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Our previously discovered monomeric aptamer for SARS-CoV-2 (MSA52) possesses a universal affinity for COVID-19 spike protein variants but is ultimately limited by its ability to bind only one subunit of the spike protein. The symmetrical shape of the homotrimeric SARS-CoV-2 spike protein presents the opportunity to create a matching homotrimeric molecular recognition element that is perfectly complementary to its structural scaffold, causing enhanced binding affinity. Here, we describe a branched homotrimeric aptamer with three-fold rotational symmetry, named TMSA52, that not only possesses excellent binding affinity but is also capable of binding several SARS-CoV-2 spike protein variants with picomolar affinity, as well as pseudotyped lentiviruses expressing SARS-CoV-2 spike protein variants with femtomolar affinity. Using Pd-Ir nanocubes as nanozymes in an enzyme-linked aptamer binding assay (ELABA), TMSA52 was capable of sensitively detecting diverse pseudotyped lentiviruses in pooled human saliva with a limit of detection as low as 6.3 × 10

Oncolytic virotherapies have shown excellent promise in a variety of cancers by promoting antitumor immunity. However, the effects of oncolytic virus-mediated type I interferon (IFN-I) production on antitumor immunity remain unclear. Recent reports have highlighted immunosuppressive functions of IFN-I in the context of checkpoint inhibitor and cell-based therapies. In this study, we demonstrate that oncolytic virus-induced IFN-I promotes the expression of PD-L1 in tumor cells and leukocytes in a IFN receptor (IFNAR)-dependent manner. Inhibition of IFN-I signaling using a monoclonal IFNAR antibody decreased IFN-I-induced PD-L1 expression and promoted tumor-specific T cell effector responses when combined with oncolytic virotherapy. Furthermore, IFNAR blockade improved therapeutic response to oncolytic virotherapy in a manner comparable with PD-L1 blockade. Our study highlights a critical immunosuppressive role of IFN-I on antitumor immunity and uses a combination strategy that improves the response to oncolytic virotherapy.

We report on a unique DNA aptamer, denoted MSA52, that displays universally high affinity for the spike proteins of wildtype SARS-CoV-2 as well as the Alpha, Beta, Gamma, Epsilon, Kappa, Delta and Omicron variants. Using an aptamer pool produced from round 13 of selection against the S1 domain of the wildtype spike protein, we carried out one-round SELEX experiments using five different trimeric spike proteins from variants, followed by high-throughput sequencing and sequence alignment analysis of aptamers that formed complexes with all proteins. A previously unidentified aptamer, MSA52, showed K

Contains fulltext : 248705.pdf (Publisher’s version ) (Open Access) Since the vast majority of species solely rely on innate immunity for host defense, it stands to reason that a critical evolutionary trait like immunological memory evolved in this primitive branch of our immune system. There is ample evidence that vaccines such as bacillus Calmette-Guérin (BCG) induce protective innate immune memory responses (trained immunity) against heterologous pathogens. Here we show that while BCG vaccination significantly reduces morbidity and mortality against influenza A virus (IAV), it fails to provide protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In contrast to IAV, SARS-CoV-2 infection leads to unique pulmonary vasculature damage facilitating viral dissemination to other organs, including the bone marrow (BM), a central site for BCG-mediated trained immunity. Finally, monocytes from BCG-vaccinated individuals mount an efficient cytokine response to IAV infection, while this response is minimal following SARS-CoV-2. Collectively, our data suggest that the protective capacity of BCG vaccination is contingent on viral pathogenesis and tissue tropism.

Immune checkpoint therapy has shown great promise in the treatment of cancers with a high mutational burden, such as mismatch repair-deficient colorectal carcinoma (dMMR CRC). However, many patients fail to respond to immune checkpoint therapy. Using a mouse model of dMMR CRC, we demonstrated that tumors can be further sensitized to immune checkpoint therapy by using a combination of low-dose chemotherapy and oncolytic HSV-1. This combination induced the infiltration of CD8+ and CD4+ T cells into the tumor and the upregulation of gene signatures associated with the chemoattraction of myeloid cell subsets. When combined with immune checkpoint therapy, the combination promoted the infiltration of activated type 1 conventional dendritic cells (cDC1s) into the tumor. Furthermore, we found this combination strategy to be dependent on cDC1s, and its therapeutic efficacy to be abrogated in cDC1-deficient Batf3−/− mice. Thus, we demonstrated that the adjuvanticity of dMMR CRCs can be improved by combining low-dose chemotherapy and oncolytic HSV-1 in a cDC1-dependent manner.

Background The ongoing COVID-19 pandemic has resulted in 185 million recorded cases and over 4 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. Results Here, we describe a novel, intranasally delivered COVID-19 vaccine based on a helper-dependent adenoviral (HD-Ad) vector. The vaccine (HD-Ad_RBD) produces a soluble secreted form of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and we show it induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. Conclusion Our approaches provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

Medicinal chemistry optimization of a previously described stilbene inhibitor of HIV-1, 5350150 (2-(2-(5-nitro-2-thienyl)vinyl)quinoline), led to the identification of the thiazole-5-carboxamide derivative (GPS491), which retained potent anti-HIV-1 activity with reduced toxicity. In this report, we demonstrate that the block of HIV-1 replication by GPS491 is accompanied by a drastic inhibition of viral gene expression (IC50 ~ 0.25 µM), and alterations in the production of unspliced, singly spliced, and multiply spliced HIV-1 RNAs. GPS491 also inhibited the replication of adenovirus and multiple coronaviruses. Low µM doses of GPS491 reduced adenovirus infectious yield ~1000 fold, altered virus early gene expression/viral E1A RNA processing, blocked viral DNA amplification, and inhibited late (hexon) gene expression. Loss of replication of multiple coronaviruses (229E, OC43, SARS-CoV2) upon GPS491 addition was associated with the inhibition of viral structural protein expression and the formation of virus particles. Consistent with the observed changes in viral RNA processing, GPS491 treatment induced selective alterations in the accumulation/phosphorylation/function of splicing regulatory SR proteins. Our study establishes that a compound that impacts the activity of cellular factors involved in RNA processing can prevent the replication of several viruses with minimal effect on cell viability.

The SARS-CoV-2 pandemic is an ongoing threat to global health, and wide-scale vaccination is an efficient method to reduce morbidity and mortality. We designed and evaluated two DNA plasmid vaccines, based on the pIDV-II system, expressing the SARS-CoV-2 spike gene, with or without an immunogenic peptide, in mice, and in a Syrian hamster model of infection. Both vaccines demonstrated robust immunogenicity in BALB/c and C57BL/6 mice. Additionally, the shedding of infectious virus and the viral burden in the lungs was reduced in immunized hamsters. Moreover, high-titers of neutralizing antibodies with activity against multiple SARS-CoV-2 variants were generated in immunized animals. Vaccination also protected animals from weight loss during infection. Additionally, both vaccines were effective at reducing both pulmonary and extrapulmonary pathology in vaccinated animals. These data show the potential of a DNA vaccine for SARS-CoV-2 and suggest further investigation in large animal and human studies could be pursued.

A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying mechanism responsible for this imbalanced immune response remains incomplete. Processing bodies (PBs) are cytoplasmic membraneless ribonucleoprotein granules that control innate immune responses by mediating the constitutive decay or suppression of mRNA transcripts, including many that encode proinflammatory cytokines. PB formation promotes turnover or suppression of cytokine RNAs, whereas PB disassembly corresponds with the increased stability and/or translation of these cytokine RNAs. Many viruses cause PB disassembly, an event that can be viewed as a switch that rapidly relieves cytokine RNA repression and permits the infected cell to respond to viral infection. Prior to this submission, no information was known about how human coronaviruses (CoVs) impacted PBs. Here, we show SARS-CoV-2 and the common cold CoVs, OC43 and 229E, induced PB loss. We screened a SARS-CoV-2 gene library and identified that expression of the viral nucleocapsid (N) protein from SARS-CoV-2 was sufficient to mediate PB disassembly. RNA fluorescent in situ hybridization revealed that transcripts encoding TNF and IL-6 localized to PBs in control cells. PB loss correlated with the increased cytoplasmic localization of these transcripts in SARS-CoV-2 N protein-expressing cells. Ectopic expression of the N proteins from five other human coronaviruses (OC43, MERS, 229E, NL63 and SARS-CoV) did not cause significant PB disassembly, suggesting that this feature is unique to SARS-CoV-2 N protein. These data suggest that SARS-CoV-2-mediated PB disassembly contributes to the dysregulation of proinflammatory cytokine production observed during severe SARS-CoV-2 infection.

In December 2019, SARS-CoV-2 emerged causing the COVID-19 pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises ACE2 and TMPRSS2 host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147, and GRP78 may also function as receptors for SARS-CoV-2. To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples. We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147, and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa. Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternate receptors for SARS-CoV-2 exist to facilitate initial host cell infection., ACE2 gene and protein expression is low to absent in airway and alveolar epithelial cells in human lungs. Our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung or other receptors for SARS-CoV-2.

The role of bats in the enzootic cycle of Lyme disease and relapsing fever-causing bacteria is a matter of speculation. In Canada, Borrelia burgdorferi sensu stricto (ss) is the genospecies that is responsible for most cases of Lyme disease in humans. In this study, we determined if big brown bats, Eptesicus fuscus, have been exposed to spirochetes from the genus Borrelia. We collected serum from 31 bats and tested them for the presence of anti-Borrelia burgdorferi antibodies using a commercial enzyme-linked immunosorbent assay (ELISA). We detected cross-reactive antibodies to Borrelia spp. in 14 of 31 bats. We confirmed the ELISA data using a commercial immunoblot assay. Pooled sera from ELISA-positive bats also cross-reacted with Borrelia antigens coated on the immunoblot strips, whereas pooled sera from ELISA-negative bats did not bind to Borrelia spp. antigens. Furthermore, to identify if bat ectoparasites, such as mites, can carry Borrelia spp., we analyzed DNA from 142 bat ectoparasites that were collected between 2003 and 2019. We detected DNA for the Borrelia burgdorferi flaB gene in one bat mite, Spinturnix americanus. The low detection rate of Borrelia burgdorferi DNA in bat ectoparasites suggests that bats are not reservoirs of this bacterium. Data from this study also raises intriguing questions about Borrelia infections in bats, including the role of humoral immunity and the ability of bats to be infected with Borrelia burgdorferi. This study can lead to more sampling efforts and controlled laboratory studies to identify if bats can be infected with Borrelia burgdorferi and the role of bat ectoparasites, such as S. americanus, in the transmission of this spirochete. Furthermore, we outlined reagents that can be used to adapt ELISA kits and immunoblot strips for use with bat sera.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently emerged to cause widespread infections in humans SARS-CoV-2 infections have been reported

Streptococcus suis serotype 2 is an important porcine bacterial pathogen and emerging zoonotic agent mainly responsible for sudden death, septic shock, and meningitis, with exacerbated inflammation being a hallmark of the infection. However, serotype 2 strains are genotypically and phenotypically heterogeneous, being composed of a multitude of sequence types (STs) whose virulence greatly varies: the virulent ST1 (Eurasia), highly virulent ST7 (responsible for the human outbreaks in China), and intermediate virulent ST25 (North America) are the most important worldwide. Even though type I interferons (IFNs) are traditionally associated with important antiviral functions, recent studies have demonstrated that they may also play an important role during infections with extracellular bacteria. Upregulation of IFN-β levels was previously observed in mice following infection with this pathogen. Consequently, the implication of IFN-β in the S. suis serotype 2 pathogenesis, which has always been considered a strict extracellular bacterium, was evaluated using strains of varying virulence. This study demonstrates that intermediate virulent strains are significantly more susceptible to phagocytosis than virulent strains. Hence, subsequent localization of these strains within the phagosome results in recognition of bacterial nucleic acids by Toll-like receptors 7 and 9, leading to activation of the interferon regulatory factors 1, 3, and 7 and production of IFN-β. Type I IFN, whose implication depends on the virulence level of the S. suis strain, is involved in host defense by participating in the modulation of systemic inflammation, which is responsible for the clearance of blood bacterial burden. As such, when induced by intermediate, and to a lesser extent, virulent S. suis strains, type I IFN plays a beneficial role in host survival. The highly virulent ST7 strain, however, hastily induces a septic shock that cannot be controlled by type I IFN, leading to rapid death of the host. A better understanding of the underlying mechanisms involved in the control of inflammation and subsequent bacterial burden could help to develop control measures for this important porcine and zoonotic agent.

Positron emission tomography (PET) allows detection of functional changes in malignant tissue. Establishment of an immortalized immunocompetent breast cancer mouse model would provide a useful platform for the analysis of novel cancer treatment strategies. This study describes a comparative functional evaluation of murine breast cancer models established from polyoma virus middle T antigen (PyMT)-derived tumors using small animal PET imaging with [(18)F]FDG and [(18)F]FLT. Primary PyMT tumor-derived cells and a cell line derived from these tumors (MTHJ) were injected subcutaneously into immunocompetent FVB mice to generate breast cancer xenografts. Tumor growth rates were comparable in both models and tumors were analyzed after 4-5 weeks post-injection. [(18)F]FDG uptake in vitro followed a comparable trend in both models but reached higher uptake levels in primary PyMT cells vs. MTHJ cells after 120 min. At all time points, [(18)F]FLT uptake was significantly higher in MTHJ compared to primary PyMT cells. Dynamic small animal PET imaging with [(18)F]FDG revealed standardized uptake values (SUVs) of 2.5±0.1 (n=8) in tumors from primary cells and 2.8±0.4 (n=6) in MTHJ tumors after 60 min p.i.. The corresponding tumor-muscle-ratios were 9.3±1.5 and 10.4±0.9, respectively. Uptake of [(18)F]FLT resulted in slightly higher SUV(60min) in MTHJ tumors (1.1±0.1, n=6) compared to tumors from primary cells (SUV(60min)=0.9±0.05, n=8, p=0.07). The tumor-muscle-ratio was comparable in both tumors (2.1±0.2 and 1.8±0.1, respectively). The PET imaging data demonstrates that the functional profile of immunocompetent murine breast tumor model MTHJ remains the same as in primary-derived PyMT tumors in vivo. Metabolic and proliferative rates as assessed with [(18)F]FDG and [(18)F]FLT are comparable in both tumor models. The observed high SUV(60min) of 2.8±0.4 with [(18)F]FDG in MTHJ tumors allows one to monitor efficacy of therapeutic interventions connected with changes in metabolic response of the tumor by means of small animal PET.

A defining hallmark of the type 1 interferons (IFNs) is their ability to interfere with virus replication. As such, viruses have evolved diverse mechanisms to subvert the antiviral effects of interferons. Herpes simplex virus type 1 (HSV-1) is a large deoxyribonucleic acid virus best known for its ability to cause cold sores in infected individuals. In cultured cells, HSV-1 is relatively resistant to the effects of IFNs. Plaque reduction assays were used to determine that the immediate early HSV-1 gene product ICP0 functions in part to ensure successful replication in the presence of an IFN-induced antiviral response. Northern blot analysis showed that in the absence of ICP0, HSV-1 transcript accumulation is significantly decreased. Finally, nuclear run-on assays demonstrated that ICP0 functions to overcome IFN-induced blocks to virus transcription. The methods used in these studies are described in detail in this section.

IL-17 and its receptor IL-17RA have received much attention as the defining cytokine of a new class of T helper cells termed Th17. Mounting evidence suggests that IL-17 causes pathology in autoimmunity, but surprisingly little is known about mechanisms of IL-17 signal transduction. Although produced by T cells, IL-17 activates genes typical of innate immune cytokines such as TNFα and IL-1β, despite little sequence similarity in their respective receptors. A prior bioinformatics study predicted a subdomain in IL-17-family receptors with homology to a Toll/IL-1R (TIR) domain, termed “SEFIR.” However, the SEFIR domain lacks motifs critical for bona fide TIR domains, and its functionality has never been verified. Here, we used a reconstitution system in IL-17RA-null fibroblasts to map functional domains within IL-17RA. We demonstrate for the first time that the SEFIR domain mediates signaling, independently of TIR adaptors such as MyD88 and TRIF. Moreover, we identified a novel “TIR-like loop” (TILL) also required for activation of NF-κB, NF-κB-dependent genes and upregulation of C/EBPβ and C/EBPδ. We further identified a distinct domain required for activation of C/EBPβ and induction of a subset IL-17 target genes. This is the first structure-function analysis of any IL-17R superfamily receptor, and reveals unique structural and functional properties in IL-17 signaling.

In non-infectious settings with minimal inflammatory stimulus, CD4 T cell help is needed for dendritic cells (DC) to cross-prime antigen in order to elicit a CD8 T cell response. Activated CD4 T cells express CD40-ligand (CD40L) and lymphotoxin-α1β2 (LTαβ), which interacts with their respective TNF receptors, CD40 and lymphotoxin-β receptor (LTβR) on DC, thereby significantly increases the DC immunogenic potential. Our early work showed that LTβR signaling induces Type I IFN expression in DC which facilitates CD8 T cell expansion against soluble protein antigen. In this study, we identified that LTβR signals through TNF receptor-associated factor (TRAF)-3 for interferon regulatory factor (IRF)-3 phosphorylation and Type I IFN production. Furthermore, using an inducible diabetes model through the transfer of activated DC, we found that LTβR signaling on DC is absolutely required for the proper priming of CD8 T cells and diabetes induction. We further showed that LTβR-induced Type I IFN affect the expression of the adhesion molecule VLA-4 on antigen specific CD8 T cells and it affects their ability to infiltrate into the pancreas. LTβR deficient DC fail to induce diabetes, and the provision of exogenous IFN-α was sufficient to rescue the diabetes status. Together, these results describe a novel role for LTβR in the induction of Type I IFN by DC, and demonstrate its relevance in an autoimmune setting.

We showed previously that histone deacetylase (HDAC) inhibition with MS-275 in the context of boosting oncolytic immunotherapy can drive heightened antitumor responses, leading to increased survival in mouse intracranial melanoma models. However, it is currently unclear how the co-administration of MS-275 directly impacts tumor growth. Here, we investigated the role of MS-275 in preventing the outgrowth of antigen-deficient tumor variants as a result of suboptimal treatment protocols. By adoptively transferring tumor antigen-specific memory T cells (Tm) that were expanded in vivo with recombinant Vesicular Stomatitis Virus (VSV-gp33), we observed complete regression of 5-day old, intradermal B16-gp33 tumors (B16-F10 overexpressing the LCMV GP33-41 epitope); however, the tumors relapsed within a month of treatment. Relapsing tumor explants were able to grow in mice that were prophylactically immunized with recombinant Adenovirus (Ad-gp33), indicating that the tumor could no longer be recognized. Strikingly however, there was zero tumor recurrence if MS-275 was co-administered with Tm and VSV-gp33, suggesting that MS-275 may prevent the emergence and/or escape of antigen loss variants. Such a benefit is lost if the administration of the drug is delayed as little as five days post VSV treatment, suggesting that its synergistic effects coincide with early immune responses and oncolytic activity. Furthermore, transplantation studies of relapsing tumor explants showed that combination treatment was unable to provide tumor protection, confirming that the mechanisms by which MS-275 prevents tumor recurrence are unlikely through direct up-regulation of antigen presentation in low- or non-antigen-expressing variants in vivo. Indeed, CD4 depletion in the absence of MS-275 resulted in sustained tumor regression, implying that immunoregulatory cells such as CD4+ Treg play a prominent role in sustaining tumor regression. Moreover, MS-275 modulates the phenotypic status of tumor-infiltrating MDSCs toward the differentiation of inflammatory macrophages. Taken together, the data suggests that combination therapy with HDACi with oncolytic immunotherapy mediates a synergized immune attack against the tumor through subversion of immunomodulatory mechanisms. Master of Science in Medical Sciences (MSMS)

Based on the cancer stem cell (CSC) model, tumors develop as a hierarchy, much like normal tissue. At the apex, CSCs are capable of self-renewal and differentiating into non-tumorigenic cells that form the bulk of the tumor. CSCs have been isolated for many types of cancer based on cell surface marker expression. For instance in human breast carcinoma, the CD44+ CD24-/low EpCAM+ population are enriched in CSCs. These cells are more resistant to traditional chemo and radiation therapy relative to the bulk of the tumor. As such, many believe CSCs to be responsible for relapse and metastasis events. Hence, a more targeted therapy towards breast CSCs can prove to be very effective. EpCAM has been shown to be a reliable, albeit not exclusive, marker of breast CSCs. A more thorough understanding of EpCAM’s function can provide new angles for designing therapeutic agents. Originally thought to be a mere adhesion molecule, EpCAM is now known to derive a signalling pathway that promotes transcription in its target genes. We aimed to provide further insight into this novel pathway, by manipulating EpCAM’s expression and function in MCF7 breast cancer cell line. We designed a dominant negative allele of the EpCAM protein to hinder the activity of the endogenous EpCAM. However, this transgene offered no significant effect on our cell line. Furthermore, we used RNA interference technology to reduce EpCAM expression. The introduction of the EpCAM short hairpin RNA constructs into our cell line had inhibitory effects on transcription reporter expression, cellular growth, adherent colony and unattached sphere formation. The inhibition of EpCAM in MCF7 spheres was followed by signs of cell death, differentiation and an epithelial mesenchymal transition process. Sphere cultures are used because they are enriched in cancer stem cells. The adverse effects of EpCAM inhibition in MCF7 spheres provides further evidence as to the role of this protein in cancer stem cells. Master of Science (MSc)

Allergic atopic asthma is a respiratory condition that involves immune responses to specific allergens resulting in coughing, wheezing, shortness of breath and tightness in the chest. During an atopic asthmatic attack, the immune system initiates cellular infiltration of lymphocytes and eosinophils, airway hyper-responsiveness and ECM remodeling, which manifests in lung dysfunction in chronic disease. ASMC have recently been shown to play a role in the inflammatory processes of asthma through the production of inflammatory mediators. Various cytokines and chemokines serve as stimulants for these pathways and therefore require further attention to examine inflammatory signaling. OSM, a member of the gp130 family of cytokines, is secreted by inflammatory cells and has been detected in the sputum of asthmatics. Previous findings have established the potential of OSM in induction of lung inflammation, its role in increasing ECM, and its potential role in asthma. Viral or bacterial infections cause asthma exacerbations which result in increased severity of symptoms. The innate immune system relies on pattern recognition receptors including the TLRs to recognize invading pathogens and activate cells such as macrophages and natural killer cells. Although there are a number of these TLRs, this project will focus on the role of TLR3 and TLR4 in ASMC. I generally hypothesized that OSM markedly increases lung cell airway smooth muscle cell responses to external stimulae, such as products of bacteria or viruses that activate toll-like receptors. This exacerbates inflammation and extracellular matrix remodeling which contributes to pathology in asthmatic patients. Findings in this thesis have demonstrated that OSM stimulation increases the production of various cytokines and chemokines and growth factors seen in asthma. Co-stimulations with OSM and TLR-ligands augmented the production of a variety of these inflammatory mediators in comparison to ligands alone. TLR responses were shown to be associated with TLR expression, at both the mRNA and protein level, as well through the activation of the JAK-STAT and NFκB pathways. These findings implicate ASMC in immunomodulatory roles in response to TLR-ligands and OSM, and could play a role in the increased severity of asthma seen during exacerbations. Master of Science (MSc)

Crohn’s Disease is a chronic inflammatory bowel disease characterized by an overzealous immune response to a microbial trigger in genetically susceptible individuals. Although this microbial trigger is unknown, Escherichia coli with adherent and invasive properties (Adherent-Invasive Escherichia coli, AIEC) is preferentially enriched in a proportion of Crohn’s Disease patients. AIEC can adhere to and invade intestinal epithelial cells and replicate intracellularly within epithelial cells and macrophages in vitro. One important colonization factor expressed by AIEC is the type 1 fimbrial adhesin protein FimH. FimH mediates colonization of CEABAC10 transgenic mice and can bind several host cell receptors including the macrophage receptor CD48 in vitro indicating a potential role for FimH in macrophage interaction. However, it was not known whether FimH contributed to phagocytosis of AIEC or colonization of wild-type mice. Here we show that FimH enhances early intracellular AIEC levels in vitro and colonization in vivo. We found that deletion of fimH may reduce intracellular AIEC burden at 2 hours post-infection and that this effect was modulated by bacteria opsonisation. Using a competitive index assay, we show that a ΔfimH mutant is unable to chronically colonize CD-1 mice at the same levels as the parental strain. Our results demonstrate that FimH is an important AIEC colonization factor and may increase interaction with macrophages. Identifying factors such as FimH which contribute to colonization and persistence will further our understanding of AIEC survival strategies within the host. Development of therapeutics targeting FimH may provide a means to reduce harmful bacteria overgrowth particularly after surgical intervention. Master of Science (MSc)

Class A scavenger receptors are multifunctional transmembrane glycoproteins that mediate macrophage functions like phagocytosis and endocytosis. The macrophage receptor with collagenous structure (MARCO) is one such receptor. It has been shown that the extracellular cysteine-rich domain of MARCO is responsible for ligand binding, but the role of the cytoplasmic domain in ligand uptake is unclear. The aim of the studies presented in this thesis is to characterize the role of the cytoplasmic domain of MARCO and to characterize the molecular pathway of MARCO-mediated endocytosis. Full-length human MARCO (hMARCO) and Δ1-34hMARCO, which lacks the first thirty-four amino acids were created in order to determine whether amino acids 1-34 contained residues required for receptor internalization and surface expression. The constructs were stably expressed in HEK293T cells and found to have similar levels of surface expression and same rate of internalization without ligand. Interestingly, hMARCO, but not Δ1-34hMARCO, surface expression was up-regulated upon ligand incubation. In order to ascertain the importance of clathrin, dynamin and actin in MARCO-mediated endocytosis, specific endocytic inhibitors were used. MARCO-mediated ligand uptake was inhibited when clathrin and actin polymerization and, dynamin functions were impaired by these inhibitors. Furthermore, ligand uptake by Δ1-34hMARCO-expressing HEK293T was insensitive to inhibitors of clthrin and dynamin but not inhibitors of actin. In conclusion, MARCO mediates endocytosis via a clathrin-mediated, dynamin-dependent pathway that involves actin. Amino acids 1-34, are required clathrin and dynamin but not actin functions during MARCO-mediated endocytosis. Additionally, amino acids 1-34 might also be important for MARCO recycling but not receptor internalization or surface expression. Master of Science (MSc)