T-2 toxin (T-2), produced by the genus Fusarium, is a cytotoxic trichothecene mycotoxin, a feed contaminant, and has been shown to be immunomodulatory. It is suspected that T-2-associated immunomodulation is mediated partly through the hypothalamic-pituitary-adrenal axis. The presence of endotoxin, a bacterial product capable of activating the hypothalamic-pituitary-adrenal axis as well as the levels of several hormones, also associated with activation of the hypothalamic-pituitary-adrenal axis, were determined in both vehicle- and toxin-treated animals. Endotoxemia was evident twenty-four hours after a single oral exposure to T-2. Blood levels of adrenocorticotropic hormone and corticosterone, parameters of the stress response, also increased twenty-four hours after T-2 exposure. Hypothalamic norepinephrine and serum corticosterone levels increased in a dose-related manner after two weeks of T-2 exposure. An increased corticosteroid level was associated with thymic involution leading potentially to decreased T-dependent antibody response, a known effect of T-2. The effects of exposure to T-2 on the development of both T-dependent and T-independent antibody response were determined in nonoperated, sham-operated and adrenalectomized mice. T-2 decreased the antibody response to a T-dependent antigen and increased a T-independent response. The effects of T-2 were partially nullified by adrenalectomy. These results provide a further confirmation of the postulate that the hypothalamic-pituitary-adrenal axis plays an important role in T-2 toxin-immunomodulation. In vitro studies were undertaken to investigate the direct effects of T-2 on various populations of lymphatic cells. Exposure to T-2 after twenty-four hours caused an increase in the uptake of 3H-thymidine by mouse splenic cells. Pokeweed mitogen stimulation also increased in this system; the response to lipopolysaccharide increased to a lesser extent. However, T-cell responses to phytohaemagglutinin and concanavalin A (Con A) decreased. Thymic cells were also sensitive to T-2. The possibility of pharmacological activity of T-2 with thymocytes was investigated. Both specific and nonspecific cell associations were observed. The association of T-2 with thymocytes was altered in the presence of dexamethasone, a synthetic corticosteroid. T-2 was shown to have both indirect as well as direct activities on the immune system. Endocrine dysfunction resulting from chronic stress and possible pharmacologic activity of T-2 provide the impetus for further investigations.

No description available.

In the last 150 years, tremendous headway has been made in our understanding of the human immune system. Pioneers in the field such as Paul Ehrlich, Elie Metchnikoff, Louis Pasteur, Robert Koch and Walter Reed carried out seminal studies that established the groundwork for our understanding of humoral and cellular immunity in humans. However, this direct line of evidence into human immunology was diverted in the mid-20th century with the adoption of a model which allowed for investigators to use a reductionist-approach with the promise to resolve immunity at a molecular level. This revolutionary model was the scientific commercialization of various inbred strains of mice. It seems inconceivable how a four-legged nocturnal rodent managed to become the focus of billions of dollars of research to improve our understanding of human immunity. Nevertheless, this strange surrogate for human immunity did provide us with major conceptual advances in areas, such as identification of dendritic cell population heterogeneity, T cell help for B cell antibody production, MHC-restricted recognition of virusinfected cells, and even the discovery of cell types like NKT cells. However, these prior advances have now been prefaced with decades-worth of disappointing, non-translational findings. The best examples of such disappointments are in murine models of autoimmunity, cancer immunotherapy, and vaccinology where numerous studies have revealed promising outcomes in mice but were met with failure or limited success upon translation into humans. We do not look at this as a failure of the murine model; rather we consider it a call to arms to innovate in vitro surrogates to examine human immunity when otherwise bound by ethical limitation from working directly in humans. To overcome these challenges, we developed a system to interrogate novel immunogens that begins by generating human dendritic cells (DCs), a cell type necessary to mounting a protective immune response. DCs for research and clinical applications are typically derived from purified blood monocytes that are cultured in a cocktail of cytokines for a week or more. Because it has been iv suggested that these cytokine-derived DCs may be deficient in some important immunological functions and might not accurately represent antigen presenting cell (APC) populations found under normal conditions in vivo, there is an interest in developing methods that permit the derivation of DCs in a more physiologically relevant manner in vitro. Here, we describe a simple and reliable technique for generating large numbers of highly purified DCs that is based on a one-way migration of blood monocytes through a layer of human umbilical vein endothelial cells (HUVECs) that are cultured to confluency in the upper chamber of a Transwell device. The resultant APCs, harvested from the lower Transwell chamber, resemble other cultured DC populations in their expression of major histocompatibility (MHC) and costimulatory molecules, ability to phagocytose protein antigens and capacity to trigger primary antigen-specific T cell responses. This technique offers several advantages over the standard method of in vitro cytokine-driven DC development, including: (1) the rapidity of this approach, as DC differentiation occurs in only 2 days, (2) the differentiation process itself, which is more akin to the development of DCs under physiologic conditions and (3) the cost effectiveness of the system, since no monocyte pre-selection is required and DC development occurs in the absence of expensive recombinant cytokines. Taken together, this approach allows for the exploration of novel immunogens utilizing a physiologically representative population of APCs enriched from circulating blood. The outbreak of the swine-origin H1N1 influenza in the spring of 2009 took epidemiologists, immunologists, and vaccinologists by surprise and galvanized a massive worldwide effort to produce millions of vaccine doses to protect against this single virus strain. Of particular concern was the apparent lack of pre-existing antibody capable of eliciting cross-protective immunity against this novel virus, which fueled fears this strain would trigger a particularly far-reaching and lethal pandemic. Given that disease caused by the swine-origin virus was far less severe than expected, we hypothesized v cellular immunity to cross-conserved T cell epitopes might have played a significant role in protecting against the pandemic H1N1 in the absence of cross-reactive humoral immunity. We collaborated with bioinformaticians to develop an immunoinformatics approach to predict CD4+ T cell epitopes conserved between the 2008-2009 seasonal H1N1 vaccine strain and pandemic H1N1 (A/California/04/2009) hemagglutinin proteins that could act as novel immunogens and function as potential vaccine candidates or compliments to current vaccine formulations. We examined these peptides using T cells from human donors not exposed to the pandemic virus demonstrating that pre-existing CD4+ T cells can elicit cross-reactive effector responses against the pandemic H1N1 virus. As well, we showed the computational tools created by our collaborators were 80-90% accurate in predicting CD4+ T cell epitopes and their HLA-DRB1-dependent response profiles in donors that were chosen at random for HLA haplotype. Combined, these results confirm the power of coupling immunoinformatics to define broadly reactive CD4+ T cell epitopes with a highly sensitive in vitro model to verify these in silico predictions as a means to understand human cellular immunity, including cross-protective responses, and to define CD4+ T cell epitopes for potential vaccination efforts against future influenza viruses and other pathogens. It is thought that utilizing highly conserved peptides as novel immunogens of cellular immunity for future vaccination strategies may require an adjuvant for efficacy. However, the FDA has approved the use of only two adjuvant compounds (Alum or MPL®) which may not be compatible or offer effective immune enhancement in novel vaccine preparations, thereby soliciting the need for novel adjuvants. Nanoparticles have since been a topic of adjuvant potential. Nanoparticles harbor great potential because they possess unique physicochemical properties compared to their larger counter parts as a result of quantum-size effects and their inherent large surface area to volume ratio. These physicochemical properties govern how a nanoparticle will behave in its environment. However, vi researchers have only just begun to catalogue the biological effect these properties illicit. Moreover, little is known about the interaction between the immune system and NPs. However, in light of the recent development in new adjuvants that involves composites and coatings of polymers, lipids, ligands, TLR agonist, the ability of a simple metal oxide nanopowder to effectively induce or couple immunomodulation would provide researchers a basic alternative to costly and complex adjuvant development. Considering the evidence suggesting NPs can act as immunopotentiators, we questioned whether these materials can act not only as innate adjuvants, but as novel immunogens to cellular immunity. To accomplish this, we under took a set of studies to investigate any nanoparticle size-induced effects using TiO2, one of the most widely manufactured nanoparticles, as a model. We explored titanium dioxide synthesized into its three most commonly nanoarchitectures: anatase (7-10 nm), rutile (15-20 nm), and nanotube (10-15 nm diameters, 70-150 nm length) in comparison to a micron-sized formulation. We used the fully human autologous MIMIC® immunological construct has been utilized as a predictive, nonanimal alternative to diagnose nanoparticle immunogenicity. Cumulatively, treatment with titanium dioxide nanoparticles in the MIMIC® system led to elevated levels of proinflammatory cytokines and increased maturation and expression of costimulatory molecules on dendritic cells. Additionally, these treatments effectively primed activation and proliferation of naïve CD4+ T cells in comparison to dendritic cells treated with titanium dioxide microspheres, characteristic of an in vivo inflammatory response, providing evidence of a size induced difference between the nano-sized and micron-sized material, revealing novel immune cell recognition and activation by a crystalline nanomaterial in a size-dependent manner. Having identified nanomaterial size as a contributing feature of nanoparticle induced immunopotentiation, we became interested if additional physicochemical properties such as surface vii reactivity or catalytic behavior could also be immunostimulatory. Moreover, because we witnessed a stimulatory effect to dendritic cells following nanoparticle treatment, we were curious how these nanoparticle-touched dendritic cells would impact adaptive immunity. Since TiO2 acts as an oxidant we chose an antioxidant nanoparticle, CeO2, as a counterpart to explore how divergent nanoparticle surface reactivity impacts innate and adaptive immunity. We focused on the effect these nanoparticles had on human dendritic cells and TH cells as a strategy towards defining their impact to cellular immunity. Combined, we report that TiO2 nanoparticles potentiate DC maturation inducing the secretion of IL- 12p70 and IL-1B, while treatment with CeO2 nanoparticles induced IL-10, a hallmark of suppression. When delivered to T cells alone TiO2 nanoparticles induced stronger proliferation in comparison to CeO2 which also stimulated TReg differentiation. When co-cultured in allogeneic T cell assays, the materials directed alternate TH polarization whereby TiO2 drives largely a TH1 dominate response, whereas CeO2 drove a TH2 bias. Combined, we report a novel immunomodulatory capacity of nanomaterials with catalytic activity. While unintentional exposure to these nanomaterials could pose a serious health risk, development and targeted use of such immunomodulatory nanoparticles could provide researchers with new tools for novel adjuvant strategies or therapeutics.

Polysaccharides are complex biopolymers that play essential roles in the biological systems including energy storage, structural support, lubrication, and signal transduction. Despite their importance, the synthesis of polysaccharides has proven to be very challenging due to the presence of multiple hydroxyl groups and difficulty in controlling the stereochemical outcome of glycosylation reactions. As a conventional chemical method to synthesize polysaccharides, ring-opening polymerization of anhydrosugars enables the synthesis of stereoregular α-(1→6)-linked polysaccharides, but is less effective in preparing polysaccharides with other linkages. Enzymatic polymerizations have also been explored, however, these methods typically require expensive monomers, and suffer from a narrow scope of enzymes and small scale of reactions. The limited approaches to polysaccharides have inspired chemists to synthesize polysaccharide mimetics with achiral linkages that can be constructed efficiently. Poly-amido-saccharides (PASs) are a new type of saccharide polymers in which the O-glycosidic linkages in natural polysaccharides are replaced with (1→2)-amide linkages. With saccharide moieties inter-connected by amide bonds, PASs exhibit characteristics of both polysaccharides and polypeptides, such as possessing pyranose-backbones and lots of hydroxyl groups, and adopting a left-handed helical conformation. However, due to lack of sufficient terminal saccharide residues, previously synthesized glucose and galactose PASs display weak interactions with carbohydrate binding lectins and receptors, limiting their applications in biomedical and pharmaceutical fields. Herein, the design and synthesis of PASs with novel structures and properties is described. By pre-installing the stereochemistry in the monomer, Altrose PASs (Alt-PASs) with β-(1→2)-amide linkages are prepared via ring-opening polymerization of an altrose-based β-lactam followed by debenzylation. Circular dichroism shows that Alt-PASs adopt a right-handed helical conformation in aqueous solution. Via the polymerization of disaccharide-based β-lactams, two PASs with either 4-O-α-D-glucose branches (Mal-PASs) or 6-O-β-D-glucose branches (Gen-PASs) are obtained. Biological studies reveal that Mal-PASs are multivalent ligands to lectin Concanavalin A, while Gen-PASs activate RAW 264.7 macrophage cells by enhancing the secretion of TNF-α and NO. The anionic ring-opening polymerization of sugar-based β-lactams is a useful method to synthesize well-defined polysaccharide mimetics, and this method expands the current repertory of approaches available to complex saccharide polymers with biological activities. / 2021-02-15T00:00:00Z

No description available.

No description available.

The threat from infectious diseases dates as far back as prehistoric times. Pathogens continue to pose serious challenges to human health. The emergence and spread of diseases such as HIV/AIDS, Severe Acute Respiratory Syndrome (SARS), avian influenza, and the threats of bioterrorism have made infectious diseases major public health concerns. Despite many successes in the discovery of anti-infective medications, the treatment of infectious diseases faces serious challenges, which include (i) the emergence and reemergence of infectious pathogens, (ii) the ability of pathogens to adapt and develop resistance to drugs, and (ii) a shortage in the development and discovery of new anti-infective drugs. Host-Oriented Broad-Spectrum (HOBS) treatments have the promising potential to alleviate these problems. The HOBS treatment paradigm focuses on finding drug targets in human host that are simultaneously effective against a wide variety of infectious agents and toxins. In this dissertation, we present a computational approach to predict HOBS treatments by integrative analysis of three types of data, namely, (a) gene expression data representing host responses upon infection by a pathogen, (b) annotations of genes to pre-defined biological pathways and processes, and (iii) genes that are targets of known drugs. Our methods combine gene set-level enrichment with biclustering. We applied our approach to a compendium of gene expression data sets derived from host cells exposed to bacterial or to fungal pathogens, to functional annotation data from multiple databases, and to drug targets from DrugBank. We present putative host drug targets and drugs with extensive support in the literature for their potential to treat multiple bacterial and fungal infections. These results showcase the potential of our computational approach to predict HOBS drug targets that may be effective against two or more pathogens. Our study takes a clean-slate approach that promises to yield unsuspected or unknown associations between pathogens and biological processes, and thus discern candidate gene/proteins to be further probed as HOBS targets. Furthermore, by focusing on host responses to pathogens as captured by transcriptional data, our proposed approach stimulates host-oriented drug target identification, which has potential to alleviate the problem of drug resistance. / Ph. D.

Influenza A virus (IAV) is generally associated with the seasonal malady that causes brief respiratory illness during the winter months, known simply as "the flu." Most otherwise healthy individuals will suffer from mild fever, congestion, headaches and myalgia that are resolved within 5-7 days of onset. However, there are nearly 500,000 influenza-related deaths that occur world-wide every year. Many of these casualties and patients hospitalized with influenza also test positive for bacterial pneumonia, the most common agent being Streptococcus pneumoniae. Although all individuals are subject to this viral:bacterial synergistic disease, the young, elderly, and immunocompromised are the most susceptible. Previous studies have shown that viral infection creates a prolonged hyper-responsive pro-inflammatory state in the lungs, which increases susceptibility to secondary bacterial infection. Lethality is due to detrimental pulmonary damage from a dysregulated host inflammatory response, known as the "cytokine storm." However, the nature of dual infection has not been well-studied in the elderly demographic. Therefore, we aim to better define this disease synergy in an aged mouse model and explore potential therapeutic alternatives that could be beneficial for the aged and other vulnerable populations. Sphingolipid modulation has emerged as a potential target to ameliorate the excessive inflammation (cytokine storm) elicited by highly pathogenic influenza. There is particular emphasis on sphingosine 1-phosphate (S1P) signaling, as well as control of intracellular S1P levels via sphingosine kinases (SK). Sphingolipids are involved in a multitude of cellular processes, and are tightly regulated by their metabolizing enzymes. We hypothesize that manipulation of sphingolipid signaling and alteration of the internal sphingolipid milieu will diminish the inflammatory response elicited by IAV infection. Using fluorescence-activated cell sorting (FACS), real-time PCR and cytometric bead array (CBA) analysis, we evaluated the immunomodulatory effects of systemic sphingosine analog treatment within the lung microenvironment under homeostatic and influenza-infected conditions. FTY720 treatment caused transient, but significant lymphopenia, influx of neutrophils and efflux of macrophages in the lungs, which was enhanced during a mild influenza infectionGene expression in the lungs was generally unaltered, but protein levels showed increases in specific influenza-induced cytokines, suggesting these treatments may have post-transcriptional effects on cytokine expression. To evaluate sphingolipid modulation in specific pulmonary cell types, we next observed the effects of these compounds and sphingosine kinase (SK) inhibitors in epithelial and alveolar macrophage-like cell lines. SK inhibitors and Enigmol demonstrated anti-viral effects in A549 cells, decreasing viral loads by up to 1.5 logs. Real-time PCR and CBA analysis further demonstrated that these effects were associated with alterations in key cytokine expression, including CCL2, CCL5, CXCL10, IL-6, and IL-8. Collectively, these findings indicate that therapeutic sphingolipid modulation has the potential for creating a protective microenvironment in the lungs that could alleviate or even prevent viral:bacterial synergistic disease. / Master of Science

Yes / Bacterial cells sense their population density and respond accordingly by producing various signal molecules to the surrounding environments thereby trigger a plethora of gene expression. This regulatory pathway is termed quorum sensing (QS). Plenty of bacterial virulence factors are controlled by QS or QS-mediated regulatory systems and QS signal molecules (QSSMs) play crucial roles in bacterial signaling transduction. Moreover, bacterial QSSMs were shown to interfere with host cell signaling and modulate host immune responses. QSSMs not only regulate the expression of bacterial virulence factors but themselves act in the modulation of host biology that can be potential therapeutic targets. / Open Access Funding from the University of Dundee.Also supported by the University of Malaya High Impact Research Grants (UMC/625/1/HIR/MOHE/CHAN/01,A-000001-50001,and UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

Propionibacterium freudenreichii CIRM-BIA 129 (Pf) est une bactérie bénéfique utilisée comme levain fromager. Elle présente en outre de nombreuses potentialités probiotiques souche-dépendantes, dont la modulation de l’inflammation. Cette propriété résulte de la production de divers métabolites. Les protéines de surface S-layer (Slps), dont la protéine majoritaire SlpB, y jouent également un rôle immunomodulateur. Les propriétés « 2-en-1 », c’est-à-dire à la fois fermentaires et probiotiques, font de Pf un bon candidat pour développer des fromages fonctionnels, afin de prévenir les maladies inflammatoires intestinales. L’objectif de cette thèse était d’étudier l’impact de la matrice fromagère sur les propriétés immunomodulatrices de Pf, via ses protéines Slps, par comparaison à une culture sur ultrafiltrat de lait (UF). Les études conduites in vitro suggèrent que les bactéries provenant du fromage auraient une meilleure capacité de tolérance aux stress gastriques et duodénaux, mais elleauraient une moindre capacité de survie dans le côlon, par comparaison à des bactéries provenant d’une culture sur UF. De plus, la protéolyse digestive des protéines de surface améliore la survie de Pf dans le côlon. Parallèlement, l’étude de digestion in vitro a montré que la protéolyse des protéines de surface a seulement été réduite par la matrice fromagère. Cette protéolyse conduit à l’abolition des effets anti-inflammatoires des protéines Slps, qui ne sont pas exprimées de novo dans l’environnement colique. Ces résultats obtenus in vitro étaient cohérents avec l’étude in vivo qui a mont / Propionibacterium freudenreichii CIRM-BIA 129 (Pf) is a beneficial bacterium used as a cheese starter. It moreover displays versatile strain-dependent probiotic properties, including the modulation of inflammation. This property results from the production of various metabolites. S-layer surface proteins (Slps), including the major SlpB protein, also play an immunomodulatory role. The "2-in-1" properties, i.e. both fermentative and probiotic properties, make Pf a suitable candidate to develop functional cheeses, in order to prevent inflammatory bowel diseases. The aim of this thesis was to study the impact of the cheese matrix on the immunomodulatory properties of Pf, mediated by its Slps proteins, compared to a culture on milk ultrafiltrate (MUF). In vitro studies suggest that the bacteria from the cheese would have a better ability to tolerate gastric and duodenal stresses, but would have less ability to survive in the colon, compared to bacteria from a MUF culture. In addition, thethe digestive proteolysis of surface proteins improves survival of Pf in the colon. In parallel, the in vitro digestion study showed that proteolysis of surface proteins was only limited by the cheese matrix. This proteolysis leads to the abolition of the anti-inflammatory effects of Slps proteins, which are not de novo expressed in the colonic environment. These results, obtained in vitro, were consistent with the in vivo study, which showed that MUF culture and cheese delivered similar amounts of metabolically active bacteria to the piglets’ colon. This in vivo study showed, however, that t