induce expression of viral early genes during the external localization of Tzs and that virB5 deletion strongly altered its membrane infection. Expression of hAd-5 CR3 in the yeast Saccharomyces cerevisiae inhibits growth, as integration. Since Tzs might translocate into plant hosts and undergoes essential function(s) do the corresponding regions of the hAd-3, 4, 9, 12 and 40 E1A proteins, which represent the prior to tumor formation, we hypothesize that in virB5 deletion strain, the abolished virulence of remaining five hAd sub-groups. A. tumefaciens to plant hosts is partly attributed to the altered surface localization of Tzs and Growth inhibition is alleviated by disruption of the SAGA transcriptional regulatory complex, the abnormal fractionation pattern with other bacterial proteins. indicating that CR3 targets the SAGA complex. In yeast, transcriptional activation by the various CR3s regions requires the yGcn5 acetyltransferase component of SAGA. We also demonstrate that the different hAd-5 CR3 regions interact with pCAF, a mammalian orthologue POSTER #: 4 of yGcn5. RNA interference directed against pCAF is being tested to see what effect it has on NITRIC OXIDE INDUCES DOSE-DEPENDENT AND hAd5 CR3 dependent transcriptional activation in mammalian cells. This will demonstrate whether the interaction of CR3 with pCAF contributes to E1A function. Surprisingly, there are REVERSIBLE CHANGES IN THE dramatic differences in transcriptional activation by all the different CR3 regions in mammalian ELECTROPHYSIOLOGICAL PROPERTIES OF AXONS cells when fused to a heterologous DNA binding domain. The requirement for pCAF by the other representative CR3s to activate transcription will also be discussed. Our results provide IN THE GUINEA PIG SPINAL CORD the first comparison of transcriptional activation by the CR3 regions of the E1A proteins of different subgroups and identify pCAF as a new functional target of CR3. Negin Ashki The University of Western Ontario POSTER #: 2 Dr. Keith C. Hayes Lawson Health Research Institute,The University of Western Ontario The contribution of the HIV-1 Tat protein to adverse Dr. Riyi Shi Center for Paralysis Research, Purdue University drug reactions induced by sulphonamides. Kemi Adeyanju University of Western Ontario Increased expression of the inducible and neuronal isoforms of nitric oxide synthase (NOS), and elevated concentrations of nitric oxide (NO) metabolites, are present within the CNS Krizova A. University of Wetern Ontario following neurotrauma and are implicated in the pathogenesis of the accompanying neurologic Rieder M. Robarts and UWO deficits. We tested the hypothesis that elevated extracellular concentrations of NO introduced by the donor Spermine NONOate, induce reversible axonal conduction deficits in neurons of Dekaban G. A. Robarts and UWO the guinea pig spinal cord. The compound action potential (CAP) and compound membrane potential (CMP) of excised ventral cord white matter were recorded before, during, and after, Background: HIV infection is characterized by severe immune dysfunction resulting in a bathing the tissue (30 min) in varying concentrations (0.25-3.0 mM) of Spermine NONOate. multitude of opportunistic infections, the most common of which is Pneumocystis pneumonia. The principal results were a rapid onset, dose-dependent, reduction in amplitude of the CAP Therapy for this lung infection requires use of the combination drug trimethoprim- (p<0.05) accompanied by depolarization of the CMP during NO exposure. These effects were sulphamethaxazole, the sulpha component of which is associated with a high incidence of largely reversible on washout, at low concentration of the donor (0.5 mM), but were only adverse drug reactions. When compared to the general population, adverse drug reactions to partially reversed at higher concentrations. Changes in the electrophysiological properties SMX are 10-fold more common among people living with HIV. Our previous studies have were not evident when the donor had been a priori depleted of NO. The results extend implicated the HIV regulatory protein Tat together with SMX-HA (sulphamethaxazole- previous reports that NO induces reversible axonal conduction deficits. They provide new hydroxylamine), a reactive metabolite of sulphamethaxazole, in the pathogenesis of these evidence of dissociation of the effects of NO on CAP and CMP during washout and after adverse drug reactions. prolonged exposure to the donor. They add support to the emerging concept that immune- Method: The first exon of Tat consisting of 72 amino acids was fused to green fluorescent mediated axonal conduction failure contributes to reversible neurologic deficits following protein. This expression cassette was subsequently introduced into an inducible vector used to neurotrauma and aid in understanding clinical phenomena such as spinal cord shock and establish a stably-transfected Jurkat T cell line. Using flow cytometry and western blots, the neurologic recovery. cell line was used as a model to assess toxicity and mechanism of cell death upon exposure to SMX-HA. The correlation between changes in glutathione (GSH) and sensitivity to SMX-HA- mediated cell death in the presence of TatGFP expression was measured by a monobromobimane fluorimetric assay. Results: TatGFP induced to different levels showed an increase in cell death on exposure to increasing concentrations of SMX-HA (p<0.05). This was accompanied by the release of cytochrome-c and caspase-3 activation. However, there was no appreciable difference in either the GSH levels or the GSSG/ GSH ratio when the expression of TatGFP was induced, regardless of the presence or absence of SMX-HA treatment. Conclusion: The data presented here suggests that Tat potentiates, in a dose-dependent manner, the sensitivity of T cells to SMX-HA-mediated cell death. Hence Tat expression during the progression of the HIV infection could predispose the infected individual to ADRs related to SMX. This appears to be due to altered cellular responses to SMX. http://www.uwo.ca/mni/MNI/IIRF/ 7 POSTER #: 5 POSTER #: 6 Cigarette Smoke Impairs Induction of Type I Anti-citrulline immune responses in acute and Interferons in Primary Lung Fibroblasts chronic arthritis in DR4 tg mice. C. Bauer Centre for Gene Therapeutics and Department of Pathology W Brintnell Department of Microbiology and Immunology and and Molecular Medicine Department of Medicine, Division of Rheumatology, University of Western Ontario K. Hornby Centre for Gene Therapeutics and Department of Pathology and Molecular Medicine D A Bell Department of Microbiology and Immunology and Department of Medicine, Division of Rheumatology, C. Zavitz Centre for Gene Therapeutics and Department of Pathology University of Western Ontario and Molecular Medicine A M Jevnikar Department of Microbiology and Immunology and M. Stampfli Centre for Gene Therapeutics and Departments of Department of Medicine, Division of Nephrology, University Pathology and Molecular Medicine,and Medicine of Western Ontario K. Mossman Centre for Gene Therapeutics and Departments of W H Robinson Division of Immunology and Rheumatology, Department of Pathology and Molecular Medicine, and Biochemistry and Medicine, Stanford University School of Medicine Biomedical Sciences Multiple viruses have been implicated in the exacerbations associated with cigarette E Cairns Department of Microbiology and Immunology and Department of Medicine, Division of Rheumatology, smoking related diseases such as Chronic Obstructive Pulmonary Disease (COPD). University of Western Ontario Therefore, we hypothesized that cigarette smoke weakens innate immune responses against viral agents; specifically, responses mediated by interferon regulatory factor 3 (IRF-3) and the Background: Research in our laboratory focuses on Rheumatoid Arthritis (RA), an type I interferon pathway. autoimmune disease characterized by chronic inflammation in the joint which leads to cartilage Human embryonic lung fibroblasts (HEL) were cultured in cigarette smoke-conditioned destruction and bone injury. RA is strongly associated with the expression of MHC class II medium for 2 hours. Trypan blue dye exclusion and MTT assays established that cells were molecules (eg. DR4) containing the consensus amino acid sequence QKRAA/QRRAA called viable and metabolically active. Cigarette smoke-conditioned medium dose-dependently the shared epitope (SE). Anti-citrulline immune responses are the most specific serologic inhibited induction of an antiviral state in fibroblasts following delivery of polyI:C, a structural marker for this disease. These responses target peptides in which arginine has been mimic of viral dsRNA and a toll-like receptor 3 (TLR-3) ligand. Transfer of culture supernatants postranslationally modified to citrulline by an enzyme peptidylarginine deiminase (PAD). We to Vero cells, in a standard plaque reduction assay, showed that significantly less type I have been using humanized DR4 tg mice (on the C57BL/6 (B6) background) to study the role interferons were produced in the context of cigarette smoke. Furthermore, cigarette smoke- of citrullinated proteins in RA pathogenesis. We have shown that citrullinated fibrinogen conditioned medium significantly attenuated expression of the interferon-stimulated genes ISG- induces T and B immune responses in DR4 tg mice and 40% of these mice also develop 15 and IRF-7, and almost entirely abrogated nuclear factor kappa B nuclear translocation chronic arthritis. The B6 wild type (wt) mice never developed arthritis following immunization following polyI:C treatment. with this citrullinated antigen. Other groups have demonstrated that intra-articular (IA) injection The effects that cigarette smoke had on antiviral responses was reversible; regardless of of Streptococcal cell wall (SCW) induces expression of PAD, citrullination of synovial antigens treatment with smoke, we observed similar antiviral responsiveness 24 hours following removal and acute arthritis in the B6 wt mice. of the smoke-conditioned medium. Understanding the impact of cigarette smoke on interferon Objective of this study was to examine the immune response to and arthritogenic potential immuno-biology and its consequences to antiviral immunity may advance our understanding of of citrullinated autoantigens produced following intra-articular injection of SCW in DR4 tg mice. the increased prevalence of viral infections in COPD. Methods: DR4 tg and B6 wt mice were injected with either 25 g of SCW or 5.0 l of PBS into opposite knee joints and monitored for signs of acute and chronic This study was supported by CIHR, and Philip Morris USA Inc. and Philip Morris arthritis. The B and T cell immune responses, and joint pathology were examined in these mice International at various times post-SCW injection. A separate group of DR4 tg mice was immunized twice with 100 g citrullinated human fibrinogen (CithFib) in CFA and IFA at day 0 and 21 respectively. At day 31 these mice also received a single IA injection of either 25 g of SCW antigen or 5.0 l of PBS into opposite knee joints. Mice were monitored for signs of acute and chronic arthritis, sacrificed at day 70 and their anti-citrulline responses determined. Results: Intra-articular injection of SCW caused increased PAD expression, deposition of citrulline and acute arthritis in both DR4 tg and B6 mice. However, T and B cell immune responses to citrullinated fibrinogen peptide (CitFib79-91) occurred exclusively in DR4 tg mice. The course of the acute arthritis remained unaltered in DR4 tg mice immunized with citrullinated human fibrinogen prior to intra-articular injection of SCW antigen. Within 10- 14 days of cessation of signs of acute arthritis approximately 70% of CithFib and SCW injected DR4 tg mice developed chronic arthritis in the ankle joints similar to that of CithFib induced arthritis. Upon sacrifice B cell immune responses to the immunizing antigen and citrullinated synovial antigens were demonstrated in these mice. Conclusions: Intra-articular injection of SCW antigen induces an acute inflammatory arthritis resulting in citrullination of synovial proteins in DR4 tg and B6 wt mice but B and T cell immune responses to a citrullinated fibrinogen peptide were observed only in DR4 tg mice. The combination of CithFib and SCW was more effective than CithFib alone in inducing arthritis in DR4 tg mice. The mechanisms responsible for this have yet to be determined. POSTER #: 7 IRF-3 PROTEIN ABUNDANCE IN VERO CELLS UNDERLIES THEIR DIFFERENTIAL RESPONSE TO VIRUS-DERIVED STIMULI . Tracy Chew McMaster University Meaghan Hancock McMaster University Brian D. Lichty McMaster University Karen L. Mossman McMaster University Interferon Regulatory Factor 3 (IRF-3) is a transcription factor that is critical for the production of type I interferon (IFN) and crucial to the innate immune response against viral infection in non-immune cells. The immune response to virus particle entry in the absence of viral gene expression has been shown to require IRF-3 but not IFN production. In addition, IRF-3 has demonstrated tumor suppressor activity in vitro and in vivo. Thus, IRF-3 activity has important implications in both antiviral and anticancer immunity. Relatively little is known about the regulation of IRF-3 signalling in the absence of IFN activity. In addition, distinct cellular components are required for IRF-3 activation depending on the nature and origin of the stimulus. In contrast to classically responsive cells such as primary fibroblasts, Vero cells, derived from monkey kidney epithelia, fail to respond in a standard antiviral assay to both UV- inactivated virus particles (UV virus) and polyinosinic:polycytidylic acid (pIC), a dsRNA mimetic. Analysis of IRF-3 in these cells revealed differences in both amino acid sequence and protein abundance compared to human cell lines tested. Interestingly, overexpression of IRF-3 derived from either human or Vero cells restores antiviral gene expression and functional protection from virus challenge in the context of pIC but not UV virus. This data demonstrates an impaired antiviral response in Vero cells over and above their inability to produce IFN, and suggests that abundance levels of IRF-3 underlie its activation and antiviral activity in response to pIC. In addition, a novel cellular component is required for antiviral signalling against non-replicating enveloped viruses that is inactive or absent in this cell line. http://www.uwo.ca/mni/MNI/IIRF/ 8 POSTER #: 8 POSTER #: 10 Immune Responses to Cellular Xenotransplants are Succinate Selection for rpoS Mutations in Modified by Sertoli Cells Verocytotoxin-Producing Escherichia coli Laura Copeman Department of Pathology, University of Western Ontario Tao Dong McMaster University and Transplantation, Robarts Research Institute Herb Schellhorn McMaster University David White Transplantation, Robarts Research Institute and Department of Pathology, University of Western Ontario Verocytotoxin-producing Escherichia coli (VTEC) strains are enteric pathogens that can cause severe human syndromes including diarrhea, hemorrhagic colitis and hemolytic colitis. A Purpose: This study was designed to evaluate the humoral immune responses to general stress response regulator, RpoS has been recently implicated in pathogenesis of xenotransplants of neonatal porcine islets, Sertoli cells or a mixture of these two cell types in VTEC as it regulates expression of LEE pathogenic island. Surprisingly, in view of its the rat. A comparison of the immune responses generated when adult Sertoli cells were used importance in cell survival, rpoS polymorphisms are common among VTEC strains and the in place of neonatal Sertoli was also done. reason for this remains unknown though it may be due to selection for loss of function. We Methods: Female Lewis rats were transplanted with 400,000 neonatal or adult porcine have previously found that succinate selects for loss of RpoS in E. coli laboratory strains. In Sertoli cells, 2000 neonatal porcine islet equivalents or a mixture of these two cell types. this study, we tested if this selection is also operant in VTEC strains. Ten representative VTEC Transplants were done either under the kidney capsule or into neovascularized subcutaneous strains from 5 different pathotypes were tested for growth on succinate minimal media. Mutants autologous collagen pouches that had been previously created by implanting polypropylene (Suc++) that formed large colonies could be isolated from 7 of the 10 VTEC strains while the mesh chambers with central Teflon rods 4 weeks pretransplant. No immunosuppression was other 3 strains grew uniformly well on succinate so that no Suc++ mutants were selected. The given at any time to the recipient animals. Weekly serum samples were collected for 5 weeks rpoS presumptive status of Suc++ mutants was determined by catalase plate assay (catalase and rat anti-pig IgG responses were measured using flow cytometry. expression is highly RpoS-dependent). Most of the Suc++ mutants were defective for catalase All test sample fluorescence measurements were compared to a control serum and data indicating RpoS was likely to be impaired in these mutants. This was confirmed by sequencing analysed as a ratio of maximum fluorescence of the test sample to maximum fluorescence of the rpoS region of 3 Suc++ mutants deficient in catalase from each VTEC strain. Independent the control. mutations identified in rpoS included transitions, transversions, single and multiple base Results: There was no significant difference in the rat anti-pig IgG response when islets deletions and duplications resulting in impaired RpoS function. Western blot analysis revealed were transplanted into collagen pouches compared to under the kidney capsule (peak immune that RpoS was diminished in derivative Suc++ mutants. Consequently, production of RpoS- response ratio (pirr) kidney 4.758±1.523 vs pirr pouch 4.089±0.654). There was a significant regulated proteins including KatE (HPII) and AppA were substantially reduced. In conclusion, decrease in this immune response when neonatal Sertoli cells were transplanted into these two consistent with our findings in laboratory K12 strains, rpoS mutants of VTEC strains exhibited locations (pirr kidney 5.375±0.874 vs pir pouch 1.151±0.783, p<0.01). Cotransplanting growth advantage and was selected for on succinate media. This selection may contribute to neonatal Sertoli cells with islets into collagen pouches did decrease the generated immune virulence and adaptation in E. coli VTEC strains. response compared to transplanting islets alone however this decrease failed to achieve statistical significance (pirr neonatal Sertoli cells+islets 2.158±1.137). Rats transplanted with adult Sertoli cells alone into collagen pouches failed to POSTER #: 11 generate a measurable immune response compared to their pretransplant levels (pretransplant ratio 0.049±0.011 vs pirr 0.0536±0.009). When adult porcine Sertoli cells were cotransplanted The Role of RIP2 in Vascularized Allograft Rejection with islets they were able to significantly decrease the generated immune response compared with cotransplantation with neonatal Sertoli cells (pirr adult Sertoli cells + islets 0.244± 0.139, p<0.0001) Todd Fairhead University of Western Ontario Conclusion: In the rat, transplanting porcine Sertoli cells into subcutaneous autologous collagen pouches results in a decrease in the amount of anti-pig IgG compared to Dameng Lian University of Western Ontario transplanting under the kidney capsule. Adult porcine Sertoli cells elicit no immune response when they are transplanted into collagen pouches. Further, the immune response to porcine Robert Zhong University of Western Ontario islets transplanted into collagen pouches is significantly decreased when adult porcine Sertoli cells are cotransplanted. Studies of cotransplanting adult porcine Sertoli cells and islets in Joaquin Madrenas University of Western Ontario diabetic rats are in progress. Allograft rejection remains a major obstacle in renal transplantation, mainly due to cellular rejection which is characterized by the development of a Th1 immune response. As such, POSTER #: 9 modulating the Th1 immune response may protect the allograft. To address this issue, we have focused on the putative ser/thr kinase, RIP2. The RIP2 knockout mice show deficiencies in ELUCIDATION OF THE BIOCHEMICAL FUNCTION OF both innate and adaptive immunity in their ability to mount a Th1 immune response. Based on CJ1123c, A PUTATIVE ACETYL-TRANSFERASE IN the Th1 deficiency of RIP2 knockout mice, we hypothesized that these mice would not reject a vascularized allograft. CAMPYLOBACTER JEJUNI To test this, we used a well characterized model of heart allograft rejection. Intra-abdominal heterotopic C3H mouse hearts (H-2k) were transplanted into either wild-type or RIP2-deficient C57BL/6 mice (H-2b). Beating of the grafted heart was monitored by abdominal palpation. Melinda Demendi Microbiology and Immunology, University of Western Tissue sections of the allograft were collected for pathology and further study. Microscopic Ontario sections were examined in a blinded fashion by a pathologist for severity of rejection. Carole Creuzenet Microbiology and Immunology, University of Western We found that there was no difference in allograft survival between wild-type versus RIP2- Ontario deficient recipient mice. C3H allografts survived a median of 7 days in wild-type recipients versus 7 days in RIP2-deficient recipients. Similarly, RIP2-deficient hearts transplanted into C3H recipients rejected at the same rate as C57BL/6 wild-type hearts transplanted into C3H recipients. Pathology of the rejecting C3H allografts was characterized by vasculitis, intravascular thrombosis, and interstitial hemorrhage. In conclusion, RIP2-deficient mice reject vascularized allografts as efficiently as wild-type mice. Acute vascular rejection appears to be integral to rejection in RIP2-deficient mice. We are presently characterizing the alloresponse in RIP2-deficient mice by examining cytokine expression profiles and antibody response in the rejecting graft in both wild-type and RIP2- deficient recipients. These experiments will help clarify the role of RIP2 and the Th1 immune response in allograft rejection. http://www.uwo.ca/mni/MNI/IIRF/ 9 POSTER #: 12 POSTER #: 14 Burkholderia cenocepacia requires a periplasmic Investigating the consequences of VSV-M HtrA protease for growth under thermal and osmotic interaction with mitochondria stress and for survival in vivo Stephen Hanson McMaster University, Centre for Gene Therapeutics R. S. Flannagan Infectious Diseases Research Group, Department of Brian Lichty McMaster University, Department of Pathology and Microbiology and Immunology, Siebens-Drake Research Molecular Medicine Institute, University of Western Ontario, London, Ontario, Canada D. Aubert Infectious Diseases Research Group, Department of Microbiology and Immunology, Siebens-Drake Research Vesicular Stomatits Virus (VSV) has recently been identified as a potential oncolytic Institute, University of Western Ontario, London, Ontario, therapeutic as a result of deficiencies in innate antiviral pathways in tumour cells. VSV is an Canada enveloped virus with a negative sense single stranded RNA genome coding for five proteins. The matrix protein is thought to mediate most of the virus-host cell interaction. The C. Kooi Department of Microbiology and Infectious Diseases, pathogenicity of VSV has mostly been attributed to the ability of the matrix protein to block University of Calgary, Calgary, Alberta, Canada nucleo-cytoplasmic export of host cell mRNAs through its ability to bind NUP98. We have P. A. Sokol Department of Microbiology and Infectious Diseases, recently identified a novel function of the matrix protein as it localizes to mitochondria upon University of Calgary, Calgary, Alberta, Canada infection or transfection. I have identified the T67M mutation in VSV-M that abrogates this targeting and have generated a recombinant VSV containing this mutation. This mutant virus M. A. Valvano Infectious Diseases Research Group, Department of appears to more quickly induce cytopathic effect (CPE) in mouse embryonic fibroblasts. I plan Microbiology and Immunology, and Medicine, Siebens- to investigate the potential mechanisms behind this increased cytopathogenicity. Drake Research Institute, University of Western Ontario, London, Ontario, Canada Burkholderia cenocepacia, a member of the B cepacia complex, is an opportunistic POSTER #: 15 pathogen causing serious infections in patients with cystic fibrosis. We have identified a six- The Biosynthesis of 6-deoxy-heptose sugars in gene cluster located on chromosome 1 that encodes a putative two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829), hypothesized to play a Yersinia pseudotuberculosis. role in the B. cenocepacia stress response. Reverse transcriptase-PCR analysis of these six genes confirmed they are co-transcribed and comprise an operon. Genes in this operon, including htrA were insertionally inactivated by homologous recombination. Genetic analyses Nathan Ho University of Western Ontario and complementation studies revealed that HtrABCAL2829 was required for growth of B. Carole Creuzenet University of Western Ontario cenocepacia upon exposure to osmotic (NaCl/KCl) and thermal stress (44oC). In addition, alanine substitution of the serine residue (S245A) within the active site and deletion of the HtrABCAL2829 PDZ domains demonstrated that they are required for protein function. The HtrABCAL2829 also localizes to the periplasmic compartment as shown by western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung Lipopolysaccharide (LPS) are complex surface polysaccharides that are essential virulence infection, we also demonstrate that inactivation of the htrA gene is associated with a bacterial factors for numerous pathogenic bacteria. The LPS of Yersinia pseudotuberculosis contain survival defect in vivo. Together, our data demonstrate that HtrABCAL2829 is a virulence unusual 6-deoxy-heptoses. Since these sugars are found in several pathogenic bacteria, but factor in B. cenocepacia. not in mammals, the enzymes necessary for their synthesis could be targets for the development of novel therapeutic agents or of diagnostic tests usable in food or medical samples. Little is known about the biosynthesis of these sugars, however genes encoding a POSTER #: 13 putative GDP-D-glycero-D-mannoheptose dehydratase (DmhA), and the associated reductase (dmhB) are thought to be involved in 6-deoxy-heptose biosynthesis as they have been Functional characterization of MsbA from identified in a cluster that contains additional O-antigen biosynthetic genes. Pseudomonas aeruginosa In Y. pseudotuberculosis, 6-deoxy-heptose sugars are branching off the main O-antigen chain. We hypothesize that dmhA and dmhB perform a critical role in synthesizing these unique sugars for incorporation into the LPS O-antigen, and that dmhA or dmhB mutants would Hamed Ghanei University of Guelph have a different O-antigen composition. We have disrupted dmhA and dmhB individually in Y. pseudotuberculosis O:2a and observed through Silver Staining an alteration in O-antigen Priyanka D. University of Guelph characteristics. To investigate whether the O-antigen composition is altered in both mutants, Abeyrathne we assessed recognition of LPS by serotype O:2 specific antibodies and have observed a decrease in reactivity to the antibodies in our mutants by western blot. We plan to analyze the Joseph S. Lam University of Guelph sugar components by HPLC to verify the change in LPS O-antigen structure. Mass spectrometry and NMR will give us insight into the specific changes in O-antigen structure. The results indicate that these genes play a role in O-antigen synthesis and show promise as diagnostic indicators for bacterial contamination. Proteins of the ATP-binding cassette (ABC) transporter superfamily have received considerable attention in recent years as they have been shown to be associated with multidrug resistance. These ABC proteins hamper effective treatment of patients with infectious POSTER #: 16 diseases and cancer, by actively transporting a wide range of structurally distinct substrates Visualization of Xanthomonas campestris pv. out of the cell. MsbA is an ABC transporter that has been shown to be essential in most Gram- negative species. vesicatoria infection of tomato using GFP and MsbA behaves as a flippase for the transport of lipid A-core portion of lipopolysaccharide confocal laser scanning microscopy (LPS) across the inner membrane as well as a drug-efflux pump for erythromycin. We have identified an open reading frame, PA4997, from the P. aeruginosa PAO1 genome that shares 65% sequence similarity to msbA from E. coli (msbAEc). To characterize the function of the Amber Jarvis- University of Western Ontario putative msbA from P. aeruginosa (msbAPa), we cloned the gene by an PCR-based approach. Several attempts to produce a knockout mutant of msbAPa were not successful, insertion of a Metcalfe gentamicin cassette to disrupt could only occur if a wild-type copy of msbAPa is provided in trans indicating that this gene is indeed essential. Interestingly, msbAEc could not substitute Diane Cuppels Agriculture and Agri-Food Canada for msbAPa, suggesting that there are differences between the two genes or their products. MsbAPa was overexpressed and purified, a portion of which was reconstituted in liposomes, and the two forms of MsbAPa were assayed for their ATPase activity. The kinetics of ATPase activity of MsbAPa versus substrate concentration fitted well with Michaelis-Menten curves. Bacterial spot, a destructive disease of tomato and pepper plants, is caused by The effect of LPS on ATPase activity of MsbAPa was monitored in purified and reconstituted Xanthomonas campestris pv. vesicatoria (Xcv). Symptoms include leaf spotting and yellowing, MsbAPa. Our results show that wild-type core-lipid A is a potent stimulator of ATPase activity defoliation, and severely-spotted fruit. Xcv strains possess hrp genes which encode a type III with a half stimulatory concentration of 2 µM. Furthermore, our data also show that the secretion system that mediates delivery of bacterial effector proteins into the host cell and are phosphate moieties of LPS could play an important role in this stimulation of ATPase activity of required for pathogenesis. It is not known how and where Xcv colonize tomato seed or if the MsbAPa. hrp genes are expressed during seed development and germination. Promoter-Probe Fusions (PPFs) of the hrpB promoter and gfp gene were constructed to study hrp gene expression during seed/plant development. A plasmid conferring constitutive GFP expression also was constructed and moved into representative Xcv strains. The expression of GFP by PPF- containing bacteria was confirmed by using confocal laser scanning microscopy (CLSM) to examine 48-hr cultures grown on the hrp-inducing medium XVM2. The GFP-tagged strains were followed in planta on tomato leaves for 14 days. Time course experiments with seeds that were infested with GFP-labelled bacteria are in progress to determine the pattern and distribution of hrp gene expression as well as the localization of Xcv within the seed during germination. The induction of these genes will be verified by RT-PCR. http://www.uwo.ca/mni/MNI/IIRF/ 10 POSTER #: 17 POSTER #: 19 INVESTIGATION OF THE WZY ALPHA AND BETA O- A Dual Reporter System for Simultaneous ANTIGEN POLYMERASES IN PSEUDOMONAS Monitoring of Bacterial Growth and Viability AERUGINOSA. Simon Lam Katarina Kaluzny University of Guelph Dr. Jana Jass Dr. Priyanka D. University of Guelph Abeyrathne Dr. Joseph S. Lam University of Guelph Bacterial growth is traditionally measured by optical density and bacterial viability by colony forming units (CFU) or fluorescent staining techniques. We have designed a dual reporter system that can simultaneously monitor both bacterial growth and viability within the same culture. Bacterial growth is monitored by the constitutive production of monomeric Red B-band, the serotype-specific form of lipopolysaccharide (LPS) produced by Pseudomonas Fluorescent Protein (mRFP) which remains fluorescent even after the bacteria is no longer aeruginosa is crucial to the proliferation and virulence of this bacterial species. The assembly viable. Viability is separately monitored by the constitutive expression of the Lux genes of B-band LPS involves several proteins including the O-antigen polymerase Wzyα, which links (LuxCDABE cassette) which results in ATP dependent bioluminescence. Upon death of the the O-antigen repeat units. Since the trisaccharides of the O-antigen units in serotype O2 and bacterium, ATP is no longer available and bioluminescence stops, although mRFP continues to O16 are connected by a β-glycosidic linkage, we hypothesized that a wzyβ gene that is derived fluoresce. Using a combined luminometer/flurometer, both can be monitored simultaneously on from D3 bacteriophage is present in the chromosome of these two serotypes and localized microtitre plates. In addition, the promoter controlling mRFP can be easily exchanged to outside the B-band biosynthesis cluster. Using primers designed from the D3-phage genome monitor the expression of other genes, such as virulence factors, without affecting viability sequence and primer walking, we have amplified, cloned and sequenced wzyβ in measurements. This construct represents a versatile method of monitoring both bacterial Pseudomonas aeruginosa serotype O2 and O16. The O2 and O16 wzyβ genes are identical, viability and the expression of virtually any gene of interest in situ. and they share 87.42% sequence identity to that of wzyβ from the D3 phage. The P. aeruginosa O2/O16 wzyβ gene is predicted to encode a protein with a size of 41.8 kDa, and its primary amino acid sequence is 90.41% identical and 97.15% similar to D3 Wzyβ, which is POSTER #: 20 42.6 kDa. An O16 wzyβ chromosomal knockout mutant was made, and the LPS produced by Characterization of the mechanism used by the mutant is devoid of B-band O antigen. Interestingly, when both wzyα and wzyβ chromosomal knockout mutants were complemented with wzyβ in trans, production of β-linked Burkholderia cenocepacia to persist within O antigen was restored. These results confirm that Wzyβ from P. aeruginosa is a functional O- macrophages antigen β-polymerase and an orthologue of the previously characterized D3 phage β-O- polymerase. Since wzyα is localized within the B-band cluster of serotypes O2 and O16, we were curious whether this gene is functional or behaves like a pseudogene. Results from RT- Julie Lamothe Department of Microbiology and Immunology, Siebens PCR showed that wzyα in both O2 and O16 were expressed. Since both O2 and O16 Drake Research Institute, Schulich School of Medicine, serotypes have β-linked O antigen, a Wzyα inhibitor is likely present in the chromosomes of University of Western Ontario these serotypes. Further analysis, such as PCR amplification or genomic DNA sequencing, is predicted to uncover a Wzyα inhibitor, likely a homologue of Iap, the D3 phage-encoded Kassidy K Huynh Division of Cell Biology, Research Institute, Hospital of Sick Children, University of Toronto inhibitor previously characterized by our group. The data from this study clearly point to the contribution of bacteriophage to the diversity of LPS biosynthesis and assembly in P. Sergio Grinstein Division of Cell Biology, Research Institute, Hospital of Sick aeruginosa. Children, University of Toronto Miguel A. Valvano Department of Microbiology and Immunology, Siebens POSTER #: 18 Drake Research Institute, Schulich School of Medicine, University of Western Ontario Stomatin-Like Protein 2 Stabilizes TCR-Dependent Signalling Microclusters In Effector T Cells Cystic fibrosis (CF) is the most common lethal autosomal recessive disease in the Caucasian population affecting approximately 70,000 individuals worldwide. Chronic bacterial infections of the lungs leading to sustained inflammation and tissue deterioration are the major Mark G. Kirchhof FOCIS Centre for Clinical Immunology and causes of mortality and morbidity in the CF community. Recently, Burkholderia cepacia Immunotherapeutics, Robarts Research Institute, The complex (BCC) isolates have emerged as important opportunistic pathogens in CF patients. London Health Sciences Centre, and The University of BCC members are multi-drug resistant bacteria and are rarely cleared from CF airway. In Western Ontario addition, patients infected with BCC isolates present a more rapid decline of lung function than patients infected with other CF opportunistic pathogens. To date very little is known about the Luan A. Chau FOCIS Centre for Clinical Immunology and virulence factors involved in the persistence of the bacteria within the CF airway. Our Immunotherapeutics, Robarts Research Institute, The laboratory and others have previously shown that BCC isolates can persist within phagocytes. London Health Sciences Centre, and The University of This study focuses on characterizing the strategy by which B. cenocepacia, a member of the Western Ontario BCC, survives within macrophages. Using different fluorescent probes and immunolabelling, Peter J. Darlington FOCIS Centre for Clinical Immunology and we show that B. cenocepacia survive within poorly acidified vacuoles that delay the Immunotherapeutics, Robarts Research Institute, The accumulation of the late endosomal/lysosomal marker LAMP-1. We also demonstrate that the London Health Sciences Centre, and The University of B. cenocepacia-containing vacuoles (BcCV) avoid fusion with lysosomes but maintain an Western Ontario interaction with the incoming endosomal pathway. These results demonstrate that B. cenocepacia actively modulate the maturation of their vacuole. Our data suggest that BcCVs Kamilia Rizkalla FOCIS Centre for Clinical Immunology and experience a delay in their progression into phagolysosomes. It is conceivable that such a Immunotherapeutics, Robarts Research Institute, The delay is advantageous for the bacteria, enabling them to activate genes that could confer London Health Sciences Centre, and The University of resistance to the hostile environment of lysosomes and may explain the persistence of the Western Ontario bacteria within the CF lungs. Joaquin Madrenas FOCIS Centre for Clinical Immunology and Immunotherapeutics, Robarts Research Institute, The London Health Sciences Centre, and The University of Western Ontario T cell activation involves the formation of a highly organized interface between the T cell and the antigen-presenting cell (APC) known as the immunological synapse (IS). The signaling cascades initiated upon TCR engagement require coordinated recruitment, assembly and stabilization of signaling complexes or signalosomes that provide the sustained signaling needed for full T cell activation. How these signalosomes are assembled remains unknown. In order to identify the molecular machinery involved, we performed a proteomic analysis of lipid rafts from T cells during TCR signaling. This led us to the identification of stomatin-like protein 2 (SLP-2) as a candidate organizer of TCR-dependent signalosomes. SLP-2 is a member of the highly conserved stomatin family of proteins that also include stomatin, SLP-1, and SLP-3. The function of stomatins is unknown, but has been linked to the organization of the peripheral cytoskeleton and the function and structure of ion channels and mechanosensation receptors. Here, we report that stomatin-like protein-2 (SLP-2) is a key element for signalosome assembly and stabilization by linking the individual components of the signaling microclusters to the actin- cytoskeleton. This role is particularly apparent in effector T cells. Consistent with this finding, up-regulation of SLP-2 expression is observed in clinical conditions of lymphocyte activation whereas knocking-down SLP-2 expression in human effector T cells correlates with loss of sustained TCR signalling and down-regulation of effector responses. Our current evidence leads us to propose that SLP-2 is a key structural protein within the T cell that contributes to the assembly of the TCR signalosome and sustains signaling from these complexes. http://www.uwo.ca/mni/MNI/IIRF/ 11 POSTER #: 21 POSTER #: 23 MODIFIED HEPTOSE SYNTHESIS IN THE Serotonin receptor signaling triggers early T cell CAMPYLOBACTER JEJUNI CAPSULE AND THEIR activation events INVOLVEMENT IN VIRULENCE Matilde Leon- Robarts Research Insitute, London, ON Dirk Lange Department of Microbiology & Immunology, University of Ponte Western Ontario Dinath Ratnayake Department of Microbiology & Immunology, University of Gerard P Ahern Georgetown University, Washington DC, USA Western Ontario Peta O'Connell University of Western Ontario, Robarts Research Institute Julie Lamothe Department of Microbiology & Immunology, University of Western Ontario Miguel Valvano Department of Microbiology & Immunology, University of Serotonin (5-HT) is a neurotransmitter and vasoactive amine involved in the regulation of Western Ontario different physiological functions such as sleep, appetite, and behaviour. 5-HT has been shown to have immunomodulatory effects when released by dendritic cells, platelets and mast cells at Carole Creuzenet Department of Microbiology & Immunology, University of site of injury and inflammation, or from sympathetic nerves that innervate lymphoid tissues. Western Ontario Although T cells have been reported to respond to 5-HT and to express different types of the G- Campylobacter jejuni infections are the leading cause of enteritis world-wide, and have been protein-coupled 5-HT receptors, the precise role of 5-HT in T cell activation and function is implicated in Irritable Bowel Disease (IBD) and Immunoproliferative Small Intestinal Disease unclear. We tested the hypothesis that T cells express functional 5-HT receptors and that 5- (ISID). The endemic character of C. jejuni infections in developing countries, and the HT signaling may promote T cell activation processes. emergence of antibiotic resistant clinical isolates of C. jejuni emphasize the need for the First, we demonstrated that mouse splenic T cells express mRNA for the type 7 5-HT (5-HT7) discovery of new potential therapeutic targets. C. jejuni expresses surface capsular receptor and levels of this protein were up-regulated upon T cell activation. Also T cell polysaccharides. A role for the capsule has been established previously in the adhesion and activation leads to expression of 5-HT1B and 5-HT2A receptors. Further, using qPCR we invasion of intestinal epithelial cells, which may be essential for the pathogenesis of C. jejuni. showed that activation lead to a -30 fold increase in mRNA for the 5-HT synthetic enzyme, Furthermore, C. jejuni has the ability to survive within macrophages, possibly causing tryptophan hydroxylase type 1(TPH-1). 5-HT was visualized within activated T cells by sustained macrophage activation. The resulting inflammatory responses triggered by C. jejuni immunofluorescence microscopy, and this monoamine was detected in the supernatants of may contribute to the pathogenesis of IBD and ISID. The aim of this study is to identify cultured naïve and activated T cells by ELISA. Exogenous 5-HT was found to activate the components of the capsular polysaccharide of C. jejuni and to establish their role in extra cellular signal-regulated kinase (ERK) and NF-kB pathways in naïve T cells. Addition of pathogenicity. a 5-HT7 specific antagonist was capable of fully inhibiting ERK phosphorylation. Inhibition of Modified heptoses, unique to C. jejuni and few other intestinal pathogens, are major the autocrine synthesis of 5-HT in vivo with para-chlorophenylalanine (PCPA), a specific components of the C. jejuni capsule. From the C. jejuni cluster of capsular biosynthesis genes, inhibitor of TPH-1, resulted in decreased levels of T cell proliferation upon CD3 ligation in vitro. we have identified three candidates, Cj1427, Cj1428, and Cj1430, which encode proteins that Furthermore, immunolabeling and flow cytometry revealed that expression of the IL-2Rα may be involved in the biosynthesis of capsular modified heptose sugars. The genes of chain was significantly inhibited in both CD4+ and CD8+ T cells from PCPA-treated mice. interest have been insertionally inactivated and the mutants phenotypically characterized for Addition of exogenous 5-HT fully restored T cell proliferation. Together this data strongly capsule production by SDS-PAGE, reduced serum resistance, and differences in susceptibility support the hypothesis that 5-HT is an autocrine stimulus that normally promotes T cell to bile salts. The results suggest that the enzymes encoded by these genes are required for activation and proliferation. the synthesis of capsular components and in their absence the capsule, although present on Moreover, these results highlight an important pro-inflammatory role for 5-HT signaling in the bacterial surface, cannot function as efficiently as in the wild-type (WT) strain. Additional immune cell function and the potential immune-related consequences of commonly used drugs experiments using intestinal epithelial cells (Caco-2) have shown that the Cj1427 mutant as which modulate 5-HT uptake and release. well as a capsule-less mutant can adhere and invade better, while reduced adhesion and no invasion were observed for the Cj1428 and Cj1430 mutants. Furthermore, experiments aimed at determining bacterial phagocytosis and survival within murine RAW macrophages also POSTER #: 24 revealed differences between WT and mutant strains. Our results support the hypothesis that Contributions of lipopolysaccharide to the motility of modified heptoses in the capsular polysaccharide contribute to its function in protection and virulence. These studies are now being followed by structural analyses of Cj1427, Cj1428, and Pseudomonas aeruginosa Cj1430 mutant capsules and WT capsule using gas-liquid chromatography and mass spectrometry, and will help us to conclusively establish the role of capsular modified heptoses in C.jejuni pathogenesis. We anticipate that the enzymes involved in the biosynthesis of these Theresa Lindhout University of Guelph components may serve as targets for novel therapeutic agents. Joseph S. Lam University of Guelph POSTER #: 22 Characterization of SLP-2 in lymphocyte signaling Flagellar-mediated motility is an important physiological function of Pseudomonas and activation in vitro and in vivo aeruginosa that plays a role in colonizing the lungs of cystic fibrosis (CF) patients. Interestingly, many of the clinical isolates of P. aeruginosa from the CF lung have been found to lack motility once a chronic infection has been established. Furthermore, chronic isolates of Caitlin D. Lemke Robarts Research Institute P. aeruginosa have also been found to possess a truncated form of lipopolysaccharide (LPS). Our hypothesis is that LPS plays a role in the motility of P. aeruginosa. Swimming and Luan A. Chau Robarts Research Institute swarming motility (both of which are governed by flagellar movement) were studied using the wild-type strain P. aeruginosa PAO1 and knockout mutants defective in one of two Joaquin Madrenas Robarts Research Institute rhamnosyltransferases (MigA and WapR) essential for adding L-rhamnose to the outer core of LPS, as well as a mutant defective in dTDP-L-rhamnose biosynthesis (RmlC). When examining swimming motility in an aqueous environment microscopically, it was discovered that the wapR and rmlC mutants exhibited considerably greater attachment to glass surfaces Development of a stable signalosome is crucial for sustained and efficient activation of T- compared to the wild-type strain and the migA mutant. In contrast, swimming speeds were and B-cells via their antigen (Ag) receptors. However, the exact mechanisms/events of statistically similar. When examining both swimming and swarming motility on the surface of a signalosome formation have not been elucidated. Thus, there is great interest in identifying substratum, as measured by the size of the colony on soft-agar surfaces, it was discovered proteins involved in this structure, and characterizing their role in signaling and activation that the mutants exhibited drastically smaller colony diameters as compared to that of the wild- downstream of the T- or B-cell receptor (TCR or BCR). One such protein that our laboratory type strain PAO1. These results indicate that flagellar function and assembly were not impaired recently isolated and identified from signalosomes of activated T-cells is stomatin like protein-2 due to mutation in the LPS genes, but rather the change in cell surface hydrophilic charges in (SLP-2). SLP-2 belongs to the stomatin family of proteins, which also includes stomatin, the mutants impedes the ability of the mutant bacterial cells in swimming and swarming motility stomatin like protein-1 (SLP-1) and stomatin like protein-3 (SLP-3). We have shown that SLP- on the surface of a substratum. 2 is upregulated in activated T- and B-cells, and that in vitro inhibition of expression in T-cells negatively affects TCR-mediated activation. On a biochemical level, we have begun to elucidate interactions between SLP-2 and other proteins involved in signaling downstream of the TCR. This area is still being examined and current studies are focusing specifically on the interaction of SLP-2 with integrins expressed by T-cells, particularly LFA-1. Furthermore, studies in the near future will focus on the molecular requirements for SLP-2 homo- and heterotypic protein interactions and overall function in lymphocyte activation. Lastly, murine studies are ongoing and current data from these indicate that loss of SLP-2 results in embryonic lethality, suggesting this protein’s importance not only for lymphocyte activation but also for embryonic development. http://www.uwo.ca/mni/MNI/IIRF/ 12 POSTER #: 25 POSTER #: 28 Mechanisms of antimicrobial peptide resistance in Identification and analysis of non-conventional Burkholderia cenocepacia nuclear import signals using Adenovirus E1. Slade A. Loutet University of Western Ontario Kris Marshall UWO Miguel A. Valvano University of Western Ontario Nikita Avvakumov Stephanie Derbyshire Burkholderia cenocepacia is an opportunistic pathogen that causes severe lung infections in Cystic Fibrosis patients. The organism is highly resistant to the killing effects of antimicrobial Joe Mymryk UWO peptides and our group studies the determinants of this resistance. We have previously shown that a B. cenocepacia mutant (SAL1) that produces severely truncated lipopolysaccharide E1A, the first adenoviral gene to be transcribed upon infection, alters the host cellular molecules is much more sensitive to antimicrobial peptides than the parental strain. To follow- environment to optimize viral replication via physical interactions with multiple host nuclear up on this study, we have begun to investigate levels of protein expression in both the parental protein factors involved in gene expression and cell growth. E1A has been heavily utilized as a strain K56-2 and SAL1 in the presence or absence of antimicrobial peptides. To date, we have tool to understand mechanisms involved in transcription, cell cycle control, oncogenic observed increased expression of an outer membrane protein and secreted proteins in SAL1 transformation and tumorigenesis. Many cellular activities rely on the transport of cellular upon treatment with polymyxin B. Additionally, we are developing a high-copy suppressor proteins into the nucleus. Protein import into the nucleus is usually mediated by physical screen in order to identify genes that can suppress the antimicrobial peptide sensitive interaction with soluble cytosolic receptor proteins (importin α) via nuclear localization phenotype of SAL1. signals (NLS). Human adenovirus type 5 E1A contains a well studied monopartite NLS (KRPRP) at the C-terminus that interacts preferentially with importin α3. Several additional regions of E1A have been shown to mediate nuclear import, but they do not POSTER #: 26 resemble a canonical NLS and the mechanism by which they function is unknown. Using an adapted transcriptional based assay to detect active NLSs we have identified and Protection from Type 1 Diabetes by iNKT Cells May characterized nuclear import functions of E1A in yeast. Functional NLSs of E1A were identified Require Interactions with CD4+CD25+ Regulatory T through testing of different E1A serotypes and mapped using deletion mutants. Import activity was localized to several regions of the E1A proteins, including the N-terminus, despite a lack Cells of basic residues. The N-terminal region of E1A also interacts with importin α suggesting that nuclear import can occur via an importin α dependent pathway in the absence of a conventional targeting signal. Dalam Ly UWO, Robarts Research Institute Qing-Sheng Mi UWO, Robarts Research Institute POSTER #: 29 Shabbir Hussain Robarts Research Institute Probiotic-treated Macrophages Release Cytokines Terry L. Delovitch UWO, Robarts Research Institute Capable of Influencing the Maturation of Dendritic Cells: A Role for G-CSF Invariant natural killer T (iNKT) cells comprise a subset of regulatory T cells characterized by their co-expression of NK cell markers and an invariant TCR that recognizes a lipid antigen in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by the Andrew Martins Department of Microbiology and Immunology, The sphingoglycolipid alpha-galactosylceramide (a-GalCer) protects against type 1 diabetes (T1D) University of Western Ontario in NOD mice. This protection involves the polarization towards a Th2-like immune response accompanied by increased IL-4. As potent activation of iNKT cells also trans-activates other Haroon I. Sheikh Department of Microbiology and Immunology, The University of Western Ontario immune cells, we further analyzed whether iNKT cells influence the function of CD4+CD25+ regulatory T cells. We found that iNKT cell activation does not alter the functional properties Sung O. Kim Department of Microbiology and Immunology, The of CD4+CD25+ regulatory T cells, as CD4+CD25+ regulatory T cells from mice given a-GalCer University of Western Ontario are still able to suppress the proliferation of T cells in vitro and prevent the transfer of T1D in vivo. In contrast, protection from T1D mediated by activated iNKT cells requires the presence of CD4+CD25+ T cells, as inactivation of CD25+ T cells during iNKT cell activation prevents protection from T1D. Inactivation of CD25+ T cells resulted in an increased activation state as Strains of bacteria referred to as probiotics have been reported to have beneficial effects on measured by increases in CD69 expression, and elevated levels of cytokine secretion. These human health; however, the mechanisms of these effects remain to be fully elucidated. data suggest that even though iNKT cells may not influence CD4+CD25+ T cell activity, iNKT Modulation of host immune responses has been suggested as a possible mode of probiotic cell-mediated protection appears to require regulation by CD4+CD25+ T cells. action. Many probiotics can colonize the intestine, where they may interact with macrophages and dendritic cells, key cells of the innate immune system which are well populated in the POSTER #: 27 lamina propria of the intestine. Our previous study has shown that bone-marrow derived immortalized macrophages exposed to Lactobacillus rhamnosus strain GR-1 produce high SEARCHING FOR THE FUNCTION OF MGTC USING levels of granulocyte-colony stimulating factor (G-CSF), a cytokine known to have anti- inflammatory effects on monocytes, yet low levels of inflammatory cytokines such as tumor IN VITRO AND EX VIVO METHODS necrosis factor alpha (TNF?) and interleukin (IL)-12. As the maturation of DCs can be influenced by the cytokine milieu, and this maturation can affect the outcome of the immune response, the objective of the current work is to investigate the effect of cytokines released by Maloney, K.E. University of Western Ontario GR-1-treated macrophages on DC maturation. To accomplish this, spent cell culture media (conditioned media, CM) from GR-1 or E.coli treated macrophages was tested for its ability to Valvano, M.A. University of Western Ontario induce maturation in mouse bone marrow derived dendritic cells (DCs), as measured by surface expression of the costimulatory molecules CD80 and CD86 and production of the cytokines IL-10 and IL-12. Activation of mitogen activated protein kinases (ERKs, JNKs, and p38) in DCs were measured after exposure to the macrophage conditioned media. GR-1- Burkholderia cenocepacia, a bacterium found ubiquitously in the environment, is emerging induced CM was found to drive lower expression of CD-86 and IL-12 than media from E.coli as an important opportunistic pathogen in patients with cystic fibrosis (CF). Very little is known treated macrophages. IL-12 expression in DCs derived from G-CSF receptor knockout (GCSF- about the mechanisms by which this pathogen can cause disease or how it survives within the R-/-) mice is found to be higher than in wild type (WT) DCs when treated with GR-1-induced airways of CF patients. Our lab has previously shown that B. cenocepacia is capable of CM. E.coli-induced CM, but not GR-1-induced CM, was found to activate JNK, and the intracellular survival within poorly acidified vacuoles distinct from lysosomes. In addition, differential activation of JNK in G-CSFR-/- versus WT DC's when treated with GR-1-induced signature tagged mutagenesis identified a gene, mgtC, that is required for survival of B. CM is found to play an important role in the modulation of IL-12 production. The results show cenocepacia in a rat agar bead model of lung infection. mgtC has been shown to be important that lack of activation of JNK when DC are exposed to GR-1-induced CM as opposed to E.coli- for the survival of other intracellular pathogens although its physiological function remains induced CM results in lower IL-12 production by the DCs, and G-CSF present in the GR-1- unknown. In this study, we show that mgtC in B. cenocepacia is required for growth in induced CM is important for the observed reduced IL-12 response. magnesium-depleted medium and is essential for survival within murine macrophages. Using fluorescence microscopy we have demonstrated that B. cenocepacia mgtC mutants, unlike the parental isolate, co-localize with the fluorescent lysosomal probe LysoTracker Red. Furthermore, by 4h post-infection mgtC mutants expressing monomeric red fluorescent protein cannot retain the protein within the bacterial cytoplasm. Together, these results demonstrate that mgtC mutants reach the lysosomal compartments where they are destroyed. In addition, B. cenocepacia mgtC mutants are rapidly targeted to lysosomal compartments after phagocytosis, suggesting that the gene is involved in more than adaptation to the environment. We therefore set out to determine if we could identify another function for mgtC. We tested various conditions that B. cenocepacia may encounter within the phagosome. With a combination of in vitro experiments and ex vivo techniques using inhibitors specific phagocytic processes we have demonstrated that mgtC-deficient B. cenocepacia are not sensitive to reactive oxygen species, reactive nitrogen species, cationic peptides or low pH. We believe the role of mgtC in intracellular survival is a critical factor in the pathogenesis of B. cenocepacia. http://www.uwo.ca/mni/MNI/IIRF/ 13 POSTER #: 30 POSTER #: 32 Topological mapping and Functional analysis of the Characterization of CD11d leukocyte integrin E.coli protein WaaL expression Megan McGarry University of Western Ontario W. Monty University of Western Ontario Cristina Marolda McKillop Miguel Valvano University of Western Ontario G.A. Dekaban University of Western Ontario Lipopolysaccharide (LPS) is produced by Gram-negative bacteria as an integral cell wall The primary injury, mechanical trauma to the spinal cord, results in death to neurons and component. It is a well-known endotoxin and possesses diverse structures. One of the activation of various cellular responses that contribute to secondary spinal cord injury (SCI) components of the LPS, the O antigen polysaccharide, is synthesized by an independent and further neuronal loss. The most potent of these cellular responses is the inflammatory pathway from that involved in the synthesis of the rest of the LPS molecule (lipid A-core response at the site of injury involving cells resident in the spinal cord, as well as infiltrating oligosaccharide). The final step in the biosynthesis of smooth LPS involves the ligation of the leukocytes. As leukocytes leave blood vessels they release pro-inflammatory molecules, O antigen to the lipid A-core oligosaccharide on the periplasmic side of the cytoplasmic including free radicals in the form of an oxidative burst. These free radicals are meant to membrane. Our lab has shown that without the membrane protein WaaL this ligation does not cleanse the area of potential infectious agents, but also contribute to further cell death. This occur. Computer generated models of the E.coli K12 WaaL predict 12 transmembrane domains cellular infiltration of leukocytes into a site of inflammation involves the CD11d/CD18 integrin with multiple cytoplasmic and periplasmic loops. Here, I characterize the WaaL protein in E. as monoclonal antibodies to CD11d block this process and result in reduced secondary SCI. coli K12, with emphasis on topology and function. We show using GFP fusion that the C- Very little is known about the CD11d/CD18 integrin itself other than CD11d transcription is terminus of WaaL resides on the cytoplasmic face of the inner membrane. Further studies highly regulated, and extensive surface expression of the protein is limited to leukocytes using TEV proteases and cysteine scanning are underway to further confirm the topology. With present at regions of local inflammation, such as the site of SCI or atherosclerotic plaque. Our emphasis on the predicted periplasmic loop 5 (El5), which is predicted to be 84 amino acids in study focuses on identifying the mechanisms that regulate CD11d surface expression on length. Deletion of this loop results in a loss of function. Site-directed mutagenesis performed leukocytes. within this region has identified key residues required for addition of O antigen transfer. Taken Other members of the leukocyte-specific family of *2 integrins (CD11a,b,c) require together the results demonstrate a key role of EL5 in the ligation of O antigen to the Lipid A heterodimerization with CD18 for surface expression. Thus it is expected that CD11d will also core. be expressed on the surface of leukocytes expressing both CD11d and CD18. An analysis of the CD11d amino acid sequence revealed a potential Casein Kinase II (CKII) phosphorylation POSTER #: 31 motif. Numerous proteins have their cellular localization controlled by CKII phosphorylation, but no integrin has previously been reported to contain an active CKII phosphorylation site. We Altering Monocyte Responses Through Chemokine hypothesize that CKII phosphorylates CD11d, and that this phosphorylation contributes to the complex regulation of the CD11d/CD18 integrin’s intracellular distribution. Our specific aims Modulation. are to determine if prior formation of a CD11d/CD18 heterodimer is required for CD11d to localize to the cell surface, if CKII phosphorylates CD11d, and to elucidate how the interplay between these two factors relates to CD11d surface expression. Dana McIvor UWO, Robarts Research Institute When visualized with confocal microscopy it appears that CD11d remains confined to intracellular structures likely to be the endoplasmic reticulum (ER)/golgi complex in HEK293 Yunming Sun Robarts Research Institute cells which stably express CD11d, but do not express CD18. CD18 protein stably expressed in Erbin Dai Robarts Research Institute HEK293 cells, in the absence of CD11d, does not appear on the cell surface either. HEK293 cells stably expressing CD11d or CD18, and transiently expressing the other half of the Ganesh M.Ramanujam Robarts Research Institute heterodimer, display co-localization of the two proteins at the cell surface when observed by confocal microscopy. In vitro kinase assays show Casein Kinase II phosphorylates a purified Alexandra Lucas UWO, Robarts Research Institute GST-CD11d fusion protein. Furthermore, CKII has been shown to phosphorylate CD11d protein immunoprecipitated from lysate of HEK293 cells stably expressing CD11d. Future Chemokines control cell migration and activation in inflammatory diseases, such as studies will determine if CKII phosphorylates CD11d in living tissue culture cells, and will atherosclerosis, and its inhibition represents a potential therapy. Mouse models deficient in investigate the effect of mutating this CKII recognition site on CD11d/CD18 heterodimerization, chemokines and their receptors have reduced atherosclerosis and transplant vasculopathy. and surface expression. A series of fusion proteins linking CD11d’s C-terminus, the region Chemokine modulating proteins (CMPs) are part of a very effective and very potent DNA virus containing the predicted regulatory Casein Kinase II motif, to CD25 have been constructed. immune evasion mechanism that disrupts chemokine gradient formation and inflammatory cell Such a system has been used in the past to study plasma membrane protein trafficking, and activation. The CMPs M-T1, M-T7 and M3 reduce mononuclear cell invasion and plaque we are using these constructs in a parallel manner to identify mechanisms regulating CD11d growth in animal models. M-T7’s unique activity is via a postulated disruption of gradient surface expression. A furthered understanding of CD11d biology gained from this study may formation through interaction with glycosaminoglycan (GAG) binding domains of chemokines. lead to improved neuroprotective strategies for therapy of secondary SCI. We are investigating inhibitory peptide sequences and chemokine binding domains responsible for anti-inflammatory activity of M-T7 through loss-of-function mutations. M-T7 binds non-species specific chemokines and rabbit-specific IFN-γ and a proposed POSTER #: 33 structure has been determined. C-terminal deletions and point mutations have been prepared in a Baculovirus expression system. Protein has been tested with CC and CXC chemokines in The role of protein glycosylation in the virulence of a membrane fluidity assay for effects on THP-1 monocyte activation and in a mouse ascites monocyte migration assay. MCP-1 (CCL2), MIP-1α (CCL3), RANTES (CCL5) and IL-8 the gastric pathogens Helicobacter pylori and (CXCL8) all had a significant effect on the membrane fluidity of THP-1 cells (p<0.05). Viron M- Campylobacter jejuni. T7, Baculovirus M-T7 and deletion mutant S M-T7 significantly reduced chemokine-mediated human monocyte activation (p<0.05). M-T7 administered systemically significantly reduced monocyte migration in BALB/C mice in response to MCP-1 activation (p=0.002). RANTES Alexandra Merkx- Microbiology and Immunology, University of Western Ontario significantly increased cell migration into the peritoneal cavity when compared to a saline control (p=0.0450). Systemic M-T7 and intraperitoneal M-T7 treatment significantly reduced Jacques RANTES activity (p=0.0007, p<0.0001) and were significantly different from each other and saline (p<0.05). Further testing with point mutations of M-T7 and NDST-1 conditional mice are Carole Creuzenet Microbiology and Immunology, University of Western Ontario currently underway. M-T7, a unique C-terminal CMP, alters cell migration into mouse ascites and modifies monocyte activation in vitro. The chemokine binding site of M-T7 remains unknown. H. pylori and C. jejuni are Gram-negative gastro-intestinal pathogens whose virulence is highly affected by protein glycosylation. The former causes gastric ulcers and cancer, while the latter causes enteritis and neurological disorders. Due to emerging drug-resistant strains, new treatments are needed. In both bacteria, the flagellins are essential virulence factors glycosylated by pseudaminic acid (PA). We have identified and disrupted genes required for PA synthesis in both bacteria, and shown that this affects flagellin production. Further analysis and glycoprotein staining revealed that in H. pylori, the PA pathway is necessary for the glycosylation of proteins other than flagellins and for the synthesis of additional virulence factors, including LPS and urease. Enzymatic deglycosylation analysis uncovered a second set of H. pylori proteins glycosylated with an unknown sugar synthesized by a PA-independent pathway. We have identified one as a membrane-associated isoform of an immunodominant antigen that protects H. pylori from oxidative stress. Numerous C. jejuni glycoproteins also possess a heptasaccharide containing diacetamidobacillosamine (DAB). Our DAB biosynthesis mutant was deficient in at least one glycoprotein and was avirulent in a chicken model, underscoring the role of this pathway in virulence. We have shown that both PA and DAB pathways are present in C. jejuni cell extracts and are investigating through activity-based assays and glycoprotein blotting how different growth conditions related to pathogenesis affect the relative activities of both pathways. Understanding how each pathway affects virulence will reveal the best targets for the development of glycosylation inhibitors to treat these major infections. http://www.uwo.ca/mni/MNI/IIRF/ 14 POSTER #: 34 POSTER #: 36 Inhibition of Indoleamine-2,3 dioxygenase (IDO) Characterization of the IRF3-mediated antiviral Activity in Renal Tubular Cells Prevents Renal response to virus particle entry Ischemia Reperfusion Injury Ryan Noyce Biochemistry and Biomedical Sciences Kanishka Mohib Karen Mossman Biochemistry and Biomedical Sciences and Pathology and Molecular Medicine Qiunong Guan Hong Diao Caigan Du Viral infection elicits the activation of numerous cellular signal transduction pathways, leading to the induction of both innate and adaptive immune responses in the host. In Anthony M. Jevnikar particular, both interferon regulatory factor 3 (IRF-3) and the viral activated kinase, TANK Indoleamine 2,3 dioxygenase (IDO) catalyses tryptophan to kynurenine and is up-regulated binding kinase 1 (TBK-1) have been shown to be essential for the induction of an antiviral by the pro-inflammatory cytokines IFN-g and TNF-a. In placental tissue it provides immune response following infection with enveloped viruses. More recently, we have shown that the privilege to the fetus through enhanced sensitivity of T cells to Fas mediated apoptosis. We activity of a novel phosphatidylinositol 3-(PI3) kinase family member is required for the previously have demonstrated that IFN-g and TNF-a can induce Fas/FasL dependent fratricide induction of an IRF3-mediated antiviral response following entry of virion particles from a in renal tubular epithelial cells (TEC). As Fas promotes ischemia-reperfusion injury (IRI) in diverse array of enveloped virus families. Treatment of human fibroblast cells with a broad PI3- kidneys, we tested whether IDO plays a role in TEC apoptosis and renal ischemia-reperfusion kinase inhibitor, LY294002, prevents the induction of interferon-stimulated gene (ISG) 56 and injury through sensitizing Fas. IDO expression was analyzed by RT-PCR and Western blot. an antiviral response following treatment with replication-deficient enveloped viruses. Cell death was measured by FACS analysis with Annexin-V-FITC and PI staining and further Interestingly, the LY294002 inhibitor fails to prevent IRF-3 homodimerization or nuclear confirmed by TUNEL assay. IRI was induced by renal artery clamping for 48 minutes at 32oC translocation upon virus particle entry, suggesting that this PI3-kinase-like molecule is having in uni-nephrectomized B57BL/6 mice. IDO in TEC was markedly increased by IFN-g/TNF-a. an affect on IRF3 post nuclear translocation. Initial studies of IRF3 activation have led to a Both RT-PCR and Western Blot analysis showed it increased at 4 hrs to maximal level at 8 to model in which RNA virus replication results in the activation of IRF3 via hyperphosphorylation 24 hrs. Inhibition of IDO by 1-methyl-d-tryptophan (1-MT) reduced IFN-g/TNF-a induced C3H of critical C-terminal serine/threonine residues. Although in vitro studies identified two main and B57BL/6 TEC "fratricide" from 50% to 33% at 24 h, compared to 8.5% in controls. clusters of residues, controversy remains over which residues are essential for IRF3 activation Conversely induced (ecdysone) transgenic expression of IDO in TEC resulted in increased in vivo. In our system, although IRF3 is essential for the induction of an antiviral response, apoptosis. Next we showed renal IDO expression was strongly upregulated by IRI. Treatment virus replication is not required and we fail to detect IRF3 hyperphosphorylation in the absence of the mice with 1-MT subsequent to IRI prevented kidney injury, indicated by a lower plasma of Newcastle Disease Virus (NDV) replication. Using two-dimensional gel electrophoresis and creatinine in treated mice (33.7 ± 8.7 mmole/L, n=10) compared vehicle treated (86.4 ± 24.8 native PAGE we have started to characterize post-translational modifications of IRF3 following mmole/l, n=11, p = 0.031) and lower urea levels (27.4± 6.8 mmol/L, n=10) versus (43.4 ± 8.3 treatment with NDV and human cytomegalovirus (HCMV), two viruses previously shown to mmol/L, n=11, p = 0.036) respectively 48 h post perfusion. Also, IDO KO mice subjected to IRI activate IRF3 in primary fibroblasts. Using these techniques, we hope to determine the minimal displayed similar trends with creatine levels (32 ± 2 mmol/L, n = 6) versus (123 ± 30 mmol/L, n post-translational modifications required to activate IRF3 and to investigate how different viral = 9, p = 0.006) and urea levels (13 ± 2 mmol/L, n = 6) versus (49 ± 11 mmol/L, n = 9, p = stimuli have the capacity to differentially modify IRF3 during a virus infection. Elucidating the 0.008) compared to wild type mice. These data suggest that TEC expression of IDO promotes signal transduction pathways that are triggered in response to the incoming virus particle will injury, perhaps through sensitizing TEC to Fas/FasL mediated apoptosis. For the first time, we allow us to further investigate the relationship between a virus and its host cell. have shown that IDO expression is enhanced by IRI and that inhibition of IDO can protect kidney function. Thus, attenuation of IDO during inflammation may represent a novel strategy to promote kidney graft function and survival. POSTER #: 37 A lipopolysaccharide modification gene cluster POSTER #: 35 encoding the synthesis and transfer of 4-amino- The evaluation of pro and anti inflammatory cytokine arabinose is essential for survival of Burkholderia gene polymorphisms in patients with Behcet’s cenocepacia disease Ximena Ortega UWO Enayat Nikoopour Department of Immunology, Tehran University for Medical Silvia Cardona Sciences, Tehran, Iran Farhad Shahram Rheumatology Research Center, Tehran University for Slade Loutet UWO Medical Sciences, Tehran, Iran Ronald Flannagan UWO Ali Akbar Amirzargar Department of Immunology, Tehran University for Medical Miguel Valvano UWO Sciences, Tehran, Iran Modification of the lipopolysaccharide (LPS) molecule by the addition of 4-amino arabinose Behrouz Nikbin Department of Immunology, Tehran University for Medical (Ara4N) in response to environmental signals is a well-established phenomenon associated Sciences, Tehran, Iran with acquisition of cationic antimicrobial peptide resistance in many Gram-negative bacteria. Fereydun Davatchi Rheumatology Research Center, Tehran University for However, it is not absolutely essential for bacterial survival under standard laboratory Medical Sciences, Tehran, Iran conditions. Burkholderia cenocepacia belongs to a group of emerging opportunistic pathogens that display an extraordinary resistance to cationic antimicrobial peptides at concentrations Introduction: Cytokines have important roles in the pathogenesis of Behcet’s disease (BD). that usually kill other bacteria even in the presence of Ara4N-modified LPS. Burkholderia Genetic polymorphism may be responsible for cytokine production and their levels. This study species contain Ara4N as a substituent of the lipid A component of the LPS molecule and also was designed to look for some of these cytokine gene polymorphisms in Iranian patients with as a component of the core oligosaccharide. Here, we have identified the Ara4N gene BD. biosynthesis cluster in a clinical isolate of B. cenocepacia and demonstrate that the genes in Patients & methods: Genomic DNA was extracted from peripheral blood samples of 147 the cluster are organized in two transcriptional units. Repeated attempts to mutagenize these Iranian patients with BD and 140 healthy control subjects using polymerase chain reaction- genes by homologous recombination with a suicide integration plasmid were unsuccessful. We sequence specific primers (PCR-SSP). Gene polymorphisms for pro-inflammatory cytokines constructed a new mutagenesis vector that enabled us to create conditional mutants by placing (TNF-α, IL-6, IFN-γ, IL-12) and anti-inflammatory cytokines (IL-10, TGF-β, IL- a rhamnose-inducible promoter upstream of any targeted gene. With this strategy we isolated 4) were determined. The frequency of different alleles was compared between the 2 groups by conditional mutants in the two transcriptional units, which were only viable in growth medium chi squared test. with rhamnose, but failed to grow under non-permissive conditions. Depletion experiments Results: There was no difference in allele frequency at positions -308 for TNF-α, p demonstrated that bacterial cells lose viability as indicated by the entry of propidium iodide into nt565 for IL-6, UTR 5644 for IFN-γ and -1188 for IL-12 between BD and control groups. the bacterial cytoplasm. Collectively, our results indicate that in contrast to the current dogma, Allele G at the p-238 of TNF-α gene showed significant increase in BD patients (P< Ara4N plays an essential role for bacterial viability in B. cenocepacia and likely other 0.001) with odds ratio of 4.3 for G/G genotype (high producers). Frequency of G/G genotype Burkholderia species. We propose that inhibitors of the Ara4N biosynthesis pathway could be (high producers) at p-174 of IL-6 was increased in patients group (P< 0.03, Odds ratio: 5). effective targets for novel antimicrobials. Polymorphism in IL-10 gene at positions -1082, -819, -592 didn’t show significant difference between patients and controls. Genotype C/T of TGF-β at codon 10 (low producer) had lower incidence in patients group (P<0.01). No difference was seen in codon 25 of TGF-β gene. There was a significant increase in the frequency of genotype CC while a decrease in the frequency of genotype TC for IL-4 gene at position -33 in BD patients (P<0.001). In BD patients, G/G genotype at p-330 of IL-2 gene was higher while G/T genotype was lower than control group. There were significant differences between the genotypes of IL-1 receptor antagonist in BD patients and normal controls. Genotype C/T was increased while genotype T/T was decreased (P≤0.02). Conclusion: Iranian BD patients showed a higher frequency of those genotypes associated with high production of TNF-α, IL-6, IL-2 and TGF-β and low production of IL-4. This may suggest the role of these genotypes in increasing the risk of susceptibility to BD in these individuals. http://www.uwo.ca/mni/MNI/IIRF/ 15 POSTER #: 39 POSTER #: 41 Identifying the regions of WbaP critical for function MOLECULAR BASIS OF T CELL RECEPTOR and interaction with O-antigen biosynthesis proteins SELECTIVITY, CROSS-REACTIVITY AND ALLELIC in Salmonella enterica DISCRIMINATION BY A BACTERIAL SUPERANTIGEN: INTEGRATIVE FUNCTIONAL AND Kinnari B. Patel Western ENERGETIC MAPPING OF THE SpeC-Vbeta2.1 Soledad M. Saldias Western MOLECULAR INTERFACE Miguel A. Valvano Western A. K. M. Nur-ur Microbiology and Immunology Rahman Lipopolysaccharide (LPS) is produced by Gram-negative bacterial as an integral cell wall Christine Herfst Lawson Health Research Institute component. The most variable LPS constituent is the O-antigen which protects Gram-negative bacteria against host defenses such as complement mediated serum killing and cationic Joaquin Madrenas Robarts Reserach Institute peptides. O-antigen is synthesized by an independent pathway from that involved in the synthesis of the rest of the LPS molecule (lipid A-core oligosaccharide). Eric Sundberg Boston Biomedical Research Institute In Salmonella enterica O-antigen biosynthesis is initiated by the membrane protein WbaP which catalyzes the transfer of Galactose-1-phosphate onto undecaprenol phosphate (Und-P). John McCormick Microbiology and Immunology This differs from initiation of O-antigen in other members of the Enterobacteriaceae family in Superantigens (SAgs) are potent immunostimulatory molecules of microbial origin that which WecA transfers N-acetyl glucosamine-1-phosphate onto Und-P. WbaP shares no function to activate large proportions of T cells. SAgs activate T cells by circumventing the sequence homology to WecA and instead is homologous to proteins involved in the initiation of conventional mode of T cell activation by concurrently binding directly to T cell receptors capsule synthesis. WbaP is predicted to have five transmembrane helices, a large periplasmic (TCRs) and major histocompatibility complex II (MHC II) molecules as intact proteins, using loop between transmembranes IV and V, and a large cytoplasmic tail. surfaces that allow for T cell activation that is independent of the TCR specificity for the loaded Previous work has proposed function for regions of WbaP. A ∆wbaP strain complemented antigenic peptide in MHC II molecule. SAgs target the alpha-beta TCR through the variable with a WbaP mutant lacking the periplasmic loop resulted in altered O-antigen chain modality. domain of the beta-chain (termed Vbeta), which subsequently leads to the activation of T cells Complementation of ∆wbaP with other WbaP mutants has shown that transmembrane V as in a Vbeta-specific manner. Previous co-crystallization studies of the bacterial SAg well as the C-terminus are required for galactosyltransferase activity of WbaP. We hypothesize streptococcal pyrogenic exotoxin C (SpeC) with either human MHC II molecule HLA DR2a, or that the periplasmic loop of WbaP is involved in chain regulation via interaction with other O- the human TCR beta chain Vbeta2.1, revealed a novel SAg-mediated T cell activation model. antigen biosynthesis proteins and that the conserved C-terminus residues encode the catalytic In this model, SpeC formed a bridge precluding direct contacts between the TCR and MHC II domains of WbaP. My project will involve over expressing the WbaP periplasmic loop in molecules. Herein, we mutated every SpeC residue involved in the SpeC-Vbeta2.1 molecular Salmonella enterica, deleting critical periplasmic loop and C-terminus residues, and identifying interface to alanine in order to provide a comprehensive understanding of Vbeta2.1 protein-protein interactions between WbaP periplasmic loop and other O-antigen biosynthesis engagement by SpeC. We determined the binding affinities of these mutant and wild type proteins. SpeC proteins to soluble Vbeta2.1 by surface plasmon resonance (SPR) analysis, as well as their functional readouts upon stimulation of both bulk T cells, including T cells expressing POSTER #: 40 exclusively Vbeta2 TCRs, and an engineered V2.1+ T cell line. Collectively, our energetic and functional data define the ‘functional epitope’ on the SpeC molecular surface Nek9, a NIMA-related kinase is a novel target for and provide the molecular basis for SpeC-TCR Vbeta domain selectivity, cross-reactivity and allelic discrimination. Of the 12 residues of SpeC involved in interacting with Vbeta2.1, adenovirus E1A proteins residues Tyr15 and Arg181 were critical for activation of all human CD3+ T cells. Next, we evaluated the effect of the SpeC mutants on Vbeta2+ T cells. SpeC mutants Y15A and R181A Peter Pelka McMaster University were drastically impaired in activating Vbeta2+ T cells. In contrast, most of the other SpeC mutants did not markedly differ from wild type except for F75A and L78A which activated only a Peter Whyte McMaster University subset of Vbeta2+ cells, indicating that specific mutations in SpeC can discriminate against different alleles within a specific Vbeta family. Next, we engineered the non-TCR expressing Jurkat T cell line to exclusively express the Vbeta2.1 chain to mimic physiological conditions as closely as possible. Activation was assessed by measuring IL-2 secretion by ELISA and IL-2 expression profiles correlated to the measured affinities of the SpeC proteins. Finally, to A monoclonal antibody raised against protein complexes consisting of the adenovirus E1A examine this interaction biochemically, we determined the binding affinity of each mutant to and associated cellular proteins recognized Nek9, a member of the NimA-related family of recombinant Vbeta2.1 by surface plasmon resonance. None of the mutants bound Vbeta2.1 kinases. Sub-cellular fractionation and immunofluorescence indicated that Nek9 was primarily with higher affinity than did wild type SpeC (KD = 13 µM). Based on the functional and cytoplasmic with only a small portion located in the nucleus whereas E1A is primarily nuclear. biochemical studies, we define the SpeC 'functional epitope' specific for Vbeta2.1 as a cluster Although co-immunoprecipitation experiments indicated that nuclear Nek9 interacted, directly of four residues each individually critical for this interaction. Three of these residues (Tyr15, or indirectly, with E1A, the major effect of E1A was to diminish the amount of Nek9 in the Phe75 and Arg181) contact the complementarity determining region (CDR) 2 loop residue nucleus. In E1A-expressing HeLa cells, the levels of nuclear Nek9 were reduced suggesting Ser52a, a non-cannonical residue insertion found only in Vbeta2 and Vbeta4 chains, and that E1A alters the subcellular distribution of Nek9 and that the interaction is transient. Nek9 evidence indicates that plasticity of this loop is important for recognition by SpeC. Although consists of an N-terminal kinase domain, a central RCC1-like domain and a C-terminal region SpeC interacts with the Vbeta2.1 hypervariable CDR3 loop, our data indicate these contacts that includes a coiled-coil domain. Unlike full length Nek9, a Nek9 deletion mutant lacking the have little to no influence on the functional interaction with Vbeta2.1. SAgs have evolved to RCC1-like domain interacted stably with E1A and accumulated in the nucleus in the presence engage their TCR ligands through a variety of diverse architectures. Despite their anticipated of E1A, possibly representing an intermediate stage of the normally transient Nek9/E1A diversity, CDR2 plasticity may have a common and important functional significance for Vbeta interaction. Examination of Nek9 interaction with a panel of E1A mutants indicated that Nek9 recognition by SAgs. interacts with the amino terminus of E1A. Attempts to stably overexpress either Nek9 or the kinase-inactive mutant in various cell lines were unsuccessful suggesting that overexpression was toxic or otherwise incompatible with continued cell proliferation. In contrast, both Nek9 and the kinase-inactive mutant could be stably overexpressed in 293 cells and in E1A- expressing HeLa cells. These results suggest that E1A disrupts a nuclear function of Nek9. http://www.uwo.ca/mni/MNI/IIRF/ 16 POSTER #: 42 POSTER #: 44 Magnetic resonance imaging of innocuously labelled Vesicular Stomatitis Virus expressing IL-15 as a dendritic cells in vivo. potential oncolytic virus Brad Shrum UWO Kyle Stephenson McMaster University Center for Gene Therapeutics PJ O'Connel Robarts Research Institute / UWO Byram Bridle McMaster University Center for Gene Therapeutics PJ Foster Robarts Research Institute / UWO Yonghong Wan McMaster University Center for Gene Theraeutics GA Dekaban Robarts Research Institute / UWO Ali Ashkar McMaster University Center for Gene Therapeutics Dendritic cells are professional antigen presenting cells and the exclusive in vivo activators Brian Lichty McMaster University Center for Gene Therapeutics of naïve T cells. Delivery of antigen-loaded DC with an appropriate stimulus results in antigen- Viruses have started to come to the forefront of targeted therapies for use in cancer specific immunity and can be used as a cell-based vaccine. DC vaccines have proven treatment. One such virus, vesicular stomatitis virus (VSV), is tropic to cancer cells but unable themselves effective at protecting against chronic disease and are in clinical trials as potential to replicate in normal tissues due to its sensitivity to interferon. In normal tissues there is a therapies of neoplasias. The clinical success of DC vaccines in cancer therapy has been sufficient interferon response to inhibit VSV replication. However, in tumour cells parts of the disappointing thus far; however, a better knowledge of DC migration in a vaccine setting in vivo interferon response are usually defective, therefore there is no interferon present to inhibit the will lead to improved immune responses. Magnetic resonance (MR) imaging can be used to replication of VSV. We have inserted interleukin-15 (IL-15), a cytokine that is able to attract image the in vivo migration of vaccine antigen-loaded DC following injection and provide the and activate NK cells and CD8+ T cells, into recombinant VSV to attempt to illicit an immune necessary information for refinement. To image cells with MR, it is necessary to label the cells response against the tumours being treated. The IL-15 may also further attenuate VSV in vivo with an MR contrast agent. To achieve meaningful and relevant results the labelling must not due to the infiltration of immune cells at the sites of replication. In this report we first looked at alter the normal function of the DC. In this study, we have labelled DC with various super the attenuation of the recombinant VSV/IL-15 virus in C57BL/6 mice. We have also begun parmagnetic iron oxide (SPIO) contrast agents in concentrations sufficient for in vivo imaging looking at the ability to of VSV/IL-15 to destroy tumours in a B16F10 brain tumour model. and investigated their impact on DC phenotype and function. We have found that the commercially available contrast agent, Feridex, is taken up intracellularly and has minimal impact on the size and expression of the DC cell surface maturation markers, MHC II, CD40 POSTER #: 45 and CD86. After delivery, the labelled DC were imaged in vivo in the draining lymph nodes of injection sites by MR and confirmed by histological methods showing they maintain their Elucidation of the catalytic mechanism of migratory ability. These results suggest that (1) DC can be labelled innocuously with MR SPIO phosphoheptose isomerase (GmhA), a drug target contrast agent; (2) following injection SPIO-labelled DC can be imaged in vivo and (3) that the resulting migration and distribution of these labelled DC is directly comparable to that of for combating Gram negative bacterial infection. unlabelled DC. These results will greatly improve the current tools available for enhancing DC vaccines which will lead to improved clinical efficacy as well as an increase in DC vaccine applications. Patricia L. Taylor Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario POSTER #: 43 Gladys P. de Leon Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario Role of HP0366 in the secretion and biosynthesis of Miguel A. Valvano Department of Microbiology and Immunology, University of virulence factors in Helicobacter pylori Western Ontario, London, Ontario Gerard D. Wright Department of Biochemistry and Biomedical Sciences, Vijayakumar University of Western Ontario McMaster University, Hamilton, Ontario Somalinga Murray S. Junop Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario Carole Creuzenet University of Western Ontario Lipopolysaccharide (LPS) is a major component of the Gram-negative bacterial cell wall. LPS is comprised of three components: lipid A, core polysaccharide (Kdo and one or more heptose residues), and polysaccharide side chains. LPS biosynthesis that is halted prior to heptose addition results in a deep-rough phenotype and membrane permeability to antibiotics Helicobacter pylori is the leading cause of chronic gastritis, gastro-duoduenal ulcers and is increased. For this reason, the heptose biosynthetic pathway has been marked as a gastric cancer in humans. Although numerous virulence factors (flagella, urease, potential drug target. Our goal is to determine the mechanism of GmhA, an isomerase that lipopolysaccharide (LPS) and vacuolating cytotoxin) have been identified in H. pylori, the exact catalyzes the first committed step of ADP-L-glycero-ß-manno-heptose biosynthesis. We mechanism of its pathogenesis is still not clearly understood. HP0366 is predicted to encode elected to use Escherichia coli as our Gram negative model system and have crystallized E. for an aminotransferase. Biochemical characterization of over-expressed and purified enzyme coli GmhA in the absence (1.95 Ǻ) and presence (2.89 Ǻ) of substrate, shows that it uses UDP-2-acetamido-2,6-dideoxy-4-keto-arabinose as its substrate. For optimal sedoheptulose 7-phosphate. Using this structure, potential active site residues were identified enzymatic activity HP0366 requires PLP as an external cofactor and glutamate as an amino and corresponding mutations were generated in the E. coli gmhA sequence. The resulting donor. The biological function of HP0366 is unknown. As a first step in deciphering its proteins were purified and the activity characterized using a spectrophotometric coupled biological function, we inactivated it by inserting a kanamycin resistance cassette. The effect of enzyme assay, recently developed in our lab. Reactions were initiated using enzymatically HP0366 inactivation on the production of virulence factors was then investigated. LPS analysis synthesized sedoheptulose 7-phosphate. To compliment these in vitro studies, low copy revealed a loss of O-antigen and urease activity in the HP0366 mutant. The loss of O-antigen plasmids expressing the mutants were generated and transformed into E. coli gmhA knock-out and urease activity in the HP0366 mutant was successfully complemented in a gene-specific cells. Cells were tested for increased susceptibility to novobiocin, which can only permeate E. manner. Electron micrographs of a HP0366 mutant revealed the loss of flagella and coli exhibiting a deep rough phenotype. These studies identified two active site residues, E65 subsequent motility assays show that HP0366 mutant is non-motile. To ascertain if the loss of and H180, which are necessary for GmhA activity and, ultimately, complete LPS biosynthesis. flagella is due to lack of flagellin synthesis or export, we performed immunoblot with an anti- Based on the importance of these residues and the knowledge of the active site imparted by flagellin A antibody. The presence of flagellins in the HP0366 mutant indicated that the lack of the crystal structure, we postulate an enediol-intermediate mechanism of action for GmhA, flagella in the mutant may be due to a defect in flagellin export. Since LPS, urease and flagella similar to that of triosephosphate isomerase (TIM). Determination of this mechanism will aid in are important virulence factors, these results suggest that HP0366 may play a crucial role in H. the rational design of inhibitors of GmhA as a means to combat Gram negative infection. pylori colonization and pathogenesis. http://www.uwo.ca/mni/MNI/IIRF/ 17 POSTER #: 46 POSTER #: 48 Inverse agonist activity of soluble CTLA-4 ligands Heme Recognition by a Staphylococcus aureus requires oligomerization of this receptor and CD28 NEAT Domain expression Christie L. Department of Microbiology and Immumology, University of Western Ontario Wendy Teft Robarts Research Institute Vermeiren Joaquin Madrenas Robarts Research Institute Jason C. Grigg Department of Microbiology and Immunology, University of British Columbia Michael E.P. Murphy Department of Microbiology and Immunology, University of British Columbia Under certain circumstances T cell activation can occur in an antigen-independent manner. We have recently shown that CTLA-4 can initiate T cell responses upon its engagement with a David E. Heinrichs Department of Microbiology and Immunology, University of human-specific recombinant ligand, known as 24:26, in the absence of TCR ligation and CD28 Western Ontario costimulation. To understand the ability of CTLA-4 to act as both an inhibitor and an activator we examined the requirements for CTLA-4-mediated T cell activation. We observed that 24:26 Successful pathogenic organisms have developed mechanisms to thrive under extreme and soluble B7.1 Ig, but not B7 molecules on APCs, induced the formation of a CTLA-4 levels of iron restriction. Heme-iron represents the largest iron reservoir in the human body and oligomer revealed biochemically by the disappearance of this receptor from the soluble cell is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains lysate fraction and subsequent appearance in the insoluble cellular fraction. Oligomerization are found exclusively in a family of cell surface proteins in Gram-positive bacteria. Many NEAT occurred upon engagement of CTLA-4 homodimers and was necessary but not sufficient for T domain-containing proteins, including IsdA in Staphylococcus aureus, are implicated in heme cell activation. The inverse agonist activity of these soluble ligands also required the presence binding. Here, we show that over-expression of IsdA in S. aureus enhances growth and an of the cytoplasmic domain of CTLA-4 since TLESS CTLA-4 molecules did not induce T cell inactivation mutant of IsdA has a growth defect, compared to wild-type, when grown in media activation upon 24:26 ligation. Here we also show that CD28 expression is necessary for the containing heme as the sole iron source. Furthermore, the heme-binding property of IsdA is function of CTLA-4 as both an inhibitory and activating receptor suggesting that these two contained within the NEAT domain. Crystal structures of the apo-IsdA NEAT domain and in receptors work in unison. Therefore, the functional outcome of CTLA-4 engagement depends complex with heme were solved and reveal a clathrin adapter-like-sandwich fold with a large on the architectural oligomerization induced by the ligand it encounters which likely initiates hydrophobic heme binding pocket. Heme is bound with the propionate groups directed at the the assembly of key signaling molecules to act as inhibitors or activators on the CD28 molecular surface and the iron is coordinated solely by Tyr166. The phenol groups of Tyr166 pathway. The ability of soluble B7.1 Ig to act as an inverse agonist of CTLA-4 suggests that and Tyr170 form an H-bond that may function in regulating heme binding and release. An CTLA-4 may act as a costimulatory molecule in vivo. analysis of IsdA structure-sequence alignments indicate that conservation of Tyr166 is a predictor of heme binding by NEAT domains. POSTER #: 47 POSTER #: 49 Finding Novel Inhibitors for the Antibiotic Resistance Enzyme APH(3’)-IIIa Glu-Arg salt bridges are critical for an interaction between iron-hydroxamate binding protein FhuD2 Tushar Shakya Graduate Student and the FhuBGC2 ATP-binding cassette transporter in Staphylococcus G.D. Wright P.I. Enrique D. Vinés University of Western Ontario Antibiotic resistance has become widespread in several clinical settings and poses an Craig D. Speziali University of Western Ontario obstacle for current antibiotic therapies in use. The aminoglycoside (AG) antibiotics are one such group that has fallen victim to resistance mechanisms. These antibiotics work by binding David E. Heinrichs University of Western Ontario to the 30S bacterial ribosomal subunit and perturb protein synthesis leading to cell death. AG antibiotics have a broad spectrum activity against both Gram-positive and Gram-negative organisms and were an antibiotic of choice prior to the emergence of resistance mechanisms. The features that govern the interaction of ligand binding proteins with membrane spanning The principal method of resistance that occurs against AGs is aminoglycoside modifying domains of cognate ABC transporters are largely unknown. Using sequence alignments and enzymes (AMEs), which work by phosphorylating, adenylylating or acetylating a specific structural modeling based on the structure of the BtuCD vitamin B12 transporter, we identified hydroxyl or amino group on the target antibiotic. Aminoglycoside phosphotransferase (3’)-IIIa six conserved basic residues in the transmembrane domains, FhuB and FhuG, of the ferric- is a protein kinase that works by phosphorylating the 3’-OH of the target AG, and is the major hydroxamate uptake (Fhu) transporter in Staphylococcus aureus. Alanine scanning reason for the decline in use of AGs such as kanamycin, amikacin and isepamicin. To counter mutagenesis was used to mutate, individually and in various combinations, all six positively this, AGs lacking 3’-OHs have been designed, such as tobramycin and gentamicin. However, charged residues in FhuB and FhuG, and the two negatively charged residues in FhuD2, Glu- the rise of APH(2”), a family of enzymes that modifies these frontline drugs, has brought into 97 and Glu-231. Our results demonstrate that only two of the positively charged residues, question how long they will last. In order to rescue their activity new strategies are being FhuB Arg-71 and FhuG Arg-61, play a dominant role in the function of the transporter, as devised to rescue their activity with the use of synergistic therapies, consisting of inhibitors of assessed by growth of S. aureus on iron-hydroxamates as a sole source of iron. Mutation of APHs and the AGs. In order to implement such therapies, it is necessary to identify lead the positive charges in FhuB and FhuG to Ala, or mutation of the negative charges in FhuD2 to compounds that can potentially become drugs. Currently, 2100 small molecules have been Ala, abrogated transport. We further demonstrated the importance of salt bridge interactions in screened against APH(3’)-IIIa using high throughput screening techniques. Of these the transporter by showing that transport of iron-hydroxamates was restored in a strain that compounds, seven have been identified as inhibitors of APH(3’)-IIIa and validated through contained the Glu-97 and Glu-231 residues in FhuD2 substituted for Arg and the positive kinetic analysis. Identification of such inhibitors will aid in the development of effective charges in FhuB and FhuG substituted for negatively charged Glu. These results provide the synergistic aminoglycoside therapies to treat various multi-drug resistance bacterial infections. first direct demonstration of the functional importance of conserved basic pockets on the surface of transmembrane domains of ABC transporters in the iron/cobalamin family of ABC transporters. http://www.uwo.ca/mni/MNI/IIRF/ 18 POSTER #: 50 POSTER #: 52 Dendritic cell-mediated immune modulation: Characteristtics of Three Classes of Targeted gene silencing using siRNA-bearing Immunoglobulins from the Avian Egg immunoliposomes R. Alejandra Ward U. Of Windsor Costin Vladau University of Western Ontario, Microbiology and Andrew Martins U. Of Windsor Immunology Xiufen Zheng University of Western Ontario, Department of Surgery Dr. Hugh Fackrell U. Of Windsor Xushang Zhang University of Western Ontario, Department of Surgery Avian IgA, IgM and IgY have all been reported in the unfertilized chicken egg. IgY, present in high concentrations in the yolk is extensively documented. However, the IgA and IgM of the The key to preventing transplant rejection and autoimmune disease lies in coaxing the host egg white remain poorly characterized. In this study we developed simple methods for the immune system to recognize grafted or healthy tissues as “self” through a process called isolation, purification, and characterization of all three classes of immunoglobulins. Up to 80% “tolerance induction”. Immune tolerance can be induced by tolerogenic dendritic cells (Tol- of the antibody to a specific antigen can be IgM and/or IgA. All classes agglutinate bacteria DCs), which generally exhibit an immature phenotype and express decreased levels of co- more efficiently at 1.5M NaCl than at low salt concentrations. IgA and IgM still function after stimulatory molecules, such as CD40, and pro-inflammatroy cytokines. Tol-DCs can also be extensive proteolysis in the presence of bile and in 4M urea and even after lyophilization and generated by employing various artificial methods, including short interfering RNA (siRNA), to pasteurization. These characteristics are interpreted in light of companion studies on 3D down-regulate expression of immune-stimulatory molecules in DCs. siRNA is a newly structure modeling. developed, Nobel-winning genetic tool used to potently suppress gene expression at the post- transcriptional level. Although siRNA is safer and more potent than other methods, a barrier to clinical application, and the generation of Tol-DC in vivo, is the current lack of an effective and POSTER #: 53 cell-specific delivery system. Herein, we investigated whether antibody-directed, “stealth” Regulation of growth factor-stimulated chemotaxis liposomes (stealth immunoliposomes, SILs) can be used to specifically deliver siRNA to DCs in vivo, in order to generate Tol-DCs. and chemokinesis of primary murine keratinocytes We have encapsulated fluorescence-labeled CD40 siRNA within stealth liposomes by rapid extrusion and freeze/thawing techniques. The DC-specific monoclonal antibody, NLDC-145, which targets an internalization receptor (DEC-205), was conjugated to the surface of the Alanna Watson Department of Microbiology and Immunology, UWO liposome as a DC-targeting mechanism. Liposome diameter was determined to be ~70nm by quasi-elastic light-scattering, and siRNA encapsulation efficiency within liposomes was Dr. Vince Morris Department of Microbiology and Immunology, UWO analyzed by spectrofluorometry and found to be 6-8 %. Epifluorescence microscopy and flow cytometric analysis demonstrated that CD40 siRNA-SILs were able to specifically bind DCs, in Dr. Bosco Chan Department of Microbiology and Immunology, UWO comparison with control (L929) cells, which do not express DEC-205. The gene silencing capacity of CD40 siRNA-SILs was determined in vitro and in vivo by quantitative real time PCR and flow cytometry. In addition, splenic DCs from mice injected with CD40 siRNA-SILs We compared the migratory response of primary keratinocytes to stimulation by growth showed a significantly increased uptake of fluorescent siRNA and greater inhibition of factors on cutaneous matrix proteins. On collagen I, both epidermal growth factor (EGF) and allogenic T cell proliferation in MLR, compared with splenic DCs from control mice. hepatocyte growth factor (HGF) stimulated chemokinetic (random) and chemotactic In summary, these results suggest that siRNA-SILs may be an effective means of delivering (directional) migration of keratinocytes. On fibronectin, the migratory response to the siRNA and a potential tool for generating Tol-DCs to modulate immune responses. corresponding growth factor was reduced. On provisional matrix, a combination of fibronectin and fibrin found in the early phase of wound healing, neither EGF nor HGF stimulated POSTER #: 51 significant cell movement. Therefore, the interaction with ECM substrate can dramatically modulate the migratory response to stimulation by growth factors. Blocking mAbs to integrin Preclinical efficacy of CD28 signaling inhibitor 6-thio- a2b1 and a5b1 effectively inhibited the stimulated cell movement on collagen I and fibronectin, respectively. When the a2b1 integrin-mediated migratory response on collagen I was GTP in prolongation of cardiac allograft survival assessed in the presence of the MEK-specific inhibitor PD98059, the stimulation of chemotaxis by EGF or HGF was significantly reduced, whereas PD98059 had little or no effect on the Shuang Wang Depts of Med, Microbiol & Immunol stimulated chemokinesis. In contrast, PD98059 had no effect on the a5b1 integrin-mediated migratory response to EGF and HGF on fibronectin. The presence of phosphoinositide 3- Hong Diao LHSC kinase (PI3K)-specific inhibitor LY294002 reduced the stimulation of migration (chemotaxis and chemokinesis) by EGF or HGF on both matrix proteins, collagen I and fibronectin. However, Qiunong Guan LHSC the two inhibitors in combination consistently yielded greater extents of inhibition than either inhibitor alone. Thus, both PI3K and extracellular-signal regulated kinase (ERK) activation Anthony M. Jevnikar Depts of Med, Microbiol & Immunol; LHSC contribute to the migratory response of keratinocytes to EGF and HGF. Our results also show that the ERK signaling pathway was differentially utilized depending on the mode of response Caigan Du Depts of Med, Microbiol & Immunol; LHSC (chemotaxis versus chemokinesis) and the specific integrin (a2b1 versus a5b1) for interaction Background: Vav-dependent small GTPase Rac1 activity is pivotal in CD28 signaling, which with the corresponding matrix substrate. is required for optimal T cell activation as a co-stimulatory receptor. 6-Thio-GTP, a metabolite of azathioprine, specifically inhibits GTPase Rac1 activation upon CD28 costimulation. The current study is to test the immunosuppressive efficacy of 6-thio-GTP in the prevention of cardiac allograft rejection in a murine model. Methods: T cell proliferations were measured by 3H-Thymidine uptake. T cell phenotype was assessed by FACS analysis. Immunosuppressive efficacy of 6-thio-GTP was tested in an allogeneic C57BL/6 (H-2b) to BALB/c (H-2d) heterotopic heart allograft mouse model. Results: In vitro 6-thio-GTP not only inhibited TCR/alloantigen stimulated T cell proliferation and CD28-dependent T cell survival but also induced T cell anergy. Administration of 6-thio- GTP (0.5 mg/kg/day) prolonged graft survival to 13.8 ± 2.39 days (n = 10) as compared to 8.3 ± 0.48 days in PBS controls (n = 10, p < 0.0001 logrank). Combination therapy of 6-thio-GTP (0.5 mg/kg/day) with CsA (15 mg/kg/day) enhanced graft survival from 15.0 ± 1.61 days in CsA monotherapy treated recipients (n = 11) to 36.8 ± 2.17 days in those received 20 days of both CsA and 6-thio-GTP (n = 9, p < 0.0001), or to 42.7 ± 16.63 days including 4 long-term surviving grafts in the group treated with 20 days of CsA and 60 days of 6-thio-GTP (n = 10, p < 0.0001). Further studies demonstrated that the 6-thio-GTP treated recipients with long-term surviving grafts had a higher frequency of Treg populations and lower proliferative responses to alloantigen-restimulation. Conclusion: 6-Thio-GTP prolongs cardiac allograft survival by suppression of alloreactive T cell activation, induction of T cell anergy and increase in Treg populations. These findings provide the evidence of immunosuppressive mechanism of azathioprine and suggest that disruption of GTPase Rac 1 signaling has therapeutic potential for transplant rejection. http://www.uwo.ca/mni/MNI/IIRF/ 19 POSTER #: 54 POSTER #: 56 Biochemical characterization of WbpI, a putative Wrestling with SUMO: Human Adenovirus E1A’s UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid interaction with the SUMO conjugase UBC9. 2-epimerase from Pseudomonas aeruginosa serotype O5 Ahmed F. Yousef Dept. of Microbiology and Immunology, University of Western Ontario, London Regional Cancer Program Erin Westman Department of Molecular and Cellular Biology, University of Joe S. Mymryk Dept. of Microbiology and Immunolgy, Dept. of Guelph Experimental Oncology, University of Western Ontario, London Regional Cancer Program David McNally Institute for Biological Sciences, National Research Council Martin Rejzek Center for Carbohydrate Chemistry, University of East Anglia Viral oncogenes have proved to be invaluable tools for probing the regulation of cellular Wayne Miller Department of Molecular and Cellular Biology, University of processes. The adenovirus E1A proteins are the first viral proteins produced during an Guelph adenovirus infection. E1A is an oncogene and can immortalize primary rodent cells or fully transform them in co-operation with a second oncogene, such as activated Ras. Numerous Joseph Lam Department of Molecular and Cellular Biology, University of cellular proteins targeted by the E1A oncoproteins have already been identified and the list Guelph keeps growing. However, the effects of E1A on the function of many of these proteins have not been determined. Saccharomyces cerevisiae is a simple eukaryote that has been used Pseudomonas aeruginosa is an opportunistic pathogen that frequently infects individuals extensively to understand the control of cellular processes. Some strains of this yeast undergo whose natural defenses are compromised, for instance those with severe burn wounds, or pseudohyphal differentiation when starved for nutrients. The changes in growth and those who have the genetic disease cystic fibrosis. An important virulence factor of this morphology that accompany pseudohyphal differentiation are easily visualized and the organism is lipopolysaccharide (LPS), which is a tripartite glycolipid anchored to the outer- pathways regulating this type of growth are well characterized. I have defined a six amino-acid leaflet of the outer membrane. In P. aeruginosa serotype O5, the long O-antigen side chain of sequence in E1A (EVIDLT), spanning residues 118-123, that stimulates yeast pseudohyphal LPS contains the unusual sugar 2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid (D- differentiation. I have also determined that this portion of E1A targets yeast UBC9 and its Man[2NAc3NAc]A). We have proposed and are investigating a 5-step pathway for the mammalian equivalent Ubc9 (a sumo conjugase). Mutational analysis revealed that all biosynthesis of UDP-D-Man(2NAc3NAc)A, through which UDP-D-GlcNAc is converted to 2,3- residues except for E118 are required for this interaction. Interestingly, this same amino acid diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-D-Glc[2NAc3NAc]A) and then epimerized at sequence is also present in a plethora of mammalian and yeast proteins that serve as SUMO C-2 by WbpI. Our objective in this study is to test the hypothesis WbpI is an epimerase that E3 ligases. This raises the interesting possibility that this sequence represents a novel motif can convert UDP-D-Glc(2NAc3NAc)A to its C2 epimer UDP-D-Man(2NAc3NAc)A. WbpI was that is present in at least some SUMO E3’s. The SUMO pathway has been implicated in many overexpressed as a His6-tagged fusion protein and purified by FPLC to >85% purity. Capillary important cellular functions including cellular localization, transcriptional regulation, and cell electrophoresis was used to assess the reaction progress, and our results showed that WbpI cycle control. can catalyze a maximum of 11% conversion of the substrate to generate a new product peak. In addition, the substrate specificity of WbpI was examined by using sugar-nucleotides from commercial sources and from chemical syntheses. WbpI exhibited no activity when UDP-D- POSTER #: 57 GlcNAc, UDP-D-GlcNAcA, or UDP-D-Glc(2NAc3NAc) were used as substrates, but it clearly possesses activity when reacting with its putative substrate, UDP-D-Glc(2NAc3NAc)A. To Lymphocyte responses in human smokers and elucidate the chemical structure of the product produced by WbpI, we used the approach of Schoenhofen et al. (JBC 281:723-732, 2006) whereby the progression of the catalytic reaction model animals of WbpI was monitored over time directly in aqueous reaction buffer by high-field nuclear magnetic resonance spectroscopy (NMR). Our results showed unequivocally that the product of the WbpI reaction is UDP-D-Man(2NAc3NAc)A. Caleb CJ Zavitz Department of Pathology and molecular medicine, McMaster University, Hamilton, Ontario Gordon J Gaschler Department of Pathology and molecular medicine, POSTER #: 55 McMaster University, Hamilton, Ontario EFFECT OF LACTOBACILLI ON PLACENTAL COX2 Clinton S. Robbins Department of Pathology and molecular medicine, McMaster University, Hamilton, Ontario AND PGDH EXPRESSION IN LPS-ACTIVATED TROPHOBLASTS P. Gerard Cox Division of Respirology, and Firestone Institute for Respiratory Health, St. Joseph's Healthcare and McMaster University, Hamilton Ontario Maryam Yeganegi Dept. of Physiology & Ob/Gyn, Univ. of Toronto, Toronto, Martin R Stampfli Department of Medicine, and Department of Pathology and Canada and Samuel Lunenfeld Research Institute, Mount Sinai Hosp.,Toronto,Canada Molecular Medicine, McMaster University, Hamilton, Ontario Carole Watson Samuel Lunenfeld Research Institute, Mount Sinai Smoking is widely understood to be the dominant etiologic factor for “smokers’ diseases” including lung, tracheal and bronchial cancers, and chronic obstructive pulmonary disease Hosp.,Toronto,Canada (COPD). Although smoke’s direct effects should not be discounted, it has been proposed that Sung Kim Dept. of Microb. & Immun., Univ. of Western Ontario, many of smoke’s deleterious health effects may be caused, at least in part, by its effect on the London, Canada immune system. Prior animal studies using high levels of smoke exposure have suggested that Gregor Reid tobacco exerts its effects on the immune system by exhausting cellular signal transduction John RG Challis Dept. of Physiology & Ob/Gyn, Univ. of Toronto, cascades, making lymphocytes unresponsive to stimulation. In this study however, we Toronto, Canada demonstrated that impaired immune responses in a well-studied model of moderate smoke exposure can not be explained by unresponsiveness to receptor-mediated signalling. On the Alan Blocking Dept. of Physiology & Ob/Gyn, Univ. of Toronto, Toronto, contrary, lymphocytes from smoke-exposed animals proliferated and secreted effector Canada and Samuel Lunenfeld Research Institute, Mount molecules equivalently to their sham-exposed counterparts. In lung, draining lymph node and Sinai Hosp.,Toronto,Canada splenic lymphocytes from smoke- and sham-exposed animals, and in magnetically sorted B cells, anti-IgM, anti-CD40, and LPS each caused equivalent proliferation and immunoglobulin Objective: Infection or inflammation is associated with 30% of preterm births. In addition, secretion. Similarly, lymphocytes and sorted T-cells proliferated and secreted IFN Bacterial Vaginosis (BV), which is characterized by the presence of gram-negative bacteria, equally in response to agonistinc anti-CD3 antibodies. We recapitulated these findings in such as Prevotella, and the absence of endogenous Lactobacillus, is associated with a 1.4-fold PBMCs isolated from human smokers, and found equivalent proliferative responses to anti- increased risk of preterm birth. Pathogenic bacteria associated with BV are known to up- IgM, CD40L and IL4, and anti-CD3 in asymptomatic smokers, in agreement with our murine regulate pro-inflammatory cytokines, which in turn cause an increase in synthesis and a results. Interestingly however, PBMCs from COPD patients were hyperproliferative to anti- decrease in degradation of prostaglandins through regulation of enzymes such as CD3. We propose that the immune dysfunction observed in human smokers is not a result of cyclooxygenase (COX2) and prostaglandin dehydrogenase (PGDH), a process which may lymphocyte unresponsiveness, and may be a result of APC dysfunction or other presently eventually lead to labour. Therefore, the aim of this study was to determine the effect of unidentified mechanisms. Lactobacillus rhamnosus GR-1, as a potential probiotic, on COX2 and PGDH protein expression in placental tissues. Methods: Term placentae were collected from women undergoing elective Caesarean section. Placental trophoblast cells were isolated using a percoll gradient and grown in 6 cm dishes at a density of 2x106 cells/ml. After 72 h, cells were serum starved for 24 h. Cells were then pretreated for 3 h with different dilutions of the supernatant from lactobacilli cultures, and subsequently treated for a further 8 hours with 200 ng/ml of LPS. As control, different dilutions of lactobacilli supernatant alone were used to treat cells. Protein was extracted and PGDH and COX2 expression levels were measured by Western Blot analysis. Results: As expected, LPS caused an increase in expression of COX2 and a decrease in expression of PGDH in trophoblast cells. Preliminary results indicate that lactobacilli decreased the LPS-stimulated increase in COX2 expression in a dose-dependent manner, while lactobacilli alone had no effect. In addition, lactobacilli at a 1:20 dilution increased the expression of PGDH. Conclusion: These results suggest that probiotic lactobacilli could inhibit the cascade leading to increased prostaglandin levels stimulated by pathogenic bacteria, by decreasing COX2 expression and up-regulating PGDH expression in placental trophoblast cells. This study has demonstrated a potential mechanism by which probiotic lactobacilli help to maintain a healthy pregnancy. http://www.uwo.ca/mni/MNI/IIRF/ 20