Th17 cells are implicated in a variety of inflammatory disorders including Spondyloarthritis (SpA). Ankylosing spondylitis (AS), the most common SpA, is genetically associated with HLA-B27 and IL-23R polymorphisms, although the link remains unexplained. In addition to classically folded heterotrimers, HLA-B27 can form β2m-free homodimers (B272). B272, as well as free heavy chains, are able to interact with KIR3DL2. I have shown an expansion of KIR3DL2+ CD4+ T cells in peripheral blood of AS patients and HLA-B27+ healthy controls. Although KIR3DL2+ CD4+ T cells comprise a minority of CD4+ T cells in peripheral blood, KIR3DL2+ CD4+ T cells account for the majority of peripheral blood CD4+ T cell IL-23R expression. KIR3DL2+ CD4+ T cells from synovial fluid are also enriched for IL-23R expression compared to matched peripheral blood samples. Moreover, treatment exposure to rIL-1/23 significantly increases IL-17 production by KIR3DL2+ CD4+ T cells in AS patients but not RA patients and healthy controls. KIR3DL2+ CD4+ T cells from AS patients produce more IL-17 than KIR3DL2+ CD4+ T cells from HLA-B27- healthy controls. KIR3DL2+ CD4+ T cells from AS patients and healthy controls are also enriched for dual production of IL-17 and IFNγ, consistent with the theory that AS is a Th17 or a Th17/1 driven disease. I have shown that naïve CD4+ T cells express KIR3DL2 de novo after activation. Interaction with B272-expressing cells acts to maintain and/or further increase KIR3DL2 expression. Interaction with B272-expressing cells also increases expression of Bcl-2. I propose that interaction of KIR3DL2+ CD4+ T cells with B272 preferentially promotes the survival of these pro-inflammatory cytokine-producing KIR3DL2+ CD4+ T cells in SpA. HD6, a B272-specific monoclonal antibody, decreases IL-17 production by PBMCs from AS patients, making it an attractive candidate therapeutic reagent in the treatment of AS and other HLA-B27-associated SpA.

Immunomodulatory drugs (IMiDs) are a class of drugs, derived from the teratogenic compound thalidomide, that have made a major impact on treatment of many diseases, from multiple myeloma to assorted inflammatory diseases. From its dark beginnings as a teratogenic agent that caused phocomelia in newborn infants, thalidomide has resurged decades later as a potent immunomodulatory agent with widespread anti-inflammatory and anti-tumor effects. Research examining Thalidomide’s effects in vitro on malignant myeloma cells has led to the development of newer analogs, lenalidomide and pomalidomide, both of which are now available on the market. Clinically, these drugs have had a tremendous impact on patient progression-free survival, especially when administered in conjunction with other novel agents. Despite the numerous properties that have been reported for IMiDs, until recently, little was known about their mechanism of action. Knowledge of likely only one of IMiDs’ direct mechanism of action has not only opened up opportunities for additional discoveries, but also propelled research to better characterize genetic profiles of multiple myeloma patients and potential biomarkers of disease progression and response to treatment. This thesis will attempt to review the history and literature behind the biological mechanisms of IMiDs, the clinical risks and benefits of using such drugs as treatment for cancer, and future directions for areas of research.

Engineering therapeutic approaches to wound healing can be divided into two major categories of fibrous and non-fibrous approaches. There has been significant progress in designing artificial skin products to replace autografting. For patients with non-healing/hard-to-heal wounds, there is an unmet clinical need for inexpensive skin substitutes to be transplanted. In skin regeneration area of research, electrospinning is a very commonly used method of production of grafts for wound healing applications, owing its popularity to the fibrous nature of the resultant product, which mimics the extracellular matrix of the native skin. Despite the high degree of porosity in conventional electrospun scaffolds, the small pore size effectively limits the penetration of cells into the scaffold. Transplantation of such scaffolds with poor cell infiltration abilities may lead to a range of negative consequences, from prolongation of the first/destructive phase of inflammation to rejection of the scaffolds. Several experimental approaches have been developed to generate interfibrillar space in the electrospun scaffolds, including but not limited to modifications of the electrospinning set-up and inclusion of sacrificial components. It has been reported that scaffolds with larger pore diameters in the range of ~ 40-100 μm can modulate, moderate and reduce acute inflammatory responses of the body, by influencing macrophages biological behavior, and direct the course of the wound healing process to the tissue remodeling phase. Macrophages are the major cell component of innate immune system and play critical roles in clearance of pathogens, resolution of inflammation and wound healing following an injury. Macrophages are characterized by their diversity and plasticity. In response to environmental stimuli, they acquire different functional phenotypes of pro-inflammatory (M1) or anti-inflammatory (M2). In this thesis, we developed a novel unique gradient porous structure from a plant-based “green” soy protein isolate (SPI) with improved pore size for macrophages to infiltrate. We further showed the ability of the scaffold to modulate phenotype switch in macrophages in vitro and in vivo. The proposed scaffold, moreover, appeared to support transition of the inflammation process from the destructive to the constructive phase in vivo. Based on the promising results of this thesis, we propose our newly developed scaffold has the ability to be used as a new therapeutic modality for treatment of non-healing chronic wounds. / Bioengineering

Immunomodulatory drugs (IMiDs) could enhance both direct anti-tumour and graft-versus-tumour effects after allogeneic haematopoietic stem cell transplantation (AHSCT). However, clinical experience with IMiDs after AHSCT using adult peripheral blood (APB) as a stem-cell source has been limited by graft-versus-host disease. Characterization of the mechanisms by which IMIDs modulate alloresponses of T cells and identification of differential effects on T cells from different cell sources could facilitate more effective use of these drugs in the setting of AHSCT. Using in vitro modelling, multi-parameter flow cytometry and gene expression analysis, I have determined the impact of the widely used IMiD lenalidomide on alloresponses of APB and umbilical cord blood (UCB)-derived T cells. Lenalidomide-treatment potentiates net alloproliferation of APB-derived T cells by selectively enhancing proliferation of CD8+ T cells. These CD8+ T cells have enhanced effector memory differentiation, are enriched for polyfunctional effectors, have enhanced direct-cytotoxicity against heamatopoietic target-cells and have a distinct gene expression profile with altered expression of key immunoregulatory-genes and depletion of cellular ikaros. Importantly, while effects on CD8+ T cells derived from UCB are similar, lenalidomide has contrasting effects on allospecific proliferation of APB and UCB-derived CD4+ T cells. While lenalidomide-treatment has no effect on alloproliferation of APB-derived CD4+ T cells, it reduces alloproliferation of UCB-derived CD4+ T cells. The reduction in UCB-derived CD4+ T cell alloproliferation is accompanied by selective expansion of CD4+CD25+FOXP3+ regulatory T cells (Treg), resulting in an overall reduction in UCB-derived T cell alloproliferation. These findings demonstrate that lenalidomide has a differential impact on alloresponses of T cells from different cell sources; alloresponses of APB-derived T cells are increased via selective expansion of polyfunctional CD8+ effectors, while alloresponses of UCB-derived T cells are limited by expansion of tolerogenic Treg. These findings have important implications for the future use of IMiDs in the setting of AHSCT.

Immunomodulatory effects of lactic acid bacteria vary with strain and may vary with growth phase and medium. The ability of different lactobacilli strains (Lactobacillus helveticus R0052, L. rhamnosus R0011, L. rhamnosus GG) at different growth phases to modulate macrophage and intestinal epithelial cell cytokine production following a pro-inflammatory challenge was examined. Modulation of cytokine production by human macrophage cell lines (U-937) and intestinal epithelial cells (HT-29) induced by Tumor Necrosis Factor α was assayed by ELISA for interleukin-8 (IL-8). Granulocyte-macrophage colony stimulating factor (GM-CSF) production was assayed by ELISA in the HT-29 cell line. Strain-dependent differences were observed in the ability of viable bacteria and spent de Mann-Rogosa- Sharpe (MRS) broths from log versus stationary growth phase in HT-29 and U-937 cells. Overall, variation in the immunomodulatory activity of these lactic acid bacteria and spent broths reflects not only strain variation but potentially also differences in growth phase and substrate. / UOIT

Cationic antimicrobial peptides (CAMPs) are produced by plants, animals and bacteria to protect their host against antagonistic microbes. The antitheses of selective antibiotics, these peptides are drawn by electrostatic and hydrophobic interactions to targets as diverse as the bacterial membrane, nucleic acids and serum proteins. This lack of specificity is their greatest strength, as mutations to single genes rarely lead to bacterial resistance. Resistance may be conferred by large scale alterations in cell envelope composition, which generally reduces bacterial fitness in the absence of peptide. Clinical applications of natural CAMPs are limited, as the peptides are toxic to mammalian cells and rapidly inactivated in vivo by serum albumin and proteases. Faced with these challenges we have prepared a number of CAMP analogues, with the goal of creating lead compounds for further development of antibacterial therapeutics. Much of our work has focused on ultrashort lipopeptides and lipopeptoids, which have properties similar to natural CAMPs and extremely abbreviated sequences. The simple structure of these scaffolds allows rapid creation of CAMP analogues in a brief period of time, allowing us to rapidly explore the structural requirements for CAMP activity. The balance of this work focuses on imparting CAMP-like behaviour to known antibiotics, in order to expand their spectrum of susceptible bacteria and combat the development of drug-resistant bacteria. In particular, the aminoglycosides neomycin and tobramycin have been fused to phenolic disinfectants such as triclosan and biclotymol, in order to improve their diffusion across the bacterial envelope and activity against Gram-negative bacteria.

Cationic antimicrobial peptides (CAMPs) are produced by plants, animals and bacteria to protect their host against antagonistic microbes. The antitheses of selective antibiotics, these peptides are drawn by electrostatic and hydrophobic interactions to targets as diverse as the bacterial membrane, nucleic acids and serum proteins. This lack of specificity is their greatest strength, as mutations to single genes rarely lead to bacterial resistance. Resistance may be conferred by large scale alterations in cell envelope composition, which generally reduces bacterial fitness in the absence of peptide. Clinical applications of natural CAMPs are limited, as the peptides are toxic to mammalian cells and rapidly inactivated in vivo by serum albumin and proteases. Faced with these challenges we have prepared a number of CAMP analogues, with the goal of creating lead compounds for further development of antibacterial therapeutics. Much of our work has focused on ultrashort lipopeptides and lipopeptoids, which have properties similar to natural CAMPs and extremely abbreviated sequences. The simple structure of these scaffolds allows rapid creation of CAMP analogues in a brief period of time, allowing us to rapidly explore the structural requirements for CAMP activity. The balance of this work focuses on imparting CAMP-like behaviour to known antibiotics, in order to expand their spectrum of susceptible bacteria and combat the development of drug-resistant bacteria. In particular, the aminoglycosides neomycin and tobramycin have been fused to phenolic disinfectants such as triclosan and biclotymol, in order to improve their diffusion across the bacterial envelope and activity against Gram-negative bacteria.

This thesis investigated the pharmacology and phytochemistry of Achillea millefolium L. (yarrow) flowers, roots, stems, and leaves based on ethnobotanical reports in North America, with a focus on applications in a respiratory model. Seasonal changes in the phytochemical profile of yarrow were also assessed. A comprehensive dataset of medicinal Asteraceae was created after collecting ethnobotanical reports from the Native American Ethnobotany (NAEB) database. Using residual and binomial analyses, 14 tribes of Asteraceae were quantitatively evaluated and ranked within ten therapeutic categories as either over- or under-selected for treatment by North American indigenous peoples. Flora belonging to the Anthemideae tribe were over-utilized as pulmonary aids, particularly species of Achillea. Yarrow was selected for further analysis in the subsequent chapters of this thesis. The respiratory pharmacology of yarrow was examined by testing the immunomodulatory effects of four plant parts in an in vitro assay using BEAS-2B human bronchial epithelial cells. Concentrations of the pro-inflammatory cytokines IL-6 and IL-8 were quantified using ELISA kits. Flowers demonstrated significant anti-inflammatory activity at 40 μg/ml in both assays, and also at 20 μg/ml in the IL-8 assay, suggesting a dose-dependent response. Roots displayed significant pro-inflammatory activity at all concentrations. A second mechanism of action via the endocannabinoid system was tested through inhibitory enzyme assays for fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), in which the flowers and roots were most active. Since extracts of the four plant parts exhibited significantly different bioactivities, active metabolites previously identified in yarrow were quantified in each part through the targeted profiling of phenolics and alkylamides using analytical chromatographic techniques. Phenolic compounds were found at highest concentrations in the flowerheads, while alkylamides were detected predominantly within roots. An accompanying phenological analysis of alkylamide and phenolic levels in all parts was explored. Collectively, this research provides the first integrated comparison of yarrow ethnobotany, bioactivity, and phytochemistry across different parts of the plant, contributing novel insights into the traditional, contemporary, and future uses of one of North America’s most important medicinal plants.

Made available in DSpace on 2014-06-11T19:23:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-07Bitstream added on 2014-06-13T20:49:53Z : No. of bitstreams: 1 orsatti_cl_me_botib.pdf: 325043 bytes, checksum: 59d94b66f5ac476afaa842ccd0fa808e (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A própolis tem sido motivo de intensa investigação científica e sua ação imunomoduladora vem sendo mencionada. A recente descoberta e caracterização da família dos receptores semelhantes a Toll (TLR) tem desencadeado grande interesse no campo da imunidade inata. Tal interesse deve-se ao fato de estes receptores proporcionarem um papel vital no reconhecimento microbiano e no desenvolvimento da resposta imune adaptativa. Tendo em vista que há muito a ser investigado no tocante à relação imunomodulação-TLR, o objetivo deste projeto foi avaliar o efeito da própolis na expressão dos mesmos, bem como determinar a produção de citocinas próinflamatórias (IL-1b e IL-6) por macrófagos e esplenócitos de camundongos BALB/c. A expressão e a produção de citocinas do perfil Th1 (IL-2 e IFN-g) e Th2 (IL-4 e IL-10) por esplenócitos também foram avaliadas. A própolis induziu aumento na produção basal de IL-1b e na expressão de TLR-2 e TLR-4 em macrófagos peritoneais. A expressão de TLR-2 e TLR-4 e a produção de IL-1b e IL-6 também apresentaram valores aumentados em esplenócitos de camundongos tratados com própolis. Estes dados sugerem que a administração de própolis a curto prazo a camundongos pode estimular os eventos iniciais da resposta imune. A própolis não afetou a expressão e produção de IL-2, IL-4 e IL-10; contudo, a produção basal e estimulada de IFN-g foi inibida após a administração deste produto apícola, o que sugere seu efeito antiinflamatório in vivo / Propolis has been the subject of intense scientific research and its immunomodulatory action has been mentioned. The recent discovery and characterization of Toll like receptors (TLR) has triggered a great interest in the field of innate immunity. This interest is due to the fact that these receptors provide a vital role in microbial recognition and development of the adaptive immune response. Considering that there is much to be investigated regarding the relationship TLRimmunomodulation, the goal of this project was to evaluate propolis effect on TLR-2 and TLR-4 expression, and to determine the production of proinflammatory cytokines (IL-1b and IL-6), in macrophages and spleen cells from BALB/c mice. Th1 (IL-2 and IFN-g) and Th2 (IL-4 and IL-10) cytokines’ expression and production were also assessed in spleen cells. IL-1b basal production and TLR-2 and TLR-4 expression were increased in peritoneal macrophages of mice treated with propolis. TLR-2 and TLR-4 expression and IL-1b and IL-6 production were also increased in spleen cells of propolis-treated mice. These data suggest that propolis administration over a short-term to mice stimulates the initial steps of the immune response. Propolis did not affect IL-2, IL-4 and IL-10 expression and production, but both basal and Con A-stimulated IFN-g production were inhibited after propolis administration, what suggests its antiinflammatory action in vivo

Orientador: José Maurício Sforcin / Banca: Renata Dellalibera-Joveliano / Banca: Maria Terezinha Serrão Peraçoli / Resumo: A própolis tem sido motivo de intensa investigação científica e sua ação imunomoduladora vem sendo mencionada. A recente descoberta e caracterização da família dos receptores semelhantes a Toll (TLR) tem desencadeado grande interesse no campo da imunidade inata. Tal interesse deve-se ao fato de estes receptores proporcionarem um papel vital no reconhecimento microbiano e no desenvolvimento da resposta imune adaptativa. Tendo em vista que há muito a ser investigado no tocante à relação imunomodulação-TLR, o objetivo deste projeto foi avaliar o efeito da própolis na expressão dos mesmos, bem como determinar a produção de citocinas próinflamatórias (IL-1b e IL-6) por macrófagos e esplenócitos de camundongos BALB/c. A expressão e a produção de citocinas do perfil Th1 (IL-2 e IFN-g) e Th2 (IL-4 e IL-10) por esplenócitos também foram avaliadas. A própolis induziu aumento na produção basal de IL-1b e na expressão de TLR-2 e TLR-4 em macrófagos peritoneais. A expressão de TLR-2 e TLR-4 e a produção de IL-1b e IL-6 também apresentaram valores aumentados em esplenócitos de camundongos tratados com própolis. Estes dados sugerem que a administração de própolis a curto prazo a camundongos pode estimular os eventos iniciais da resposta imune. A própolis não afetou a expressão e produção de IL-2, IL-4 e IL-10; contudo, a produção basal e estimulada de IFN-g foi inibida após a administração deste produto apícola, o que sugere seu efeito antiinflamatório in vivo / Abstract: Propolis has been the subject of intense scientific research and its immunomodulatory action has been mentioned. The recent discovery and characterization of Toll like receptors (TLR) has triggered a great interest in the field of innate immunity. This interest is due to the fact that these receptors provide a vital role in microbial recognition and development of the adaptive immune response. Considering that there is much to be investigated regarding the relationship TLRimmunomodulation, the goal of this project was to evaluate propolis effect on TLR-2 and TLR-4 expression, and to determine the production of proinflammatory cytokines (IL-1b and IL-6), in macrophages and spleen cells from BALB/c mice. Th1 (IL-2 and IFN-g) and Th2 (IL-4 and IL-10) cytokines' expression and production were also assessed in spleen cells. IL-1b basal production and TLR-2 and TLR-4 expression were increased in peritoneal macrophages of mice treated with propolis. TLR-2 and TLR-4 expression and IL-1b and IL-6 production were also increased in spleen cells of propolis-treated mice. These data suggest that propolis administration over a short-term to mice stimulates the initial steps of the immune response. Propolis did not affect IL-2, IL-4 and IL-10 expression and production, but both basal and Con A-stimulated IFN-g production were inhibited after propolis administration, what suggests its antiinflammatory action in vivo / Mestre