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Hypersensitivity pneumonitis (HP) is a pulmonary disease caused by repetitive exposure to some components of bioaerosols. HP is characterized by neutrophil recruitment in the airways, B cell-enriched tertiary lymphoid tissue formation in the lung and antigen-specific antibody accumulation that can lead to pulmonary fibrosis. HP diagnosis is challenging since causal agent cannot be identified in 30% of cases and there is no distinct clinical sign for the disease. Given the late diagnosis and the inability of current treatment to restore normal lung function in chronic HP, there is no effective treatment for chronic HP. The objective of this thesis was to improve the knowledge on persistent inflammation mechanisms in HP and to identify new therapeutic targets. Sphingosine-1-phosphate receptor 1 (S1P₁) modulators control lymphocyte migration between lymphoid organs and are currently used or under investigation to treat some inflammatory diseases. The objective of the first study was to determine the impact of S1P₁ modulators on experimental HP progression. Mice were exposed three consecutive days a week for three weeks to the immunogen archaea Methanosphaera stadtmanae (MSS) and treated with an S1P₁ agonist, RP001. In experimental HP, RP001 prevents neutrophil recruitment, TLT reactivation and antigen-specific antibody accumulation induced by MSS rechallenge. Because HP is caused by environmental agents, patients are exposed to both T-dependent and T-independent antigens. Thereby, in a second study, the objective was to evaluate the impact of T-independent antigen on experimental HP. In MSS-sensitized mice, lipopolysaccharide (LPS) challenge induces an increase in neutrophilic inflammation, activates pulmonary lymphocytes and induces MSS-specific IgG1 accumulation in the bronchoalveolar lavage. Since we showed that S1P₁ ligands interfere with antigen-specific antibody accumulation, the second objective of this study was to determine the impact of S1P₁ ligands on T-independent antigen-induced B cell activation and functions. An S1P₁ agonist inhibits LPS-induced cytokine release and co-stimulatory molecule increase leading to a decrease in their ability to activate T cells. Even though it is assumed that B cells and their production of soluble mediators are central to HP pathogenesis, few studies were interested in their regulation in this disease. In a third study, the objectives were to characterize circulatory lymphocyte sub-populations in HP and to determine the impact of S1P₁ ligands on human B cell activation induced by T-independent antigens. Venous blood of ten chronic HP patients and ten control subjects paired for age, sex and smoking status were analyzed. The proportion of circulating memory B cells are reduced, and B cell-related soluble mediators are increased in chronic HP patients compared to control subjects, while T cell populations were similar between the two groups. Furthermore, an S1P₁ ligand prevents co-stimulatory molecule increase and cytokine release (TNF, IL-6), which is insensitive to dexamethasone. In conclusion, we unravel circulating B cell alterations in chronic HP patients. We also identify two potential actors implicated in HP pathogenesis, the T-independent response and the S1P₁ modulators. We showed that an S1P₁ agonist prevents the reactivation of experimental HP and inhibits T-independent antigen induced B cell functions. Thus, this thesis improves the knowledge regarding persistent inflammation in HP and identifies the S1P₁ receptor as a potential target to interfere with HP-related B cell functions.

Pancreatic ductal adenocarcinoma is an extremely lethal cancer that is difficult to treat. A better understanding of the biology of pancreatic ductal cancer will help to develop targeted therapies that may improve clinical outcomes. Recently, the lipid signaling molecule sphingosine-1-phosphate (S1P) has emerged as a driver of malignant behavior in many types of cancer. Its role in pancreatic cancer remains unknown. Pancreatic cancer cells express high levels of the S1P receptor known as S1PR1, which is the receptor most important for mediating growth and migration through S1P signaling. In addition, the subcellular expression of the sphingosine kinases is altered in pancreatic cancer cells, which may contribute to their malignant behavior. Exogenous S1P increases pancreatic cancer cell migration, while inhibition of S1P signaling decreases the metabolic activity of pancreatic cancer cells as well as their ability to invade and migrate. Taken together, these results demonstrate the importance of S1P signaling in maintaining malignant behavior in pancreatic cancer cells. In addition, inhibition of S1P signaling represents a potential therapeutic target in pancreatic ductal cancer.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains ix, 109 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Sphingosine kinase 1 (SphK1) is the key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), which is an important signaling molecule that regulates multiple biological process including inflammatory responses. Elevated SphK1 activity as well as upregulated S1P levels is linked to various diseases such as cancer, fibrosis and sickle cell disease. Therefore, there is a growing interest in studying SphK1 as a potential target for these diseases. Through high-throughput screening, various SphK1 inhibitors have been discovered, among which PF-543 is the most potent and selective inhibitor reported to date (Ki=3.6 nM, >100 fold selectivity for SphK1). Previous research indicated that SphK1 inhibitor PF-543 is effective in reducing S1P levels and slowing down the development of sickle cell disease in vivo. However, the lack of in vivo stability of PF-543 still makes it necessary to develop inhibitors with an improved pharmacokinetic profile. In this study, PF-543 was employed as the lead compound, and the influence of different tails groups and head groups on binding affinity and in vivo stability were investigated. In brief, (R)-prolinol-based derivatives with various tail groups including alkyl, alkoxy and biphenyl groups were synthesized. Their inhibition potency was tested in a broken-cell assay, and hit compounds were further evaluated in a yeast cell assay to determine EC50 values. The U937 cell line and mice model were utilized for hit compounds to quantify S1P reduction in vitro and in vivo. Our preliminary results indicated compound 2.14d was the best hit discovered, with 88% SphK1 inhibition at 1 μM. In addition, compound 2.14d with a Ki of 0.68 μM and an EC50 of 0.15 μM, reduced the S1P of U937 cells by 90% at 1 μM. Its analog with a shorter tail group, 2.14a, reduced plasma S1P levels by 20% in mice (10 mg/kg, 3 h). Further modification of the head group of 2.14d produced compound 3.14c bearing a secondary benzylamine head group, with an EC50 value of 0.39 μM and less in vivo activity (14% plasma S1P reduction at 10 mg/kg, 6 h). / Doctor of Philosophy / Sphingosine-1-phosphate (S1P) is a molecule related to various diseases, such as cancers and inflammatory diseases. Elevated levels of S1P promote the development of these diseases, thus making it necessary to reduce the production of S1P in patients. Since S1P is generated in human body by an enzyme called sphingosine kinase (SphK), inhibiting the activity of SphK can be beneficial for reducing S1P levels. Developing inhibitors for SphK is also a promising strategy for curing such diseases. A very potent inhibitor has been reported, but it could be metabolized quickly into other inactive metabolites in human, which renders it ineffective. To develop better drug candidates, a series of compounds with similar structure has been synthesized and tested for their potency and metabolic stability. Based on analysis of the relationship between the compound structures and activities, several compounds with less potency and different metabolic stability has been prepared and their efficacy in reducing S1P levels has been tested.

Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaves 62-68). Also available via the World Wide Web.

Les bases esfingoides, aminoalcohols de cadena llarga, sovint es troben enllaçades a un àcid gras formant les anomenades ceramides. Aquestes ceramides constitueixen la part hidrofòbica essencial dels glicolípids, com -GalCer, iGB3 o KRN7000, que es troben a la membrana de les cèl·lules eucariotes i que participen en processos fisiològics importants involucrats en la prevenció de malalties com infeccions virals, infeccions microbianes, càncer, diabetis, malalties de Parkinson i Alzheimer entre d’altres. Dins el marc de la necessitat de desenvolupar noves metodologies de síntesi d’aquestes bases esfingoides, en la present tesi doctoral s’han desenvolupat tres nous mètodes sintètics, sent els mètodes estereoselectius més eficients fins el moment, basats en reaccions catalitzades per metalls de transició. / Glycosphingolipids are commonly found in eukaryotic cell membranes, plasma membranes and some intracellular organelles. They play a critical role in communication as shown promising bioactivities against diverse pathologies. Glycosphingolipids are constituted by a mono- or oligosaccharide moiety link to the ceramide part, which is a sphingoid base link to a fatty acid. Due to its importance, it is necessary to develop new methodologies to synthesize them. In this thesis, we developed three new synthetic methods based on stereoselective processes catalyzed by transition metal reactions.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [95]-101).

Le remodelage pulmonaire cause une altération de la structure du poumon et peut mener à la diminution de la fonction respiratoire. Ce remodelage peut aussi bien affecter les voies respiratoires en obstruant le flot de l’air, comme il est observé dans l’asthme, que la compliance du parenchyme pulmonaire, tel qu’observé dans la fibrose pulmonaire idiopathique (FPI). Dans le cas de l’asthme allergique, la forme la plus commune d’asthme, une inflammation chronique de type allergique induit le remodelage bronchique, dont l’épaississement du muscle lisse bronchique. Ces deux composantes contribuent directement à l’hyperréactivité bronchique. Dans le cas de la FPI, les dommages tissulaires et l’inflammation semblent être impliqués dans l’induction de la maladie, mais la progression de la FPI est maintenue même en absence d’inflammation. Tant en asthme qu’en FPI, le remodelage est persistant et les options thérapeutiques sont partiellement efficaces, limitées ou absentes. La voie de signalisation de la sphingosine-1-phosphate (S1P) est reconnue non seulement pour son rôle dans la régulation de l’immunité et de l’inflammation, mais aussi pour ses impacts majeurs dans les phénomènes de prolifération et de survie cellulaire. Ces effets sont souvent associés à l’activation de cinq récepteurs membranaires, nommés S1P1 à S1P5. D’ailleurs, ces récepteurs peuvent être activés par les composés pharmacologiques analogues à la S1P. Bien que ces analogues répliquent ou modifient la plupart des effets de la S1P, ils possèdent aussi des activités intracellulaires indépendantes des récepteurs qui sont plutôt associées à la modulation de la survie cellulaire. Ainsi, les sphingolipides et leurs analogues sont susceptibles de moduler plusieurs mécanismes impliqués dans le remodelage pulmonaire. Le but de cette thèse était donc d’explorer l’impact des sphingolipides et leurs analogues sur divers aspects impliqués dans le remodelage pulmonaire. Dans une première étude, nous avons déterminé l’effet de l’analogue de la sphingosine AAL-R sur l’inflammation observée dans un modèle murin d’asthme allergique. AAL-R diminue l’accumulation pulmonaire des lymphocytes et induit leur apoptose au poumon. La réduction du nombre de lymphocytes pulmonaires est associée à une diminution importante de l’éosinophilie et de l’hyperréactivité bronchique. Ainsi, nous résultats montrent qu’AAL-R interfère avec l’inflammation et l’hyperréactivité bronchique dans un modèle d’asthme allergique aigu par un mécanisme impliquant l’apoptose des lymphocytes. Dans un second chapitre, l’impact d’AAL-R sur l’épaississement du muscle lisse bronchique a été évalué dans un modèle murin d’asthme chronique. AAL-R réduit l’épaisseur du muscle lisse bronchique dans le tissu remodelé, ce qui est associé à une diminution de l’hyperréactivité bronchique. Nous montrons d’ailleurs que AAL-R interfère avec la prolifération des cellules musculaires lisses, in vitro. Cette étude montre que des analogues de la sphingosine, tel qu’AALR, pourraient contrecarrer le remodelage tissulaire tel qu’observé en asthme, ce qui ouvre de nouvelles pistes précliniques pour cette maladie incurable. Troisièmement, nous avons étudié l’influence de l’analogue de la sphingosine FTY720 sur la phase inflammatoire ou la phase fibrotique d’un modèle murin de fibrose pulmonaire induit par une blessure cellulaire aiguë. En concordance avec la littérature, l’administration de FTY720 en phase inflammatoire diminue la fibrose pulmonaire. Toutefois, lorsqu’administré en phase fibrotique, FTY720 exacerbe cette dernière. Cette exacerbation ne semble pas impliquer l’augmentation de la perméabilité vasculaire, mais plutôt l’augmentation de la transcription du connective tissue growth factor (CTGF). Similairement, FTY720 stimule la transcription du CTGF par les fibroblastes in vitro. Contrairement au dogme actuel, notre étude montre que FTY720 promeut la fibrose pulmonaire ce qui serait médié, du moins en partie, par la transcription accrue du CTGF. Dans une dernière étude, nous avons exploré le lien entre la voie de signalisation de la S1P, le CTGF et les dérégulations des fibroblastes issus de patients atteints de FPI. Ces fibroblastes expriment plus de CTGF que des fibroblastes de patients ne souffrant pas de FPI, ce qui est exacerbé par la S1P. De plus, un antagoniste de S1P3 renverse partiellement cette augmentation. Nous postulons donc que la S1P et le S1P3 pourraient être impliqués dans la pathogenèse de la FPI. Dans son ensemble, cette thèse démontre que les sphingolipides et leurs analogues influencent des mécanismes du remodelage pulmonaire pathologique. Nos résultats suggèrent que certains acteurs de la voie de signalisation de la S1P pourraient être modulés de manière bénéfique dans le contexte de l’asthme et de la fibrose pulmonaire. / Pulmonary remodelling causes the alteration of the lung structure and can lead to reduced respiratory function. Remodelling can affect the lung airways by obstructing the airflow, as observed in asthma, as well as the lung parenchyma by reducing lung compliance, as observed in idiopathic pulmonary fibrosis (IPF). In the most widespread form of asthma, allergic asthma, chronic allergic inflammation induces lung remodelling, including airway smooth muscle thickening. Both components directly contribute to airway hyperresponsiveness. In IPF, acute lung injury and inflammation seem to be involved in its pathogenesis, yet the progression of the disease is maintained even in the absence of inflammation. Both in asthma and IPF, lung remodeling is persistent and therapeutic treatments are either partially effective, limited or simply unavailable. The sphingosine-1-phosphate (S1P) pathway is recognized not only for its role in the regulation of immunity and inflammation, but also for its major involvement in the events of cellular proliferation and survival. These effects are often associated with the activation of five G protein-coupled receptors, termed S1P1 to S1P5. Furthermore, these receptors can be activated by pharmacological compounds analogous to S1P. Although these analogs replicate or modify most S1P effects, they also possess intracellular activities independent of S1P receptors that are rather associated with the modulation of cell survival. Therefore, sphingolipids and their analogs likely modulate several mechanisms involved in pulmonary remodelling. The aim of this thesis was thus to explore the impact of sphingolipids and their analogs on various aspects of pulmonary remodelling. In a first study, we determined the effect of the sphingosine analog AAL-R on the inflammation observed in a murine model of allergic asthma. AAL-R reduces pulmonary accumulation of lymphocytes and induces their apoptosis specifically in the lung. The reduction of pulmonary lymphocytes number is associated with reduced lung eosinophilia and airway hyperresponsiveness. Our results show that AAL-R interferes with allergic inflammation and airway hyperresponsiveness in an acute allergic asthma model by a mechanism involving the apoptosis of pulmonary lymphocytes. vi In a second chapter, we evaluated the impact of AAL-R on airway smooth muscle thickening using a murine model of chronic asthma. AAL-R reduces the thickness of the airway smooth muscle in remodelled bronchi, which is associated with diminished airway hyperresponsiveness. Furthermore, we show that AAL-R interferes with airway smooth muscle cells proliferation in vitro. This study shows that sphingosines analogs, such as AAL-R, could reverse tissue remodelling as observed in asthma, offering new preclinical targets for this incurable disease. Thirdly, we studied the influence of the sphingosine analog FTY720 on the inflammatory phase or the fibrotic phase of a murine model of pulmonary fibrosis induced by an acute injury. In concordance with literature, FTY720 administration during the inflammatory phase reduce lung fibrosis. However, when administered during the fibrotic phase, FTY720 exacerbates fibrosis. This exacerbation does not involve increased vascular permeability, but rather increased connective tissue growth factor (CTGF) transcription. Similarly, FTY720 stimulates CTGF transcription by fibroblasts in vitro. Contrarily to the actual dogma, our study shows that FTY720 promotes pulmonary fibrosis which is mediated, at least in part, by increased CTGF transcription. Finally, we explored the link between the S1P signalling pathway, the transcription of CTGF and the deregulations observed in lung fibroblasts isolated from IPF patients. These fibroblasts express more CTGF than lung fibroblasts from patient without IPF, which is exacerbated by S1P. Moreover, a specific S1P3 antagonist partially reverses this exacerbation. We postulate that S1P and S1P3 could be involved in idiopathic lung fibrosis pathogenesis. As a whole, this thesis demonstrates that sphingolipids and their analogs influence mechanisms underlying pathological pulmonary remodelling. Our results suggest that certain components of the S1P signalling pathway could prove beneficial in the context of asthma and lung fibrosis. These results support the hypothesis that phosphorylatable sphingosine analogs interfere with inflammation and ASM thickening observed in allergic asthma. However, they also stimulate CTGF transcription by fibroblasts, thus possibly exacerbating pathologies involving these cells, such as IPF. Identification of the mechanisms modulated by S1P analogs could provide insights regarding putative targets in the context of pulmonary remodelling.

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