β-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of β-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma β-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and β-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of β-glucan and the results confirmed that β-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

(1→3)-β-D-Glucans are carbohydrate polymers that are present in the cell wall of various fungi and bacteria; they are pathogen associated molecular patterns that circulate during infection and modulate immunity. Our laboratory has previously established the pharmacokinetics of intravenously and orally administered glucans; the present studies investigated the renal excretion of (1→3)-β-D-glucans following intravenous and oral administration. Three fluorescently-labeled glucans were administered to adult male rats in the presence or absence of toxic challenge. Urine specimens were collected and analyzed by fluorescence spectroscopy, size-exclusion chromatography and GPC/MALLS. 71 ± 3% of fluorescence remained in the >5K MWCO fraction; this fraction showed a minor peak with a molecular mass (171 ± 11K) corresponding to injected glucan (~150K). Most excreted glucans were of lower molecular mass (13 ± 8.5K), indicating most (1→3)-β-D-glucans are excreted by the kidneys as smaller polysaccharides. The presence of urinary glucans may be an important indicator of fungal infection.

Using various synthetic strategies, we designed several libraries of novel polysaccharide mixed ester derivatives for oral drug delivery applications. Cellulose and cellulose esters have been extensively studied and utilized for different applications such as separation membranes, sustainable plastics, and enteric coatings in oral drug delivery carriers. We sought to exploit the ring-opening of cyclic anhydrides, succinic and glutaric anhydride, to append ω-carboxyl groups to commercially available cellulose and cellulose ester substrates. We used scalable synthetic strategies and widely available and cheap reagents to show a proof-of-concept for the manufacturability of these different polymer derivatives. We incorporated different degrees of substitution of ω-carboxyl groups to impart a range of water solubility in these polymers. The derivatives displayed excellent <i>T</i>g values for ASD applications, adequate water solubility, and good amphiphilic properties. We designed very effective amorphous solid dispersion (ASD) oral drug delivery polymers that prevented recrystallization of felodipine for hours and had excellent congruent polymer-drug release from the formulation at 20% drug loading. During the ring-opening reactions of the cellulose derivatives with glutaric anhydride we discovered that crosslinking and gelation can occur, especially with cellulose and cellulose ester substrates with a high degree of substitution (DS) of hydroxy groups. We isolated and characterized these gelled products using rheology, and solid-state 1D and 2D NMR spectroscopy, to evaluate whether the gels are physical or chemical in nature and proposed a mechanism for gelation. We determined that the gels are mostly physical but can proceed to chemical crosslinking over time. We designed a library of cellulose ester derivatives, and we investigated their performance as amorphous solid dispersion (ASD) drug delivery vehicles for the lipophilic drug felodipine, through <i>in vitro</i> experiments. Aside from felodipine, many other active pharmaceutical ingredients (APIs) are also highly crystalline and poorly water-soluble. ASDs are used to disrupt the crystalline packing of these drugs through dispersing them in amorphous polymeric carriers, facilitating their water-solubility, and preventing their recrystallization. We showed that our polymers performed remarkably well in the <i>in vitro</i> studies and inhibited crystallization of model compound felodipine for several hours while providing optimal drug release, affording highly promising ASD polymers. If company formulators are unable to develop an effective oral-delivery carrier to prevent a drug from recrystallizing, then the drug cannot be tested in <i>in vivo</i> toxicology studies, and therefore cannot be brought to market because of its poor aqueous solubility and subsequent low bioavailability. To test the robustness of our polymers, we also performed <i>in vitro</i> ASD experiments at the pharmaceutical company AbbVie with their most rapidly crystallizing pipeline compounds, and several commercially available drugs (Compound A, axitinib, and ziprasidone). We demonstrated that our polymers could also prevent drug recrystallization with these rapid crystallizers, outperforming commercial polymers like FDA-approved hydroxypropyl methyl cellulose acetate succinate (HPMCAS (MF)), even at exceptionally high drug loading ratios of 40 times the concentration of polymer. α-1,3-Glucans are an emerging class of polysaccharides and are structurally different than cellulose due to their α (1→3) linkage versus the cellulose β (1→4) glycosidic linkage. We demonstrated that we could modify these derivatives using a variety of esterification strategies and TEMPO-mediated C6 selective oxidation, affording a myriad of different novel polymer products, some of which are structural analogs of the cellulose ester derivatives we previously created. The polymers had higher <i>T</i>g values than the cellulose ester polymers, which may be useful for applications where heat resistance is desired. In the future, we will screen some of these α-1,3-glucan derivatives with poorly water-soluble enzalutamide, posaconazole and celecoxib model drugs, to evaluate their crystallization inhibition properties and the influence of polymer morphology upon structure-property relationships. We expect that these synthetic polymer strategies will offer scalable routes to novel ASD polymers, which we demonstrated to be highly effective drug crystallization inhibitors against a variety of different hydrophobic pharmaceutical compounds. / Doctor of Philosophy / Polysaccharides are polymers comprised of many linked sugar molecules and are an incredibly abundant and renewable resource. They are found everywhere in nature such as the wood from trees, the shells of crabs, the exoskeletons of bugs, and the mushrooms that sprout in damp forests. The research in this dissertation focuses on the use and chemical modification of polysaccharides for designing new, polysaccharide-based oral drug delivery systems called amorphous solid dispersions (ASDs), which significantly aid in the solubility and bioavailability of important medications. We started with the chemical modification of cellulose, the most abundant plant polysaccharide on planet Earth, and previously modified commercial cellulose substrates (known as cellulose esters) to create novel polymers for ASDs. We successfully modified these polymers, characterized them, and evaluated their potential as oral drug delivery vehicles by formulating them with several different classes of potent drugs used to treat a variety of diseases such as hypertension and schizophrenia. We showed that our designed cellulose ester polymers kept these hydrophobic drugs water-soluble for long-enough so that they can be adequately absorbed in the human body through the gastrointestinal tract, significantly outperforming commercial polymers in many cases. During the chemical modification of the cellulose esters, we also observed that they were prone to form gels, and we investigated this gelation phenomena in more detail through rheometry, 1D and 2D solid-state nuclear magnetic resonance spectroscopy (similar in principle to the medical diagnostic method, magnetic resonance imaging or MRI). We discovered that these gels can be physically and/or chemically linked together, and that different gelation mechanisms can dominate depending on the polysaccharide substrate and the esterification reagent used. We extended our research to other polysaccharide derivatives called α-1,3-glucans, which can be sourced from fungi, and/or enzymatically synthesized in the lab. Using various synthetic esterification and oxidation chemical methods to functionalize this polysaccharide, we designed a library of entirely novel polymers with different physical structures relative to the cellulose ester polymers. The polymers displayed thermal properties that show promise in drug delivery vehicle applications and in applications where high heat resistance is required. Overall, we developed next-generation polymers for amorphous solid dispersion oral drug delivery applications. We displayed the versatility of using a select few chemistry strategies to create a variety of different polymers with very different physicochemical properties. We hope that this work will help researchers design sustainable, plant-based polymers for ASD applications and we hope to nurture future structure-function studies to improve ASD performance for the benefit of patients in need.

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In the last decade, the potential of β-glucan, a fungal cell wall component, to induce epigenetic and functional modification of innate immune cells, signified as trained innate immunity (TII) has been demonstrated in several pre-clinical and clinical studies. Parenteral administration of β-glucan has resulted in centrally induced TII in the bone marrow/circulating monocytes. Such trained innate immune cells play a critical role in protection against secondary infections. However, there are now indications that inducing local long-lasting immunity at mucosal barrier tissues such as the lung is warranted for protective immunity against respiratory pathogens. Currently, it remains unclear whether respiratory mucosal administration of β-glucan will induce long-lasting resident-memory macrophages and TII and if so, what are the underlying mechanisms of development and maintenance of memory macrophages at respiratory mucosa. To address this, and kinetics of immune responses in the lung were studied. Profound changes in airway macrophage (AM) pools were observed starting from 3 days post-exposure, which was associated with monocyte recruitment, and this was followed by a series of phenotypic shifts in AMs. The altered AM phenotype profile persisted for up to 8 weeks post-exposure. Importantly, β-glucan-trained AMs demonstrated heightened MHC II expression, enhanced responses to secondary stimulation and improved capacity to perform bacterial phagocytosis. Furthermore, mice with, β-glucan-trained AMs displayed higher rates of survival and improved bacterial control, in the lung and periphery, following a lethal S. pneumoniae infection. Our findings together indicate that a single intranasal delivery of β-glucan is able to train AMs. Further work into epigenetics, metabolism, and the contribution of AMs in protection is needed. / Thesis / Master of Health Sciences (MSc) / The immune system has been classically divided into two major compartments known as the innate and adaptive immune system. For decades, the predominant consensus amongst the field was that only the adaptive immune system can form memory against any pathogens encountered. It has been well established that plants and invertebrates only possess an innate immune system and still show boosted responses and enhanced protection against previously encountered as well as new pathogens. Recently, such capacity for innate immune memory has also been demonstrated in humans and pre-clinical animal models. Innate immune memory provides non-specific, broad- spectrum protection whereas adaptive memory is specific to a singular pathogen. Inducing broad-spectrum protection can be crucial for the future of human medicine. Activation of both adaptive and innate immune arms could prove to be extremely beneficial in vaccination strategies. Through the use of a pre-clinical model, we have found that administering β-glucan, a component of fungal cell wall, directly into the lung significantly alters the phenotype and functionality of lung immune cells, and also provides enhanced protection against a heterologous infection.

Resumo: Exopolissacarídeos do tipo β-glucanas são polímeros produzidos por uma grande variedade de microrganismos e podem possuir diferentes propriedades físicas, químicas e aspectos estruturais. Esses biopolímeros apresentam atividades biológicas interessantes (antitumor, antiviral, anticoagulante) e aplicações comerciais como produtos em alimentos, cosméticos e farmacêuticos. Entretanto, para a aplicação dessas moléculas, é necessário primeiramente o conhecimento de suas estruturas químicas. Assim, o objetivo deste trabalho foi a produção, caracterização química de quatro exopolissacarideos (EPSGRAVIOLA, EPSMANGA, EPSPINHA e EPSLARANJA) de isolados de Botryosphaeria obtidos de frutas tropicais em decomposição e crescidos em sacarose como única fonte de carbono, determinando o melhor EPS para realizar testes de atividade anticoagulante. A homogeneidade de cada EPS foi determinada por cromatografia de filtração em gel, os quais eluíram como um único pico. Hidrólise ácida total e análise por HPAEC/PAD mostrou glucose como constituinte básico. Dados de metilação e RMN de 13C indicaram que os EPSMANGA, EPSPINHA e EPSLARANJA são glucanas lineares unidas por ligações do tipo β(1®6) e o EPSGRAVIOLA é uma glucana com ligações β(1®3) e com ramificações em C-6 de resíduos glucopiranosídicos. O espectro de FT-IR mostrou uma banda em 891 cm-1, e a espectroscopia de 13C NMR mostrou que todas as ligações eram do tipo β. Estudos realizados com o corante Vermelho Congo indicaram que os EPS possuem conformação em tripla hélice. O EPSLARANJA, uma b- D-(1®6)-glucana, foi submetido a sulfatação visando induzir a atividade anticoagulante e melhorar a solubilidade da molécula em solução, importante para a atividade biológica. Espectros de FT-IR mostraram bandas em 808 and 1252 cm-1, indicando a entrada dos grupos sulfato e as análises de RMN de 13C mostraram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Exopolysaccharides (EPS) as β-glucans are polymers produced by a great variety of microorganisms and can possess different physical and chemical properties, and structural features. These biopolymers having interesting biological activities (antitumor, anti-viral, anticoagulant), and commercial applications in foods, cosmetics and pharmaceutical products. However, for the applications of these macromolecules, it is first necessary to understand their chemical structures. Therefore the goal of that study was the production, chemical characterization and biological activity of four exopolysaccharides (EPSGRAVIOLA, EPSMANGO, EPSPINHA and EPSORANGE) obtained from Botryosphaeria strains isolated from rotting tropical fruit grown on sucrose as carbon and the best EPS was used for the anticoagulant activity The homogeneity of each EPS was determined by gel filtration chromatography, which was eluted as a single peak. Total acid hydrolysis and HPAEC/PAD analysis of each EPS yielded only glucose. Data from methylation analysis and 13C NMR spectroscopy indicated that the EPSMANGO, EPSPINHA and EPSORANGE consisted of a linear chain of (1-6)- linked glucopyranosyl residues and EPSGRAVIOLA consisted of a main chain of glucopyranosyl (1-3) linkages substituted at O-6. FTIR spectra showed one band at 891 cm-1, and 13C NMR spectroscopy showed that all glucosidic linkages were of the β-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. The EPSORANGE, a β-(1®6)- D-glucan was submitted to a sulfation to induce anticoagulant activity and also to make this EPS more soluble, which is in favor to its biological action. The FT-IR spectrum showed bands at 808 and 1252 cm-1 indicating insertion of sulfonyl groups and the 13C NMR analysis showed that the sulfonyl groups were inserted mainly in C-4 of the b(1®6)-D-glucan... (Complete abstract click electronic access below) / Orientador: Roberto da Silva / Coorientador: Maria de Lourdes Corradi da Silva / Banca: Gabriela Alves Macêdo / Banca: Maria Inês Rezende / Banca: Jonas Contiero / Banca: Eleonora Cano Carmona / Doutor

Idiopathic Pulmonary Fibrosis (IPF) is a debilitating respiratory disorder that is characterized by a progressive decline in lung function. Originating through unknown etiology, it is essentially an unchecked wound healing response that causes the build-up of excessive scar tissue in the lung interstitial tissue with a heavy toll on the patient’s respiratory capacity. Pro-fibrotic alternatively activated macrophages (M2) have been linked as an important contributor to the fibrotic remodeling of the lung. Previous Ask research indicates that targeting M2 macrophages is possible through the use of the Dectin-1 receptor, a transmembrane cell surface receptor found in high abundance on M2 macrophages. Activating the Dectin-1 receptor through the use of beta-glucan, a ligand the receptor has a high affinity for, initiates a pro-inflammatory response within the naturally immunosuppressive macrophage and can alter its activity to be less fibrogenic. Our data suggest that M2 polarization of naïve macrophages can be inhibited in vitro by beta-glucan microparticles. Additionally, we have found that polarized M2 macrophages adopt M1-like characteristics when treated with beta-glucan microparticles, in a process that is largely Dectin-1 dependent. M2 cell surface marker CD206, increased levels of which are associated with rapidly progressing IPF, shows significantly decreased frequency of expression in M2 macrophages treated with beta-glucan microparticles. Our assessment for cell-specific uptake of beta-glucan microparticles suggests an important role of the Dectin-1 receptor for significantly increased uptake in murine wild-type M2 macrophages relative to their Dectin-1 knockout counterpart. The use of beta-glucan microparticles as a potential anti-fibrotic therapeutic was assessed in the bleomycin model of fibrotic lung disease. Mice given bleomycin and treated with beta-glucan displayed decreased soluble collagen content and TGFB expression within lung homogenate relative to fibrotic bleomycin control mice. Overall, these results provide insight into the use of beta-glucan as a potential activity modulator of macrophage function in IPF and the possibility of its use as a therapeutic. / Thesis / Master of Science (MSc)

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with worsening respiratory symptoms and physiological impairment. Pulmonary fibrosis is a chronic lung disease characterized by forming scar tissue (fibrosis) in the lungs. Alternatively activated macrophages (M2) known as pro-fibrotic are known to contribute to the fibrotic remodeling of the lung. In addition to the polarization of slices from naïve to pro-fibrotic, the addition of anti-fibrotic therapeutics reprogram slices back to a naïve condition. To polarize the slices, naïve slices are incubated with a previously investigated method in the lab known as the polarization cocktail. The polarization cocktail can be achieved by adding of IL-4, IL-6 and IL-13 to naïve(M0) slices in the Precision Cut lung slice (PCLS) model. For the therapeutic model, slices are incubated with the polarization cocktail and subsequently with the therapeutic. Our results have shown that the precision cut lung slice model can mimic previously investigated in-vivo experiments with the polarization cocktail. Secondly, the addition of therapeutics result in the slices exhibiting lower amounts of M2 markers and arginase activity concluding the model is suitable for the polarization and reprogramming of macrophages. / Thesis / Master of Science in Medical Sciences (MSMS)

The cholesterol-lowering effect of mixed linkage (1→3) (1→4)-β-D-glucans (β-glucan) from barley has been documented, yet the underlying mechanism responsible for this action and factors influencing it, such as physicochemical properties of β-glucan and genetic background of an individual, remain unclear.As a component of dietary fibre, β-glucan also has the potential to shift the gut microbial community, however, whether alterations in the gut microbiota are associated with the physiological effects of β-glucan have yet to be determined. This study was designed to assess the effects of β-glucan molecular weight (MW) and dose on loweringserum cholesterol levels and to elucidate its mechanism of action in human subjects. Additionally, this study examined gene-diet interactions as well as changes in the gut microbiota profile following consumption of barley foods. In a controlled four phase crossover trial, mildly hypercholesterolemic but otherwise healthy subjects (n =30) were randomly assigned to receive breakfasts containing 3g high MW (HMW), 5g low molecular weight (LMW), 3g LMW barley β-glucan or a control diet with wheat and rice (WR control), each for 5 weeks. The washout period between the phases was 4 weeks. The consumption of 3g/d HMW diet lowered total cholesterol (TC) compared with WR control diet (P =0.0046), but not the LMW diet at either 3g/d or 5g/d. Individuals with the SNP rs3808607-G allele of CYP7A1 had greater TC reduction in response to 3g/d HMW β-glucan diet compared to the individuals carrying homozygous TT alleles (P<0.01). Cholesterol absorption and synthesis were not changed, but bile acid synthesis increased by 3g/d HMW diet compared to the control. Consuming 3g HMW/d β-glucan altered gut microbiota at the phylum and genus levels and the impacted microbial members was correlated with favorable shifts of cardiovascular disease risk factors. In conclusion, physicochemical properties of β-glucan play critical roles in the cholesterol-lowering effect and gut microbiota alteration ability of β-glucan. The results suggest the increasing bile acid synthesis rather than inhibiting cholesterol absorption and synthesis is the mechanism responsible for the cholesterol reducing property of β-glucan.The altered microbiota profile by HMW β-glucan is associated with its physiological effect. / February 2016